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MICR2024 Practical Manual 2017
Contents
Introduction – Microbes in the Environment ……………………………………………………………………………. 2
Overview ……………………………………………………………………………………………………………………………. 3
Practical timetable ……………………………………………………………………………………………………………….. 4
List of Exercises ………………………………………………………………………………………………………………….. 6
Practical Course Objectives ………………………………………………………………………………………………….. 7
Course Structure ………………………………………………………………………………………………………………….. 7
Laboratory Rules …………………………………………………………………………………………………………………. 8
Assessment …………………………………………………………………………………………………………………………. 9
Textbooks …………………………………………………………………………………………………………………………. 11
Equipment Requirements ……………………………………………………………………………………………………. 12
PRACTICAL WEEK 1 ………………………………………………………………………………………………………. 13
PRACTICAL WEEK 2 ………………………………………………………………………………………………………. 23
PRACTICAL WEEK 3 ………………………………………………………………………………………………………. 49
PRACTICAL WEEK 4 ………………………………………………………………………………………………………. 67
PRACTICAL WEEK 5 ………………………………………………………………………………………………………. 79
PRACTICAL WEEK 6 ………………………………………………………………………………………………………. 86
PRACTICAL WEEK 7 ………………………………………………………………………………………………………. 94
WEEKS 8-13: Forensic Microbiology Project ………………………………………………………………………. 96
Appendix 1– Microbial growth media ………………………………………………………………………………… 104
Appendix 2 – Use and care of the microscope ……………………………………………………………………… 106
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Introduction – Microbes in the Environment
Microbiology is a very broad scientific discipline. This is due to the ubiquity of microorganisms and
their incredible physiological diversity. Microorganisms influence virtually all aspects of life on our
planet, playing vital roles in food production, environmental health, human health and a surprisingly
wide range of industries. Microbiologists find employment in fields as diverse as health,
environmental engineering (especially wastewater), mining, chemical engineering, pharmaceuticals,
food processing, agriculture, aquaculture and many more. Despite the very wide range of applications
for microbiology that already exist, one of the most exciting aspects of microbiology is how much
we don’t know.
The defining feature of microorganisms is that they are small. Historically this has presented two
distinct technological barriers to study of microbes:
(1) They are hard to see. The invention of the microscope marks the foundation of microbiology as a
scientific field.
(2) They are hard to characterize. There is limited morphological diversity observable with a
microscope. They do show amazing physiological diversity, but physiological processes that occur
within a single cell are at rates that are typically below the sensitivity of detection of biochemical
techniques. The development of pure culture techniques provided a means to perform physiological
tests and the basis of a classification system. Collectively, microscopy and pure culture techniques
opened the door to the world of microorganisms and led to the diverse applications that we currently
see.
However, there is also a third technical barrier to the study of microorganisms –
(3) They are difficult to isolate in pure culture. Over the past 100 years tens of thousands of microbial
strains have been isolated and thousands of species described. This global culture collection includes
the causative agents of major infectious diseases and microbial strains that support multi-billion dollar
industries. The physiological and biochemical diversity of these cultivated microorganisms exceeds
that of all the plants and animals put together. Nevertheless, environmental microbiologists have
always been aware that this is just the tip of the iceberg. Even in exhaustively studied environments
such as the human body it is estimated that around 70% of the microbes are still unknown. In soil
environments the proportion isolated in pure culture is less than 1% and in some marine environments
it is as low as 0.001%. If the biotechnological value and importance of the organisms we know about
is great, imagine the opportunities that will emerge when we finally explore the great bulk of
microbial diversity.
This intermediate microbiology subject is aimed at introducing you to the field of microbiology with
an emphasis on environmental (non-clinical) aspects of the discipline; particularly soil, water,
agricultural and veterinary microbiology. In this series of practicals you will learn a number of
fundamental concepts and techniques of microbiology. You will see how the techniques of
microscopy and pure culture opened the field of microbiology and can be applied to either laboratory
characterization or environmental surveys of microorganisms. We will examine various approaches
for selective recovery of physiologically diverse microorganisms from natural environments with a
focus on those that may have industrially important properties such as antibiotic production or
nitrogen fixation. You will see some of the limitations to recovering all microorganisms in pure
culture. Finally you will carry out a mini research project aimed at identifying a pathogen.
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Overview
LABORATORY RULES
You are expected to have completed the pre-lab tasks (reading and questions) before practical
classes in weeks 2, 3, 4, 6 and 8.
To comply with occupational, health and safety rules it is compulsory to wear a lab coat and
covered footwear for all classes. You will be asked to leave the class if you fail to comply.
All microorganisms should be treated as potential pathogens. Follow directions for disposal of
microbiological waste carefully. Report all spills immediately to your demonstrator.
ASSESSMENT:
The practicals contribute a total of 40% to the assessment in this unit of study.
Peerwise assessment questions. All students will make use of the Peerwise web tool for creation,
sharing, evaluation and discussion of assessment questions. During the semester you will be
required to:
• create at least two multiple choice questions in Peerwise, and
• answer at least ten multiple choice questions in Peerwise.
The Peerwise tool is a very productive way to revise and share your knowledge. Although
participation in Peerwise is not marked, students who do not meet the requirements above will not
receive marks for the online quizzes below.
Lab quizzes (15%) – In weeks 5 and 10 there will be short online quizzes on the practical material,
which must be completed BEFORE the practical session in each week. Note that if the Peerwise
requirement above is not completed, students will receive 0 % for the Lab quizzes
Project report (20%) – In week 13 you must submit a scientific report on your project. This will
be submitted online through Blackboard, and will be assessed using plagiarism detection software
to check for originality.
Skills Competency (5%) – You will be assessed on your microscopy, aseptic technique and data
recording skills throughout the semester.
LAB MANUAL ORGANIZATION
For each week’s class there is:
(1) A checklist that describes which exercises (or follow-up activities) will be performed and
on what pages you will find the exercise described.
(2) A list of the AIMS of the exercises being commenced in this practical.
(3) A list of any pre-lab tasks that should be completed BEFORE the practical class.
For each exercise:
(1) The complete exercise is described in the week where it is started. In order to complete
follow-up parts of the exercise in subsequent weeks you must refer back to the pages in the
manual where the exercise is described.
(2) There are a series of questions that you should answer in the practical class. You will need
to have completed your pre-lab tasks, as well as the practical to answer these questions.
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Practical timetable
Material highlighted by shading should be covered before class
Practical Exercises Page
Week 1 Introduction to course, assignation of lab space, microbiological safety
A Microscopy (overview)
A1: Setting up the microscope and examination of fixed specimens
Phase contrast microscopy
B Winogradsky column (overview)
B1: Set up column
14, Appendix
14
16
31 July
Week 2 PRE-LAB TASKS
A Microscopy (cont’d)
A2: Preparation of material for microscopy: wet mount, Gram stain
B Winogradsky column (cont’d)
B2: Day 7 observations (discuss cellulose breakdown)
C Macroscopic examination of microbes (overview)
D Aseptic technique (overview)
D1: Inoculating media in tubes
D2: Inoculating media on plates
E Environment, growth limits and sterilisation (overview)
E1: Physical factors limiting growth (Demonstration)
E2: Sterilisation techniques (Demonstration)
E3: General principles of media for microbial growth (Demonstration)
24
25
25
19
29
33
34
33
37
37
40
42
7 Aug
Week 3 PRE-LAB TASKS
B Winogradsky column (cont’d)
B2: Day 14 observations (discuss sulphate reduction)
D Aseptic technique (cont’d)
D1 and D2: examination of plates – FOLLOW-UP
F Distribution of microbes in the environment (overview)
F1: Miniproject – enrichment and isolation of distinctive bacteria
A: Halotolerant organism (Staphylococcus)
B: Nitrogen fixer (Azotobacter, Beijerinckia)
C: Cellulose-degrader (Cytophaga)
D: Methylotrophic organism (Methylobacterium)
F2: Isolation of plant-associated microbes
50
19
36
51
51
63
14 Aug
Week 4 PRE-LAB TASKS
B Winogradsky column (cont’d)
B2: Day 21 observations (discuss oxygenic and facultative phototrophy)
F Distribution of microbes in the environment (cont’d)
F1: examine plates and transfer of enrichment cultures – FOLLOW-UP
F2:– examination of colonies on plates FOLLOW-UP
G Enumerating microorganisms (overview)
G1: Viable count of pure culture
G2: Direct count (Demonstration)
G3: Viable counts of soil bacteria and effect of media
68
20
56
64
69
70
71
74
21 Aug
Week 5 QUIZ 1
PRE-LAB TASKS
B Winogradsky column (cont’d)
B2: Day 28 observations (discuss purple and green sulphur bacteria)
F Distribution of microbes in the environment (cont’d)
F1: Plate out from enrichments – FOLLOW-UP
G Enumerating microorganisms (cont’d)
G1 and G3: Count colonies on plates – FOLLOW-UP
G4: Growth curve in liquid batch culture
G5: Enumeration of indicator bacteria in water samples (Demonstration)
80
20
57
71
81
84
28 Aug
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Week 6 PRE-LAB TASKS
B Winogradsky column (cont’d)
B2: Day 35 (final) observations
F Distribution and diversity of microbes in the environment (cont’d)
F1: Characterization of isolates from plates and enrichments– FOLLOW-UP
F1: PCR amplification of 16S gene of single isolates
F1: Group data collation
H Antagonism and antibiosis
H1: Chaetomium subculture
H2: Soil dilutions and Actinomycete plating.
87
20
58
60
91
4 Sep
Week 7
F Distribution and diversity of microbes in the environment (cont’d)
F1: Group discussion (if time permits)
H Antagonism and antibiosis (cont’d)
H1: Inoculate cellophane plates with susceptible fungus – FOLLOW-UP
H2: Inoculate Actinomycetes with agar overlay – FOLLOW-UP
PROJECT: Isolation of a pathogen & Koch’s Postulates, including introductory tutorial
90
92
96
11 Sep
Week 8 PRE-LAB TASKS
H Antagonism and antibiosis (cont’d)
H1 and H2: Examine plates – FOLLOW-UP
Project: isolation of a pathogen & Koch’s Postulates (cont’d)
90, 92
93-100
18 Sep
Mid semester break – No Practical this week
Week 9
2 Oct
Public Holiday (Labour Day)
Week 10 QUIZ 2
Project: isolation of a pathogen & Koch’s Postulates (cont’d)
Tutorial : Features of A Good Report
96-103
9 Oct
Week 11
16 Oct
Project: isolation of a pathogen & Koch’s Postulates (cont’d)
Tutorial: Setting Criteria for Report
96-103
Week 12
23 Oct
Project: isolation of a pathogen & Koch’s Postulates
96-103
Week 13
30 Oct
Project: Submission of report via Blackboard 102
Lab Quizzes
Lab Quizzes will take place in weeks 5 and 10. These are online multiple choice quizzes in
Blackboard, which will be made available one week before the cut-off date and must be completed
BEFORE the start of the practical session on the appropriate date. The quiz content will be as
follows:
Quiz 1 – Exercises A, C, D, E, F
Quiz 2 – all exercises, but especially Exercises B, F and H.
The Quizzes will test understanding of background and results for the relevant exercises. Make sure
you have read the Practical Handbook and the Background material available on the Blackboard
site, and be prepared to think about your results in the context of this background. Writing your own
Peerwise questions is a great way to revise this material, and answering the shared questions from
other students will help make you confident in your own knowledge.
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List of Exercises
Exercise Weeks
A. Microscopy: Setting up microscope, looking at fixed specimens,
preparation of ‘live’ specimens (wet mount), simple stains.
1 and 2
B. Winogradsky column 1 to 6
C. Macroscopic examination of microbes: Colony morphology of bacteria
and fungi
2
D. Aseptic technique: Inoculation of broth, inoculation of plate, streak
dilution plates.
2 and 3
E. Environment, growth limits and sterilization: Effect of carbon source,
electron acceptor. Effect of oxygen. Effect of temperature. Effect of pH.
Sterilization techniques.
2
F. Distribution of microbes in the environment: Targeted enrichment
culture for distinctive microbes in diverse habitats. Isolation of microbes.
Preliminary characterization of isolates and 16S rRNA gene PCR/sequencing.
Skills test
3, 4, 5, and 6
G: Enumerating Microorganisms: Plate counts of soil bacteria on different
media, plate counts of pure culture, Direct count, MPN counts for coliforms
from water, Growth curve.
4 and 5
H: Antibiosis and antagonism: Preliminary characterization of endophyte
antagonism of plant pathogen. Isolation of antibiotic-producing soil bacteria.
6, 7, and 8
PROJECT: Forensic Microbiology: Isolation of a pathogen from a
‘diseased environment’ and fulfilment of Koch’s postulates.
7 and then weekly
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Practical Course Objectives
General objective: To introduce the student to the world of microorganisms and to provide
familiarity with the basic techniques that are necessary for their study.
Specific objectives:
1. To train the student in the skills necessary for the handling of micro-organisms in a safe and
competent manner.
2. To illustrate and reinforce the basic concepts of microbiology, which are examined in the lecture
course.
3. To develop an understanding of the interrelationships between microorganisms and other forms of
life.
4. To train the student in the design, implementation and interpretation of scientific experiments.
To these objectives the practical course divides into several sections:
• basic techniques
• isolation and identification procedures
• growth of microorganisms and control of growth
• microbial ecology – interactions in soil, water, food, health and disease.
Course Structure
1. PRACTICAL SESSIONS:
The practical classes are held each week on Mondays in ATP Biomedical Building Lab 240. Two
sessions will be run each week, from 12-3 pm and from 3-6 pm. Students will be allocated to one
of the two sessions. Because experiments continue from week to week, it will not be possible for
students to switch sessions during the term.
All practical classes will begin with a brief introductory talk explaining:
• what is to be done
• rationale and theoretical considerations
• location of materials
• safety warnings and precautions
• continuous assessment instructions
It is therefore imperative that all students are present and punctual for this pre-laboratory talk. Feel
free to ask questions to clarify any of the procedures described.
2. WORKING UNITS: Experiments are generally performed in groups of five students. Each group
should divide the workload among themselves, ensuring that each member is completely familiar
with what the others are doing. Make sure that results are exchanged and seen by all members of
the group.
3. DEMONSTRATORS: Two to three demonstrators are available at all times. They are not
allocated to specific groups, but circulate throughout the laboratory. Consult your demonstrator if
you have any difficulties understanding the method or object of a particular exercise. They are
there to answer your questions.
4. PLANNING: In order to use laboratory time effectively, it is essential that the appropriate section
of the practical book be read PRIOR to class.
5. PRE LAB TASKS: To ensure that you gain maximum benefit from the laboratory work and to
encourage preparation for prac classes a pre-lab task has been set. This must be completed prior
to coming to class. A complete set of answers will be available on the course Blackboard website
following the practical class.
6. QUESTIONS: Each exercise includes a number of questions. These should be answered during
the class as the exercise is completed.
NB The prelab tasks and questions are not directly assessed. However completion of these tasks is
essential to provide study material for the lab quizzes. These quizzes are worth a total of 15% and
you will be need to use your lab manual (with the previously answered questions) as a reference in
the quiz.
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Laboratory Rules
Many of the microorganisms that will be studied in class can cause disease if mishandled. Therefore,
great care should be taken in handling cultures, slides and other materials that have been in contact
with living microorganisms. The following precautions should be observed for the safety of everyone
working in the laboratory:
PROTECTIVE CLOTHING AND DRESS
1. A laboratory coat must be worn at all times in the laboratory. It should be donned on entry to
the laboratory and removed on leaving to reduce the risk of contaminating clothing.
2. Substantial shoes must be worn. Thongs and some sandals are unsatisfactory protection and
should not be worn in the laboratory.
3. Long hair should be tied back to reduce the risk of accidental burning in a Bunsen flame.
SAFETY CONSIDERATIONS
1. Do not eat, drink or smoke in the laboratory and never place pencils, pens, labels or other
materials in the mouth.
2. Mouth pipetting of liquids is banned. A rubber teat or filler must be used at all times.
3. Store bags, coats, umbrellas etc. away from the work area.
4. Cultures are never to be taken from the laboratory.
5. Inoculated media must be properly labelled (i.e. with name, date and the nature of the specimen)
and put in the appropriate box for incubation.
6. Do not sit on benches.
7. Turn gas burners down or off when not in use during the laboratory period to keep the laboratory
as cool as possible.
8. Any personal accident must be reported to a demonstrator immediately.
9. Any spillage of culture material must be reported to a demonstrator immediately so that
appropriate action may be taken.
10. At beginning of each Practical, the work area must be scrubbed with disinfectant to minimise
contamination.
11. At the end of each Practical:
(a) Discard all used tubes, pipettes, Petri dishes etc. into the designated receptacles.
(b) Clear the bench top of all equipment except stain bottle rack, pipette canister and
disinfectant bottle.
(c) Sponge bench top with disinfectant.
(d) Wash hands with the skin disinfectant supplied before leaving the laboratory at any stage.
(e)
12 Ensure that you know where the nearest emergency exit is.
NB Failure to compliance with all laboratory rules will result in penalty marks.
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Assessment
The assessment for this course consists of the following components:
1. Theory paper (2 hrs) 60%
2. Practical 40% total
Composed of:
Quizzes 15%
Skills and competency 5%
Project report 20%
1. THEORY PAPER
The theory examination will take place in the examination period at the end of the Semester.
2. PRACTICAL COMPONENT
(i). Lab Quizzes (15%):
(a) Quizzes will be available on the Blackboard site for one week before each of the indicated
dates in the timetable. Cut-off time for quiz submission is the beginning of the first prac
session (12 pm). Feedback on the quiz answers will be provided on Blackboard, and in
discussion with demonstrators during the practical class.
(b) Questions in the quizzes may pertain to the theoretical background (pre-lab reading) as
well as the results of each exercise.
(c) The quizzes are “open book” assessments and a lab manual in which pre-lab tasks and
exercise questions have been completed will be an invaluable resource.
(d) A zero mark will be allocated to a student missing any quiz for other than a valid medical
reason or as a result of serious misadventure.
(e) Each quiz is worth equal value (Blackboard allocates different total marks for different
quizzes depending on the number of questions – the overall % mark for each quiz will be used
for assessment purposes).
(f) Students are required to contribute at least two shared questions to the Peerwise
assessments tool during the term, and to answer at least ten shared questions. Students who
fail to fulfil this minimum requirement will be allocated a zero mark for the Lab Quizzes.
(ii). Skills and Competency (5%)
Exercise F and the Forensic Project involves mini-projects where you will isolate and
characterise bacteria and fungi. You will be assessed on your technical competency in (i)
microscopy of fungi, (ii) microscopy of bacteria, (iii) aseptic transfer of fungi, (iv) aseptic
transfer of bacteria.
(iii). Project Report (20%)
In the last five weeks of the practical class you will conduct a microbiology research project.
You must submit a written report in the style of a scientific paper in the last week. Further
details on the assessment criteria will be given in tutorials.
(iv). Practical Manuals
Your practical manual should be brought to all sessions and may be examined by the
demonstrator at any practical session. The pre-lab tasks should be completed before each
practical. This includes answering the questions in the spaces in the manual. You may find it
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useful to incorporate photocopies or printouts of additional reading material into the manual.
Accurate and detailed experimental results should be recorded directly into the spaces
provided. Each exercise is to be completely written up by the practical class following the
completion of the exercise. This includes answering the associated questions. It is strongly
recommended that you answer the questions at the time of writing up the exercise. The
questions are designed to help reach conclusions as well as to stimulate discussion. Answers
to these questions are available on the Blackboard site. Your records of practical work
should be complete and up to date at all times. The results and discussion material in
your lab manual will be necessary to answer some quiz questions.
(3) PENALTIES
(i). Lateness of work due (without documented explanation).
Reports – Reduction of awarded mark by 5% for each day overdue.
(ii). Cheating (ie copying another student’s work without acknowledgment.). Project Reports
will all be analysed with Plagiarism detection software via Blackboard. If plagiarism is
detected, students will be reported to the University, in accordance with the University’s
Academic Honesty in Coursework Policy 2015.
(iii). Absences. Nonsubmission of any quiz, continuous assessment task etc which is not
covered by an approved special consideration request will result in a zero mark being
recorded.
What to do if you miss a practical class
(i) It is your responsibility to obtain all information and results for the class and to fill in your
Practical Manual. Where new techniques have been taught, you must take steps to learn the
necessary skills. Make sure that you understand all the material covered during your
absence, since it may be assessed later in the course.
(ii) Inform your demonstrator of the reason for your absence and, if necessary, submit a Special
Consideration form to the unit co-ordinator.
(iii) If you have missed two or more practicals, or been affected in your performance in an exam
or assessable task, it is necessary to submit an Application for Special Consideration.
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Textbooks
The recommended textbooks for the course are:
• Willey, J. M., Sherwood, L. M., and Woolverton, C. J. (2014) Prescott’s Microbiology.
9
th
edition, McGraw-Hill
.
Other useful Microbiology textbooks are:
• Madigan, M. T. and Martinko, J. M. (2006) Brock Biology of Microorganisms 11
th
edition, Prentice-Hall.
•
Most topics in the course are adequately covered in these texts. Occasionally, however, a lecturer
may need to rely on other specialised texts. In this case you will be clearly advised and the text will
be available on “closed reserve” in Badham and/or SciTech libraries. Alternatively you will be
supplied with printed handouts.
Students wishing to read beyond the recommended text are directed to the following:
• Adl, S. M. (2003) The ecology of soil decomposition. CABI Publishing. Also available as
an e-book from the University Library website.
• Schumann, G. L. and D’Arcy, C. J. (2010) Essential Plant Pathology, 2
nd
edition, APS Press.
• Waller, J. M., Lenné, J. M. and Waller, S. J. (2003) Plant Pathologists Pocketbook, 3rd
edition,
CABI Publishing. Also available as an e-book from the University Library website.
• Holt, J. G. (1994) Bergey’s manual of determinative bacteriology, 9th edition, Williams
and Wilkins.
The Microbewiki. Maintained by Kenyon College, Ohio.
http://microbewiki.kenyon.edu/index.php/MicrobeWiki
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Equipment Requirements
Supplies Furnished by the Student:
Note book
Laboratory Coat
Camera or mobile phone with camera function.
Equipment provided in your kit box in the laboratory:
1 x plastic jar distilled water
1 x plastic jar 70% v/v alcohol
1 x box tissues
1 x chopping board
1 x box matches
1 x box slides
1 x box cover slides
1 x inoculating needle holder
2 x inoculating loops
1 x mounted needle
1 pair forceps
1 x scalpel handle with blade (covered)
2 x plastic Pasteur pipettes
1 x razor blade
1 x permanent marker pen
Permanent equipment in the laboratory:
Compound microscopes
Dissecting microscopes
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PRACTICAL WEEK 1
Exercise A Microscopy
A1 Use and care of the microscope – Bright field microscopy
Exercise B Winogradsky Column
AIMS
After completing this week’s practical the student should:
1. Know the basic safety rules for a microbiology laboratory
2. Be aware of the requirement to fulfil pre-lab tasks before each weeks practical class.
3. Have a basic appreciation of the use and care of a bright-field microscope.
After completion of Exercise A the student should:
4. Understand that different types of microscopes, or different microscopic techniques on the same
microscope, are used for different tasks in microbiology.
5. Be aware of the level of detail needed in collecting and recording microscopic observations –
this skill will be tested in exercise F.
After completion of Exercise B the student should:
6. Appreciate the concept of ecological succession.
7. Be able to describe the roles of sulphate reducers and anoxygenic phototrophs in the
biogeochemical cycle of sulphur.
8. Be able to use examples from the Winogradsky column system to explain the spatial and
temporal distribution of microbes in different habitats.
9. Have developed skills in recording and interpreting macroscopic observations in terms of
microbiological phenomena – these skills are essential for the project in MICR2024.
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Exercise A1
THE MICROSCOPE
————————————————————————————————————————Microorganisms may be defined as living organisms which are invisible to the unaided human eye.
The smallest distance between two objects which can be resolved by the unaided eye is about 0.1 mm
(100 µm). The majority of microorganisms are smaller than this and in order to see them as distinct
objects it is necessary to use a microscope. The light microscope (bright field use) can resolve objects
down to 0.2 µm in diameter and this includes all bacteria, protozoa, algae and fungi, but not viruses,
most of which are smaller and can only be seen with the electron microscope.
Description of the Microscope
The microscope consists of the following parts:
1. The stand – made up of a heavy base which carries a curved limb bearing the observation tube
and a stage. At the bottom of the observation tube is a revolving nosepiece which carries the
objective lenses.
2. Light source – is a built in lamp equipped with a field iris diaphragm.
3. The optical system consists of three parts:
(a) Substage condenser fitted with an iris diaphragm – the function of the condenser is to collect
the light from the wide source produced by the electric bulb and condense it to provide an
intense cone of light just wide enough to fill the objective being used. The width of the cone
is controlled by the sub-stage iris diaphragm ie controls the Numerical aperture of the
condenser.
(b) Three objective lenses:
– Low power lens, magnifies 10X. This lens is used to screen the specimen to locate
the bacteria.
– High Power lens, magnifies 40X. This lens is used to examine large
microorganisms such as fungi and yeasts and is often used for wet mounts of
unstained viable organisms with a coverslip.
– Oil immersion lens magnifies 100X. This lens is used to examine bacteria.
(c) Eyepiece lens at the upper end of the observation tube. It usually has a magnification of
10X. The total magnification of the optical system is obtained by multiplying the
magnification of the objective by that of the eye piece.
4. Focussing mechanisms – There are three mechanisms used for focussing:
(a) Course adjustment for raising or lowering the stage.
(b) Fine adjustment for raising or lowering the stage.
(c) Knob for raising or lowering the substage condenser.
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Method
I. EXAMINE THE MICROSCOPE AND FAMILIARISE YOURSELF WITH THE
VARIOUS PARTS (DEMO/REVISION)
The Abbé condenser – receives the light and is used to focus and centre it on the object.
The iris diaphragm of the condenser – controls the N.A. of the optical system (ie of the
condenser).
The objective lenses – give the initial magnification to the object being viewed.
Three objectives provided are –Low power (10X), High power (40X) and Oil immersion
(100X)
The ocular or eyepiece – magnifies the image formed by the objective lens. This double
magnification accounts for the term ‘compound microscope’.
Lamp switch and light intensity control – on/off switch for light source. Light intensity must
be increased as the power of the objective increases. Use this switch not the iris diaphragm.
II MICROSCOPIC EXAMINATION OF SPECIMENS.
(1) Refer to the Appendix (page 104), for notes on the optics and setting up the microscope.
(2) Examine the specimens provided.
(3) Record your observations for each specimen.
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III Phase Contrast Microscopy
Various structures of the cell, the cell itself, and the background have different refractive
indices.
The rays of light passing through the object are retarded a fraction of a wavelength compared
with the rays passing through the suspending medium. This produces a difference in ‘phase’
between the two types of emerging rays. The phase contrast microscope converts refractive
index differences (ie differences in phase) into differences in intensity (differences in
amplitude) so producing light and dark contrast in the image. Thus transparent objects show
contrast where differences in refractive index or thickness occur.
The phase set up is used for unstained preparations and has clarified much that had previously
been inferred from killed and stained specimens, allowing distinction between fine structure
and artefacts caused by staining. One of the main advantages is that it allows the observation of
living cells.
Compare the images viewed with and without phase contrast (not all the microscopes have
phase contrast available – ask your demonstrator). Record your observations in the table
below.
Results (phase contrast microscopy)
SAMPLE Observations
(IV) Dark Field Microscopy
By means of a special condenser which in its simplest form is fitted with a central circular stop,
the object is illuminated with a cone of light without permitting any direct rays to enter the
objective. Only light diffracted by the object is able to enter the objective with the result that
the object appears self-luminous against a dark background.
Dark field illumination has 2 specific uses:
(i) It permits visualisation as points of light of objects too small to be seen by direct
illumination. Such small objects can be counted even though their true shape and size is
difficult to ascertain.
(ii) It enables examination of larger objects which are invisible under direct illumination or
which cannot be stained, eg spirochaetes. Above all, as live organisms can be examined,
knowledge of true shape and motility can be acquired.
17
EXERCISE B
WINOGRADSKY COLUMN
Rarely does a natural environment contain only a single type of microorganism. In most cases, a
variety of organisms are present and it is particularly challenging for the microbiologist to devise
methods and procedures which will permit the isolation and culture of organisms of interest,
especially if they are relatively low in numbers or slow growing.
The most common approach to this goal is the enrichment culture technique. In this method, a medium
and set of incubation conditions are used that are selective for the desired organism and that are
counterselective, or even inhibitory, for the undesired organisms.
The Winogradsky column; named after the famous Russian enrichment culture microbiologist, Sergei
Winogradsky, was devised by him in the 1880’s to study soil microorganisms. The column is a
miniature anaerobic ecosystem, and if properly established, will provide a range of habitats that allow,
depending on individual metabolic capabilities, various kinds of microorganisms to grow and cycle the
nutrients present in the soil.
In this experiment, you will set up a Winogradsky column and make observations over a period of 5
weeks. This will facilitate the study of conversions and recycling of nutrients in the environment as
well as illustrate varying metabolic capabilities of several types of prokaryotes. Discussions each week
focussing on different processes will enable you to interpret the observations to that point and to relate
these to the lecture material.
Materials (per group)
1. Mixing container
2. Tap water
3. 1 x 250ml beaker, 1 x 1 litre beaker
4. 1 x 1 litre glass measuring cylinder
5. 3 rubber bands
6. Chemicals – cellulose, CaSO4, CaCO3 , (NH4)2 SO4 (preweighed)
(General)
1. Soil – a range of different soils and sediments
2. Sand
3. Spatulas
4. Aluminium foil
5. “Cling film”
Method
1. One column per group. Use a large measuring cylinder for construction of your Winogradsky
column.
2. At the side of the laboratory we have provided a number of soil/sediment samples from different
sources. Choose one of these, and put approximately 100 mL of it in a beaker (enough to
provide a depth of approx. 3cm in your column)\. Mix it with a little tap water to give the
consistency of thick cream.
3. To the material in the beaker add:
1g cellulose powder
1g CaSO
4
1g (NH
4
)
2
SO
4
1g CaCO
3
Mix thoroughly and then transfer the supplemented material to the bottom of the measuring
cylinder.
18
4. In an icecream container or large beaker, thoroughly mix some more of your chosen
soil/sediment with sand (1 part soil to 5 parts sand). Add tap water to make a cream-like
consistency.
5. Carefully add the soil-sand mixture to your column, being very careful not to disturb the bottom
sediment layer. It is vitally important to trap as little air in the column as possible, so add more
tap water if necessary, use a glass rod to fill evenly, and add the sand mixture in portions. Fill
until the surface is approx. 4cm from the top of the cylinder. Add more tap water to within 2
cm of the cylinder rim.
6. Cut and fold a piece of aluminium foil and attach it with rubber bands such that half of the
cylinder will be shielded from light. Label the foil, not the cylinder, and place the column in
position next to the windows in the lab. Cover the open end of the column with cling film and
secure with a rubber band.
7. The demonstrators will remove excess water from the column if necessary the next day, leaving
a layer only 1cm deep on the surface of the mud.
Follow up (Weeks 2-8)
1. Inspect your column over the succeeding weeks and record any changes you see (see below).
Compare your column with those of other groups. Try to disturb the aluminium foil as little as
possible. Top up with tap water if necessary.
2. Discuss any changes noted with your demonstrator.
19
Results
Record the appearance of the Winogradsky column every week. Note particularly:
(i) Black coloured patches – time of appearance, position in column, size/extent of patch
(ii) Bright coloured patches (pink, red, purple, rust-red, green) – time, position, extent
(iii) White/chalky patches – time position extent
(iv) Production of gases (bubbles or smell)
(v) Do particular features (e g coloured patches) disappear after a period?
(vi) The spatial and temporal relationship of different features (e.g. are black patches seen at same
depth but different sides to purple ones)
USE COLOURED PENCILS IF POSSIBLE & LABEL FULLY. WHERE POSSIBLE INCLUDE EXPLANATION FOR
OBSERVATION IN THE SPACES PROVIDED.
Day 7.
Illuminated Side Summary of Reactions Dark Side
Day 14.
20
Day 21.
Day 28.
Day 35.
21
Discussion
Evaluate the development of the Winogradsky column. Describe the changes observed.
Relate the following changes that are potentially observed in the column to the metabolic processes
that may have occurred and the environmental conditions that favoured/selected those particular
metabolic processes. Give an example of an organism that can carry out each of the conversions noted.
Purple/pink/red coloured patches in subsurface regions of light side only:
Dark green patches in subsurface regions of light side:
Dark green patches at surface regions of light side
Chalky grey/white deposits on either light or dark side near surface:
Black in subsurface regions of dark side:
Red/brown patches in surface/subsurface of light side only.
Also describe any other essential reactions that must be taking place, but which are not evidenced by
observable effects.
22
23
PRACTICAL WEEK 2
Exercise A Microscopy
A2 Preparation of material for microscopy – Gram stain (page 25)
Exercise B Winogradsky column
B2 Week 1 observations and discussion of cellulose breakdown
Exercise C Macroscopic examination of cultures (page 29)
Exercise D Aseptic technique
D1 Tube to tube inoculation (page 34)
D2 Streak dilution (page 34)
Exercise E Environment, growth limits and sterilization (Demonstration)
E1 Physical factors limiting microbial growth (page 37)
E2 Sterilization techniques (page 40)
E3 Principles of media for microbial growth (page 42)
AIMS
After completing this week’s practical the student should be able to:
1. Be familiar with the basic equipment of a bacteriology laboratory;
2. Understand the principles of aseptic technique and be able to apply these in:
(i) inoculating an agar slope
(ii) streaking out bacterial growth onto an agar plate to obtain separated colonies;
3. Describe bacterial colonies using standard terminology.
4. Distinguish yeasts, fungi and various bacteria when growing on solid medium.
5. Understand some of the reasons behind particular growth characteristics of microorganisms on
solid media.
6. Prepare heat fixed, Gram stained smears of bacteria suitable for microscopic examination.
7 Differentiate a mixture of bacteria into Gram positive and Gram negative cells.
8. Understand the rationale underlying the Gram stain.
9. Use a microscope to observe bacteria with the oil immersion objective and bright field
illumination at maximum resolution.
10. Describe the appearance of living bacteria under dark field illumination and phase contrast
illumination.
11. Understand the basic principles underlying the choice of ingredients in laboratory media;
12. Understand the usage of different types of media for general growth, for selective growth or for
differentiation of bacteria.
24
PRE LAB TASKS – (TO BE COMPLETED BEFORE WEEK 2)
1. Read the section of manual, “General Principles of Aseptic Techniques”, (page 33).
2. Read Blackboard notes on “Isolation of Pure Cultures”.
3. Read the Blackboard notes and animation on “Building a Winogradsky column”, and
Prescott p. 664
4. Answer the following questions:
(i) What is the most common source of contamination in a laboratory?
(ii) Why is it best to work close to a Bunsen flame when manipulating microorganisms?
(iii) Why must only sterile equipment be used in these procedures?
(iv) What is a pure culture?
(v) How did the introduction of solid media simplify the isolation of pure cultures?
(vi) What was the first “solid medium” ever used?
5. Read Blackboard notes on the Gram stain and Microscopy.
6. Answer the following questions.
(i) The Gram reaction of a cell is dependent on three separate factors.
(a)……………………………………………………………….
(b) ……………………………………………………………….
(c) ……………………………………………………………….
(ii) What is the size of a typical bacterial cell/ eg Staphylococcus epidermidis?
7. How many times should it be magnified to make it comfortably visible to the human eye?
8. Which objective, therefore, must be used to adequately visualise bacteria?
9. Which method of microscopy would be most appropriate for use in determining the size of
bacteria.
10. Read introductory remarks “General Principles of Media Making”, (page 43).
11. Read Blackboard notes on Media and Growth of Organisms.
12. Fill in the tables and blank spaces for Nutrient agar, Salt Agar and MacConkey Agar in
Exercise E3, (page 44).
25
Exercise A2 MICROSCOPIC EXAMINATION OF BACTERIA
——————————————————————————————————-THE GRAM STAIN
Staining serves two roles in microbiology – to visualize cells and to provide histochemical information. Firstly, Bacteria
are very small and their cells are almost transparent, differing only slightly in refractive index from water or culture media.
Hence in order to observe bacteria clearly with the ordinary light microscope it is necessary to stain them. Secondly,
Bacterial morphology is simple. To recognize diversity we need to look at the molecular level. Histochemical stains reveal
diversity that is not otherwise detectable. One of the most widely used staining procedures in microbiology illustrates
both aspects – the Gram stain.
This staining procedure differentiates bacteria into two broad groups, Gram-positive and Gram-negative, representing
respectively those which retain the crystal violet-iodine complex and those which lose it in the presence of a differentiating
agent, eg alcohol or acetone.
Materials
(per pair)
(1) nutrient agar (NA) plate of Staphylococcus epidermidis
(2) nutrient agar plate of Escherichia coli
Method
(1) Use a fresh slide for each mount.
(2) Flame the slide by passing it several times through the strong flame to remove any grease. Allow the slide to cool
completely.
(3) Prepare a fixed smear as follows:
(a) Label the slides clearly before preparing the smear. During the staining procedure below, ensure that the label
is not washed away by e.g. alcohol.
Label
26
(b) Place a small loopful of tap water on the slide. With a cool sterile loop, take a small amount (3-5 colonies) of
each of the cultures to be examined and emulsify (mix them thoroughly) together in the water using the loop.
Flame the loop between cultures.
(c) Spread to form a thin smear about the size of a 5 cent piece. Aim at obtaining a thin, even smear which is
only just visible when dry. Do not repeatedly lift the loop in and out of the suspension as this releases aerosol
droplets containing bacteria into the air. If the suspension cannot be spread out but aggregates into droplets,
the slide is greasy and should be discarded.
(d) Flame the loop before returning it to the block.
(e) Allow the slide to air dry.
(f) Fix the smear by passing it rapidly 3 times through the Bunsen flame, smear uppermost. Fixing:
* kills the bacteria
* causes them to adhere firmly to the slide so that they do not wash off during the staining procedure,
* inactivates autolytic enzymes so that the cells do not degenerate after staining, and
* the cells are made more permeable to the stain.
(g) Allow the slide to cool.
(h) Stain the smears by placing the slide on the rack over the sink and covering the smears with staining solution
as required.
(4) Gram stain the smear as follows:
(a) Cover the smear with 2% crystal violet for approx. 1 min.
(b) Gently rinse with water and drain off the excess.
(c) Cover the slide with Gram’s iodine and leave for 1 min (approx.).
(d) Wash with water. Drain and wipe the underneath of the slide.
This is the critical step.
(e) Decolourise with 95% ethanol. Do this over a light background. Holding the slide at an oblique angle, let the
alcohol run gently over the smear until no large amounts of purple wash out (up to 30 seconds). The degree of
alcohol decolourization depends on the thickness of the smear as well as the properties of the organism.
Amount of culture:
Much less than a
whole loopful
27
Alternatively, decolourization can be achieved using acetone: alcohol (1:1) for 3-5 seconds only (depending
on thickness of smear as well as on the properties of the cells being stained).
Have the tap running. Washing with water stops the decolourisation process.
Take care not to over decolourize. Experience is the only way to determine how long to decolourize. The time
required varies with thickness of smear and properties of the organism.
(f) Wash gently with water.
(g) Counter-stain with safranin for 3 min.
(h) Wash with water.
(5) Blot dry.
(6) Examine under low power to focus (40 X objective), then use the oil immersion lens 100 X objective) to resolve
the detail. Refer to Appendix 1C for instructions on use of the microscope
Notes on the Gram stain:
Gram-positive cells appear dark purple in colour. Gram-negative cells are pink.
(a) If a smear is thick it may have areas where Gram-negative cells fail to be completely decolourized. The
overcrowded cells also make it difficult to observe shapes and characteristic cell arrangements.
(b) Cells from physiologically young cultures generally are the best material for definitive Gram staining. Older
cells of Gram-positive types may lose the ability to retain the crystal violet-iodine complex and hence the smear
may appear to contain a mixture of Gram-positive and Gram-negative organisms. This is particularly the case
for endospore formers.
(7) Remember to clean the oil immersion objective after use, to remove all traces of oil.
Results
(1) Record and draw all observations.
(2) Drawings should include sufficient cells to indicate their shape and arrangement (i.e. accurately draw 1-5 cells, each
at least 0.5 cm in width).
(3) Use scale bar to show relative sizes. (One cell of S. epidermidis is 1µm in diameter.)
(4) Label drawing fully noting Gram reaction, shape and arrangement if any.
28
Discussion
a) Discuss the basis of the Gram stain
You should be able to relate the staining results to cell wall structure and predict the different responses of Gram-negative
and Gram-positive bacteria to cell-wall targeting antibiotics and chemical agents.
b) Evaluate the importance of the Gram stain in taxonomy.
Conclusion
Summarise the steps of the Gram stain procedure (use three words (max) for each step).
1.
2.
3.
4.
5.
6.
29
EXERCISE C
MACROSCOPIC EXAMINATION OF MICROORGANISMS
———————————————————————————————————————–There are several distinct groups of microorganisms that you will learn to differentiate on the basis of macroscopic
appearance in these classes. These are the filamentous fungi, yeasts and the different types of bacteria. Each has a
characteristic appearance when growing on a solid agar medium.
In the characterisation of a BACTERIAL COLONY, isolated colonies only should be considered and the following
features should be observed:
Size (in mm)
Shape Punctiform, circular, filamentous etc.
Elevation Flat, raised, convex, etc
Margin Entire, undulate, lobate, etc
Colour Presence of pigment and whether it is diffusible (extends into the
medium) or non-diffusible (confined to the colony)
Surface Smooth or rough, glistening or dull, etc.
Density Opaque, translucent or transparent
Consistency dry, moist, mucoid, brittle, viscous.
Refer to p. 32 for diagrammatic representation and descriptive terminology of bacterial colony morphology on agar.
Materials (per bench)
(1) Plate culture of bacterium Pseudomonas sp.
(2) Plate culture of bacterium Staphylococcus sp.
(3) Plate culture of bacterium Streptomyces sp.
(4) Plate culture of yeast Saccharomyces sp.
(5) Plate culture of fungus Penicillium sp.
NOTE: Genus and species names are either italicised or underlined.
Method
(1) Examine each organism macroscopically and characterise the colony ie describe in the specified terms as shown
opposite.
(2) Notice particularly the features which distinguish each of these organisms from the other, eg smell, size, colour,
surface etc.
(3) Record results.
30
Results
1. Appearance of bacterial colonies
Pseudomonas sp Staphylococcus sp Streptomyces sp
Size
Shape
Elevation
Margin
Colour
Surface
Density
Consistency
Other
Characteristic
(eg. smell)
Growth
parameters:
Medium Nutrient Nutrient Yeast
Agar Agar Malt Extract
Agar
Temp 25
o
37
o
25
o
Time 24hrs 48hrs 5 days
31
2. Appearance of filamentous fungal colony (Penicillium sp., 7 days, 25°C, Potato dextrose agar)
Size…………………………………………………………..
Surface………………………………………………………..
Pigment ………………………………………………………
Any other feature ……………………………………………
NB Do not smell filamentous fungi as spores can cause allergies.
3. Appearance of yeast colony (Saccharomyces sp., 3 days, 30
°
C, Sabourauds agar)
Size…………………………………………………………
Smell…………………………………………………………
Surface……………………………………………………….
Colour……………………………………………………….
32
Discussion
After discussing the characteristics of the supplied cultures with your demonstrator, summarize the major differences
between the organisms examined.
Compare your colony descriptions with those of other members of your bench. Discuss with your demonstrator the most
common flaws in recording macroscopic observations. If accurate colony descriptions are recorded under adequately
controlled conditions it is frequently possible to presumptively identify a microbe to species level from these data
alone. Are your records to this standard?
Suggest reasons for the obvious differences between how microbes behave in culture – particularly consider microscopic
morphology (how might cell shape and arrangement affect colony density or opacity?), growth requirements, growth
conditions and habitat (to what extent does where organisms come from reflect their growth requirements?).
33
EXERCISE D
ASEPTIC TECHNIQUES
——————————————————————————————————-General principles of aseptic techniques
Aseptic techniques are methods of handling materials which minimise the chances of microbial contamination. They
are used when manipulating pure cultures of microorganisms in order to:
* keep them pure and
* prevent the environment from being contaminated.
The techniques are based on the knowledge that all natural materials, including air, water, dust, clothing, soil, faeces, etc.,
contain microorganisms that may infect cultures, sterile solutions or other sterile items.
Aseptic techniques include the following:
(i) Using only sterile equipment when manipulating cultures or sterile materials – eg inoculating loops, pipettes, other
glassware etc.
(ii) Use of microbe-impermeable barriers to exclude air-borne microorganisms – eg
* use of cotton wool plugs in tubes and pipettes. (Note that cotton wool is only an effective microbial filter when
it is dry.)
* use of caps on bottles;
* wrapping of items in paper.
(iii) Working close to the Bunsen flame at all times, because the upward draft near the flame reduces the chance of
microbes in air from falling into open containers.
(iv) Passing the neck of open containers, pipettes and other items briefly through the flame at the start and if possible at
the end of the manipulation.
(v) Opening containers for the minimum time necessary.
(vi) Holding open containers in a near horizontal position to reduce the open area exposed to vertically falling particles.
(vii) Cleaning work surfaces with disinfectant solution (don’t use a dry cloth as this tends only to disperse dust into the
air) before and after aseptic manipulation.
(viii) The careful disposal of unwanted cultures and contaminated equipment to reduce the general level of microbial
contamination in the working environment eg used pipettes immediately placed in jars containing disinfectant
solution.
(xi) Use of a ‘sterile’ inoculating/dispensing room – a room provided with sterile air (filtered) and an ultra-violet lamp
in which manipulations can be done with a minimum risk of contamination.
The laboratory study of microorganisms is based mainly on pure cultures (i.e. cultures containing only one species of
organism) and it is therefore essential to avoid contaminating such cultures with other microorganisms.
The following techniques will be demonstrated after which you will carry them out yourself:
A. Inoculation of a tube or bottle.
B. Streaking of a culture onto the surface of an agar plate so as to obtain well separated colonies on the plate after
incubation. Various methods of streaking give satisfactory results in experienced hands, but the following method is
recommended as being particularly suitable for beginners.
34
Materials
(per student)
(1) NA plates
(2) Nutrient broth tubes
(per pair)
(4) NA slope of Escherichia coli
Method
A. TUBE TO TUBE TRANSFER (slope to liquid)
(i) Label the bottle to be inoculated with the name of the organism, the date and your name.
(ii) Place the bottle to be inoculated, the culture and the block with loop in front of the Bunsen and
convenient to the right hand.
(iii) Loosen the caps of both bottles.
(iv) Sterilize the loop by heating in the Bunsen flame to red heat, starting from the hand end and
drawing the wire through the flame towards the top of the loop.
(v) Allow to cool.
(vi) Still holding the loop in your right hand, remove the cap of the culture bottle with the little
finger of this hand.
(vii) Keep the bottle as nearly horizontal as possible and flame the neck of the bottle.
(viii) With the cooled loop, remove a small amount of the culture.
(ix) Flame the neck of the bottle.
(x) Replace cap and put the culture bottle down on the bench.
(xi) Now remove the cap of the culture medium flaming the neck.
(xii) Inoculate the broth by inserting the inoculated loop into the medium, and rubbing gently on
the side of the bottle. Look to see that some inoculum leaves the loop and is left in the liquid
medium.
(xiii) Reflame the neck of the bottle before replacing the cap.
(xiv) Reflame the loop as in (4) before returning it to the block.
(xv) Place in appropriate incubation box.
(xvi) To test your aseptic technique, repeat the process with a sterilized loop, without inoculating the
loop (step 8). If you have worked carefully, the medium should show no growth next week!
B. STREAKING THE PLATE
(i) The purpose of streaking plates is to distribute the inoculum over the surface of the medium in
such a manner as to separate the bacterial cells from each other in order to obtain well isolated
colonies, on at least a portion of the plate.
(ii) Using the plate supplied, label the base with name, date and the organism.
(iii) Place the plate to be inoculated base uppermost and convenient to the left hand.
(iv) Sterilize the loop.
(v) Allow to cool.
(vi) Still holding the loop in your right hand pick up the culture bottle and remove the cap as
before, flaming the neck of the tube.
35
(vii) With the cooled loop remove a small amount of culture.
(viii) Flame the neck and recap the bottle.
(ix) Pick up the base of the Petri plate to be inoculated with the left hand.
(x) Hold the plate at about 45o to the horizontal and spread the specimen over the primary
inoculation site (1) and out from it (2). See diagram below.
(xi) Return the base of the plate to the lid. Rotate the plate through approximately 180
0
.
(xii) Flame the loop, then allow to cool.
(xiii) (Again pick up the base of the plate and streak as in 3 and 4 of the diagram flaming the loop
between each change of direction.
(xiv) Return the plate to the lid, flame the loop and return it to the block.
(xv) Place the plate in the correct incubator box in the inverted position.
Plate held for streaking:
Streaking out colonies on an agar plate is an essential technique to learn for all microbiological work.
There are additional NA plates available – keep practising your aseptic technique until you are happy
with it.
Lid
45º
The aim is to achieve more than
one well-isolated colony. An ideal
result gives 10 to 20 well-isolated
colonies on the 3
rd
or 4
th
set of
streaks. It is still acceptable if
there is no growth on these but
more than one well-isolated colony
is on the 2
nd
set or last streak.
36
Follow up (week 3)
(1) Inspect the mock-inoculated NA broth for absence of bacterial growth.
(2) Inspect the agar plate for regions of confluent growth and for separated colonies.
(3) Record the colony morphology using standard terms.
(4) If unsure submit the cultures to the demonstrator for comment.
Results
(1) Appearance of streak plate and of colonies:
(describe single colonies only)
Size …………………………………………………..
Shape …………………………………………………
Elevation …………………………………………….
Margin ……………………………………………….
Colour ……………………………………………….
– pigment ……………………………………………
Surface ………………………………………………
Density ………………………………………………
Consistency …………………………………………
Conclusion
Summarise the essential steps of aseptic technique. What are the most important aspects to think about when performing
these steps?
(a) Tube to tube transfer.
(b) Streaking a plate.
37
EXERCISE E1
THE EFFECT OF PHYSICAL FACTORS ON THE GROWTH OF BACTERIA AND FUNGI
—————————————————————————————————————————————-Temperature
The temperature range within which living organisms can grow is an extremely wide one, extending from about -5 °C to
about 110 °C. Since living organisms consist largely of water, the lower limit for their development is set by the
temperature at which water freezes. In a living cell, this is slightly below 0 °C owing to the depression of the freezing
point of water by the dissolved organic and inorganic compounds. The upper limit is set primarily by the thermal lability
of the chemical constituents of living matter, the proteins and nucleic acids; these substances are rapidly destroyed at
temperatures in the range between 50 °C and 90 °C. Exposure to temperatures above the range for growth causes rapid
cell death, except in the special case of heat resistant endospores.
Microorganisms are classified into three primary groups on the basis of preferred temperature: psychrophiles
(cold-loving microbes), mesophiles (moderate-temperature-loving microbes) and thermophiles (heat-loving microbes).
Some of these primary groups can be subdivided (eg psychrotrophs). Most bacteria grow only within a limited range of
temperatures, and their maximum and minimum growth temperatures are only about 30
o
C apart. They grow poorly at the
extremes within their range.
Water
Microorganisms, like all other living organisms, require water for growth, are made up of 80-90% water, and in
situations where water availability is limited microbial growth is slowed or prevented entirely. Among the situations
where limited water availability is an important factor in restricting microbial growth are intrinsically dry materials and
solutions containing very high concentrations of solutes. Examples of dry materials are grains, other seeds and
pharmaceutical powders and tablets. High concentrations of sucrose occur in nectar, honey, preserved foods such as jams
and jellies and in pharmaceutical syrups. Near-saturated solutions of sodium chloride occur naturally in some salt lakes
and strong solutions are used in the salting of foods such as bacon and fish. The osmotic loss of water causes plasmolysis
and death of the cell.
In these environments the only microorganisms able to grow are those specially adapted to growth in the
presence of low concentrations of available water (and to high concentrations of the particular solute). Most
microorganisms, however, must be grown in a medium that is nearly all water. For example, the concentration of agar
used to solidify growth media is about 1.5%. If markedly higher concentrations are used, the growth of some bacteria can
be inhibited by the increased osmotic pressure.
pH
Most bacteria grow best in a narrow pH range near neutrality, between 6.5 and 7.5. Very few bacteria grow at an acidic
pH below about pH 4. This is why a number of foods, such as sauerkraut, pickles, and many cheeses, are preserved from
spoilage by acids produced by bacterial fermentation. Nonetheless, some bacteria, acidophiles, are remarkable tolerant of
acidity. Moulds and yeasts will grow over a greater pH range than bacteria will, but the optimum pH of moulds and
yeasts is generally below that of bacteria about pH 5-6.
In acid or alkaline environments the growth of microorganisms is slowed or prevented, particularly in the presence of
weak acids or bases. It appears that this is a consequence of the nature of the lipoprotein cell membrane which is
relatively impermeable to charged ions but considerably more permeable to uncharged molecules. Once the uncharged
molecule has entered the cell it ionises, and the resultant release of H+
disrupts the internal cell pH value. This results in
protein denaturation and death to the cell. Thus, weak acids such as acetic, propionic and benzoic acids are used as
preservatives in foods as well as pharmaceuticals.
Oxygen
Oxygen functions as the terminal electron acceptor in aerobic respiration. Hence organisms that possess no other means
of generating energy are inhibited in its absence. However, many bacteria and some fungi can generate energy and grow
in the absence of oxygen by the processes of fermentation (in which an organic compound serves as terminal electron
acceptor) or anaerobic respiration (in which an inorganic molecule other than oxygen serves as electron acceptor, i.e.
NO3
-, SO4
2- or CO3
2-).
In terms of oxygen requirements, microorganisms can be classified into three distinct types:
(a) Organisms requiring oxygen for growth are called aerobes .
(b) Organisms that are able to grow in the presence or absence of oxygen are called facultative anaerobes.
(c) Organisms which grow only in the absence of oxygen are called anaerobes.
38
DEMONSTRATION
Materials
(i) The plates presented to you have been inoculated with four different organisms as shown below:
(ii) The plates were then incubated under the conditions specified in the Table below, varying temperature, osmotic
stress and aerobiosis.
Method
Examine the plate for growth. Record the amount of growth in each case using ++++, +++ etc to indicate the effect
altered environmental conditions have on each organism.
Results
Effects of environmental factors on growth of potential spoilage organisms
Conditions of Growth Growth of
MediumpH AtmosphereTemperatureSucroseNaClS. aureusPs. fluorescens B. stearothermophilus
Penicillium
sp.
NA
NA
NA
NA
NA
NA
NA
7.5
7.5
7.5
3.5
7.5
7.5
7.5
Air
Air
Air
Air
H2/CO2
Air
Air
5°
30°
60°
30°/60°†
30°/60°†
30°/60°†
30°/60°†
—–50%
——-10%
† B. stearothermophilus cultivated at 60 °C, other organisms at 30 °C.
Staphylococcus
aureus
Pseudomonas
fluorescens
Bacillus
stearothermophilus
Penicillium
sp.
39
Discussion
(1) For each organism identify the conditions it grew (a) best and (b) least and explain each case,
eg S. aureus grew best on: …………………………………………………………………
least on: …………………………………………………………………
because: …………………………………………………………………
…………………………………………………………………
P. fluorescens grew best on: …………………………………………………………………
least on: …………………………………………………………………
because: …………………………………………………………………
…………………………………………………………………
B. stearothermophilus grew best on: …………………………………………………………………
least on: …………………………………………………………………
because: …………………………………………………………………
…………………………………………………………………
Penicillium sp. grew best on: …………………………………………………………………
least on: …………………………………………………………………
because: …………………………………………………………………
…………………………………………………………………
(2) Discuss the practical implications of your observations in food storage and spoilage..
Consider which physical conditions can be managed to preserve foods?
Which organisms would be most likely, or least likely, to continue to grow despite preservation methods?
What are the likely environmental sources of organisms with these properties?
40
EXERCISE E2
STERILISATION WITH AUTOCLAVE AND HOT-AIR OVEN
————————————————————————————————————————-The autoclave and hot-air oven are the two most widely used devices for sterilising laboratory equipment. The conditions
necessary for sterilisation, particularly the time and temperature, are influenced by many factors, including the numbers
and kinds of contaminating spores. This exercise should serve two purposes:
1) to acquaint you with the process of the autoclave and hot air sterilisation,
2) to demonstrate that the number of bacterial spores, as well as the kind of bacterial spores must be considered in
establishing sterilisation times and temperature.
DEMONSTRATION
Materials
1. Paper strips impregnated with Bacillus subtilis spores at the following concentration were prepared:
Strip A = 10
7
cfu/ml
Strip B = 10
6
cfu/ml
Strip C = 10
5
cfu/ml
2. A separate set of strips was subjected to each of the following treatments:
Autoclave 10 minutes
Autoclave 15 minutes
Hot air 10 minutes
Hot air 60 minutes
3. Following heat treatment each strip was transferred to a tube containing Nutrient Broth (NB) and the broth was
incubated under appropriate conditions to detect the presence of survivors of the heat treatment.
4. Steps 1-3 were repeated for three other organisms as follows:
Bacillus stearothermophilus cells
Escherichia coli cells
Penicillium sp. spores
Method
1. Examine the broth for evidence of survivors. Remember that only one cell surviving will
multiply to produce visible turbidity.
2. Tabulate the results.
41
Results
Survival of microorganisms following heat treatment.
GROWTH following:
Species Cell Concentration
cfu/ml
Autoclave
10 min
Autoclave
15 min
Hot air
10 min
Hot air
60 min
Escherichia coli
10
7
10
6
10
5
B. stearothermophilus
10
7
10
6
10
5
Bacillus subtilis
10
7
10
6
10
5
Penicillium sp.
10
7
10
6
10
5
Growth = + No growth = -Discussion
Compare (a) the relative heat resistances of the organisms tested. Account for these.
(b) the relative usefulness of the heat treatments tested.
Account for any anomalous results.
42
43
EXERCISE E3
GENERAL PRINCIPLES OF MEDIA FOR MICROBIAL GROWTH
————————————————————————————————-Until around 1950, laboratory workers had to spend a good deal of time preparing laboratory media
from various raw materials. If a medium contained five or six ingredients, it was not only necessary
to measure out the various materials but also, in many instances, to fabricate some of the components
such as beef extract or veal infusion by long tedious methods. Today dehydrated media have
revolutionised media preparation techniques. For most routine bacteriological work media
preparation has been simplified to the extent that all that is necessary is to dissolve a measured
amount of dehydrated medium in water, adjust the pH, dispense into tubes and sterilise.
The consistencies of media vary according to the purpose for which they are used. The following
types are available:
1. Solid media have agar-agar or gelatin added to them for solidification. Agar-agar, which is an
extract of marine kelp, is superior to gelatin because it does not liquefy at temperatures below boiling
and it is not liquefied by many bacteria. Solid media are used for the study of colony morphology,
isolation of pure cultures and the prolonged storage of pure cultures.
2. Semi-solid media have a smaller amount of agar than the solid media, thus they have a jellylike consistency. These media are used in sugar utilisation tests and motility tests.
3. Liquid media are broths such as nutrient broth (NB) and tryptic soy broth (TSB), which lack
the solidifying agents.
REQUIREMENTS FOR MICROBIAL GROWTH
Microorganisms vary widely in their requirements for growth. Therefore culture media can support
normal growth only if the nutritional requirements of the organism are satisfied. These include:
1. A suitable energy source.
2. Suitable carbon and nitrogen sources.
3. Adequate amounts of certain mineral salts.
4. Many organisms also require certain additional factors such as amino acids and vitamins.
When the nutritional requirements of the test organism are well established it is possible to devise a
medium having carefully defined constituents; such a medium is known as a chemically defined
media. However, for the primary isolation of organisms from unknown mixtures, complex media
such as meat extract agar or blood agar are commonly employed. These contain a sufficient variety
of nutritional requirements for most bacteria. Where the existence of particularly exacting organisms
is suspected, however, certain special media must be used.
Types of Culture Media
Media serve many different purposes in bacteriological work. The following types will be used at
different times in this course. They may be defined in various ways as general purpose, complex,
enriched, synthetic, selective or differential, or any combination of these. May want to link to e.g.
44
A. General Purpose Media
Bacteriological media may be designed for various purposes and the commonest one is to sustain the
growth of as wide a variety of bacteria as possible. A medium of this type goes back to the earliest
days of bacteriology when the common medium was a meat broth or infusion to which, later,
“peptones” were added. “Peptones” are produced commercially by acid or enzymatic hydrolysis of
protein. The source of protein is sometimes a commercial secret, but animal tissues or casein are
commonly used. In some media, commercially prepared meat extracts in paste or powder form can be
substituted for fresh meat and, in fact, a wide variety of general and special purpose media are available
in dehydrated form.
They may be modified in a variety of ways by the addition of specific chemicals or complex
supplements to produce a medium with some desired characteristic. For example, nutrient agar is
enriched by the addition of sterile blood. It will then support the growth of nutritionally fastidious
bacteria. Either nutrient broth or nutrient agar may be supplemented with a specific substrate for use
as a biochemical test medium.
Nutrient agar
Ingredient % Function* in the Medium
Yeast
extract
Peptone
NaCl
Agar
0.5
1.0
0.5
1.5
* Function in medium could be e.g. to supply
carbon or other nutrient, or to act as an
inhibitor.
1. Fully define nutrient agar using the
terms general purpose, selective, complex etc.
……………………………………………………….
……………………………………………………….
……………………………………………………….
2. For what purpose is it used?
……………………………………………………….
……………………………………………………….
B. Selective Media
If it is desired that only certain types of bacteria in a mixture shall be allowed to grow, a selective
medium will be used. A medium may be made selective by eliminating certain growth requirements
or by adding toxic substances to them. The use of salt agar in the isolation of staphylococci is a good
example of the application of a selective medium. This medium contains considerable sodium chloride
(7.5%) which inhibits most bacteria, yet allows staphylococci to grow albeit at a reduced rate.
MacConkey agar containing bile salts is another example.
45
Salt agar
Ingredient % Function* in Medium
Nutrient
agar
+
additional
NaCl
7.5
1. Fully define salt agar.
……………………………………………………….
……………………………………………………….
……………………………………………………….
2. For what purpose is it used?
……………………………………………………….
……………………………………………………….
C. Differential Media
Media of this type cause different species of bacteria to grow differently, making their identification
easier. Differential media are often also selective. MacConkey agar is a good example of a differential
medium. It is used in water and milk analysis to differentiate the lactose fermenting coliform bacteria
from the non-lactose fermenters. The coliforms (E. coli and K. pneumoniae) ferment the lactose in
MacConkey medium producing acid which causes their colonies to turn pink, due to the indicator
present in the medium. Pale colonies are produced by non-lactose fermenting organisms such as
Pseudomonas.
MacConkey Agar
Ingredient % Function* in Medium
Peptone
Lactose
NaCl
Bile salts
Neutral
red
Crystal
violet
Agar
2.0
1.0
0.5
0.15
0.005
0.0001
1.5
1. Fully define MacConkey agar
……………………………………………………….
……………………………………………………….
……………………………………………………….
2. For what purpose is it used?
……………………………………………………….
……………………………………………………….
46
D. Synthetic (Defined) Medium
If the exact chemical composition of a medium is known, it is said to be a synthetic medium.
Winogradsky’s medium which consists of measured amounts of phosphates, magnesium sulphate,
CaCO3 and minerals, is a synthetic medium.
n.b. As agar is an extract from kelp it may contain traces of undefined organic matter. Thus, strictly
speaking, agar cannot be used in true synthetic media, and other solidifying agents must be used
instead.
Winogradsky agar
Ingredient % Function* in Medium
KH2PO4
MgSO4
NaCl
MnSO4
FeSO4
Na2MoO4
CaCO3
Sucrose
Agar
Trace
amounts
”
”
”
”
”
0.01
1.0
1.5
1. Fully define Winogradsky’s medium -……………………………………………………….
……………………………………………………….
……………………………………………………….
2. For what purpose is it used?
……………………………………………………….
……………………………………………………….
47
BACKGROUND OF SELECTED BACTERIA:
Escherichia coli
* Named after Theodor Escherich who first isolated the type species of this organism in
1885; colum = colon, large intestine.
* Found in the gastrointestinal tract of animals and is common in soil and water.
* E. coli can cause urinary tract and other infections and certain serotypes cause enteric
diseases.
Bacillus subtilis
* Bacillus = a small rod (L.); subtilis = slender (L).
* Common soil bacterium used as an inoculant in agriculture. Not a human pathogen
* Produces extremely heat and chemical resistant spores.
Staphylococcus aureus
* Staphyle = bunch of grapes (Gr) appearance of clusters of cells; coccus = berry (Gr.),
spherical shape of cells; aureus = golden (L.), colonies on agar media often have
yellow pigment.
* Causes notorious “golden staph” infections.
* Staphylococci are commonly found on the skin and mucous membranes of animals
including humans.
* Survives on dry areas of the body such as skin and is tolerant to high salt
concentrations arising from sweat.
Sinorhizobium meliloti
* Sino= China (L); rhizo = root (Gr); bium = life (L); meliloti = clover. A bacterium
living on the roots of a Chinese clover plant.
* Sinorhizobium is a symbiotic bacterium capable of fixing atmospheric nitrogen in
root nodules on the roots of clover.
48
DEMONSTRATION
Method
Examine the plates provided. Record the presence or absence of growth on each medium. Tabulate
the results
as ++++, +++, ++, -, etc according to relative amount of growth.
Media organism Nutrient agar MacConkey agar Salt agar Winogradsky
agar
Escherichia coli
Bacillus subtilis
Staphylococcus
aureus
Sinorhizobium
meliloti
Describe the colony morphology of E. coli and S. meliloti on each medium. Where individual wellisolated colonies are not visible be sure to record any limitations or caveats that will affect your
observations (consider this practice for Exercise F skills test).
Discussion
Relate the presence or absence of growth of each organism to the composition of the medium,
nutritional requirements and the physiological properties of the organism.
In what ways did the medium affect colony morphology (e.g. size only or did it also affect, colour,
shape, consistency etc).
49
PRACTICAL WEEK 3
Exercise B Winogradsky column
B2 Week 2 observations and discussion on sulphate reduction (page 19)
Exercise D Aseptic technique (Follow-up see page 36 to record your results)
Exercise F Distribution of microbes in the environment (page 51)
F1 Selective culture of distinctive bacteria (page 52)
F2 Isolation of plant-associated microbes (page 63)
AIMS
At the completion of Exercise F the student will:
1. Appreciate that different environments are typically inhabited by different suites of organisms.
2. Appreciate that organisms can be selectively isolated from environments by enrichment culture –
even if they are normally of low abundance.
3. Understand use of rational enrichment culture techniques for the isolation of specified bacteria.
4. Be skilled in the use of aseptic techniques to obtain pure cultures.
5. Be skilled in the use of macroscopic and microscopic observations to characterize and identify
bacteria and fungi.
50
PRE LAB TASKS (TO BE COMPLETED BEFORE WEEK 3)
1. Read introductory remarks to Exercise F.
2. Answer the following questions:
a. Define “enrichment culture” (to be distinguished from “enriched medium”).
b. What is the advantage of an enrichment in liquid culture method over conventional
direct isolation methods for some organisms in the natural environment?
c. Explain why the Winogradsky column can be considered to be an enrichment culture.
3. Review Prescott “The nitrogen cycle” p636
4. List the five (5) discrete processes that make up the environmental Nitrogen Cycle. Write a
simple equation to describe the conversion carried out in each.
5. Read “Soils as a microbial habitat” (Prescott p. 680-684), and review your reading on the
Winogradsky column (Blackboard site)
(i) List two bacterial genera that are significant contributors to aerobic cellulose degradation?
(ii) Name a bacterial genus that is a major contributor to anaerobic cellulose degradation and
briefly describe how the end products of anaerobic decomposition differ from those of aerobic
cellulose decomposition?
6. Read Prescott Section 31.3 “Microbe-Plant Interactions” (p. 684-698).
(i) Why are methylotrophic bacteria often found on plant leaves?
(ii) Plant-associated microbes may be epiphytic (on the plant surface) or endophytic
(within the plant). Where would you expect to find more microbial diversity, and
why?
51
EXERCISE F
DISTRIBUTION OF MICROBES IN THE ENVIRONMENT
————————————————————————————————————————-The study of microbial ecology focuses on two major issues:
(1) biodiversity, including identification, and quantification of microorganisms in various habitats,
and;
(2) microbial activity, that is, what are microorganisms doing in their habitats.
One of the oldest ideas in microbial ecology is (“…everything is everywhere, but the environment
selects” Baas-Becking, 1934). The implication of this notion is that the abundance and ease of
dispersal of microbes is such, that even in inappropriate environments, organisms may still be
present in very low numbers. Therefore the presence of an organism does not mean it is ecologically
active. However, should environmental conditions become appropriate then organisms rapidly begin
growth and may become abundant. The dominant organisms in a sample are most likely to be those
adapted to the environmental conditions and the rarer organisms those that only occur by chance. We
can exploit these ideas when attempting to recover organisms of interest. By sampling environments
most likely to favour the desired organism we improve our chances of success since it is more likely
to be relatively abundant here. By using highly selective culture conditions we further increase our
chances of success by restricting the growth of unwanted organisms. In this exercise we will test
these ideas by examining 4 different environments for distinctive bacteria. Relative abundance in
each environment will be estimated by attempts at direct isolation of organisms as plate cultures.
Enrichment cultures will also be established from each environment in liquid cultures. Each group
will attempt to isolate one microbe in pure culture and identify it to genus level using simple
tests.
In order to take an organism from its natural habitat and successfully grow it in the artificial and
relatively selective conditions in a laboratory a large number of factors need to be considered. The
requirements, both physical and chemical of the organism must be known and satisfied. If requirements
are not fully known, conditions that are as non-selective as possible are applied. Traditionally this
involved use of a rich complex medium which attempts to mimic in composition the natural habitat
and incubation under conditions that mimic the physical environment. Modern microbial ecologists
are now aware that use of dilute nutrient media and very long incubation times (up to 3 months) is
more successful in recovering a broad sample of microbial diversity.
Having successfully grown the (generally) mixed population from a natural environment, purification
of the desired type must precede any attempt at identification. Microbial diversity is greatest with
respect to biochemical activity. As a consequence these are the most useful characteristics to exploit
in identification. Since individual microbial cells are too small to perform biochemical tests on, and in
mixed cultures it is impossible to interpret which cells are performing which activity – isolation in pure
culture is an essential part of performing biochemical tests for identification. Over the past 20 years
nucleic acid probes have become important tools to identify and quantify organisms in their natural
environment. This approach is especially useful as many of the organisms of ecological significance
have proven very difficult to study.
52
EXERCISE F1 SELECTIVE CULTURE AND ISOLATION
Each group will carry out a selective enrichment for one of the groups of organisms below, using samples taken from the
source environment listed. As a comparison, you will also test samples prepared by one of the other groups from a different
environment, to study the relative abundance of your target group in the two environments.
For example, group 1 will select for Halotolerant organisms in the nose environment, but will also test one sample from
e.g. compost under halotolerant-selective conditions.
Materials
Per group
(1) sterile swabs
(2) sterile distilled water
(3) 10ml sterile phosphate-buffered saline (PBS)
(4) 5 ml sterile phosphate-buffered saline (PBS)
(5) Leaves
(6) Soil
(7) Compost
Work in groups of 5
Group 1
(8) 7 x Nutrient agar + 6% NaCl plates
(9) 2 x Nutrient medium + 6% NaCl liquid
Group 2
(10) 7 x Minimal medium N-free + sucrose agar plates
(11) 2 x Minimal medium N-free + sucrose flasks
Group 3
(12) 7 x Minimal medium + nitrate + cellulose agar plates
(13) 2 x Minimal medium + nitrate + cellulose flasks
Group 4
(14) 7 x Minimal medium + nitrate + methanol agar plates
(15) 2 x Minimal medium + nitrate + methanol flasks
F1a HALOTOLERANT ORGANISM – GROUP 1
Osmotolerant and osmophilic organisms are those that can either survive or prefer to grow under conditions of low water
activity. Low water activity can arise via a variety of means including high solute concentrations (dissolved salts such as
NaCl, or small molecules such as sugars) and low water concentration (due to evaporation). Environments that permanently
have low water activity include salt lakes, those that typically have low water activity include aerial surfaces such as skin
and leaves, and those that frequently have low water activity are arable soils. As you might expect diverse osmotolerant
organisms are widespread in nature and are the dominant groups in environments that typically experience low water
activity. The halophiles are a subset that is specifically adapted to high salt environments. Here we will select for them by
using high salt nutrient agar and compare their distribution in environments that vary in water activity and salt
concentration.
SAMPLING – Animal skin surfaces
Provide your own Nose
a) Moisten a swab stick with sterile water and swab the inside of the nose (Do not use your finger).
b) Using the same swab inoculate the 10 ml PBS bottle by vigorously mixing. Discard the swab by returning it to its
tube and breaking the stick. Label this bottle 10
-1
NOSE
c) With a sterile, 1 ml pipette tip (blue colour) aseptically transfer 500 ul into the bottle with 5 ml PBS. Label this
bottle 10
-2
dilution-NOSE.
d) Using a sterile 200 ul pipette tip (yellow) transfer 50 ul of the 10
-2
dilution into a new bottle with 5 ml PBS. Label
10
-4
dilution-NOSE.
e) Repeat step d) to make a 10
-6
dilution.
53
F1b NITROGEN-FIXING ORGANISM – GROUP 2
Although molecular nitrogen (N2) is abundant, constituting about 80 per cent of the earth’s atmosphere, it is chemically
inert and therefore not a suitable source of the element for most living forms. All plants and animals as well as most
microorganisms, depend on a source of combined, or fixed, nitrogen in their nutrition. Since combined nitrogen in the form
of ammonia, nitrate, and organic compounds is relatively scarce in soil and water, it often constitutes the limiting factor
for the development of living organisms.
N2 ? NH3 ? organic N
Nitrogen fixation is a critical, but highly specialised reaction. It is very energy-demanding and highly sensitive to oxygen.
We can broadly divide nitrogen-fixing organisms into two categories:
(i) Symbiotic – the energy and oxygen control is provided by a host organism.
(ii) Free-living (non-symbiotic) – here the cell must either be adapted to grow in the absence of oxygen or have adaptations
to avoid oxygen inhibition of the nitrogenase enzyme, such as high respiratory rate, modified cell walls, or presence of a
capsule.
Non-symbiotic nitrogen fixation is accomplished by a surprisingly wide range of free-living organisms, the best known
and possibly the most effective being Azotobacter and Beijerinckia. Other, organisms include heterotrophs such as
Azospirillum, Clostridium and Klebsiella, photosynthetic bacteria such as Anabaena, Nostoc, Rhodospirillum,
Rhodopseudomonas, Chlorobium and Chromatium.
In this exercise we will attempt to isolate free-living nitrogen fixing bacteria by the simple expedient of growth in a defined
medium with a high concentration of carbon (sucrose) and no form of fixed nitrogen. [NOTE – although such media are
conventionally referred to as N-free unless the flask is hermetically sealed they do in fact have large amounts of atmospheric
nitrogen present].
SAMPLING – Plant surfaces
Use Leaf samples provided
a) Using sterile, forceps place 3-5 leaves into a bottle containing 10 ml sterile PBS.
b) Shake vigorously for 3 minutes. Label this bottle 10
-1
LEAF
c) With a sterile, 1 ml pipette tip (blue colour) aseptically transfer 500 µl into the bottle with 5 ml PBS. Label this
bottle 10
-2
dilution-LEAF.
d) Using a sterile yellow pipette tip transfer 50 ul of the 10-2
dilution into a new bottle with 5 ml PBS. Label 10
-4
dilution-LEAF.
e) Repeat step d) to make a 10
-6
dilution.
54
F1c CELLULOSE-DEGRADING ORGANISM – GROUP 3
Polysaccharides and particularly plant-derived cellulose is the major reservoir of carbon in terrestrial environments.
Decomposition of cellulose is therefore a vital process in terrestrial environments – and in the digestive tracts of all
herbivores. As you might expect for such a fundamental process there is a tremendously wide range of organisms involved,
including Bacteria and Fungi.
A universal constraint for all cellulose-degraders is that cellulose is insoluble. Organisms must secrete enzymes
extracellularly to breakdown the cellulose (or in the case of digestive system inhabitants rely on host chewing to aid the
process). Otherwise the nutritional and environmental requirements of the cellulolytic organisms are extremely diverse and
their activity is influenced by many factors. The most important of these are moisture, pH and nitrogen content. Aerobic,
mesophilic organisms (Cytophaga, Sporocytophaga and a very wide range of fungi) account for most of the cellulose
decomposition in agricultural and forest soils. Here we will target aerobic bacterial cellulolytic organisms by enriching for
them in a defined medium in which cellulose is the only form of organic carbon. NOTE – it is conventional to describe
such media as having a single carbon source but in fact atmospheric carbon gases (notably carbon dioxide) are also present.
SAMPLING – Soil
a) The soil samples provided have been finely ground with a mortar and pestle. Add ~1 g with the small spatula (a full
teaspoon would be ~5 g) to 10 ml sterile PBS.
b) Shake vigorously for 3 minutes. Label this bottle 10
-1
SOIL
c) Allow the particles to settle for about 15 seconds (large particles may block your pipette tip).
d) With a sterile, 1 ml pipette tip (blue colour) transfer 500 ul into the bottle with 5 ml PBS. Label this bottle 10
-2
dilution-SOIL.
d) Using a sterile yellow pipette tip transfer 50 ul of the 10-2
dilution into a new bottle with 5 ml PBS. Label 10
-4
dilution-SOIL.
e) Repeat step d) to make a 10
-6
dilution.
F1d METHYLOTROPHIC ORGANISM – GROUP 4
The vast majority of carbon metabolism occurs with organic compounds that include carbon-carbon bonds. However
compounds without such bonds (C1 compounds) are a vital part of the global carbon cycle. These include carbon dioxide
and various organic compounds the most important of which are methane and methanol. Since these compounds are
produced in very large amounts their decomposition is a critical process. It is also widely exploited in biotechnology.
Organisms capable of utilizing C1 compounds have specialised metabolic pathways. They can be broadly subdivided into
Obligate methylotrophs (of which the methanotrophs are a subset) and facultative methylotrophs. The obligate
methylotrophs tend to prevail in environments where C1 compounds are present in stable and high concentrations. The
best example of this is bogs, where methane is produced in the sediments and provides a carbon source for methanotrophs
growing in the overlying sediments. Another example is plant surfaces. Many plants produce large quantities of methanol
as a metabolic by product. On the leaves, where there are relatively few other carbon sources available, the methanol is
one of the more important carbon sources for epiphytes. Here we will attempt to isolate methylotrophs by growth in a
defined medium with methanol as the sole carbon source.
SAMPLING – Compost
a) The soil samples provided have been finely ground with a mortar and pestle. Add ~1 g with the small spatula (a full
teaspoon would be ~5 g) to 10 ml sterile PBS.
b) Shake vigorously for 3 minutes. Label this bottle 10
-1
COMPOST
c) Allow the particles to settle for about 15 seconds (large particles may block your pipette tip).
d) With a sterile, 1 ml pipette tip (blue colour) transfer 500 ul into the bottle with 5 ml PBS. Label this bottle 10
-2
dilution- COMPOST.
55
d) Using a sterile yellow pipette tip transfer 50 ul of the 10-2
dilution into a new bottle with 5 ml PBS. Label 10
-4
dilution- COMPOST.
e) Repeat step d) to make a 10
-6
dilution.
ALL GROUPS
1). Using the inoculum prepared by your own group (e.g. group 1 nasal suspension, etc), label four plates as 10
-1
, 10
-2
,
10
-4
, and 10
-6
. Inoculate each by transferring 100 µl of the appropriate suspension to the centre of the plate. Use a
sterile spreader to spread the inoculum evenly over the surface.
2). Using the 10
-2
and 10
-4
dilutions only from one of the inocula prepared by the other groups (your demonstrator will
advise), inoculate one plate for each sample as above. Make sure you record which sample is on the plate.
3). Each group should have a total of 6 plates for incubation – 4 with your inoculum and two from another inoculum.
Place them in the box for incubation.
4) Label the two flasks of liquid media with 10
-1
or 10
-4
and your group name.
5). From the 10
-1
and 10
-4
dilutions that you prepared – aseptically transfer 100 µl into the appropriate flask of selective
medium liquid. Place them in the box for incubation.
6). The samples from the body surfaces will be incubated at 37° (1-3 days), and those from environmental surfaces at
25° (1 week).
56
FOLLOW UP (Week 4)
The aim of the enrichment is to purify and characterize the dominant microbes that (a) grow under the
tested selective conditions, and (b) are present in the environmental samples tested. To achieve this,
refer to this flow chart and follow the instructions below – you have already carried out the first 2 steps.
3. EXAMINATION OF CULTURES
a) Liquid cultures – examine your cultures for signs of growth. Record your observations. Look
particularly for the following:
(i). turbidity – a clear indicator of microbial growth.
(ii). pellicle – a film on the surface of unshaken flasks.
(iii). bubbles – indicates production of gases (metabolism)
(iv). odour – indicates transformation of organic compounds (metabolism)
If you can see clear signs of growth then use your loop to inoculate a sterile agar plate of your
selective medium. Streak out the inoculum to maximise your chances of observing well-isolated
colonies. This will be referred to as PLATE-L (for plate from Liquid). NOTE – good streak
technique is vital. If you had strong growth in the liquid culture there will be billions of cells in a
single loopful. You will not get isolated colonies unless you streak them out carefully.
If you do not see clear signs of growth then transfer 1 ml to a fresh flask of the selective liquid
medium and INCUBATE BOTH the original flask and the newly inoculated flask.
Dilution series of
environmental
sample
Plate cultures (4 of
your dilutions, 2
from another group
Liquid cultures
(2 of your dilutions)
Growth?
Growth?
Y
N
Y
N
Transfer some of the
liquid to a second liquid
culture, and incubate
BOTH cultures
Streak out to a
selective plate
(Plate-L)
Streak out to a
selective plate
(Plate-P2)
Restreaksingle
colony to a selective
plate (Plate-P1)
Incubate
further
Pure
culture?
Pure
culture?
Growth?
Further
characterization
Further
characterization
Y
Y
Restreakto
selective plate till
pure!
N
N
Pure
culture?
Y
Y N
57
b) Plate cultures – examine the plates and record the numbers of colonies. (NOTE – on some plates
there will be so much growth that you cannot see individual colonies. Record this as “confluent
growth”). This gives you an indication of how many cells in that environment are capable of growth
under your selective conditions.
It is likely there will be diverse colony types appearing. RECORD THE COLONY MORPHOLOGY
of the most abundant morphotypes. This will enable you to determine if the same species were
recovered by direct isolation and isolation after enrichment in liquid.
Using a sterile loop transfer a well-isolated colony to a new plate of the same selective medium and
streak out. This will be referred to as PLATE-P1. Incubate under the same conditions.
Seal the original plate with a Parafilm strip. This original plate will be stored at 4 °C.
FOLLOW UP (Week 5)
a) Liquid cultures – examine your cultures as per last week. Select one of the enrichments from
which to attempt your isolation (even if there are no obvious signs of growth you must do this, this
week).
Use your loop to inoculate a sterile agar plate (PLATE-P2) of your selective medium. Streak out the
inoculum to maximise your chances of observing well-isolated colonies.
b) Plate cultures
PLATE-L: You are likely to see more than one colony type on this plate. Examine the plate and
record the different types of colonies found. NOTE – selective media are not perfect and it is
possible for organisms to ‘cheat’. For example, there may only be one organism capable of using
methanol, but many species that are capable of either scavenging trace amounts of carbon or stealing
carbon from the methylotroph. The most abundant colony-type is most likely to be the organism of
interest (even if it is not the biggest, most spectacular colony).
PLATE-P1: If your technique last week was good then you should see only one type of colony.
Consult your demonstrator for further feedback on your plating technique.
58
FOLLOW UP (Week 6)
Examine the plates. Are the colonies you isolated on plates P1, P2 and L similar?
Perform the following characterization tests on your isolate:
(i) Catalase test (tests for presence of the enzyme catalase – a characteristic of aerobic organisms).
Method: Place a drop of hydrogen peroxide solution onto a slide. Pick a colony, and resuspend
it in the hydrogen peroxide solution. If bubbles form, the organism is catalase-positive (i.e.
decomposition of H2O2 to oxygen is taking place
(ii) Oxidase test (tests for cytochrome c oxidase – a characteristic of organisms with certain
respiratory chains).
Method: The test is done with test strips that contain an alternative electron acceptor that
turns blue-purple when reduced. Dampen the strips with a little distilled water, then load a
loopful of bacterial cells onto the strip. A positive test (oxidase +) will result in a color
change to pink, through maroon and into black, within 10–30 seconds. A negative test
(oxidase -) will result in a light pink coloration or absence of coloration.
(iii) Gram stain (distinguishes cell wall type and also reveals cell morphology and arrangement).
(iv) Motility Test (Hanging drop slide to test for motility)
Method:
1. Hold a clean coverslip by its edges and carefully dab a very little Vaseline on its corners
using a toothpick. (Don’t use too much Vaseline – it will be squeezed toward the center and
mix with the drop).
2. Place a loopful of the culture to be tested in the centre of the prepared coverslip (if using a
colony, resuspend it in a little water).
3. Turn the clean depression slide upside down (depression side down) over the drop on the
coverslip so that the Vaseline seals the coverslip to the slide around the depression. Note that
you need to use the special depression slides for this, not a normal microscope slide.
4. Turn the slide over so the coverslip is on top.
5. Place the preparation onto the microscope slide holder. Close the condenser diaphragm, and
align the slide under low magnification so that the edge of the drop is in the centre of the field
of view.
6. Without raising or lowering the eyepiece, swing the 40X objective into position (Be sure the
objective is clean).
7. Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the
drop can be seen as a thick, usually dark line.
8. Focus the edge of the drop carefully and look at each side of that line for very small objects
that are the bacteria.
9. Adjust the light using the diaphragm lever to maximize the visibility of the cells.
10. Observe the cells to determine whether true motility can be observed. Motile cells may be
seen swimming to and from the air-liquid interface. This can be distinguished from Brownian
movement, due to the impact of energized water molecules on the cells.
11. Do not discard the depression slide with the other waste slides (expensive!!). Place the used
depression slide into the separate beaker provided.
59
Identification of Isolate from Morphology and Biochemistry.
Identification is about excluding possibles just as much as it is finding candidates. Each group should
have done sufficient background reading to have a short list of possible organisms (e.g. a list of 4-5
nitrogen-fixing bacteria from soil for group 3). Try to write down at least one genus that you can
exclude for each test (for example a rod-shaped cell means you can exclude any genus that is typically
a coccus).
For more detailed bacterial identification guides, you may want to consult:
• Holt, J. G. (1994) Bergey’s manual of determinative bacteriology, 9th edition, Williams and
Wilkins.
Selected tables from Bergey’s Determinative Bacteriology have been uploaded as Excel files onto the
Course Blackboard website. Use the filter functions in each column of the relevant tables to select for
the characteristics you have identified for your isolate (e.g. Gram type, shape, motility, catalase activity
etc).
List 4 genera that typically possess the properties you are targeting (e.g. 4 halophiles; 4 nitrogenfixers; 4 methylotrophs; or 4 cellulose-degraders). Indicate those you are most likely to have
encountered in your sample.
1.
2.
3.
4.
Tests Results and interpretation (ie what your organism was and any candidate
genera you exclude on this basis)
1. Shape
2. Gram stain
3. Spore shape (if
applicable)
4. Motility
5. Colony Morphology
6. Catalase
7. Oxidase
Possible Genera
Confirmatory tests (Check
‘The Prokaryotes’ or
‘Bergey’s Manual’)
60
4. IDENTIFICATION OF ISOLATE BY MOLECULAR CHARACTERIZATION OF
THE 16S RIBOSOMAL RNA SEQUENCE.
Traditionally, strain identification has been carried out by measuring a list of typical enzyme activities
to determine the characteristic physiology of the isolate However, in the early 1990’s, Norman Pace
and his colleagues discovered that the phylogenetic assignment of bacteria aligns well with the nucleic
acid sequence of a conserved RNA molecule, the 16S RNA molecule that forms part of the ribosome
in all bacterial cells. This molecule is about 1500 nucleotides long, and its nucleotide sequence is very
similar in all species, to ensure that it can fulfil its structural role. However, it also possesses several
regions that vary in sequence between species – sequencing the part of the gene that encodes these
regions allows us to rapidly identify the species of any given isolate.
The 16S gene from a bacterial isolate can be amplified from cell material using the polymerase chain
reaction (PCR). Bacterial cells are mixed with a thermostable DNA polymerase, nucleotide
triphosphates, and specific oligonuclotide primers that bind to highly conserved regions in the 16S
rRNA gene, in an appropriate buffer. We will use primers that bind on the forward DNA strain at
position 27 and on the reverse strand at position 1492 (27f/1492r), which are commonly used for
bacterial phylogenetic analysis. The mixture is then put through a heating/cooling sequence as follows:
Overview of the Polymerase Chain Reaction
(Maier et al, 2009
Env. Microbiology)
25
cycles
5 min. 94°C
1 min. 94°C
1 min 50°C
1.5 min. 72°C
5 min. 72°C
Boils cells and releases DNA
denatures DNA (strand separation)
Specific primers anneal to denatured DNA
DNA synthesis from specific primers
final elongation step
61
Materials (per group)
(1) Sterile water
(2) PCR tubes
(3) PCR Master mix (contains buffer, dNTPs (dATP, dCTP, dTTP, dGTP), DNA polymerase,
oligonucleotide primers)
(4) 1.5 mL Eppendorf tubes
(5) Benchtop Eppendorf centrifuge
(6) Boiling water bath
Method
DNA extraction:
1. Choose a single colony of the dominant colony morphotype from your last selective plate.
Ask a demonstrator to check your choice of colony before starting this procedure
2. Using a pipette tip, resuspend this colony in 100 µL of sterile water in a 1.5 mL Eppendorf
tube.
3. Centrifuge the tube in a benchtop centrifuge for 1 minute (max speed) to pellet the cells.
Discard the supernatant (use a pipette and pipette tip to remove all of the supernatant).
4. Resuspend the cell pellet in 100 µL sterile water.
5. Use an Eppendorf clamp to heold the lide of the tube closed, or make a hole in the lid of the
closed Eppendorf tube with a needle (why do we need to do this?).
6. Incubate the Eppendorf tube and cells in a boiling water bath (use a float) for ten minutes.
7. Place tube on ice for a few minutes.
PCR reaction
1. Place a PCR tube (the small tubes!) on ice. Label the lid of the tube with a felt tip pen so that
you can see it in the icebath.
2. Add 24 µL PCR master mix to the tube. Always use a fresh pipette tip!
3. Add 1 µL of your boiled cell suspension to the tube.
4. Close the tube, and mix by flicking gently. Tap tube on the bench to ensure sample collects at
the bottom of the tube.
5. Place the tube in the icebath next to the PCR thermocycler. When all samples are ready, the
demonstrators will start the PCR thermocycler, using the thermal program outlined above
Sequence analysis
When the PCR run is complete, the demonstrators will analyse the samples by electrophoresis on an
agarose gel, and a printout of the gel will be mounted on the course Blackboard site. Samples that
show successful amplification (~1.5 kb product on the gel) will be purified and submitted for DNA
sequencing to the Australian Genome Research Facility. The returned DNA sequences will be
available on the Blackboard site.
62
To identify your organisms by 16S rDNA gene analysis, copy the DNA sequence obtained to the
BLAST search page (http://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST stands for “Basic Local
Alignment Search Tool” and the BLAST program carries out an alignment of your sequence with all
known sequences in the online gene databases.
• Choose “nucleotide blast” as the Blast program to run
• Paste your sequence into the query box.
• Choose the nonredundant nucleotide database – “Nucleotide collection (nr/nt)”, and tick the
box to exclude uncultured/environmental sequences.
• Press the “BLAST” button.
• The results (may take a minute or two to appear) show a colour-coded diagram with
similarities, and below this a Table of sequences that are closely related to your sequence.
The best hits are at the top of the table. Click on the “Max Score” value to see how similar
your sequence is to the best hit in the database. Is this a 16S sequence? What is the species?
Discussion
Evaluate the success of the isolation based on the relative proportion of cultivable organisms in the
site investigated (compare with data from Exercise G3 or literature values)
We would expect to find differences in isolation success from different environments. Which
environment was most successful for your organism – suggest reasons for any differences found
between the different environments.
If you obtained a 16S rRNA sequence for your isolate, did this correlate with a species that you
expected? Was the molecular species identification consistent with the selection method you used
(e.g. does it indicate a known methylotrophic organism if you used methanol as enrichment
substrate)?
Highlight the potential significance of the organism you have recovered – including ecological
importance, biotechnological importance and potential problems (e.g. food spoilage, disease).
63
EXERCISE F2
PLANT-ASSOCIATED MICROBES
——————————————————————————————————Exercise F2a Rhizobium and root nodules
In dry-land agriculture as well as in most terrestrial ecosystems, the greatest amount of nitrogen is fixed symbiotically by
bacteria of the genus Rhizobium growing in the root cells of various Legumes. The relationship is symbiotic since the
plants benefit from the supply of fixed nitrogen to them; the bacteria benefit in that they are heterotrophic and receive their
nutrients and energy supply from the plant; and neither organism is harmed by the association. The bacteria enter the plant
through root hairs, and the plant responds with the formation of root nodules. The Rhizobium-Legume association is strain
specific in that only certain strains of Rhizobium are effective nitrogen fixers in the roots of specific plant species. “Cross
inoculation groups” are groups of legumes which develop nodules on exposure to the same strains of bacteria. It is
considered good practice, when planting such crops not to assume that the most active bacteria are already in the soil. Their
presence is assured by the “inoculation” of seed surfaces with the proper organism. Symbiotic nitrogen fixation is a very
important part of the maintenance of the proper nitrogenous reserves in agricultural soils.
1. Effective and ineffective nodulation – (demonstration experiment)
Legume plants will be supplied that have been inoculated with a variety of different rhizobial species.
1) Examine the plants, noting differences in the growth of the plants (plant size)and health of the plants (plant colour)
2) Carefully remove single plants from the soil, and estimate the size and number of nodules on the roots.
3) Tabulate the results. Can you see a correlation between inoculation groups, nodulation efficiency and plant health?
2. Isolation of Rhizobium from root nodules
In this exercise you will examine nodules from legume plants and attempt to isolate the organism present in the nodules.
Materials (per bench)
(1) YMA plate of Sinorhizobium meliloti.
(2) 1 nodulated clover plant.
(per group)
(3) Yeast-Mannitol agar (YMA) plate.
(4) Petri dishes (sterile)
(5) McCartney bottles (sterile).
(6) 5 ml sterile water.
(7) Scalpel blade.
(8) 3% H2O2
(9) 95% alcohol in dropper bottle.
(10) 1 pasteur pipette.
Method
(i) Examine the plant material for nodules, noting the position and colour.
(ii) Select a nodule and remove it with a portion of plant. Place it in a sterile McCartney bottle.
(iii) Defat by pouring a little alcohol from the staining rack over the piece for 10 seconds. Drain.
(iv) Surface sterilise the nodule by immersion in H2O2 for 3 min swirling occasionally. Drain
(v) Wash with the sterile water and drain.
64
(vi) Crush the nodule by placing it in a drop of sterile water in an empty petri-dish and, crushing
it with the cooled flamed end of a microscope slide.
(vii) With a sterile cooled loop, streak a portion of the crushed material on to the YMA plate.
Incubate at 25 °C for 3-5 days. (Plates will be refrigerated after appropriate time.)
(viii) Prepare a Gram stain of the “crush” and of the pure culture of S. meliloti side by side on the
same slide (i.e. not mixed together, but as two mounts on one slide).
(ix) Compare the Gram morphology of these organisms with Gram stains of the issued culture.
(x) Do not discard this slide. Label the slide appropriately, and give it to your demonstrator, who
will arrange for its storage until week 4.
Follow up (Practical week 4)
(1) Gram stain the isolate growing on the YMA and compare its morphology with that seen in the original plant material.
(2) Tabulate all results.
Results
(b) Gram morphology of Sinorhizobium meliloti
YMA S. meliloti culture Organism from nodule (bacteroids Plant isolate (cultured on YMA)
DISCUSSION
Consider:
1. The differences between effective and ineffective nodules and what physical or chemical factors account for these
differences.
2. The difference between S. meliloti in agar culture and in the nodule. Suggest reasons for these differences
65
Exercise F2b Isolation of Fungal Endophytes from leaves
Introduction
Many microbes live on (epiphyte) and within (endophyte) most plant surfaces. Few of these
microbes cause disease. The function of the remaining microbes remains largely unknown. A few
fungi will grow from within tissue and their fruiting structures may be found above the stomata of
leaves. The aim of this exercise is to:
(i) compare the communities of fungi found on and in one leaf to determine whether the
communities differ, and
(ii) learn some introductory methods to manipulate and examine fungi in the laboratory,.
Methods (per group)
Week 3
(i) Isolation of epiphytic fungi (those growing on the leaf). Take a fresh leaf and cut it
into 1 x 1 cm fragments. Take one fragment and press both sides firmly onto V8 agar
containing antibacterial agents (repeat of technique used above). Remove the leaf
fragment from the agar, and place in bleach solution (see step (ii).
(ii) Isolation of endophytic fungi (those growing within the leaf). Place a second leaf
fragment into the bleach solution together with the fragment used in step (i), and
incubate for 2 minutes. Make sure the fragments sink in the bleach to ensure bleach
contacts the entire surface of the leaf fragments. This will kill all epiphytic organisms
on both leaf fragments.
(iii) With sterile forceps, transfer the surface-sterilised leaf fragments to a container of
sterile water, to rinse away the bleach.
(iv) Transfer fragments to a second fresh plate of V8 agar. Press each fragment to the
surface using sterile forceps, and leave them in place. This allows isolation of
endophytic species (i.e. the fungi growing in the leaf will grow out onto the agar).
(v) Incubate both plates for one week at 16 – 20 °C.
(vi) Think about why you have plated both leaf fragments – what differences might you
expect to see between between them?
FOLLOW UP (Week 4)
Examine your plates for fungal colonies. Note that among the epiphytes yeasts may be pink, and
many of the filamentous fungi are dark green to black. The pink to black colour is due to the pigment
melanin, a complex of compounds that provide protection from UV radiation. Are the same colours
evident among the endophytic fungi? As yeasts require molecular methods to identify, subculture a
filamentous fungus. Search your plates for spores among fungal colonies. Spores may be difficult to
detect if lots of water drops are present. Note (sketch) the arrangement of spores in relation to one
another and the hyphae bearing the spores so that you know what to look for under higher power.
Subculture: Use a sterile scalpel (dip blade in 70% ethanol and pass through the flame. DO NOT
HEAT TO RED HOT. RED HOT BLUNTS THE BLADE PREVENTING A CLEAN CUT
THROUGH HYPHAE). Allow the blade to cool (20 sec). Select a fungus that has formed spores.
Remove the lid from the Petri dish. Cut a tiny wedge from the growing edge of ONE colony. Lift the
wedge and aseptically transfer to a fresh plate of V8 agar. Incubate the subculture plate at 16 – 20 C.
66
Visulalization: Take a second thin (less than 1mm) slice from the mature part of the SAME colony
where you saw spores. The aim here is to have a thin enough section so that you do not have to
ratchet the stage up and down to visualise the spores and their attachments microscopically. Lay the
slice on a drop of stain (Cotton Blue in lactic acid) on a glass slide. Stain may be taken up by either
the walls or cytoplasm of hyphae. Thus structures are more easily visualised. Place a cover slip over
the section of fungus. Examine using the light microscope at 10x. Search the upper surface of agar
for spores and the structures from which they are formed. To examine them more carefully, remove
the slide from the microscope stage, press the coverslip down to flatten the fungus. Examine again,
this time at 10 x and then the points of interest at 40 x. Again, make notes to enable you to identify
your fungus using an identification key.
FOLLOW UP (Week 5)
Examine your subculture microscopically (first under a dissecting microscope and then using stained
material on a slide the compound microscope). Again, determine whether the fungus is sporulating,
and whether you have the same fungus identified last laboratory.
Why are the fungal communities on and in leaves different?
Why are so many yeasts found on the leaf surface but not in the leaf?
Did you notice any differences between the colonies from isolation plates and the same fungus after
subculturing?
67
PRACTICAL WEEK 4
Exercise B Winogradsky column
B2 Day 21 observations and discussion on oxygen tolerant photosynthesis
Exercise F Distribution of microbes in the environment
F1a-d Follow-up on all cultures to examine colony diversity (page 56)
F2a Follow-up on nodule squash isolations (page 64)
F2b Follow up on endophyte isolations (page 65)
Exercise G Enumerating microorganisms
G1 Viable count of pure cultures (page 70)
G2 Demonstration of other counting methods – direct count (page 71)
G3 Viable counts of soil bacteria – effect of media (page 74)
AIMS
After completing Exercises G1-3, the student should:
1. Appreciate that there are many ways of estimating bacterial numbers;
2. Be familiar with the application and limitations of some of these;
3. Be able to prepare serial dilutions of a bacterial suspension correctly;
4. Be competent in the Plate Count technique and turbidity measurements;
5. Be able to calculate cell concentration using raw data from a plate count and a Petroff Hausser
count.
68
PRE LAB TASKS (TO BE COMPLETED BEFORE WEEK 4)
1. Read Prescott “Microbial Growth” (p141-171)
2. Read the introductory remarks to Exercise G.
3. Answer the following questions.
(i). Why must culture be diluted in order to carry out a viable (pour plate) count?
(ii). Give 3 causes of “low results” in the plate count technique.
(iii). The direct microscopic method of counting bacteria invariably gives a
considerably higher count of bacteria than the Plate Count. Explain.
(iv). What main problem would be encountered in applying counting techniques to
cultures of Enterococcus and Staphylococcus?
(v). All counting techniques are prone to large errors both statistical and technical.
(a) How can the random sampling error be minimised.
(b) Give 2 ways in which pipetting errors can be minimised.
(vi). The viable count is specifically prone to errors which reduce the viable count (see
(ii) above).
State how one of these may be minimised.
4. Give the three advantages of the Petroff-Hausser chamber for counting bacteria over the use of
stained smears.
(a)
(b)
(c)
69
EXERCISE G
ENUMERATING MICROORGANISMS
————————————————————————————————————————There are several methods available for counting the number of bacteria in a suspension:
1. Total Cell Count. This is a microscopic procedure and gives the total number of cells – dead or alive, as single cells
or in clusters.
The total count can also be estimated indirectly by spectrophotometry. Bacterial suspensions scatter light to an extent
dependent on cell size and numbers. When working with a particular species of bacterium at a particular stage of
growth it is possible to construct a calibration curve which permits the rapid estimation of total cell numbers from a
spectrophotometer reading. We will use this method in exercise G4
2. Viable Count. This measures the number of viable cells (but does not distinguish between clumps of cells and single
cells) and can be done either by the Colony Count Method on nutrient agar plates (Exercises G1 and G2) or by the
Extinction Dilution Method in broth medium (demonstration G5).
In these methods, error may arise from two sources:
1. Random sampling error – (statistical). This arises because only a very small proportion of a randomly distributed
population is counted. To illustrate statistical error in a colony count, consider that the inoculum per plate contains
only a small number of organisms, taken from a suspension where they are randomly distributed. Therefore the
actual number of organisms per plate differs by chance. The error which arises by chance can be calculated. The
relative importance of the error to the count decreases as more colonies occur per plate or more replicate plates
are counted.
2. Technical error The random sampling error cannot be eliminated, but the magnitude of the error may be
calculated and minimized by certain sampling practices, in particular, adequate replication of samples. Technical
error may be readily minimized by adopting a standardized procedure which minimises the error inherent in each
step. Technical errors common to the Total Count and Viable Count methods will be discussed here. Errors
specific to the different methods will be discussed in the appropriate sections.
a) Pipetting
Technical errors in pipetting may arise because of (a) incomplete delivery, and (b) adsorption of bacteria to
the wall of glass pipettes. This is relatively more important in the narrow bore pipettes: 9% per ml in 1 ml
pipettes; 2% per ml in 10 ml pipettes.
Technical error in pipetting can be minimized by:
i) Suck the bacterial suspension up and down at least 5 times
ii) After filling drain 3 seconds against side of container
iii) Transfer to next dilution vessel and blow out contents.
iv) Touch tip against side of vessel and allow to drain 3 seconds.
v) Blow out the accumulated drop.
b) Serial Dilution
Technical errors in the serial dilution process may be used by (a) Use of the same pipette for several
consecutive dilutions, causing inflation of count at higher dilutions due to carry-over of cells, (b) Cumulative
pipetting error from each dilution step, since each pipetting step involves some error.
Technical error in serial dilution can be minimized by:
i) Using a fresh pipette for each dilution step. The tip of the pipette may only be dipped in the
liquid in one vessel, i.e. the lower dilution (higher concentration)
ii) Minimising the number of steps e.g a single 1 to 100 dilution, rather than two 1 in 10 dilutions.
70
EXERCISE G1. THE PLATE COUNT
This technique is widely used to determine viable counts. Serial dilutions are prepared and a suitable
aliquot of each dilution is either spread over the surface of the agar with a sterile spreader or
incorporated into the agar. In the version described here, 1.0 ml of each dilution is pipetted into
duplicate Petri dishes and molten agar (10 mL) at 50
°
C is poured into each dish and mixed with the
bacterial dilution. After incubation, the resulting colonies are counted
The count is based upon the assumption that each bacterium placed in or on nutrient agar medium will
multiply and produce a visible colony. The number of colonies present should therefore be the same
as the number of viable bacteria inoculated into or onto the agar. However, it is not always possible to
be absolutely sure that each colony arose for a single cell especially in species that form specific
arrangement (eg chains, clumps, pairs etc.) For this reason the results to the viable count are always
expressed in terms of colony forming units (cfu) rather than number of bacterial cells.
Materials
1. Culture of Escherichia coli
2. 2 x 99 mls 0.1% peptone diluent
3. 3 x 9 mls 0.1% peptone diluent
4. Pipettes and pipette tips
5. 6 sterile petri dishes
6. 6 nutrient agar pourers
Method
1. Draw a dilution diagram, showing use of pipettes and diluents to prepare 10-4
, 10
-5
, 10
-6
and 10
-7
dilutions of the E.
coli culture and the plating out of the 10
-5
, 10
-6
and 10
-7
dilutions.
2. Label the containers of diluent and arrange them in order on the bench.
3. Label the six plates on the lids. Label 2 for each dilution to be plates i.e. 10
-5
, 10
-6
and 10
-7
.
4. Prepare dilutions according to your dilution diagram.
5. Dispense the dilutions according to your diagram, adding 1 mL of dilution to each plate.
71
6. Pour the plates according to the described procedure. Note that agar should be at ~50 °C and not hotter – if they are
too hot, the hot agar will kill the bacteria in the pour plate procedure
7. Incubate plates at 37
º
C.
Follow up (Week 5)
1. Count the colonies on those plates having between 30 and 300 colonies. Count both submerged and surface colonies.
Plates with more than 300 colonies exhibit competition for nutrient inhibition, crowding, coalescing of colonies and
thus a lower result is obtained.
Plates with fewer than 30 colonies carry a high statistical error.
2. Dot each colony with a marker pen as you count it so that the possibility of counting a colony twice is avoided.
43. Calculate the concentration of viable cells cfu/ml in the original suspension.
Results
Plate count of E. coli culture
Colonies/ Dilution
Plates
10
-5
10
-6
10
-7
Plate 1
Plate 2
(1) Calculate the number of E.coli cfu per ml of the suspension.
Plate 1: ………………………………………………………
Plate 2: ………………………………………………………
Mean: ………………………………………………………..
(2) Compare your results with the other pairs of students working on the same E. coli suspension.
…………………………………………………………………
…………………………………………………………………
Sources of error in viable count determination
The Plate Count method gives only an approximate estimate. Even if it were possible to eliminate
technical error and to standardise other specified sources of error (see below) the random sampling
(statistical error) would remain. This could contribute to an error of 10-30% in a viable count. The
main sources of technical error in viable count determination are:
1) Clumping of Cells
Many bacteria are distributed in chains or groups of varying sizes. Therefore, colonies developing
on plates are derived from aggregates or varying numbers of bacteria. This can be minimized by
shaking suspensions for a standard period of time and by the use of dispersing agents in the
72
diluents. Suck suspensions up and down 5 times in pipettes.
2) Incorrect choice of diluent
Distilled water, especially copper-distilled, is bactericidal to most bacteria (though it also
sometimes gives a higher count, due to dispersive effect on clumps). The use of cold diluent
causes death due to cold shock, in many bacteria. Use a non-toxic and osmotically suitable
diluent, eg Liquid medium with carbon source omitted or Ringer’s solution or a buffer containing
a balanced mixture of CaCl2, MgSO4 and FeCl3. The Aust. Standard Method uses 0.1% (w/v)
peptone solution. Do not use diluent straight from the refrigerator. Measure out diluents after
sterilisation – volume may be lost during autoclaving, leading to incorrect dilutions
3) Incorrect choice of medium and medium temperature
The type of medium may affect the number of colonies that develop – sometimes even the
particular brand of meat extract, yeast extract or peptone can have an effect. Although higher agar
temperature encourages better mixing with bacteria suspensions, too high a temperature is lethal.
To minimize variation, use 10 mL of a medium of standard composition for plate counts, and
preequilibrate agar at 50 ºC before mixing with bacterial suspension for viable counts.
4) Poor distribution of cells in the agar due to method of pouring and mixing plates.
Settling of bacteria and adhesion of cells to the plate before addition of agar can be a major cause
of error, as can inefficient mixing with agar. To avoid these errors, pour 10 ml of agar medium
into the plate within 15 min. of dispensing the bacterial dilution (see practical details below), and
mix immediately as follows:
• 5 to and fro movements, followed by five to and fro movements at right angles to the first
set,
• 5 centrifugal movements in a clockwise direction
• 5 centrifugal movements in an anticlockwise direction.
• This should all take only 5-10 seconds and the plate remain flat on the bench throughout.
5) Number of colonies per plate
Too few colonies per plate produce high variability, while on plates with too many colonies the
colonies suffer from overcrowding – this reduces the count, due to coalescing of adjacent colonies
and self-inhibition due to competition and antagonism. A range of 30-300 colonies per plate is
widely accepted and ensures that at least one dilution will produce countable plates in a ten-fold
dilution series. For statistical reasons plates should be prepared at least in
duplicate and their counts should be reasonably close. If the counts vary greatly, some aspect of
the technique must be suspect.
73
EXERCISE G2. TOTAL CELL COUNT -DEMONSTRATION
The total number of bacteria in a sample can be estimated either using an electronic particle counter, or by counting bacterial
cells under the microscope. Both of these methods only count the total number of cells, and do not differentiate between
living and dead cells (and in some cases non-biological particles). Microbes may be counted directly after drying a known
volume of a cell suspension onto a slide and staining the cells appropriately, but is more usually carried out in a calibrated
counting chamber of known dimensions, eg.Petroff-Hauser counter. The cells are generally counted unstained under the
phase microscope.
.
Bacterial counting chambers have a variety of names, but the best known is the Petroff-Hausser Counting Chamber. The
chamber consists of a central depression of known depth (1/50 mm) covered by a thick coverslip supported by the main
part of the slide. The surface of the depression is ruled into a pattern of squares of known area – large squares with sides
of 1/5 mm, enclosing 16 “smallest squares” with sides 1/20 mm (see diagram below). The volume of liquid over one
smallest square is 5 x 10
-8
ml.
Sources of error in Petroff Hauser total count method
1. Overfilling.
Overfilling the chamber means that the actual depth exceeds 1/50 mm. Use the minimum fluid required to fill the
chamber, so that the coverslip is firmly held by capillary force. The chamber must never be filled to the extent
that liquid overflows into the moat.
2. Agglutination of cells.
Resuspending cells in acidic solutions such as eg. unbuffered NaCl solution that has been exposed to air can lead
to clumping. Use buffered saline (pH 7.5) containing a trace of anionic detergent.
3. Adhesion of cells to glass
Adhesion prevents a random distribution of cells. Use buffered saline with detergent. Examine the chamber
microscopically before use, to ensure that no cells have dried onto the Wash and dry immediately after use.
4. Sampling errors
Counting errors arise from counting cells twice, or missing cells completely, and from counting an inadequate
number of samples (statistical errors). To avoid double counting, count cells lying across ruling – e.g. count only
those which lie on the bottom and right hand lines of each square. Allow cells to settle before counting, and kill
motile organisms by heat or suspension in a diluent containing formalin (0.5% v/v) – this is essential when counting
pathogenic bacteria. Use a hand tally counter if available to avoid losing count. To avoid statistical errors, count
samples giving 5-15 cells per smallest square, and count a total of at least 600 organisms, setting up the chamber
at least 4 times, and counting at least 150 organisms each time.
74
The PETROFF-HAUSSER counter is used to count bacteria. This counter is ruled in the same way as the
Haemocytometer but is only 0.02 mm. deep. Using this ruling, the total number of organisms, living and dead, may
be counted.
Method:
1. Examine the demonstration sample provided on the microscope at the front of the class. Count the number of bacteria
in several different larger squares (as described above). Average these to obtain the number of cells in the smaller
squares, and enter the result in the table.
2. Calculate the overall average count per small cell
No. of cells per smallest square
3. If the suspension provided was 10
-3
dilution of the original, calculate total number of cells per mL in the original culture.
75
EXERCISE G3. ENUMERATION OF SOIL MICROORGANISMS
The total count of organisms in the soil varies constantly and is a reflection of the constantly changing environment, e.g.
water content, temperature, presence of organic material such as leaves. These factors contribute only partly to the variation
of soil population. Many other unknown factors must be involved. This total count of bacteria can be counted directly
under the microscope, but this does not differentiate between viable and dead cells, and is made more difficult by the
presence of soil particles.
The viable count of organisms in the soil varies according to ecological conditions. However, it is also dependent on the
growth medium chosen to grow the bacteria, since soil contains an enormous diversity of bacteria (thousands of species
per gram of soil), each of which has evolved to fit a specific microniche in the soil, and each of which has specific growth
requirements. Since no medium is completely non-selective, the choice of medium will inevitably bias the count towards
one group of organisms or another, and a range of media are commonly used. In addition, the conditions of incubation
(temperature, humidity) impose a further selective force.
In this experiment the viable counts of soil microorganisms obtained using different growth media are compared.
These are:
• Tryptone soy agar – a general purpose medium, containing digests of casein (tryptone) and soybean, at pH 7.3.
It is relatively high in nutrients, and is used for the cultivation of fastidious organisms
• R2A agar – a semi-synthetic medium which contains a range of energy sources and salts at pH 7.2, together with
small amounts of peptone and yeast extract. It is a low nutrient medium that was originally developed to study
slow-growing bacterial species that will not readily grow on fuller, complex organic media.
• Sabouraud’s agar – a peptone medium containing glucose at pH 5.6, which promotes the growth of fungi.
• Glycerol yeast extract plus cycloheximide – this selects for actinomycetes (the antibiotic cycloheximide inhibits
growth of fungi).
Materials
(per group)
(1) Garden soil – 1 g
(2) 99 ml sterile water
(3) 4 x Tryptone soy agar (TSA) pourers (each pourer contains about 20 mL agar).
4 x R2A agar pourers.
4 x Sabouraud’s agar (Sab) pourers.
4 x Glycerol yeast extract + cycloheximide (GYC) pourers.
(per group)
Each group choose one of the selective agar media above
(4) 5 x 1 ml pipette (sterile).
(5) 4 Petri dishes (sterile).
(6) 1 x 99 ml sterile water.
(7) 4 x 9 ml sterile water.
Method
(per group)
(1) Add 1 g soil to the 99 ml water (i.e. soil suspension, 10-2
dilution).
(2) Stopper the bottle and shake vigorously for 2 min.
(3) Shake the soil suspension again just prior to sampling and allow it to settle for 30 seconds.
(4) Prepare serial dilutions of the soil suspension to 10
-6
(10
-3
to 10
-6
will be plated).
76
(5) Dispense 1 ml aliquots into prelabelled plates as appropriate for your media:
10
-6
, 10
-5
, 10
-4
, 10
-3 into TSA plates
10
-6
, 10
-5
, 10
-4
, 10
-3
into R2A plates
10
-6
, 10
-5
, 10
-4
, 10
-3
into Sab plates
10
-6
, 10
-5
, 10
-4
, 10
-3
into GYC plates
(6) Pour the plates with the correct medium, using sterile technique. Remember that the agar needs to be kept at 50 °C
until immediately before pouring, or it will clump. Pour the plates so that the surface of the plate is covered (about
20 mL) – see comments on sources of error for plate counts in Exercise G1 (page 70).
(7) Incubate at 25 °C for 1 week.
Follow up (Week 5)
Method
(1) Select a suitable dilution and count the total number of colonies. Describe the predominant colonial types that
appear on each of the media.
Results Viable Count of Soil Microorganisms
Medium Dilution Number of colonies Microorganisms per
gram
Predominant Type
Tryptone Soy Agar
R2A Agar
Sabouraud’s Agar
Glycerol Yeast Extract
(+ cycloheximide) agar
77
Discussion
What does this tell you about the accuracy of viable counts?
Compare your data here with the outcome of the direct plating on selective media in Exercise F1. Estimate the approximate
proportion of total heterotrophic bacteria in your soil sample that are nitrogen fixers, cellulolytic, methylotrophic or
halophilic (ie approx count on selective medium divided by approx count on general medium).
Give two simple things you could do to increase the viable count from this soil sample.
78
79
PRACTICAL WEEK 5
Exercise B Winogradsky column
B2 Day 28 observations (discuss purple and green sulphur bacteria)
Exercise F Distribution of microbes in the environment
F1 Plating out of isolates (follow-up pages 57 and 66)
Exercise G Enumerating microorganisms
G1 Count the E. coli colonies (follow-up page 71)
G3 Count the soil colonies on different media (follow-up page 76)
G4 Growth curve in liquid culture (page 80)
G5 Enumeration of indicator bacteria in water (page 84)
AIMS
After completing Exercises G4 and G5 the student should be able to:
1. Describe and explain the growth curve of bacteria in batch culture;
2. Plot raw data and use the graph to compare generation time;
3. List factors that affect the shape of the bacterial growth curve (eg duration of each phase, slope
of exponential phase, maximum yield of cells)
4. Know the desirable characteristics and the limitations of using indicator organisms in
enumeration procedures
5. Be able to describe several methods for the quantification of microorganisms in the natural
environment (MPN, viable count, metabolic activity) and know the shortcomings and
advantages of each.
6. Describe the Most Probable Number (MPN) technique for the enumeration of bacteria and know
the advantages and shortcomings of this method.
80
PRE LAB TASKS (TO BE COMPLETED BEFORE WEEK 5)
1.Review introductory remarks to microbial growth curves (“Growth curve: when one becomes
two and two becomes four”, Prescott p. 160-164).
2.Review Introductory animation on Microbial growth on the Blackboard site.
3. Answer the following questions:
a. Why is a bacterial growth best visualized on a semi log plot?.
b. Why do bacteria experience a lag phase before growth starts?
c. Do all bacteria die during the death phase?
4. Review the modes of action of the two antibiotics chloramphenicol and penciliin (Prescott,
p. 191)
a. Which part of the bacterial cell and which cell process is targeted by
chloramphenicol?
b. Which part of the bacterial cell and which cell process is targeted by penicillin?
5. Read the introduction to the most probable number (MPN) technique, Prescott p. 648
6. In using the MPN technique, explain why one dilution may yield no growth, but a higher
dilution (ie more dilute) may yield growth. An example of an MPN code of this type is 2.0.1.
7. Read background to the “multiple tube fermentation test” for coliforms, Prescott, p. 1000
a. What gas do you think is produced during growth of E. coli on MacConkey broth?
b. Why is a confirmatory test needed for E. coli detection?
81
EXERCISE G4
BACTERIAL GROWTH IN LIQUID MEDIA
————————————————————————————————-Growth is the orderly increase in all of the components of an organism. The cell increases in size and ultimately results
in cell division. In a batch culture, a bacterial growth curve consists of four major phases: the lag phase, exponential
phase, stationary phase and the death phase. The generation time, the duration and shape of the growth curve depend on
both the genetic constitution of the organism and the interaction of the organism with its environment (e.g. nutrients,
temperature, acidity of the medium and aeration).
Vibrio natriegens is a Gram-negative marine bacterium, which was first isolated from salt marsh mud. It requires
approximately 2% NaCl for growth, but under optimal growth conditions, with all nutrients provided, it reproduces
extremely quickly. The aims of the following experiments are:
• to examine the phases of a normal growth curve for Vibrio natriegens ;
• to calculate the mean generation time for V. natriegens under optimum growth conditions.
• to examine the effects of temperature and antibiotics on the growth of this organism.
Materials (per bench)
(1) 4 x 40mls BHI (brain-heart-infusion broth) + 2% NaCl in conical Erlenmeyer flasks.
(2) Vibrio natriegens (exponential phase culture).
(3) 1 mL pipettes and tips.
(4) Shaker at 37 °C or Shaker at room temperature (ca 25 °C)
(5) Spectrophotometer
(6) Chloramphenicol solution (1 mg/ml)
(7) Penicillin solution (1 mg/ml)
Method
(1) The groups at different benches will examine the growth of V. natriegens under 4 different sets of conditions.
Group 1. growth at 37 °C (optimal growth temperature)
Group 2. growth at 25 °C
Groups 3 & 4 – the effect of antibiotics: on growth under optimal conditions (37
o
C)
Group 3 – the effect of chloramphenicol (20 µg/ml)
Group 4 – the effect of penicillin (20 µg/ml)
(1) Pre-warm the media at the appropriate temperature for 10 minutes.
(2) Inoculate with 2.0 mL culture (5 % (v/v) inoculum).
(3) Immediately read the absorbance at 600 nm against uninoculated blank
(4) Return flask to shaker immediately.
For optimal growth, the cells need not only nutrients and the correct temperature, they also need oxygen. It is therefore
very important to return the flask to the shaker as soon as possible after sampling, since even a short period without shaking
will reduce the oxygen supply and reduce the growth rate
(5) Carry out absorbance readings at 20 min intervals for the first hour then 10 min reading. (USE THE SAME
SPECTROPHOTOMETER THROUGHOUT THE EXPERIMENT).
TRY TO GET AS MANY TIME POINTS AS POSSIBLE, BUT DON’T BE AFRAID TO MISS
SOME – EVERY 30 MINUTES WILL STILL WORK FINE. IF YOUR TIMES ARE DIFFERENT
FROM THOSE SHOWN IN THE TABLE BELOW, NOTE THE ACTUAL TIME OF SAMPLING
82
Groups 3 and 4 only
(6) When culture is in mid exponential phase and showing a reading of at least A600 0.3, add 1 mL of the appropriate
antibiotic solution.
Determine the absorbance immediately after the addition of the antibiotic to define the effect of the dilution.
ENTER ALL RESULTS ON GROUP RESULT SHEET (NEAR SPECTROPHOTOMETER), AND
THEN COPY THEM INTO THE TABLE BELOW FOR ANALYSIS
(1) A600 readings for growth of Vibrio natriegens
time
(minutes)
37
o
25
o
37
o
37
o
+ Pen + Cam
0
20
40
60
70
80
90
100
110
120
130
140
150
Plot the growth curve for V. natriegens in terms of absorbance, on the semi log graph paper below.
DO NOT USE COMPUTER TO GENERATE GRAPH.
Time (min)
A600
83
By using a logarithmic scale on the A600 axis of your plot, you can use the linear portion of the plot to calculate the
maximum growth rate. This is because time and absorbance during a growth curve are related to the generation time as
follows:
Calculate the mean generation time (i.e. the doubling time) for V. natriegens at 25 °C and at 37 °C. If your data suffice,
average the growth rate values at 37 °C from the Group 1 experiment with those of Group 3 and 4 experiments prior to
antibiotic addition – how reproducible are your data?
Compare and explain the effects of the two antibiotics on the exponential growth rate. How do the results reflect the
mechanism of bacterial growth inhibition for these two antibiotics?
Discussion
Account for the shape of a growth curve for a batch culture, identifying the phases of batch growth
Describe several ways in which the shape of a growth curve can be altered e.g. lag phase extended,
log phase extended, slope of exponential phase changed etc.
t
2
-t
1= g x log(A2/A
1
)
log2
g= generation time
A1, A
2
are absorbancesat time t1
, t
2
84
EXERCISE G5
ENUMERATION OF INDICATOR BACTERIA IN WATER
Of the classic water-borne diseases, cholera is absent from developed regions of the world, while typhoid fever, bacillary
dysentery and amoebic dysentery have been reduced to low levels. Control of these intestinal infections became feasible
when it was realised, in the latter part of the nineteenth century, that they could be transmitted by sewage-contaminated
water. This made obvious the need to protect water sources, and promoted the development of water treatment processes
and methods for judging the bacteriological quality of water intended for human consumption. The success of these efforts
is evident in public health statistics. We tend to take the provision of safe water for granted, but the potential for waterborne epidemics is ever present, and must be constantly guarded against.
It is possible to detect faecal contamination in a water supply by testing the water for the presence of the coliform group
of organisms, which includes Escherichia coli, Enterobacter aerogenes and a number of related species of bacteria. E. coli
is a normal inhabitant of the intestinal tract of man and, when found in water, indicates pollution of the water with intestinal
discharges. Such pollution is usually sufficient to condemn that water supply for drinking purposes.
Generally, coliforms are detected by their ability to produce acid and gas from lactose during growth at 37 °C. Coliform
bacteria can therefore be detected using any medium that is selective for coliforms and contains lactose. In New South
Wales, MacConkey’s broth is widely used as it is selective and is relatively easily prepared. Fully defined media such as
Formate lactose glutamate broth have been recommended as they appear to be even more selective than MacConkey’s broth
and have the added advantage of complete standardisation in terms of composition.
• The presence of coliforms is usually initially tested in a presumptive medium, measuring acid and gas production
in MacConkey broth. The coliform count is determined by applying the multiple tube method and using Most
Probable Number tables.
• The presence of coliforms is then confirmed with a confirmatory test, in this case growth in Brilliant Green Bile
Broth.
o Turbidity and gas at 37 °C confirms the presence of coliforms,
o Turbidity and gas at 44 °C is an indicator of faecal coliforms (i.e. E. coli).
DEMONSTRATION OF MULTIPLE TUBE METHOD
Materials (per bench)
(1) 9 x 9 mls MacConkey broths (presumptive medium), with Durham tubes. Durham tubes are simply smaller test
tubes inserted upside down in the outer test tube, to collect gas that has been generated by the culture during growth.
These presumptive broths have been inoculated with a water sample – the nine tubes represent triplicates of serial
ten fold dilutions (10
0
, 10
-1
, and 10
-2
) of the original water sample.
(2) Brilliant Green Bile (BGB) Broth with Durham tubes. These tubes have been inoculated with the original water
sample and incubated at 37 °C or 44 °C overnight.
Method
(1) Examine the presumptive broths for the presence of acid and gas (presumptive coliforms). Record the number of
positives at each dilution in the table overleaf. Note that a positive test for the presence of a coliform organism
requires both gas and acidification (colour change).
(2) Use the Most Probable Number tables provided to determine the most probable number of coliforms per ml of
original sample
(3) Examine the tubes for the confirmatory test BGB broths. Do the results confirm fecal coliforms?
85
RESULTS (per bench)
Dilution no of tubes showing
turbidity
no of tubes showing
gas production
no. of tubes with
presumptive
coliforms
Confirmed fecal
coliforms (E.coli)?
(yes/no)
Potability*
1
10
-1
10
-2
“Potability” = Safe drinking water quality. The bacteriological standard for potability is less than one coliform per 100 mL.
NOTES
Value of the MPN for 3 tubes inoculated from each of 3 successive 10-fold dilutions.
Positive tubes at
each dilution
M.P.N. (per
inoculum of
first dilution)
Confidence
Limits (95%)
0.0.0 0.0
0.0.1 0.3 0.064-1.4
0.1.0 0.3 0.084-1.4
0.1.1 0.6 0.13-2.8
0.2.0 0.6 0.13-2.8
1.0.0 0.4 0.085-1.9
1.0.1 0.7 0.15-3.3
1.0.2 1.1 0.24-5.2
1.1.0 0.7 0.15-3.3
1.1.1 1.1 0.24-5.2
1.2.0 1.1 0.24-5.2
1.2.1 1.5 0.32-7.0
1.3.0 1.6 0.34-1.75
2.0.0 0.9 0.19-4.2
2.0.1 1.4 0.30-6.6
2.0.2 2.0 0.45-9.4
2.1.0 1.5 0.21-7.0
2.1.1 2.0 0.43-9.4
2.1.2 3.0 0.64-14
2.2.0 2.0 0.43-9.4
2.2.1 3.0 0.64-14
2.2.2 3.5 0.75-16
2.2.3 4.0 0.85-19
2.3.0 3.0 0.64-14
2.3.1 3.5 0.75-16
2.3.2 4.0 0.86-19
3.0.0 2.5 0.53-12
3.0.2 6.5 1.4-30
3.1.0 4.5 0.96-21
3.1.1 7.5 1.6-35
3.1.2 11.5 2.5-54
3.1.3 16.0 3.4-75
3.2.0 9.5 2.0-44
3.2.1 15.0 3.2-70
3.2.2 20.0 4.3-94
3.2.3 30.0 6.4-140
3.3.0 25.0 5.3-120
3.3.1 45.0 7.6-300
3.3.2 110.0 24-520
3.3.3 140.0 30-660
MPN calculation and Confidence Limits
The MPN is a statistical result and represents the most
likely value that will be obtained. However, the actual
result will fall between the limits 95% of the time
tested.
Select the dilution giving zero growth in all three tubes
(“dilution to extinction”), and the two previous
dilutions. If none of the dilutions give 3 negative tubes,
choose the highest dilution.
Example:
Nine tubes incubated with 1 ml of 10
0
dilution (3
tubes), 10
-1
dilution (3 tubes), 10
-2
dilution (3 tubes).
Results – 10
0
– 2 tubes positive; 10
-1
– 1 tube positive;
10
-2
– 0 tubes positive. Recorded as “2.1.0”.
The MPN is quoted as 1.5 organisms per 1 mL of 10
0
dilution with 0.21-7.0 confidence levels.
95% of the time, the true numbers of organisms will fall
between 0.21 and 7 organisms per ml. The result is
usually then expressed as organisms per 100 mL.
See Prescott’s Microbiology, 9
th
edition, p. 649 for
further explanation.
86
PRACTICAL WEEK 6
Exercise B Winogradsky column
B2 Day 35 (final) observations
Exercise F Distribution of microbes in the environment
F1 Preliminary characterization of selected isolate (page 58)
F1 Identification of isolate by PCR and sequencing of the 16S rRNA gene (page 60)
Group discussions and data collation
Exercise H Antagonism and Antibiosis (page 77)
H1 Fungal antagonism – Chaetomium inoculation (page 89)
H2 Bacterial antibiotics – Soil dilution and plating (page 91)
AIMS
After completing Exercise H, the student should:
1) Understand the diverse response of microbes to antagonistic metabolites;
2) Be familiar with two processes used to demonstrate in vitro antagonism;
3) Be able to quantify response to antagonistic metabolites;
4) Understand the application and limitations of measures to assess antagonism
87
PRE-LAB TASKS (TO BE COMPLETED BEFORE WEEK 6)
1. Read the Introductory notes to Exercise H (Antagonism and Antibiosis).
2. Review Prescott “Microbial Interactions (Chapter 30).
3. Read the article on Antibiotics and Streptomyces on the Blackboard site.
4. View the animation on “Isolating antibiotic producers” on the Blackboard site.
5. Answer the following questions:
a) List two antagonistic interactions and two symbiotic interactions between microbes,
or between microbes and other organisms.
i.
ii.
b) Most antibiotic compounds are microbial secondary metabolites. Define a “secondary
metabolite”.
c) If one microbial species of microbe inhibits the growth of a second species, this may
be due to competition or antagonism. Explain the difference.
d) Of 12,000 bioactive secondary metabolites isolated in the 1940’s-1960’s, only 160
reached clinical use. Give three reasons why so many candidate drugs were not
developed further
i.
ii.
iii.
88
EXERCISE H
ANTAGONISM AND ANTIBIOSIS
————————————————————————————————————————–Microbes compete by one or more of at least four mechanisms:
1. Competitive exclusion – Usually via faster growth and reproduction;
2. Niche Specialization – For example tolerance of environmental stresses, or metabolism of
substrates, that other microbes can not;
3. Inhibition – For example release of waste products that other organisms can not tolerate (e.g. acid
production) or synthesis of secondary metabolites that are specifically targeted against competing
organisms (e.g. bacteriocins, antibiotics);
4. Symbiotic/Mutualistic association – with other organisms can improve any of the above
mechanisms and thus increase competitiveness;
Microbes in the first category usually only use simple sugars, sexual reproduction tends to be
uncommon, survival structures tend to be uncommon, and the size of populations increases and
decreases dramatically. These organisms are the most easily recovered microbes in the environment.
Extremes of temperature, osmotic potential, water availability, salinity, barometric pressure,
concentrations of toxins (see below), and pH can all reduce the number of microbes that exist in a
habitat. The microbes found in extreme environments either contain molecules (e.g. proteins) that are
adapted to function best under the particular conditions where the organism lives (e.g. thermostable
proteins) or have evolved mechanisms that maintain the intracellular environment distinct from the
external environment (e.g. many acidophiles and halophiles maintain a ‘normal’ internal
environment).
By accessing a nutrient source not widely used by other organisms, a microbe can limit the number
of competitive interactions it must deal with. Two general rules of thumb are that compounds that are
more difficult to use (more enzymatic steps required to convert a substrate into a molecule able to
enter central metabolic pathways) or that are energetically unfavourable (low or no net yield of
energy in their breakdown) are used by fewer organisms. Such specialist organisms are usually not
growth-limited (they don’t grow any faster when more nutrients are provided) but they do grow
slowly.
Antagonism is the use of chemical and physical interactions by one organism that results in the
replacement of another in the environment. The chemicals are known as secondary metabolites
because they are formed from metabolic pathways that are not essential for the functioning of the
producer. In fungi, enzymic pathways that are used for production of secondary metabolites include:
polyketide synthase, peptide, aromatic compound synthesis. Oligosaccharides, ß-lactam rings and
fatty acids may also be attached to the metabolites. In most cases, the enzymic pathways used to
form the secondary metabolites are unknown and the conditions under which the formation of
metabolites is initiated are unknown.
Association with other organisms is an extremely successful and common mechanism to increase
competitiveness. All plants and animals have many symbionts and saprotrophs growing in
association, some for short and others for long periods of time. For example, the most common
group of fungi in soil, arbuscular mycorrhizal fungi, are obligately associated with the roots of
plants. In virtually all plant-microbe associations the plant supplies energy (photosynthate) and a
consistent environment. The microbe associate may supply diverse benefits to the host plant
including various nutrients. Here we examine one possible function, the antagonism of pathogens.
89
EXERCISE H1 ANTAGONISM OF FUNGAL PATHOGENS BY CHAETOMIUM
———————————————————————————————————————–In this exercise, we will quantify the effect of the release of an unknown array of secondary metabolites
from one widespread endophytic fungus, Chaetomium globosum, on a fungal pathogen. C. globosum
forms small colonies in leaves of a wide variety of wheat cultivars. C. globosum releases various
metabolites that may be involved with competitive interactions, and diverse primary metabolic
enzymes necessary to digest and absorb organic substrates.
The most common preliminary approach to determining presence of inhibitors is to culture two
microbes together on agar. Once an interaction is observed, the next step is to determine whether the
interaction was due to chemical or physical interference or antagonism. We will test for the presence
of chemical inhibitors.
Materials
Four plates of V8 juice agar.
Sterile cellophane
Sterile scalpel
Four different fungal pathogen cultures (choose one of these for your experiment)
Method
1. Co-culture of antagonist and sensitive fungus
(i) Subculture C. globosum and your chosen pathogenic fungus onto a plate of V8 agar by
cutting a tiny wedge from the growing edge of each colony, and transferring them to a new
plate approx.. 4 – 6 cm apart.
(ii) As a control, subculture C. globosum and the same pathogenic fungus by itself onto two
separate plates, transferring each into the centre of the plate.
(iii) Incubate the plates for one week at 16 – 20 °C.
2. Consecutive culture of antagonist and sensitive fungus.
(i) Place a piece of sterile cellophane onto the surface of a V8 agar plate, using sterile
technique.
(ii) Inoculate the plate by transferring a plug of C. globosum into the centre of the cellophane.
Incubate for four days at 16-20 °C, but do not let the fungus to grow over the edge of the
cellophane onto the plate itself.
AFTER 4 DAYS, REMOVE THE FUNGUS AND CELLOPHANE. YOU WILL NEED TO
COME INTO THE LAB TO DO THIS. THIS STEP IS CRITICALLY IMPORTANT, AS IT
PREVENTS THE FUNGUS FROM DIGESTING THE CELLOPHANE AND THEN
PENETRATING THE AGAR BELOW.
90
Follow up (Week 7)
1. Co-culture of antagonist and sensitive fungus.
(i) Measure the growth radiii of both fungi, both between the two colonies, and away from
each other. Compare with the growth of each fungus on agar alone. Collate data from the rest
of the class as replicates, and determine whether there are significant differences.
2. Consecutive culture of antagonist and sensitive fungus
(i) Collect the plate that had the “cellophane culture” of Chaetomium globosum, and also a
new V8 agar plate.
(ii) Pour a thin layer of sterile V8 agar onto the surface of the plate that had the “cellophane
culture”.
(iii) Pour a thin layer of sterile V8 agar onto the surface of the new V8 plate as well.
(iv) Once cool, inoculate both plates with the pathogenic fungus.
Follow up (Week 8)
(iii) Measure the growth radius of the pathogenic fungus on both the Chaetomium-treated and
the untreated plates. Compare using class data as the replicates.
Discussion
Briefly discuss possible reasons for inhibition of the fungal pathogen in the co-culture experiment.
Briefly indicate the reasons why Chaetomium globosum is grown on a layer of cellulose in the
consecutive culture experiment.
Briefly indicate the reasons for pouring a layer of fresh agar over the treated agar
Differences between rates of growth may not be evident even though inhibitors are produced. Why?
If a reduction in disease is noted in wheat when Chaetomium globosum is present, what reasons other
than direct antagonism may explain the reduction (hint: Dingle & McGee, 2003, Mycol Res 107:
310-316)?
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EXERCISE H2
ISOLATION OF ANTIBIOTIC-PRODUCING MICROORGANISMS FROM SOIL
The search for new and better antibiotics continues. No antibiotic is effective against all microbial
agents and there are some pathogenic organisms for which no effective antibiotic has yet been
discovered. Many antibiotics are produced by filamentous bacteria belonging to the genus
Streptomyces. These organisms may readily be isolated from soil, with different soils yielding various
species capable of producing different antibiotics.
The object of this exercise is to screen a number of soils for actinomycetes capable of producing
antibiotics against two test organisms, Staphylococcus aureus and E. coli. The soil actinomycetes will
be isolated on glucose-salts agar with nitrate as the sole nitrogen source and 250 ppm cycloheximide
added to inhibit fungal growth, a partially selective medium which favours actinomycetes (see page
76).
In industrial practice, the antibiotic-producing organisms would be isolated and the antibiotics
characterized. Many antibiotics are discarded on the grounds that they have already been discovered,
have high mammalian toxicity or low anti-microbial activity in vivo. After a long period in which very
few completely new antibiotics were being discovered, recent revolutions in molecular biology and
isolation strategies are leading to a new generation of bioactive compounds from microorganisms.
Materials
(per group)
(1) 12 plates of glucose-nitrate agar
(+ cycloheximide)
(2) 2 x 99 ml sterile distilled water (SDW)
(3) 2 x 9 ml SDW
(4) Pipettes and pipette tips
(5) sterile plastic spreaders
(6) 95% ethanol (in beaker)
(7) Soil sample
Method
(1) Add approx. 5g soil to 99 ml SDW.
(2) Prepare soil dilutions of approx 5 x 10
-4
, 1 x 10
-4
and 1 x 10
-5
, as depicted below.
(3) Working from the most dilute to the most
concentrated soil suspension, pipette 100 µL of
each dilution onto 4 plates of medium.
(4) Working from most dilute to most concentrated,
use the spreader to evenly spread the soil
suspension over the surface of the plates.
(5) Incubate the plates at 30
o
C for 4 – 6 days.
99 mL 99 mL
9 mL
9 mL
1 mL 2 mL 1 mL
Approx. 5 x 10
– 4
1 x 10
– 4
1 x 10
– 5
dilution
5g
soil
92
Follow up (Week 7)
Materials
(per group)
(1) 6 tubes of 5-ml molten nutrient top agar at 50
o
C
(2) Overnight broth cultures of S. aureus and E. coli
Method
(1) Select the 6 best plates. Aim for plates with 30-300 well separated colonies, and a range of
colony morphologies.
(2) Overlay each plate with nutrient agar containing one of the test organisms (S. aureus or E. coli)
as follows:
(a) Transfer 1 loopful (about 50 µL) of a suspension of the test organism to 5 ml molten
nutrient agar at 50
o
C, and mix by rolling the tube between the palms of your hands.
(b) Rapidly pour the inoculated agar over the soil dilution plate and tilt to ensure an even
thickness of agar over the entire plate. Do not let the agar cool down before pouring!
(c) Incubate overnight at 37
o
C.
Follow up (Week 8)
(4) Examine the plates for zones of inhibition around actinomycete colonies.
(5) Assuming that 5g soil was added to the first dilution bottle, calculate the number of
actinomycetes per g soil and the proportions that produce antibiotics inhibitory to each test
organism.
Discussion
(i) What factors would influence the size of the inhibition zone around each actinomycete
colony?
(ii) Were the active actinomycetes on different plates similar or different? How can you
distinguish between different strains on the plates?
(iii) What steps do you think might be taken to maximize the chance of isolating novel strains of
actinomycete – that are potentially producing novel antibiotics?
93
94
PRACTICAL WEEK 7
Exercise H Antagonism and Antibiosis
H.1 Inoculate plates with susceptible fungus (follow-up page 90)
H.2 Inoculate Actinomycete plate with test organisms in agar overlay (follow-up page 92)
PROJECT: Isolation of a pathogen and Koch’s Postulates (page 96)
Tutorial: Introduction to the project
PRE-LAB TASKS (TO BE COMPLETED BEFORE WEEK 7)
Read the introduction to the Project.
95
96
WEEKS 8-13: Forensic Microbiology Project
You will largely work at your own pace over weeks 8-12 according to the general guidelines for the
project. There will be tutorials during this period on how to prepare your report.
PRE-LAB READING
Disease diagnosis based purely on symptoms can be tricky as the symptoms tell you about the plant’s
response to infection rather than the pathogen. For example, a wilt disease could be caused by a
virus, bacterium or fungus. Just as microorganisms can be isolated from air, soil or dead plant tissue,
they can be isolated from diseased plant tissue. However, when you isolate organisms from a
diseased plant you are likely to find more than one microorganism present – saprophytes as well as
the pathogen – and it may well be unclear which organism is causing the disease symptoms.
Identification of the real pathogen is extremely important for advising control measures. Books and
host indices are a help if the symptoms are well described and the pathogen is a common one, but
what if you are trying to identify a new disease? How can the effective pathogen be unambiguously
identified?
Koch’s Postulates
Robert Koch, a German physician famous for his study of tuberculosis, developed a routine
procedure to identify the cause of a disease. In 1882 he devised four steps to ensure correct
identification of the causal pathogen:
1. Determine whether the suspected pathogen is always associated with the disease (symptom
complex) in question. For instance, if some plants are found that show all symptoms but in/on which
the suspected pathogen is not present, the cause of the disease may be something else than the
suspected pathogen.
2. Isolate the suspected pathogen, grow it in pure culture on an artificial medium, and describe it.
3. Inoculate the organism onto a healthy plant of the same variety and species, and determine
whether the symptoms obtained are the same as those of the original disease.
4. Reisolate the pathogen from the inoculated plant and compare with the original isolate. This rule
was added in 1901 by the American plant pathologist E. F. Smith, and serves to confirm that the
organism actually spread and multiplied in the host.
For rapid diagnosis in a diagnostic clinic, Koch’s postulates usually are not followed, since most
diseases are well known to trained personnel and the described procedures are time consuming. For
previously unknown diseases, however, these rules of proof have to be satisfied. For pathogens that
cannot be grown on artificial media, such as viruses, the rules have to be modified; for example,
viruses have to be isolated, purified, and characterised to satisfy rule 2.
Symptomatology
Symptomatology is the study of symptoms and signs as evidence in disease diagnosis. The purpose
of this exercise is to develop a practical understanding of symptomatology for use in diagnosis or
other areas of pathology. Individual diseases are recognised largely by characteristic symptoms and,
possibly, signs. Familiarity with symptoms and signs is essential in forensic diagnosis. Familiarity
with the terminology of symptomatology is necessary in use of diagnostic reference books etc.
Disease — A deviation from normal structure and/or function resulting from an interaction with a
causal agent, and characterised by certain symptoms and signs.
97
Symptom — A visible or otherwise detectable expression of abnormal physiology, development, or
behaviour resulting from disease. Symptoms often involve changes in form, colour, odour, texture,
and structural integrity.
Sign — Any observable part or remnant of the causal agent (pathogen) in disease. Common signs
include vegetative or reproductive structures of pathogens, such as fungal mycelium, parasite eggs or
spores.
Syndrome — A specific group of symptoms associated with and characteristic of a disease. These
symptoms may appear all at once or in a sequence.
The use of symptoms and signs in the diagnosis of diseases
Symptoms and signs are the most important evidence used in diagnosis, although information about
environmental, cultural, and other conditions must be considered as well as these may lead to noninfectious diseases. The roles of symptoms and signs in diagnosis are different. Signs can point directly
to causal agents. However, one must be careful in evaluating them since they may belong to secondary
agents or saprophytic colonisers. Symptoms alone are often less conclusive than signs because they
may be shared by different diseases or pest-related injuries. Nevertheless, symptoms can indicate how
a disease develops and thereby point to potential causal agents whose mechanisms of pathogenesis
(such as toxins or rot enzymes) cause the observed effects. Symptoms may be the only evidence of
causes (such as mineral deficiencies) that do not produce signs, or may call attention to secondary or
contributing causal factors. Symptoms support more complete interpretation and evaluation of a
disease than do signs and thereby contribute both to diagnosis and to the prescription of more effective,
integrated control measures that address all participating causes of disease.
Each disease is characterised by certain symptoms although the expression of symptoms for a given
malady may vary. Symptoms rarely occur singly but instead occur in groups called syndromes.
Symptoms may vary in order of occurrence or in their intensity or extent of development; they may
be altered or absent in a syndrome; or they may be altered or hidden (masked by other symptoms or
effects). Symptoms can change in nature and intensity over the course of a disease, and they may be
modified by environmental conditions.
Symptoms can reflect localised or systemic effects of disease. Primary symptoms, such as rot, are
those which occur close to the site of active infection and pathogenesis, while secondary symptoms,
such as wilt in the case of root disease, appear at sites remote from infection and reflect systemic
stresses such as translocated toxins or water deprivation. For these reasons, it is essential that
symptoms be studied as results or effects caused by mechanisms of pathogenesis (disease
development). Symptoms can be external or internal, macroscopic or microscopic. Symptomatology
is best learned by examination of actual diseases.
98
AIMS
To become familiar with techniques used in diagnosis of plant diseases, including:
? Macroscopic and microscopic investigation of symptoms and pathogen structures
? Isolation of microorganisms and handling of cultures
? Inoculation of healthy plants to reproduce symptoms of natural infection
? Disease and pathogen identification by comparison with descriptions in the literature
METHOD
Read through the whole exercise before starting so that you know exactly what you will be doing.
Make sure you bring a notebook to class so that you can make and annotate the detailed drawings
you will need for your report.
Work in groups of 5
Week 1:
You can either bring your own specimen (check with your demonstrator first), or use the diseased
sample provided. Samples should be diseased plants or plant parts. Do not bring contaminated or
infected meat products – the pathogens that infect these substrates are quite likely to also infect
humans, and constitute a severe health risk.
1. Fully describe the symptoms.
The fact that you will have only one or a few specimens to study will limit your ability to confirm
“constant association”. Record the name of the host. Information about a disease has no scientific
value if we don’t know the patient. Include the name of the cultivar if this information is available.
Describe the symptoms and signs in detail at both macroscopic and microscopic level, and if possible
take photos. Later, after you have isolated the pathogen and inoculated it into a healthy host, you will
have to compare the resulting symptoms and signs with those of the original disease. This will be
possible only if you have an accurate description of the original symptoms (see Koch’s 3
rd
postulate
above).
The description should proceed in three phases:
Macroscopic symptoms and signs. Examine the diseased specimens and determine if the symptoms
they display are of non-infectious or infectious origin. Compare diseased and healthy plants. Make
sketches. Include information about the colour of the tissues, the shape and size of the lesions, any
characteristic odours, and the consistency of the tissue (soft, watery, leathery, hard, etc.).
1. Study the specimen under low magnification (hand lens or dissecting microscope).
Particularly, look for signs of the pathogen: mycelium, spore stalks with spores, fruiting
bodies. Describe and sketch.
2. Microscopic investigation. Prepare microscope mounts by cutting thin tissue sections with a
razor blade, or by making preparations of fungal structures, either removed from the tissue
with a scalpel or needle or by using the cellophane-tape technique. Look for fungal
mycelium, spores, and spore-producing structures, and for masses of bacterial cells streaming
from tissue when placed in a drop of water.
DOCUMENT YOUR OBSERVATIONS WITH PHOTOGRAPHS OR WITH DETAILED
SKETCHES – THESE ARE PARTICULARLY IMPORTANT FOR MICROSCOPIC
OBSERVATIONS, WHICH MAY BE MORE DIFFICULT TO PHOTOGRAPH.
99
This investigation might give you some additional clues about whether the disease is caused by a
fungus or bacterium, whether it is caused by a type of fungus that presently cannot be cultured, or
whether the disease is caused by a virus, mycoplasma, phytoplasma or abiotic cause that should be
investigated by other techniques.
2. Look for signs of the pathogen
Cut the diseased area from the plant and examine under low magnification for mycelium, spores or
bacterial ooze when placed in a drop of water.
3. Attempt to isolate the pathogen
? Immerse a few small samples of your specimen containing both healthy and diseased tissue
in 0.5% sodium hypochlorite solution for 30-60 seconds, then rinse in sterile distilled water
and dry on sterilised filter paper.
? If you suspect a fungus, cut small (5x 5 mm) sections from the edge of the lesion (see
diagram) and plate onto a suitable medium (consult your demonstrator).
? If you suspect a bacterium, use a sterile scalpel to chop a piece of the affected tissue in a drop
of sterile water in a petri dish. Using a sterilised inoculating needle, and the “16-streak
method” (see p. 34), streak a drop of the macerated tissue onto a plate of King’s B agar. Label
the plate appropriately with group name, sample name, and date. The plate will be incubated
(inverted) at 30
o
C until colonies appear, then refrigerated.
Week 2
Examine and describe the colonies that grow. If several different organisms grow on a streak plate or
from plated-out tissue, you should prepare a pure culture of each of them, so that you can inoculate
each separately to a host. Photograph the colonies, and count the frequency of each colony type -sometimes the pathogen is the dominant organism isolated from diseased tissue, but often the most
frequent isolates are actually just saprophytes!
If you have several organisms, restreak each organism onto a separate agar plate to ensure purity.
However, fungal or bacterial colonies that originate at a distance from streaks or plated-out tissue are
airborne contaminants. Such contaminants will often sporulate profusely and the plate must then be
handled with extreme care to avoid shaking or blowing the spores all through the plate. Try opening
the plate carefully and placing a few drops of 95% ethanol on the contaminant colony with a Pasteur
pipette or dropper. This may kill the contaminant while wetting the spores so they won’t dust around.
1. Transferring to a pure culture
1. Sterilise transfer loop or needle in a flame, and allow to cool. Open the plate slightly, insert the
loop or needle, and pick up a bit of material from the culture. With a sporulating fungus or a bacterial
colony, it may be sufficient to touch the colony with the needle to pick up enough cells to transfer. If
the culture is not sporulating, cut out a small piece (1-3 mm diameter) of agar at the edge of the
100
colony. Close the plate and transfer the sample to a fresh plate. After transfer, be sure to save the
isolation plate until you are certain that your transfer has been successful and the organism is
growing in the new plate. Record any observations on the appearance and rate of growth of the
organism.
2. Examine your isolate
? Measure the size of the cells to determine whether your organism is a prokaryote (bacterium)
or a eukaryote (fungus or yeast).
? If you find a bacterium, observe a colony smear in a drop of water on a clean microscope
slide, under a coverslip, using phase contrast. Prepare a Gram stain of the organism to
determine cell wall structure (page 25). Prepare a hanging drop preparation of the organism
to determine motility (page 58).
? Give your isolates identifying strain names (e.g. a strain with a big yellow colony could be
named BY1). This will make it much easier to refer to them unambiguously when writing
your report.
? If you isolate a fungus, describe and photograph the colonies. Identify the pathogen as far as
possible based on the colony morphology and spores you find.
3. Re-inoculate healthy host tissue
Mock-inoculate healthy host specimens and inoculate others with the culture you isolated. Use the
least damaging and most natural inoculation technique possible.
Consult your demonstrator, or try several methods, for example:
? Rub the host surface with 95% ethanol and let dry. With a scalpel or razor blade, make a cut
in the host tissue and insert a piece of agar culture. Tape a piece of agar culture to the intact
host surface; place a drop of water at the edge of the agar.
? Place drops of a spore suspension or bacterial cell suspension on the host surface. Prick with
a needle through some of the drops into the host tissue while leaving intact the tissue under
other drops
There are a number of factors that determine the success of an inoculation. Some experimenting may
be necessary:
? The host should be at a susceptible stage. Many hosts vary in their susceptibility with age.
For instance, only young seedlings may be susceptible to damping-off pathogens; ripe fruits
may be more susceptible to certain pathogens than unripe fruits.
? The inoculum must be viable and in sufficient quantity. Although a single fungal spore or
bacterial cell may cause disease, the likelihood is usually small. Thus, if a first inoculation
fails, you should try again with a larger amount of inoculum. Some pathogens may lose
virulence in culture, but since you have a freshly isolated pathogen, this is not too likely to be
a problem.
? The inoculum must be placed in a suitable infection court. Inoculate the organism into the
same tissue from which it was isolated. Some pathogens may be able to attack only roots, or
leaves, or flowers, or fruits. Many fungi can penetrate through intact plant surfaces, but other
fungi and all plant pathogenic bacteria require wounds or natural openings for infection.
Think about the number of replicates you need to make to be sure about your result, and provide an
appropriate number of host samples for inoculation (and mock inoculation).
Environmental conditions must be suitable for infection. Free moisture or high humidity is required
by the majority of pathogens. You can maintain a humid environment around the inoculated host
specimen by enclosing it in a plastic bag or a jar with a moist paper towel. Leave the jar lid slightly
loose or the plastic bag not tightly closed in order to avoid anaerobic conditions.
101
4. Check your specimen daily
The disease may develop very rapidly, or the specimen may be over-run with secondary invaders.
Compare symptoms with those recorded on your original specimen. Record your observations on the
rate of disease development. You need to record your observations as soon as symptoms appear.
Week 3 or 4
Observe any symptoms that appear on your inoculated specimens, by comparing to the shaminoculated specimens.
5. Reisolation of the Pathogen (Rule 4)
? Describe symptoms and signs and compare with your original description.
? As soon as disease symptoms are clearly evident, isolate from the edge of the lesion, away
from the inoculation site, since you want to show that the pathogen has actually spread
through the host tissue. Follow the procedures as described in Week 1.
? Prepare a pure culture of the reisolated pathogen for comparison with the original isolate and
identification of the pathogen and disease. At the same time, you should transfer the original
isolate to a new plate so that you can compare cultures of the same age of the two isolates.
6. Identification of the pathogen.
This is the hardest part of the experiment, but several types of reference materials may help you in
trying to identify your pathogen to genus. There are basically two approaches:
1. Start with references that describe diseases of specific hosts or groups of hosts (e.g. diseases of
citrus fruits), and look for symptom descriptions or pictures that match your disease. This will
hopefully help you to narrow the search down to a few genera or, ideally, one genus. Then you
should look up that genus or those genera in the references that contain descriptions of the fungi or
bacteria to verify your conclusion.
2. You can also start with the pathogen you isolated and key it out using one of the identification
keys provided. After identifying the pathogen you should verify your conclusion by checking
whether an organism of that name has been recorded to cause a disease of the host in question and, if
possible, by checking the symptom description.
Not all students will be successful completing Koch’s postulates. A host of things can go wrong
despite the best attempts. However, even if you cannot come up with the “right” pathogen, you can
still draw some conclusions. If you are unable to identify your isolate, you can still give a detailed
description of its characteristics. If your inoculation results in different symptoms than you observed
originally, or your reisolation yields a different organism, you can make a comparison of the two
diseases or organisms. Reasonable attempts to complete the exercise, to understand the procedures,
and to interpret the results are what make this exercise valuable.
Questions
1. What obvious limitation is there to the application of Koch’s Postulates to obligately biotrophic
pathogens?
2. What control treatments are essential with your inoculation?
3. How could you make sure that you have isolated a pure culture?
4. If you have completed the steps of “Koch’s postulates” successfully, does your evidence leave any
doubt that the organism isolated is the cause of the original disease? Describe any scenarios with
another cause that you can think of.
5. If the reinoculation does not result in any disease symptoms, what are the possible explanations?
List as many as you can.
102
References
• Agrios, G. N. (2005). Plant Pathology 5
th
edition , Academic Press
• Brown J. F. and Ogle H. J. (1997) Plant pathogens and plant diseases, Rockvale Publishing.
• Schumann, G. L. and D’Arcy, C. J. (2010) Essential Plant Pathology, 2
nd
edition, APS Press.
• Waller, J. M., Lenné, J. M. and Waller, S. J. (2003) Plant Pathologists Pocketbook, 3rd
edition,
CABI Publishing. Also available as an e-book from the University Library website
Report
Write an individual report based on your group’s attempts to isolate the pathogen from the diseased
specimen. Briefly outline the techniques used in your isolation and identification. Discuss the
biology of the pathogen and the importance of the disease.
The report is due by 9 am, Monday 6th
November. It should be submitted online using the
MICR2024 Blackboard site.
Note that the report will be submitted as a Turnitin assignment. The text of the report will
therefore be automatically scanned for text that appears significantly similar to text from
elsewhere. Reference lists are excluded from this scan.
The report should be no more than 4-6 pages (excluding drawings), and should be written up as if for
publication as a scientific article (i.e. Title, Author, Abstract, Introduction, Materials and Methods,
Results, Discussion and References – see following page). Drawings and diagrams are welcomed
where they add to the clarity of the report.
A model report can be seen at the Blackboard site. There are also a number of useful guides to
scientific writing that you should consult before handing in your report. These include:
• Northey, M. and von Aderkas, P. (2011). Making sense : life sciences : a student’s guide to
research and writing, Oxford.
• Pechenik, J. A. (2010) A short guide to writing about biology. 7
th
edition,
Pearson/Longman.
103
Recommended Format
Title: The title should be suitable, accurate and sufficiently descriptive.
Abstract: The abstract should be a clear and concise statement of the content and main conclusions
of the paper. It should preferably be limited to 200 words.
Introduction: The introduction should not be too long, and it must clearly state the aims or objectives
of the work.
Methods: The methods used in the work must be described well enough to allow the work to be
repeated. The active ingredients and chemical names should be clearly specified. There
should be no ambiguity in denoting the composition of mixtures and solutions. The
methods used should be adequate to support the conclusions made.
Results: Results must be clearly stated. Statistical treatment should be adequate. The tabular
material should be an essential part of the paper, support the claims made in the text, and
be in suitable form. If the information can be presented as effectively in the text, it
should appear there or be presented as a graph. Tables should be scanned for irrelevant
or unnecessary data. The captions/legends of tables and figures must contain adequate
information. Illustrative material that is not clear or that does not aid materially in the
presentation of the results should be omitted.
Discussion: The discussion should provide a reasonable interpretation of the results and place them
in the context of the work described. The author’s deductions and conclusions should be
logical and clearly stated. Alternative interpretations should be considered and evaluated.
The discussion should not repeat the results except to introduce or clarify deductions or
discussion.
References: The references cited must be appropriate to the experimental or other work described.
Ensure that all the important references are cited.
Presentation: The report should be well organised and presented clearly and logically.
Based on feedback from previous years, important points to remember include:
? Be concise: excessive wordiness is annoying to readers. Do not submit a rambling essay.
? Address the question posed
? The “Results” should be your own observations. Other people’s observations should be
compared to yours in the “Discussion”.
? Be aware of, and discuss, the limitations imposed by the experimental design
? Be careful not to speculate – you can extrapolate if you have sufficient justification from your
results, and results from relevant work by others. Remember “Occam’s razor” – the best
explanation is usually the simplest.
? Check for spelling and grammatical errors
? Check that all Latin binomials are italicised or underlined
? Check that you use singular and plural forms correctly e.g. bacterium (-ia), conidium(-ia),
fungus(-i), mycelium(-ia), sporangium(ia), medium(ia), etc.
104
Appendix 1– Microbial growth media
PDA Potato Dextrose Agar (1 L)
• 250 g potatoes diced, boiled and strained
• 500 mL potato pure
• 20 g Dextrose
• 20 g Agar
Autoclave, cool to 50 °C, add 4 drops lactic acid to prevent bacterial growth.
V8 juice agar medium (1 L)
• 200 mL V8 juice
• 3.0 g CaCO3
• 20 g Agar
Adjust pH to 7-7.5.
Autoclave, cool to 50 °C, add 4 drops lactic acid to prevent bacterial growth.
Nutrient Agar (1 L)
• 5.0 g Yeast extract
• 10.0 g Peptone
• 5.0 g Sodium Chloride
• 15.0 g Agar
Adjust pH to 7.4, Autoclave
King’s B Agar (1 L)
• 20 g Proteose peptone No 3
• 1.5 g K2HPO4
• 1.5 g MgSO4.7H2O
• 15.0 mL Glycerol
• 15.0 g Agar
Adjust pH to 7.2, Autoclave
105
Tryptone Soy Agar (1 L)
• 15 g Tryptone
• 5 g Soytone
• 5 g NaCl
• 15.0 g Agar
Adjust pH to 7.2, Autoclave
R2A Agar (1 L)
• 0.5 g Yeast extract
• 0.5 g casein hydrolysate
• 0.5 g Glucose
• 0.5 g soluble starch
• 0.3 g K2HPO4
• 0.2 g sodium pyruvate
• 0.5 g peptone
• 0.024 g MgSO4.7H2O
• 15.0 g Agar
Adjust pH to 7.2, Autoclave
Sabouraoud’s Agar (1 L)
• 10 g Peptone
• 40 g Glucose
• 15.0 mL Agar
Adjust pH to 5.6, Autoclave
Glycerol yeast extract plus cycloheximide (1 L)
• 5 g Yeast extract
• 50 mL Glycerol
• 15 g Agar
• Adjust pH to 7.2. Autoclave. Add 100 mg cycloheximide before pouring.
MacConkey Agar (1 L)
• 20 g Peptone
• 10 g Lactose
• 5 g NaCl
• 1.5 g Bile salts
• 50 mg Neutral Red
• 1 mg Crystal violet
• 15.0 g Agar
Adjust pH to 7.2, Autoclave
106
APPENDIX 2 – USE AND CARE OF THE MICROSCOPE
THE USE AND OPTICS OF THE COMPOUND MICROSCOPE
107
Optics
The microscope comes equipped with an eyepiece (10X magnification), and four objective lenses.
These are two low power objectives (4X and 10X magnification) a 40X magnification objective, and
a 100X magnification objective, which is used with oil immersion. The total magnification may be
calculated by multiplying the magnification power of the eyepiece with that of the objective lens used.
Further optical details are given below:
Objective
Property Objective
Magnification 4X 10X 40X 100X
Numerical aperture
(N.A.)
0.10 0.25 0.65 1.30
Working distance
(W.D.)
19.87 5.40 0.39 0.11
Focal length (mm) 29.20 15.98 4.31 1.81
Resolving power (µm) 3.4 1.3 0.52 0.26
With
eyepiece
(10X)
Total magnification 40X 100X 400X 1000X
Focal depth (µm) 172.5 27.6 3.03 0.66
Field of view (mm) 4.5 1.8 0.45 0.18
• Working distance: The distance from the specimen or cover glass to the nearest point of the
objective.
• Numerical aperture: The N.A. represents a performance number which could be compared to
the relative aperture of a camera lens. The quantity of light which the objective receives from
the object- increases with the square of the performance number.
• Resolving power: The resolving power of a lens is measured by its ability to separate two
points.
• Focal depth: The distance between the upper and lower limits of sharpness in the image
formed by an optical system. As you stop down the aperture iris diaphragm, the focal depth
becomes deeper. The larger the N.A. of the objective the shallower the focal depth.
• Field number: A number that represents the diameter in mm of the image of the field
diaphragm that is formed by the lens in front of it.
• Field-of-view diameter: The actual size of the field of view in mm.
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SETTING UP THE MICROSCOPE
1. FOCUSSING
a) Place the slide to be examined on the stage so that the specimen to the stage opening.
b) Rack the condenser upwards to its highest setting.
c) Open fully the condenser iris and the field iris diaphragms.
Focus on the object:
d) Raise the stage until the low power lens is less than 5 mm from the slide, and while observing
through the ocular, slowly move the stage and objective apart until the object is in focus.
2. ADJUSTING THE CONDENSER
a) Close down the field iris diaphragm to about 1/3 field of view.
b) Adjust the height of the condenser so that the image of the field diaphragm is sharply
focused in the same plane as the object, ie object and field diaphragm in focus
simultaneously.
c) The correct adjustment is achieved when the image of the field diaphragm opening has a
sharp edge and is as small as possible.
d) Centre the diaphragm image if necessary by using the centring screws on the condenser.
e) Reopen the field diaphragm until the image of the diaphragm coincides with the edge of the
field of view.
f) Regulate the iris diaphragm of the condenser to give adequate but not excessive illumination
and contrast:
Remove the ocular and while viewing the back lens of the objective adjust the diaphragm so
that the illuminated circle is open to approximately 7/8 of its maximum diameter.
3. USING OIL IMMERSION OBJECTIVE (X100)
The oil immersion objective is necessary for the microscopic examination of stained bacterial
smears.
a) Swing aside the low power objective.
b) Place a single drop of immersion oil directly on top of the smear.
c) Rotate the nosepiece so that the oil immersion lens clicks into position.
NB: (Rotate from x10 to x100)
d) Use the fine focus knob to refocus. (This should require much less than 1 turn of the fine
focus knob).
e) Adjust the condenser diaphragm as follows:
Remove the ocular, and adjust the iris diaphragm of the condenser to optimal contrast by
opening it completely and then closing it slightly so that it matches the back lens of the
objective (approx. 7/8 of maximum diameter open.)
f) Replace the ocular and examine object.
Note: Unstained wet mounts may require further adjustment of the condenser iris diaphragm
to improve contrast.
4. CLEAN-UP
a) When your observations are complete clean the microscope. Use lens tissue to wipe oil from
the oil immersion lens and the stage
Store with the low power objective in position over the stage opening.
Jordan is planning a party and deciding how much of the budget to devote to Beverages (B) and how much to comestibles (C). Guests at the party will have the utility function
“The Pacific Crest Trail wasn’t a world to me then. It was an idea, vague and outlandish, full of promise and mystery. Something bloomed inside me as I traced its jagged line with my finger on a map”