National Centre for Marine Conservation
and Resource Sustainability
MICROBIOLOGY LABORATORY MANUAL
C.M. Burke 2008
1
TABLE OF CONTENTS
Table of contents
2
Practical marking schedule & references
4
Techniques completion list
5
Microbiology Project
6
Sub-Project 1: A survey of microbes in several environments.
7
Sub-project 2: Taxonomy: how microbes are identified and catalogued.
10
Sub-project 3: Does the environment affect the survival or growth of microbes?
13
METHODS MANUAL
18
LABORATORY RULES
1
PERSONAL PROTECTIVE EQUIPMENT AND PROCEDURES
2
PRACTICAL REPORTS
3
METHOD 1: Optimising Illumination For Brightfield.
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Method 2:
Phase Contrast Microscopy
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Method 4:
Measurement Of Microorganisms.
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STAINING OF BACTERIA
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Method 5:
Preparation Of The Film
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Method 6:
Simple Staining
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Method 7:
Gram Staining
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Method 8:
Spore Stain
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Method 9:
Capsule Stains
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Method 10:
Ziehl-Neelsen Stain
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Method 11:
Albert's Stain
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Method 12:
Flagella Stain
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MANUFACTURE AND USE OF CULTURE MEDIA
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Method 13:
Preparing a Blood Agar plate Ошибка! Закладка не определена.
Method 14: Streak-Plate Method For Isolation Of Pure Cultures Ошибка! Закладка
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Method 15:
Colonial Morphology
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Method 16:
Preparation Of Lawn Plates
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Method 17: Comparison Of The Inhibitory Effects Of Common Disinfectants And
Antibiotics.
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COUNTING BACTERIA
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Method 18: Preparing A Serial Decimal Dilution Series Ошибка! Закладка не
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Method 20:
The Spread Plate Method
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Method 21:
Miles And Misra Plate Count Ошибка! Закладка не определена.
Method 22: Making A Calibrated Pasteur Pipette
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Method 23:
Total Counts
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Cell Counts With A Haemocytometer
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IDENTIFYING BACTERIA - BIOCHEMICAL TESTS
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Ammonification
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Camp Test
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Carbohydrate Fermentation
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The Catalase Test
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Citrate Utilization
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Coagulase Test
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Decarboxylation Of Amino Acids
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Method 31:
Indole Test
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Method 32:
KCN
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Method 33:
Macromolecule Hydrolysis
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Method 34:
Methyl Red (Mrvp) Test
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Method 35:
Motility Test (Craigie Tube) Ошибка! Закладка не определена.
Method 36:
Nitrate Reduction
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Method 37:
O/129 Sensitivity
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Method 38:
ONPG
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Method 39:
Oxidase
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Method 40: O - F Test (Oxidation - Fermentation
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Method 41:
Salt Tolerance
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Method 42:
Urease Production
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Method 43:
Voges - Proskauer (Vp) Test Ошибка! Закладка не определена.
Method 44:
Inoculating Biochemical Media Ошибка! Закладка не определена.
Identification of an unknown culture.
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Results
Biochemical testing of unknown cultures#. Ошибка! Закладка не
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Method 45:
Macfarland's Standards
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Method 46:
Salmonella Serology
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Method 47:
Taking Microbiological Samples
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Method 48:
Aesculin (Esculin) Hydrolysis Ошибка! Закладка не определена.
Method 49:
Enrichment Culturing
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COUNTING BACTERIA & OTHER MICROBES IN ENVIRONMENTAL
SAMPLES.
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Method 50: Total Count By Epifluorescence Microscopy Of Acridine OrangeStained Cells (Abbreviated Method).
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Method 51: Nalidixic Acid Direct Viable Count By Epifluorescence Microscopy
Of Acridine Orange-Stained Cells.
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Method 52:
Demonstration Of Microbial Activity.
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COLIFORM COUNTS FOR DETERMINING SANITARY QUALITY. Ошибка!
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INTRODUCTION
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Method 53:
Most Probable Number Method Ошибка! Закладка не определена.
Method 54: Abbreviated Method Of The Membrane Filtration Technique For
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Method 24:
Method 25:
Method 26:
Method 27:
Method 28:
Method 29:
Method 30:
3
Practical marking schedule & references
Sub-project
Due
Individual marks:
Sub-project 1 (4% of total mark)
Week 5 practical
Laboratory report on the survey of microbes in the environment.
Subproject 2 (8% of total mark)
Week 9 practical
Laboratory report with results of taxonomic classification and a discussion of the
methods.
Subproject 3 (8% of total mark)
Week 12 tutorial
Laboratory report with results and discussion of the effect of the environment on
growth and survival of bacteria.
Completed Practical Schedule (5%)
Week 11 practical
Group marks:
Laboratory diary (5% of total mark)
Week 11 practical.
References
Bergey’s Manual of Systematic Bacteriology, 2nd edition, vol. 1. Springer.
Bergey’s Manual of Systematic Bacteriology, 1st edition, vols 1 – 4. Williams and
Wilkins.
Bergey’s Manual of Determinative Bacteriology, 1st edition. Williams and Wilkins.
Buller, N.B., 2004. Bacteria from fish and other aquatic animals: a practical
identification manual. CABI publishers, 341 pp. 579.3176 B936b 2004
Cowan and Steel’s Manual for the identification of medical bacteria, 2nd ed.
Cambridge University Press.
Encyclopedia of Microbiology vols 1 - 4. J. Lederberg (ed.) Academic Press.
Ref.579.03 E56 2000.
Madigan, M.T. and Martinko, J.M., 2006. Brock Biology of Microorganisms. 11th
edition. Prentice Hall International New Jersey USA.
Prescott, L.M., Harley, J.P. and Klein, D.A., 2002. Microbiology. 5th edition. WCB
McGraw-Hill Boston. 1026pp + appendices.
4
Techniques completion list
Dated signature of demonstrator indicates task successfully completed.
Technique
Prepare a Blood Agar
plate
Plate mixed culture to
achieve isolated
colonies
Gram stain
Spore stain.
MacConkey Agar
interpretation
TCBS Agar
interpretation
Identification of
unknown culture
Pour plate technique
Spread plate technique
Miles Misra technique
Viable plate count
Comments
Sterile
Evenly mixed blood
Flat surface
Labelled
All species have well isolated
colonies
Loop not flamed
Streaks crossover
Distinguishes purple Gram (+)
& pink Gram (-)
Over decolourised
Under decolourised
Smear too thick
Smear too thin
Spores & cells stained
Spores insufficiently stained
Smear too thick
No spores in cells
Correctly identify lactose
fermentation
Understand selective mechanism
Understand differential
mechanism
Correctly identify sucrose
fermentation
Understand selective mechanism
Understand differential
mechanism
Shows cell dilution
Correct colony description
Correct Gram stain
Correct primary tests results
Correct primary identification
Correct secondary tests results
Correct final identification
Plate with countable colonies
Shows cell dilution.
Plate with countable colonies
Shows cell dilution
Plate with countable colonies
Shows cell dilution
Correct calculation
Demonstrator
Date
5
Microbiology Project
The laboratory class is closely integrated with the theory discussed in the lectures and
tutorials. By using a hands-on enquiry approach you will experience microbiology
and how it is carried out. The enquiry approach requires the full involvement of all
students in the class, for everyone will have much to contribute and indeed this
contribution will be essential, not only for your individual understanding, but also to
enable all students to develop their own understanding of the concepts and practice of
microbiology. Full involvement means contributing to the generation of experimental
hypotheses, determination of the methods to be used and the careful carrying out of
experiments to produce results which enable the evaluation of the hypotheses.
Throughout the semester, students will work in groups of three to investigate three
major sub-projects, which are linked to form the whole project. The overall aim of the
project is to purify a microbe, to identify it as far as possible and to determine the
effects that the environment has on the growth and survival of this organism.
Each student in each group will maintain a detailed laboratory diary of all work
carried out and leadership will rotate for each of the three sub-projects. The diary will
stay in the laboratory at all times. Additionally, all students will produce a report
summarising the experiments in standard scientific format for each sub-project.
Details of how to prepare diaries and portfolios are given on pages 7 and 8 of the
microbiology methods manual. The project to be carried out has been designed to
ensure that all students practise all the basic microbiological techniques necessary for
growth and study of microbes. To check progress on this there is a practical schedule
on page 2 of the practical guide. Students should ensure that the demonstrator signsoff on each technique as it is successfully completed. The practical schedule is worth
5% of the final mark.
The general process that the class will follow each sub-project is as follows:
1.
2.
3.
4.
5.
Formulate hypotheses to be tested or determine an aim of your research and
record these in the laboratory diary.
Students design how to test the hypothesis, decide appropriate methods and
record in diary.
Methods will be demonstrated with controls.
Students carry out methods and record results in the laboratory diary.
Evaluate results and write discussion (notes only in diary).
The three major sub-projects address the following questions:
1.
How common are microbes and are there very many different types?
2.
How do you distinguish microbes and name them?
3.
Does the environment affect microbial survival or growth?
6
Sub-Project 1: A survey of microbes in
several environments.
Introduction
Microorganisms are indispensable to the efficient functioning of Earth as a lifesupporting planet. As the earliest life forms they have developed over thousands of
millions of years and have an incredible adaptability, which enables them to colonise
almost every habitat and utilise a vast assortment of substrates. These microscopic,
living microbial cells are everywhere, on mountain tops, in hot sulphur springs, soil,
water, food, air and in and on the bodies of animals and plants. For this reason it is
necessary to use extreme care not to introduce unwanted organisms into culture media
or into cultures of organisms to be studied. Many procedures are available to
demonstrate the presence of microorganisms in the environment and some of these
will be described throughout this course. Each procedure will, of itself, demonstrate
only a particular type, or group, of microbes because of the great variation in physical
and chemical growth requirements among them.
The preparation of culture media is an integral part of microbiology as it enables us to
grow masses of microbes so that we can visualise them and/or the characteristic
effects that they have on the media. It requires an ability to handle reagents
aseptically, that is, without contaminating them. This is necessary because many
current microbiological techniques require pure cultures, which means that there
must be a total absence of contaminating microbes from the media; otherwise it is not
possible to separate the desired microbes from the contaminants. (Think: if you can
not see either the desired microbe or a contaminant initially, how do you know which
one successfully grows on the medium?). Solid media enable the growing of microbes
in isolated masses called colonies, which can be used in isolation and identification of
microbes. The contents of different culture media can be changed to reflect the
diversity of nutritional requirements of various microbes.
Because of their smallness, microbes are commonly observed as macroscopic
colonies on agar and/or the cells are stained so that they can be observed
microscopically and their characteristic features determined. Simple stains enable
observation of the shape, size and arrangement of cells. The Gram stain is the basis
of all bacterial taxonomy (i.e. the classification of bacteria) and is usually the first test
done by a microbiologist on an unidentified culture. It not only enables the shape, size
and arrangement of cells to be determined, but also enables the microbiologist to
determine the type of cell wall. This can either be Gram positive (purple) or Gram
negative (pink) depending on the chemical structure of the cell wall. Furthermore,
numerous staining techniques have been developed to demonstrate different structures
of microbial cells, so that they may be identified or be more completely described.
Therefore, because of their microscopic nature, studying microbes requires various
issues and difficulties to be considered:
1. As they are microscopic how might you see them?
2. Is microbial cellular morphology useful for distinguishing them one from
another?
7
3. What advantages accrue if you grow organisms in cultures?
A. Is it better to use a broth or a solid agar medium? Why?
B. What disadvantages occur by culturing?
C. How long does is it take for a culture to grow?
4. Is colonial morphology a useful characteristic for distinguishing different
microbes?
5. How are cellular features determined for microbes?
A. What types of cellular features can be determined?
B. Can you use these features to help identify the microbe?
6. Because you can not usually see microbes in situ, how do you know whether
different sites have the same or different microbes associated with them?
7. Are the microbes associated with a site growing there, or merely surviving?
Sub-project 1 aim:
To survey microbes present in a variety of environments (soil, laboratory, sea water,
humans).
{NB. if you wish to provide your own sample of soil, talk to the demonstrator a day or
two before hand.}
Background reading
Madigan and Martinko., 2006. Sections 1.1 – 3; 2.4 – 6; 5.1 – 3.
Weeks 2 - 4:
1) First, the group will determine the leader for this sub-project.
2) The group should generate a question that they are interested in examining.
Essentially, this means deciding what type of samples would you like to study.
Record the question (the aim of the experiment) in the laboratory diary.
3) Once the aim has been generated, use the questions raised above to consider how
you may go about the experiment – in particular, determine which laboratory
methods you need to be taught. Record these in the laboratory diary.
4) Watch the demonstration of particular techniques and carry these out on control
organisms and the samples.
5) Distribute tasks amongst group members.
6) Carry out the techniques on the samples to be analysed.
7) Record any observations or data required by the techniques (for example, the
nature of each sample).
8) Label and incubate the samples appropriately.
9) Investigate and answer any questions given. This can be done by the group, but the
answers are to be used for the discussion of each sub-project write-up.
10) Reflection: what do you know about the sub-project now and what is the next step
to be carried out?
11) Practical weeks 2 and 3 will be used to carry out follow-up experiments or tests to
complete sub-project 1.
12) Determine the necessary techniques by consideration of the questions raised at the
beginning of this sub-project.
13) Watch the demonstrations of these techniques and carry them out on control
organisms.
14) Distribute the day’s tasks amongst group members.
15) Carry out techniques on the experimental organisms.
16) Sub-culture a selection of organisms in order to obtain a pure culture.
17) Record all observations in the laboratory diary.
18) Reflection: each week, consider what the group now knows and what needs to be
8
done next.
19) Distribute new tasks to group members.
20) Investigate and answer any questions given. This can be done by the group, but the
answers are to be used for the discussion of each sub-project write-up.
1.
2.
3.
4.
5.
6.
7.
8.
Questions to consider when discussing the results:
What is the purpose in using solid media?
What are the advantages and disadvantages of agar as a solidifying agent?
Comment on how useful colonial morphology is for identifying a microbe.
Which type of environment(s) generally had the most growth? Why do you
think this was so?
Microscopy:
a. Distinguish between resolution and magnification.
b. Define the term “numerical aperture”.
c. What impact does the numerical aperture of a lens have on resolution?
d. For brightfield viewing of unstained microbes either the iris diaphragm
can be closed slightly or the substage condenser can be slightly
lowered from the usual position. Why? Which is preferable?
What type of information about a cell can be gained from a simple stain, such
as methylene blue?
What type of information about a cell can be gained from the Gram stain?
Why is this important (or put another way, how is this used?)
Why bother doing special stains such as spore stains and capsule stains,
particularly if some of them will give variable results depending upon the
incubation conditions?
Assessment rubric
Component
Title
Student number
Assignment cover sheet signed
Assessment rubric signed, included
Introduction
1. Question or aim stated
2. Rationale described
Mark
0.5
2
Materials and methods
1. Described in detail or
2. Listed from methods manual
3. Any alterations noted
2
Results
1. Data set is complete
2. Tables and figures have correct format
3. Microscopy figures include a scale
3
Discussion
1. Addresses the aim in relation to the data described in the results
2. Answers subproject 1 questions given in laboratory guide
3
References
1. Appropriate format
2. Complete list
0.5
9
SUB-PROJECT 2: TAXONOMY: HOW MICROBES ARE
IDENTIFIED AND CATALOGUED.
Introduction
Many microbes are indistinguishable on basic media such as TSA, so to separate
mixtures selective and/or differential media are used. These enable either only a few
types to grow, or cause microbes to appear differently from one another. Selective and
differential media enable microbes to be isolated and identified faster and more
efficiently than basic media.
Microbes exhibit diverse physiologies and so their growth requirements will also vary
considerably. Therefore, it is necessary to consider the environmental conditions that a
culture may be subjected to. An important parameter is the concentration of oxygen
present in, and above, the culture medium. Some microbes have an obligatory
requirement for oxygen, whereas others require its absence, and a third group is
indifferent to oxygen. Both the medium and the incubation environment can be used
to selectively grow particular microbes, thus purifying the culture.
Once a culture has been purified (it is said to be axenic), then the usual practice is to
identify it. This is done by combining the colonial morphological characteristics on
standard media, by staining (e.g. Gram stain), by determining the presence or absence
of metabolic reactions (biochemical characterisation) and by serological or genetic
techniques. The latter is a form of genotype testing, whereas all the others are
phenotypic tests, that is they examine expressed characters of the organism. Thus, a
series of characters can be ascribed to the culture and then taxonomic guides, such as
Bergey’s Manual or Cowan and Steel, are consulted to determine the most likely
identification.
There are 2 different approaches to taxonomy (the identification of an unknown
organism). In the first, a taxonomic (dichotomous) key is used in which questions are
answered sequentially. Each answer narrows the number of possibilities until only one
remains. In the second approach, called numerical taxonomy, a standard battery of
tests is performed and the results compared to a database. The identification is then
made as the organism showing greatest similarity to the experimental results.
Issues to consider within sub-project 2
1. Different media and/or culture conditions grow different organisms. How
might you use this to purify a culture.
2. Different animals metabolise in different ways. Does this apply to microbes
also?
A. How might you determine this?
B. Why is it important to have pure cultures?
C. How do you know that the tests are working properly?
3. How are microbial characteristics collated and evaluated and then used?
Background reading:
Madigan etal., 2005. Sections 6.15; 11.10 – 13; 24.1 – 4; Chapter 12 has specific
information on individual bacterial groups.
10
Sub-project 2 Aim:
To purify and identify an unknown bacterium obtained from one of your samples.
Weeks 5 – 8
Determine a group leader for this sub-project.
Subproject 2 outline and questions for consideration during the project
and afterwards when evaluating it
1) Generate hypotheses regarding the likelihood of finding a desired bacterium in a
particular sample. That is, from the available information think about what
bacteria are likely to be present in a particular sample. {Each student is to consider
a particular target organism after consultation with the demonstrator.}
2) Purify at least one culture from the environmental samples that you have been
working with.
A) Can you distinguish between selective and differential media. Give 2 examples
of each and explain the mechanisms?
B) How do you combine the use of selective and differential media with nonselective media to isolate a pure culture on a bacterium from a mixed culture.
C) Reflection:
I) In which medium (NB or FTM) would you expect an obligate aerobe, an
obligate anaerobe and a facultative aerobe to grow? Why?
3) Determine the initial tests needed to start the identification process on your pure
culture.
A) What types of controls should always be used for phenotypic tests?
4) Watch demonstrations of phenotypic tests and carry these out on controls and your
test culture.
5) Incubate under appropriate conditions. (What are these?)
6) Record results in a table.
7) Evaluate results against a taxonomic key. (This will be demonstrated first).
A) Do you have enough information to identify the unknown organism?
B) If not, then what further tests need to be carried out?
8) Inoculate any further tests, with appropriate controls and incubate.
9) Repeat until you are able to identify the unknown.
10) Reflection for the discussion:
A) Are any biochemical tests more useful than others in differentiating bacteria?
B) Was your identification of the unknown uncomplicated; i.e. Did all tests
produce results that gave an unequivocal answer for the unknown according to
Cowan and Steel tables or Bergey’s manual?
C) Do you think that, if one test result out of ten (say) disagrees with the others,
that this obviates the identification? Discuss within your group and explain
your answer in no more than one page for your individual report.
11
Assessment rubric
Component
Title
Student number
Introduction
3. Question or aim stated
4. Rationale described
Mark
1
Materials and methods
4. Described in detail or
5. Listed from methods manual
6. Any alterations noted
1.5
Results
4. Data set is complete
5. Tables and figures have correct format
6. Microscopy figures include a scale
3
Discussion
3. Addresses the aim in relation to the data described in the results
4. Answers subproject 2 questions given in laboratory guide
5. Provides a critical evaluation of your personal learning in microbiology
4
References
3. Appropriate format
4. Complete list
0.5
12
Sub-project 3: Does the environment affect
the survival or growth of microbes?
Introduction
Heating and ultraviolet irradiation are commonly used for sterilisation or
disinfection. That is, they can be used either to completely kill all living agents
(bacteria, viruses, spores, fungi) or to reduce the load of such agents in a sample.
With respect to heat, the intensity of heat applied and for how long will determine
whether the sample is sterilised or disinfected. Ultraviolet irradiation is unlikely to
sterilise a sample, because of the capacity of microbes at self-repair, and because UV
has very poor powers of penetration. It is therefore a means of disinfection, which is
defined as a reduction in microbial load, particularly of pathogens. Light wavelength
and intensity affect the amount of damage inflicted on the cell. Particular
wavelengths are more dangerous because they are more strongly absorbed by
chemicals within the cell and thus more damage is possible. Intensity measures the
amount of UV light being radiated and increases with either or both the energy of the
source and the length of time the target is exposed. Increasing intensity will increase
the amount of damage inflicted, especially when the intensity is such that repair
mechanisms are simply overwhelmed.
Sterilisation is defined as the complete killing of all life forms including bacteria,
fungi, spores and viruses. Thus, an object is either sterile or it is not. It is an absolute
term so it is nonsensical to talk of something being nearly or partly sterile. Heat
sterilisation processes within industry are often tested by using Bacillus
stearothermophilus, which produces remarkably heat-resistant spores; the rationale
being that, if these spores are killed, then any other infectious agent should be killed
as well. So for example it is used in autoclave testing. By their nature, autoclaves
produce wet heat in the form of steam, whereas ovens produce dry heat via radiation
from the heating element. Wet heat is more effective in killing cells because it
transfers heat by both radiation and conduction. Also, wet heat causes coagulation of
proteins, inflicting further damage that does not occur with dry heat.
Antibiotics are chemicals naturally produced by some microbes to enhance their
competitiveness in the environment. Commonly, soil microbes such as fungi and
species of the bacterial genera Streptomyces and Bacillus form antibiotics.
Disinfectants are used to reduce the microbial load of an object. Microbiologists need
techniques to broadly survey the effectiveness of different compounds as killing
agents and then to quantify the effect of inhibitors.
In its simplest format, the effectiveness of a killing agent can be determined by the
presence or absence of viable cells after treatment. On the other hand, the degree of
effect requires that the actual number of cells before and after treatment be
determined.
Bacterial biomass (and hence growth or death if the biomass changes in response to a
treatment) can be measured as:
1.
The total mass of bacterial protoplasm in a sample by the use of direct
methods such as dry weight determination. As dry weight determination is a
13
lengthy procedure it is usually used to calibrate a more rapid, indirect method
such as light scattering.
OR
2.
The number of organisms in a sample. Two types of counts are of interest:
(a)
Viable count - for the purpose of counting, viability is equated with
the capacity to form a macroscopic colony on nutrient agar or to
produce visible turbidity in broth.
(b)
Total count - the number of living and dead organisms in a sample.
{It should be remembered that mass and number can vary independently
during bacterial growth. The above principles are also applicable to other
microorganisms.}
Therefore, it is necessary to first decide what type of data will be sufficient to answer
your question:
1) Simple presence or absence of viable cells of the test organism, which can be
determined by the ability to grow.
2) A quantitative count, which will allow you to draw conclusions on the relative
effects of a treatment on survival or growth.
Background reading:
Madigan etal., 2005. Sections 6.1 – 8; 20.1 – 5; Additional specific information on
antibiotics can be found in sections 20.6 – 9.
Aims of sub-project 3:
1) To determine the survival of a culture of bacteria in sterile seawater in comparison
to E. coli.
2) To determine the efficacy of heat as a sterilising agent.
3) To determine the efficacy of ultraviolet light as a killing agent.
4) To determine the spectrum of activity of common disinfectants and antibiotics
against a variety of different microbes.
Weeks 9 – 11:
1) Determine the group leader for this sub-project.
2) Generate individual hypotheses to examine each of the 4 listed aims.
3) Determine the methods to be used for each.
a) Consider whether it is necessary to quantitatively determine the number of
bacteria, or whether it will be sufficient to determine presence or absence of
live bacteria?
b) Record your decisions and the reasons supporting them.
c) What would be the problem if you did not have a pure culture?
4) Bacterial survival in seawater:
a) You will be provided with a volume of sterile aged seawater in week 8.
b) Design a verifiable hypothesis on survival of bacteria in sea water.
c) Design an experiment to determine how well your bacterial culture will
survive in this sea water for one week.
d) Will the viable count technique chosen affect the outcome of the experiment?
e) When should you sample?
f) What controls should you use?
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g) How much of your culture will you inoculate into the water?
h) How will you incubate your inoculated seawater sample?
i) Inoculate your sea water in week 8 and incubate for one week to determine
survival.
j) Reflection: what factors will affect the survival of a bacterium?
5) Heat sensitivity:
a) The equipment available to you includes hot air ovens at 120°C and 160°C as
well as an autoclave operating at 120°C and 1.1 kg cm-1 (about 1 atmosphere)
and sterile sealable glass bottles.
b) Estimate the length of time that would be worth testing to determine lethality
of each treatment.
c) Compare Bacillus stearothermophilus versus one of your cultures.
d) What controls should you include?
e) Reflection: consider the mechanisms of how damage is caused by heat to
explain why wet heat is more effective at killing cells than is dry heat.
6) Ultraviolet light.
a) Design an experiment to determine the effect of wavelength and intensity of
UV on survival of one of your pure cultures. {Hint: the plastic of petri dishes
is opaque to UV light}.
b) You have available to you light sources emitting UV light at 256 nm or 366
nm.
c) The class can collaborate to test a variety of organisms, including control
cultures.
d) Reflection: what properties of bacteria influence their ability to resist UV
light?
7) Antibiotics and disinfectants.
a) Design an experiment to survey the activity of a range of antibiotics and
disinfectants against several different bacterial cultures.
b) You are provided with filter paper discs impregnated with antibiotics, sterile
filter paper discs and solutions of some common disinfectants.
c) How could you determine if the agent was microbicidal or merely
microbistatic?
d) Reflection: by what mechanisms can bacteria resist antibiotics?
Questions to consider for writing the discussion
1) Define accuracy, precision and sensitivity of analytical techniques.
2) What was the survival of your culture after exposure to 45 C.
3) Examine the coefficients of variation. Compare across groups and methods. What
part of the methods appears to introduce the largest amount of variability in the
estimates? Does any method appear more precise than the others? Explain your
answer.
4) Is any method more accurate?
5) Which method would you expect to be the most sensitive?
6) How do the different viable count methods compare? Which is the most accurate,
precise or sensitive? For each method, comment on the ease of performance, the
amount of materials used and which technique to determine which would be best
for routine use on a large number of samples in relation to the usefulness or
otherwise of the data obtained.
7) Distinguish between sterilise and disinfect.
8) Discuss the results in terms of the factors affecting the efficacy of UV light as a
disinfecting agent.
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9) Define disinfection, minimum inhibitory concentration and minimum microbicidal
concentration. Was the disinfectant you sub-sampled bacteriostatic or
bactericidal? Explain your answer.
10) In relation to antibiotic activity, what is meant by the terms: narrow spectrum and
broad spectrum? Give examples of each. Could any of the antibiotic activities that
you have observed in this practical be considered to have a broad spectrum?
11) What do synergism and antagonism mean? Have you observed these in this
experiment or earlier in your first survey of environmental microbes? If so,
between which antibiotics?
Assessment rubric
Component
Introduction
Mark
1. Hypothesis stated
2. Rationale described
2
Materials and methods
7. Described in detail or
8. Listed from methods manual
9. Any alterations noted
2
Results
7. Data set is complete
8. Tables and figures have correct format
9. Microscopy figures include a scale
3
Discussion
6. Addresses the hypothesis in relation to the data described in the results
7. Answers subproject 3 questions given in laboratory guide
8. Personal evaluation.
3.5
References
5. Appropriate format
6. Complete list
0.5
Mark = / 11 = / 8
16
17
Name:
MICROBIOLOGY
METHODS MANUAL
National Centre for Marine Conservation
and Resource Sustainability
C.M. Burke 2008
18
LABORATORY RULES
ACCESS
 Access to the laboratory is restricted to individuals with an understanding of the safety
practices employed in the laboratory.

The laboratory doors are to be kept closed at all times, and furthermore the outer door must be
locked. This is to deter thieves.

Undergraduate students are not permitted to work without a staff member being present in the
laboratory complex. They may only work without direct supervision when authorised in
writing by a Microbiology Safety Officer. The staff member designated to supervise the work
must sight such authorisation.
LABORATORY RULES
1.
Regard all organisms and biological materials used in this laboratory as potentially infectious
and pathogenic to humans or to other animals such as fish.
2.
Coats, jackets and other outer apparel should be left outside the laboratory, together with
3.
4.
5.
6.
7.
8.
9.
10.
bags and books not required for the laboratory session.
Long hair should be tied back neatly, away from the shoulders and enclosed footwear should
be worn - (thongs and open sandals are not allowed).
Avoid placing any object in your mouth - (pencils, pens, fingers etc). Mouth pipetting is
strictly forbidden in the microbiology laboratory.
Eating and drinking are not allowed anywhere in any of these laboratories.
Laboratory gowns must be worn inside the laboratory. They are not to be worn outside the
laboratory. When leaving the laboratory, please return the gown to the hook, neatly folded,
inside out. If your gown becomes soiled during a class, please advise the demonstrator.
No slides or cultures of any kind are to be taken from the laboratory.
If you have an accident of any kind call the instructor immediately. A spilled culture can
be covered with paper towels and laboratory disinfectant poured over the towels and
contaminated area; leave for 10 minutes before mopping up.
The working area should be wiped with disinfectant at the beginning and end of the
laboratory session.
Always wash your hands before leaving the laboratory, even when you are only leaving for a
brief period.
WASTE DISPOSAL
The waste disposal protocol in the Microbiology Laboratory is designed to separate the noninfectious from the infectious waste. The infectious waste needs to be disposed of in a manner that
minimizes the risk to both staff and students and facilitates the recycling of reusable material.
Please follow the instructions carefully and if in doubt - ASK!
1
Sharps:
One dedicated yellow sharps container per laboratory.
Used for needles, scalpel blades and infectious broken glass
Do not wander around the laboratory carrying sharps, ALWAYS take the
container to the sharps, NOT the sharps to the container!
Biogram buckets:
Located on each bench.
Used for contaminated waste, eg used swabs, wet preparations, capillary
tubes, contaminated reagent strips and pipettes.
Not to be used for Gram stains, paper, matches or chemicals.
Carefully, place pipettes into the Biogram, tip first. Draw up some Biogram
and then remove the pipette bulb.
Biohazard bin:
Located in the middle of the laboratory.
Used for contaminated waste, eg used culture plates and contaminated paper
towels.
Not to be used for paper or for glass or other sharps.
Billy cans
Two of these are located at the front of the laboratory.
1) Used for recyclable glass or plastic tubes/bottles.
Please ensure that they are capped and all sticky labels are removed.
2) Used for fixed and stained slides (not for wet preps).
Paper bin
For non contaminated paper only, eg paper towel from hand washing or
blotting Gram stains.
PERSONAL PROTECTIVE EQUIPMENT AND PROCEDURES
Gowns


Front buttoning laboratory coats are not suitable. Rear-opening, wrap-around gowns are
provided, as they offer greater protection from contamination. The gowns are supplied so that
the laboratory maintains control over their cleaning. Gowns are laundered fortnightly.
Any gowns that are soiled in excess of normal use are to be removed from circulation until
laundering can be arranged.
Safety Glasses and Eye Protection




All students and staff are required to have safety glasses on hand and are to use them when
procedures are undertaken that involve significant risk of splashing with infectious, toxic or
corrosive liquids. Most bacteriological procedures do not require safety glasses.
Safety glasses must be worn when opening the autoclave.
An eyewash station is located in the anteroom.
The eyewash fluid should be replaced with fresh sterile distilled water every month. The date
of “renewal” shall be recorded on the bottle.
Gloves


Gloves are not considered essential for routine work in the microbiology laboratory.
Gloves should be worn when:
 mopping up a spill.
 performing procedures where there is a high risk of contaminating hands.
 open cuts or skin conditions are present as these increase the risk of infection from
accidental contamination.
2
Handwashing



The standard handwashing procedure is to use running water and Hibiclens. Taps that do not
require hand operation are preferred. As are soap dispensers, rather than bars of soap. Use the
back of you hand to operate the soap dispenser. These procedures reduce the contamination of
the sink and soap.
Hands must always be washed before leaving the laboratory.
The Hibiclens dispensers at all handwashing sinks within the laboratory must always be
sufficiently full. Report any empty dispensers to the demonstrator.
High Risk Individuals /Antenatal Considerations

Persons who are immunocompromised or otherwise particularly susceptible to infection need
to be identified so that additional precautions for microbiological safety can be taken when
necessary.

Within this context, pregnant women are known to be at high risk if they become infected with
Listeria monocytogenes. Therefore, for their own safety, any female student or staff member
who is, or thinks that she may be pregnant, should discuss the matter with the academic in
charge of the unit prior to commencing work with Listeria monocytogenes.
USE OF THE LABORATORY: Students will be marked on the care with which they use
equipment, and how they CLEAN UP afterwards.
Students should check with the demonstrator if they finish early and wish to leave, so that bench
areas can be checked. There may also be a post-laboratory discussion.
3
PRACTICAL REPORTS
1) Use a book for your laboratory diary. Some advice is given on the next page.
2) An integral part of the scientific process is to reflect on what you are doing. That is to ask
questions such as: what do I know, what qualifications must I place on my data, what questions
remain to be answered and what do I need to do next? Some questions have been listed in the
laboratory notes to give you some guidance of relevant issues, and these should be seriously
considered, but do not restrict yourselves to these questions only.
3) Each group will prepare reports on each of the three sub-projects with all group members
contributing. These reports will have the format of: title, introduction, aim(s), materials and
methods, results and discussion with a conclusion. Reports are to be typed and submitted
electronically via MyLO. {Note the comments on plagiarism in the unit outline and student
handbook.}
4) DIAGRAMS: properly drawn, these are very valuable to you. All diagrams and descriptions
are to be completed prior to leaving the laboratory. Some general hints on drawing cells
seen with the microscope are:
(a)
(b)
(c)
(d)
(e)
(f)
(g)
label the diagram fully.
include a scale bar and the total magnification (e.g. X400).
draw clear diagrams of a few ( 4) representative cells of any one type.
be careful of the shape of the cells; e.g. are the ends rounded, pointed or
square? The differences are significant.
in a series of drawings at one magnification, try to keep the scale constant,
e.g. at X1,000, 1cm = 1µm.
5) SCIENTIFIC NOTATION: Genus species or Genus species, i.e. a Capital letter for the initial
letter of the genus only. The whole name should be underlined or italicized. Appropriate
abbreviations can be used in any particular report, after the full name has been written once.
Other taxonomic groups (family, order, class, phylum) are neither underlined nor italicized.
Some examples:
a)
b)
c)
d)
e)
f)
Staphylococcus aureus, Staph. aureus, S. aureus
Streptococcus pyogenes, Strep. pyogenes, St. pyogenes
Bacillus cereus, B. cereus
Escherichia coli, E. coli
Pseudomonas aeruginosa, Ps. aeruginosa
Families : Micrococcaceae, Enterobacteriaceae, Bacillaceae.
Advice to group leaders
For each sub-project one of the group members will be nominated as group leader, in turn. The
leader will be held responsible for the group's work and for organising the submission of the
sub-project report. The diary will be checked, feedback given and signed by the
demonstrator as much as is possible. An example of what the demonstrator will look for in
the diary and in the group's understanding is given on the next page.
As group leader you are responsible for organising the group's work, not doing it all yourself!! This
means discussing issues with your partners and setting up a work plan of jobs to do, who will
do them and when. Afterwards, the leader must check that everything has been done and has
4
been properly recorded, so that it is obvious what has been done, how it was done, when and
by whom. This does not require an essay. Notes are fine.
LABELLING: This is an essential part of microbiology as many media or cultures are
indistinguishable from others. Therefore, include your name, the name of the organism or
sample, the date and any special incubation conditions. Buy an indelible marker for labelling
Petri Dishes. However, DO NOT use indelible markers on reusable containers (including plastic
tubes), rather use stick-on labels and REMOVE these before discarding.
Project laboratory diary.
******The diary is to remain in the laboratory between practical classes.******
****** The diary will be assessed as an integral part of the project.******
A diary or laboratory notebook is an essential component of research or monitoring work. It is used
to record all relevant information about the work (aims, methods, results, conclusions, who did
what and when). It is NOT a good idea to write out the information on scrap paper with a view to
writing it out neatly later on. DO NOT do this as it leads to inaccuracy as notes or memories can
be faulty. Information that should be noted includes, but is not limited to, the following:
1
2
3
Has the aim of the project been clearly stated in the form of a verifiable hypothesis?
Has the group found relevant background information?
Is there a workable experimental design?
a) Sufficient replication if necessary?
b) Appropriate controls?
c) Best possible technique chosen?
4 Has the work carried out been fully described? For example:
a) Samples examined.
b) Media recipes listed or references given, techniques described or referred to?
c) Cultures inoculated and incubated.
d) Summaries of information gained from the literature.
5 Are the results clearly expressed? Preferably in tables or diagrams where possible.
a) Does the group have a pure culture?
b) Do the members know whether their culture is pure or not?
c) Have the experiments enabled verification of the hypothesis?
6 Is detailed descriptive information concisely summarised?
7 Are the results interpreted?
a) Are the interpretations correct?
b) Have any qualifications on the results been recorded?
8 Does the group know what it will do next?
a) Have they found out how to carry out relevant technical procedures.
b) Does the group have a timetable for future work?
c) Has the group allocated jobs to different group members,
d) Has the group allocated a time line for completion of these jobs?
i) Flow charts may be useful.
9 Have individual group members signed-off on jobs that they have carried out?
a) Failure to sign-off will be taken as evidence that nothing has been done by that person or
persons.
b) Has the group leader signed off and listed the new leader?
10 Demonstrators will give a group mark out of 10, and will note students who are not listed as
having contributed to the project.
5