Professional Documents
Culture Documents
University of
Lethbridge
BIOLOGY 3200
Principles of Microbiology
LABORATORY MANUAL
Spring, 2005
Written by: L. A. Pacarynuk and H. C. Danyk
Revised: January, 2005
TABLE OF CONTENTS
Exercise Page
Biology 3200 Laboratory Schedule 2
Grade Distribution 3
Exercise 7 – Virology 34
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BIOLOGY 3200 LAB SCHEDULE
SPRING, 2005
Jan. 11 Introduction, Microscopy
Jan. 13 General Lab Procedures, Biosafety
Mar. 1 Virology
Mar. 3 Virology
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Laboratory Grade Distribution:
The laboratory component of Biology 3200 is worth 50% of your course mark. It is distributed as
follows:
• Assignments 7.5%
• Lab Report
• Wine Fermentation 15%
• Due Thursday, Apri.l 7 by 4:30 PM
• In-Lab Skills Tests 7.5%
• Lab Exam 20%
Performance: Up to 10% of laboratory grade (5 marks out of 50) will be subtracted for poor
laboratory performance. This includes (but is not limited to) failure to be prepared for the
laboratory, missing lab notebook or lab manual, poor time management skills, improper
handling and care of equipment such as microscopes and micropipettors, and unsafe practices
such as not tying hair back, chewing gum, applying lipstick, eating, drinking, or chewing on
pencils, and sloppy technique leading to poor results.
Unannounced skills tests will be given during the semester. Students are expected to work
independently on some technical aspect of microbiology and will be graded based on their
techniques and their results.
Late Assignments will be penalised as follows: After 4:30 pm but prior to 9:00 am the next day
- -25% (eg. if the assignment is out of 50 points, you will lose 12.5 marks); between 9:00 am and
4:30 pm –50%; etc.
Extensions will only be considered upon application to your lab instructor no less than two days
prior to the due date of the assignment. This application should include documentation and the
portion of the assignment completed at that point. Failure to include any evidence of work
completed will result in no extension being granted.
The lab exam (April 14) is comprehensive, covering all aspects of the laboratory. It may contain a
practical as well as a theoretical component.
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THE UNIVERSITY OF LETHBRIDGE
Policies and Procedures
Occupational Health and Safety
1. Unknown/Known Release
2345 SECURITY
331-5201 CHEMICAL RELEASE OFFICER
2301 ADMIN. ASSISTANT
394.8937 OCCUPATIONAL HEALTH AND
394.8716 SAFETY
If the area must be evacuated all employees will be evacuated to the North Parking
Lot.
4
• GUIDELINES FOR SAFETY PROCEDURES
EMERGENCY NUMBERS
EMERGENCY EQUIPMENT:
Know the location of the following equipment which will be indicated to you at the beginning of
the first lab:
5
SPILLS
BACTERIA SPILLS: If necessary, remove any contaminated clothing. Prevent anyone from
going near the spill. Cover the spill with dilute bleach and leave for 10 minutes before wiping
up.
DISPOSAL
Bacterial Cultures:
Tubes and flasks containing liquid cultures are placed in marked trays for
autoclaving.
Bacterial Slides
Used microscope slides are placed into the trays of bleach found at the end of each of the
laboratory benches.
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EXERCISE 1
INTRODUCTION TO MICROSCOPY
MICROSCOPY
Students should be familiar with all names and functions of the components of their
compound light microscopes as demonstrated in Appendix 1.
1. Magnification
Magnification is a measure of how big an object looks to your eye. The number of times that an
object is magnified by the microscope is the product of the magnification of both the objective
and ocular lenses. The magnification of the individual lenses is engraved on them. Your
microscope is equipped with ocular lenses that magnify the specimen ten times (10X), and four
objectives which magnify the specimen 4X, 10X, 40X, and 100X. Each lens system magnifies the
object being viewed the same number of times in each dimension as the number engraved on the
lens. When using a 10X objective, for instance, the specimen is magnified ten times in each
dimension to give a primary or "aerial" image inside the body tube of the microscope. This image
is then magnified an additional ten times by the ocular to give a virtual image that is 100 times
larger than the object being viewed.
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2. Resolution
Resolution is a measure of how clearly details can be seen and is distinct from magnification. The
resolving power of a lens system is its capacity for separating to the eye two points that are very
close together. It is dependent upon the quality of the lens system and the wavelength of light
employed in illumination. The white light (a combination of different wavelengths of visible
light) used as the light source in the lab limits the resolving power of the 100X objective lens to
about 0.25 µm. Objects smaller than 0.25 µm cannot be resolved even if magnification is
increased. Spherical aberration (distortion caused by differential bending of light passing
through different thicknesses of the lens center versus the margin) results from the air gap
between the specimen and the objective lens. This problem can be eliminated by filling the air
gap with immersion oil, formulated to have a refractive index similar to the glass used for cover
slips and the microscope's objective lens. Use of immersion oil with a 100X special oil immersion
objective lens can increase resolution to about 0.18 µm. Resolving power can be increased further
to 0.17 µm if only the shorter (violet) wavelengths of visible light are used as the light source.
This is the limit of resolution of the light microscope.
The resolving power of each objective lens is described by a number engraved on the objective
called the numerical aperture. Numerical aperture (NA) is calculated from physical properties of
the lens and the angles from which light enters and leaves.
Examine the three objective lenses. The NA of the 10X objective lens is 0.25. Which objective lens
is capable of the greatest resolving power?
3. Working Distance
The working distance is measured as the distance between the lowest part of the objective lens
and the top of the coverslip when the microscope is focused on a thin preparation. This distance
is related to the individual properties of each objective.
4. Parfocal Objectives
Most microscope objectives when firmly screwed in place are positioned so the microscope
requires only fine adjustments for focusing when the magnification is changed. Objectives
installed in this manner are said to be parfocal.
5. Depth of Focus
The vertical distance of a specimen being viewed that remains in focus at any one time is called
the depth of focus or depth of field. It is a different value for each of the objectives. As the
microscope is focused up and down on a specimen, only a thin layer of the specimen is in focus at
one time. To see details in a specimen that is thicker than the depth of focus of a particular
objective you must continuously focus up and down.
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Observing Bacteria
Bacteria generally occur in three shapes: coccus (round), bacillus (rod-shaped), and
spirillum (spiral-shaped). Size of bacterial cells used in these labs varies from 0.5 µm to 1.0
µm in width and from 1.0 µm to 5.0 µm in length, although there is a range of sizes which
bacteria demonstrate. Association refers to the organization of the numerous bacterial cells
within a culture. Cells may occur singly with cells separating after division; showing
random association. Cells may remain together after division for some interval resulting in
the presence of pairs of cells. When cells remain together after more than a single division,
clusters result. Cell divisions in a single plane result in chains of cells. If the plane of cell
division of bacilli is longitudinal, a palisade results, resembling a picket fence. Both bacterial
cell shape and association are usually constant for bacteria and hence, can be used for
taxonomic identification. However, both properties may be influenced by culture condition
and age. Further, some bacteria are quite variable in shape and association and this may also
be diagnostic.
Micrometry
When studying bacteria or other microorganisms, it is often essential to evaluate the size of the
organism. By tradition, the longest dimension (length) is generally stressed, although width is
sometimes useful for identification or other study.
An ocular micrometer can be used to measure the size of objects within the field of view.
Unfortunately, the distance between the graduations of the ocular micrometer is an arbitrary
measurement that only has meaning if the ocular micrometer is calibrated for the objective being
used.
1) Place a micrometer slide on the stage and focus the scale using the 40x objective.
2) Turn the eyepiece until the graduations on the ocular scale are parallel with those on the
micrometer slide scale and superimpose the micrometer scale.
3) Move the micrometer slide so that the first graduation on each scale coincides.
4) Look for another graduation on the ocular scale that exactly coincides with a graduation on
the micrometer scale.
5) Count the number of graduations on the ocular scale and the number of graduations on the
micrometer slide scale between and including the graduations that coincide.
6) Calibrate the ocular divisions for the 40x and the 100x objective lenses. Note that immersion
oil is not necessary for calibration.
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Stage Micrometer
0 (each division =
0.01 mm)
Ocular Micrometer
0 5 10
Figure 1. Calibration of an ocular micrometer using a stage micrometer. The mark on the
stage micrometer corresponding to 0.06 mm (60 µ m) is equal to 5 ocular divisions (o.d.) on the
ocular micrometer. ∴ 1 ocular division equals 60 µm/5 ocular divisions or 12 µ m.
Once an ocular micrometer has been calibrated, objects may be measured in ocular divisions and
this number converted to µm using the conversion factor determined.
Bacterial size is generally a highly heritable trait. Consequently, size is a key factor used in
the identification of bacterial taxa. However, for some bacteria, cell size can be modified by
nutritional factors such as culture media composition, environmental factors such as
temperature, or other factors such as age.
EXPERIMENTAL OBJECTIVE
In this first exercise, you will calibrate the 40x and 100x objectives of your compound
microscope. Then you will use the compound light microscope to assess the shape and
associations of bacteria that have already been fixed to slides and stained. You will also use
your determined calibration factors to evaluate sizes of organisms viewed.
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METHODS:
1) Use the diagram in Figure 1 to calibrate the 40x and the 100x objectives on your
compound microscopes. Record these values in your lab book as you will then use these
values when measuring cells and structures for the rest of the lab.
Note: Do NOT use immersion oil when calibrating the 100x objective. This is the ONLY
time during the term that you will not use immersion oil with this objective.
2) Use the compound microscope to observe the prepared slides of bacteria using the 10x
and 40x objective lenses. Observe the same slides under the 100x objective using
immersion oil.
3) Diagram two of the organisms viewed following the instructions found in Appendix 2.
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EXERCISE 2
GENERAL LABORATORY PROCEDURES AND BIOSAFETY
A primary feature of the microbiology laboratory is that living organisms are employed as part of
the experiment. Most of the microorganisms are harmless; however, whether they are non-
pathogenic or pathogenic, the microorganisms are treated with the same respect to assure that
personal safety in the laboratory is maintained. Careful attention to technique is essential at all
times. Care must always be taken to prevent the contamination of the environment from the
cultures used in the exercises and to prevent the possibility of the people working in the
laboratory from becoming contaminated. Ensure that you have read over the guidelines on
Safety, and those on Aseptic technique (Appendix 3). As well, you should be familiar with the
contents of the University of Lethbridge Biosafety web site:
http://www.uleth.ca/fas/bio/safety/biosafety.html
EXPERIMENTAL OBJECTIVES
Students will use fluorescein dye-labelled E. coli cultures to perform a series of exercises
designed to illustrate the potential for contamination that is always present when working
with microorganisms. As well, students will become familiar with using aseptic techniques
to handle microorganisms.
METHODS
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7) Remove lids from agar plates and set aside.
8) Hold pipette tip 30 cm from glass plate and allow 10 drops to fall (one drop at a time)
onto the glass plate. Put any remaining bacterial culture back into the original culture
tube.
9) Remove glass plate to disinfectant tray and cover agar plates. Place on a tray on the side
bench.
10) Use the hand-held UV lamp in C741 to inspect your bench coat, gloves, and lab coat.
What do you observe?
11) Your plates will be incubated for 16-20 hours at 37oC, and then refrigerated at 4oC.
During the next laboratory period, evaluate your plate results and record the number of
colonies present.
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EXERCISE 3
BACTERIAL and YEAST MORPHOLOGY
Prior to viewing bacteria, two procedures must be performed: 1) fixation and 2) staining.
Fixation performs 2 functions: (i) immobilises (kills) the bacteria; and (ii) affixes them to the
slide. The most common fixation procedure for bacteria is heat fixation, whereby the slide
containing a drop or smear of bacterial culture is passed rapidly once or twice through the
heat of a Bunsen flame.
Staining
Bacteria are almost transparent and hence, unstained bacteria are not readily visible without
special techniques such as phase contrast microscopy (see: Madigan et al, 2003, pp. 56-63) or
dark-field microscopy, which is also referred to as negative staining (Negative staining will
be utilised later on this laboratory). Any procedure that results in the staining of whole cells
or cell parts is referred to as positive staining.
Most positive stains used involve basic dyes where basic means that they owe their coloured
properties to a cation (positively charged molecule). When all that is required is a general
bacterial stain to show morphology, basic stains such as methylene blue or carbol fuchsin
result in the staining of the entire bacterial cell.
Differential stains are used to distinguish bacteria based on certain properties such as cell
wall structure. Differential stains are useful for bacterial identification, contributing to
information based on bacterial size, shape, and association. Differential staining relies on
biochemical or structural differences between the groups that result in different affinities by
various chromophores (Appendix 4).
Gram staining behavior relies on differences in cell wall structure and biochemical
composition. Some bacteria when treated with para-rosaniline dyes and iodine retain the
stain when subsequently treated with a decolourising agent such as alcohol or acetone.
Other bacteria lose the stain. Based on this property, a contemporary of Pasteur, Hans
Christian Gram, developed a rapid and extremely useful differential stain, which
subsequently bears his name - the Gram stain used to distinguish two types of bacteria,
Gram positive and Gram negative. Gram negative forms, which are those that lose the stain
on decolourisation, can be made visible by using a suitable counterstain. The strength of the
Gram stain rests on its relatively unambiguous separation of bacterial types into two groups.
However, variables such as culture condition, age or environmental condition, can influence
Gram staining of some bacteria.
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The bacterial cell wall is very important for many aspects of bacterial function and hence, the
Gram stain also provides valuable information about the physiological, medicinal and even
ecological aspects of the bacteria.
Members of the genus Mycobacterium contain groups of branched-chain hydroxy lipids called
mycolic acids. Robert Koch first described this property; it allowed him to determine the
organisms present in lesions resulting from tuberculosis. As a result of the presence of these
lipids, these organisms are not readily stained via Gram staining. Instead, cells require heat
treatment so that a basic fuchsin and phenol dye penetrate the lipids. Once stained, these
lipids resist decolourisation when treated with acid.
PHB granules are common inclusion bodies in bacteria. Monomers of β-hydroxybutyric acid
are connected by ester linkages forming long polymers which aggregate into granules. As
these granules have an affinity for fat-soluble dyes such as Sudan black, they can be stained
and then identified with the light microscope. These granules are storage depots for carbon
and energy.
Endospore Staining
Endospores are highly resistant to heat, chemical disinfectants and to desiccation and
therefore allow the bacterial endospore to survive much more rigorous conditions than the
vegetative cells. Endospore resistance is due to several factors, including:
• A decrease in the amount of water compared to vegetative cells
• An increase in the amount of dipicolinic acid and calcium ions
• Enzymes which are more resistant to heat
• A spore coat which is impermeable to many substances
Endospores may be formed in a central, terminal, or sub-terminal position in the cell and
their shape varies from ellipsoidal to spherical. The location of the endospore in the cell is
usually characteristic of the species. For example, the location and shape of the Bacillus
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subtilis endospore is different from the location and shape of the Clostridium endospore.
Therefore, the presence or absence of endospores and the description of the endospore is
useful to a microbiologist as an aid in identification.
The resistant properties of endospores make them difficult to stain, hence heat is used in
conjunction with staining to enable the stain to penetrate into the spore coat.
EXPERIMENTAL OBJECTIVE
The objective of this series of exercises is to perform specialised staining procedures in order
to examine different properties of microorganisms, both bacteria and yeast. These exercises
will also reinforce proper techniques for handling of microorganisms.
METHODS:
Equipment
• microbiology kits
• compound microscopes
• slides
Bacteria
Mycobacterium smegmatis
Bacillus thuringiensis
Escherichia coli
Staphylococcus epidermidis
Yeast
Saccharomyces bayanus
Follow the guidelines for each stain as described below. Work individually.
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Prepare scientific diagrams (Appendix 2) showing results from each stain on separate pieces
of paper. These will be collected and graded. If the stain is for a specific structure, ensure
this structure is diagrammed and labelled.
Gram positive organisms stain purple; Gram negative organisms, red (pink).
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4) Decolourise with 20% sulfuric acid until no more stain comes out. Wash with tap water
to remove excess.
5) Counterstain with methylene blue for 1 minute.
Acid fast organisms retain the red stain while others are stained blue.
Endospores will stain green and the rest of the cell pink.
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Thought Questions:
References:
Atlas, R. M. 1997. Principles of Microbiology. Wm. C. Brown Publishers, Toronto.
Madigan, M. T., Martinko, J. M., and Parker, J. 2000. Brock Biology of Microorganisms
Ninth Edition. Prentice-Hall of Canada, Inc., Toronto.
Ross, H. 1992-1993. Microbiology 241 Laboratory Manual. The University of Calgary Press,
Calgary.
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EXERCISE 4
BACTERIAL REPRODUCTION
MEASUREMENT OF BACTERIAL GROWTH (See Madigan, et. al., 2003. Chapter 6 Pg.137-
151)
Most bacteria reproduce by an asexual process called binary fission. In this process a single
mother cell produces two identical daughter cells. Cell growth is often equated with increase in
cell number due to the difficulty in measuring changes in cell size. Under ideal conditions
populations of bacterial cells grow exponentially as cell number doubles at a regular interval or
generation time (td). For example Escherichia coli has a generation time of 20 minutes under
optimal conditions (e.g., 37°C, vigorous aeration and a rich growth medium).
In the laboratory, pure cultures are routinely grown as batch cultures in test tubes and
Erlenmeyer flasks. A batch culture is prepared by inoculating a fixed amount of liquid medium
with the bacteria then the resulting culture is incubated for an appropriate period of time with no
further addition of microorganisms or growth substrates.
Cell growth in batch cultures can be divided into four phases. Initially the culture is in a lag
phase where cells are preparing to reproduce. During this time cells are adjusting their
metabolism to prepare for a new cycle of growth. There is an increase in cell size without
increasing numbers. As cells begin to divide and their growth approaches the maximal rate for
the particular set of incubation conditions established, the culture enters the exponential growth
phase (log phase). One cell gives rise to two, two cells give rise to four, and so on. In this phase,
cells are growing and dividing at the maximum growth rate possible for the medium and
incubation conditions. Growth rate is determined by a number of factors, including available
nutrients, temperature, pH, oxygen and other physical parameters as well as genetic
determinants. As nutrients become limiting or waste products accumulate, the growth rate once
again slows and the culture enters the stationary phase. During this phase, there is no further
net increase in cell number, as growth rate equals the rate of cell death. The final phase of a batch
culture is the death phase. During this phase, there is an exponential decline in viable cell
numbers. This decline may be reversed if environmental parameters are modified by the
addition of nutrients, for example.
The rate of growth of bacterial cells is usually monitored by measuring the increase in cell
number. Bacterial cell numbers may be enumerated by a number of methods. Direct count
methods enumerate all cells whether they are viable or not. The most common direct count
method uses a microscope and a specialized counting chamber (e.g., Petroff-Hauser chamber) to
count the number of cells in a known volume of culture. Automated systems such as Coulter
counters may also be used to determine cell number.
In contrast, indirect count methods require the growth of cells in culture in order to enumerate
cell numbers. The most common method for enumerating living cells is the viable plate count.
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Serial dilutions of a cell suspension are prepared and spread on to the surface of a solid agar
medium (spread plate) or incorporated into molten agar that is then poured into sterile petri
dishes (pour plate). Following a suitable incubation time, the number of colonies growing on and
in the inoculated agar are counted and used to determine the number of viable cells in the
original suspension. This method makes the assumption that each colony arose from a single
viable cell or colony forming unit (CFU).
Turbidimetric methods can be used to rapidly assess biomass (e.g., cell numbers). The amount of
light passing through a cell suspension can be determined with a spectrophotometer. The optical
density (OD) is a measure of the amount of light passing through the suspension. A calibration
curve can be generated using suspensions of known numbers of bacteria.
EXPERIMENTAL OBJECTIVE
In this experiment you will monitor the growth of an E. coli culture by the viable count and
turbidimetric methods. You will determine the number of bacteria (CFU) present in your culture
following various time points of incubation. You will establish a growth curve and calibration
curve for OD using the viable count data you collect.
Prelab preparation: Turn on the spectrophotometer and set to 600 nm at least 15 minutes prior
to taking readings.
METHODS
• 100 mL bottles of molten Luria-Bertani (LB) agar
• 10% bleach
• Test tube racks
• Sterile Petri dishes
• Sterile 5 mL pipettes
• Pipette pump
• 10-100 µL micropipettor
• 100-1000 µL micropipettor
• Sterile tips for micropipettors
• Container of sterile microfuge tubes
• Microfuge tube racks
• 65 oC water bath
• Sterile d2H2O
• Spectrophotometer blank containing TB broth
• Bacterial waste container
• Vortex
• Cuvettes
• Spectrophotometer
• Culture flask of E. coli
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Please work in groups of four. At 20 minute intervals, monitor the growth of your E. coli culture
by determining viable counts as well as optical density following the procedures outlined below.
A. Culture sampling
1) For laboratory sections 1 and 2, each group of four will be assigned a culture flask. Please
mark the flask with your bench number and lab number. Groups in laboratory sections 3
and 4 will continue to sample from the flask corresponding to your bench. Data from all four
lab sections will be pooled and posted on the Biology 3200 web site.
2) Everyone in the laboratory will be sampling at the same time. Samples will be collected three
times at 20 minute intervals. For labs 1 and 2, these correspond to: 9:45 am, 10:05 am, 10:25
am, and for labs 3 and 4: 11:10 am, 11:30 am, and 11:50 am. Your laboratory instructor will
set a timer so that everyone is coordinated. Prior to beginning, designate two individuals in
your group to be responsible for obtaining optical density (OD) readings at each time point.
The other two individuals will prepare and plate appropriate serial dilutions for viable
counts.
3) At 20 minute intervals aseptically obtain one 5 mL sample of culture and immediately place it
in a spectrophotometer tube. This material will be used to measure optical density (OD)
(Section B). After reading, dispose of your 5 mL sample of culture in the waste beaker
provided. Rinse the spectrophotometer tube using the squirt bottle of bleach provided and
then dispense the solution into the waste beaker.
4) Remove another 100 µL of the culture and place it into a sterile microfuge tube. Label this
tube Tube 1. Use this culture for Section C.
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C. Enumeration of viable bacteria
1) Remove four sterile microfuge tubes from the container on the side bench. In order that
you don’t contaminate all of the tubes, gently tap out four tubes from the container rather
than using your hand to grab tubes.
2) Set up your serial dilutions according to the information in Table 4.1. Aseptically pipette
900 µL of TB into Tube 1 that already contains 100 µL of bacterial culture. You have now
created a 1:10 dilution. Mix well using the vortex mixer. Create the remaining serial
dilutions (tubes 2-4) in the same manner. Use fresh tips for each transfer.
Table 4.1. Preparation of serial dilutions from E. coli culture sampled at 20 minute
intervals
Tube Amount of Amount of Final Dilution
Number µL)
sterile TB (µ Culture Factor
1 900 100 µL from 10-1
culture flask
2 990 10 µL from tube 10-3
1
3 990 10 µL from tube 10-5
2
4 900 100 µL from 10-6
tube 3
5 (for labs 900 100 µL from 10-7
3 and 4 tube 4
only)
The dilution sequence will be set up each time you take a sample from your culture flask.
3) Labs 1 and 2 will be plating the contents of Tube 3 and Tube 4 (10-5 and 10-6 dilutions).
Labs 3 and 4 will be plating the contents of Tube 4 and Tube 5 (10-6 and 10-7). Obtain 2
sterile Petri dishes. Label the bottom (not the lid) of the plate with the time the sample
was taken, your group name, and the dilution. 20 mL corresponds to where the bottom
edge of the lid is when the lid is on the Petri dish.
4) Add the contents of Tube 3 to the appropriately labelled sterile Petri dish. Obtain a bottle
of molten LB agar from the water bath at the side of the lab, and add approximately 20
mL of molten agar (after flaming the mouth of the bottle) to the diluted culture. Swirl
carefully to mix the inoculum evenly with the medium. Label the bottle of molten agar
with your group name and replace it immediately in the water bath.
5) Follow the instructions provided in step 4 above to plate out the contents of Tube 4.
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6) When the agar has solidified, place the inverted plates on a tray at the side of the lab.
The plates will be incubated for 16 – 20 hours at 37°C and refrigerated until the next lab
session.
Prepare a Results and Discussion section upon conclusion of this laboratory according to the
information found in Appendix 6.
Thought Questions:
• Use your graph(s) to calculate generation time of E. coli.
• Compare your value to that from the literature. Do the values differ? Why might this be?
• Compare and contrast indirect and direct methods of counting bacteria.
• Use your standard calibration curve to calculate the CFU/mL of culture for an undiluted
sample in which the OD was 0.75.
• Based on the differences in ingredients, what are the differences between growing cells on LB
versus TB? Why is TB used for generating growth curves of E. coli rather than LB?
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EXERCISE 5
THE AMES TEST
MUTATION AND RECOMBINATION (See Madigan, et. al., 2003. Chapter 106 Pg. 265-276)
You have learned about some of the advantages of using a model system in your study of the
effect of UV light on DNA in Biology 2000 (Introduction to Genetics). The Ames test also makes
use of a model system in order to measure the mutagenic potential of compounds. This test is a
reversion mutagenesis assay and uses strains of the bacterium Salmonella that have point
mutations in various genes in the histidine operon. These His- mutants are unable to synthesise
histidine and therefore unable to grow on minimal media lacking histidine. When the His- tester
cells are cultured on a minimal agar medium containing trace amounts of histidine, a small and
relatively constant number of cells per plate spontaneously revert to His+ and subsequently
reproduce and form colonies. Incorporation of a mutagen into the agar increases the number of
revertant colonies per plate, usually in a dose dependent manner.
EXPERIMENTAL OBJECTIVE
You will make use of the Ames test in order to evaluate the mutagenicity of a selection of
compounds.
PRE-LAB PREPARATION
Each class should bring in a total of three household compounds they would like to test. These
will be decided in advance. Note that these compounds must be known (ie “mystery liquid”
from the garage is not acceptable) and they must be taken home again once Period 1 of the lab is
finished.
METHODS:
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• Forceps
• 3x micropipettors (10 – 100 µL)
• Sterile tips
• 5x beakers with biohazard bags
• Small vials containing 95% ethanol for flaming
1) For each plate, you will be creating an overlay using a single strain mixed with the top
agar. The top agar has had a trace amount of histidine and biotin added. Using the
Table as a guide, obtain and label the appropriate number of minimal salts plates.
2) Have your plates labelled, and take to the station set up at the back bench. Set a
micropipettor to 50 µL. Remove one tube of agar overlay from the waterbath, and
aseptically add 50 µL of liquid culture to the tube. Vortex to mix and pour over the
surface of your agar plate. Clean up your work surface prior to going back to your
bench.
Note: you must work very quickly in order to avoid the top agar solidifying.
4) Flame forceps to sterilise. Note that this does not mean holding forceps in the flame of
your Bunsen burner until redhot! Rather, dip the forceps in ethanol, and wave through
the flame. Allow the ethanol to burn off. Pick up a sterile filter paper disk and dip in the
appropriate mutagen. For the cigarette extract, you will need to go to the fume hood to
do this.
26
7) Tap the filter paper several times to remove excess liquid. Hold the filter paper for a few
moments to ensure that liquid doesn’t drip all over your plates. Place the filter paper in
the centre of the plate with the solidified overlay. Tap gently to ensure that the filter
paper stays in place.
8) Incubate your plates for 48 hours at 37 oC. In the next lab, enumerate the number of
colonies on each plate and record the results on the board.
Thought Questions:
• What specific mutations in the His operon do each of the Salmonella strains used
contain?
• Evaluate the compounds tested for mutagenicity. What kind of mutations are
being caused by the compounds tested? (use the information from the first
Thought Question to answer this)
• Typically, mutagens are first mixed with liver extract prior to carrying out the
Ames test. What would be the purpose of this step?
References:
Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.E. 1973. Carcinogens are mutagens: a
simple test combining liver homogenates for activation and bacteria for detection. Proc.
Natl. Acad. Sci. U.S.A. 70:2281-2285.
Ames, B.N., Lee, F.E., and Durston, W.E. 1973. An improved bacterial test system for the
detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A.
70:782-786.
Ames, B.N., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and
mutagens with the Salmonella-microsome mutagenicity test. Mutational Research 31:347-
364.
Madigan, M. T., Martinko, J. M., and Parker, J. 2003. Brock Biology of Microorganisms
Tenth Edition. Prentice-Hall of Canada, Inc., Toronto.
27
EXERCISE 6
BIOCHEMICAL TESTS (Selective and Differential Media; IMViC Tests)
Normally, the coliform group of bacteria is used to indicate the pollution of water with fecal
wastes of humans and animals, and thus, the suitability of a particular water supply for
domestic use. The term coliform is used to describe aerobic and facultatively anaerobic Gram
negative rods that ferment lactose with gas formation. Most, but not all organisms within
this group are intestinal in origin; for instance, Escherichia coli. Consequently, presence of
lactose fermentors in a sample of water provides circumstantial evidence of pollution by fecal
wastes, and may suggest the presence of pathogenic bacteria such as members of the genera
Salmonella and Shigella. These pathogens, in addition to non-pathogens such as E. coli are
members of the Enterobacteriaceae family. In order to identify the organisms present in the
water, several biochemical tests that rely on differences in the chemical composition of media
used may be performed (see Appendix 4 and Appendix 8 for more details).
A strategy for bacterial isolation involves the use of selective media, media with specific
components that promote the growth of some bacteria and inhibit the growth of others.
Selectivity may be achieved in three ways:
• by adding something to the medium to discourage the growth of species not
required
• by altering the pH of the medium
• by omission of some ingredient required by most bacteria, but not by the organism to
be isolated
Differential media contain specific biochemical indicators that demonstrate the presence of
certain substances characteristic of certain bacteria. Thus, differential media are useful for
bacterial identification.
EMB agar contains the two indicators, eosin Y and methylene blue as well as the
carbohydrate lactose. Eosin (an acidic dye) reacts with methylene blue (a basic stain) to form
a compound of either acidic or neutral nature. The acid produced by lactose fermentors is
sufficient to cause this dye compound to be taken up by the cells. Non-lactose fermentors are
colourless because the eosin and methylene blue compound cannot be taken up by the cells.
The basic stain methylene blue inhibits bacterial growth, particularly that of Gram positive
28
bacteria (due to their cell wall composition). Eosin methylene blue (EMB) agar is thus
selective for Gram negative bacteria.
MacConkey Agar
MacConkey agar is a differential and selective plating medium recommended for use in the
isolation of Gram-negative bacilli and the differentiation of lactose fermentors from non-
lactose fermentors. The differential action of the MacConkey agar is indicated by the colonies
of coliform bacteria becoming “brick red” in colour. This occurs when the coliforms utilise
the lactose producing acids. The decrease in pH results in the uptake of the indicator neutral
red by the cells. Non-lactose fermentors are colourless and transparent. Production of acid
may also result in a zone of precipitated bile surrounding the colony. Bile salts and crystal
violet present in the medium inhibit Gram-positive bacteria from growing.
As demonstrated with MacConkey Agar, bacteria vary in their ability to ferment various
sugars. Products of fermentation are often acids and hence, pH changes can demonstrate
successful fermentation. In addition, gas (usually but not always CO2) is often produced
during fermentation, offering another indicator.
Hugh and Leifson’s method for demonstrating the presence of the products of fermentation
consists of a semi-solid medium containing peptone (short chains of amino acids), the
carbohydrate of interest (usually glucose or lactose), and a pH indicator, Bromothymol blue.
Tubes are stab-inoculated all the way to the bottom of the tube, so as not to introduce oxygen
into the medium. Several reactions may be observed. Facultative organisms will produce an
acid reaction (the indicator changes to yellow) throughout the entire tube of medium. The
acid reaction produced by oxidative organisms is apparent first at the surface, extending
gradually downwards into the medium. Note that organisms that oxidise glucose are
generally unable to ferment any carbohydrate. Strict fermentors will produce an acid
reaction at the bottom of the tube.
Organisms unable to use the carbohydrate may be able to grow using the peptone in the
medium. Production of alkaline products result in the formation of a blue colour at the top of
the tube (although this does not indicate that the organism is aerobic).
This medium contains triphenyl tetrazolium chloride and a small concentration of agar in
order to make the medium semi-solid. TTC is reduced when broken down by the organism,
and the TTC turns red where this has occurred. If the organism is facultative and motile, it
moves throughout the entire tube of medium and the whole tube becomes red. If the
organism is aerobic and motile, the top of the tube becomes red.
29
METHODS
IMViC TESTS
Only preliminary taxonomic assessment of bacteria can be made on the basis of microscopic
size, shape, association, and Gram staining. Information regarding natural occurrence is also
valuable since bacteria generally occur in specific habitats. This is particularly the case for
fastidious bacteria, those with very specific nutritional and environmental requirements.
However, even when supplemented with habitat information, bacterial identification based
on microscopic assessment is generally incomplete.
Confident bacterial identification can be made based on biochemical tests, and for certain
pathogens, or for examining microbial presence in specific environments, series of diagnostic
tests have been developed. For example, the IMViC tests are used routinely to confirm the
presence of coliform organisms in water. “IMViC” is an acronym for ‘Indole, Methyl Red,
Voges-Proskauer, and Citrate utilisation’ tests (the “i” is inserted for ease of pronunciation).
30
When cultured on peptone water, a liquid medium containing tryptophan, certain bacteria
will produce indole. The presence of this indole is readily revealed through addition of
Kovak's reagent, producing a pink colour. This reagent contains the organic solvent amyl
alcohol that extracts the coloured (pink) substance.
II. The Methyl Red (MR) Test - Mixed Acid Fermentation Pathway
Fermentation of glucose via the mixed acid fermentation pathway results in the formation of
a number of organic acids such as lactic and acetic acid. If this is a primary fermentation
pathway of a bacterium, a noticeable drop in pH will occur with incubation on MRVP media.
This decrease in pH can be revealed by a methyl red solution which is yellow under neutral
conditions and red at a pH less than 5.
The nutritional requirements of different bacteria vary considerably and these can provide
useful information contributing to biochemical identification. In Simmon's citrate agar,
citrate, in the form of sodium citrate, is the sole carbon source. Organisms able to utilise the
citrate grow on the surface of the medium and due to oxidative formation of sodium
carbonate, raises the pH of the medium changing it from green to blue (bromothymol blue is
the indicator).
V. Urea Hydrolysis
Some bacteria can produce urease, an enzyme which hydrolyses urea into ammonium and
carbon dioxide. The presence of this enzyme is detected by growing the bacteria in a
medium containing urea and a pH indicator, phenol red. If ammonium is produced as a
result of urea hydrolysis, the increase in pH will turn the medium to a violet-red colour.
31
METHODS:
1) Inoculate 6 Indole broth tubes separately with the 6 bacteria. After 48h of incubation at
37 oC, add 20 drops (1 mL) of Kovak's reagent (work in the fume hood and leave your
tubes in the fume hood to develop and observe). Shake and look for the formation of a
pink colour in the top (organic) phase; it may take 20 minutes to develop. The pink
colour is a positive result, indicating the ability to use tryptophan.
Note, please place tubes containing amyl alcohol in a separate rack in the fume hood as
this material needs to be disposed of separately.
2) A single culture solution (peptone, glucose, potassium phosphate) will be used for both
the methyl red and Voges-Proskauer tests. Inoculate 6 MRVP tubes with the 6 bacteria
provided, one culture into each tube.
• After 48h of incubation at 37°C, remove about 1/4 of the broth (=2 mL = 40 drops)
from the MRVP tube and transfer that to another test tube. Add 3-5 drops of methyl
red solution. An immediate red reaction provides a positive response to the test,
indicating the presence of mixed acid fermentation. A yellow or orange colour
represents a negative response.
• As the same solutions are used for the MR and VP, remove an additional 2 ml of
culture solution and add 1 ml α-napthol (Barritt's reagent A - 1 ml is about 20 drops)
and 1 ml 40% KOH (Barritt’s reagent B; caution - this is caustic). Shake vigorously for
30 seconds.
• Shake the tubes frequently and observe for up to 30 minutes for the formation of a
red colour that represents a positive VP test. A yellow or brown colour is a negative
result.
3) Inoculate 6 Simmon's citrate agar slants separately with the 6 bacteria. For these
inoculations, use your loop to smear cells along the surface of the slant. Incubate tubes
for 48 h at 37°C.
32
• After incubation, observe colours on the surface and down through the tubes. A dark
blue colour is a positive result while green indicates a negative test for citrate
utilisation.
4) Inoculate 6 urea slants separately with the 6 bacteria. After incubation for 48h at 37°C,
observe for the development of a violet-red colour.
5) After completing the Indole, MR, VP, Citrate and Urea tests, collaborate with the other
students at your bench to generate in your lab book tables of results for all bacteria in all
tests.
Thought Questions:
• Identify your unknown. Can it be any of the knowns? Why or why not? Provide
evidence to support your choice of organisms.
• Compare and contrast chemically defined and complex media. Provide two examples
of complex media used in this exercise and explain why these media are considered
complex.
• Provide two examples of compounds responsible for buffering in media.
• Is agar a nutritionally complete substrate for microbes? Why or why not?
• Design a defined medium for an organism that can grow aerobically on acetate as a
carbon and energy source.
• In this laboratory, would you classify the organisms used as photoautotrophs,
photoheterotrophs, chemoautotrophs, or chemoheterotrophs? Explain your choice(s).
33
EXERCISE 7
VIROLOGY
(Please review the material on sewage treatment posted on the Biology 3200 web page)
EXPERIMENTAL OBJECTIVE
The objectives of this series of exercises are first to isolate coliphage from filtered raw and treated
sewage obtained from the Lethbridge Wastewater Treatment Plant, to examine the plaque
morphologies, and to prepare phage isolate from one particular plaque. Using this phage isolate, the
phage titre will be determined, and the host specificity of the phage will be examined using several
enteric bacterial strains. These exercises will demonstrate standard techniques in phage isolation and
manipulation.
Prior to the laboratory, sewage samples were collected at the areas indicated on the schematic posted
o
on the web page. Both samples were stored at 4 C prior to filtering, for up to 1 week. On the morning
of the lab, samples were filtered twice using 0.45 µm filters.
PART A - ISOLATION
METHODS:
Work in groups of 4.
Note that sewage filtrate contains human pathogens. Work very carefully. Students who are
clearly unprepared or are sloppy will be asked to leave the lab.
Procedure
1) Obtain a tube of culture of E. coli K12.
34
2) Obtain 5 Luria Methylene Blue agar plates, and 5 sterile test tubes. Label your 5 tubes according
to Table 7.1.
1 500 0 0
2 0 500 0
3 0 0 500
4 500 500 0
5 500 0 500
3) Pipette the appropriate amount of filtrate and/or cells into each of your labeled test tubes. Leave
the tubes at room temperature on your bench to incubate for 20 minutes to allow the phage to
adsorb to the cells.
4) While your cultures are incubating, label your Luria Methylene Blue plates according to Table 7.1.
Mark the level of 4 mL on each of your tubes using the marked test tube on the side bench as a
guide.
5) Starting with Tube 1, aseptically pour molten agar into the tube up to the level of 4 mL. Vortex to
mix, then immediately pour the contents over the surface of the appropriately labeled plate. Swirl
the plate gently to ensure that the entire surface is covered with the agar.
6) Repeat step 5 for the remaining tubes and plates.
7) After 10 minutes, the overlay should be set. Invert your plates and place them on a tray on the
o o
side bench to be incubated. Plates will be incubated at 37 C for 16 – 20 hours, then stored at 4 C
until the next laboratory period.
MATERIALS
• Pasteur pipettes
• Bulbs
• Chloroform (in the fume hood)
• Vortex mixer
• Phage dilution buffer
• Plates from last lab
• 1 dissecting microscope per bench
35
• Microfuge tubes (sterile)
• 1 mL pipettes and propipettors
• Microfuge racks
• Labeled microfuge rack on the side bench for class tubes
5) Obtain your plates. Examine them carefully. Record the number of plaques present for both raw
and treated filtrate. Is there any difference?
6) Make detailed observations of plaque morphology. Features to look for include size, shape, and
turbidity (clear vs cloudy). Use the dissecting microscopes for your observations.
7) After making observations, obtain a microfuge tube and aseptically add 1 mL of phage dilution
buffer to your tube. Label with your group designation.
8) Use a Pasteur pipette (with a rubber bulb attached) to remove a plaque (squeeze the bulb, insert
pipette into the agar over a plaque, gently release bulb to remove a plug of agar containing the
plaque). Note that for each group of 4, two morphologically distinct plaques should be chosen.
Release plaque into the prepared tube of phage dilution buffer.
9) Vortex vigourously to disperse the agar.
10) Move to the fume hood and use a Pasteur pipette to add a drop of chloroform to your tube. Vortex
the mixture once again.
What does the chloroform do?
o
Place your tubes in the rack on the side bench. The tubes will be stored at 4 C allowing the phage to
elute from the agar into the buffer.
METHODS
36
• Microbiology kits
Thought Questions:
• Based on the schematic found on Dr. Brent Selinger’s web site, what step(s) is/are most likely
responsible for the difference in coliphage numbers between raw and treated sewage?
• Have you isolated more than one type of phage? How might you be able to tell?
• To what components of the bacterial cell to phage typically adhere?
37
EXERCISE 8
SOIL AND COMPOST MICROBIAL ECOLOGY
Soil Bacteria
The microflora of the soil exist as a complex food chain that brings about the release of
nutrients from dead plant material on the surface. The surface layer of newly fallen plant
material is called the litter and chemically it is composed of insoluble materials such as
cellulose, hemicellulose and lignin. Only a few organisms, usually fungi, are able to utilise
these high molecular weight compounds since carbohydrates, amino acids, vitamins and
other growth factors are lacking. However, once microorganisms do begin to decompose the
litter, the chemical structure of the litter is modified and the organisms produce end-products
that are released into the environment and become available for use by others. Death of these
organisms also provides new small molecular weight compounds that may then be utilised.
Bacteria are able to utilise the end-products of fungal metabolism. Nematodes and protozoa
feed on the bacteria and mites and other animals live on the nematodes and protozoa. In this
way nutrients are recycled.
Compost Bacteria
EXPERIMENTAL OBJECTIVES
In this experiment, you will prepare serial dilutions of compost and of soil samples and plate
out the appropriate dilutions. After incubation, you will determine the number of bacteria
isolated in your two samples, and assess the microorganisms for their ability to utilise
carboxymethylcellulose, casein, starch and xylan. You will choose one organism from either
soil or compost, use biochemical tests to identify the microorganism you have chosen, and
use the class results to compare and contrast microbial diversity in soil and in compost.
38
METHODS:
• Microbiology kits
• Soil
• Compost
• Balance
• Bottles containing 100 mL sterile water
• Vortex
• Petri dishes
• 250 mL bottles of molten peptone yeast extract agar in 60oC water bath
• 9 mL water blanks
• Sterile pipettes, propipettors
• Crystal violet
• Safranin
• Carbol fuchsin
• Gram’s iodine
• PYE broths
• Sudan black
• 95% ethanol
Out of your group of four, one pair will prepare enumeration plates for soil, while the other
pair will prepare enumeration plates for compost.
1) Weigh 1 g of soil or of compost provided and add to 100 mL of sterile distilled water.
This is dilution #1 (1:100). Shake the suspension for 5 minutes.
For those pairs working with soil, please follow the instructions outlined in steps 2-5; those
pairs working with compost, please follow steps 6-9.
2) Use the sterile 9 mL distilled water blanks provided to create serial dilutions of your
soil. Please ensure that you vortex your samples well prior to making each new
dilution. You will require 10-4 and 10-5 dilutions of soil.
3) Add 1 mL of the 10 -4 dilution to each of two sterile, labeled Petri dishes and 1 mL of
the 10 -5 dilution to two labeled Petri dishes.
4) Obtain a 250 mL bottle of molten peptone yeast extract agar – label with tape and leave in
waterbath when not in use.
5) Use the sterile 9 mL distilled water blanks to create serial dilutions of your compost. Please
ensure that you vortex your samples well prior to making each new dilution. You will require
-4 -5 -6
10 , 10 , and 10 dilutions of your compost.
39
6) Add 1 mL of the 10 -4 dilution to each of two sterile Petri dishes, 1 mL of the 10 -5
dilution to two sterile Petri dishes, and 1 mL of the 10-6 dilution to two sterile Petri
dishes.
7) Obtain your labeled 250 mL bottle of molten peptone yeast extract agar from the
water bath.
8) Add approximately 20 mL of medium to the plates prepared in Step 7. Swirl
carefully to mix the inoculum evenly with the medium.
9) The plates will be incubated for 24 hours at 30oC and refrigerated until the next lab
session.
Two classes will identify organisms from compost bacteria plates while the remaining classes
will identify organisms from soil bacteria plates. Your instructor will indicate what plates to
remove your unknown from.
Choose a morphologically distinct colony from the plate provided by your instructor and
prepare a streak plate for single colonies on peptone yeast extract agar (Appendix 3).
The plates will be incubated for 24 hours at 30oC and then stored at 4oC.
Prepare a Gram stain of the pure culture and record the cell morphology. Record the colony
morphology of the culture (see Appendix 6). Prepare a liquid culture of a single colony using
PYE broth. Use this culture to inoculate all of the biochemical test media you use.
Use the dichotomous key provided to develop a detailed outline of the series of steps you
plan to take to identify your unknown. This outline should include tests to carry out as well
as dates when you intend to do these tests. This outline must be handed in and approved
before you will be allowed to proceed.
The following media and reagents will be available for you to utilise as you attempt to
identify your unknown:
40
• nutrient agar
• endospore stain reagents
• capsule stain reagents
• hydrogen peroxide (catalase test)
• oxidase reagent (oxidase test)
• IMViC reagents
• indole broths
• MRVP broths
• citrate slants
• urea broths
• litmus milk broths
• mannitol broth (with phenol red indicator)
• H & L medium containing glucose
• H & L medium containing lactose
• sucrose agar
• motility medium (with TTC)
41
1. Gram stain
Gram positive ......................................................................................…………………...... 2
Gram negative...........................................................................................………………….16
2. Cell morphology
bacillus or spirillus……...................................................................………….........………...3
coccus….................................................................................................……………………..12
ovoid….....................................................................................……………………Azotobacter
3. Endospore stain
positive.......................................................................................................……..……………..4
negative ...................................................................................…………………................... 6
1
4. True endospores......................................................................................…………………........... 5
2
Special spore types..........................................................................................……………………6
1
Endospores are seen within the vegetative cells on an endospore stain after growing on sporulation agar for 48
hours.
2
Rod shaped cells break up into coccoid shapes or conidia after growing on an agar plate for several days
42
14. Large, mucoid colonies on sucrose agar; microaerophilic or
facultative anaerobic; capsule present.......................................………………..Leuconostoc
Small, round (1-2 mm) colonies on sucrose agar, no capsule.........………………Streptococcus
21. Bent bacilli, methyl red (-), Voges Praskauer (+), catalase (+)......................……………..Vibrio
Spiral bacilli, no growth in peptone water (indole broth)
without cellulose strip....................................................................…………………Spirillum
27. Non-motile...........................................................................................…………………….Klebsiella
Motile..............................................................................................……………………..Enterobacter
43
30. Motile................................................................................……………………............................... 31
Non-motile.............................................................................................…………………….Shigella
Once your outline has been approved, carry out your tests using materials available in your kits
or on the side bench. You will have four lab periods.
Record all of your results in your lab book. Include diagrams of all staining results, as well as
descriptions of cell and colony morphology and tables of biochemical test results. Include the
results from Part C below.
Use reference material to identify your organism to species (note, identify all possible
species).
Note: you will be pooling your results with those from the other classes and examining class
results as well.
Methods
Each bench will require:
• 2 casein agar plates
• 2 NA plates containing carboxymethylcellulose
• 2 NA plates containing starch
• 2 NA plates containing xylan
• Each individual will require:
• 1 casein agar plate
• 1 NA plate containing carboxymethylcellulose
• 1 NA plate containing starch
• 1 NA plate containing xylan
• 4 replica-plating templates per bench
• sterile toothpicks – 1 beaker per bench
• waste beakers for used toothpicks
• Enumeration plates (of soil and of compost bacteria)
Each group of 4 is responsible for replica-plating 40 random colonies from plates of soil or
compost bacteria onto plates containing one of the 4 substrates of interest
44
(carboxymethylcellulose, starch, casein or xylan). Note that plates containing CMC, starch or
xylan all contain these substrates added to a nutrient agar base whereas plates containing
casein are composed of skim milk and agar only.
1) Using a sterile toothpick for each new colony, carefully scrape a well-defined colony
from one of your soil or compost bacteria enumeration plates.
2) Stroke the toothpick across square number 1 on each of the three different labelled
plates (CMC, starch or xylan). For plates containing casein as the substrate, you may
need to estimate placement of the colonies as you may not be able to see the template
through the plate. Place the used toothpick into the beaker provided.
3) Select a fresh toothpick and repeat steps 1-2 for 40 different colonies of bacteria.
4) Obtain 4 plates, each containing a different substrate. Label with your name and
with the name of the substrate.
5) Use an inoculating loop to streak out your unknown (from your PYE plate of pure
culture) onto each of the 4 plates (you want to streak for single colonies).
Invert all of the plates. These plates will be incubated at 30oC for 48 hours.
Thought Questions:
45
• For enumeration, why do you only count plates having between 30 and 300 colonies?
• Why do you incubate soil and compost bacteria at 28-30oC?
• Provide one specific example of a differential medium used in the current exercise.
• What is the differential component in the medium in your answer in (a)?
• How is the medium differential?
• Is this medium type selective also? Why or why not? How would you make this
medium selective for the carbon source in question?
• In addition to manipulating nutrients, how else could you make culture conditions
selective? Provide a specific example.
References:
Atlas, R.M. and Richard Bartha. 1998 Microbial Ecology: Fundamentals and
Applications, Fourth Edition. Benjamin/Cummings Publishing Company, Inc.
640 pp.
Poulsen, O. M. and Petersen, L. W. 1989. Electrophoretic and enzymatic studies on the crude
extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC21399.
Biotechnol. Bioeng. 34: 59-64.
Ross, H. 1993. Cellular, Molecular and Microbial Biology 343 Laboratory Manual. The
University of Calgary press, Calgary AB.
Teather, R. M. and Wood, P. J. 1982. Use of Congo red polysaccharide interactions in the
enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl.
Environ. Microbiol. 43: 777-780.
46
EXERCISE 9
APPLICATIONS OF MICROBIOLOGY
A number of industrial processes make use of the end products of bacterial and fungal
fermentations. For instance, in the presence of acid producing bacteria, and often the enzyme
renninase, milk will form curds (solid) and whey (liquid). Once the solids are compressed,
salted, and aged, the resulting product is cheese. Different cheeses are produced by varying
the bacterial inoculum, varying the milk used, or even by introducing fungi such as certain
species of Penicillium into the curds.
Some Streptococcus species and some Lactobacillus species produce only lactic acid as a result
of reduction of pyruvic acid. These organisms are responsible for the production of yogurt.
Yogurt can be made from milk simply by inoculating with a starter culture of yogurt that
contains live bacterial culture. Conversely, yeasts produce alcohol and CO2 rather than lactic
acid as a result of the reduction of pyruvic acid.
EXPERIMENTAL OBJECTIVE
This experiment will illustrate fermentation pathways and organisms involved in the
production of alcohol.
A Alcohol Production
Prior to your lab period, grape juice, water and yeast cells were added to a sterile container. Over
the next two weeks, you will be responsible for sampling the fermenting juice at various time
intervals. The primary fermentor is inoculated with a high cell density (~106 yeast cells/mL).
The bulk of the must (grape juice medium) is rapidly depleted of oxygen by the yeast and
remains anaerobic, despite the primary fermentor remaining open to the atmosphere. Yeast cells
continue to reproduce by acquiring the needed energy and carbon through fermentation. The
fermentation is an ethanolic fermentation because ethanol and CO2 are the fermentation
endproducts.
Growth of the yeast culture can be monitored by measuring optical density and enumerating
CFU/mL (Recall Exercise 4 - Bacterial Reproduction). Ethanol concentration can be estimated
indirectly by measuring the specific gravity of the wine must with a hydrometer. The specific
gravity of the must decreases as the grape juice sugars are converted to ethanol and CO2. A
"specific gravity to percent ethanol" conversion chart supplied with the hydrometer is then used
to determine ethanol content of the must.
Note: this exercise requires some out of lab participation. Failure to sign up will result in a
deduction of 5% from your lab grade.
47
Please sign up for a time slot when at least half of your group members can attend.
At each time point the following data must be collected by each group:
• Temperature
• OD600
• pH
• Specific gravity
• Viable counts
Procedure – Work very carefully. These results will form the basis of your major lab report.
1) Mix the culture well, then remove a sample from the primary fermentor.
2) From the sample (not the primary fermentor), measure and record the sample
temperature.
3) Measure and record the specific gravity.
4) Measure and record the pH of the sample.
5) Measure and record OD600. Use the blank provided to zero the machine. Read the optical
density of at least 5 mL of the sample. If the OD600 exceeds 0.7, you will have to dilute the
sample with the sterile must provided (start by creating a 1:1 dilution). Read the OD600 of
the diluted sample, then multiply by the dilution factor to obtain your corrected reading.
Record the corrected reading on the sheet provided.
48
Please save the blank for the next group.
5) For viable counts, Table 8.1 provides you with dilutions to create depending upon your
sample time, as well as guidelines for what dilutions to plate out. Ensure that you plate
out duplicates of each dilution.
6) For your dilutions, prepare the required number of 900 µL dilution blanks = 900µL of
filter sterile wine must in 1.5 mL microfuge tubes.
7) Clearly label plates with time, name and dilutions. Spread plate (in duplicate) 100 µL of
suggested dilutions on YPD agar.
8) Invert plates and incubate at 28ºC for 48 hours.
9) Tidy up work area.
Thought Questions:
• Numerous data relating to alcohol fermentation were collected by the class over the sampling
period, including measurements of pH, temperature, specific gravity, optical density, and
CFU’s/mL of culture. Design and construct a series of figures to graphically represent the
data that were collected.
• Calculate the generation time and the specific growth rate of the yeast cells in the culture.
• Name two factors that control the final ethanol concentration in a culture.
• Although we stirred our culture each time before sampling, winemakers do not. Why would
winemakers not stir the culture?
• Why did the pH of the culture change as fermentation proceeded?
• Why did the specific gravity of the culture change over time?
49
Table 8.1 Dilutions of yeast mixture to create and to plate out for all time points.
Time point Dilutions to Create Dilutions to Plate
(hours)
Spread plate 100 µL of filter
2 None sterile wine must in duplicate on
YPD
Spread plate 100 µL of each of
3 10-3; 10-4; 10-5 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-3 -4 -5
7 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-3 -4 -5
12 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-4 -5 -6
24 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-4 -5 -6
28 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-4 -5 -6
32 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-4 -5 -6
48 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-4 -5 -6
53 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
79 10-4; 10-5; 10-6 the 3 dilutions in duplicate on
YPD
Spread plate 100 µL of each of
-4 -5 -6
217 10 ; 10 ; 10 the 3 dilutions in duplicate on
YPD
50
APPENDIX 1
THE COMPOUND LIGHT MICROSCOPE
As you label Figure 1, your Instructor will review the use of this microscope with you. Locate the
ocular lens (eyepiece); there will be one if the microscope is monocular, or two if it is binocular.
Then locate the objective lenses, the ones nearest the object to be studied. These two lenses
(ocular and objective) are connected by the body tube of the microscope. The objective lenses
(there will be two or more, the smallest being that with the least magnifying power, and the
largest being that with the greatest magnifying power) are mounted on a revolving nosepiece
above a flat stage on which the study specimen (slide) is placed.
Your microscope is equipped with a mechanical stage. This consists of a clip to hold the slide in
place (the clip is spring-loaded; the Instructor will demonstrate how it works) and two knobs at
the side of the microscope body to move the slide side-to-side, or forward-to-back. Note also the
two micrometer scales on the mechanical stage, which allow you to note the coordinates of a
particular object on the slide you are viewing.
51
Place a slide on the stage and center it over the hole in the stage. Adjust the distance between the
oculars to match your interpupillary distance (distance between your pupils). Revolve the
nosepiece so that the lowest power objective lens (generally the 10x power lens) is in position. To
focus the microscope, locate the coarse and fine adjustment knobs at the base of the microscope,
and use the coarse adjustment to move the slide close to, but not touching, the objective lens.
Look at the stage from the side as you do this. On most microscopes this involves raising the
stage, but on some the lenses are lowered. Also, on most microscopes an automatic stop will
prevent you from moving the stage closer than about one centimeter from the lens. Now, look
through the ocular lenses, and move the slide away from the objective lens until the specimen
becomes clear (is in focus). Finish focusing with the fine adjustment knob. Once you have
focused with the low objective power lens, you may switch over to the next higher power lens
with only fine focus adjustments (the microscope is said to be parfocal).
As you switch from one objective lens to another, you will notice that the working distance, the
clearance between lens and stage, decreases with increasing lens power. This is illustrated in
Figure 2 below.
Figure 2: The working distance (above) and the field of view (below) change
with magnification of objective lens.
It should be obvious to you why, on high power objective lenses (40x or 100x), you must use only
the fine focus knob to adjust focus; otherwise the risk of (damaging) contact between lens and
slide becomes great. Also illustrated in Figure 2 is the diminishing field of view as objective lens
power increases; this is due to a smaller and smaller aperture at the bottom of the lens through
which light enters. This means that [a] things are harder to find on a slide when you are using
high power since only a small fraction of the slide can be seen, and [b] less light enters your eye
and everything in the field appears darker. As a consequence, you will learn to [a] switch back to
52
a lower power objective lens when you want to "scan" around the slide, and [b] manipulate the
amount of light coming into the lens so that you can see the objects clearly.
The amount and concentration of light coming through the specimen and hence to your eye can
be adjusted in several ways. First, of course, is the on/off light switch, generally located at the
base of the microscope, and often associated with a rheostat to control light intensity. A
condenser lens is mounted below the stage, and concentrates the light on to the specimen; it
generally needs no adjustment of position. An iris diaphragm is located below the condenser
lens. Find the lever which controls the diaphragm; it can be very useful in adjusting illumination
and contrast.
Biology 3200 microscopes are binocular, containing two eyepieces. To correct for the slight
difference in the focus of your two eyes, precisely fine focus a specimen using only your one eye
which is at the non-focusing ocular (if your microscope contains two focusing oculars, either may
be used to begin). Next, open the other eye and bring the image into focus for that eye using only
the ocular focus. Since other students use these same microscopes during the semester, this
exercise of binocular focusing should be performed at the onset of each microscope session.
Electron Microscopy
Bacterial size places them at the limits of resolution of the light microscope. Even with the best
quality lenses, magnification can only be increased slightly beyond 1,500x. Much higher
magnification can be achieved with the electron microscope with the scanning electron
microscope (SEM) reaching about 100,000x magnification and the transmission electron
microscope (TEM) capable of 1,000,000x magnification.
53
APPENDIX 2
PREPARATION OF SCIENTIFIC DRAWINGS
54
9) Example of a drawing:
Figure 1. A chain of Bacillus subtilis cells stained with methylene blue (23 000x)
• Notice that in the figure, enough organisms are shown such that the arrangement can be
seen.
• Drawing magnification is calculated based on length or width, not both of only one of the
organisms (not the whole chain).
• Figures are given numbers - Figure 1, Figure 2, etc.
• As much detail as possible is provided in the title (eg Gram reaction seen, type of stain used,
type of organism etc.).
55
APPENDIX 3
ASEPTIC TECHNIQUE
A. Aseptic Technique
Much microbiological work, and to some extent biochemical work, depends on the maintenance
of pure cultures of microorganisms. Therefore, there are various essential precautions that MUST
be observed to exclude unwanted organisms. Accidental contamination may ruin your results
completely.
Aseptic technique is largely a matter of common sense, but it is essential to realise that bacterial
and fungal spores are present everywhere, and a high standard of technique must be attained.
Correct methods of handling cultures and apparatus will be demonstrated. These methods
should be followed.
1. Clean air contains many bacterial and fungal spores carried on dust particles or in water
droplets. Any surface exposed to air quickly becomes contaminated, and if material is to
be kept sterile it should be exposed only as much as is absolutely necessary for
manipulation. Instruments which can be sterilised by heating in a bunsen flame (e.g.
inoculating loops) can be left exposed, but they must be flamed thoroughly before use,
and again before being replaced in the holder.
Items of equipment that cannot be treated in this way (e.g. pipettes) are sterilised in
wrappings or containers from which they must not be removed until actually needed.
They must not be allowed to touch unsterile surfaces during use. Plugs and caps of
tubes and bottles must not be laid on the bench nor must sterile containers be left
open to collect falling dust.
2. Clothes, hair, skin and breath all carry a heavy microbial load and where strict asepsis is
essential, sterilised gowns, caps, gloves etc. are worn. Even in normal microbiological
work care must be taken to prevent contamination from the above mentioned sources. A
clean laboratory overall is advised for all lab work.
Microbial contamination in the lab is most often due to currents of unsterile air. The chief merit
of inoculation chambers and screens therefore lies in the protection they give from drafts. This
protection can be supplemented by keeping all windows and doors shut and by cutting down
personal movement within the laboratory. These precautions can be offset by careless use of
burners that create convection currents.
56
3. Before any operation is started, all necessary materials should be assembled in
convenient order with provision for protecting sterile objects until needed, and for
disposing of used apparatus (so as not to contaminate other material).
Purposes: 1) To prevent the contamination of the environment and people working in the
laboratory from the cultures used in the exercises
2) To prevent accidental contamination of cultures of microorganisms and of
solutions and equipment used in the laboratory
Correct methods of handling cultures and apparatus will be demonstrated. These methods
should be followed. Consider carefully and remember the following points:
• Prior to starting any work in the laboratory, wash hands with soap, and wash down
bench area using 10% bleach. This procedure should be repeated after the lab is
complete.
• Clean laboratory coats must be worn. If you have long hair, tie it back before working in
the laboratory environment.
• Eating or drinking are not permitted in the laboratory. Do not place pencils, fingers or
anything else in your mouth.
• Clean air contains many bacteria and fungal spores carried on dust particles or in water
droplets. Any surface exposed to air quickly becomes contaminated. If material is to be
kept sterile, it should be exposed only as much as is absolutely necessary for
manipulation.
Plugs and caps of tubes, tops of Petri dishes and bottles of solutions, (even water!!) must not
be laid on the bench nor must sterile containers and cultures be left open and exposed to the
air.
57
Inoculation of Culture Tubes
Again, the important thing to remember is that exposure of sterile liquids or bacterial cultures to
air must be minimised.
-Ensure that you have the tubes, plate of inoculum, inoculating loop and a sterile tube of medium
available within easy reach.
-Flame the inoculating loop until red hot. When removing inoculum from a tube, remove the cap
from the tube by grasping the cap between the last finger and the hand which is also holding the
inoculating needle (Figure 1). Do not place the cap on the bench!!
-Flame the mouth of the tube by passing it rapidly through the Bunsen burner 2-3 times. This
sterilises the air in and immediately around the mouth of the tube.
-Cool the loop on the inside of the tube, remove the inoculum.
-Reflame the mouth of the tube and replace the cap
-Flame the inoculating loop before replacing
-Note, when removing inoculum from a plate, cool the loop in the agar before picking up the
bacteria
58
Streaking for Single Colonies
-A loop of liquid culture or a small amount of bacterial growth from a plate culture is transferred
aseptically to a sterile plate in the area shown by Figure 2A.
-Once the first set of streaks has been made, the inoculating loop is reflamed until red hot. DO
NOT REINTRODUCE THE LOOP INTO THE ORIGINAL CULTURE!!!
-Cool the loop, and make a second set of streaks as shown in Figure 2B, only crossing over the
initial set of streaks once.
-Flame the loop again, cool, and repeat for three more sets (Figure 2C). Note, try not to gouge the
agar while streaking the plate.
59
Preparation of Spread Plates:
Generally, volumes of culture greater than 100 µL are NOT plated as it takes too long for the
liquid to dry.
• Use aseptic technique to obtain 100 µ L of culture and place in the middle of a plate of
medium.
• Use a sterile glass spreader (this may involve dipping a spreader into a beaker of alcohol
and waving it through a Bunsen Burner flame. If this is the case, DO NOT hold the
spreader in the flame and avoid tipping the spreader so that flaming alcohol runs over
your hand. Once the flame has burnt out, the spreader is ready to use).
• Use the same hand that holds the spreader to lift the lid of the plate and keep it just above
the plate the entire time.
• Gently touch the spreader to the side of the medium (not directly in the culture in case the
spreader is still a bit warm). Smooth the culture evenly over the surface of the plate
ensuring that you cover the entire plate.
• Invert the plates and place in the incubator when dry.
C. Sterilisation
Media must be sterilised after distribution into tubes, flasks or bottles. Sterilised media may later
be transferred aseptically to previously sterilised containers, but this should only be done when
really necessary, e.g. in preparing "plate" cultures, since some risk of contamination is
unavoidable.
Methods of Sterilisation
1. Most media (including agar) can be sterilised by treatment with steam under pressure in
an autoclave, the usual treatment being 15-20 minutes at a pressure of two atmospheres.
This raises the steam temperature to 121°C. When using an autoclave, the water should
be allowed to boil, and the steam to fill the autoclave before shutting the valve. This
allows the material to heat up and ensures that the correct steam pressure is attained.
Never overfill an autoclave since this will upset the pressure/volume relationship and
the correct temperature will not be attained. Materials that might be adversely affected
by this treatment may sometimes be treated for a short time or at a lower temperature,
but this will not be effective if the material is heavily contaminated to begin with. Screw
caps on bottles must be left slightly open during sterilisation and screwed down on
removal from the steriliser.
2. Media that are difficult or impossible to autoclave satisfactorily, e.g. gelatin media and
some sugar media, may be sterilised by intermittent steaming. Objects to be sterilised are
heated over boiling water in a steamer (steam temperature 85°-95°C) for 15-20 minutes
on each of three or more successive days. Time must be allowed for the medium to reach
the same temperature as the steam. Between treatments the material must be kept at a
temperature allowing spores to germinate (30°-37°C) and so lose their heat resistance.
60
3. It is often necessary to sterilise some ingredients of a medium separately and to add them
to the rest of the medium before use. Heat-labile ingredients, e.g. urea, serum, etc. must
be sterilised by filtration through a bacteria-proof filter, i.e. Seitz filters or membrane
filters.
4. Dry glassware, e.g. glass petri dishes, empty flasks, pipettes may be sterilised in the
autoclave and then dried or may be sterilised in a hot air oven. Any oil material
must also be sterilised in a hot air oven. The minimum effective treatment is 1 hour
at 150°C. This should be increased to 160°C or the time of heating prolonged to 2 or 3
hours wherever possible.
61
APPENDIX 4
THE CULTIVATION OF BACTERIA
(Please read Madigan et. al., 2003; Chapter 5)
Nutritional Classification:
The nutritional classification of organisms is based on three parameters: the energy source, the
principal carbon source and the source of reducing power. With respect to energy source,
phototrophs are photosynthetic organisms that use light as their energy source and chemotrophs
are organisms that depend on a chemical energy source. Organisms able to use CO2 as a
principal carbon source are autotrophs. Heterotrophs depend on an organic carbon source. To
designate the source of reducing power, the term lithotroph or organotroph is applied.
Lithotrophs use inorganic compounds as their source of reducing power, and organotrophs use
organic compounds as their source of reducing power.
To summarise:
source of
energy source carbon source reducing power
photoautotroph light CO2 inorganic
(photolithotroph) oxidizable
substrate
photoheterotroph light organic organic
(photoorganotroph)
chemoautotroph chemical (oxidation of CO2 inorganic
(chemolithotroph)* reduced inorganic
compounds e.g. NH3,
NO2- and H2)
chemoheterotroph chemical organic organic
(chemoorganotroph)
62
*All chemoautotrophs are chemolithotrophs, but not all lithotrophs are autotrophic. For
example, the methylotrophic bacteria can use organic carbon as their carbon source.
Common Media Constituents (see Table 5.4, Madigan et. al (2003) for examples):
Nitrogen sources:
• Inorganic sources such as ammonia or nitrate
• Nitrogen fixing organisms use atmospheric N2
• Extracts, infusions
• Peptone – hydrolysis of proteins produces mixtures of short-chains of amino acids
(peptides). Sources of peptones may include meat, fish, blood, or soybeans
• Tryptone – pancreatic digestion of casein
Micronutrient Sources:
• May not be necessary to add as these are required in such small concentrations.
Growth Factors:
• Some organisms are able to synthesise all growth factors from precursors. Other
organisms require these compounds already synthesised
• For example – thiamine, biotin
Buffering Components
Buffers, which prevent large changes in pH, are often required to facilitate growth. This is
particularly true of media composed of simple compounds or in which acid-producing bacteria
are cultivated. Mixtures of sodium and potassium phosphates are often employed. In complex
media, buffering is provided by the peptides and amino acids.
63
Gelling Agents
For a solid medium, agar, a water soluble polysaccharide, is added to the medium. First
discovered in 1658 in Japan, agar was first used for microbiological purposes by R. Koch in 1882.
It is extracted from members of Class Rhodophyceae (a group of red-purple marine algae). Agar is
particularly suited to microbial propagation because:
• It lacks metabolically useful chemicals such as peptides and fermentable carbohydrates
(it cannot be broken down by bacterial enzymes)
• It melts at a high enough temperature (85 oC) to support growth of different temperature
requiring microbes
• It lacks bacterial inhibitors
Below are two examples of media used for cultivation of microbes. TY is an example of a
complex medium whereas VMM is an example of a minimal or defined medium:
For the next example – VMM – three different mixtures (Solutions A, B and C) of ingredients
are made up separately, autoclaved separately, and then combined. Finally, a carbon source is
added just prior to pouring.
64
VMM (Vincent’s Minimal Medium - Vincent, 1970) (used for the study of nutritional
requirements of Rhizobium leguminosarum)
Solution A:
Compound Amount (/L) Source of?
K2HPO4 1.0 g Buffering agent/
Macronutrients
KH2PO4 1.0 g Buffering
agent/Macronutrients
KNO3 0.6 g Macronutrients (nitrogen in
particular)
For Solid Medium: 12.5 g Gelling agent
Agar
Solution B (10x):
Compound Amount (/L) Source of?
FeCl3 0.1 g Macro/Micronutrients
MgSO4 2.5 g Macronutrients
CaCl2 1.0 g Macronutrients
Autoclave and add to a final concentration of 1x
Solution C (100x)
Compound Amount for: 1 L Source of?
Biotin 0.01 g Growth factors
Thiamine 0.01 g Growth factors
Calcium Pantothenate 0.01 g Growth factors
Autoclave and add to a final concentration of 1x.
Carbon sources: Depending on the organism studied, a variety of carbon sources may be added.
For instance, when studying genes required for catabolism of a certain carbon source, a scientist
will often first create a mutant or auxotroph unable to catabolise that carbon source. To confirm
presence of the mutation, it is necessary to plate the putative auxotroph on medium containing
the carbon source of interest, and plating on a medium containing a carbon source that the
organism is able to utilise. In Rhizobium leguminosarum, some examples of carbon sources that are
useful for these types of experiments are mannitol, sorbitol (both are sugar alcohols), or
rhamnose. Each carbon source is prepared as a stock solution, filter sterilised, and added to a
final concentration of 0.4% (w/v).
Many species of bacteria are facultative aerobes, i.e. they can grow under aerobic or anaerobic
conditions, the latter ability being dependent upon the presence of some substance that can be
utilised as an electron acceptor by the species concerned. Some bacteria are obligate aerobes,
unable to use anything but oxygen as a final electron acceptor. Others are obligate anaerobes that
cannot use oxygen as an electron acceptor. A few bacteria are somewhat intermediate, growing
65
best in low oxygen tensions. These are called microaerophilic bacteria. During growth in liquid
culture, microorganisms tend to utilise all available oxygen and so reduce the medium. Thus, the
oxidation-reduction potential (Eo) of the medium may become low enough to allow anaerobic
growth to occur. One example of this is found in the fermentation of sugar to produce alcohol by
yeast (Exercise 8 part C). Unless the mixture is stirred frequently, the little oxygen available in
the grape juice solution is utilised rapidly by the growing culture. Organisms then switch to
anaerobic growth.
In order to sample material containing anaerobes, specimens must be obtained and immediately
placed into an environment containing an oxygen-free gas and an indicator that changes colour
when oxidised to indicate when oxygen has contaminated the sample. Organisms may then be
cultured in sealed jars containing gas mixtures of N2 and CO2 or even by cultivation in an
anaerobic chamber.
Cultures should be incubated at the temperature most favourable to growth or the specific
activity being studied. Human pathogens and commensal species grow best at body
temperature, i.e. 37°C. Soil organisms and plant pathogens are normally incubated at 20-30°C.
The optimum temperature is that temperature at which the growth rate is maximal for a
particular organism. Note that for every organism, there is also a minimum temperature below
which no growth occurs, and a maximum temperature, above which no growth occurs.
The terms used to describe microorganisms according to their temperature requirements are as
follows:
References:
Difco Manual. 1998. Difco Laboratories, Division of Becton Dickinson and Company,
Maryland.
Madigan, M. T., Martinko, J. M., and Parker, J. 2003. Brock Biology of Microorganisms 10th
Edition. Prentice-Hall Canada Inc., Toronto.
Ross, H. 1992/3. Microbiology 241 Lab Manual. University of Calgary Press, Calgary.
66
APPENDIX 5
BACTERIAL OBSERVATION
Colony Morphology is that of a single isolated colony on the plate, not the morphology of the
entire bacterial growth on the plate. Colony morphology is influenced by medium composition;
type of medium organism is grown on (defined, complex, specific type) should be noted in
conjunction with the description of colony morphology.
The following characteristics are those most commonly used to describe colony morphology:
1) Shape or form
3) Elevation:
67
APPENDIX 6
LABORATORY REPORTS
Lab reports shall be in the style of scientific papers published in refereed journals. This scientific
style is relatively similar across journals although specific formats vary, including the form of
literature citations. The journals Microbiology or Canadian Journal of Microbiology will be used as
models for the specific format of Biology 3200 reports. Please do not use formats from journals
such as Nature or Proceedings of the National Academy of Sciences as this will result in loss of marks.
For detailed information on preparation of scientific reports, please refer to the Biology 3200 web
site.
The text should be in prose form and standard rules of grammar apply. Check spelling,
including technical terms and names of bacterial species which are italicised or underlined; for
instance, Escherichia coli or Escherichia coli.
The reports shall be double-spaced, single-sided and typed. Staple the report together and do not
submit it in a cover.
The reports shall contain the normal components of a scientific paper including:
Title - the title should identify the experimental topic as completely as possible.
Abstract - the abstract is an abbreviated version of the complete report. Typically containing no
more than 250 words, the abstract picks out the highlights of the introduction, methods, results
and discussion. The abstract should be complete enough that it can be removed from the report
and will still provide a meaningful description of the study.
Introduction - The introduction serves to (i) provide background information and a description
of what is known prior to the study, and (ii) offer a justification for the study. This justification
describes why the experiment was performed - how does it fit into science and are there any
applied aspects of the knowledge (i.e. is it relevant to medicine, agriculture or other disciplines).
Relevant literature is used and cited.
Methods - The methods or 'Materials and Methods' describes the materials involved in the study,
including biological materials (bacteria, etc.), and outlines the procedures used in the study.
Reference must be made to this laboratory manual (Pacarynuk and Danyk, 2004). Other
references, the text by Madigan et. al., (2003), or other published materials may be cited. Global
referencing (“All of the following methods are taken from…”) should be avoided. The methods
section should be adequate for the reader to completely understand what was done and also to
be able to repeat fully the study.
Results - The results describe the observations or experimental outcomes, providing figures,
tables or other data as suitable. This section answers the question “What Happened?” The author
should decide what is the most suitable format for experimental information and draft the report
accordingly. Figures may include drawings that should be in pencil. Graphs or other figures
may also be included as appropriate. Experimental results should be presented only once. If
68
information is presented in a figure then it should not be repeated in a table. Each figure and
table must have a caption which is complete enough that the figure and caption can be removed
from the report and still be understandable. Figures and tables must be referred to in the text
and described so that if the reader did not have the figure or table, trends or highlights of the
results would still be evident. Never include a figure or table without referring to it and
describing it; to do so will result in loss of marks. Avoid evaluating or interpreting your results
in this section.
Discussion - The discussion should refer to concepts or questions posed in the introduction and
relate these concepts from the literature to the results. Do not restate the results in this section.
Your discussion will be graded based on your evaluation of the results with respect to the
literature. Any time you use information from another source, it must be immediately cited
within the text. Failure to do this constitutes plagiarism and may result in a mark of zero being
assigned for the entire document. For examples of how to cite properly, refer to peer-reviewed
journal articles in Microbiology or Canadian Journal of Microbiology.
Never include quotations, such as phrases from the course text or this lab manual. Direct quotes
are inappropriate in scientific writing. Always introduce relevant concepts using your own
wording and then cite using the format found in Microbiology or in Canadian Journal of
Microbiology.
Literature Cited – This section only includes references cited within the body of the text.
Again, use the format found in Microbiology or the Canadian Journal of Microbiology.
References will include journal papers, books and most likely, Holt (1989) or Holt (1994)
(Bergey’s Manual of Systematic Bacteriology). It is important to note that Bergey did not
write Bergey’s Manual of Systematic Bacteriology; the proper formats for referencing are as
follows:
Holt, J. G. (editor-in-chief). Bergey’s Manual of Systematic Bacteriology, Vol. I, 1984; vol. II,
1986; vols, III and IV, 1989. Williams and Wilkins, Baltimore.
69
APPENDIX 7
USE OF THE SPECTROPHOTOMETER
Many procedures for the quantitative analysis of compounds in biological fluids are based on the
fact that such compounds will selectively absorb specific wavelengths of light. For example, a
solution that appears red to us (such as blood) absorbs the blue or green colours of light, while
the red is reflected to our eyes. The eye, however, is a poor quantitative instrument, and what
appears bright red-orange to one person may appear dull red-purple to another. A
spectrophotometer is one instrument that will objectively quantify the amount and kinds of light
that are absorbed by molecules in solution. A source of white light is focused on a prism to
separate it into its individual bands of radiant energy (Figure 1). One particular wavelength is
selected to pass through a narrow slit and then through the sample being measured. The sample,
usually dissolved in a solvent, is contained in an optically selected tube or cuvette, which is
standardized for wall thickness and has a light path exactly one centimeter across (these tubes are
therefore expensive!).
After passing through the sample, the selected wavelength of light strikes a photoelectric tube. If
the substance in the cuvette has absorbed any of the light, the light transmitted out the far side
will then be reduced in total energy content. When it hits the photoelectric tube, it generates an
electric current proportional to the intensity of the light energy striking it. By connecting the
photoelectric tube to a device that measures electric current (a galvanometer), a means of directly
measuring the intensity of the light is achieved. The galvanometer has two scales: one indicates
the % transmittance, and the other, a logarithmic scale with unequal divisions graduated from 0.0
to 2.0, indicates the absorbance.
70
Zeroing the Spectrophotometer
Because most biological molecules are dissolved in a solvent before measurement, a source of
error can be the absorption of light by the solvent. To assure that the spectrophotometric
measurement will reflect only the light absorption of the molecules being studied, a mechanism
of "subtracting" the absorbance of the solvent is necessary:
1) Align the needle to 0 on the transmittance scale using the knob on the left hand side of
the machine (as you face the machine). Note, this step should be performed prior to
placing any tubes into the machine.
2) Insert the reagent "blank" (the solvent) into the instrument, and align the needle to 0 on
the absorbance scale using the knob on the right hand side of the machine (as you face
the machine).
3) The sample, containing solute plus solvent, is then inserted. Any reading on the scale
that is less than 100% transmittance (or greater than 0.0 absorbance) is considered to be
due to absorbance by the solute only.
Units of measurement: The transmittance scale is a % number; a ratio of the light exiting
the sample tube to the light entering the tube. However, this number is not a linear reflection of
the concentration of the solute molecules (Figure 2). The absorbance scale, on the other hand,
does reflect a linear relationship. Although you do not necessarily know the exact concentration
of the solute molecules in your sample, you do know that if the absorbance value doubles, the
concentration of solute in your sample has doubled. Absorbance has no units, but the
wavelength of the light is usually indicated by a subscript.
Figure 2. The relationship between % transmittance and solute concentration (on the left), and
absorbance and solute concentration (on the right).
71
APPENDIX 8
Media, Reagents and pH Indicators
MEDIA:
Composition (g/L):
Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, and autoclave
for 15 min at 121oC.
Composition (g/L):
Dissolve in distilled water to a final volume of 1 L, autoclave for 15 min at 121oC, and
pour into sterile Petri dishes.
72
LB Medium (Luria-Bertani Medium):
Composition (g/L):
Tryptone 10.0 g
Yeast extract 5.0g
NaCl 10.0 g
Dissolve in distilled deionised H2O to a final volume of 1 L, autoclave for 20 minutes at
15 psi (1.05 kg/cm2) on liquid cycle, and pour into sterile Petri dishes.
Composition (g/L)
Tryptone 12.0 g
Yeast Extract 24.0 g
Glycerol 4.0 mL
Dissolve in distilled deionised H2O to a final volume of 900 mL, autoclave for 20 minutes
at 15 psi (1.05 kg/cm2) on liquid cycle. Allow the solution to cool to 60 oC or less, and
then add 100 mL of a sterile solution of 0.17M KH2PO4, 0.72M K2HPO4 (this is the
solution resulting from dissolving 2.31 g of KH2PO4 and 12.54g of K2HPO4 in 90 mL of
deionised H2O. After the salts have dissolved, adjust the volume of the solution to 100
mL with deionised H2O and sterilise by autoclaving for 20 minutes at 15 psi on liquid
cycle).
73
Nutrient Agar:
Composition (g/L)
Peptone 5.0 g
NaCl 5.0 g
Yeast extract 2.0 g
Beef extract 1.0 g
Agar 15.0 g
Dissolve in distilled water to a final volume of 1 L, autoclave for 15 min at 121oC, and
pour into sterile Petri dishes.
TY Agar
Composition (g/L):
Tryptone 5.0 g
Yeast Extract 3.0 g
CaCl2 0.5 g
MgSO4 0.1 g
Agar 13.0 g
Add distilled water to a final volume of 1 L, autoclave for 15 min. at 121 oC, and pour
into sterile Petri dishes.
Composition (g/L):
Tryptone 10.0 g
Yeast Extract 5.0 g
NaCl 5.0 g
Glucose 1.0 g
Methylene Blue 0.02 g
Agar 15.0 g
74
Dissolve in distilled deionised H2O to a final volume of 1 L, autoclave for 20 minutes at
15 psi (1.05 kg/cm2) on liquid cycle, and pour into sterile Petri dishes.
Composition (g/L):
Tryptone 10.0 g
NaCl 5.0 g
Glucose 1.0 g
CaCl2 0.11 g
Agar 6.0 g
Add 3 mL of NaOH per L and check for a pH of 7.2. Add agar, dissolve, then autoclave
for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle.
Composition (g/L)
Peptone 10.0 g
NaCl 5.0 g
Yeast Extract 5.0 g
For solid medium:
Agar 15.0 g
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Yeast Peptone Dextrose Medium (YPD; YEPD)
Composition (g/L):
Composition (g/L):
Autoclave 20 minutes on liquid cycle (121 oC; 15 psi). Dispense into sterile test tubes to a
final volume of 5 mL per tube. Note, could be dissolved by heating first, dispensed, then
the tubes autoclave to sterilise.
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Eosin Methylene Blue Agar:
Used for selection of Gram negative bacteria, and differentiation of lactose fermenting
organisms.
Composition (g/L):
Peptones 10.0 g
Di-potassium hydrogen phosphate 2.0 g
Lactose 5.0 g
Sucrose 5.0 g
Eosin Y, yellowish 0.4 g
Methylene blue 0.07 g
Agar 15 g
Dissolve in distilled water to a final volume of 1 L, autoclave 15 min at 121oC, and pour
plates.
MacConkey Agar:
Used for selection of Gram negative bacteria, and differentiation of lactose fermenting
organisms.
Composition (g/L):
Peptone 20.0 g
NaCl 5.0 g
Lactose 10.0 g
Bile salts 5.0 g
Neutral red 0.075 g
Agar 12.0 g
Dissolve in distilled water to a final volume of 1 L, autoclave 15 min at 121oC, and pour
plates.
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Casein Agar:
Composition (g/L):
Agar 10.0 g
Skim milk 100.0 g
Add distilled water to a final volume of 1 L, autoclave for 15 min at 121 oC and pour into
sterile Petri dishes.
Indole Broth:
Used for the differentiation of organisms that can metabolise tryptophan to produce
indole.
Composition (g/L):
K2HPO4 15.65g
L-Tryptophan 5.0 g
NaCl 5.0 g
KH2PO4 1.35g
Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, and autoclave
15 min at 121oC.
Used for the differentiation of bacteria based on acid production (methyl red test) or
acetoin production (Voges Proskauer reaction).
Composition (g/L):
Glucose: 5.0 g
KH2PO4 5.0 g
Pancreatic digest of casein 3.5 g
Peptic digest of animal tissue 3.5 g
Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, and autoclave
15 min at 121oC.
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Simmons Citrate Agar:
Used for the detection of citrate degradation by microbes. Organisms able to utilise
citrate exhibit growth and the medium changes from green to blue. Citrate negative
organisms do not grow and the medium remains green.
Composition (g/L):
Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, autoclave 15
min at 121oC, and cool in the slant position.
Urea Broth:
Urea broth supports the growth of microorganisms that can utilise urea as their sole
carbon source. Organisms that are able to metabolise urea change the incorporated
indicator to red.
Composition (g/L):
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Sucrose Agar:
Used for differentiation of bacteria based on their ability to produce dextran – a polymer
of sucrose.
Composition (g/L):
Dissolve in distilled water to a final volume of 1 L, autoclave for 15 min at 121oC, and
pour into sterile Petri dishes.
Litmus Milk:
Used for the maintenance of lactic acid bacteria and the differentiation of bacteria based
on their action in milk.
Composition (g/L):
Dissolve in distilled water to a final volume of 1 L, heat to boiling and dispense into test
tubes. Autoclave for 20 min at 115oC.
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Motility Medium S:
Composition (g/L):
References:
Atlas, R.M., and Parks, L.C. 1993. Handbook of Microbiological Media. CRC Press, Inc.
Boca Raton, Florida.
Difco Manual: Dehydrated Culture Media and Reagents for Microbiology. 10th Ed.
(1984). Difco Laboratories, Detroit, Michigan.
Ross, H. 1992/3. Microbiology 241 Lab Manual. University of Calgary Press, Calgary.
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REAGENTS:
Ethanol, 70%:
Barritt’s Reagents:
Gram’s Iodine:
Kovac’s Reagent:
Nigrosin Solution:
Add 10 g of nigrosin (water soluble) to 100 mL of distilled water. Boil for 30 min, and
add 0.5 mL of formaldehyde (40%). Filter twice through double filter paper. Store under
aseptic conditions.
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Oxidase Test Reagent:
Phloxine B:
Safranin:
Dissolve 0.3 g of Sudan Black in 100 mL of 70% ethanol. Shake before each use.
References:
Clark, G. (1984) Staining Procedures. 4th Ed. Williams and Wilkins, Baltimore, Maryland.
83
pH INDICATORS:
84
APPENDIX 9
Care and Feeding of the Microscopes
85