Applied Microbiology Fall 2015

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CONTENTS
Schedule…………………………………………………………………………………………….1
Introduction…………………………………………………………………………………………3
Overview of the Laboratory
Grading
Attendance
Best Laboratory Practices
Guidelines for Laboratory Notebook
Guidelines for Lab Reports
Exercises
1: Microscopy ………………………………………………………………………………………..7
2: Isolation of Actinomycetes……………………………………………………………………...10
3: Effect of Temperature on Growth……………………………………………………………...12
4: Evaluation of Commercial Yeast Products…………………………………………………...17
5. Microbial Food Spoilage………………………………………………………………………..21
6. Isolation of Salmonella………………………………………………………………………….23
7. Water Analysis…………………………………………………………………………………..26
8. Yeast Metabolism………..………………………………………………………………….…..31
9. Preparation of a Malt Beverage…………………………………………………………….…..35
10. Comparison of Microbial Populations: RFLP Analysis of 16s DNA……………………….39
11. Kirby Bauer Method…………………………………………………………………………….42
Appendix A: Culture Collection……………………………………………………………………45
Appendix B: Media Ingredients……………………………………………………………………46
Appendix C: Gram Staining………………………………………………………………………..52
Appendix D: Quadrant Streak Plating ……………………………………………………………53
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LABORATORY SCHEDULE
Faculty Coordinator: Dr. Ines Rauschenbach

inesrau@aesop.rutgers.edu
Teaching lab: 848-932-5418
Date Topic Exercise

Introduction to Lab/Biosafety
Principles of Microscopy 1
Sept. 14
Isolation of Actinomycetes 2
Microbiological Techniques - Review Appendix
Results: Isolation of Actinomycetes
Sept. 21
Effect of Temperature on Growth 3
Results: Effect of Temperature on Growth
Sept. 28
Evaluation of Commercial Yeast Products 4
Results: Evaluation of Commercial Yeast Products
Oct. 5
Microbial Food Spoilage 5
Results: Microbial Food Spoilage
Oct. 12
Isolation of Salmonella on Hektoen, XLD Agar & Bismuth 6
Results: Identification of Salmonella
Identification of Salmonella – TSI 6
Oct. 19
Microbial Source Tracking Water Analysis: Microbial 7
Indicators of Fecal Contamination
Results: Identification of Salmonella – TSI
Oct. 26 Results: Microbial Source Tracking Water Analysis:
Microbial Indicators of Fecal Contamination
Yeast Metabolism – Inoculation 8
Nov. 2 Preparation of a Malt Beverage 9
Midterm Quiz
Results: Yeast Metabolism (Analysis of Cultures)
Nov. 9
Beer (Rack and Bottle)
Results: Yeast Metabolism (Analysis of Data)
Comparison of Microbial Populations (Preparation of 10
Nov. 16 samples and set up PCR for RFLP analysis)
Culture Collection: Gram Stains & Wet Mounts, Appendix
Differential and Selective Media
Set Up Restriction Digests of PCR Products 10
Kirby Bauer Method 11
Nov. 23
Culture Collection – Finish: Gram Stains & Wet Mounts, Appendix
Differential and Selective Media
Results: Kirby Bauer Method
Electrophoretic Analysis of Digests 10
Nov. 30
Culture Collection – Finish: Gram Stains & Wet Mounts
Appendix
Microbial Products
Dec. 7 Culture Collections Due
Final Quiz
Applied Microbiology Lab Website: http://www.rci.rutgers.edu/~microlab/
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OVERVIEW OF APPLIED MICROBIOLOGY LABORATORY
Students taking this class are expected to have already mastered the basic laboratory
techniques that will enable them to work efficiently and safely in a microbiology
laboratory. They will know, understand, and use the concept of "aseptic technique",
which is fundamental to working with microorganisms. In this course, students will
augment these basic concepts and develop and improve their laboratory techniques
through exercises and experiments in a variety of areas including mycology, microbial
physiology, microbial biochemistry, and applied immunology as they relate to applied
microbiology and microbial biotechnology in the areas of food, environmental, industrial
and medical microbiology. Early exercises will focus on honing traditional skills such as
microscopy and developing techniques for measurement of cell populations. The
kinetics of microbial growth and thermal death will be demonstrated experimentally.
Some time will be devoted to the study of traditional food and beverage fermentations as
the progenitors of modern industrial microbiology and biotechnology. Later exercises
will explore the use of immunological tests and gene probes for detection of microbial
cells and toxins. The compilation of data and proper keeping of laboratory notebooks
will be stressed. The overall goal is to provide students with a wide variety of
experiences which will help equip them later for working with microorganisms in
research or testing laboratories.
Applied Microbiology Website

The URL for the lab web site is http://www.rci.rutgers.edu/~microlab. Use the
“schedule” link on the site to find relevant information. Date links on the for each week’s
experiment(s). This information will be useful for your pre-lab preparation as well as
supplying a visual tool to aid in understanding the concepts of the lab.
Required Laboratory Notebook

You will be using LabArchives to keep all your notes and quizzes.
Grading
Laboratory performance will count 25% of the final course grade. The laboratory grade
will be based on 500 points, distributed approximately as follows:
Culture Collection 100 points

Notebook 150 points
Written reports, 2 @ 50 each 100 points
Weekly Quizzes 100 points
Midterm Quiz 50 points
Final Quiz 100 points
Total 600 points
Attendance
You are expected to attend every lab. We understand that there are unforeseen
circumstances or emergencies that prevent you from attending. If you need to miss lab,
you are expected to immediately contact your instructor and TA and provide appropriate
documentation for your absence. For more information regarding attendance, check the
course website (http://www.rci.rutgers.edu/~microlab.)
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BEST LABORATORY PRACTICES
We will maintain safety standards at the highest level. The following list of best practices
is printed here to remind you of the safety discussion presented during the first
laboratory meeting. If you have further questions please see the website for more
information.
• Do not bring food or drinks into the laboratory - EVER!

• No sandals – toes must be covered.
• Long pants and skirts must be worn at all times.
• Lab coats are required. (Must be purchased in this lab!)
• Safety glasses/goggles are mandatory and provided.
• Long hair must be tied back.
• Always use aseptic technique when working with organisms in culture.
• Wash your hands before leaving the laboratory.
• Place coats, books, and other personal possessions in assigned cabinet.
• Cleanliness: Before the beginning and at the end of your laboratory work, wipe
down the benchtop with a microbiocide (e.g. Staphene - 1%).
• Instructions for disposal will be provided during your lab period. NEVER THROW
ANYTHING DOWN THE SINK DRAIN. The following will help when you are
unsure:
General Instructions for disposal:

• Microscope slides and cover slips ONLY go in the red “sharps” containers
located at intervals along benches.
• Place used pipettes (tips down) and all other non-glass waste in labeled
plastic buckets on bench.
• Old media are disposed of in the fume hood at the front of the lab; used Petri
dishes go in the autoclave bag placed in the bin; used culture tubes in the
test tube racks.
• Pour excess stains into hazardous waste containers provided near the sink in
the front of the lab.
• Your Teaching assistant will remind you where to dispose of items as they
are used.
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LABORATORY NOTEBOOK GUIDELINES
Your notebook is an account of what you do in lab each week. If you work in a lab,
whether an industrial lab or a research lab it is critical that you keep an accurate and
complete notebook. The information in your notebook will be what you use to write your
formal lab reports. Just as the information in a lab notebook provides the information for
journal articles and/or patents.
1. Take down all pertinent information such as changes in procedure, observations, etc.
Make your entries clear and intelligible, so that when you look at your notes again in a
month or two you will still understand them.
2. Although artistic ability does not count, your drawings should be clear and reasonably
large and should show the important structural features of your specimen, the
approximate shapes, relative sizes and relationships among the parts. Also, include with
your drawings information on size, color, and texture of the specimen as well as the
magnification used. This information is also important when taking photomicrographs.
3. Results/observations should be labeled clearly with the conditions under which they
were observed (elapsed time, type of treatment, etc.)
4. Do not hesitate to include anything you think is important or interesting in your

notebooks. If you have a question regarding your notebooks please ask your instructor.
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GENERAL INSTRUCTIONS FOR LABORATORY REPORTS
Following are some basic guidelines for writing the formal laboratory reports that will be
required this semester. We expect them to be of good scientific merit and grammatically
correct. In addition, they should be brief but complete, generally no more than three
pages.
1. Abstract. Although the abstract is the first section, it is written AFTER the report is
finished. It provides a brief (one paragraph) overview of the key facts presented in your
report.
2. Title. The title should briefly and accurately describe the experiment so that even
someone who has not done the experiment would understand what it was about.
3. Introduction. The introduction should contain some background material to the

experiment, why the problem or topic is significant, a purpose for doing the experiment,
and your objectives. You should state your hypothesis in this section.
4. Materials and Methods. This section need not be very long, and in describing the
METHODS you may assume the reader has some basic knowledge of microbiology (i.e.
knows what a loop is used for, how to sterilize, etc.). You need not copy the procedure
from the laboratory manual, but any deviations from that procedure and in the materials
or equipment used should be noted. The reader should be able to reproduce the
experiment from the information in your report together with the lab manual.
5. Data and Results. The data should be presented in logical progression. For clarity of
presentation, graphs and tables should be used as often as possible; however, tables
and graphs require descriptive text to draw the reader to important points and clarify the
data presented. Please show all calculations. If the same calculation needs to be
performed on different sets of data, then one sample calculation will suffice.
6. Discussion and Conclusion. This is the most important section of your report. The
discussion is not a summary and should not be a restatement of your results. This
section should convince the reader of the support or rejection of your hypothesis.
Readdress your hypothesis and explain whether it has been supported or not and
describe how you interpreted your results to reach your conclusions. The discussion
should discuss your results within the context of the known literature on this topic. It
may include suggestions about modifying or extending the experiment to get additional
information or clearer results. It may also include assumptions as to why expected
results were not observed. This section need not be lengthy, but it should be relevant.
7. References. Include any references that were used in writing your report. Do not
forget to include your laboratory manual and lecture notes as reference. Your instructor
will discuss the format (FEMS) the references must take.
8. Grading. Your report will be graded on presentation and interpretation of results, not
the results themselves.
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1. MICROSCOPY
The light is one of the most important instruments available for the study of
microorganisms. Modern light microscopes can provide considerable information about a
specimen to a trained observer.
The quality of the observed image depends on three important factors:

1. magnification – enlarging the appearance of an object
2. resolution – ability to distinguish two points as separate
3. contrast - difference in visual properties making one object distinguishable from
other objects
Achieving maximum information from an observed image requires a clear understanding

of the adjustments required for optimal alignment of the various components in the
optical system. This laboratory exercise provides intermediate microbiology students
with an opportunity to develop their skills as microscopists.
Each student will be assigned the use of a binocular light microscope equipped for
both bright field microscopy and phase contrast microscopy. These microscopes
are expensive instruments and should be treated with care. Remember bright field
microscopy will be used mainly for viewing stained specimens and phase contrast
will be used for observing unstained cells directly in wet mounts.
EQUIPMENT AND MATERIALS

• Binocular microscope with
• Glass slides and cover slips
• Suspension of commercial baker's yeast and bacterial suspensions
• Phase contrast/bright field condenser
PROCEDURE
A. General Comments on the Use of the Microscope
1. The intensity of the lamp is controlled by a power supply, built into the microscope -
never adjust light intensity with the condenser or field diaphragms.
2. Select objective lenses by rotating the nosepiece. Make sure that the lens clicks
into position.
3. The binocular head can be adjusted for inter-pupillary distance by sliding the
eyepieces towards or away from each other.
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4. If your vision is very different in each eye, look at the image with your right eye
through the right eyepiece and focus with the fine adjustment; then without further
change of the fine adjustment, look at the same image with your left eye through the left
eyepiece and focus by rotating the diopter ring at the base of the eyepiece. This
procedure will apply approximately the same correction to your vision as your glasses
do. Write these settings in your notebook so you know your correct diopter settings.
B. Adjustment for maximum resolution at 100x magnification under bright field

microscopy using Köhler illumination.
1. Turn the rotating condenser to the 0 position as indicated by the mark on the
microscope.
2. Plug in and turn on your microscope, open field diaphragm, raise condenser to its
highest position, look through objective, and adjust light intensity by using the voltage
control knob.
3. Place a slide marked with a wax pencil “X” on the stage. Center the X under the
objective using controls for moving the mechanical stage, and focus on the X image
using coarse and fine adjustments.
4. Focus the image of the lamp diaphragm in the same plane as the specimen as
follows: While looking at the image, slowly stop down (close) the field diaphragm. One
should see the leaves of the field diaphragm closing in around a brightly illuminated
circular area. If the circular area is not centered, proceed to step 5. If the image of the
field diaphragm is blurred, adjust the height of the condenser slightly up or down by
turning the condenser height adjustment knob until the edge of the circular area is in
sharp focus. The image of the wax pencil mark may interfere with this operation. If so
move the mechanical stage so that the image is just outside of the field of view, but do
not change the focus.
5. Center the image of the field diaphragm in the middle of the field of view by turning the
condenser centering screws. Focus on the iris diaphragm (i.e. edge of the illuminated
circular area) as in step 4. While continuing to look through the eyepieces, open the field
diaphragm until the illuminated circular area just fills the field of view.
6. Adjustment of numerical aperture. Remove one ocular and look down the tube. Close
or open the condenser diaphragm until approximately l/5th of the field of view is
obscured. NOTE: This operation can be confusing because the ring on the phase plate
of the condenser or the color of the wax pencil can obscure the field. Check with your
instructor. If in doubt, open the condenser diaphragm all the way and stop down just until
the light starts to dim.
7. The microscope is now adjusted for maximum resolution at l00x magnification (10x
objective x 10x ocular = 100x). If one continues to observe slides at this magnification,
no further adjustments are usually necessary other than focusing on the same or a
different slide. However, if you go to a higher magnification, the opening of the
condenser diaphragm must be changed to conform with the smaller field diameters of
the higher objectives and you may again have to focus the image of the lamp diaphragm
so that it is in the same plane as the image of the specimen.
C. Adjustment for maximum resolution at 400x.
1. Revolve the nosepiece until the 40x (high dry) lens clicks into position.
2. Lenses are parfocal, though fine adjustment focusing may be required.
3. Focus on the edges of the field (lamp) diaphragm, center, and open as in steps 4 and
5 above and adjust the numerical aperture as in step 6 above.
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D. Mounting and observation of yeast cells.
1. Place a loopful of culture or the suspension provided on the slide (Note: it is

extremely important not to add too many cells and that the amount of liquid is kept at a
minimum).
2. Place a cover slip on the drop and press down gently. The suspension should fill no
farther than the edge of the cover slip. If it floats around, too much liquid has been
added.
3. Place the slide on the microscope stage and lower the 40x objective until it almost
touches the cover slip.
4. While looking through the microscope, focus upward with the fine adjustment knob
until the yeast cells come into view. Note how the cells appear and compare them with
the cells as they appear under phase microscopy as described in section F.
5. Rotate the knurled part of the condenser so that the number 40 coincides with the
mark on the side or front of the microscope.
6. Observe the yeast cells and describe the differences in the image between the cells
as they appear under bright field vs. under phase contrast microscopy.
E. Microscopic examination of a bacterial suspension by using the 100x oil

immersion lens.
1. Aseptically prepare a wet mount of a bacterial culture and examine it under phase
contrast microscopy using the oil immersion lens at 100x.
2. Critically examine this preparation and make a drawing which describes the things
you see. The purpose of this exercise is not only to test your skills at being able to set
up the microscope properly, but also to test your abilities to observe and report your
findings in a clear and logical manner. Therefore, take your time, look at various
microscopic fields, and concentrate on what you are seeing.
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2. Isolation of Actinobacteria
The phylum Actinobacteria is a large group of Gram-positive bacteria. They form

branching filaments that somewhat resemble fungal mycelia, which resulted in their
classification under the former name, Actinomycetes. Actinobacteria are most commonly
found in fresh and marine waters, as well as the soil and play a vital part in the
decomposition or organic material and the carbon cycle. This replenishes the nutrients
and influence humus formation.
One important genus belonging to Actinobacteria is the Streptomyces. These spore-

forming bacteria are soil dwellers and are characterized by leathery, pigmented,
filamentous colonies. Streptomyces are chemoheteroorganotrophs grow best at 25°C at
pH 8-9. Because of their ability to break down complex organic materials, they are
important in agriculture for the production of fertile soil. You can even smell them, the
break down of the organic materials produce volatile compounds called geosmins, which
give soil its distinct earthy flavor and aroma.
In this experiment, we will be collecting soil samples and enrich for this group of
organisms. Once isolates are identified and in pure culture, you will have the opportunity
to test their antibiotic production against known organisms and the organisms in your
culture collection. For your isolation of Streptomyces, dried soil will resuspended in
buffered saline and plated directly onto Actinomycete Isolation Agar (AIA). This medium
is used for isolation and propagation of Actinomycetes. It contains the following
ingredients:
Sodium propionate used as a substrate in fermentation and to inhibit molds
Dipotassium phosphate provides the buffering system.
Sulphates - serve as source of sulfates and metallic ions.
Glycerol - serves as an additional source of carbon.
Materials
Soil sample
3 Saline dilution blanks (9 ml)
6 Actinomycete Isolation Agar (AIA) plates
Broth cultures of 5 test organisms
Mueller Hinton Agar
PRETREATMENT
The soil samples have been pretreated with CaCO3 (10:1 w/w) and incubated at 37°C for
4 days. The samples were then suspended in sterile Ringer solution (1/4 strength) and
diluted 1:100. The samples were then placed in a water bath at 45°C for 16 hours so that
the spores would separate from the vegetative cells.
PROCEDURE – Week 1
1. Work with a partner and inoculate the treated soil onto AIA agar.
2. Incubate the plates at 28°C for 5-7 days.
1. Examine the AIA plates and look for typical Streptomyces colonies. They are small,
opaque, compact, frequently pigmented (brown, yellow, pink, etc.), often leathery, and
appear dry and dull looking. Sometimes, a depression in the agar surface will be
observed around the colony.
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2. Prepare a wet mount and observe with phase contrast microscopy. The wet mount
should reveal a multitude of spores with a few filamentous cells.
3. Select two potential Streptomyces colonies and quadrant streak each for isolation
onto AIA. Incubate the plates at 28°C for 5-7 days.
1. Pick a well-isolated colony from each plate and streak onto TSA slants. Incubate the
plates at 28°C for 5-7 days and then store in the refrigerator for further use.
PROCEDURE – Antibiotic Testing for your Unknowns

1. Obtain two Mueller Hinton Agar Plates and cross-streak one Streptomyces isolate per
plate (down the middle of the plate – see Figure 7.1). Once streaked with
Streptomyces, use a look to inoculate your four test cultures. Start each streak near
the edge of the plate and streak toward the Streptomyces streak in the middle of the
plate. Finish the streak near, but not touching, the Streptomyces streak. Do not
reverse the direction of the streak and only streak one time!
2. Incubate at 28°C for 2 days, the store in the refrigerator. Observe for antibiotic
activity.
1. Use loop to streak Streptomyces #

down the middle of the plate.
2. Use loop to streak the test

organisms from the outside
of the plate toward the
middle. Only streak once.
Do not touch the area where
you inoculated
Streptomyces.
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3. EFFECT OF TEMPERATURE ON GROWTH
Many complicated changes occur during the course of bacterial growth, but under
favorable conditions the sequence of events generally follows an orderly pattern. The
growth of a population of cells in culture in the laboratory can be divided roughly into four
distinct phases: a lag phase, an exponential (log) phase, a stationary phase, and a death
phase. These phases are best illustrated by a growth curve in which the log of the
number of organisms is plotted against time.
Environmental factors influence the course of population growth. One of the most
important of these factors is the food supply, which is necessary for the formation of new
cells. In addition, physical factors of the environment, such as temperature, acidity (pH),
water activity (aw), oxidation-reduction potential, surface tension, and other factors affect
the growth pattern.
The growth sequence of bacterial populations, though fairly well defined under a given
set of conditions, cannot be represented mathematically by a simple equation. Growth
during the exponential phase, however, appears to follow first-order kinetics, as might be
expected of single cell organisms such as bacteria and yeasts that multiply by simple
fission or budding. The increase in number of cells during this period can be expressed
with the equation for a first-order reaction as follows:
Growth rate equation
µ = LogODn - Log ODo x 2.303 Slope = µ

tn - to 2.303
µ = growth rate constant

OD = number of cells at time n and 0
t = time in min
(2.303 is the conversion factor between natural log (ln) and base 10 log (log10): 2.303 x
log10 = ln)
During the lag phase of growth immediately preceding the exponential phase, little or no
increase in number of cells occurs; and in fact there may be a slight decrease. The
length of the lag phase can be decreased by increasing the size of the inoculum or by
making the conditions more favorable for growth, but it cannot be eliminated completely.
The stationary phase follows the log phase. During this phase the number of living cells
in the population remains more or less constant, with the number dying equaling the
number being produced. Limited sources of energy and nutrients can bring about the
stationary phase, but some factor or factors that limit populations even in the presence
of an adequate food and energy supply may be involved.
It has long been known that increasing the temperature increases the rate of chemical
reactions. Arrhenius developed the mathematical expression to describe this effect, as
follows:
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Arrhenius equation
Log µ = - H +C
2.303 RT
µ = growth rate constant

H = activation temperature
R = the gas constant
T = temperature in degrees Kelvin
C = the intercept
A plot of log of the reaction rate (µ as a function of 1/T gives a straight line with a
negative slope (- H/2.303R) and an intercept of C. The slope of this line is proportional to
the heat of activation and it is characteristic of a given reaction.
Though more complex, the kinetics of bacterial growth in exponential phase appears to
be similar to that observed with chemical reactions in the way the growth rate responds
to an increase in temperature. This observed relationship is empirical, however, and has
no theoretical basis in such complex biological systems.
If the Arrhenius equation is applied to bacterial population growth, a plot of growth rate
as a function of temperature yields a linear curve over only that part of the temperature
range permitting growth of the organism. The reason for this is that the growth rate of
any species falls off abruptly at both the upper and lower limits of the temperature range.
The abrupt fall-off at high temperatures occurs as cellular proteins essential for cellular
activity become denatured. In addition, cell structures such as membranes are destroyed
by relatively high temperatures. The maximum temperature for growth is that
temperature at which destructive reactions overwhelm the cell. This temperature is
usually only a few degrees higher than the temperature at which growth rate is greatest,
the optimum growth temperature. At the colder end of the temperature range, most
microorganisms stop growing at a point (minimum growth temperature) well above the
freezing point of water. Microorganisms can be categorized roughly as thermophiles,
mesophiles, or psychrophiles based on the temperature range over which growth
occurs.
Limiting or increasing the size of microbial populations by controlling the temperature of

their environment has many practical applications. Low temperatures, for example,
where the growth rate is much reduced, can be used to retard spoilage of food by
slowing the growth of spoilage organisms. On the other hand, temperatures close to the
optimum for growth would be employed where the production of cells or a cell product is
the goal. In this exercise you will observe the effect of temperature on the growth rate of
a mesophilic organism, Escherichia coli, in exponential phase. Growth will be measured
in a spectrophotometer as optical density (OD) at intervals over several hours, and at
several different temperatures. Growth of cultures can be monitored by
spectrophotometry because the number of photons scattered is proportional to the mass
of cells in a sample. If the cells are in exponential phase and the concentration of cells is
not too high, OD readings will correlate quite well with cell number since cells in this
phase are likely to be similar in size and shape.
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We will use the increase in optical density of a culture as a measure of growth and plot
these data as a growth curve. There are many methods that can be used to accomplish
these measurements. We will compare three variations in experimental design.
MATERIALS & EQUIPMENT

• A young culture of Escherichia coli, in exponential or early stationary phase
• 10 ml pipette
• One culture tube for the uninoculated medium blank
• Spectrophotometer
• Water baths set at 25o, 35o, 45o, 50oC.
PROCEDURE #1
• One 125 ml culture flasks, containing 75 ml trypticase soy broth (TSB)
• A set of 10 new culture tubes for (one tube for each time interval)
PROCEDURE #2
• One 250 ml culture flask containing 75 ml TSB
• One new culture tube to use as cuvette for all time points.
You will work in pairs. Each pair will work at one temperature and complete the two
variations in experimental design at that temperature. This may vary depending on the
number of students in your section.
PROCEDURE #1
1. Remove a 5 ml sample from the uninoculated 125 ml flask of TSB; set this sample
aside as an uninoculated medium blank. Set the wavelength of the spectrophotomer to
660 nm.
2. Inoculate the 125 ml culture flask (containing TSA) with 2 ml of the E. coli culture.
Swirl the flask gently to mix thoroughly and immediately pipette 5 ml amounts into each
of the ten tubes. Label the tubes 1-10 with a Sharpie.
3. Blank the spectrophotometer using the tube of uninoculated culture broth as the
reference. Place the 10 tubes in a water bath/incubator at your designated temperature.
Remove tube #1 immediately. Wipe the tube with a Kimwipe, place the tube in the
spectrophotometer and read the OD660. Record this number as the zero time reading in
a table similar to that shown below.
4. At each designated sampling time remove another tube from the bath/incubator, mix
to suspend the cells, wipe the tube with a Kimwipe and place the tube in the
spectrophotometer. Read and record the OD660 and the time.
PROCEDURE #2
1. Inoculate the 250 ml culture flask with 2 ml of the E. coli culture. Swirl the flask gently
to mix thoroughly and immediately remove a 5 ml sample to your “cuvette”. Read and
record the OD660. This is your time 0 measurement.
2. Repeat step #1 at each time interval.
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GRAPHING AND CALCULATIONS
Data Tables – Procedure 1
Time Temperature (°C)

(min) 25 35 40 45 55
0
15
30
45
60
75
90
105
120
Data Tables – Procedure 2
Time Temperature (°C)

(min) 25 35 40 45 55
0
15
30
45
60
75
90
105
120
1. After all OD readings have been recorded in the table, plot log OD versus time in min.
Share data with your classmates and plot a separate curve for each test temperature.
As an alternative, plot OD versus time on two-cycle semilog paper. We will discuss
semilog paper but if you are unfamiliar with it, checkout the explanation linked to the lab
website. From each curve, identify the exponential phase of growth as the best straight
line drawn from points on the curve that delineate the section between the lag phase and
early stationary phase. Repeat the graphing for each of the three procedures. Graphs
for procedure #1 should be done on paper. You may use the computers for the other
graphs.
2. Calculate a growth rate constant (µ) for each temperature from the equation given on
the first page of this experiment.
Values for ODn and ODo should be read directly from the graph. Use any segment from
the exponential growth phase (straight line portion) for both the OD and t values.
3. From the curves, determine a generation time for the culture at each test temperature.
In this exercise, generation time will be the time in minutes required for the cell mass to
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double and can be read directly from the graph. Alternatively, calculate the generation
time (g) at each temperature from the expression:
g = 0.693
µ
4. Plot an Arrhenius curve, with log µ versus 1/T (Kelvin). As an alternative, simply plot
µ versus T.
QUESTIONS
1. Did the variation in procedure change your results for the Escherichia coli growth
curve? Explain.
2. Discuss the errors inherent in each procedure used in this exercise. Which
procedure do you think is best? What factors are important in choosing the best
procedure?
3. If a food is considered spoiled when the bacterial count reaches 1 x 109 cells per g of
food, calculate the time it would take for spoilage to occur under the following conditions:
Initial count l x 104 cells per g; temperature, 20o; generation time, 1 hr. Assume growth is
in exponential phase.
4. What other variation in procedure might be included? How would that change the
results?
5. If the temperature of storage is raised to 30oC, would it take longer for the food to
spoil? Explain.
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4. EVALUATION OF COMMERCIAL YEAST PRODUCTS: MICROBIAL POPULATION
COUNTS
Several methods can be used to determine the size of microbial populations. Generally
these methods are based on one or more of the following types of measurements:
Cell mass: Cell mass can be determined directly by weighing dried cells. If the
microorganism is single-celled, the density of a culture or cell suspension can be
determined rather quickly with a spectrophotometer. If the cells are assumed to be all of
the same size and shape, then a standard curve can be plotted relating optical density
(OD) to cell number. Sometimes a relatively constant cell component, such as nitrogen,
is determined and correlated with cell number. In a later exercise, you will prepare a
standard curve relating cell number to optical density. We will not use cell mass
determination in this exercise.
Cell counts: Cells can be counted directly either under the microscope or with an
electronic particle counter; viable cells in a population can be counted indirectly by
colony count on an appropriate nutrient agar plate. We will be counting cells under the
microscope as well as making viable cell counts.
Cell Products: The concentration of a specific metabolic product can be measured and
correlated with cell number by reference to a standard curve. We will be measuring CO2
production.
To demonstrate several methods for counting microbial populations and assess their
application to evaluating commercial products, this exercise will have three parts.
1. Determination of the total number of cells per unit weight in one of several
commercial baker's yeast products.
2. Determination of the number of viable yeast cells in the same product as well as
the level of contamination (if any) with coliform organisms.
3. Determination the relative rate of carbon dioxide production by yeast cells from a
glucose substrate.
From these data, both your own and that of other students, you will be asked to
determine the "best value" among the various yeast products tested.
A. TOTAL CELL COUNT

Direct microscopic methods can be used to count microbial cells quickly and simply. The
chief drawbacks of this method are that the dead cells cannot be distinguished from the
viable cells in a sample and thus both are counted. Also, a relatively dense population is
needed for an accurate count. Direct counts are also possible with electronic particle
counters (such as the Coulter Counter), but this type of equipment is expensive and not
always readily available.
In this exercise a microscopic direct count will be made in a Levy counting chamber to
determine the number of yeast cells in a volume of a suspension prepared from a yeast
product. (This counting chamber was designed for counting red blood cells, which are in
the same size range as yeast cells.)
18
MATERIALS
• Several commercial yeast products, both dried and fresh
• Levy counting chamber & Microscope
• Pasteur pipettes
• Tap water and Erlenmeyer flask
PROCEDURE
1. Suspend 0.1 g of yeast, dry granules or wet cake, in 100 ml of tap water. Shake the
suspension vigorously to distribute the cells evenly.
2. Place a coverslip over the calibrated surface of the Levy counting chamber. Use the
special coverslip found in the box with the counting chamber. Do not discard this
coverslip.
3. With a Pasteur pipette, transfer a drop of the cell suspension to the edge of the
coverslip. Let capillary action pull the suspension underneath the coverslip.
4. Scan the ruled area of the counting
chamber under 100x total magnification.
Identify the 25 large squares (5 x 5) in the
center of the ruled area. Each of these 25
"large" squares is subdivided into 16
small squares. If you are unsure about the
large and small squares take another look
at the pictures on the web site. The
small squares are those referred to in
the calculations to follow.
5. Count the yeast cells in 80 small squares. To

do this most efficiently, count 5 large squares - each
corner square and one in the center. Each large
square is made up of 16 small squares so once you
have counted 5 large squares you have counted 80
small squares (5 x 16 = 80). If cells fall on a line count
those on the top and left-hand lines only. Use phase
contrast microscopy and 400x magnification. The
illustration below shows cells distributed in one of the
large squares, subdivided into 16 small squares.
6. Calculate the average number of cells per "small" square, i.e. divide the number of
cells counted in the 5 "large" squares by 80. One small square is a cube (with the
coverslip forming the top of the cube) and has dimensions of 1/20 x 1/20 x 1/10 mm so
the volume of this cube is equal to:
1/20 mm X 1/20 mm X 1/10 mm = 1/4000 mm3
So now we know the volume of liquid above each of the small squares is 1/4000 mm3.
Remember your conversions: 1 ml = 1 cm3. How many milliliters does 1/4000 mm3
represent? Since we are talking about cubic dimensions, 1 cm3 = 1000 mm3.
19
7. Calculate the number of cells per ml of suspension. If "n" is the average number of
cells per small square (1/4000 mm3), then the number of cells per ml of suspension is "n"
x 4000 x 103.
8. Calculate the number of cells per liter of suspension, i.e. per gram of original product.
(Remember in step #1 you suspended 0.1 g of yeast in 100 ml of tap water. This is
equivalent to 1 g/liter.)
9. From the label find the total weight of the product, and from this calculate the number
of cells in the package. Finally, calculate the price per cell or per gram as instructed.
B. DIFFERENTIAL PLATE COUNTS
In this part of the exercise, determine the number of viable cells in the yeast cell
suspension prepared in part A above. Remember that in a direct count under the
microscope living cells as well as dead cells will be counted. A useful product, such as a
dried yeast preparation or a fresh cake, depends upon the metabolic activity of living
cells. Thus, with baker's yeast we will assume that the product that has the greater
proportion of living cells is the better product.
To determine the number of living yeast cells, prepare and plate a dilution series based
on the direct counts obtained in part A. To aid in evaluating the quality of the product,
use an all-purpose medium such as tryptic soy agar, as well as differential media, such
as mycophil. To detect coliforms that might be present as contaminants, use MacConkey
agar as a differential medium. If many coliforms are found, it may indicate poor process
conditions.
MATERIALS
• Yeast cell suspension from part A
• Buffered water, sterile test tubes
sterile pipettes
• µ-pipettes and sterile tips
• TSA or Trypticase-glucose agar plates
• Mycophil agar plates
• MacConkey agar plates
PROCEDURE
WEEK 1
Draw appropriate dilution schemes for the viable plate counts. Use your results from the
direct plate counts as your starting population size. Plate three appropriate dilutions on
each type of medium. Use the TSA and mycophil agar for the yeast cell count. Use the
MacConkey agar for the coliform count. Plate only the most concentrated dilution on
MacConkey agar (1 plate). Incubate the plates at 30oC for 48 to 72 hours.
WEEK 2
Count colonies on all plates, differentiating between yeast colonies and bacterial
contaminants by the appearance of the colonies. Yeast colonies will be white to cream
colored and rather dense on the TSA and mycophil. On the MacConkey agar, yeast
colonies will be very small; coliform colonies will be red. Record your results in table
form.
20
Based on colony counts, calculate the number of living yeast cells in the suspension.
Compare this count to the total cell count from part A of this exercise. Express the
number of living cells present in the suspension as a percentage of the total number of
cells. Calculate the number of viable cells per gram of product; calculate the number per
package. Calculate the number of coliform contaminants, if any, in the product.
C. PRODUCTION OF CARBON DIOXIDE BY YEAST CELLS
Carbon dioxide is the product of yeast cell metabolism that leavens bread. The strains
used in baker's yeast, variants of Saccharomyces cereviseae, have been selected to
produce large quantities of carbon dioxide. In this part of the exercise, determine the
relative rate of carbon dioxide production by a commercial yeast product, the same one
used in the previous parts of this exercise. Compare your results with those of students
using other products to determine the comparative quality of the product in terms of cost
and leavening action.
MATERIALS
• Commercial dried or cake yeast
• 1 g glucose in 10 ml water
• 50 ml plastic syringe fitted with a luer-lok
PROCEDURE
1. Sprinkle 2 g of yeast cake or 1 g of dry yeast over 8 ml of water in a 50 ml beaker. Mix
thoroughly to a smooth suspension.
2. Add glucose solution to the yeast cell suspension. Mix vigorously for five seconds and
quickly draw the entire suspension up through a large gauge needle into the syringe.
3. Tip the syringe, open the lock, and expel any air above the suspension in the syringe.
Close the lock. Place on tray in incubator @ 30°C.
4. As carbon dioxide is produced, the suspension will become saturated with the excess
forcing the plunger of the syringe out from the barrel. Read the amount of expelled
carbon dioxide from calibrations on the syringe barrel. Take readings every 10 minutes
until the capacity of the syringe is exceeded.
5. Plot volume of carbon dioxide evolved versus

VCO2
time. Normalize rates to 1 g product and compare rates
commercial brands tested in the class. Decide which
product is the "best value". (Determine the amount of
carbon dioxide produced per g, and the cost per g).
Vn – V0
Note: if the rate of CO2 evolution is not linear, compare
instead volumes evolved at a set time, e.g. 20 min or 30 min. Tn – T0
Time
21
5. MICROBIAL FOOD SPOILAGE:
Effect of Improper Handling of Ground Meat - Sampling and Enumeration
Fresh meat provides an almost ideal growth medium for many microorganisms; it has a
neutral pH, abundant moisture, and is rich in nutrients. Prior to slaughter, animal tissues
are generally sterile and most of the microbial contamination of large pieces of meat is
on the surface where contact has been made with knives, work areas, handlers, and
even fecal material. It is difficult to maintain sanitary conditions in slaughter houses, and
in addition to spoilage organisms there is much concern over the possibility of
contamination by pathogens. In ground meat, the contaminating organisms on the
surface become spread throughout the product, which now because of the processing
has a considerably larger surface area. Furthermore, there is the potential that
microorganisms from infected lymph glands will be distributed throughout the product.
Unless steps are taken to stop or slow the growth of these organisms, spoilage will occur
rather quickly. Because of the high protein content of meat, proteolytic bacteria are the
most prominent spoilage organisms, causing deterioration in the texture and color of the
product and generating foul odors.
Although spoilage is slowed by refrigeration, psychrotrophic organisms, present on

almost all meat and meat products, will grow and cause spoilage even at refrigerator
temperatures. At freezing temperatures, these organisms will not grow, and while
deterioration of the product will occur eventually, it is not microbial in origin.
In this exercise you will compare the number of bacteria found in samples of ground beef
stored for one week in the refrigerator (improperly handled) with that of a similar sample
stored in the freezer for the same time period and thawed overnight in the refrigerator
(properly handled). The bacterial counts determined will be compared with control
counts made from a fresh sample of ground meat. The analysis of all the samples,
however, will be limited to those species that will grow in culture on an all-purpose
growth medium at 35°C.
When testing food (or any samples) the method used must be appropriate for the
sample. If we were testing milk (a colloidal suspension) simple dilutions would be easily
accomplished. If the sample were concentrated orange juice our sampling method would
need to be appropriate for a viscous liquid. Ground beef is not only a solid sample but
contains connective tissue making it difficult to work with. We will use a lab blending
device, the stomacher, which will both macerate and mix our sample. The “stomacher”
is so named due to the similarity of its action to that of the stomach in blending food.
MATERIALS
• Properly or improperly handled ground beef samples

• Stomacher and bags
• Bottle of phosphate buffer
• Sterile tubes for dilution
• Week 1: Plates of trypticase soy (TSA); Eosine methylene blue (EMB)

• Week 2: MacConke(MAC) with sorbitol
22
PROCEDURE
1. Eight days before lab portions of the ground beef were weighed out for each lab
section; one portion (10 g) was wrapped in foil and stored in the freezer for one week
and then thawed in the refrigerator overnight; a second portion (10 g) was wrapped and
stored in the refrigerator for eight days; a third portion (10 g) was thawed at room
temperature. You will be working with only of the samples named above.
For all samples, note evidence of deterioration in esthetic qualities, e.g. color, texture,
and odor.
2. Transfer each 10 g portion to a sterile stomacher bag containing 90 ml buffer. Blend

each 10 g sample separately using the stomacher according to the manufactures
instructions. Consider this a 10-1 dilution of the fresh ground beef.
3. Prepare four 9-ml dilution blanks in the sterile tubes. Make serial dilutions of the
ground beef sample, up to and including 105. Use the spread plate technique (0.1 ml per
plate) to plate the highest three dilutions on TSA. Plate the three lowest dilutions (most
concentrated samples) on EMB.
4. Incubate the plates at 35oC for 48 hrs and refrigerate. Calculate the number of cells
per g of ground beef.
5. Compare the number and kinds of organisms found in the hamburger under the two
conditions of storage, freezer and refrigerator, with that found in the fresh sample.
WEEK 2
Plate selected E. coli colonies on MAC agar containing sorbitol. Use a grid pattern (as
shown by your instructor) so that several colonies can be tested on one plate. Incubate
24 hrs at 35OC and observe the colonies. White colonies indicate possible
pathogenicity.
Questions:
You plated dilutions of ground beef on MacConkey agar containing sorbitol, rather than
lactose, as a carbon source.
a. Why is lactose used in the original MacConkey formulation?
b. Why has the substitution to sorbitol been used for certain applications of medium?
23
6. ISOLATION OF SALMONELLA
Procedures used in this exercise for isolating Salmonella sp. from food are based on
methods described in the Bacteriological Analytical Manual for Foods (BAM), Food
and Drug Administration, Bureau of Food, Division of Microbiology, Washington, DC.
The steps involved include the following:
1. Preenrichment (as necessary) in a non-inhibitory medium to allow recovery of

injured or inactive cells in food processed by methods such as freezing or drying.
2. Enrichment in a medium that allows salmonellae to grow, but which is somewhat
inhibitory to coliforms and other groups of microorganisms.
3. Isolation of a Salmonella sp. on differential media.
4. Identification of the species as Salmonella by biochemical tests.
The Enrichment Media

Enrichment Media - Media that have been supplemented with highly nutritious
materials, such as blood, serum, yeast extract to cultivate fastidious organisms
Selenite cystine broth

This medium is an enrichment medium for salmonellae, contains sodium selenite which
is reduced to selenium by certain microorganisms. Selenium reacts with sulfur-
containing amino acids to inhibit growth of most bacteria, but it is mainly the Gram-
positive organisms that are inhibited. In addition, the fermentation of lactose in this
medium by organisms such as the coliforms lowers the pH of the medium to the range
where the growth of Salmonellae is favored over competing organisms.
Tetrathionate broth
This medium contains bile salts, sodium thiosulfate and brilliant green, a dye. The bile
salts and brilliant green dye selectively inhibit growth of Gram-positive organisms, but
also inhibit or at least suppress the growth of some Gram-negative, non-pathogenic
enteric bacilli. Hydrogen sulfide is produced by many if not most of the salmonellae as
they hydrolyze sulfhydryl groups from inorganic sulfur-containing compounds, in this
medium, the sodium thiosulfate.
The Selective/Differential Media
Selective Media - Used to SELECT (ISOLATE) specific groups of bacteria (Include

substrates to enhance the growth of one type, but inhibit the growth of another type
of bacteria)
Differential Media - Used to DIFFERENTIATE (TELL APART) different types of
related bacteria (Incorporate a compound that produce a characteristic change in the
appearance of bacterial growth and/or the medium surrounding the colonies)
Bismuth sulfite agar

Selection: This medium contains brilliant green dye as well as freshly precipitated
bismuth. These compounds act together to selectively inhibit Gram-positive organisms.
In addition, the growth of coliform organisms as well as that of many enteric pathogens
is suppressed more than that of the salmonellae.
Differentiation: In the presence of hydrogen sulfide produced by many of the
salmonellae, metallic salts are formed in reactions with ferrous sulfate that stain colonies
and the surrounding medium black or brown.
24
Xylose Lysine Desoxycholate (XLD) Agar
Selection: This medium is a moderately selective medium recommended for isolation
and differentiation of enteric pathogens. Sodium Desoxycholate is added to inhibit Gram
positives.
Differentiation: Xylose is incorporated into the medium because it is fermented by
practically all enterics except for Shigella sp. (production of acid). Lysine is added and
can be attacked via the Enzyme lysine decarobylase; with reversion back to an alkaline
pH (Shigella reaction). In order to differentiate other enterics, sucrose and lactose are
added to produce excess acid.
Hektoen agar
Selection: This medium contains bile salts that selectively inhibit the growth of Gram-
positive organisms.
Differentiation: Lactose is incorporated into the medium to facilitate recognition of
pathogens, which do not ferment lactose, in the presence of coliforms which do. The
medium also contains sodium thiosulfate and ferric ammonium citrate from which ferric
sulfate is formed if hydrogen sulfide is produced.
Triple-sugar-iron agar
Selection: This medium is used in the selective identification of enteric bacteria.
Differentiation: TSI Agar contains three sugars (dextrose, lactose and sucrose), phenol
red for detecting carbohydrate fermentation, and ferrous ammonium sulfate for detection
of hydrogen sulfide production (indicated by blackening in the butt of the tube).
Carbohydrate fermentation is indicated by the production of gas and a change in the
color of the pH indicator from red to yellow. To facilitate the detection of organisms that
only ferment dextrose, the dextrose concentration is one-tenth the concentration of
lactose or sucrose. The small amount of acid produced in the slant of the tube during
dextrose fermentation oxidizes rapidly, causing the medium to remain red or revert to an
alkaline pH.
In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is
under lower oxygen tension. After depletion of the limited dextrose, organisms able to do
so will begin to utilize the lactose or sucrose.
Some bacteria (such as Salmonella spp.) utilize thiosulfate anion as a terminal electron
acceptor, reducing it to sulfide. If this occurs, the newly formed hydrogen sulfide (H2S)
reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a
black precipitate.
MATERIALS
• Fresh ground chicken or turkey
• Blender and sterile blender jars
• Enrichment broth; tetrathionate or selenite cysteine broth
• Differential media; bismuth sulfite, XLD agar and Hektoen agars
• Triple sugar iron agar (TSI)
Caution: Salmonella spp. are classified as BSL-2 organisms. Use the biosafety cabinet
for all inoculations and culture transfers.
25
1. In this exercise you will follow these procedures in attempting to isolate Salmonellae
from fresh and frozen chicken parts purchased in local supermarkets. Because the time
intervals between certain steps are critical, this enrichment step have been done for
you: Weigh 25 g fresh chicken into a sterile blending jar. Add 225 ml of either selenite
cystine broth or tetrathionate broth and blend for 2 min. Incubate the blended chicken
and broth for 24 hr at 35oC.
2. Isolation on differential media. Use the quadrant streak technique (See appendix) to
inoculate from either of the enrichment cultures onto each of three differential media:
bismuth sulfite agar, Salmonella-Shigella agar, and Hektoen agar. Incubate the plates
for 24 hr at 35oC.
3. After incubation, examine the plates for colonies typical of Salmonellae. The following
descriptions of typical colonies are taken from the Difco Manual:
Bismuth sulfite agar: Salmonellae colonies will be greenish-gray to black,

sometimes with a sheen.
XLD agar (xylose, lysine, desoxycholate): Salmonellae colonies appear red

with a black center indicating the production of hydrogen sulfide.
Hektoen agar: Salmonellae colonies will be greenish blue with black centers
indicating the production of hydrogen sulfide.
If colonies typical of Salmonellae are identified on any of your plates, select two colonies
at random and inoculate each onto a slant of triple-sugar-iron agar. Use a needle for
these inoculations rather than the loop; streak the slant and then stab into the butt;
incubate the cultures for 24 hr at 35oC.
After incubation, examine the triple sugar iron agar cultures for reactions typical of
Salmonellae; alkaline (red) slants and acid (yellow) butts, with or without hydrogen
sulfide production, shown as blackening of the agar. Coliform organisms will turn the
medium yellow throughout. If the reaction on triple-sugar-iron agar is consistent with
Salmonella sp., inoculate a series of biochemical tests (see handout) with cells taken
from the TSI culture.
26
7. WATER ANALYSIS: Microbial Indicators of Fecal Contamination
Monitoring the quality of drinking and recreational waters is of utmost importance as

contamination may pose high risks for human health. Waters contaminated with human
feces commonly contain human pathogens, such as Salmonella spp., Shigella spp.,
protozoans, and viruses. Waters contaminated with animal feces may also contain
pathogenic species, such as Escherichia coli or Salmonella spp. Understanding the origins
of fecal pollution is vital in the assessment of associated risks as well as any actions
necessary to clean up and prevent further contamination.
Many times, microbes when present in water are usually found in concentrations so low as
to make their detection difficult. For this reason, tests for determining the microbiological
safety of water usually rely on the detection of indicator organisms such as the coliform
group of bacteria. Because the fecal coliform organisms are always present in high
numbers in fecal matter, their presence in a water sample indicates that the water has been
contaminated in the recent past with fecal material. The water-borne pathogens, with few
exceptions, are enteric organisms that will be discharged in the feces of infected hosts,
either man or animal. Detection of fecal coliforms thus indicates that the water being
sampled might contain enteric pathogens.
The concept, that the origin of fecal contamination can be traced with the aid of
microbiological, molecular and chemical methods, is also known as “fecal” or “microbial
source tracking.” The rationale behind this concept is to characterize groups of “indicator”
microorganisms and detect subtle difference between those groups for the purpose of
identifying the host or environment from which these organisms originated.
In this exercise, you will use two tests to examine various water samples for evidence of
fecal contamination. The most probable number test (MPN) is based on the detection in
traditional tube and plate tests of lactose-fermenting, gram-negative rods of the Escherichia
coli type. The membrane filter (MF) tests are based on retention of bacterial cells from
filtered water samples on the surface of filter membranes.
In addition to these specific tests for the detection of fecal contamination, you will use a
standard plate count to get some idea of the relative number of bacteria present in the water
samples being tested.
I. Most Probable Number (MPN) Test
The MPN test for water safety has three parts: Presumptive, Confirmed, and Completed.
The presumptive test simply detects the presence of bacteria that can produce gas in
lactose broth inoculated with the water sample. Because some nonenteric organisms
(Clostridium and Bacillus are two examples) in water can ferment lactose and give false
positive results, additional tests, the confirmed and completed tests, must be performed.
The lactose broth used in the presumptive test is only slightly selective. This allows the
recovery of all coliforms in the sample; however, a more highly selective medium might lead
to an inaccurately low count.
For the confirmed test the moderately selective medium Brilliant green lactose broth
(BGLB) is used to confirm that coliforms are responsible for the fermentation; EC broth (E.
coli broth) is used in the confirmed test for fecal coliforms.
27
Any positive samples from the confirmed test are inoculated onto the highly selective
medium Eosin Methylene Blue (EMB) for a completed test. Positive colonies are tested for
standard coliform traits. To identify E. coli specifically as being present in the sample, the
IMViC tests would be performed. (See the Bacterial Analytical Manual for procedures for
the confirmed and completed tests). In this exercise we will do only the presumptive test.
MATERIALS
• 500 ml environmental water sample.
• 6 tubes lactose broth, single strength, with Durham fermentation tubes
• 3 tubes lactose broth, double strength, with Durham fermentation tubes
1. If your water sample is from a potable source (water tap, wells, or springs), use it directly
for inoculations into lactose broth. If your sample is from a source more likely to be
contaminated (rivers, lakes, sewage), dilute it in phosphate buffered water 1:100 (or as
instructed) before inoculating the lactose broth.
2. Inoculate each of 3 tubes of double strength lactose broth with 10 ml samples.
3. Inoculate each of 3 tubes of single strength lactose broth with 1 ml samples.
4. Inoculate each of 3 tubes of single strength lactose broth with 0.1 ml sample.
5. Incubate all of the lactose broth cultures at 35oC for 48 hr.
1. Read as positive those tubes that show significant gas accumulation in the Durham
fermentation tube (i.e. production should be more than just a bubble).
2. Determine the MPN of coliforms in the sample by referring to the statistical table.
II. Membrane Filter (MF) Tests

Filtration units can be used to trap bacterial cells from a water sample on the surface of a
membrane. If nutrients are supplied to these cells, visible colonies will develop on the
membrane. Nutrients for the cells can be supplied from saturated cellulose pads or from
agar media on which the membranes are incubated. If suitable media are selected,
differential counts can be made. In this exercise, you will use the water sample collected in
part I in tests for coliform organisms in general, for fecal coliforms specifically, and for fecal
streptococci. To accomplish this you will perform 9 filtrations of your sample; three sample
levels for each of the three media.
MATERIALS
• Water sample (same as for part I), up to 300 ml will be required

• Sterile filter funnel and base, with clamp
• Suction flask
• Sterile phosphate buffer, pH 7.2
• 3 small Petri dishes, MI Agar
• 3 small Petri dishes with M-Enterococcus agar
28
1. Assemble the sterile filtration unit, placing a sterile filter membrane grid side up on the
base. Clamp the funnel to the base.
2. Because the number of fecal organisms in the water sample is not known, you will need
to filter different amounts to hit the optimum range (20 to 60 colonies per membrane). If the
sample is likely to have a low bacterial count, a relatively large volume should be filtered; if a
high count is expected because of the source, smaller volumes or diluted samples should be
used. The filter holder and assembly need not be sterilized between filtrations of different
volumes of the same water sample. However, the highest dilution (lowest concentration) of
organisms should be filtered first.
2. If your sample is from a potable source and was left undiluted for the MPN test, filter 1, 10
and 100 ml volumes. If you diluted your sample for the MPN, filter 0.1, 1 and 10 ml
volumes. Start with the smallest volume; pour about 50 ml phosphate buffer into the funnel;
pipette 0.1 ml sample (or the smallest volume used) into the buffer; attach the vacuum and
pull the sample through the membrane. Rinse down the sides of the funnel with more buffer.
Remove the membrane with sterile flamed forceps (not ridged), and place on top of the MI
Agar. Place another sterile membrane on the filter base and clamp to the funnel as
before. Filter one more 0.1 ml volume for the M-Enterococcus medium, with each
filtration placing the membrane on either the saturated pad or the agar medium. Wash
down the sides of the funnel with at least 50 ml of sterile phosphate buffer between each
filtration.
3. Filter two 1 ml volumes (or 10 ml volumes for potable sources) as above, one each for
the MI Agar, and M-Enterococcus agar.
4. Filter two 10 ml volumes (or 100 ml volumes) as described; one filtration for each type of
media.
5. Incubate the MI Agar plates at 35oC for 48 hr.
6. Incubate the M-Enterococcus agar plates at 35oC for 48 hr.
1. Mi Agar - On this medium, total lactose-fermenting organisms will be fluorescent.

Colonies of E. coli will appear blue.
Remove the membrane from the Petri dish and dry on absorbent paper at room
temperature. Examine the membrane for colonies exhibiting a characteristic green metallic
sheen. Record your results for each dilution (volume) tested, but use a plate with 20 to 60
colonies (if possible) for your calculations. Express your final result as the number of
coliforms per 100 ml water.
2. M-Enterococcus Agar: This medium is selective for fecal streptococcal organisms.

Fecal streptococcal colonies will be red or pink. Examine the membrane under low
magnification if necessary. Calculate the ratio between fecal streptococci and fecal
coliforms. A high ratio may indicate contamination from an animal rather than human
source.
29
III. Standard Plate Count
This procedure will detect only those microbial cells that can grow in the laboratory on a
standard culture medium. As such it says very little about whether or not the water is safe to
drink.
MATERIALS
• 1 Bottle phosphate buffered water

• Sterile tubes
• Pipettes
• 4 plates TSA
1. Prepare three serial dilutions (101,102, 103) of the water sample from part I. Spread 0.1 ml
samples from the undiluted and the three sequential dilutions on the TSA plates.
2. Incubate the plates at 30oC for 48 hours.
1. Count the cells from the plate containing 30 to 300 colonies. Multiply with the dilution
factor. Express results as number of culturable bacteria per 100 ml water.
QUESTIONS
1. What are local standards for drinking water in relation to coliform counts? From what
governmental agency can you get this information?
2. Describe the confirmed and completed tests in the MPN series.
3. Is water with a high coliform count safe to drink? Explain your answer.
4. What is the significance of the fecal coliform/fecal enterococci ratio in relation to the
source of fecal contamination of water?
REFERENCES
Standard Methods for Water and Wastewater Analysis. 16th Ed. 1985 American Public
Health Assoc., Washington, DC
Bacterial Analytical Manual (BAM)

http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm114664.htm
http://water.usgs.gov/
30
MPN Determination from Multiple Tube Tests
# of Positive Test Tubes 95% Confidence Limit

3 of MPN Index per
3 of 10 3 of 1 mL
0.1mL 100mL Lower Upper
mL each each
each
0 0 1 3 <0.5 9
0 1 0 3 <0.5 13
1 0 0 4 <0.5 20
1 0 1 7 1 21
1 1 0 7 1 23
1 1 1 11 3 36
1 2 0 11 3 36
2 0 0 9 1 36
2 0 1 14 3 37
2 1 0 15 3 44
2 1 1 20 7 89
2 2 0 21 4 47
2 2 1 28 10 150
3 0 0 23 4 120
3 0 1 39 7 130
3 0 2 64 15 380
3 1 0 43 7 210
3 1 1 75 14 230
3 1 2 120 30 380
3 2 0 93 15 380
3 2 1 150 30 440
3 2 2 210 35 470
3 3 0 240 36 1300
3 3 1 460 71 2400
3 3 2 1100 150 4800
th
Adapted from Standard Methods for the Examination of Water and Wastewater, 12 edition.
(New York: The American Public Health Association, p. 608)
31
8. YEAST METABOLISM
The yeast Saccharomyces cerevisiae, a microorganism of extraordinary industrial

importance, will grow either aerobically or anaerobically. If oxygen is present, glucose
can be oxidized via the Krebs cycle all the way to carbon dioxide and water. Under
favorable aerobic conditions, many new yeast cells will be produced and little alcohol
accumulated. This mode of metabolism is important in the baking industry where the
leavening of dough depends on the production of large amounts of carbon dioxide by
yeast cells. If the yeast is grown under anaerobic conditions where access to oxygen in
limited, metabolism does not proceed through the Krebs cycle but terminates in the
glycolytic pathway. Under these conditions large amounts of ethyl alcohol and carbon
dioxide are produced and relatively fewer new yeast cells are formed compared to
aerobic growth. Oxygen exerts its effect on the mode of metabolism by stimulating the
formation of the respiratory enzymes and inhibiting glycolytic pathway enzymes. High
concentrations of glucose in the medium will repress the formation of respiratory
enzymes even in the presence of an ample supply of oxygen. Under these conditions
metabolism shifts to the anaerobic mode and more alcohol will accumulate.
In this exercise, the strains of S. cereviseae that will be used in brewing beer will be
grown on a glucose substrate under aerobic conditions and also under conditions where
access to air is limited. In one aerobic culture glucose will be added intermittently to
prevent exposure of the yeast cells to concentrations high enough to shift the mode of
metabolism. After incubation the cultures will be analyzed for cell concentration and
alcohol content to demonstrate the effects of aerobic and anaerobic growth. Glucose
concentration in the cultures will be analyzed to allow calculation of the relative efficiency
of aerobic versus anaerobic growth in production of cells based on amount of glucose
used.
MATERIALS
WEEK 1
• 3 sets of culture media (per group) as follows:
o Flask 1: 50 ml yeast nitrogen base (Difco)-dextrose (5%) broth in a 125
ml Erlenmyer flask, foam stopper.
o Flask 2: 50 ml yeast nitrogen base (Difco)-dextrose (5%) broth in a 50
ml Erlenmyer flask, rubber stopper equipped with a one-way valve.
o Flask 3: 40 ml yeast nitrogen base (Difco) broth (5/4 strength) to which
the first addition (2 ml) of 25% glucose has been added, in a 125 ml
Erlenmyer flask, foam stopper.
• Broth culture of S. cereviseae strains used in beer brewing
WEEK 2
• Materials for determination of ethanol via enzymatic assay
• Dinitrosalicylic acid (DNS) reagents for sugar determination
• Pipettes, test tubes, and other standard supplies and equipment.
• Glucose standard solution (2 mg/ml)
32
1. Inoculations of Yeast Cultures
PROCEDURE – WEEK 1
1. Inoculate each of the three culture media with 1 ml of yeast broth culture.
2. Remove the rubber stopper from flask 2 and flush that culture with a steady flow of
nitrogen for a minute or two before replacing the stopper. Make certain the one-way
valve is in place and working.
3. Incubate the cultures in flasks 1 and 3 at room temperature on a shaker for 5 days. If
the temperature in the room is likely to be above 20oC, place a small plastic "baggie"
over the foam stopper to prevent evaporation of the medium. (On days 2, 3, 4, and 5 of
the incubation period, additional 2 ml aliquots of the glucose solution will be added to the
culture in flask 3. A total of 10 ml glucose solution will be added to this culture overall.
The aim is to keep the glucose concentration in the culture at a relatively low level).
4. Incubate the culture in flask 2 at room temperature but without shaking.
PROCEDURE – WEEK 2
1. At the end of the incubation period, shake each culture gently to suspend the cells
evenly. Transfer 1.5 ml of each culture to microcentrifuge tubes. Pellet the cells in
microcentrifuge at 10,000 RPM for 2 minutes. You will use ~1.2 ml of the clear
supernatant solution for sugar analysis.
2. Cell Concentration
Microbial cells in suspension scatter and absorb light passing through the suspension so
that a culture of bacteria or yeast of more than 107 or 108 cells per ml will appear turbid.
As you know this property of cells in suspension can be used as a measure of cell
concentration. In cell suspensions that are not too dense, cell mass measured as optical
density (OD) is generally proportional to cell number.
PROCEDURE
You will determine the cell concentration in the yeast cultures by reading OD540 in the
spectrophotometer and converting OD into cell concentration by referring to a standard
curve relating cell number to OD. Prepare this standard curve as follows:
1. Use the protocol shown below and set up a series of yeast cells dilutions of different
concentrations. To make these solutions use the experimental flask (1, 2 or 3) that
appears to you visually to have the greatest cell number (the cloudiest flask). Make the
dilutions in dd water.
33
Protocol for cell suspensions
Tube # H2O (ml) Cells (ml) Read OD540 Calculated cells/ml*
1 5.0 0
2 4.5 0.5
3 4.0 1.0
4 3.0 2.0
5 2.0 3.0
6 1.0 4.0
7 0.5 4.5
8 0 5.0
*Calculated from direct cell count in counting chamber.
2. Read the OD540 of each suspension. Use plain YNB as a reference to set the
spectrophotometer to zero. Read dd H20 against YNB and record difference if any. Set
the spectrophotometer to 0 using H20 as a blank before taking readings. Make readings
in the same “cuvette”, starting with the least concentrated suspension. Drain it lightly on
towel between readings.
3. Determine the cell concentration in one of the dilutions in the linear range by direct
cell count in the counting chamber. By calculation, determine the number of cells in all
of the other suspensions. Plot cell concentration versus OD. Because the relationship
is not linear at high cell concentrations, do not use that portion of the curve for your
calculations.
4. Determine the OD540 of the experimental samples. They may need to be diluted so
that the OD is within the linear range of your standard curve. Read concentration from
standard curve.
RESIDUAL GLUCOSE
Dinitrosalicylic acid (DNS) is the reagent used to determine glucose concentration.

Reducing sugars, such as glucose, reduce the nitro group and cause a darkening of the
solution. The color change measured as OD at 575 nm can be correlated with sugar
concentration.
CAUTION! THE DNS SOLUTION IS STRONGLY BASIC. PROTECT YOUR HANDS

WITH GLOVES. TAKE CARE NOT TO GET THIS REAGENT ON THE
SPECTROPHOTOMETERS.
34
PROCEDURE
Standard Curve
1. Prepare a glucose standard curve for boiling using known concentrations of glucose.
Follow the protocol below.
2. After addition of the DNS solution place all the tubes in a boiling water bath for 5 min.
Cool for a minute then add 1mL of Rochelle salts to each tube to stabilize the color. Use
tube # 1 of the standard curve as a reagent blank to set OD to zero. Determine the OD
of each tube.
3. Plot OD versus glucose concentration. Because the nitro group becomes reoxidized,
the origin may not go through zero; don't force it. Read the glucose concentration for
each sample directly from the standard curve.
Protocol for preparing standard curve

Standard* H2O DNS Glucose
Tube No. OD575
(ml) (ml) (ml) (mg)
1 0 1 5 0
2 0.1 0.9 5 0.2
3 0.2 0.8 5 0.4
4 0.3 0.7 5 0.6
5 0.4 0.6 5 0.8
6 0.5 0.5 5 1
*Glucose solution -2 mg/ml
Experimental Samples
1. You will determine residual sugar in the spent culture media using the supernatant
solution you prepared by microcentrifugation of each of the three samples. Prepare
sample from the supernatant solutions from each of your three flasks as shown below.
2. After addition of the DNS solution place all the tubes in a boiling water bath for 5 min.
Cool for a minute then add 1mL Rochelle salts to each tube to stabilize the color. Use
tube # 1 of the standard curve as a reagent blank to set OD to zero.
3. Read the glucose concentration for each sample directly from the standard curve.
Protocol for glucose determination from spent media

Tube # Flask # Sample (ml) H2O (ml) DNS (ml) OD575
1 1 1.0 0 5
2 1 0.1 0.9 5
3 2 1.0 0 5
4 2 0.1 0.9 5
5 3 1.0 0 5
6 3 0.1 0.9 5
QUESTIONS
1. What is the Pasteur effect? Was it demonstrated in this exercise? Explain.
2. What is the Crabtree effect? Was it demonstrated in this exercise? Explain.
3. Give another example of the use of a tetrazolium salt in an enzyme linked assay.
35
9. PREPARATION OF A MALT BEVERAGE
(Content and Recipes adapted with permission from “Love2Brew.”)
MATERIALS
• Malt extract
• Hops
• Glass carboy (4 liters) equipped with gas trap
• Hydrometer
• Bottles and capper
• Cultures of Saccharomyces cereviseae
PROCEDURE
Week 1 – Preparation of Wort and Fermentation

Work in groups of 3 or 4; one group per bench side. Each group will ferment about 4
liters of beer. Carboys have been scrupulously cleaned and are large enough to provide
head space to accommodate foaming during fermentation. See each recipe for specific
instructions.
Week 2 – Racking and Bottling

Add one carbonation sugar cube to each bottle. Using a sanitized hose, syphon beer
into carboy, allowing no transfer of trub. Swirl carboy to mix, open hose clamp and fill
sanitized bottles to within one inch of the top. Cap and store at room temperature for at
least 4 weeks. Refrigerate overnight. Have a brew!
RECIPES
1. German Hefeweizen
Ingredients
Malts & Specialty Grains: 1 lbs. Wheat Dry Malt Extract
Hops: 9 Grams German Hallertau
Yeast: Safbrew WB-06
Procedure
1. Add 1.25 gallons of water to your boil kettle. Bring the water to a boil.
2. Remove from heat and add 1 lb. Wheat DME to your water. Stir.
3. At this point your mixture is now wort (pronounced “wert”; defined as unfermented
beer). Allow your wort to return to a boil. Be sure to observe your boil so as to avoid a
messy boil-over.
4. Once the foaming subsides, begin your 60 minute boil process. Timing is referred to
by minutes left in the boil.
50 minutes: Add 6 Grams German Hallertau
15 minutes: Add 3 Grams German Hallertau. Boil for the remaining 15 minutes.
5. After your wort is done boiling it is very important you cool it as quickly as possible to
avoid potential infections. Create an ice bath (ice and water) in your sink and set the
brew kettle in it. You need to cool your wort down to 70°F or lower.
36
2. Belgian Blonde
Ingredients
Malts & Specialty Grains: 1 lbs. Pilsen Malt Extract, 8 oz. Light Malt Extract
Hops: 7 Grams Hersbrucker (Bittering), 4 grams German Tettnang (Flavoring/Aroma)
Yeast: Safbrew T-58
Procedure
2. Remove from heat and add 1 lb. Pilsen DME and 8 oz light DME to your water. Stir.
messy boil-over.
4. Once the foaming subsides begin your 60 minute boil process. Timing is referred to
60 minutes: Add 7 grams Hersbrucker for Bittering.
10 minutes: Add 4 grams German Tettnang for aroma. Keep the mixture boiling
for the final 10 minutes
3. American Blonde
Ingredients
Malts & Specialty Grains: 1 lbs. Light Malt Extract
Hops: 3 Grams Ahtanum (Flavor), 6 grams Ahtanum (Aroma)
Yeast: Safale US-05
Procedure
2. Remove from heat and add 1 lb. Light DME to your water. Stir.
beer). Allow your wort to return to a boil. Be sure to observe your boil so as to avoid
a messy boil-over.
20 Minutes: Add 3 grams oz. Ahtanum for flavoring.
10 Minutes: Add 3 grams Ahtanum for aroma.
5 Minutes: Add 3 grams Ahtanum for aroma. Boil for the final 5 minutes
37
4. Amber Ale
Ingredients
Malts & Specialty Grains: 1.1 lbs. Amber Malt Extract
Hops: 3 grams Nugget (Bittering), 3 grams Nugget (Flavoring), 3 grams Nugget (Aroma)
Yeast: Safale US-05
Procedure
1. Add 1.25 gallons of water to your boil kettle. Bring your water to a boil.
2. Remove from heat and add 1 lbs. Amber Malt Extract to your water. Stir.
messy boil-over
60 minutes: Add 3 grams of Nugget hops for flavoring.
25 minutes: Add 3 grams Nugget hops for flavor.
5 minutes: Add 3 grams Nugget hops for aroma. Boil for the final 5 minutes.
5. American Wheat
Ingredients
Malts & Specialty Grains: 1 lbs. Wheat Malt Extract
Hops: 7 grams Cascade (Bittering/Flavor/Aroma)
Yeast: Safale US-05
Procedure
2. Remove from heat and add 1 lb. Light DME to your water. Stir.
messy boil-over.
60 minutes: Add 3 grams Cascade for bittering.
30 minutes: Add 2 grams Cascade for flavoring.
5 minutes: Add 2 grams Cascade for aroma. Boil for the final 5 minutes
38
6. Basic Stout
Ingredients
Malts & Specialty Grains: 1 lbs. Dark Malt Extract

Hops: 4 grams Magnum (Bittering)
Yeast: Safale S-04
Procedure
2. Remove from heat and add 1 lb. Dark Malt Extract to your water. Stir.
messy boil-over
60 minutes: Add 4 grams of Magnum hops for flavoring. Boil for the remaining
60 minutes.
7. Saison
Ingredients
Malts & Specialty Grains: 1lb Pilsner Malt Extract

Hops: 7 grams Kent Goldings (Bittering)
Yeast: Belle Saison
Procedure
2. Remove from heat and add 1 lb. Dark Malt Extract to your water. Stir.
messy boil-over
60 Minutes: Add 7 grams of Kent Goldings hops for flavoring. Boil for the
remaining 60 minutes.
39
10. COMPARISON OF MICROBIAL POPULATIONS – RFLP Analysis of 16S rRNA
Gene
With the use of restriction fragment length polymorphism (RFLP) we can identify
changes in the genetic sequence that occurs at a site where a restriction enzyme cuts.
Restriction enzymes recognize specific short sequences of DNA and cut the DNA at
those sites. RFLP analyses can be used for genotyping, forensics, paternity tests,
hereditary disease diagnostics etc. For our experiment, we will amplify the 16S SSU
gene from three distinct populations of organisms isolated this semester (B. cereus/toxin
producers; H2S positive, non-lactose fermenting organisms from ground turkey; putative
E. coli from water sample) compare RFLP (restriction fragment length polymorphisms)
within and among the populations.
There are basics steps to conduct this experiment:
1. Isolate DNA (single colonies) and set up Colony PCR

2. Digestion with Restriction Enzymes
3. Electrophoretic Analysis to assess where the DNA was cut and compare the
banding patterns between the different populations
WEEK 1
COLONY PCR
You will prepare samples as described and add to the reactions mix already prepared for
you. The ingredients listed below have been added to a PCR tube (200µl, thin-walled):
You will add 1µl of your DNA sample for a total of 25 µl reaction volume:
12.5µl TopTaq Master Mix
0.5µl forward primer
0.5µl reverse primer
10.5µl H2O
You will prepare your sample as follows:

Pipette 100µl sterile water into a microfuge tube. Add a small amount of culture
(~colony equivalent) with a needle. Vortex the tube vigorously to obtain a smooth
suspension. (A cloudy suspension is expected.) Add 1µl of your suspension to the
prepared PCR sample. Your TA will use the following conditions for the PCR:
• 94°C - 5 min – This initial step ruptures the cells so the DNA is exposed for
replication cycles
• (94°C - 30 sec; 50°C - 30 sec; 72°C - 90 sec) – 40x. 40 cycles of melting, primer
annealing and elongation….
• 72°C - 7 min – Final extension.
• 4°C – Until removed from thermocycler
• Stored in freezer until sample analysis
ELECTROPHORESIS OF PCR PRODUCTS (BY INSTRUCTOR)

Samples (10 µl each) will be subjected to agarose gel electrophoresis (1.5% agarose).
The PCR products (16S RNA is ~1500 bp) will be compared to standards to check size
for appropriate PCR product. A photograph of the gel will be available to aid in
estimating volume of product to be used in the restriction digest.
40
WEEK 2
RFLP ANALYSIS
10 µl* of PCR product will be added to the 5 µl of reaction mix provided. The mix
contains the following:
1.5 µl 10X restriction enzyme buffer
0.3 µl HaeIII (10U/µl)
0.3 µl MspI (10U/µl)
2.9 µl (10U/µl) RNAse free sterile water
Incubate restriction digests for 2h @ 37°C.
*Amount of product used will depend on intensity of product band on gel (above). If
band(s) are very intense amount of product used will be decreased – and water added to
bring total added to digest to 10µl.
ELECTROPHORESIS OF RESTRICTION DIGEST PRODUCTS
MATERIALS
Digested Samples: HaeIII + MspI digests of 16S PCR products
DNA standards (100bp ladder)
Agarose
TAE buffer (40mM Tris;0.11% Acetic Acis; 1mM EDTA)
Ethidium bromide
Gel boxes with Power supply
PROCEDURE - WEAR GLOVES WHEN HANDLING GEL BOXES.
1. Prepare 40 ml of 3% w/v agarose solution. Weigh appropriate amount of agarose.

Add 40 ml TAE to 125ml Erlenmeyer flask; add agarose. Dissolve the gel using the
microwave oven.
2. While the gel is being dissolved someone in the group can set up the casting tray.
3. Cool the dissolved gel slightly and add 4 µl of the ethidium bromide (EtBr) solution
provided – wear gloves. Adding the EtBr directly to the gel will allow us to follow the
progress of the electrophoresis and generate less EtBr contaminated waste.
4. Pour the gel into casting tray. DON’T FORGET THE COMB!
5. After the gel has set, rotate the tray 90°, and place it in the electrophoresis chamber
(gel box). Remove the comb carefully.
6. Add running buffer (TAE) to cover the gel slab filling both chambers to the “fill line”.
7. You will load your entire sample. Use a micropipetter with a fresh tip to deliver each
sample.
8. After the samples have been loaded, attach the terminals from the gel box unit to the
DC power supply; gels will be run @ 75V for 1.5 hr; photographed on UV light box
and analyzed.
REFERENCES
Bertagnolli, A. D., Treusch, A. H., Mason, O. U., Stingl, U., Vergin, K. L., Chan, F.,
Beszteri, B., Giovannoni, S. J. 2011. Bacterial diversity in the bottom boundary layer
of the inner continental shelf of Oregon. Aquatic Microbial Ecology. 64:15-25.
41
Bruce, K. D., and M. R. Hughes. 2000. Terminal restriction fragment length
polymorphism monitoring of genes amplified directly from bacterial communities in soils
and sediments. Molecular Biotechnology. 16:261-269.
Cabrera,J.A., Kurtz, A., Sikora, R.A., Schouten, A. 2010. Isolation and

characterization of fenamiphos degrading Bacteria. Biodegradation. 21:1017–1027.
Lueders. T., Friedrich, M.W. 2003. Evaluation of PCR Amplification Bias by Terminal
Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA
Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil
DNA Extracts. Applied And Environmental Microbiology. 69:320–326
Sfanos, K., Harmody,, D., Dang, P., Ledger, A., Pompon. S.,
McCarthy, P., Lopez, J. 2005. A molecular systematic survey of cultured microbial
associates ofdeep-water marine invertebrates. Systematic and Applied Microbiology 28:
242–264
Widmer, F., Seidler, R.J., Gillevet, P.M., Watrud, L.S., Di giovanni, G.D. 1998. A
Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus
Pseudomonas (Sensu Stricto) in Environmental Samples. Applied And Environmental
Microbiology. 64(7):2545-255
Winch, S., Mills, H.J., Kostka, J.E., Fortin, D., & Lean. D.R.S. 2009. Identification of
sulfate-reducing bacteria in methylmercury contaminated mine
tailings by analysis of SSU rRNA genes. FEMS Microbiol Ecol. 68:94–107
Xiao, L., Hlavsa, M.C.. Yoder, J., Ewers, C., Dearen, T., Yang, W., Nett, R., Harris,
S., Brend, S.M., Harris, M., Onischuk, L., Valderrama, A.L., Cosgrove, S., Xavier, K.,
Hall, N., Romero, S., Young, S., Johnston, S.P., Arrowood, M., Roy, S., Beach, M.J.
2009. Subtype Analysis of Cryptosporidium Specimens from Sporadic Cases in
Colorado, Idaho, New Mexico, and Iowa in 2007: Widespread Occurrence of One
Cryptosporidium hominis Subtype and Case History of an Infection with the
Cryptosporidium Horse Genotype. Journal Of Clinical Microbiology. 47(9):3017–3020
Zhou, J., Davey, M.E., Figueras, J.B., Rivkina, E., Gilichinsky, D., Tiedje, J.M. 1997.
Phylogenetic diversity of a bacterial community determined from Siberian tundra soil
DNA. Microbiology. 143:3913-3919.
42
11. ANTIBIOTIC DISK ASSAY: THE KIRBY-BAUER DISC METHOD
(Adapted from Davis & Rauschenbach, General Microbiology Laboratory Manual
Spring 2014)
The method of determining the susceptibility of an organism to an antibiotic is simply to

inoculate an agar plate with the culture to be evaluated and allowing the antibiotic to
diffuse into the agar medium, usually from a filter paper disc. The whole surface of a
Petri dish is swab inoculated with the test bacterium. A filter paper disk impregnated
with an antibiotic is then applied to the surface of the inoculated agar plate. The
antibiotic diffuses from the filter paper into the agar. At some particular distance from
each disk, the antibiotic is diluted to the point that it no longer inhibits microbial growth.
The effectiveness of a particular antibiotic is shown by the presence of “zones of
inhibition.” These zones appear as clear areas surrounding the disk from which the
substances with antimicrobial activity diffused. The diameter of the zones (in mm) is
readily measured with a ruler, and the relative efficiency of the antibiotic is apparent from
the size of the zone of inhibition.
The agar diffusion method is in widespread use: filter-paper disks, each containing
defined concentrations of specific antibiotics, are readily available. The relative
effectiveness of different antibiotics provides the basis for the sensitivity spectrum of the
organism. This information, together with various pharmacological considerations, is
used in the selection of an antibiotic for treatment. It should be emphasized that
chemotherapeutic agents are not chosen simply on the basis of the drug producing the
widest growth-inhibition zone. This is because of the nature of growth-inhibition
substances. The density or viscosity of the culture medium, the rate of diffusion of the
antibiotic, the concentration of the antibiotic on the filter disk, the sensitivity of the
organism to the antibiotic, and the interaction between the antibiotic and the medium
may affect the size of the zone. The disk diffusion method represents a simple
procedure for screening substances to determine if they have significant antibiotic
activity.
Results of Kirby-Bauer “antibiotic” susceptibility testing (A selection of the following

antibiotics will be available for use)
Inhibition zone diameter (mm)
Antibiotic Disk Concentration Resistant Intermediate Susceptible
Ampicillin 0.01mg ≤11 12-13 ≥ 14
Bacitracin 10 units ≤8 9-12 ≥ 13
Chloramphenicol 0.03mg ≤ 12 13-17 ≥ 18
Cefotaxime 0.03mg ≤ 14 15-22 ≥ 23
Cephalothin 0.03mg ≤ 14 15-17 ≥ 18
Ciprofloxacin 0.005mg ≤ 15 16.20 ≥ 21
Erythromycin 0.015 mg ≤ 13 14-17 ≥ 18
Gentamicin 0.01mg ≤ 12 13-14 ≥ 15
Penicillin G
Staphlococci 10 units ≤ 28 - ≥ 29
Other organisms 10 units ≤ 19 20-27 ≥ 28
Polymyxin 300 units ≤8 9-11 ≥ 12
Streptomycin 0.01 mg ≤ 11 12-14 ≥ 15
Tetracycline 0.03 mg ≤ 14 15-18 ≥ 19
Vancomycin 0.03 mg ≤9 10-11 ≥ 12
43
MATERIALS
Your unknown culture of choice
Filter disks containing a number of different antibiotics at various dosages
Ethanol
Nutrient agar pours
Ruler graduated in millimeters
Forceps
Sterile Petri plates, 150 mm in diameter
PROCEDURE - WEEK ONE
1. Uniformly “swab inoculate” the entire surface of the Petri plates with your culture.
This may be accomplished by swabbing to cover one-half of the plate area, rotating
the plate 90°, swabbing over the initial inoculum with the same swab and covering
one-half of the plate area, rotate the plate 90°, following the same procedure, and
rotate the plate one more time repeating procedure. You will be swabbing 4 different
times.
2. Aseptically place antibiotic disks on the culture plates.
3. Incubate at 35°C for 24h.
PROCEDURE - WEEK TWO
1. Measure the zones of inhibition for each antibiotic for each of the cultures. Record
your results (Figure 17-6).
2. Compare your results with the standard zones of inhibition for each antibiotic and
determine the sensitivity, resistance, or intermediary relationship of each organism to
each antibiotic.
Zone of inhibition
Antibiotic disk
No Inhibition
Kirby-Bauer plate showing antibiotic zones of inhibition.
44
SUPPLEMENTAL INFORMATION
1. Bauer, A.L., W.M.M. Kirby, J.C. Sherris, and M. Turck. 1966. Antibiotic
susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-
496.
2. Chart, H (ed.). 1994. Methods in Practical Laboratory Bacteriology. CRC Press,

Boca Raton, FL.
3. Becton Dickinson. The Difco Manual.

http://www.bd.com/ds/technicalCenter/inserts/difcoBblManual.asp
4. Murray, P. R. (Editor in Chief). 2007. Manual of Clinical Microbiology, 9th Edition.

Amer. Soc. Microbiol., Washington, DC.
5. Russell, A.D., W.B. Hugo, and G. A. J. Ayliffe. 1982. Principles and Practice of
Disinfection, Preservation and Sterilization. Blackwell Scientific Publications Ltd.,
Oxford, England.
45
APPENDIX A - CULTURE COLLECTION
Every industrial microbiology group maintains a culture collection so that all important
(and potentially important) strains are catalogued and available to the group for further
study. This is an extremely important function. When any patent involving the use of a
microorganism is filed, a culture of this organism must be deposited with the ATCC
(American Type Culture Collection). This semester you will develop and catalogue a
collection of organisms.
During the course of the experiments this semester you will isolate organisms from a
number of sources including food, water and soil. You may also bring in samples from
which to isolate organisms. You will keep a collection of some of these isolates for
characterization and identification later in the semester.
Using the skills you learned in General Microbiology; you will perform the quadrant
streak plate technique to isolate colonies and check the purity of cultures. Once
colonies are isolated you will transfer the colony to a slant to keep in your collection.
Remember that slants, because of their low surface area to volume ratio dry out much
more slowly and are appropriate for keeping viable culture for a much longer period of
time than a Petri plate.
During the course of the course of the semester, you will work independently on
establishing your culture collection. It is crucial to maintain a good record of all the
experiments you perform for your isolates. It should be detailed enough for another
research to reproduce your techniques.
The following will serve as guidelines for a successful project:
- Information about your “Organism Project”

o Growth conditions
o Media composition
o Experimental design on how to re-isolate the organism
o Suggestions for alternate growth media (for selection and differentiation, if
applicable)
- Your culture collection should contain at least 5 different organisms:

1. Isolate from Soil
2. Isolate from Beef
3. Isolate from Chicken/Turkey
4. Isolate from Water
5. From an environment of your choice
- For each organism, you should:

o Record the date of isolation
o Record the “environment” it was isolated from and the media that you first
used to isolate it
o Describe the growth conditions
o Describe cell morphology, Gram reaction, and motility
o Describe any antibiotic susceptibility/resistance
o Describe any other metabolic characteristics that you were able to study
o Suggestions for alternate growth media (for selection and differentiation, if
applicable)
46
APPENDIX B – MEDIA
(In alphabetical order; adapted from the Difco Manual)
All media recipes show amounts in g/L unless otherwise indicated.
Actinomycete Isolation Agar

Sodium Caseinate 2.0
Asparagine 0.1
Sodium Propionate 4.0
Dipotassium Phosphate 0.5
Magnesium Sulfate 0.1
Ferrous Sulfate 1.0 mg
Agar 15.0
Glycerol 5
Suspend 22 g of the powder in 1 L of purified water. Mix thoroughly. Heat with frequent
agitation and boil for 1 minute to completely dissolve the powder. Add 5 g of Glycerol.
Autoclave at 121°C for 15 minutes.
Bismuth Sulfite Agar

Beef Extract 5.0
Peptone 10.0
Dextrose 5.0
Disodium Phosphate 4.0
Bismuth Sulfite Indicator 8.0
Ferrous Sulfate 0.3
Agar 20.0
Brilliant Green 25.0 mg
agitation and boil for 1 minute to completely dissolve the powder. DO NOT
AUTOCLAVE. Evenly disperse the precipitate when dispensing. Use the medium the
same day it is prepared. Test samples of the finished product for performance using
stable, typical control cultures.
Eosine methylene blue (EMB)

Pancreatic Digest of Gelatin 10.0
Lactose 10.0
Agar 15.0
Suspend 37.4g of the powder in 1L of purified water. Mix thoroughly. Heat with frequent
agitation and boil for 1 minute to completely dissolve the powder. Autoclave at 121°C for
15 minutes.
47
Hektoen Agar
Proteose Peptone 12.0
Yeast Extract 3.0
Bile Salts No. 3 9.0
Lactose 12.0
Saccharose 12.0
Salicin 2.0
Sodium Chloride 5.0
Sodium Thiosulfate 5.0
Ferric Ammonium Citrate 1.5
Agar 14.0
Acid Fuchsin 0.1
Bromthymol Blue 65.0mg
Suspend 76g of the powder in 1L of purified water. Mix thoroughly. Heat to boiling with
frequent agitation to dissolve completely. Do not autoclave. Cool to 45 – 50°C and use
immediately.
MacConkey Agar
Bacto Peptone 17
Protease Peptone, Difco 3
Bacto lactose 10
Bacto Bile salts No. 3 1.5
Sodium Chloride 5
Bacto Agar 13.5
Bacto Neutral red 0.03
Crystal Violet 0.001
Suspend 50g in 1 liter distilled water. Heat to boiling with gentle swirling to dissolve
completely. Sterilize in the autoclave for 15 min @ 15 PSI (121°C). Avoid overheating of
the medium. Cool to 45 – 50˚C and aseptically dispense in approximately 20 ml amounts
into sterile Petri dishes. For MacConkey Agar + amp, add filter sterilized ampicillin to
cooled agar for a final concentration of 50 µg/ml.
MacConkey with Sorbitol

Peptone 15.5
Proteose Peptone 3.0
D-Sorbitol 10.0
Bile Salts 1.5
Sodium Chloride 5.0
Agar 15.0
Neutral Red 0.03
Crystal Violet 1.0 mg
agitation and boil for 1 minute to completely dissolve the powder. Autoclave at 121°C for
15 minutes. Cool.
48
Mannitol-Egg Yolk-Polymyxin Agar
Beef extract 1
Peptone 10
Mannitol 10
NaCl 10
Phenol red 0.025
Agar 15
Water 950 ml
Melt the agar and distribute the medium in bottles, 235/500 ml bottle. Autoclave @
121°C for 15 min. Cool to 50oC. Add 12 ml egg yolk emulsion (Difco - 50%), and 2.5 ml
polymyxin stock solution (5 mg/ml). Distribute in Petri dishes, about 20 ml/dish.
M-Enterococcus Agar
Yeast Extract 30.0
Peptone 10.0
Sodium Chloride 15.0
Esculin 1.0
Cycloheximide 0.05
Sodium Azide 0.15
Agar 15.0
Suspend 7.12g of the powder in100mL of purified water. Mix thoroughly. Heat with
frequent agitation and boil for 1minute to completely dissolve the powder. Autoclave at
121°C for 15 minutes. Cool to 45°C. Add 0.024 g of nalidixic acid and 1.5 mL TTC
Solution 1% (0.015g Triphenyl Tetrazolium Chloride). Adjust to pH 7.1 if necessary.
Dispense 4-6 mL into 9 × 50 mm Petri dishes. Test samples of the finished product for
performance using stable, typical control cultures.
MI Agar
Proteose Peptone No. 3 5.0
Yeast Extract 3.0
D-Lactose 1.0
MUGal (4-Methylumbelliferyl-β-D-galactopyranoside) 0.1
Indoxyl-β-D-glucuronide (IBDG) 0.32
Sodium Chloride 7.5
Monopotassium Phosphate 1.0
Sodium Lauryl Sulfate 0.2
Sodium Desoxycholate 0.1
Agar 15.0
Suspend 36.5g of the powder in 1L of purified water. Mix thoroughly. Heat with frequent
agitation and boil for 1minute to completely dissolve the powder. Autoclave at 121°C for
15 minutes and cool in a 50°C water bath. Add 5 mL of a freshly prepared 1 mg/mL
filter-sterilized solution of cefsulodin per liter of tempered agar medium (final
concentration of 5 µg/mL).
49
Mycophil
Papaic digest of soybean meal 10
Dextrose (glucose) 10
Agar 18
Suspend 38 g in 1 liter distilled water and heat to boiling to dissolve completely. Sterilize
in autoclave for 15 min @ 15 PSI (121°C). Cool the medium to 45-50°C and dispense
into sterile Petri dishes.
Tetrathionate Broth
Beef Extract 5.0
Peptone 10.0
Sodium Chloride 3.0
Calcium Carbonate 45.0
Sodium Thiosulfate (anhydrous) 38.1
Oxgall 4.7
1. Suspend 105.8g of the powder in 1L of purified water and boil gently.

2. Cool to below 45°C.
3. Add 19mL of iodine solution (20g iodine and 25g potassium iodide in 100 mL water)
and 9.5 mL brilliant green solution (0.1 g brilliant green in 100 mL water).
4. Adjust the pH of the complete medium to 7.0 ± 0.2 using 1N HCl.
5. Dispense into sterile tubes, mixing well to evenly disperse the calcium carbonate
Triple sugar iron agar (TSI)

Beef Extract 3.0
Yeast Extract 3.0
Pancreatic Digest of Casein 15.0
Proteose Peptone No. 3 5.0
Dextrose 1.0
Lactose 10.0
Sucrose 10.0
Ferrous Sulfate 0.2
Sodium Chloride 5.0
Agar 12.0
Phenol Red 24.0 mg
agitation and boil for 1 minute to completely dissolve the powder. Dispense into tubes
and autoclave at 121°C for 15 minutes. Cool in a slanted position so that deep butts are
formed.
50
Trypticase Soy (TSA)
Tryptone (pancreatic digest of casein) 15
Soytone (papaic digest of soybean meal) 5
NaCl 5
Agar 15
Suspend 40 g in 1 liter distilled or deionized water and heat to boiling to dissolve

completely. Sterilize in the autoclave for 15 min @ 15 PSI (121°C). Cool to 45-50°C.
Dispense as desired.
XLD Agar
Xylose 3.5
L-Lysine 5.0
Lactose 7.5
Saccharose 7.5
Sodium Chloride 5.0
Yeast Extract 3.0
Phenol Red 0.08
Sodium Desoxycholate 2.5
Ferric Ammonium Citrate 0.8
Agar 13.5
Suspend 55 g of the powder in 1 L of purified water. Mix thoroughly. Heat with agitation
just until the medium boils. Do not autoclave. Cool to 45-50°C in a water bath and use
immediately.
Yeast Nitrogen Base (Per Liter)
Nitrogen Source:
Ammonium Sulfate 5.0 g
Amino Acids:
L-Histidine Monohydrochloride 10.0 mg
LD-Methionine 20.0 mg
LD-Tryptophan 20.0 mg
Vitamins:
Biotin 2.0 µg
Calcium Pantothenate 400.0 µg
Folic Acid 2.0 µg
Inositol 2,000.0 µg
Niacin 400.0 µg
p-Aminobenzoic Acid 200.0 µg
Pyridoxine Hydrochloride 400.0 µg
Riboflavin 200.0 µg
Thiamine Hydrochloride 400.0 µg
Compounds Supplying Trace Elements:
Boric Acid 500.0 µg
Copper Sulfate. 40.0 µg
Potassium Iodide 100.0 µg
Ferric Chloride 200.0 µg
51
Manganese Sulfate 400.0 µg
Sodium Molybdate 200.0 µg
Zinc Sulfate 400.0 µg
Salts:
Monopotassium Phosphate 1.0 g
Magnesium Sulfate 0.5 g
Sodium Chloride 0.1 g
Calcium Chloride 0.1 g
Dissolve 6.7 g of base and 5 g of dextrose in 100 mL of purified water (with warming, if
necessary). Mix well. Filter sterilize. Store at 2-8°C.
52
APPENDIX C – GRAM STAINING
Materials
A culture of Gram-positive (Micrococcus luteus) and Gram-negative (Citrobacter freundii)
bacteria will be used as controls for this exercise.
Gram Stain Reagents

Crystal violet
Gram iodine
Safranin
Decolorizer: Acetone:Ethanol (50:50) – Red squirt bottle
Staining “KIT” (Staining tray, slide holder, clothes pin to hold slides)
Paper towels
Staining Procedure
1. Preparation of smears. We will always include positive and negative controls on all
of our Gram stains. This is the best way to evaluate our technique and learn the
Gram reaction of the organism of interest. Use your loop to add one loopful of
water to each oval drawn on the slide with wax pencil as shown below. Use your
needle to mix a SMALL sample of culture into the appropriate water drop and
spread it over the whole area of the oval. This will facilitate drying.
+ -
UNK
2. Dry the smears in the air and then gently heat fix by passing through a flame. Your
instructor will demonstrate heat fixing.
3. Crystal violet staining: Place the slide on a staining rack and flood each smear with
crystal violet. Stain for 1 minute.
4. Wash the crystal violet from the slide with water. Drain any excess water onto a
paper towel by tapping slide gently on the towel.
5. Flood the smears with iodine and retain iodine on slide for 1 minute.
6. Wash the iodine from the slide with tap water and drain off any excess water.
7. Decolorize: Place the slide on a staining rack and flood each smear with decolorizer
until there is no purple color in the draining liquid.
8. Briefly wash the smears with tap water. Drain any excess water.
9. Counterstain the bacteria with safranin for at least 2 min.
10. Wash with tap water. Blot dry.
11. Examine the slides under bright-field with no cover slip. Focus the slide beginning at
100x total magnification (10x objective), then directly switch to the 1000x total
magnification (100x objective). Don’t forget to add oil. You can add it directly to your
slide.
53
APPENDIX D – QUADRANT STREAK PLATE
The microorganisms found in any environmental niche are a diverse group consisting of
several to many species. Before studying the characteristics of any one of the species in
the laboratory, it is often desirable to isolate that species as a pure culture. A pure
culture contains only the progeny of a single microbial cell, grown in the absence of any
other cell types, in contrast to a mixed culture which may contain several different
species and strains. The quadrant streak plate (or isolation streak plate) technique is a
convenient method for obtaining an isolated colony that can be used to generate a pure
culture from a mixed culture. In this method, an inoculating loop (for a liquid culture) or
needle is used to streak cells from a mixed population across an agar surface. Large
numbers of cells are deposited along the streak lines initially; as the number on the loop
thins out the cells deposited become isolated (separated) from each other. Each
isolated colony that subsequently grows is assumed to have developed from a single,
isolated cell. The isolated colony on the plate is not a pure culture as it has not grown in
the absence of other cells types
1 2
4 3
Figure 5.1 The quadrant streak plate method for isolation of single colonies.
Quadrants are labeled 1-4. Method is described below.
To obtain a pure culture, cells are picked from an isolated colony with an inoculating
needle and transferred to a fresh culture medium. Cells that grow on the new medium
are a subculture of the cells from the original isolated colony. Subcultures are used for
subsequent study of the isolated organism. Contaminating organisms from the original
culture remain on the streak plate.
MATERIALS
Week 1
A mixed Culture
One plate of TSA, Bunsen burner/Striker, Test tube rack, Inoculating loop
PROCEDURE
1. Use your sharpie and divide your Petri dish into four quadrants. Number each
quadrant 1 – 4. Label your Petri dish with your section number – seat number.
2. Sterilize the inoculating loop in the flame of the Bunsen burner and cool for a “ten
count”. Remove a loopful of your mixed culture to the agar surface, “scrub” across
54
about a quarter section of the plate as demonstrated (Figure 5.1; #1). “Scrubbing” is
similar to “coloring” in the first quadrant - You are inoculating this area with a
continuous back and forth motion as if you were trying to rub off all of the cells
clinging to the loop.
2. Flame the loop to sterilize and cool. Touch the loop to a clean spot on the agar plate
to assure that it is cool. Streak through the area in the inoculated sector and into the
next sector four times. Use straight lines making sure to lift the needle before each
subsequent streak line. (Figure 5.1; #2).
3. Repeat as in step 2 (see Fig. 5.1; #3 & 4) until the four sectors on the plate have
been inoculated. The idea is to streak so that each succeeding sector contains fewer
cells than the preceding one, resulting in at least one sector with cells spaced far
enough apart to allow isolated colonies to develop.
4. Label the agar side of the plate; incubate the cultures, agar side up, @ 35°C. The
plates will be removed from the incubator after 24 hrs and refrigerated until the next
laboratory period.
WEEK TWO
1. Examine the pattern of growth to confirm that the procedure was followed correctly,
and that isolated colonies were obtained in one of the sectors. If you failed to get
isolated colonies, streak another plate.
2. Transfer cells from a desirable, isolated colony to an agar slant. Incubate @ 35°C for
24 hrs and refrigerate until the next laboratory period.
SUPPLEMENTAL MATERIAL
1. NSF: Micro e-guide; Laboratory Safety; Aseptic Techniques; Streaking plates.
http://www.microeguide.com/safe_at_frame.htm.
55
POCKET GUIDE: FOCUS AND RESOLUTION ADJUSTMENTS (@ 1000X TOTAL MAGNIFICATION)
a. BRIGHT FIELD: FOCUS

1. Lower stage using coarse focus; place slide securely into slide clip.
2. Rotate nosepiece to 10X objective lens; click into place making sure edge of coverslip (or stained image) is in
place under objective opening.
3. WHILE LOOKING AT THE MICROSCOPE, raise stage carefully to within 0.5 cm of 10X objective lens.
4. Raise condenser to the highest position, set condenser dial to “0” and close (stop down) the condenser
diaphragm.
5. While looking through the microscope, slowly lower the stage (using coarse focus) until image comes into or
nearly into focus. NOTE: TO LOWER THE STAGE, TURN COARSE FOCUS TOWARD YOU.
6. Use fine focus to sharpen image.
BRIGHT FIELD: MAXIMUM RESOLUTION
7. While looking through the microscope, close down field diaphragm until edges come into view.
8. Adjust condenser height to sharpen the edges of the diaphragm (sharp decagon visible) center as necessary.
9. Open field diaphragm until the edges of the decagon are at the edge of the field.
10. Adjust fine focus as necessary.
11. While looking through the microscope, open condenser diaphragm all the way and then close down until the
field just begins to dim (this is the numerical aperture [NA] adjustment).
12. While looking at the microscope, rotate nosepiece so that the 40X objective is in place.
13. While looking through the microscope, focus image: minimal adjustment of the FINE FOCUS ONLY
should be required. Repeat steps 7-11.
NOTE: IF THE IMAGE LOOKS CLOUDY OR NOTHING APPEARS TO BE IN THE FIELD OF VIEW,
CLEAN THE OIL OFF THE 40X OBJECTIVE AND REPEAT STEP 13.
14. Once in focus at 400X total magnification (40X objective), rotate nosepiece so no objective is in place.
15. Place a small drop of oil on slide according to instructions; rotate nosepiece until 100X objective is in the oil
and clicked into place.
16. Minimal adjustment of the FINE FOCUS ONLY should be required.
(Repeat NA adjustment as necessary)
b. PHASE CONTRAST: FOCUS

1. Lower stage using coarse focus.
2. Place slide securely into slide clip.
3. Rotate nosepiece so that 10X objective lens clicks into place – make sure edge of coverslip is in place under
objective opening.
4. WHILE LOOKING AT THE MICROSCOPE, raise stage carefully to within 0.5 cm of 10X objective lens.
5. Raise condenser to the highest position; set condenser dial to “10” or “PH1” (depending on your microscope).
6. While looking through the microscope, slowly lower the stage (using coarse focus) until image comes into or
nearly into focus.
NOTE: TO LOWER THE STAGE, TURN COARSE FOCUS TOWARD YOU.
7. Use fine focus to sharpen image.
PHASE CONTRAST: MAXIMUM RESOLUTION
8. While looking through the microscope, close down field diaphragm until edges come into view; center as
necessary.
9. Adjust condenser height to sharpen the edges of the diaphragm (sharp decagon visible).
10. Open field diaphragm until the edges of the decagon are at the edge of the field.
11. Adjust fine focus as necessary.
12. While looking at the microscope, rotate nosepiece so that the 40X objective is in place.
13. CHANGE THE CONDENSER DIAL SETTING TO “40” OR “PH2”.
14. Minimal adjustment of the FINE FOCUS ONLY should be required. Repeat steps 8-11.
NOTE: IF THE IMAGE LOOKS CLOUDY OR NOTHING APPEARS TO BE IN THE FIELD OF VIEW,
CLEAN THE OIL OFF THE 40X OBJECTIVE AND REPEAT STEP 14.
15. Once in focus at 400X total magnification (40X objective), rotate nosepiece so no objective is in place.
16. Place a small drop of oil on slide according to instructions received.
17. Rotate nosepiece until 100X objective snaps into place; CHANGE THE CONDENSER DIAL SETTING TO
“100” OR “PH3”.
18. Minimal adjustment of the FINE FOCUS ONLY should be required.

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5

3: Effect of Temperature on Growth……………………………………………………………...12

4: Evaluation of Commercial Yeast Products…………………………………………………...17

5. Microbial Food Spoilage………………………………………………………………………..21

9. Preparation of a Malt Beverage…………………………………………………………….…..35

10. Comparison of Microbial Populations: RFLP Analysis of 16s DNA……………………….39

11. Kirby Bauer Method…………………………………………………………………………….42

Appendix A: Culture Collection……………………………………………………………………45

Appendix B: Media Ingredients……………………………………………………………………46

Appendix C: Gram Staining………………………………………………………………………..52

Appendix D: Quadrant Streak Plating ……………………………………………………………53

Faculty Coordinator: Dr. Ines Rauschenbach

Date Topic Exercise

Applied Microbiology Lab Website: http://www.rci.rutgers.edu/~microlab/

Applied Microbiology Website

Required Laboratory Notebook

Culture Collection 100 points

• Do not bring food or drinks into the laboratory - EVER!

General Instructions for disposal:

4. Do not hesitate to include anything you think is important or interesting in your

3. Introduction. The introduction should contain some background material to the

The quality of the observed image depends on three important factors:

Achieving maximum information from an observed image requires a clear understanding

EQUIPMENT AND MATERIALS

A. General Comments on the Use of the Microscope

B. Adjustment for maximum resolution at 100x magnification under bright field

C. Adjustment for maximum resolution at 400x.

1. Place a loopful of culture or the suspension provided on the slide (Note: it is

E. Microscopic examination of a bacterial suspension by using the 100x oil

The phylum Actinobacteria is a large group of Gram-positive bacteria. They form

One important genus belonging to Actinobacteria is the Streptomyces. These spore-

PROCEDURE – Antibiotic Testing for your Unknowns

1. Use loop to streak Streptomyces #

2. Use loop to streak the test

Growth rate equation

µ = LogODn - Log ODo x 2.303 Slope = µ

µ = growth rate constant

µ = growth rate constant

Limiting or increasing the size of microbial populations by controlling the temperature of

MATERIALS & EQUIPMENT

2. Repeat step #1 at each time interval.

Data Tables – Procedure 1

Time Temperature (°C)

Data Tables – Procedure 2

Time Temperature (°C)

A. TOTAL CELL COUNT

5. Count the yeast cells in 80 small squares. To

1/20 mm X 1/20 mm X 1/10 mm = 1/4000 mm3

B. DIFFERENTIAL PLATE COUNTS

C. PRODUCTION OF CARBON DIOXIDE BY YEAST CELLS

5. Plot volume of carbon dioxide evolved versus

Although spoilage is slowed by refrigeration, psychrotrophic organisms, present on

• Properly or improperly handled ground beef samples

• Week 1: Plates of trypticase soy (TSA); Eosine methylene blue (EMB)

2. Transfer each 10 g portion to a sterile stomacher bag containing 90 ml buffer. Blend

1. Preenrichment (as necessary) in a non-inhibitory medium to allow recovery of

The Enrichment Media

Selenite cystine broth

The Selective/Differential Media

Selective Media - Used to SELECT (ISOLATE) specific groups of bacteria (Include

Bismuth sulfite agar

Bismuth sulfite agar: Salmonellae colonies will be greenish-gray to black,

XLD agar (xylose, lysine, desoxycholate): Salmonellae colonies appear red