Microbiology Exercises
For BIOL C210 –
General Microbiology
Pedro J. A. Gutiérrez, PhD
Coastline Community College
Page | 1 Microbiology Exercises
4th Edition
Copyright © 2012 by Pedro J. A. Gutiérrez, PhD
Page | 2 Table of Contents
Lab Preparation: Safety ....................................................................................................... 5
Lab Preparation: Biohazard waste disposal ........................................................................ 7
Lab Preparation: Lab Map .................................................................................................. 8
Lab Preparation: Lab Tools ................................................................................................ 9
Basic Lab Techniques: Plates and Tubes ........................................................................... 10
Exercise 1: Cultivation of Environmental Microbes.......................................................... 11
Exercise 2: Aseptic Technique ........................................................................................... 17
Exercise 3: Bacterial Growth Curves .................................................................................25
Exercise 4: Pure Culture Isolation .................................................................................... 30
Exercise 6: Microscopy – Part II ..................................................................................... 40
Exercise 7: Sample Preparation for Microscopy .............................................................. 43
Exercise 8: Identification of Bacterial Unknowns ............................................................45
Exercise 9: The Eukaryotes............................................................................................... 50
Exercise 10: The Simple Stain ...........................................................................................56
Exercise 11: The Negative Stain .........................................................................................59
Exercise 12: The Gram Stain .............................................................................................. 61
Exercise 13: The Endospore Stain .................................................................................... 64
Exercise 14: Selective and Differential Media .................................................................. 68
Exercise 15: Physical Requirements for Growth ............................................................... 72
Exercise 16: Biochemical Profiles of Microbes ..................................................................78
Exercise 17: Using BLAST to find sequence similarities .................................................. 85
Exercise 18: DNA cloning ................................................................................................. 94
Exercise 19: Chemical Transformation of Bacteria ......................................................... 109
Exercise 20: The Standard Plate Count........................................................................... 113
Exercise 21: Physical Methods of Controlling Microbial Growth ....................................117
Exercise 22: Chemical Methods of Controlling Microbial Growth ................................. 122
Exercise 23: Water Quality .............................................................................................. 133
Exercise 24: Milk Quality................................................................................................. 141
Exercise 25: A Synthetic Epidemic .................................................................................. 144
Page | 3 Exercise 26: The Capsule Stain........................................................................................ 149
Exercise 27: The Acid Fast Stain ..................................................................................... 152
Exercise 28: Biofilms ....................................................................................................... 156
Exercise 29: Getting information from PubMed ............................................................. 162
Appendix A: Microbes used in the lab ............................................................................. 165
Page | 4 Lab Preparation: Safety
The Science Department at Coastline Community College is committed to providing a safe
laboratory environment. These safety guidelines have been established for your protection
and will be rigidly enforced. Non-compliance may result in a grading penalty and/or
dismissal from the laboratory.
SAFETY GUIDELINES:
1. Be prepared! Read laboratory procedures prior to coming to that laboratory
session. By being knowledgeable on the procedure you are about to perform, you will
assure the safety of yourself and those around you.
2. Only students registered in the course will be permitted in the laboratory.
3. Lab coats must be worn at all times. The garment must cover your arms. It must
button or snap down the front so it can be removed without pulling it over your head.
Always leave lab coats in the lab and do not wear in non-lab areas. NEVER
take lab coats home unless authorized by the Laboratory Manager.
4. Absolutely no food, drinks or gum are allowed in the laboratory at any time. This
includes water bottles. Do not apply any cosmetics while in the laboratory.
5. Closed-toed shoes must be worn at all times. Sandals and open-toed shoes will
not be allowed in the laboratory.
6. Tie back long hair to avoid contact with open flames and contamination.
7. Backpacks, purses and coats must be placed in lockers before entering the lab. Keep your
workspace free of unnecessary items and clutter.
8. Wash your hands thoroughly when you arrive, after you are finished with the assigned
lab experiment and before you leave the lab for any reason. Antimicrobial soap is
provided in the lab; please do not use any other soap.
9. Wipe down your lab bench with disinfectant at the beginning of class, after you are
finished with your lab experiment, and immediately after any spills.
10. All cultures are to be treated as potential pathogens and the following
precautions are to be followed:
a. Place all cultures and media tubes upright in a test tube rack when moving
around the laboratory and when working at your lab bench.
b. Do not tilt or invert test tubes unless specifically instructed to do so. This is the
number one cause of contamination.
c. Label all media clearly with your name, date, lab section, and organism.
Page | 5 d. Do not walk about the laboratory with inoculating loops, needles, or pipets
containing infectious material.
e. Spilled cultures are to be covered with paper towels and saturated with disinfect
for 15 minutes.
f. Removal of any materials from the laboratory is strictly prohibited unless
authorized by the Laboratory Manager.
11. Be aware of safety precautions on chemical reagent bottles. Material Safety Data Sheets
(MSDS) are available for all chemicals in the laboratory.
12. Turn off Bunsen burners when not in use and make sure gas jets are tightly closed before
leaving the laboratory.
13. Familiarize yourself with the location of all safety equipment in the laboratory (e.g. eye
wash stations, fire extinguishers, first aid kit and safety goggle bin) and emergency
escape routes.
14. Report all spills, accidents, cuts and injuries to your instructor, no matter how minor.
15. Dispose of all biological waste in their proper container (see next page).
16. Any additional conditions and/or procedures set forth and communicated by Coastline
faculty and/or staff (both written and oral) must be adhered to.
Page | 6 Lab Preparation: Biohazard waste disposal
Please dispose items in special containers as indicated below:
Always remember to put biohazard bag in contaminated discard bin. When full, change out bag
and autoclave as soon as possible.
Material
Method of Disposal
Media in tubes with biological material
Place tube upright in indicated test tube rack
in “tubes to be autoclaved” bin
Biological liquid in flask or bottle
Place in container as indicated by instructor
Biological liquid in test tubes
Place tube upright in indicated test tube rack
in “tubes to be autoclaved” bin
Broken glass
Broken glass container
Contaminated swabs
Place in “contaminated discard” bin
Glass slides
Please in “slide discard” bin
Needles, syringes and other sharps,
whether contaminated or not
Sharps container
Non-contaminated paper
Regular trash
Petri dishes and contaminated solids
Place in “contaminated discard” bin
Pipets
Pipet discard tray, located by every sink
Pour tubes and other glassware that is
NOT contaminated
Wash glassware and place in racks next to
sink. Put caps in white plastic basket.
Transfer pipets
Place in “contaminated discard” bin
When test tube racks are full in “tubes to be autoclaved bin, remove and place on biohazard
waste cart. Place a new empty rack in its place for the next class.
Please sign the Laboratory Safety Agreement.
Page | 7 Lab Preparation: Lab Map
Draw a lab map below including location of lab safety equipment, waste disposal and culture
sample locations.
Page | 8 Lab Preparation: Lab Tools
Below is a list of lab items in your lab drawer box.
Draw and label each of the items in your lab drawer box in the space below.
Lab drawer Items
Bibulous paper
Lens paper
Butane lighter
Clothes pins
Immersion oil bottle
Lens cleaner bottle
Inoculating loops
Inoculating needle
Fill out the lab drawer checklist form.
Microscope cord
Ruler
Sharpie pens
Test tube clamp
Forceps
Labeling tape, white
Basic Lab Techniques: Plates and Tubes
The use of Petri plates in microbiology is primarily restricted to the growth of microorganisms
on agar with nutrients. To be able to observe growth and make sure that you can believe the
results of your experiments, the following rules must be followed when using Petri plates:
1. Label the bottom of the plate, not the lid. The bottom of the plate is the smaller “half”
and holds the agar. If you label the lid, it can rotate during transport or incubation and
you will not be able to identify your samples.
2. Write around the edge of the plate, so that you can more clearly see growth in the middle
of the plate when you hold it up to the light.
3. Your label should always include: First name Last initial, Section ID, name of organism
(Capital letter for genus, written out species name), type of media, and other important
experimental conditions.
4. To incubate plates, they are placed in the incubator (or on a designated space) with the
AGAR AWAY from the table (bottom of the plate faces the ceiling). This will make sure
that any condensation that accumulates on the plates does not fall on your colonies while
they are growing, making it impossible to interpret in your results.
For working with tubes containing liquid medium, the following rules apply:
1. Use the Sharpie to write on the tube directly, but AVOID the white factory label on the
tube (it is very hard to remove markings from this area).
2. Your label should always include: First name Last initial, Section ID, name of organism
(Capital letter for genus, written out species name), type of media, and other important
experimental conditions.
3. Do NOT TILT tubes when working with them. Even though they have caps, the caps are
not airtight so that our little microbes can take up oxygen. If you tilt the tube, the culture
or media will spill and possibly contaminate you as it lands on your hands…
Page | 10 Exercise 1: Cultivation of Environmental Microbes
Goal: To investigate whether microbes can be found in various environmental
locations and become familiar with diverse colony morphology of microbes
Introduction: Scientists have found microbes in almost every environment on earth:
deep in the ocean, many kilometers below the earth’s crusts, in hot springs and in the
permanent ice of the Arctic. By definition, most microbes (except the helminths),
cannot be seen well with the naked eye, which means that we require microscopes to
observe them well. Yet when they multiply in a single spot, such as on an agar plate that
contains nutrients, they form mounds of cells called colonies. These colonies can be
easily seen with the naked eye and can differ in size, shape, color and texture depending
on the characteristics of the microorganism.
Culture media is used to stimulate growth of microorganisms. Media is divided into two
groups: complex/non-synthetic media and synthetic media. Complex/nonsynthetic media contains plant and animal tissue extracts which provide all the essential
chemical components to support life. The media is called complex because the exact
chemical composition is unknown. Synthetic media, on the other hand, is produced
from known chemicals following specific recipes, so we are sure of its chemical
composition. Both Tryptic Soy and Nutrient Media are considered complex media
because they contain beef extract.
Materials (per student):
•
•
•
1 Tryptic Soy Agar (TSA) or Nutrient
Agar (NA) plate
1 Sterile swab
1 Tube of sterile water
Materials (per group):
•
1 broth culture of Escherichia coli,
Staphylococcus epidermidis or other
bacteria.
Procedure:
Summary
You will swab a sample site where you think there may be bacteria present and streak
the swab on a complex media plate and then observe the type of growth on the plate.
1. Record the actual bacterial strain used in this experiment:
Strain
Purpose
Page | 11 2. Label your plate with all the necessary information.
3. Divide the plate into three sectors as shown in the picture below. One sector will
be for your negative control, one will be for your sample and one will for your
positive control.
4. Decide on your sample site (must be at room temperature) and formulate your
question below:
_______________________________________________________
5. Carefully open your sterile swab and dip it in the sterile water tube. Streak this
on the appropriate control sector.
6. Go to your sample site and roll the swab on the surface of the sample back and
forth several times.
7. Transfer the sample to the agar plate using the technique presented in lab. Make
sure that you are rolling the swab on the surface of the agar and not digging into
it.
Page | 12 8. Dip your used swab into the culture tube and streak it out onto the appropriate
control sector.
9. Dispose of the swab in the biohazard bin and group all the plates from your team.
Tape them together and place them in the designated incubation area (make sure
all the plates have the agar side facing UP).
10. Plates will be incubated at room temperature (approximately 25C) for 24-48
hours.
11. Your negative control was your swab dipped in _______________ and
streaked out on the plate.
12. Your positive control was your swab dipped in _______________ and
streaked out on the plate.
Page | 13 Filamentous -­‐ threadlike
Serrate – jagged, toothlike
Undulate – wavy indentations
Lobate – clear indentations
Umbonate – raised with convex center
Convex – dome-­‐like elevation
Raised – slight elevation
Flat – no elevation seen
Rhizoid – branching, spreading growth
Irregular – dimpled edges
Circular – unbroken, continuous edge
Elevation:
Smooth – even, well-­‐defined
Shape:
Margin:
Results:
Was there growth on your negative control?
Yes
No
Was there growth on your positive control?
Yes
No
Did the growth pattern on your controls correspond to what was expected? Yes
No
If not, explain what may have happened: ________________________________
____________________________________________________________
____________________________________________________________
The total number of colonies in your sample sector is: ___________
Class results:
Source
Number
of colonies
Source
Number
of colonies
Source
Number
of colonies
Page | 15 Colony morphology
The number of different colony types in your sample sector is: ___________
Pick two different types of colonies and describe their shape, color, margin and
elevation.
Type 1
Type 2
Shape
Color
Margin
Elevation
Please write your conclusion below:
____________________________________________________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 16 Exercise 2: Aseptic Technique
Goal: To pour sterile complex media plates and aseptically transfer a bacterial
culture onto three types of media.
Introduction: In order to study any organism that is too small to see with the naked
eye, it is necessary to allow it to multiply in number to the point when it can be analyzed.
This holds true for bacteria and yeasts, whose individual cells cannot be seen without
the help of a microscope. The biggest problem though is, how do we know that we are
studying a pure population of a single type of microorganism and don’t have unwanted
contaminating microbes?
Fortunately, we have laboratory techniques that ensure we are working with a pure
population of organisms. The techniques used to minimize contaminants are called
Aseptic (from sepsis, which means rotten). These techniques allow us to grow pure
bacterial cultures on solid media and broth (liquid) media.
When bacteria are introduced into a medium this is called inoculation. As you could
guess, the media must be free from microbes to begin with for us to grow a pure culture.
The removal of all life from an object is called sterilization. Thus, we always have to
use sterilized media to be able to study a single type of microbe.
Media is made in liquid or solid form. The nutrients are the same in terms of
concentration. The only difference between media in liquid or solid form is that the
solid form contains agar, a carbohydrate derivative made from seaweed that is used as a
solidifying agent (it is also used in cooking). As with gelatin, agar melts at high
temperature and is solid at room temperature. The higher the concentration of agar in
the media, the “stiffer” the media becomes after it has solidified.
Broth cultures are placed in test tubes or larger flasks (usually Erlenmeyer flasks), while
solid media is mostly used to make plates, slants or deeps. Slants are test tubes
containing solid culture media that is at an angle. To make them, molten media is
added to a test tube and then tilted so that the media solidifies in a slanted position.
Deeps are made the same way except that the test tube is NOT tilted when it is allowed
to cool. Another difference between deeps and slants is that the concentration of agar in
deeps (0.5-0.7%) is about one-third of the concentration in slants (1.5%). (The
concentration in agar plates and slants is the same.)
What are some advantages to these different forms of media? Broth cultures are used to
grow many, many cells in a small volume. This is because the cells in liquid media have
greater access to nutrients. A big advantage of slants is that they are easy to store and
transport. Deeps are used to grow bacteria that require lower than atmospheric
concentration of oxygen. In addition, semi-solid deeps (those with 0.5-0.7% agar) can
Page | 17 be used as an assay (test) for motility. If the bacteria are motile, you will get growth
radiating from the inoculation site. The final result then looks like an inverted
Christmas tree.
Materials (per student):
•
•
•
1 nutrient (or TSB) broth tube
1 nutrient agar (or TSA) slant
1 nutrient agar semi-solid deep
•
•
•
Inoculating loop and needle
1 bacterial broth culture as provided
1 TSA pour
Procedure:
Plate pouring
To begin we will make a petri plate from a complex media agar. A pour is a test tube
containing media with molten agar which is transferred directly into a sterile petri plate.
Once solidified and dried for a few days, the plate can be used for growing microbes.
1. Label the bottom of empty petri plates with your first name and last initial,
“TSA” and the date.
2. Take a large tube with molten TSA (in the 50˚C water bath). With a paper towel,
wipe the outside so no condensation falls into the sterile petri plate.
3. Aseptically open the tube, flame the lip twice over the Bunsen burner and pour
completely into a sterile Petri plate that is clam-shelled.
4. Make sure that no edges (from the tube or plate) touch each other!
5. Replace the petri plate lid. Swirl very gently to spread the agar on the bottom of
the plate. Do **not** leave the lid off while the agar plate solidifies.
6. Rinse the test tube with tap water and place in designated test tube rack.
7. Allow plates to completely solidify on your bench before moving or storing.
Page | 18 Aseptic transfer of bacteria
To transfer bacteria, one usually uses an inoculating loop or inoculating needle. In
general, a loop transfers many more microbes than a needle.
Use a loop when going from
Plate à plate
Plate à slant
Plate à broth
Slant à slant
Slant à plate
Slant à broth
Broth à broth
Broth à plate
Broth à slant
Use a needle when going from Broth à deep, Slant à deep.
Culture
Loop
Loop
Needle
Broth Slant Deep
Each person will work with one culture and inoculate three different types of media with
either an inoculating loop or needle as shown in the picture. You will also collect data
from other members of your group, so that you are analyzing a total of four cultures.
Page | 19 1. Label your broth, slant and deep according to class instructions.
2. Inoculate the broth following these instructions:
a. Gently flick the culture tube to mix the bacteria in the broth.
b. Hold the loop in your dominant hand like a pencil and the culture tube in
the other hand
c. Sterilize the loop in the Bunsen burner flame (near the top) until it is red
hot
d. Use your little finger to gently take off the cap from the culture tube.
e. Hold the tube at around a 20º angle (near the flame) and pass the mouth
of the tube through the flame briefly three times. Make sure that the
opening of the tube does not touch anything.
f. Insert the loop into the tube and move the tube until the loop is dipped in
the culture just below the surface. If you hear a “sizzle”, the loop was too
hot. Repeat the procedure, but after sterilizing the loop, count five
seconds before you take the cap off the culture tube.
g. Flame the tube opening and turn the tube into the cap to recap it. Return
the culture tube to the rack.
h. Remove the cap from the broth tube as before, briefly flame the opening
three times.
i. Insert the loop into tube and move the tube until the loop is dipped in the
culture just below the surface. Withdraw the tube away from the loop,
flame the opening of the tube, cap it and place it in the rack.
3. To inoculate the slant,
a. Repeat instructions 2a-2g
b. Remove the cap from the slant as you did for the other tubes, briefly flame
the opening three times and insert the loop.
c. Move the tube until the loop reaches the bottom of the slant. Avoid
touching the sides of the tube!
d. Gently place the loop on the agar and then pull straight from the bottom of
the slant to the top tracing a line. The loop should glide over the agar, not
gouge it. (Note: normally when you inoculate a slant you will move the
loop in a zig-zag motion from the bottom to the top)
4. To inoculate the deep,
a. Sterilize your inoculating needle, let it cool and then straighten it out as
much as possible. The straighter the needle the better the inoculation into
the deep.
b. Repeat instructions 2a-2g, using the inoculating needle.
Page | 20 c. Remove the cap from the deep tube as before, briefly flame the opening
three times.
d. Insert the needle into tube and move the tube until the needle has been
inserted into the middle of the deep all the way to the bottom. Carefully
withdraw the tube away from the needle, flame the opening of the tube,
cap it and place it in the rack.
5. Incubate media at 37˚C until the next lab session.
6. Record the appearance of your cultures.
Page | 21 Results:
Pours
Was your plate contaminated (circle one)?
YES
NO
If so, give a possible explanation reflecting on your technique and what modification you
would make:
____________________________________________________________
____________________________________________________________
____________________________________________________________
Broth
Define the following terms
Turbidity - __________________________________________________
Pellicle - ____________________________________________________
Sediment - __________________________________________________
Floculence - _________________________________________________
Pigment - ___________________________________________________
Characterize your broth culture
Bacterium
Turbidity
(-,+,++)
Presence of pellicle,
sediment or
floculence
Pigment
For Turbidity use the following scale (use an index card with lines):
- (no turbidity/clear), + slightly turbid, ++ very turbid
Page | 22 Slants
Sketch bacterial growth appearance on slants and note culture color (pigment)
Bacterium:
________
________
________
________
Growth
Pattern:
__________
__________
__________
__________
Color:
__________
__________
__________
__________
Deeps
Draw bacterial growth in deeps
__________
Motility:
(Yes/No)
__________
__________
__________
Bacteria:
__________
__________
__________
__________
Page | 23 Conclusions:
As a conclusion, summarize the phenotypes for your individual culture.
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 24 Exercise 3: Bacterial Growth Curves
Goal: Experimentally calculate the generation time for a bacterial strain from
Absorbance measurements and from given numbers.
Introduction: Almost all bacteria reproduce via binary fission. This is a form of
asexual reproduction in which a mother cell divides into two daughter cells. Mitosis,
which is also a form of cell division, only applies to eukaryotes. The amount of time it
takes for a cell to divide into two cells is called the generation time (or doubling time).
The doubling time is a unique characteristic for prokaryotic cells, but it can vary
depending on the environmental conditions. We therefore observe that among bacteria
doubling times have wide-ranging values. For example, Escherichia coli has a doubling
time of about 20 minutes while Mycobacterium tuberculosis has a doubling time of 15
hours. Notice that when we refer to growth, we are referring to an increase in the
number of bacteria due to cell division, not to an increase in size of an individual
bacterium.
To calculate the doubling time for a strain, we need to know the starting and ending
number of cells in the culture, as well as the time that has elapsed. We also assume that
every time a cell divides, it produces two daughter cells. For example, to go from 1 to 16
cells, we need four divisions (1à 2 à 4 à 8 à 16). If these four divisions took 1 hour
(60 minutes) to complete, then each division takes 15 minutes (doubling time = time
elapsed/#of divisions). So the key is to find the number of divisions and divide the time
the culture was growing by this number. We can calculate the number of divisions (n)
from the following equation: Nf=No x 2n. This equation tells us how the cell numbers
present in a culture at the beginning (No) and at the end (Nf) are related to the number
of cell divisions (n). If we solve for n, n = log(Nf/No)/log(2). Since doubling time (Td) =
elapsed time (t)/ # of divisions (n), we get the final equation:
T d= t x
log 2
log(Nf/No)
Although doubling times can vary a lot, the way that bacteria grow when given nutrients
is very similar. We can verify this with bacterial growth curves: graphs which track
the number (or concentration) of bacteria present in a culture through a defined time
period. The shape of a typical growth curve is shown in the data section. When we carry
out this experiment, regardless of the strain, we identify four phases in a bacterial
growth curve. These are the lag, log (exponential), stationary and death
(decline) phase. Each phase gives us some knowledge of what the bacteria are doing
Page | 25 at the time. When bacteria are first placed in new medium (i.e., plenty of nutrients),
they show no growth. This phase is called the lag phase and is when the bacteria are
starting to respond to their environment. No active protein synthesis is occurring doing
this phase, and since binary fission requires the work of proteins, no bacterial cell
division is occurring either. In the log phase, bacteria are actively synthesizing
proteins (recall DNA à RNA àprotein) and all other cellular components, utilizing the
plentiful nutrients in the environment. Bacteria grow most rapidly when they are
actively metabolizing nutrients and so we observe exponential growth during this phase.
As the availability of nutrients decreases and metabolic wastes production increases, the
rate of cell growth matches the rate of cell death and curve flattens out. This since the
rate of growth does not change, we call this stationary phase. The last phase of a
bacterial growth curve is when too much waste has been produced and there are very
few nutrients left in the culture. At this point, cells begin to die more rapidly and we
observe this by the decreased growth rate of the death phase.
So how do we actually track the number or concentration of bacteria to draw a growth
curve? There are two methods: one involves directly counting the number of bacterial
cells in a broth culture and the other involves measuring increasing turbidity in a
culture. Direct counting of cells usually involves taking a small sample of the broth
culture, mixing it with a dye and placing the sample on a special microscope slide called
a hemocytometer. This slide contains a microscopic grid that corresponds to a
specific volume (usually 10-4 ml). Once you count all the bacteria in the grid, you
multiply the total number by the factor that gives you 1ml. To measure increasing
turbidity as bacterial cells grow, we use an instrument called a spectrophotometer.
This instrument shines a light through a sample and has a detector opposite the light
source, which measures the amount of light that makes it through and expresses it as a
percentage. The light that makes it through is given as % transmittance, whereas the
light that is scattered by the sample is given as % absorbance.
By understanding the dynamics of bacterial growth, we can better design strategies to
control microbial growth. A common example is the use of Penicillin. One can add
penicillin to cells in all four phases of the growth curve, but only one is able to effectively
kill cells. Penicillin inhibits the enzymes which cross-link peptidoglycan layers in cells.
This antibiotic is particularly good against gram-positive cells due to their thick
peptidoglycan layer in the cell wall. But for penicillin to be effective, the cell has to be
actively producing new peptidoglycan, which is when it would activate the
peptidoglycan linking enzymes. This only happens in the log or exponential phase,
when cells are actively dividing. If there is no cell division, the cell has no need of
making new peptidoglycan, making it pointless to treat with penicillin since the linking
enzymes are not being expressed.
Page | 26 Procedure:
Summary
You will first label the different phases of a typical growth curve. Then using absorbance
measurements of an unknown bacterial culture received through email, you will record
the absorbances in your table and draw the graph. You will determine a doubling time
for your data and then write down the absorbances that you lab partner received. Using
their absorbances you will plot the graph and calculate the doubling time. After plotting
the graphs you will calculate the doubling time using the equation presented in the
introduction.
1. Label the various phases of the growth curve
2. Place an asterisk (*) next to the phase in which bacteria are most sensitive to
penicillin. Place a diamond (t) next to the phase that is used to calculate the
doubling time.
3. Record your Absorbance measurements in the table below.
4. Graph the data
5. Determine the generation time by finding the closest time interval where the
absorbance doubled.
6. Repeat Steps 3-5 with your lab partner’s measurements.
7. Calculate the doubling time for problems listed using the equation in the
introduction.
Bacterial number (log)
Data:
Time
Page | 27 Your measurements
Time (min)
Absorbance
Time (min)
Lab partner’s measurements
Absorbance
Time (min)
Absorbance
Time (min)
Absorbance
Page | 28 TimeInitial Absorbance:_______ TimeDoubled Absorbance: ______ Doubling time: ________
Lab partner’s data:
TimeInitial Absorbance:_______ TimeDoubled Absorbance: ______ Doubling time: ________
*Note*TimeInitial Absorbance refers to the time when your cells are at the beginning of log
phase.
A culture was inoculated with 20 bacteria and incubated for 6 hours. The final number
of bacteria in the culture was 7 x 106 cells. What is the doubling time of this strain?
Doubling time: ________
Another culture was inoculated with 3 bacteria and grown for 4 hours. At the end of the
incubation, there were 48 cells in the culture. What was the doubling time of this
strain? (Make sure that you calculate the doubling time two ways: using the equation
and by reasoning it out.) Doubling time: ________
Did all your plotted curves show all phases of bacterial growth? YES
NO
If your answer was “NO” which phase was the one that was not detected?
LAG
LOG
STATIONARY
DEATH
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 29 Exercise 4: Pure Culture Isolation
Goal: To isolate single colonies from a mixed culture of bacteria using two streak
plate methods.
Introduction: Microbes, as all other living organisms, live in close proximity to other
species. This makes it difficult to study the characteristics of a particular microorganism
in its native environment. To study a specific microbe effectively, we must grow it
separately from all other microbes. A pure culture refers to a culture that contains
only one species of microbe; if other species are present, we call them contaminants.
The best way to assure ourselves that we have a pure culture is to separate out one
microbial cell and then let it grow. We may imagine that this is technically impossible,
because even with our great microscopes we don’t have micro-tweezers to pick up
exactly one microbe and then place it in culture media. Fortunately, this problem was
solved for us at the beginning of the golden age of microbiology when agar was first
combined with culture media. Agar had been used as a thickener in cooking, and in fact
it was Angelina Hesse, the wife of one of Robert Koch’s colleagues that suggested its use
in growing microbes. This allowed Koch to isolate pure colonies and finally prepare
pure cultures. So how do we separate out single cells on an agar plate? Basically, we
use a bacteriological loop to drag bacteria from one part of the plate to another part
until several single bacteria come off the loop. Each time we drag the loop across the
plate it is called a streak, and therefore this method is called the streak plate
method.
Materials (per student):
•
•
2 nutrient agar (or TSA) plates
Bacteriological loop
Materials (per pair):
•
•
•
•
1 Bunsen burner
1 practice plate (agar only or as
determined by instructor)
1 Test tube rack
1 mixed bacterial culture (strains to be
specified in class)
Procedure:
Summary
You will use two “streak plate” methods based on the “T streak” example shown in class
and another method from a web search. After incubation you will evaluate which streak
method yielded the most single colonies. The most effective streak method will be your
“personalized” method that you will use throughout the semester.
Page | 30 1. Label both plates appropriately, making sure not to label the center of the plate.
2. Go to Google images and enter “Streak plate methods”.
3. Pick a method and draw it in the Method B circle (include a * when you flame
the loop and number the streaks):
Method A: T-streak
Method B
4. Turn on the Bunsen burner, sterilize your loop and then streak the practice plate
as directed by be the instructor.
5. Streak the plate using streak plate method A following these instructions:
a. Gently flick the culture tube to mix the bacteria in the broth.
b. Hold the loop in your dominant hand like a pencil and the culture tube in
the other hand
c. Sterilize the loop in the Bunsen burner flame (near the top) until it is red
hot
d. Use your little finger to gently take off the cap from the culture tube.
e. Hold the tube at around a 20º angle (near the flame) and pass the mouth
of the tube through the flame briefly three times. Make sure that the
opening of the tube does not touch anything.
f. Insert the loop into the tube and move the tube until the loop is dipped in
the culture just below the surface. If you hear a “sizzle”, the loop was too
Page | 31 g.
h.
i.
j.
hot. Repeat the procedure, but after sterilizing the loop, count five
seconds before you take the cap off the culture tube.
Flame the tube opening and turn the tube into the cap to recap it. Return
the culture tube to the rack.
Gently slide the loop over the agar following streak pattern for streak #1.
Flame the loop, let it cool, rotate the plate 90 degrees and follow pattern
for streak #2. (Note: Do NOT dip the loop back into the culture after
flaming it!)
Repeat i. for streaks #3 and #4 as appropriate.
6. Streak the other plate using the instructions in #2, but using streak plate
method B.
7. Flame the loop after you have finished and group all the plates from your team.
Tape them together and place them in the designated incubation area (make sure
all the plates have the agar side facing UP).
8. Plates will be incubated at 35-37°C for 24-48 hours.
Results:
The two strains in the mixed bacterial culture are: __________________________
__________________________
How many single colonies do you observe with Method A?
Strain: ______________
Number of single colonies: __________
Strain: ______________
Number of single colonies: __________
How many single colonies do you observe with Method B?
Strain: ______________
Number of single colonies: __________
Strain: ______________
Number of single colonies: __________
Describe the colony morphology for each strain:
__________________________________________________________________
__________________________________________________________________
Page | 32 Conclusion:
Please write your conclusion below:
__________________________________________________________________
__________________________________________________________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 33 Exercise 5: Microscopy - Part I
Goal: To learn the various parts of the compound light microscope, how to care for
it and how to use it to view specimens using dry objectives
Introduction: Since most microbes are not immediately visible, we rely on
magnification instruments to help us observe them. These instruments are called
microscopes and can view organisms to incredible detail down to individual protein
complexes. There are a variety of microscopes, each having its range of magnifications
and different uses. We will be using compound light/brightfield microscopes.
These microscopes will let us magnify an organism to 1000 times. With this
magnification we will be able to observe bacteria very clearly, as well as internal
components in eukaryotes such as the yeasts. We call them compound microscopes
because they have several lenses rather than just one. The illumination source is visible
light, which passes through the specimen and into our eye. Two parts of the microscope
are used for magnification: the ocular lens and objective lens. The magnification of the
ocular lens is usually 10x, whereas each objective lens has its own proper magnification.
Our microscopes have four objectives: a 4x, 10x, 20x and 100x. To calculate the total
magnification that you are using to view a specimen, you would multiply the
magnification of the ocular by the magnification of the objective that you were using to
view the specimen.
Our microscopes also have some features engineered into them that facilitate locating
and viewing specimens. The objectives are both parfocal and parcentered. Parfocal
means that the specimen remains in focus when changing from one objective to another.
Therefore, only a slight adjustment with the fine focus is required once the specimen has
been focused at 40x. Parcentered means that the specimen remains in the center of the
visual field when switching between objectives. If one centers the specimen in the
middle of the field at the 40x magnification, it will be easier to find at higher
magnifications.
In this exercise we will learn how to care for the microscope and work with the dry
objectives: 4x, 10x and 40x. The 4x, 10x and 40x objectives are called “dry objectives”
because they do not use oil.
Identify the following parts of the microscope:
•
•
•
•
•
Ocular
Nosepiece
Arm
Coarse focus
Fine focus
•
•
•
•
•
•
Objective
Stage
Condenser
Illuminator
Light control
Iris Diaphragm
Page | 34 Page | 35 Materials:
•
•
•
•
Microscope
Lens paper and lens cleaner
(or isopropanol)
6 inch plastic ruler
Fine tip sharpie or pen
•
•
•
Prepared slides (per group): letter “e”,
colored threads, S. cerevisiae, bacterial
shapes
Colored pencils
Index card strip
Procedures:
General Microscope/specimen maintenance
•
•
•
•
Always use both hands to transport the microscope – one on the arm and one on
the base.
Before looking at specimens, clean all objectives with lens paper and isopropanol
or lens cleaner.
If specimen slides are dirty, you can also use lens paper and lens cleaner to clean
them
Use plastic tray to transport slides to your bench.
Verification that microscope was properly stored
Check the following when first handling your microscope during the lab session:
1)
2)
3)
4)
5)
6)
7)
8)
9)
Arm of microscope facing outside
Eyepiece lined up with front of microscope
Stage is centered and does not contain any specimen slide
4x objective in position
No oil on objectives
Power switch in “off” position
Light source at lowest intensity
Stage in lowest position
Condenser in highest position
Note any deficiencies on the Microscope Care Card.
Operating the microscope and visualizing specimens using dry objectives
1) Plug the power cord into the microscope and then into the outlet.
2) Clean all objectives with lens paper and isopropanol or lens cleaner.
3) Turn on the light source a quarter turn and make sure light is coming through
the center of the stage.
4) Place specimen on stage.
5) Center specimen on the stage.
Page | 36 6) Adjust oculars
7) Using coarse adjustment knob, focus with right eye, then adjust left eye ocular
until specimen is in sharp focus. (Try to use both eyes, using one eye to look
under the scope can cause headaches.)
8) Center specimen in field of view.
9) If desired, change the objective to view specimen under different
magnifications.
Part 1: Identifying correctly stored microscopes
Your instructor will show you 9 pictures of stored microscopes (labeled A-I). Go to the
results section to record whether these scopes were stored correctly.
Part 2: Viewing the letter “e” slide
Draw the letter “e” (in the Results section) as it appears when you place it on the stage.
Following the procedure to view a specimen, use the 4x objective to view the letter “e”
slide so you are using a 40x magnification to view the “e”.
Draw the “e” as you observe it when looking through the microscope.
Part 3: Measuring the working distance
The working distance is defined as the distance (in mm) from the specimen to the
objective when it is in focus.
We will measure the working distance using the letter “e” slide.
Using the given index card strips, draw 5 lines 1 mm apart and then two more lines 5
mm apart from each other. Your card strip should look like this:
With scissors, cut to make a triangular edge on the strip – the result should look
something like this:
Make sure the letter “e” is in focus with the 4x objective, then use the pointed end of the
card to measure the working distance and record it in the Results section. Swing the 10x
Page | 37 objective into position. Focus with the fine adjustment knob. Measure and record the
working distance. Repeat for the 40x objective.
Part 4: Colored threads slides
Following the procedure to view a specimen, use the 4x objective to view the colored
thread slide. Move the thread intersection into the center of the field of view.
Swing in the 10x objective and focus on one thread. After you have focused on the
thread, lower the stage with the fine adjustment knob (towards you) until all the threads
are out of focus.
Slowly use the fine adjustment knob to bring the stage up and observe which thread
comes into focus first, second and third. Which thread is on the top, middle and
bottom?
Results:
Part 1: Identifying correctly stored microscopes
Next to the microscope label place a check mark if it was stored correctly, or if it was
stored incorrectly, write down why.
Microscope A - _____________________________________________
Microscope B - _____________________________________________
Microscope C - _____________________________________________
Microscope D - _____________________________________________
Microscope E - _____________________________________________
Microscope F - _____________________________________________
Microscope G - _____________________________________________
Microscope H - _____________________________________________
Microscope I - _____________________________________________
What components of appropriate microscope storage cannot be determined by
analyzing the pictures?
____________________________________________________________
Page | 38 Parts 2 &3: Viewing the letter “e” slide and measuring working distance
Letter “e” on stage appears as _______
Letter “e” through the microscope appears as ________
Working distance of the 4x objective _______ (in mm)
Working distance of the 10x objective _______(in mm)
Working distance of the 40x objective _______(in mm)
As you moved from the 4x to the 40x objective, what was happening to the working
distance?
The working distance gets
LARGER
SMALLER
Part 4: Colored threads slides
The top thread is _____________, the middle thread is _____________, and the
bottom thread is ___________.
Why do we only use the fine adjustment knob for focusing when we change between
objectives?
_______________________________________________________
_______________________________________________________
Page | 39 Exercise 6: Microscopy – Part II
Goal: To learn how to view specimens using the oil objective
Introduction: Bacteria and Archaea are significantly smaller than eukaryotes such as
yeasts, molds or helminths. Even when we use the 40x dry objective, they are difficult to
see and therefore require a higher magnification to observe them. You also noticed how
when using the 40x objective the sample appears darker. This is due to less light able to
go through the specimen as the working distance decreases (more light is escaping
through the sides). To be able to use a higher magnification we need tiny working
distance and enough light to come through the specimen. We need to use oil because
the light scatters too much as it goes from the specimen to the objective. This is
accomplished by the use of a special oil objective – labeled 100x. When oil is added and
this objective is used, the oil focuses the light into the objective thus illuminating the
specimen. The resulting magnification is 1000 times the size of the original specimen.
Materials:
•
•
Microscope
Lens paper and lens cleaner
(or isopropanol)
•
•
Prepared slides (per group) S. cerevisiae,
bacterial shapes, or other slides as
determined by instructor
Colored pencils
Procedures:
Always take special care when working with the oil objective. If the
objective gets scratched due to mishandling, it is ruined and nothing can be
seen through it.
Also, take care NOT to get oil onto the 40x or 10x objectives. Oil on these
objectives can ruin them.
Visualizing specimens using the oil objective
1) As done for each session, check to see if your microscope was stored properly.
2) Clean all objectives with lens paper and lens cleaner (or isopropanol).
3) Follow the steps from the last exercise to visualize your specimen with the 10x
dry objective.
4) Make sure your specimen is at the center of the visual field.
5) Once your specimen is in focus, switch your objectives (don’t touch the focus
knobs!) so that the specimen is lined up between the 40x and 100x objective
(the 40x and 100x objectives will make a 45 degree angle with the sample).
Page | 40 6) Add a small drop of oil and slowly bring the 100x oil objective in line with the
specimen.
7) Slowly turn the fine adjustment knob away from you until the specimen
comes into focus. (Move the focus very slowly because it is easy to over or
under-focus at this magnification.)
8) If you are unable to locate your specimen, you can switch to the 10x and
refocus. To get to the 10x from the 100x, you would switch to the 4x and then
the 10x objective. NEVER rotate the dry 40x objective over a slide that has
oil.
9) Once you have observed your sample under oil, you can use the stage control
knobs to move the slide to view another specimen on the same slide without
switching out the 100x objective.
Viewing the letter yeast slide and bacterial shapes slide
Following the procedure to view a specimen, use the 10x objective and then the 100x
objective to view the Yeast slide. Draw how the yeast appears under both objectives in
the Results section.
Using the same procedure to visualize under oil, view the bacteria shapes slide.
Draw the morphology and arrangement you observed under oil (1000x). Note that once
you have focused on one bacterial shape, you can move the slide with the control knobs
without having to switch out the objective or change the focus.
Results:
Yeast slide
100x magnification (dry)
1000x magnification (oil)
Page | 41 Bacterial shapes slide
Draw each of the bacterial shapes you observe and label below:
_______________
_______________
Which is bigger – yeast or bacteria (Circle one)?
_______________
YEAST
BACTERIA
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 42 Exercise 7: Sample Preparation for Microscopy
The Wet Mount
1. Take a slide and coverslip from the appropriate boxes.
2. With the loop, take two loopfuls of the sample and place it on the slide.
3. Run the coverslip over the slide until it touches the sample. At this point, the
sample will spread on the edge of the coverslip.
4. Gently lower the coverslip on the slide. This will prevent too many bubbles from
appearing below the coverslip.
5. The sample is ready to be inspected under the scope.
6. Note: if you have a “dry” sample, just add two loopfuls of water, mix with dry
sample (like a little bit of a bacterial colony) and continue with step 3.
The Hanging Drop
1. Take a concavity (or depression) slide and a coverslip. Using the syringe filled
with petroleum jelly, place four small amounts of jelly on the corners of the
coverslip.
2. In the center of the coverslip, place one or two loopfuls of sample. Do not use too
much sample! If you do, we you prepare the slide the large volume of liquid will
touch the slide and the sample will run to the edge of the coverslip.
3. Turn the concavity slide over so that the indentation on the slide is facing down.
4. Make sure that the sample is centered in the indentation. Gently press the slide
so that the jelly on the cover slip touches the slide.
5. Turn the slide over. The sample should now be “hanging” into the indentation
space of the slide and is ready for viewing.
Page | 43 The Bacterial Smear
1. Clean the slide by flaming both side 3 times.
2. Using the Sharpie, mark the underside of the slide with circles.
3. Place three loops of water (or culture) onto a slide.
4. (From solid media) Slightly touch the loop to the bacterial growth. Mix bacteria
with water by tapping the loop gently several times and spread the drop out with
the loop.
5. (From broth cultures) After adding three loops of culture on the slide, spread the
drop out with the loop.
6. Allow the smear to AIR dry. The better the drop has been spread out, the more
quickly it will dry. Also clean slides give better smears. If your smear is taking a
while to dry or drying unevenly, you can use the loop again to spread it out.
7. Now for HEAT-FIXING the slide. Using you slide holder, and with the cells on
the slide facing up, pass the slide over the upper part of the Bunsen burner flame
two or three times. Make sure that you are heating the face of the slide that does
not have the cells! Also, be careful not to overheat the slide, as aerosols can be
produced or the slide can shatter.
8. Allow the slide to cool and then place in your slide holder.
Draw your wet mount and hanging drop sample below:
Wet Mount
Hanging Drop
Page | 44 Exercise 8: Identification of Bacterial Unknowns
Goal: To understand bacterial classification and identification. This knowledge
will be used to generate a dichotomous key that will then be employed to identify a
bacterial unknown.
Introduction: Taxonomy is the science of classification of living organisms (and
viruses) based on similarities. It is an activity that has been present for several
thousand years. Aristotle, a famous Greek philosopher, separated living things into
animal and vegetable primarily based on their ability to move. He is known for his
developments in philosophy that proposed various ways of understanding reality. He
was the original person to propose the terms genus and species as a way to recognize
and classify any material thing.
A Swedish botanist, Carolus Linnaeus (Carl von Linné) took this philosophical
understanding on how we classify things and applied it to living organisms. In his
famous work, Systema Naturae, he developed a Latin binomial nomenclature that
gives each organism two names: the first name is called the genus (pl. genera) and the
second name is species (or specific epithet). Why did he use Latin? In 1735, this was the
common language for academics around the world (as English is today), so publishing
in Latin ensured that other people would read his work! In fact, biologists still use this
Latin naming system today to refer to living organisms to know precisely which
organism is being studied.
Some examples of this naming system are Homo sapiens (humans),Fugu rubripes
(pufferfish),Mus musculus (mouse), Saccharomyces cerevisiae(baker or brewer’s yeast)
and Escherichia coli (coliform bacteria). The scientific names are underlined or written
in italics. In our class, we will always italicize scientific names. Also notice that the first
name is capitalized and the second name is not.
Robert Whittaker, extended Linnaeus’ work by developing a five kingdom
classification system in which he separated living organisms according to their cellular
organization and method of nutrient acquisition. Currently, we use a modification of
Whittaker’s five kingdom classification system.
How we classify bacteria
Classification of any organism is based on visible characteristics or phenotypes. With
the development of molecular biology, classification of bacteria has migrated from more
observable phenotypes to a classification more based on genomic sequences
(genotypes). Biologists decide which characteristics are more relevant in
Page | 45 classification, and it’s important to note that no classification system can perfectly
organize the great variety of living organisms that exist. Yet, once criteria for
classification have been determined, we can then use these to identify unknown
organisms.
Classical approaches to identification
Before the development of molecular biology, bacteria were classified using a classical
approach of grouping bacteria into various categories based on phenotypic similarities.
Some of the major phenotypes used in classical approach include:
- Morphology (Bacilli, Cocci, Spirilla)
- Cell wall type (Gram negative or Gram positive)
- Growth requirements (Oxygen concentration, pH, temperature, Nutrients)
- Metabolic reactions (production of certain enzymes or byproducts)
In addition, the variety of microorganisms requires us to go beyond the genus/species
classification to more detailed classifications that include strain, variant, serotype or
phage type.
Molecular approaches to identification
The development of molecular biology techniques allows for the clear identification of
organisms based on their unique DNA or rRNA sequences. Most of these techniques are
based on genetic homology, which is the amount of similarity among different
organisms. Thus, if two organisms show perfect DNA sequence homology, then we
assume that they are from the same species. Some of these techniques include:
- DNA hybridization (A fragment of DNA from a known organism is matched up
with an unknown organism. The closer the match, the more highly related the
organisms are.)
- %GC content (The proportion of G-C basepairs in the genome is calculated. If the
%GC is the same as another organism, they are likely related.)
- DNA or rRNA sequencing (DNA or rRNA is isolated and sequenced. The result is
a string of sequence that can be entered into databases for identification.
We will be using a combination of a classical approach and a molecular approach to
identify our unknowns. Usually these traits are summarized in flow charts called
dichotomous keys. Generally, these keys begin with more general characteristics
(like morphology or gram stain status) and then become more specific.
Page | 46 Materials (per group)
• 6-8 objects to be handed out by the instructor
• List of bacterial phenotypes
• List of gram positive or gram negative bacteria
• (From previous lab session) TSA plate with isolated unknown or unknown
culture
Procedure:
1. Using the objects handed out by the instructor, construct a dichotomous key to
uniquely identify each of the objects. You will work together as a group.
2. Draw your dichotomous key on the board.
3. You will also receive a list of bacterial phenotypes and a list of gram positive or
gram negative bacteria. All of the phenotypes on the list can be tested in the lab.
Using the Bergey’s manual and online sources (like Todar’s book of bacteriology)
you will design a dichotomous key for all the bacteria on the list. You will share
your key with other groups and using those keys you will design an experimental
strategy to identify your unknowns.
4. Streak out your unknown for single colonies.
5. Incubate your plate at 37˚C for 48 hours.
6. Pick a single colony from the plate with your loop and streak a slant. Label this
slant working stock.
7. Pick another single colony from the plate with your loop and streak another slant.
Label this slant backup stock.
8. Place your slants in the designated location for incubation.
Bring a small notebook where you will record all the experiments that you run on your
unknown as well as the controls that you used.
Page | 47 Results:
Draw your dichotomous key below.
Dichotomous key
Your unknown number is:
Describe the colony morphology of your unknown (after isolation):
____________________________________________________________
____________________________________________________________
____________________________________________________________
Page | 48 Phenotype
Result (pos or neg)
Phenotype
Result (pos or neg)
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 49 Exercise 9: The Eukaryotes
Goal: To become familiar with major classes of eukaryotic microbes using
standard microscopy techniques.
Introduction: While most of the emphasis in microbiology is on prokaryotes,
eukaryotic microbes also have a substantial impact on human wellbeing. They belong to
the Eukarya domain and are divided in four general classes: algae, protozoa, fungi and
helminths.
Algae
Algae live in water environments and are photosynthetic organisms. They can be
unicellular or multicellular and usually contain cellulose in their cell walls, just as plants
do. They can reproduce both sexually and asexually. It is estimated that 80% of the
Earth’s oxygen is produced by algae.
Protozoa
Protozoa (“first animals”) are free-living or parasitic unicellular organisms. They are
motile thanks to pseudopods, flagella or cilia. They are chemoheterotrophs, either
absorbing or ingesting their nutrients from the environment. They can reproduce both
sexually and asexually.
Fungi
The fungi are divided into three categories: molds, yeasts and dimorphic fungi. They
can be either uni- or multi-cellular and have cell walls composed of chitin, which is the
same molecule that makes up insects’ exoskeleton. Fungi are aerobic (molds) or
facultative anaerobes (yeasts). Reproduction in fungi can take many forms. They can
reproduce asexually, by producing spores or budding, or sexually by producing spores.
In terms of nutritional type, fungi are chemoheterotrophs, acquiring their nutrients
from their surroundings. Unlike animals that gain their nutrients through ingestion,
the fungi absorb their nutrients, and thus are labeled saprophytes.
Fungi are very beneficial. They are our source for antibiotics such as penicillin and
cephalosporins, and they are critical for the production of many food products such as
beer, cheese and bread. A number of fungi are detrimental to animals and plants.
Fungal diseases are referred to as mycoses. Some common fungal infections are
Athlete’s foot, ringworm and yeast infections. Some fungi can also produce strong
toxins (mycotoxins) that when ingested can lead to hallucinations, cancer or paralysis.
Page | 50 When compared to bacteria, several differences can be highlighted. The fungi are
generally more resistant to osmotic stress, grow better in acidic conditions, tolerate
lower moisture and are larger in size. They also can metabolize complex carbohydrates
for energy.
Molds are composed of long, multicellular, branched filaments called hyphae. As the
hyphae grow and intertwine, they become visible without the aid of a microscope and
are then termed mycelia (sing. mycelium).
Yeasts are unicellular, non-filamentous fungi that are commonly found in nature as a
white powdery coating on leaves and fruit. They are typically oval or spherical in shape,
and can reproduce either sexually or asexually. They undergo mitosis either by budding
or fission. When buds do not break off in a dividing yeast cells, short chains of cells
called pseudohyphae are produced. Diploid yeast cells can also produce spores
through a process called sporulation, when they are starved for nutrients. As
environmental conditions become favorable again, these spores will germinate and
produce new yeast cells.
Unlike the aerobic molds, yeasts are facultative anaerobes that undergo fermentation.
Ethanol and carbon dioxide are their most common fermentation products, which have
graced humanity for thousands of years in the forms of bread, wine and beer.
Yeasts, particularly the budding yeast Saccharomyces cerevisiae and the fission yeast
Schizosaccharomyces pombe, have been invaluable as model organisms in research.
Many basic cell processes are identical in lower and higher eukaryotes, and so
discoveries made in yeasts have benefited us.
Some fungi are dimorphic (or two-shaped) which means that they can grow in either a
mold or yeast form. Whether the fungus grows as a mold or yeast depends on some
environmental condition like temperature or carbon dioxide concentration. This trait is
common among pathogenic fungi. At lower temperatures (outside the body) they will
grow as molds, but they grow as yeasts once they have been internalized.
To culture yeasts Sabouraud agar is used. This selective medium has glucose,
peptone and a low pH, thus discouraging the growth of most other microbes.
Helminths
Most of the animals that spend part of their life cycles in humans are worms that fall
into two phyla: Platyhelminthes and Nematoda. The Platyhelminthes are commonly
known as flatworms and the Nematoda are usually known as roundworms. These
Page | 51 worms are referred to as helminths. Since they are animals, they possess the following
characteristics: they are multicellular; they are chemoheterotrophs; and they contain
organ systems such as digestive, circulatory, nervous, excretory and reproductive
systems. Interestingly, even though parasitic worms possess these organ systems, they
do not function as well as their free-living counterparts. Sometimes they can lack a
digestive system, their nervous systems are reduced and their means of locomotion is
highly limited. In addition, they tend to have complex reproductive systems that can
involve intermediate hosts for each larval stage of development.
Helminths can be dioecious or monoecious. Dioecious organisms have either male or
female reproductive organs, while monoecious (or hermaphrodites) organisms have
both male and female reproductive organs. In nature monoecious organisms can mate
amongst themselves and simultaneously fertilize each other or they can self-fertilize.
The Platyhelminthes contain two classes: the Trematodes (also known as flukes) and
Cestodes (also known as tapeworms). Trematodes are flat and look like leaves. They
absorb food through an outer covering called a cuticle (remember this is an animal so a
cuticle cannot be considered a cell wall). Cestodes typically are intestinal parasites.
They completely lack a digestive system and feed themselves by absorbing food through
their cuticle (they do not ingest host tissues). The general body plan of the Cestodes is a
head region, called the scolex, which has suckers to attach to the intestinal mucosa of
the host. The neck region of the scolex produces a segment called a proglottid. This
segment contains both male and female reproductive organs. As new proglottids are
produced by the scolex, the worm’s length will increase. The proglottids farthest away
from the scolex mature first and essentially become bags of fertilized, infective eggs.
Nematodes or the round worms have a complete digestive system (mouth, intestine and
anus) which means that they ingest the host’s tissues. Most nematodes are dioecious
and display sexual dimorphism in that females are usually larger than males. Either
their eggs or larvae are infective for humans. Enterobius vermicularis (pinworm)
produces eggs that are infectious for humans, while the larvae of Trichinella spiralis are
the infectious agent.
Materials (per group)
• Prepared yeast, mold and helminth
slides
• Slides and coverslips
• Concavity slides (not disposable)
•
•
•
•
Syringe with petroleum jelly
Methylene Blue
Pond water
S. cerevisiae and S. pombe cultures
Page | 52 Procedure:
Summary
You will use basic microscopy techniques to observe samples of algae, fungi and
helminths.
Note: You do not need to use the oil immersion objective for this exercise
1. Algae:
a. Prepare a pond water sample using the hanging drop technique
b. Heat fix and stain provided pond water slides with Methylene Blue.
c. Draw and identify three different organisms using the pond atlas pictures
2. Fungi:
a. Using the 4x magnification, observed prepared slides of yeasts.
b. Prepare wet mounts of Schizosaccharomyces pombe and Saccharomyces
cerevisiae using Methylene Blue. Label a bud and a mother cell.
c. Under 4x and 10x magnification, observe prepared mold slides and draw
three different molds. Label the Conidiospores/Sporangiospores and
hyphae.
3. Helminths:
a. Observe prepared slides of Clonorchis sinensis and Enterobius
vermicularis.
b. Draw and label structures as instructed in the Data section.
Results:
Algae
Sample
View under scope
Source (circle one):
Hanging Drop
Heat fixed slide
Organism ID: __________________
Source (circle one):
Hanging Drop
Heat fixed slide
Organism ID: __________________
Page | 53 Sample
View under scope
Source (circle one):
Hanging Drop
Heat fixed slide
Organism ID: __________________
Fungi/Yeasts
Sample
View under scope
Prepped slide:
______________
Prepped slide:
______________
Wet mount:
______________
Wet mount:
______________
Page | 54 I. Trematodes
(Clonorchis sinensis)
- Sketch (as seen under microscope)
- Identify and label the following structures:
• Uterus
• Yolk Gland
• Eggs
• Ovary/Seminal Recepticle
II. Nematodes
(Enterobius vermicularis)
- Sketch (as seen under microscope)
- Identify and label the following structures:
• Head
• Tail
• Esophagus
What is the sex of the pinworm?__________
What is the identifying characteristic between
male and female pinworms?
_______________________________
_______________________________
Page | 55 Exercise 10: The Simple Stain
Goal: To learn basic bacterial staining techniques.
Introduction: Bacteria and yeasts tend to be transparent and therefore are hard to
visualize under the microscope. To solve this difficulty, stains (or dyes) are used to
add color to the cells, which is then readily visible under the microscope. Stains contain
a charged color molecule (ion) called a chromophore. The chromophore can either be
negatively (anionic) or positively (cationic) charged. Since bacteria have a slightly
negative charge, cationic chromophores will be attracted to the cell while anionic
chromophores will be repelled. The result is that stains/dyes with cationic
chromophores will stain the bacterial cell, while stains/dyes with anionic chromophores
will stain the background.
As a matter of convention, basic stains/dyes contain cationic chromophores, while
acidic stains/dyes contain anionic chromophores.
Stains can be described as simple or differential stain. A simple stain refers to the
use of a single dye, whereas a differential stain allows one to detect differences between
organisms (as in cell wall differences). Using a differential stain will not stain all the
cells alike, even though we follow the same procedure.
Materials (per pair)
•
•
•
Unknown cultures
Slides with previously prepped smears
Bibulous paper
•
•
•
Staining tray
Clothespins
Sterile water
Materials (per group)
• Methylene blue, Safranin and Crystal Violet
• Small plastic squeeze bottle with water
Procedure:
(Note: Each slide will be stained with one dye.)
1. Place clothespins on either side of the slide, and place on staining tray.
2. Add 3-5 drops of Methylene Blue on each smear.
3. Wait 2 minutes and then gently rinse by allowing the water to run through the
smear (don’t point the water stream on the smear directly, as you could peel off
the bacteria!)
Page | 56 4. Rinse the back of the slide and blot it in the bibulous pad to dry.
5. You have now finished your first simple stain.
6. Now, repeat steps 1-4 using Safranin to stain another slide.
7. Repeat steps 1-4 using Crystal Violet to stain a third slide.
8. The staining time for Safranin is 1.5-2 minutes and for Crystal Violet it’s 1 minute.
Results:
Use your best stain to draw the morphology and arrangement of your bacterial samples:
Strain/Dye:
_________________
_________________
Morphology: _________________
_________________
Arrangement:_________________
_________________
Page | 57 Strain/Dye:
_________________
Unknown #___/________
Morphology: _________________
_________________
Arrangement: _________________
_________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 58 Exercise 11: The Negative Stain
Goal: To identify bacterial morphology using the negative stain
Introduction: The negative stain is an extension of the simple stain, but uses an
anionic chromophore (acidic) instead of a cationic one. Therefore the background will
stain but the cell will not, which allows visualization of cell morphology. No heat fixing
is used in this technique, therefore the cells remain alive on the slide and should be
disposed of as biohazardous material.
Materials (per pair)
• Slides
• Inoculating loop
•
•
Nigrosin
Unknown bacteria
Materials (per group)
• Cultures of Staphylococcus aureus and Bacillus cereus
Procedure:
1.
2.
3.
4.
Clean two slides per person using the Bunsen burner.
Place a pea-sized drop of ink with your loop on one end of the slide.
Mix one loopful of S. aureus (or B. cereus) culture into the ink.
Set the slide with your ink sample on the bench and use the push slide technique
(see figure below) to make a gradient smear on the slide.
5. Allow the slide to completely dry. Visualize under oil.
Page | 59 Results:
Draw the morphology and arrangement of your bacterial samples as viewed in the
negative stain:
Strain:
_____________
Morphology/
Arrangement: _____________
______________
______________
Unknown #_____
_______________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 60 Exercise 12: The Gram Stain
Goal: Use the Gram stain to determine cell wall type
Introduction: The Gram stain is the most common differential stain used in
microbiology. It was developed by Christian Gram in 1884 to differentiate between
Streptococcus pneumoniae and Klebsiella pneumoniae. It allows us to tell the
difference between cells that have specific cell wall structures (i.e., Gram positive or
Gram negative). Gram positive cell walls are composed of a thick layer of
peptidoglycan and teichoic acids, while Gram negative cell walls have two lipid bilayers
that sandwich a thin layer of peptidoglycan. The outer membrane in the Gram negative
cell wall also contains Lipopolysaccharide (LPS). During cell lysis of Gram negative
organisms, a portion of the LPS called Lipid A is released and causes a strong immune
response that can lead to shock. Lipid A is also known as an endotoxin.
The basic procedure begins with the addition of the primary stain (Crystal Violet) to
the bacterial smear. After rinsing, the mordant (Gram’s iodine) is added. The purpose
of the mordant is to increase the intensity of the Crystal Violet staining. Iodine enters
the cell and combines with the Crystal Violet to form a Crystal Violet – Iodine (CV-I
complex). Due to its increased size, this complex will not readily leave the cell. The next
step, decolorization, is the most critical one of the procedure. The decolorizer
(Ethanol or acetone) will dissolve lipids in the outer membrane of Gram negative
bacteria and makes the cell wall porous. This allows the CV-I complex to exit the cell,
turning it colorless. Then the counterstain (Safranin) is added to restain Gram
negative cells. When the decolorizer is added to Gram positive cells, the thick layer of
peptidoglycan is dehydrated and becomes more compact as a result. This ends up
trapping the CV-I complexes in the cell and therefore the cell remains purple.
Counterstaining with Safranin does not override the purple pigment of the Crystal
Violet. Therefore one expects Gram negative cell to stain pink/red and Gram positive
cells to stain purple.
Materials (per group)
• A bottle of Crystal Violet, Gram’s Iodine, Safranin and 95% Ethanol.
• Known cultures of Gram positive and Gram negative bacteria (18-24h old)
• Distilled water
Materials (per pair)
•
•
•
Staining tray
Loops
Clothespins
•
•
Marking pen
Bibulous paper
•
•
Clean slides
Unknown culture
Page | 61 Procedure:
1. Prepare smears of your unknown with controls on either side as drawn in the
diagram.
Gram
Positive
control
Unknown
Unknown
Gram
Negative
control
2. Cover the smear with Crystal Violet for 1 minute. Gently rinse the slide with
water.
3. Cover the smear with Iodine and stain for 1 minute. Gently rinse with water.
4. Decolorize until the runoff is clear (about 5-15 sec). Gently rinse with water.
5. Cover the smear with Safranin for 2 minutes. Gently rinse with water.
6. Blot with Bibulous paper. Observe under the sample under oil.
Results:
Draw the morphology and arrangement of your unknown and control samples:
Strain:
Unknown __________
________/_________
Morphology: _________________
_________________
Arrangement:_________________
_________________
Do the morphology and arrangement of your Unknown match your simple stain?
YES
NO
Page | 62 If your results from the Gram Stain do not match your Simple Stain, please explain
below:
____________________________________________________________
____________________________________________________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 63 Exercise 13: The Endospore Stain
Goal: To identify whether a bacterial strain produces endospores
Introduction: Each organism has its own set of growth requirements to thrive. An
actively growing and metabolizing bacterium (or yeast) is called a vegetative cell. If
these actively growing cells encounter adverse environmental conditions, such as a
significant depletion of nutrients, the usual result is cell death. Yet some bacterial
genera possess a survival mechanism that allows them to package their DNA into an
extremely resistant, dormant cell type called a spore.
Due to the thick spore coat, these spores are highly resistant to dehydration, radiation,
heat and toxic chemicals. They have low metabolic activity and are known to survive
hours of boiling and high doses of radiation that would kill humans. The process by
which these spores are generated is called sporulation (or sporogenesis), which is
usually triggered by low carbon or nitrogen in the environment. When more favorable
conditions are encountered, these spores will undergo germination, which will
regenerate the vegetative cell.
A single bacterial vegetative cell will form a single spore; when this spore germinates it
will produce one vegetative cell. Thus, sporulation in bacteria is not a reproductive
process since there has been no increase in the number of bacterial cells. After
sporulation, the spore remains inside of the cell until cell lysis. When the spore is inside
the dying bacterial cell, it is called an endospore; when the bacterial cell lyses, the
endospore is released and is then called an exospore.
The principal genera of bacteria that can sporulate are Bacillus, Clostridium and
Sporosarcina. Bacillus and Clostridium are bacilli and Sporosarcina is a coccus. Of
these, Bacillus and Clostridium have significant impact in the health care environment
and food industry because they are not removed with typical disinfection procedures.
Materials (per group)
•
•
•
•
“Young” Bacillus subtilis slant
“Old” Bacillus subtilis slant
“Young” and “Old” samples of
unknowns
Malachite green and Safranin
•
•
•
•
Culture tube with sterile water
Squirt bottle of distilled water
Heating plate
2 Beakers with boiling chips
Page | 64 Materials (per pair)
•
•
•
•
Glass slides
Inoculating needle
Marking pen
Bibulous paper squares
•
•
•
Bibulous paper
Clothespins to hold slides
Staining tray
Procedure:
1. Place water in the beaker and set to boil.
2. You need to make smears of your samples (clean slides, air dry, etc.). On one
slide place two smears: “Old” and “Young” B. subtilis. On the other slide, make
two smears from your “Old” and “Young” Unknown culture.
3. After you have heat fixed your smears, clip the slides with the clothespins and
place them over the beaker with boiling water.
4. Place a bibulous paper square over the smears. This square is used to filter out
large crystals in the malachite green, the primary stain.
5. Moisten the entire bibulous square by gently expelling the dye and rubbing the
dropper over the square.
6. Add more malachite green to the bibulous square until completely moist. (NOTE:
be very careful not to get malachite green on your clothes or anywhere else.
Staining with malachite green is permanent.
7. Do not let the malachite green dry on your slide! Keep on adding dye so that it
remains moist.
8. Heat the slide for 5 minutes. Take the slide off the beaker and remove the
bibulous square with your loop.
9. Wait for 3 minutes to let the slide cool.
10. Gently rinse both sides with water.
11. Cover the smear with Safranin (the counterstain) and let sit for 2 minutes.
12. Rinse with water and blot the slides in the bibulous paper pad.
13. Examine under oil.
Page | 65 Results:
Draw your endospore stains here:
Strain:
_________________
Are endospores
present?
YES
Strain:
NO
_________________
Are endospores
present?
YES
NO
_________________
YES
NO
_________________
YES
NO
Page | 66 Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 67 Exercise 14: Selective and Differential Media
Goal: To determine the gram status and other species characteristics using
selective and differential media.
Introduction: In the same manner that some bacterial strains give different results in
differential stains, by adding different chemicals to growth media one can also observe
differences in bacterial cultures. Media can be classified into four major groups:
General Purpose media, Selective media, Differential media and Enriched media. Some
media can be made to have several properties, such as being Selective and Differential
or Enriched and Differential.
General Purpose media is the media that is used routinely in the cultivation of
microbes. Tryptic Soy Agar and Nutrient Agar are commonly for this purpose.
Selective media contain toxic compounds that completely inhibit or slow down the
growth of certain species of bacteria, while others are not affected.
-
Eosin Methylene Blue Agar (EMB) contains two dyes that inhibit the growth of
gram positive bacteria, but permits the growth of gram negative bacteria.
Therefore, we say that EMB selects for gram negative bacteria. The color of the
media is a dark pink or purple. This medium is also a good way to verify gram
stains of gram negative organisms.
-
Phenylethyl Alcohol agar (PEA) contains an alcohol that helps denature the
outer membrane of gram negative cell walls. This allows gram positive bacteria
to grow, while the growth of gram negative is inhibited. PEA therefore selects for
gram positive bacteria, but selects against gram negative bacteria. The color of
the media is yellow-tan, as it is for TSA. This medium is also a good way to verify
gram stains of gram positive organisms.
-
Mannitol Salt Agar (MSA) contains 7.5% NaCl. This high concentration of salt
provides osmotic pressure that is too high for many bacteria, and thus inhibits
their growth. Some species, especially in the Staphylococcus genus, can grow in
high salt and are termed salt tolerant. Therefore MSA selects for salt tolerant
bacteria. The plates have a pink appearance (but this is NOT due to the salt!)
Page | 68 Differential media contain chemical compounds that affect the colony appearance
(usually by color) of different bacterial species. Just like in the differential stain where
using the same procedure results in bacteria appearing differently under the
microscope, you can visually observe differences among bacterial species on the same
medium.
- EMB also contains a compound that allows us to observe whether or not a
bacterial species ferments lactose. Bacteria that ferment lactose produce either
dark purple colonies or a metallic green sheen. Bacteria that are non-lactose
fermenters will produce pink colonies.
-
MSA also contains mannitol and phenol red, a pH indicator. It is this pH
indicator that gives the pink color to the plates. If bacteria ferment mannitol,
they produce acids that lower the pH of the media. A drop in pH turns the phenol
red yellow, which then produces a yellow halo around the streak. Therefore a
yellow halo around the streak means that the bacteria ferment mannitol.
-
Note that both EMB and MSA are selective and differential media.
Enriched media contains growth factors, usually from blood or serum, which
encourage the growth of bacteria that do not grow easily on general-purpose media.
-
Blood Agar (BA) contains 5% sheep blood added to normal TSA plates. The
blood provides proteins and hormones that help bacterial cells grow. In addition,
BA is also differential. Certain bacterial strains can lyse red blood cells to varying
degrees. These are referred to as hemolytic patterns. On BA this is observed
as either a clear halo around the bacterial streak or a greenish-brown area around
the streak. If cells are able to completely lyse red blood cells, forming a clear are
around the streak, they are called ß- (or beta) hemolytic. If their ability to lyse
red blood cells is limited, they form a greenish-brown zone around the streak and
are called α- (or alpha) hemolytic. If a bacterial species cannot lyse red blood
cells, it will not produce any zone around the streak and is called γ- (or gamma)
hemolytic.
Materials (per pair)
• Unknown cultures
• 2 plates of EMB and MSA
• 1 plate of PEA and BA
• Inoculating loop/student
Materials (per group)
• Cultures of Bacillus cereus, Lactococcus
lactis, Escherichia coli, Staphylococcus
aureus, Salmonella typhimurium,
Staphylococcus epidermidis or other
organisms used as controls
Procedure:
Page | 69 1. Divide EMB, PEA, MSA and BA plates into quadrants.
2. Decide on positive and negative controls for each of the media.
3. Remember that if you are using a differential and selective medium that you will
need a positive/negative control for the selective aspect and differential aspect.
4. To inoculate your unknown, use your loop to aseptically transfer from your
working stock. When you go into your slant, just touch the agar in a place where
there are cells. Do not scrape to get cells from the slants!
5. For EMB, PEA and BA, make a 2 cm streak on the plate. For MSA make a 0.5 cm
streak.
6. Incubate plates for 48 hours at 35C.
*Tips* Dot Bacillus cereus on Blood Agar and Staphylococcus aureus on MSA.
Results:
Record your results in the following tables:
0 = no growth
Growth + = slight growth
++ = good growth
EMB
R = Purple colonies
K = Pink colonies
MSA
NH = No halo (pink around colony)
YH = Yellow halo
Blood
Agar
BrH = brown/green halo (alpha hemolysis)
ClH = clear halo (beta hemolysis)
NH = No halo (gamma hemolysis)
EMB (selective)
Sample
EMB (differential)
Strain
Growth
Sample
Unknown
Unknown
+ control
+ control
- control
- control
MSA (selective)
Sample
Strain
Color
(R/K)
MSA (differential)
Strain
Growth
Sample
Unknown
Unknown
+ control
+ control
- control
- control
Strain
Halo
(NH/YH)
Page | 70 PEA (selective)
Sample
BA (differential)
Strain
Growth
Sample
Unknown
Unknown
+ control
+ control
- control
- control
Halo
(BrH/ClH/NH)
Strain
Did all of your positive and negative controls behave as expected?
YES
NO
If not, explain what may have happened: ________________________________
____________________________________________________________
Conclusions:
As a conclusion, summarize the phenotypes for your unknown.
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 71 Exercise 15: Physical Requirements for Growth
Goal: To identify oxygen and temperature requirements for optimal
bacterial growth
Introduction: Bacteria require more than just the correct growth factors for optimal
growth. They also require physical factors such as specific oxygen concentrations, pH
levels and temperature. In this exercise, we will focus on analyzing oxygen and
temperature requirements.
Oxygen requirements
Microbes have a variety of oxygen requirements ranging from no oxygen to highly
oxygenated environment. Obligate aerobes (also called strict aerobes) require
atmospheric oxygen levels to live. Microaerophiles also require oxygen, but at lower
than atmospheric levels. Facultative anaerobes can grow without oxygen but prefer
an oxygenated environment. Aerotolerant anaerobes do not use oxygen in their
metabolism and therefore grow the same in the presence or absence of oxygen. Oxygen
is toxic for obligate anaerobes (or strict anaerobes), so they cannot grow in its
presence.
Several methods are used to investigate the oxygen requirements for different microbes.
The most common are: 1) culture tubes with thioglycollate medium, 2) anaerobic jars or
envelopes and 3) Brain Heart Infusion shakes. The principal component in
thioglycollate medium is thioglycollate, a reducing agent that reacts chemically with
the free oxygen, effectively decreasing the free oxygen concentration. Thioglycollate
creates an oxygen gradient in the tube with the portion of the medium closest to the
surface being highly oxygenated and the bottom of the tube being anoxygenic.
Observing growth patterns in thioglycollate can help characterize the oxygen
requirements of various bacteria. Frequently an oxygen indicator such as resazurin or
methylene blue is also added to thioglycollate to indicate the level of oxygen in the
tube. Resazurin and methylene blue are colorless in the absence of oxygen and pink and
blue respectively in its presence.
Brain Heart Infusion shakes work similarly to thioglycollate tubes. BHI agar
contains various reducing agents to reduce the oxygen concentration in the tube. The
agar is kept melted until inoculation. Once inoculated, the culture is mixed throughout
the tube and the quickly solidified in ice water. This traps the bacteria throughout the
tube and generally yields clearer results than thioglycollate tubes.
Page | 72 Below is a schematic of the growth patterns observed in BHI agar corresponding to a
microbe’s oxygen requirement.
Anaerobic jars or packs can also be used. These packs contain a catalyst and an
envelope that produces carbon dioxide and hydrogen gas. Hydrogen combines with free
oxygen in the presence of a palladium catalyst to produce water. Carbon dioxide also
helps provide an anaerobic environment. These jars/packs will also contain an
indicator strip with methylene blue or resazurin to ensure that the containers maintain
an anaerobic environment.
Temperature requirements
Optimal microbial growth is characterized by actively metabolizing cells. Metabolism is
dependent on the wide range of enzymes that facilitate metabolic reactions. Therefore
enzyme activity is directly linked to microbial growth. Enzyme activity is profoundly
influenced by temperature. As the temperature increases, substrates and enzymes
collide more frequently and the rate of enzymatic reactions increase. This continues
until increased temperature begins to denature the three-dimensional conformation of
the enzymes. Once enzymes unfold, they are rendered inactive. Conversely, enzymatic
activity decreases when the temperature drops, which leads to a decrease in metabolic
activity and slows growth.
Page | 73 Bacteria have a minimum, optimum and maximum growth temperature. This range is
typically from 20 to 30 degrees. Bacteria that grow in cold temperatures from 0°C to
20°C are termed psychrophiles (cold loving). These are the bacteria that can spoil
food in the refrigerator, which are usually set around 5°C. Mesophiles (middle loving)
are bacteria that grow optimally from 20-45°C. Since normal body temperature is 37°C,
it stands to reason that most human pathogens are mesophiles. A group of mesophiles
that can grow around 4°C and cause food poisoning are called psychrotolerant. At
the other end of the temperature spectrum, we have thermophiles which grow
optimally from 50-60°C and hyperthermophiles (or extreme thermophiles) which
grow at 80°C or higher. Interestingly, even though the rate of chemical reactions is
increased at high temperatures, thermophiles tend to grow slowly. Because of their high
temperature requirements for growth, they are not pathogenic to humans.
Materials (per pair)
Oxygen Requirements
• 2 BHI molten agar shakes
• Inoculating loop
•
•
2 x 60mm TSA plates
Unknown cultures (in broth)
Temperature Requirements
• 8 TSB tubes
• Inoculating loop
•
•
Unknown cultures (in broth)
Index card with lines
Materials (per group)
Oxygen Requirements
• 3 BHI molten agar shakes
• Cultures of Clostridium sporogenes, Escherichia coli and Micrococcus luteus
Temperature Requirements
(1 set for 3 groups)
• 12 TSB tubes
• Cultures of Staphylococcus aureus, Escherichia coli and Geobacillus
stereothermophilus or others provided by instructor
Page | 74 Procedure:
Oxygen Requirements
1. Make labels for your unknown and assigned known culture.
2. Using a sterile transfer pipette, inoculate 0.5 ml of your unknown into a shake
tube with molten BHI agar.
3. Roll the tube 15 times between the palms of your hands.
4. Place in the ice bath for 5 minutes until the agar has solidified.
5. Repeat #1 and #2 for your assigned known culture.
6. Divide the TSA plates into five sectors. Label one plate “Aerobic” and the other
“Anaerobic”.
7. Using your loop, make a small streak for your unknowns and the three known
cultures.
8. Bring “Anaerobic” plates to the front to be placed in the anaerobic packs.
9. Incubate for 48 hours at 35°C.
Temperature Requirements
1. Label your TSB tubes with the bacterial strain and incubation temperature (0°C,
25°C/Room Temp, 35°C and 55°C.
2. Using a loop, aseptically inoculate your unknown and the three known cultures
into the TSB tubes.
3. Place in the appropriate racks so that they can be incubated at the labeled
temperature. Incubate for 48 hours.
Page | 75 Results:
Oxygen Requirements
Draw the results of your Aerobic and Anaerobic plates below:
_________________
_________________
1 1 5 5 2 2 4 4 3 3 Quadrant 1
Quadrant 2
Quadrant 3
Quadrant 4
Quadrant 5
Strain
Draw/shade the growth patterns that you observe in the BHI shakes:
Strain:
_________
_________
_________
_________
Page | 76 Interpret your results from your plates and shakes:
Strain
Oxygen Requirement
Plates
Conclusion
Shakes
Clostridium sporogenes
Micrococcus luteus
Escherichia coli
Unknown #
Temperature Requirements
Use the lined index card to gauge turbidity. Record level of growth in each of the TSB
tubes with the following key:
- no turbidity/clear, + slightly turbid, ++ very turbid
Strain
0 °C
25 °C
(Room Temp)
35 °C
55 °C
Geobacillus
stereothermophilus
Staphylococcus aureus
Escherichia coli
Unknown #
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 77 Exercise 16: Biochemical Profiles of Microbes
Goal: Touse various tests to identify the biochemical fingerprint in various
microbes.
Introduction: Bacteria can be differentiated by the variety of metabolic processes that
they can carry out. Each microbe can therefore be said to have a biochemical fingerprint
that can aid in its identification. Among many biochemical assays, we can observe the
production of secreted enzymes; detect the fermentation of specific carbohydrates and
the production of various metabolic byproducts.
Exoenzymes
Exoenzymes are enzymes that are secreted by the bacterial cell into their surroundings.
This allows cells to absorb nutrients that are too large to import through the cell wall
and plasma membrane. These exoenzymes use hydrolysis reactions to breakdown
carbohydrate molecules such as amylose, or protein molecules such as casein. The
molecules that the enzymes act on are called substrates. Interestingly, ß-lactamase is
an exoenzyme that breaks down penicillin related antibiotics. When released from a
cell, it can break down the antibiotic in the environment, permitting penicillin-sensitive
cells in the vicinity to survive and grow. In our lab, we will be determining whether our
bacteria produce catalase (turns hydrogen peroxide into water and oxygen) and
gelatinase (hydrolyzes gelatin). If a bacterial strain is catalase positive, bubble will
appear immediately when the hydrogen peroxide is added. If the strain is gelatinase
positive, the gelatin will be broken down and will appear liquefied after incubation.
Carbohydrate fermentation
There is a great deal of microbial diversity in their ability to ferment various
carbohydrates, which we can also use for identification purposes. Fermentation is a
process used by different microorganisms as a low-energy yielding alternative when
cellular respiration is inhibited. Fermentation does not need oxygen, but can occur in
its presence. When bacteria ferment carbohydrates, many of the byproducts are acidic
and/or gaseous. We can use a well-established assay in the lab to detect fermentation.
This entails in using phenol red broth, a carbohydrate depleted medium with the pH
indicator phenol red. We add the carbohydrate of interest to this medium and inoculate
it with the bacteria. The culture tube containing the phenol red broth also has a smaller
inverted tube called a Durham tube. This tube will catch any microscopic gas bubbles
produced by the bacteria, and will result in an easily observable bubble. Phenol red is
Page | 78 red at neutral pH (about pH 7). As acid products build up due to bacterial fermentation,
the pH indicator will turn the medium yellow.
The IMViC tests
This is a group of classical tests used in microbiology for identifying unknowns,
especially within the enterics. There are four tests which make up the acronym: I is for
indole, M is for Methyl Red, V is for Voges-Proskaeur and C is for Citrate utilization.
The small “i” is added for easier pronunciation.
-
The Indole test is a test for tryptophan hydrolysis. If a bacteria strain produces
tryptophanase, it breaks down the amino acid tryptophan into indole,
ammonia and pyruvate. Cells are grown in tryptone broth, which contains a high
concentration of tryptophan. As tryptophan is broken down the indole escapes
into the media. When Kovac’s reagent is added to the culture tube, it reacts
with the free indole and produces a red organic layer at the top of the culture.
-
The Methyl red test detects mixed acid fermentation. Some bacteria produce
large quantities of acid products when they ferment glucose, lowering the pH of
the medium to pH 5 or less. Cells are inoculated into MR-VP medium, which
contains glucose, peptone and dipotassium phosphate. After a 48 hour
incubation, methyl red is added directly to the broth and color change is
observed. Methyl red is another pH indicator that behaves conversely to phenol
red. At pH 6.4 or below, methyl red is red, above this pH methyl red is yellow.
Therefore if a bacterial strain produces mixed acids, methyl red will turn a deep
red.
-
The Voges-Proskaeur test detects the production of a non-acid byproduct of
glucose metabolism called acetoin. Cells are inoculated into MR-VP medium
and after incubation for 48 hours, the culture broth is mixed with two VP
reagents. After about 30 minutes, these reagents will signal the presence of
acetoin by producing a pink/red color in the broth tube.
-
The Citrate test determines whether a bacterial strain can use citrate as a sole
carbon source. Bacteria are inoculated on a Simmons citrate agar slant, which
contains the pH indicator Bromothymol blue. If bacteria can use citrate as a sole
carbon source, they will produce alkaline products that raise the pH of the
medium, turning the pH indicator from green to Prussian blue.
Page | 79 Materials (per pair)
Exoenzymes
• Unknown cultures (slants and
broth)
• Inoculating loop
• Slides
•
•
•
•
2 gelatin deeps
Inoculating needle
Marking pen
Two sterile transfer pipets
Carbohydrate fermentation
• Unknown cultures
• Inoculating loop
• Marking pen
•
•
•
2 phenol red broth + lactose
2 phenol red broth + glucose
2 phenol red broth + fructose
IMViC tests
• Unknown cultures
• Inoculating loop
• 2 Tryptone broth tubes
•
•
•
2 MR-VP tubes
2 Citrate slants
(2nd lab period) two clean culture tubes
Materials (per group)
Exoenzymes
• Dropper bottle with hydrogen peroxide
• Cultures of Escherichia coli, Bacillus cereus and Staphylococcus aureus(on
slants)
• Broth cultures of Escherichia coli and Bacillus cereus
• 2 gelatin deeps
Carbohydrate fermentation
• Cultures of Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa,
Salmonella typhimurium and Staphylococcus epidermidis
• 4 each: phenol red broth + lactose, phenol red broth + glucose, phenol red broth
+ fructose
IMViC tests
• Cultures of Enterobacter aerogenes and Escherichia coli
• 2 Tryptone broth tubes
• 2 MR-VP tubes
• 2 Citrate slants
• (2nd lab period) Dropper bottle of methyl red
• (2nd lab period) two clean culture tubes
• (Added to cultures by instructor: Kovac’s reagent and VP reagents)
Page | 80 Procedure:
Exoenzymes
1. Clean a slide and draw three circles with the marking pen.
2. Aseptically remove a loopful of your positive control from the slant and place on a
clean glass slide. Repeat with your negative control.
3. Aseptically remove a loopful of your unknown from your slant and place on a
clean glass slide.
4. Place 2-3 drops of hydrogen peroxide on each sample and observe for
effervescence.
5. Record your results.
6. Take an inoculum from your unknown with the inoculating needle and stab the
gelatin deep. Repeat four times in different parts of the deep.
7. As controls, your group will inoculate one gelatin deep with E. coli (negative
control) and B. cereus (positive control).
8. Incubate for 48 hours at 35˚C.
9. After incubation, place in refrigerator for 30 minutes. After cold incubation,
observe for liquefaction of gelatin by tilting the tube. Make sure the cap is
screwed on well.
10. Record your results.
Carbohydrate Fermentation
1. Label each phenol red broth tube before you begin. (Please do not mix them up –
they all look the same!)
2. Inoculate your unknown using the loop into each of the three phenol red broth
tubes.
3. As a group, inoculate the positive and negative controls for both acid and gas as
instructed.
4. Incubate for 48 hours at 35°C.
5. Record your results.
IMViC tests
1.
2.
3.
4.
Label the tryptone and MR-VP culture tube so as not to confuse them.
Using the loop, inoculate your unknown into the tryptone broth and MR-VP tube.
Using your loop, streak your unknown on the citrate slant.
As a group, inoculate E. coli and E. aerogenes into the tryptone, MR-VP and
citrate media.
5. Incubate for 48 hours at 35°C.
6. After incubation, take half the volume in the MR-VP tube from your unknown
and place it in a clean culture tube. Label this tube “VP”. Repeat for the controls.
Page | 81 7. Gently flick your VP tubes and bring them to the instructor so that VP reagents
can be added to them. Gently flick the tube every 5 minutes and read the tubes
after 30 minutes and at the end of the lab. A positive result is a pink or rose
color. This reaction can be very slow.
8. Gently flick your tryptone tubes and bring them to the instructor so that Kovac’s
reagent can be added to them. A red layer on the surface is a positive result. The
reaction should be immediate, but allow 15 minutes to pass before you consider it
a negative result.
9. Add 3-5 drops of methyl red to your “MR” tubes. Do not add methyl red to
your VP tubes!
10. Gently swirl your “MR” tubes and observe the color. A positive reaction is a
reddish color (yellowish or no change is a negative result). The reaction should
be immediate.
11. Observe the color of the Citrate slant. A Prussian blue color is a positive result
(green is a negative result).
12. Read and record your results.
Results:
Answer YES or NO in result blank
Exoenzymes
Catalase
Sample
Unknown
+ control
- control
Strain
#
Bubbles?
Gelatinase
Sample
Unknown
+ control
- control
Strain
#
Liquefaction?
Carbohydrate Fermentation
Glucose - Acid
Sample
Strain
Unknown
+ control
- control
#
Yellow
media?
Glucose - Gas
Sample
Strain
Durham
bubble?
Unknown #
+ control
- control
Page | 82 Lactose - Acid
Sample
Strain
Unknown
+ control
- control
#
Fructose - Acid
Sample
Strain
Unknown
+ control
- control
Yellow
media?
Lactose - Gas
Sample
Strain
Unknown
+ control
- control
Yellow
media?
#
#
Fructose - Gas
Sample
Strain
Unknown
+ control
- control
Durham
bubble?
Durham
bubble?
#
IMViC
Indole
Sample
Unknown
+ control
- control
Strain
#
Voges-Proskaeur
Sample
Strain
Unknown
+ control
- control
Red
halo?
#
Methyl red
Sample
Unknown
+ control
- control
Rose
color?
Citrate
Sample
Unknown
+ control
- control
Strain
Red
color?
#
Strain
Prussian
blue
color?
#
Conclusions:
As a conclusion, summarize the biochemical profile for your unknown.
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Page | 83 Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 84 Exercise 17: Using BLAST to find sequence similarities
Goal: To identify similar gene sequences using the BLAST program.
Enzymes in Glycolysis
• Hexokinase
• Phosphoglucose
Isomerase
• Phosphofructokinase
• Aldolase
• Triose phosphate
isomerase
• Glyceraldehyde-3phosphate dehydrogenase
• Phosphoglycerate kinase
• Phosphoglycerate mutase
• Enolase
• Pyruvate kinase
Enzymes in Krebs
•
•
•
•
•
•
•
•
Citrate synthase
Aconitase
Isocitrate dehydrogenase
α-Ketoglutarate dehydrogenase
Succinyl-CoA synthetase
Fumarase
Succinate dehydrogenase
Malate dehydrogenase
1. Pick an enzyme from glycolysis or the Krebs cycle. Enzyme name: ____________
2. Go to the National Center for Biotechnology Information (NCBI).
http://www.ncbi.nlm.nih.gov/ (Note: you can enter “NCBI” into Google to find it
quickly.)
Or use the following QR code:
Page | 85 3. Open another tab and bring up the BLAST page from NCBI. Click on the BLAST link.
4. The following page will appear:
5. Go back to the NCBI tab and click on the “All Databases” menu and select “Protein”.
Page | 86 6. Enter the name of the enzyme you want to find followed by the name of your
unknown (as an example we’ll look for DNA ligase in Escherichia coli). Then click
Search.
7. When the following page appears, click on a record.
Page | 87 Protein BLAST
8. Scroll to the bottom of the record to locate the protein sequence.
9. Go the BLAST page and click on protein BLAST.
Page | 88 10. Go to the NCBI-Protein tab and copy the first 40 amino acids from the sequence. Go
the Protein-BLAST page and paste it in the “Enter Query Sequence” Box.
11. Scroll down and click on BLAST.
12. The following screen will appear after several moments.
Page | 89 13. Scroll below the multicolored bars to the “Descriptions” table.
14. For the highest ranking entry that appears record the Organism, Max score and E
value.
Organism: ____________ Max score: _________ E value: ________
Did the highest Max score correspond to the lowest E value?
YES
NO
15. Now repeat #8-14 but BLAST 60 amino acids.
Organism: ____________ Max score: _________ E value: ________
Did the highest Max score correspond to the lowest E value?
YES
Did the E value increase or decrease?
DECREASE
INCREASE
NO
Nucleotide BLAST
16. Go back to the protein record in Step #8 and click on “CDS”. At the bottom of the
page a box will open. Click on “FASTA”.
Page | 90 17. Copy 70 nucleotides from the first line of the DNA sequence (entire line).
18. Go to the BLAST results page and click on “Home”.
19. Then select “nucleotide blast” and paste the sequence.
Page | 91 20. Scroll to Choose Search Set, click “Others” so that you search the entire nucleotide
collection and not just human or mouse sequences. Verify that the dropdown menu
says “Nucleotide collection (nr/nt)”. Scroll down the bottom and click on “BLAST”.
21. For the highest ranking entry that appears record the Organism, Max score and E
value.
Organism: ____________ Max score: _________ E value: ________
Did the highest Max score correspond to the lowest E value?
YES
NO
22. To determine if your sequence was unique to your organism, read down the
description table until to find a change decrease in the Max score. Once you have
found the boundary, look at the organisms on the left and read up to the top of the
list. If only your organism appears, then your sequence was unique.
In our example, the Max score was 130 and most of the organisms found were E. coli.
Unfortunately, a few Shigella species were also found and therefore this 70
nucleotide sequence was not unique to E. coli.
Page | 92 Was your search sequence unique for your organism?
YES
NO
Explain. ____________________________________________________
23. Repeat #17-20 but search with 140 nucleotides instead of 70.
Did the E value increase or decrease?
INCREASE
Was this search sequence unique for your organism?
DECREASE
YES
NO
24. If this sequence identified your unknown, email it to yourself for use in your
unknown report.
Page | 93 Exercise 18: DNA cloning
Goal: Design a strategy to create a recombinant DNA molecule.
Introduction: One of the greatest advances in biotechnology is the ability to “mix-andmatch” genes from two different organisms. Recombinant DNA refers to a molecule
of DNA that comes from two different sources. Since all living things contain a common
DNA “code” that is turned into protein through transcription and translation, it was
thought that by introducing the DNA code for a protein (genes) from one organism into
another organism, this new organism would start producing this foreign protein. This is
indeed the case; as long as you introduce a complete gene, the new organism will
transcribe and translate it. In many cases the organism will also fold the protein
correctly so that it can function. What are the implications and some current
applications? Medically important proteins, such as insulin or human growth hormone,
were very expensive to isolate and purify from original sources (e.g., you need many
pituitary glands to isolate hGH). Sometimes substitutes were found, as in the case of
insulin dependent diabetics who injected themselves with porcine insulin. Now these
proteins can be expressed and produced in bacteria and are much more accessible to
patients.
To introduce genes from one organism into another you need to follow several steps.
You need to know where your gene is located on the DNA, generate many DNA copies of
the gene, add sequences to the ends so that it can be enzymatically “glued” into an
appropriate host vector and finally introduced into a host organism where the gene can
be transcribed and translated by host proteins.
This involves DNA “cutting and pasting” with enzymes. Enzymes that digest (cut) DNA
or RNA are called nucleases. The most common enzymes used to cut DNA are
restriction endonucleases. Once the vector and gene of interest have been digested
with the same restriction endonuclease, they can be “glued” to each other with an
enzyme called ligase. This is the same type of enzyme that works to connect the
discontinuous fragments generated during DNA replication. Once the gene and vector
have been glued together, they new recombinant molecule is introduced by
transformation into bacteria (or transfection if the recipient is a plant or animal
cell). At this point the recombinant DNA molecule is confirmed to have the right DNA
sequences and the new therapeutic protein is analyzed and collected.
When designing a strategy to clone a gene into another organism we need to:
1) Find the entire sequence for your gene and which restriction enzymes cut
OUTSIDE of the gene (if the restriction enzyme cuts in the middle of the gene,
you will lose part of gene!)
Page | 94 2) Pick an independently replicating circular DNA molecule (called a vector) and
find restriction enzymes that only cut ONCE within it.
3) Choose a common restriction enzyme between 1 and 2.
4) Make many copies of the gene using the Polymerase Chain Reaction (PCR). This
requires designing primers with restriction sites at the end (chosen in #3) that
amplify the entire gene.
5) Digest the PCR product (containing your gene)and the vector with the chosen
restriction enzyme.
6) Perform gel electrophoresis (i.e., run a gel) on the gene fragment and vector to
confirm correct sizes.
7) Isolate the gene fragment and vector from the gel and glue them together with
ligase.
8) Insert into your host organism using transformation or transfection.
9) Isolate the DNA molecule and make a restriction map to confirm your gene is
inserted into the vector correctly.
We can visualize the following steps below:
Page | 95 Tools used in DNA cloning
Restriction enzymes
Enzymes that cut DNA (or RNA) are called endonucleases or exonucleases.
Endonucleases can cut intact DNA while exonucleases need a free DNA end to cut. The
“cutting” reaction is a hydrolysis reaction that breaks the phosphate-oxygen bond in the
DNA backbone. Restriction endonucleases are a large category of enzymes used to
cut DNA. These enzymes naturally occur in many bacterial strains and function as a
defense mechanism against viral infection since they recognize palindromic DNA
sequences. These nucleases first recognize a specific sequence in the DNA and then cut
it. Since DNA is double-stranded, three types of “cuts” are produced, depending on
where the cut on the top strand occurs relative to the bottom strand: 5’ overhangs, 3’
overhangs and blunt ends.
5’-GT
TATAAC-3’
3’-CAATAT
TG-5’
5’ Overhang
5’-TGGCC
A-3’
3’-A
CCGGT-5’
5’-GGAC
3’-CCTG
3’ Overhang
Blunt
GTCC-3’
CAGG-5’
Even though the DNA backbone has been cut, this does not affect the ability of the
nucleotides to hydrogen bond with one another. Therefore overhangs can hydrogen
bond with single-stranded complementary sequences. When overhangs have sequences
that can hydrogen bond with one another they are called compatible.
PCR
PCR stands for the Polymerase Chain Reaction. This method was an inspiration of Gary
Mullis while he was returning from a surfing expedition. His insight is now one of the
most valued techniques in Biotechnology and garnered for him the Nobel Prize. PCR is
essentially DNA replication in a test tube. It is a way to make many copies of a DNA
template using a high temperature DNA polymerase, dNTPs and short primer
sequences. By going through hot-warm cycles, the DNA separates, finds a primer,
synthesizes new DNA and the process then begins again.
Gel Electrophoresis
Gel electrophoresis is a method used to separate DNA fragments based on length in base
pairs. The theory is that larger DNA strands have a hard time travelling through a
matrix made of small gel particles (agarose or polyacrylamide). They will therefore
remain close to where they were originally added to the matrix. Smaller fragments on
the other hand can find the nooks and crannies between the gel beads and can travel
farther. When an electric field is applied to these fragments, they will travel towards the
Page | 96 positive end since DNA is negatively charged. Longer fragments will migrate more
slowly than smaller fragments and this difference in migration can be observed when
the DNA is stained with a DNA-binding dye such as ethidium bromide.
Materials (per person)
• Computer with internet connection (DSL preferred)
Procedure:
Summary
You discover that the catalase plays an important role in handling toxic intermediates.
To study this enzyme further you will design a strategy to introduce its gene from a
given microbe into a vector from Escherichia coli; thus making a recombinant DNA
molecule. NOTE: the procedure below is a sample using a different strain
from the one you were assigned.
Becoming Familiar with PCR and Gel electrophoresis
PCR
1. Go to the “Life Sciences Learning Center – PCR Virtual Lab”:
https://www.urmc.rochester.edu/MediaLibraries/URMCMedia/life-scienceslearning-center/media/PCR_final.swf?ext=.swf
2. Do the PCR lab.
3. (Optional) For another animation, you can view this
http://learn.genetics.utah.edu/content/labs/pcr/
Page | 97 Gel Electrophoresis
Go to http://learn.genetics.utah.edu/content/labs/gel/
Go through the animation to understand electrophoresis.
Generating PCR primers specific to gene of interest
1) Open two tabs in your browser:
a. one for National Center for Biotechnology Information (NCBI)
http://www.ncbi.nlm.nih.gov/
b. another for NEBcutter from New England Biolabs
http://nc2.neb.com/NEBcutter2/
Page | 98 2) In the NCBI webpage, select the “Protein” Database from the drop down menu
and type the enzyme and name of the microbe (including the strain
name/number) into the search box. Click search.
3) A list of enzymes will appear. Click on the one that has the correct amino acid
length (as given).
Page | 99 4) A reference page for the enzyme will appear.
5) Scroll to the bottom of the reference page to the protein sequence. Click on
“CDS” on the left hand side.
6) A box will appear near the bottom. Record the locus name (gene name – 4
letters) in the Results section. Then click on “FASTA”
Page | 100 7) The nucleotide sequence for the gene will appear on the page. Write down the
chromosome coordinates for the gene (nucleotide numbers). Subtract them and
give an exact length (in bases) for the gene.
8) After recording the gene length, go the “Change region shown box” and subtract
200 bp from the left value and add 200 bp to the right value. Click the “Update
View” button. This will return the gene sequence with 200 bp on each side.
9) After pressing “Update View” copy the entire sequence, click on the tab for NEB
Cutter and paste it into the NEB Cutter box. Then click the “Submit” button.
10) Click back on the NCBI tab and click on “Pick Primers”
Page | 101 11) The following page will appear. In the Primer parameters box, there is a
parameter called “PCR product size”. This is where you enter a range for the
desired PCR product. Remember that you want to amplify the entire gene so you
need to add 200 bases to the original size. Your Minimum value will be: gene
length + 200 and your Maximum value will be: gene length + 400. Then click
the “Get Primers” button at the bottom.
Page | 102 12) This page will take a while to process. Once it has finished you will get a similar
page to this:
13) Print the first page of the results and tape it to your lab manual. Then scroll down
the page and locate the first set of primers. Write the forward and reverse primer
sequence (5’-3’) and the final product size in the Results section.
For these primers to be ready to use in PCR, we would add the recognition sequence
from our chosen restriction enzyme at the 5’ end of each primer. For example, if we had
Page | 103 chosen EcoRI, GAATTC would be added to the 5’ end so that the new Forward primer
would read GAATTCCGATTCC… (we would also add GAATTC to the Reverse primer).
14) Go back to the NEB Cutter tab and you will see a page that contains a linear map
with restriction sites. Print this page and attach to your lab manual. Then in the
“List” box click on “0 cutters”.
15) A list of enzymes that do NOT cut will appear. Print this list, highlight the
enzymes that are in the results tables and attach to your lab manual. Then use it
to fill the tables in the results section.
16) Click “Back to main display” and repeat #13 with the “1 cutters” list.
17) To get information on the restriction sites in pBR322, go back to the NEB
homepage and select “Products”. Then click on “DNA Plasmids”. Under “Related
Technical Resources heading, click on the “DNA sequences and Maps tool” link.
18) Scroll down to find pBR322 and click on “Site” on the far right column.
19) Print out the sites page for pBR322, highlight the restriction enzymes present in
the results table and use the information to complete the tables in the results
section.
20) After filling out the table, pick an enzyme that is not present in your PCR gene
product but that is present once in pBR322. This will be the enzyme that you use
to make your recombinant DNA molecule.
21) Now pick an enzyme that cuts once in your PCR product and once in pBR322.
This will be the enzyme you use to confirm your new recombinant molecule.
22)Make a map of your recombinant molecule in the circle pictured in the results
section label the gene name and restriction sites where you inserted the gene into
the vector. Also write the total size of the new molecule in the middle of the
Page | 104 circular map. In addition, label the location of the restriction site you will use to
confirm the molecule.
23)In the gel pictured in the results section, draw the band(s) at the appropriate level
that would expect for your PCR product (lane A), repeat for your vector (lane B)
and in lane C draw the band(s) you would expect after your confirmation digest.
24)Finish by filling in the Cloning Summary
Results:
Generating PCR primers specific to gene of interest
Enter your microbe (including the strain) here: ________________________
The name of the gene locus is: _________
Starting nucleotide number: _______
Ending nucleotide number: _______
Gene length: ______ Gene length + 200 = ______ Gene length + 200 = ______
Forward primer: ________________
Reverse primer: ________________
Final product size from PCR (in bp): __________
In the space below, tape your results from your Primer-Blast showing your primers.
Page | 105 Choosing appropriate restriction enzyme for cloning PCR product
List 3 enzymes that cut once in your gene: _______________________________
Using your results in NEBcutter and the pBR322 map, fill in the tables below:
Enzyme information
Enzyme
Recognition
Sequence
BamHI
BglII
EcoRI
HinDIII
KpnI
NdeI
PstI
SmaI
XhoI
Overhang
(3', 5' or Blunt)
Enzyme
Confirmation table
Cuts ONCE in
Cut site in gene
gene
(number)
(Place "X")
BamHI
BglII
EcoRI
HinDIII
KpnI
NdeI
PstI
SmaI
XhoI
Enzyme
Cloning table
Cuts ONCE in
Cuts in gene?
pBR322
(Place "X" if NO)
(Place "X")
Cut site in pBR322
(number)
BamHI
BglII
EcoRI
HinDIII
KpnI
NdeI
PstI
SmaI
XhoI
From your data above, choose an appropriate restriction enzyme to clone your gene:
__________
From your data above, choose an appropriate restriction enzyme to confirm you have
cloned your gene correctly: __________
Page | 106 Paste your Restriction map from NEB cutter and the “0 cutter list” on this page.
Page | 107 Restriction map and gel electrophoresis
bp (KB)
Lane A
Lane B
Lane C
8 6 4 3 1 0.5 0.2 Summary of cloning strategy
In order to clone the _________ gene from _____________ into E. coli, I will use a
forward primer with the following sequence: ________________ and a reverse
primer with the following sequence:__________________ to perform PCR on
genomic DNA from my microbe.
I will then digest the PCR product and my vector pBR322 with _________. When I
analyze these samples with gel electrophoresis, I expect to observe ______ band(s) in
my PCR product with _______ bp(s) in size. My vector should produce ______
band(s) with __________ bp(s) in size.
To confirm my new recombinant molecule, I will digest it with _______ and expect to
observe _________ band(s) of _______________ bp(s) when analyzed in gel
electrophoresis.
Page | 108 Exercise 19: Chemical Transformation of Bacteria
Goal: To introduce a plasmid bearing an ampicillin resistance gene using chemical
transformation into Escherichia coli that is sensitive to ampicillin
Introduction: One of the most significant processes discovered with the advent of
molecular biology is that cells can take up foreign DNA and express the genetic
information contained therein. This has allowed scientists to isolate genes that are of
medical or industrial interest and insert them into various types of cells. For example,
human insulin used to treat diabetics is currently produced in Escherichia coli. Many
crops are now pesticide and herbicide resistant due to genes that were introduced to
allow them to cope with these insults. Transformation is the process by which
bacteria take up DNA and begin to express the information carried on the DNA. Some
bacteria, such as Acinetobacter sp. can take up DNA from the environment without
additional manipulation. This is referred to as natural transformation.
There are two major (artificial) methods for bacterial transformation: electrical and
chemical transformation. In electrical transformation (also called electroporation),
bacteria are treated with an electrical shock that opens many tiny hole in the cell walls.
This allows the plasmid to enter the cell. In chemical transformation, a cation like
calcium is used to bring the plasmid DNA close to the cell. A brief heat shock opens up
holes in the cell wall and allows the plasmid to enter. Cells that are prepared to take up
DNA are called competent cells. Even the best competent cells do not give perfect
transformations, therefore you will notice that although you are plating millions of
bacteria on the plate, only several hundred cells will actually take up the plasmid.
Materials (per group)
• 1 plate with single DH10B E. coli colonies
• 2 tubes (16mm) with 0.7 ml of 0.05M CaCl2
• 1 tube with 5ml of Luria Broth
• 2 sterile test tubes (or Eppendorf tubes)
• 1 x 1 ml sterile pipette
•
•
•
•
•
4 sterile transfer pipettes
Glass beads or two bent
glass rods
1 beaker with ice
2 LB-amp plates
1 LB agar plate
Procedure:
Summary
You will resuspend fresh bacterial colonies in cold calcium chloride. To this mixture you
will add plasmid DNA, then heat shock the tubes and plate the mixtures on LB-amp and
LB agar plates.
Page | 109 1. Take your sterile tubes and label one with a “+” and the other with a “-”.
2. Using the transfer pipette, place 0.25 ml of CaCl2into each tube. Do not discard
the transfer pipette as you will use it to mix the bacteria.
3. With your loop collect 2-3 single E. coli colonies from the plate (approximately
the diameter of a pencil eraser) and transfer into the sterile tubes with CaCl2.
4. Mix cells and CaCl2by pipetting up and down with the transfer pipette.
5. Return the “-” tube to the ice and bring up the “+” tube to the instructor.
6. Your instructor will add 10 µl of DNA to your “+” tube.
7. Mix the contents well with the transfer pipette and incubate both tubes on ice for
15 minutes.
8. During incubation, label both LB-amp plates with Class ID and Team Number.
Then label one with “LB-amp + plasmid” and the other one with “LB-amp –
plasmid”. Label the LB agar plate with Class ID, Team Number and “LB agar +
plasmid”.
9. After the ice incubation, place both tubes in the 42˚C water bath, making sure
that the bottoms of the tubes are submerged below the water line. Keep them
there for exactly 90 seconds, while you shake them gently. This is the heat
shock step.
10. Place both tubes back on ice for 2 minutes.
11. Add 1 ml of Luria broth with the sterile 1 ml pipette using the blue pipetter.
12. Place in 37˚C water bath for 20 minutes.
Glass beads
13. If your class is using glass beads to spread the bacteria on the plate, take up all
the labeled plates to the front so that the instructor can dispense the glass beads
into your plates.
Page | 110 14. Using the sterile transfer pipet, pipette 0.2 ml from the “+” tube onto one LBamp plate. Gently shake the plate (with the beads facing up) to spread the
bacteria over the plate. Make sure that the beads are rolling over the entire
surface of the plate. Correct technique should produce the sound of maracas.
15. After the plate has absorbed the culture (the plate looks dry), turn the plate over
so that the beads fall on the lid. Gently remove the lid and place the beads in the
appropriate waste container (as specified by instructor)
16. Using the same sterile transfer pipet, pipette 0.2 ml from the “+” tube onto the
LB agar plate and spread the cells as described above.
17. Repeat steps 14 and 15 with the “-” tube and plate this sample on the other LBamp plate.
18. Go to step 24.
Bent glass rods
19. Using the sterile transfer pipet, pipette 0.2 ml from the “+” tube onto one LBamp plate.
20. Dip the bent glass rod in the 70% isopropanol and pass once through the Bunsen
burner flame to sterilize.
21. Using the rod, spread the culture over the entire surface of the agar plate – make
sure that you spread the culture all the way to the edges of the plate! Rotating
the plate while you move the glass rod back and forth may help.
22. Continue to spread the bacteria on the plate until the culture has been absorbed
and the plate looks dry. Dip the glass rod in the isopropanol and flame after
finishing.
23. Using the same procedure above, spread 0.2 ml from the “+” tube onto the LB
agar plate and spread 0.2 ml from the “-” tube onto the other LB-amp plate.
24. Tape your group’s plates together and place in designated incubation location.
The plates will be incubated at 35-37˚C for 48 hours.
Page | 111 Results:
Plate
Sample type*
Result^
LB-amp + Plasmid
LB agar + Plasmid
LB-amp - Plasmid
* Test sample, positive control or negative control
^ Lawn, countable colonies, no growth
In this experiment, the LB agar - plasmid was omitted. What is a good reason to omit
this control? What type of results would require that we have this control?
____________________________________________________________
____________________________________________________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 112 Exercise 20: The Standard Plate Count
Goal: To estimate the number of bacteria in various food and
environmental samples
Introduction: It is critical to know the number of bacteria in food samples to
determine whether food is safe to consume. In addition, knowing the number of
bacteria in water is also useful to determine whether there has been fecal contamination.
The most common method for determining the number of bacteria in a sample is the
Standard Plate Count. It combines two techniques: the pour plate method and the
serial dilution method. The pour plate method combines molten agar (held around
45˚C) with a sample and is then poured into a petri dish. In addition to allowing the
growth of aerobes, this method permits some microaerophiles and facultative anaerobes
to grow since some cells will be embedded in the agar. Since we do not know how many
cells are originally present in the sample, we use serial dilutions in which we take 1/100,
1/1000, 1/10,000, etc. of the sample. One (or more) of these dilutions will produce
countable colonies on the plate (between 30-300 colonies). Remember that not all
bacteria present in the sample will grow because they may have special physical or
chemical requirements for growth. This also means that our Standard Plate Count
method is an estimate of the number of bacteria in the sample. We therefore use the
number of colonies and call them Colony Forming Units (CFUs) per gram of sample
as our final estimate.
To estimate the number of bacteria in the sample we use the following equation:
# of Colony Forming Units (CFUs) x amount 1 g of sample was diluted (Dilution factor)
= #CFUs/gram of sample.
If you diluted your sample 1/100, then your dilution factor is the reciprocal of this
number (100). If you diluted your sample 1/7, your dilution factor would be 7. If
several dilutions give you countable colonies (CFUs) then you could calculate the
CFUs/g of sample for each of the dilutions and logically would expect similar results.
Interestingly, the Standard Plate Count method could be used to design experiments
that could test hypotheses such as the “5 second rule” or find out how long mayonnaise
can stay out in the sun before it goes bad.
Page | 113 Materials (per pair)
• 1 milk dilution bottle with 99ml sterile water
• 3 screwcap test tubes each with 9ml sterile
water
• 4 melted TSA pours held at 55°C in the water
bath
• 4 empty sterile petri plates
Materials (per group)
• 1 milk dilution bottle with 99ml sterile water
• 3 screwcap test tubes each with 9ml sterile
water
• 6 melted TSA pours held at 55°C in the water
bath
• 6 empty sterile petri plates
•
•
•
•
•
1ml pipetters
2 x 1ml pipettes
A food sample
1 weighing dish
1 spoon/spatula
•
•
•
•
•
•
1 digital balance
1 soil sample
1 weighing dish
1 spoon/spatula
2 x 1ml pipettes
1ml pipetters
Procedure:
1. With labeling tape, label the three tubes with sterile water 10-3, 10-4 and 10-5.
Label the petri dishes 10-3, 10-4, 10-5 and 10-6.
2. Weigh out 1 gram of your sample and add it to the milk dilution bottle containing
99ml of water.
3. Screw the cap tightly and shake the bottle up and down 30 times.
NOTE: All transfers are aseptic.
4. Transfer 1ml of the sample to the tube labeled 10-3. Screw the cap on tightly and
invert the tube 6 times to mix.
5. Using the same pipette, transfer 1 ml from the 10-3 tube to the 10-4 tube. Screw
the cap on tightly and invert the tube 6 times to mix.
6. Using the same pipette, transfer 1 ml from the 10-4 tube to the 10-5 tube. Screw
the cap on tightly and invert the tube 6 times to mix.
7. Dispose of the pipette in the biohazard waste.
8. Using a new 1ml pipette, transfer 0.1 ml from the 10-5 test tube to the petri dish
labeled 10-6. By taking 1/10 of sample (0.1 ml), you are effectively making
another 1/10 dilution. Your lab partner will pour the molten agar into the plate
and gently swirl to mix the sample.
9. With the same 1 ml pipette, transfer 1 ml from the 10-5 test tube to the petri dish
labeled 10-5. Your lab partner will pour the molten agar into the plate and gently
swirl to mix the sample.
10. With the same 1 ml pipette, transfer 1 ml from the 10-4 test tube to the petri dish
labeled 10-4. Your lab partner will pour the molten agar into the plate and gently
swirl to mix the sample.
Page | 114 11. With the same 1 ml pipette, transfer 1 ml from the 10-3 test tube to the petri dish
labeled 10-3. Your lab partner will pour the molten agar into the plate and gently
swirl to mix the sample.
12. As a group, you will repeat the procedure (steps 1-11) with a soil sample.
13. As a group, you will use two pours to make two TSA plates without any sample.
Label the plates A and B.
14. Allow all plates to solidify, tape them together and place in correct area for
incubation. Plates will be incubated for 48 hours at 35C.
Results:
Count all the colonies on your plates. If you get above 300 colonies on any plate, you
can record it as “TMTC” (Too many to count).
Sample: _____________
Plate
Dilution Factor
Colonies on plate
CFUs/g of sample
10-3
10-4
10-5
10-6
Average of CFUs/ g of sample: _____________
Control Sample: _____________
Plate
Dilution Factor
Colonies on plate
CFUs/g of control
10-3
10-4
10-5
10-6
Average of CFUs/ g of control sample: _____________
Page | 115 Control Sample: _____________
Plate
Dilution Factor
Colonies on plate
CFUs/sample
A
B
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 116 Exercise 21: Physical Methods of Controlling Microbial Growth
Goal: To investigate how various physical methods can be used to control
microbial growth
Introduction: Although pathogenic microbes are in the minority, they are of primary
concern to us if we are to remain healthy. Methods to control the growth of microbes
can be divided into two strategies: physical and chemical methods of control. Among
physical methods, the most common methods used today are heat
sterilization/disinfection, radiation, osmotic pressure, filtration and freezing. In this
exercise we will determine the effectiveness of moist heat and osmotic pressure
methods. Sterilization refers to the removal of all life forms, including hardy
endospores. Disinfection refers to the destruction of vegetative cells.
Moist Heat Methods
Methods that use heat to control microbial growth can be divided into two groups: dry
heat and moist heat methods. Dry heat methods kill microbes by burning (oxidation) or
denaturing protein, and require high temperatures. Moist heat has a great advantage in
terms of penetration. Since cells are mainly composed of water, hot steam directly
transmits energy to the cell and cellular proteins will denature. Heat can also damage
cell membranes and nucleic acids. Therefore using moist heat is more effective at lower
temperatures than dry heat. There are four types of moist heat methods generally used:
boiling, tyndallization, autoclaving and pasteurization.
•
•
•
Boiling in water (100˚C) for 15 to 3o minutes kills most vegetative cells but does
not kill endospores. Because endospores survive, boiling is technically a
disinfection method.
Tyndallization uses steam or boiling (100˚C) for 30 minutes, followed by
incubation at 37˚C overnight. This process of steaming/boiling followed by
incubation is repeated three times. The incubation allows for germination of
spores, which will then be killed in the next steam/boiling treatment. This
method can therefore be classified as a sterilization method.
Autoclaving uses steam that is heated to 121˚C for 15 minutes. To reach 121˚C,
the steam is pressurized to 15 pounds/square inch (PSI). The increase in
pressure increases the temperature. This treatment kills all vegetative cells and
endospores and therefore is considered a sterilization method. Interestingly, one
way to test if your autoclave is functioning properly is to include an enclosed
capsule containing bacterial spores from a thermophile, autoclaving the capsule
Page | 117 •
and then incubating in TSB. If there is growth, the autoclave is not working
properly.
Pasteurization is used to kill vegetative pathogens and some microbes that cause
food spoilage in food products such as milk, fruit juices, beer and wine. This
treatment does not eliminate endospores and therefore is considered a
disinfection method. There are several variations of the pasteurization treatment
in which temperature is increased and treatment time is decreased. The Low
Temperature Holding (LTH) method, in which samples are heated to 63˚C for 30
minutes, is the one we will use in the lab.
Osmotic pressure
The movement of water across a semi-permeable membrane is called osmosis. As the
water moves in or out of the cell, it exerts a force that is referred to as osmotic
pressure. Since many metabolic reactions are dependent on certain concentration of
water in the cell, increasing or decreasing the water in the cell can affect cell
metabolism. This situation is especially true when water leaves the cell due to a
hypertonic environment. In this situation, the solute concentration is much higher on
the outside of the cell, and water inside rushes out to “balance” the water concentration.
This results in water leaving the cell and the plasma membrane pulling away from the
cell wall, a process that is termed plasmolysis. The low concentration of water in the
cell will prevent many metabolic reactions from taking place and the cell stops growing.
This is the rationale behind using salt (or sugar) as a food preservation technique. Of
course some organisms are halophiles and survive quite nicely in hypertonic
environments.
In a hypotonic environment, where water rushes into the cell because the solute
concentration is higher on the inside than on the outside, the cell wall provides
resistance against cell lysis. If the cell wall has been compromised, as when cells have
been treated with penicillin, then the cells will burst.
Moist heat methods
Materials (per group)
• 18 tubes with TSB
• 600ml beaker with boiling chips
• Inoculating loops
• Test tube holder
• Hot plate
• Index card with lines
• Broth cultures of Bacillus cereus, Bacillus subtilis spores, Escherichia coli and
Mycobacterium smegmatis
Page | 118 Procedure:
1. Fill beakers half full with tap water and boil on hot plate (include 5 boiling chips).
2. Each person should work with one culture. Label your tubes with your organism
and the treatments (3 moist heat treatments & control).
(Note: make sure your label goes around the tube so it doesn’t fall off during
treatment.)
3. Inoculate your culture into each of the 4 TSB tubes. Two uninoculated TSB tubes
will remain as controls.
4. Treat your TSB tube with the appropriate method (coordinate with your group).
a. For boiling, place your tube in the beaker with boiling water and boil for 10
minutes.
b. For Pasteurization, place your tube in the 63C water bath for 30 minutes.
c. For the autoclave treatment, bring your tube to the rack labeled
“autoclave” in the front.
d. You will not treat your control tube.
5. After treatment (don’t worry about the autoclave samples), place all tubes in the
appropriate location for incubation. Make sure to include two uninoculated TSB
tubes with your samples as well.
6. After a 48-hour incubation, score the level of growth in your tubes. To gauge
turbidity, use the provided index card with lines. Use your control tube with
most turbidity as your ++++ value. Your uninoculated tubes will be your – value.
Osmotic Pressure
Materials (per group)
Materials (per pair)
• 6 TSB broth tubes
• Inoculating loops
• 6 TSB broth + 6.5% NaCl
• Individual unknown cultures
• 6 TSB broth + 10% NaCl
• Index card with lines
• Cultures of Escherichia coli and Staphylococcus aureus
Page | 119 Procedure:
1. Inoculate your unknown using a loop into each of the three TSB tubes containing
different salt concentrations.
2. As a group, inoculate a control set with the known cultures.
3. Incubate for 48 hours and note your results. To gauge turbidity, use the provided
index card with lines. Use your positive control tube with most turbidity as your
++++ value. Your negative control tube will be your – value.
Results:
Moist heat methods
Score for turbidity in your samples – make sure that you mix the tube well before noting
your results!
Organism
63°C, 30
min
100°C,
10min
121°C, 15 Positive
min
ctrl
Negative
ctrl
Bacillus cereus
Bacillus subtilis spores
Escherichia coli
Mycobacterium smegmatis
According to your results, which methods disinfect? (Circle all that apply)
Pasteurization
Boiling
Autoclaving
According to your results, which methods sterilize? (Circle all that apply)
Pasteurization
Boiling
Autoclaving
Page | 120 Osmotic Pressure
Score for turbidity in your samples – make sure that you mix the tube well before noting
your results!
Sample
Strain
TSB
TSB +
6.5% NaCl
TSB +
10% NaCl
Unknown
Positive control
Negative control
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 121 Exercise 22: Chemical Methods of Controlling Microbial Growth
Goal: To investigate how various chemical methods can be used to control
microbial growth
Introduction: In addition to physical methods to control microbial growth, chemical
methods are also used frequently in health care settings. Chemical methods can be
classified into two general categories: 1) Disinfectants/Antiseptics and 2)
Antimicrobials. In this exercise we will determine the effectiveness of a variety of
household disinfectants/antiseptics and antimicrobials on elimination microbial
growth.
Disinfectants/Antiseptics
Disinfectants refer to chemical substances applied to nonliving objects in order to kill
or inhibit microbial growth. Antiseptics are substances that can be applied to living
tissue. Many chemical compounds can be used both on living tissue and inanimate
objects, thus serving both as disinfectants and antiseptics. A summary chart of common
chemical agents is listed below:
Class of agent
Surfactants
Quaternary
compounds
Phenolics
Peroxygens
Mode of
action
Lower surface
tension;
physical
removal
Microbial
targets
General
Disadvantages
Examples
No killing action
Soap
Protein
denaturation/
Disrupt plasma
membranes
Disrupt plasma
membranes/
Denature
enzymes
Gram positive
Enveloped
viruses
Fungi
Mycobacteria;
good to use on
body fluids
Inactivated by
soaps and anionic
substances
Mouthwash
Oxidation
General, even
sporocidal
Hydrogen
peroxide should
not be used on
wounds
Lysol
Hydrogen
Peroxide
Page | 122 Alcohols
Denature
proteins and
dissolves lipids
Bacteria
Fungi
(not sporocidal)
Does not sterilize;
need correct
concentration
Isopropanol
Halogens
Oxidizing
agents
Iodine targets
bacteria, spores
and fungi
Without tincture,
poor wetting
action
Bleach,
betadine
Antimicrobials
Competition and survival in nature can be observed even on the molecular level.
Various organisms can secrete substances that inhibit growth or even kill their
competitors. One notable example, which led to the discovery of the first antibiotic, was
the observation that a mold Penicillium notatum inhibited the growth of gram-positive
bacteria. This observation was made by Alexander Fleming and led to the discovery of
Penicillin. Antimicrobial substances that are naturally secreted by microorganisms are
termed antibiotics. Antimicrobials refer to any substance that kills or inhibits the
growth of microbes. Antimicrobials may be semi-synthetic, denoting the chemical
modification of naturally occurring antibiotics so that they are more effective.
Synthetic antimicrobials are substances that are chemically synthesized in their
entirety.
The spectrum (narrow or broad) of an antimicrobial refers to the types of microbes
against which it is effective. For example, narrow spectrum antimicrobials may only be
active against several gram-negative species, whereas broad spectrum antimicrobials
may be effective against mycobacteria and gram-positive bacteria. For our discussion,
the main types of classes of bacteria are gram-positive, gram-negative, mycobacteria
and chlamydiae (intracellular pathogens).
Measuring microbial susceptibility
To determine the susceptibility of microbes to a particular chemical agent, several
different assays (tests) can be carried out. They are classified as diffusion or broth
dilution methods. Two common diffusion methods are the disk-diffusion method
(or Kirby-Bauer test) and the E test. The disk diffusion method uses filter disks that
are soaked with a specific concentration of an antimicrobial. The disk is then placed on
a petri plate that has been previously inoculated with a test organism spread over the
entire surface of the plate. After incubation, one measures the zone of inhibition around
the filter disk. The size of the zone is compared to standards to determine whether the
organism is resistance, intermediate or susceptible to the antimicrobial agent. In the E
test, a strip containing a gradient of antimicrobial concentrations is placed on a
Page | 123 organism inoculated petri plate. After incubation, the zone of inhibition looks like an
ellipse around the strip. At the point where the zone of inhibition disappears, one can
determine the minimal inhibitory concentration (MIC), or the lowest
concentration of the antimicrobial that prevents visible microbial growth.
To measure the zone of inhibition, use millimeters (mm) and measure including the
disk:
Zone of inhibition
Disinfectants/Antiseptics
Materials (per group)
• 4 different disinfectants/antiseptics
in small beakers or petri dish lids
• Test tube with sterile water
• 8 TSA plates
• 8 sterile transfer pipets
• 4 nonsterile transfer pipets
•
•
•
•
1 dish of sterile paper disks
Glass beads or bent glass rods
1 jar of 70% Isopropanol
Broth cultures of Bacillus cereus,
Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa
Materials (per pair)
•
•
Individual unknown cultures (in broth)
2 clean forceps
Page | 124 Procedure:
1. Using your marker, divide each TSA plate into five sectors, labeling four sectors
with the agents you will test and a “control” sector. Also label the culture that will
be spread on the plate. (See sketch on next page.)
2. Each student will work with a known culture and their unknown.
Glass beads
3. If your class is using glass beads to spread the bacteria on the plate, take up all
the labeled TSA plates to the front so that the instructor can dispense the glass
beads into your plates.
4. Using the sterile transfer pipet, pipette 0.2 ml of your known culture onto the
TSA plate. Gently shake the plate (with the beads facing up) to spread the
bacteria over the plate. Make sure that the beads are rolling over the entire
surface of the plate. Correct technique should produce the sound of maracas.
5. After the plate has absorbed the bacterial culture and it looks dry, turn the plate
over so that the beads fall on the lid. Gently remove the lid and place the beads in
the appropriate waste container (as specified by instructor).
6. Go to step #11.
Bent glass rods
7. Using the sterile transfer pipet, pipette 0.2 ml of your known culture onto the
TSA plate.
Page | 125 8. Dip the bent glass rod in the 70% isopropanol and pass once through the Bunsen
burner flame to sterilize.
9. Using the glass rod, spread the culture over the entire surface of the agar plate –
make sure that you spread the culture all the way to the edges of the plate!
Rotating the plate while you move the glass rod back and forth may help.
10. Continue to spread the bacteria on the plate until the culture has been absorbed
and the plate looks dry. Dip the glass rod in the isopropanol and flame after
finishing.
11. Now spread your unknown on the other TSA plate as described above.
12. Sterilize your forceps by dipping into the isopropanol and flaming. Pick up one
sterile paper disk and dip it into the beaker/lid containing your
disinfectant/antiseptic.
13. Shake off excess liquid and place it on the agar surface in the appropriate sector.
14. Repeat this process with the three other agents. As a control, dip the sterile disk
into sterile water.
15. Tape your team’s plates all together and place in incubation station.
16. Your plates will be incubated at 35˚-37˚C for 24-48 hours.
17. After incubation, measure the zone of inhibition around each disk in mm and
record your results.
Page | 126 Antimicrobials
Materials (per group)
•
•
•
•
•
1 large Mueller-Hinton plate
1 sterile transfer pipet
2 forceps
1 dish with 2 sterile paper disks
Glass beads or bent glass rods
•
•
1 jar of 70% Isopropanol
One broth culture of either
Escherichia coli, Staphylococcus
aureus, Bacillus cereus or
Pseudomonas aeruginosa
Procedure:
1. Mark your Mueller-Hinton plate with the bacterial strain you will spread on it
and with dots that will serve as guides for antimicrobial disks.
Glass beads
2. If your class is using glass beads to spread the bacteria on the plate, take up your
Mueller-Hinton plate to the front so that the instructor can dispense the glass
beads into it.
3. Using the sterile transfer pipet, pipette 1.0 ml of your known culture onto the
Mueller-Hinton plate. Gently shake the plate (with the beads facing up) to
spread the bacteria over the plate. Make sure that the beads are rolling over the
entire surface of the plate. Correct technique should produce the sound of
maracas.
4. After the plate has absorbed the bacterial culture and it looks dry, turn the plate
over so that the beads fall on the lid. Gently remove the lid and place the beads in
the appropriate waste container (as specified by instructor).
5. Go to step #10.
Bent glass rods
6. Using the sterile transfer pipet, pipette 1.0 ml of your known culture onto the
Mueller-Hinton plate.
Page | 127 7. Dip the bent glass rod in the 70% isopropanol and pass once through the Bunsen
burner flame to sterilize.
8. Using the glass rod, spread the culture over the entire surface of the agar plate –
make sure that you spread the culture all the way to the edges of the plate!
Rotating the plate while you move the glass rod back and forth may help.
9. Continue to spread the bacteria on the plate until the culture has been absorbed
and the plate looks dry. Dip the glass rod in the isopropanol and flame after
finishing.
10. Sterilize your forceps by dipping into the isopropanol and flaming. Pick up one
sterile paper disk, dip it into the test tube with sterile water and place it on one of
the dots you made on the Mueller-Hinton plate.
11. Repeat this process with the antimicrobial disks. As you place the disks on the
plate, gently tap the disk to make sure that it completely touches the agar.
12. Tape your team’s plates all together and place in incubation station
13. Your plates will be incubated at 35˚-37˚C for 24-48 hours.
14. After incubation, measure the zone of inhibition around each disk in mm and
record your results.
Page | 128 Results:
Disinfectants/Antiseptics
Agent name
Class of agent
Measure the zone of inhibition using your plastic ruler.
Zone of Inhibition (mm)
Organism
Control
Agent:
Agent:
Agent:
Agent:
Bacillus cereus
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Bacterial Unknown #
In the table below, assess antiseptic/disinfectant effectiveness and bacterial resistance.
MOST
LEAST
Agent effectiveness
Bacterial resistance
According to your results, which disinfectant/antiseptic was the most effective against
your unknown? ________________________________
Which control was not included in the experiment? What would you use for this
control?
____________________________________________________________
____________________________________________________________
Page | 129 Antimicrobials
Record the zone of inhibition (ZOI) in millimeters for each antimicrobial and use the
table below to determine whether the bacterial strain is resistant, intermediate or
susceptible to the antimicrobial. Write your results on the board for the class. Record
the other groups’ data in your table as well.
Antimicrobial
Ampicillin
(Am)
Chloramphenicol
(C)
Novobiocin
(NB)
Penicillin G
(P)
Polymyxin B
(PB)
Streptomycin
(S)
Tetracycline
(Te)
Trimethyloprimsulfamethoxazole
(SXT)
Conc.
INTERPRETATION (mm)
Resistant
Intermediate Susceptible
(R)
(I)
(S)
10µg
≤ 13
14-16
≥17
30µg
≤12
13-17
≥18
30µg
≤17
18-21
≥22
10 units
≤11
12-21
≥22
300 units
≤8
9-11
≥12
10µg
≤11
12-14
≥15
30µg
≤14
15-18
≥19
25µg
≤10
11-15
≥16
Mode of
action
Divide the antimicrobials used according to the mode of action.
Mode of action
The zone of inhibition for the control disk is: __________ (mm)
Page | 130 Organism
Ampicillin
ZOI (mm) R/I/S
Chloramphenicol
ZOI(mm) R/I/S
ZOI(mm)
Organism
Novobiocin
ZOI (mm) R/I/S
Penicillin G
ZOI(mm) R/I/S
Polymyxin B
ZOI(mm) R/I/S
Organism
Streptomycin
ZOI (mm)
R/I/S
Tetracycline
ZOI (mm)
R/I/S
R/I/S
Trimethyloprimsulfomethoxazole
ZOI (mm) R/I/S
Page | 131 Totals
Organism
R
I
S
In the table below, record which strain is most/least resistant.
MOST
LEAST
Bacterial resistance
Which antimicrobial has the broadest spectrumof activity? _________________
Which antimicrobial has the narrowest spectrum of activity? ________________
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
Page | 132 Exercise 23: Water Quality
Goal: To investigate whether sewage indicators can be found in various sources of
potable water.
Introduction: One of the principal routes of disease transmission is through nonliving sources such as air, water and food. This type of transmission is generally known
as vehicle transmission. To be more specific, we can use airborne, waterborne or
foodborne transmission to denote the source. We are especially concerned with
waterborne transmission since many infectious diseases such as cholera, amoebic
dysentery and giardiasis can be transmitted when potable (drinking) water is
contaminated with fecal matter. The ease of disease transmission to many people is also
one of reasons we are greatly concerned when natural disasters happen in developing
areas of the world. A huge earthquake may not only kill many thousands; it also
compromises water quality likely leading to disease outbreaks.
We currently do not have the resources or technology to test for all the specific
pathogens that pollute drinking water, so we rely on sewage indicators. These are
organisms commonly present in the GI tract and their detection in our water sources is a
strong indication that fecal contamination has occurred. Currently, the most common
sewage indicators tested for are Enterococcus faecalis (Streptococcus faecalis in a
previous life) and coliform bacteria, which belong to the Enterics. Coliform bacteria
are non-spore forming, gram negative, facultative anaerobic rods that ferment lactose to
produce acid and gas.
To calculate the number of coliform bacteria in a potable water sample, we will use the
Most Probable Number (MPN) method. The name already implies that the method
does not determine the exact number of coliforms in a water sample, but estimates it.
For statistics geeks, the method is derived using the Poisson distribution. This method
is comprised of three different tests: the presumptive, confirmed and completed
tests. These tests are briefly described below.
The starting point is 100 ml water sample. In the presumptive test, a portion of the
water sample is added to each of 5 tubes containing double-strength lactose broth and a
Durham tube. This test serves as the first screen for coliforms in the water sample. If
coliforms are present in the water sample, they should ferment the lactose and produce
acid and gas. Since there is no pH indicator in lactose broth we cannot assess for acid
production, but we can assess for gas production using the Durham tube. A tube is
considered positive if it is turbid (remember that turbidity indicates growth) and if there
is a gas bubble in the Durham tube. To determine the presumptive MPN, we then look
Page | 133 up the number of positive tubes on the MPN index table, which gives us the MPN in a
100 ml water sample.
In the confirmed test, EMB plates are inoculated with a sample from a positive lactose
tube. EMB will help us determine if the bacteria growing in the lactose broth is gram
negative (selective), as well as whether it in fact ferments lactose (differential).
In the completed test, colonies from the EMB plates are inoculated onto a nutrient
agar slant and into a Phenol red broth tube containing lactose and a Durham tube.
Growth on the nutrient agar slants will be used for a Gram stain. The Gram stain
verifies whether colonies on EMB are in fact gram negative and a yellow phenol red
broth tube with a gas bubble would verify that the colonies produce acid and gas from
lactose.
Positive results in all tests would indicate that coliforms were present in our potable
water.
Session I: Materials (per class)
• 7 milk dilution bottle with sterile
water (3 positive and 4 negative
controls)
• 35 tubes (20x150mm) with 10 ml of
2x lactose broth & Durham tube
• 7 sterile 10 ml pipettes
• 7 sterile 1 ml pipettes
•
7 sterile transfer pipettes
7 TSA or Nutrient agar pours
7 sterile petri plates
3 x 1ml broth culture of Escherichia
coli or other enteric.
4 x 1ml TSB (for negative control)
•
•
•
•
1 sterile 10 ml pipette
1 sterile 1 ml pipette
1 sterile petri plate
1 TSA or Nutrient agar pour
•
•
•
•
Session I: Materials (per pair)
•
•
1 sterile milk dilution bottle (for
sample collection)
Five 20x150mm tubes with 10 ml of
2x lactose broth and Durham tube
Session II: Materials (per class)
•
•
20 EMB plates (60 mm)
Broth culture of Alcaligenes faecalis
Session III: Materials (per class)
•
•
•
20 nutrient agar slants
20 Phenol red broth tubes with
lactose and Durham tube
Cultures of Escherichia coli and
Pseudomonas aeruginosa
Page | 134 Session IV: Materials (per pair)
•
•
Gram stain reagents
Broth culture of Staphylococcus aureus and Escherichia coli
Procedure:
Summary
You will remove 50 ml from your water sample, 10 mls at a time, and transfer it to each
of five lactose tubes. You will then take 1 ml from your water sample and place it in the
empty sterile petri plate to calculate the number of bacteria/ml in your sample. In the
second session you will inoculate a loop from any positive tubes onto EMB and streak
for single colonies. In the third session, you will pick purple or metallic green colonies
and inoculate them into phenol red broth with lactose. You will also take a metallic
green or purple colony and inoculate a slant. In the fourth session, you will perform a
Gram stain from the slant.
Session I: Presumptive test
1. Label your lactose broth tubes and the empty sterile petri plate with your class
ID, team number and water source (5 lactose broth tubes/sample or control).
2. Transfer 10 ml from your water sample into each of the five lactose tubes using
the 10 ml sterile pipette and the green pipetter.
3. Repeat steps 1 and 2 with your assigned controls.
(Note: Spike positive controls with 0.5 ml of E. coli using the sterile transfer
pipette and negative controls with 0.5 ml of TSB.)
4. Pipette 1 ml from your sample into the sterile (empty) petri plate. Add the pour
on top of the water drop and swirl gently on the bench to mix the sample well.
5. Repeat steps 4 and 5 for your assigned control.
6. Place the lactose tubes in the designated incubation racks and after your plates
have solidified, tape them together. Tubes and plates will be incubated at 35˚37˚C for 48 hours.
Page | 135 Session II: Confirmed test
7. Determine the number of positive tubes in your samples and controls, and assign
an MPN value for them using the MPN index table. Also record the number of
colonies on your plate and calculate the CFUs in 100ml of your sample.
8. Record the MPN values for the class.
9. Choose one tube that is turbid and contains a gas bubble. Aseptically transfer
one loop from the tube onto an EMB plate. Streak out for single colonies.
(Note: If your sample had an MPN of < 2.2, then borrow a sample from a group
that had a higher MPN)
10. Repeat step 10 with the positive control.
11. Tape your team’s plates together and place in designated incubation area. Plates
will be incubated at 35˚-37˚C for 48 hours.
Session III: Completed test
12. Record whether you observed any growth on EMB and whether those colonies
were purple or metallic green.
13. If you had purple or metallic green colonies, label a nutrient agar slant and
phenol red broth lactose tube as in step 1.
14. Pick a purple or metallic green colony with your loop and inoculate a nutrient
agar (or TSA) slant.
15. Pick from the same purple or metallic green colony and inoculate the phenol red
broth-lactose tube. Repeat for controls.
16. Place tubes in designated incubation area. Tubes will be incubated at 35˚-37˚C
for 48 hours.
Session IV: Completed test - B
17. If possible, perform a Gram stain at 24 hours. Use Staphylococcus aureus as
your negative control and Escherichia coli as your positive control.
Page | 136 Results:
Presumptive Test
MPN Index Table
Number of Positive Tubes
MPN per 100 ml water
0
1
2
3
4
5
<2.2
2.2
5.1
9.2
16
>16
From US Dept. of Health and Human Services, MPN calculator
Calculate the MPN in you controls.
Sample: Positive control
Tube
Growth (Y/N)?
Bubble (Y/N)?
Sample: Negative control
Tube
I
I
II
II
III
III
IV
IV
V
V
Total
Total
Growth (Y/N)?
MPN:
MPN:
# colonies on plate:
# colonies on plate:
# of CFU/100 ml:
# of CFU/100 ml:
Did your controls match your expected results?
YES
Bubble (Y/N)?
NO
If not, explain: __________________________________________________
Page | 137 Now report your results for your samples:
Sample: _____________
Tube
Growth (Y/N)?
Bubble (Y/N)?
Sample: _____________
Tube
I
I
II
II
III
III
IV
IV
V
V
Total
Total
Growth (Y/N)?
MPN:
MPN:
# colonies on plate:
# colonies on plate:
# of CFU/100 ml:
# of CFU/100 ml:
Bubble (Y/N)?
Does your MPN value agree with the number of CFUs you calculated from your plate?
(circle one)
YES
NO
Record class MPN values below:
Water source
MPN value
Water source
Positive control 1
Negative control 1
Positive control 2
Negative control 2
Positive control 3
Negative control 3
MPN value
Negative control 4
Page | 138 Did both of your samples have < 2.2 MPN?
YES
NO
If YES, then write the name of the water source with a > 2.2 MPN that you will use for
the confirmed and completed tests: _________________________
Confirmed Test
Did your inoculum from your water sample grow on EMB?
YES
NO
If NO, then write the name of the water source showing EMB growth that you will use
for the completed tests: _________________________
If you observed growth, what was the color of the colonies observed?
PURPLE
METALLIC-GREEN
BOTH
OTHER: _______
Number of EMB plates (from your sample) showing positive colonies: _______
Completed Test
Gram reaction
Sample
Phenol red broth w/lactose
+ or -
Morphology/
Arrangement
Sample
Water sample
Water sample
+ control
+ control
- control
- control
Color
Bubble?
Conclusions:
__________________________________________________________
__________________________________________________________
__________________________________________________________
__________________________________________________________
Page | 139 Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 140 Exercise 24: Milk Quality
Goal: Test the quality of milk using the methylene blue reductase test
Introduction: Since the 1920s methylene blue has been used to test the quality of
milk samples. The premise behind this test is a color change that occurs in methylene
blue when its oxidation state is changed. When oxidized, methylene blue is blue,
whereas when it is reduced, it turns colorless and is called leucomethylene blue.
Different aspects of cellular metabolism, such as reductions carried out by cytochromes
in the electron transport chain, can reduce methylene blue. This test for cellular
respiration is therefore an indirect way to estimate how many bacteria are in a milk
sample. In good quality milk, it will take six hours or more for the methylene blue to
turn colorless.
Materials (per group)
• Two milk samples
• 6 Sterile screw cap tubes
• 2 x 1ml pipets and 1 x10ml pipet per
sample
• 10 ml of Methylene Blue (4.4
mg/100 mL)
•
•
1 test tube with 12 ml of TSB
3 ml overnight Escherichia coli
culture
Materials (per class)
• Ice water bath
• 35˚C water bath
Procedure:
Summary
To determine milk quality, you will add the methylene blue to your milk samples (and
controls) and record the amount of time it takes for the sample to turn white.
1. Label your screw cap tubes that will hold your milk samples.
2. Using aseptic technique, add 10ml of your milk samples to each tube.
3. Add 1 ml of TSB to each of your tubes except for the positive control. To this
tube, add 1 ml of the E. coli culture.
4. Add 1 ml of the methylene blue reagent to each tube, close tightly, and invert 3-4
times to mix.
Page | 141 5. Place the negative control tube in the ice water bath.
6. Place your samples in the 35˚C water bath for 5 minutes to prewarm the tubes.
After this, remove the samples, invert twice to mix and record the starting time.
7. Replace tubes in the water bath and keep them there between readings.
8. Observe the color of the samples every 30 minutes by comparing to your controls
and record when each sample turns white. The time interval tested will be up to
2.5 hours.
9. Make a final recording from an overnight incubation.
Results:
Milk quality variable being tested: ___________________________________
Sample
Start time
End time*
Elapsed time
Quality of Milk
Positive control
Negative control
* Time when the sample turns white
Conclusions:
__________________________________________________________
__________________________________________________________
__________________________________________________________
__________________________________________________________
Page | 142 Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 143 Exercise 25: A Synthetic Epidemic
Goal: To learn basic concepts of epidemiology in a simulated epidemic
Introduction: From the recent outbreaks of E. coli O157:H7 in spinach to the tracking
of the H1N1 virus, it becomes apparent that epidemiology (the science of disease
transmission) is extremely valuable. In the U.S. the task of tracking outbreaks falls on
the Centers for Disease Control (CDC). The CDC also closely collaborates with the
World Health Organization by providing disease transmission information.
This exercise simulates an epidemic that is spread through contact transmission.
Contact transmission is divided into three categories: direct contact transmission,
indirect contact transmission and droplet transmission. Direct contact transmission is
exemplified by personal contact such as shaking hands or giving a “high five”. Indirect
contact is when microbes are deposited on an inanimate object (a fomite) that can then
be passed on when someone else touches the object. Droplet transmission, as the name
would suggest, describes transmission from cough or sneezes.
In this exercise, we will be playing the role of the epidemiologist by identifying the
source or sources of a bacterial infection.
Materials (per person)
• 1 nutrient agar (or TSA) plate
• 1 disposable glove
• 1 unknown test tube with swab
• Sharpie for labeling
Materials (per group)
• 1 nutrient agar plate
• 1 disposable glove
• 1 swab
• Broth culture of Micrococcus luteus
Procedure:
Summary
One to several people will have a contaminated swab with Micrococcus luteus, which
produces yellow colonies. This organism will be spread through contact with infected
palms. Before you begin the “epidemic”, you will need to determine whom you will
come into contact with for each round. This will be critical to find the source or sources
of infection.
Page | 144 1. Divide your nutrient agar plate into five sectors, labeling each with I, II, III, IV
and V as shown below:
2. Label the other plate in the same way but instead of numbers I-V, write the
names of each person in your lab group. In the sector left over, write a “+”.
3. Record the number of your swab and for each round, determine the classmate
with whom you will come into contact and write their swab numbers in the chart.
Also write the swab number and name on the nutrient agar plate.
4. Wait for the instructor to verify that everyone has five different people on their
contact chart.
5. Pick a person in your group to flick the broth culture of M. luteus, open one of the
swabs and place it in the broth culture. This will be your __________ control.
6. Place the glove on your left hand and using your index and middle finger gently
touch the sector on the control plate labeled with your name.
7. Pick one person in your group to swab their gloved fingers with the swab dipped
in the M. luteus broth culture. This person should then gently touch the sector
on the control plate labeled “+”. After touching the plate, this person should
dispose of their glove (in the biohazard trash) and get a new one.
8. Carefully unwrap your swab, making sure that the cotton does not touch
anything. Rub the swab (held in your right hand) on your left (gloved) palm.
9. Discard the swab in the appropriate container – not the regular trash!
Page | 145 10. When the instructor signals Round I, use your gloved fingers (middle and index)
to touch the palm of your classmate. Your classmate will also touch your palm.
11. Gently touch sector I with your fingers on your nutrient agar plate.
12. Repeat steps 10 and 11 for rounds II-V.
13. Discard the glove in the autoclave bag and incubate your plate at room
temperature until the next lab period. Record your results.
Results:
Your swab # is _________.
Control plate
Sector
Amount of growth
(-,+,++,+++)
Name
I
II
III
IV
V
“+”
Experiment plate
Sector
Name
Swab #
Amount of growth
(-,+,++,+++)
I
II
III
IV
V
Collect the infection data for the class on the next page:
Page | 146 Example
Swab #
Round I
Round II
Round III
Round IV
Round V
3
- (2)
- (27)
+++ (17)
++ (20)
++ (5)
The number in parentheses is the person that you came into contact with in that round.
Swab #
Round I
(
(
(
(
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Round II
(
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)
)
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)
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Round III
(
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)
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)
Round IV
(
(
(
(
(
(
(
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Round V
(
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Page | 147 The source of infection was swab #___________.
Diagram the infectious scheme:
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 148 Exercise 26: The Capsule Stain
Goal: To determine whether a bacterial strain produces a capsule using the
capsule stain
Introduction: Many bacteria secrete a sugar coating on their surface called a
glycocalyx. It is a sticky gelatinous polymer made up of polysaccharides and/or
proteins. A bacterial glycocalyx can take two forms – it can appear as a slime layer
which is irregular and loosely attached, or as a capsule with is organized and firmly
attached to the cell wall. Since dyes cannot stain the bacterial capsule, we can stain the
cell and the background to visualize the capsule as a “halo” around the cells. No heat
fixing is used in this technique because this would distort the appearance of the capsule.
Therefore the cells remain alive on the slide and should be disposed of as biohazardous
material.
Materials (per pair)
• Two slides
• Inoculating loop
• Congo red (1%)
Materials (per group)
• Broth cultures of Klebsiella
pneumoniaeand Escherichia coli
• Maneval’s stain
Procedure:
1. Clean two slides using the Bunsen burner.
2. Place a loop of Congo red on each end of the slide two make smears as below:
Congo red
alone
E. coli
Congo red
alone
K.
pneumonia
e
3. Mix one loopful of E. coli (or K. pneumoniae) culture into the Congo red and
make a smear.
4. Allow the slide to air dry completely.
5. Add Maneval’s stain to the slide for 1 minute.
Page | 149 6. Place the slides in coplin jar with distilled water. Slowly invert6 times (about 3
seconds per inversion) to wash and let air dry.
7. While the slide is air-drying, view a previously prepared capsule stain under oil.
8. Visualize under oil. Capsules are colorless against a red background with cells
staining red to red/brown.
Results:
The smear with the congo red alone is my ________________ control.
Draw and color what you observed in the prepared slide and bacterial samples:
Prepared slide
Strain:
_________________
_________________
Capsule?
_________________
_________________
Page | 150 Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 151 Exercise 27: The Acid Fast Stain
Goal: Use the acid fast stain to identify bacteria in the Mycobacterium
genus
Introduction: The acid fast stain is a differential stain primarily used for identifying
bacteria in the Mycobacterium and Nocardia genera. These genera contain several
species that are pathogenic in humans. Some significant examples are Mycobacterium
tuberculosis, the etiological agent of tuberculosis, Mycobacterium leprae, which causes
leprosy and Nocardia asteroides, which is common in soil and causes opportunistic
lung infections. A recent research study reported that Mycobacterium aviumintracellulare was a common inhabitant of plastic showerheads. This bacterium is
known to cause pulmonary infections in immunocompromised individuals.
The acid fast stain works by staining a component in the cell wall of these genera called
mycolic acid. Mycolic acid is a hydrophobic, waxy lipid present in the cell wall that
prevents the uptake of dyes. It forms an external layer to a thin peptidoglycan layer in
acid-fast bacteria. Carbolfuchsin, the primary stain, penetrates the waxy mycolic acid
layer. The decolorizer is a mixture of alcohol and hydrochloric acid that readily removes
the carbolfuchsin from cell walls without mycolic acid, but does not remove it from cell
walls containing this lipid. Therefore cells with mycolic acid are resistant to
decolorization by acid-alcohol and are termed “acid-fast”. Methylene blue is used as the
counterstain to visualize cells that are non acid-fast.
Materials (per pair)
•
•
•
•
•
Two slides
Inoculating loop and needle
Bibulous paper squares
1 beaker with boiling chips
Clothespins and Staining tray
Materials (per group)
•
•
•
•
Slant or plate cultures of
Mycobacterium smegmatis and
Micrococcus luteus
1 bottle of Carbolfuchsin, Acidalcohol and Methylene Blue
1 hot plate
Distilled water for smear
Page | 152 Procedure:
Summary
In this exercise you will use a mixed culture smear to note the difference between acidfast and non acid-fast bacteria. We will use the Ziehl Neelsen acid fast staining method
which involves heating the primary stain. After the smear is prepared, a bibulous
square is added to the top as a filter for the carbolfuchsin. The carbolfuchsin is heated
for 5 minutes, cooled, decolorized with acid-alcohol and counterstained with methylene
blue.
1. Clean one slide using the Bunsen burner.
2. Add 300 ml of tap water to the beaker and place on hot plate to boil the water.
3. Make the following smears:
(Note: when making the smears with M. smegmatis, use an inoculating needle to
pick up a small amount, then tap 20-30 times to break up clumps.)
M. luteus
Mixed
culture
M.
smegmatis
Mixed
culture
4. Air dry and heat fix as usual.
5. Place a bibulous square over the smear and wet it with the carbolfuchsin.
6. Place the slide on top of the beaker with the screen to steam.
7. Steam the slide for 5 minutes. Remove the slide and let it cool on your bench.
8. While your sample slide cools, pull out the microscope and visualize a prepared
acid-fast stain slide.
9. Remove the bibulous square and rinse both sides of the slide with tap water.
10. Hold the slide at an angle and drip acid-alcohol across the smear until the red
color stops running.
11. Rinse with tap water.
12. Cover the smear with methylene blue and stain for 2 minutes.
13. Rinse both sides of the slide with tap water, dry in the bibulous pad and observe
under oil.
Page | 153 Results:
Draw and color your bacterial sample as it appears in the Acid Fast stain. Label which
bacterium is acid-fast and which is not.
Strain:
Acid Fast?
Prepared slide
Mixed culture
________________
________________
_____________
_____________
Page | 154 Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 155 Exercise 28: Biofilms
Goal: Observe biofilms from an environmental setting and from a health care
setting. Test for predisposition to dental caries.
Introduction: Ever since Robert Koch and his postulates tracing a single type of
microbe as a disease-causing agent, microbiologists have typically focused on single cell
characteristics or the characteristics of single populations (i.e., pure cultures). Within
the last ten years, scientists have turned their attention to a structure known as a
biofilm. A biofilm is a community of microorganisms that thrive on solid surfaces
exposed to water. They are not just layers of cells, but also have structural components
that allow water to bring nutrients and take away waste products.
Microbes in biofilms are usually protected by extracellular polysaccharides that they
themselves synthesize. As the polysaccharide grows, other cells can attach and begin to
grow as well. This allows a biofilm to host a community of organisms which can include
many different types of microbes such as bacteria, algae and fungi.
Growth in a biofilm is also greatly influenced by quorum sensing. Quorum sensing is
a process that modifies gene expression of bacterial cells as they grow which allows
them to communicate and coordinate behavior. One example of quorum sensing is the
production of a signaling chemical called an inducer as bacterial cells grow. As the
inducer leaks into the surroundings, it serves as a chemoattractant bringing other
bacterial cells to the biofilm. These additional cells in turn make more inducer.
A notable example of a biofilm occurs on teeth. Our mouths contain more than 500
different species of microorganisms, most of which are bacteria. The total population of
bacteria in our mouths is estimated to be between 50 and 100 billion. Among these,
various Streptococcus species (particularly Streptococcus mutans) are very skilled at
synthesizing biofilms that lead to tooth decay. These strains convert sucrose into
dextran, which is a highly branched polysaccharide that is sticky. Dextran allows the
bacteria to stick to the surface of the tooth and also traps food particles, which are then
fermented. This is called dental plaque. As Streptococcus mutans ferments these
food particles, especially sucrose, it produces acid as a by-product (recall that acid
production is a frequent sign of fermentation). This acid eats away at the tooth enamel
and results in a cavity.
One can therefore investigate the likelihood of getting cavities by testing for acidproducing bacteria present in saliva.
Page | 156 This is done using a special agar called Snyder agar, which has the following
characteristics:
- a low pH to inhibit growth of non acidophiles
- contains dextrose (glucose) which can be fermented
- contains Bromcresol green as a pH indicator (turns from green to yellow when the
pH decreases due to acid production)
Materials – Environmental Biofilm
(Per group)
• 5 slides
• Plastic slide holder
• Wide rubber band
• 5 feet of twine
• Methylene Blue (3rd lab session)
Materials – Human Biofilm
(Per student)
• Toothpick
• Glass slide
• 1 tube of melted Snyder agar held at 55C
• ½ of sterile 60mm Petri dish
• 1 sterile transfer pipet
(Per team)
• Cultures of Escherichia coli and Lactobacillus acidophilus
• Methylene Blue
• Sterile water
Procedure:
Summary – Environmental Biofilm
You will use slides as a surface to encourage growth of a biofilm from an environmental
locale such as a stream or pond. Then the slides will be stained with methylene blue and
analyzed for microbes.
Page | 157 1. Clean 5 slides and place them in an open slide holder. Place a rubber band
around the slides and tie the string to the slide holder cap.
2. Place the slides in a pond or stream that is easy to get to and won’t be disturbed.
3. Note the location in your data and mark the location so you don’t forget!
4. The next lab period, remove one slide from the holder and label it “1”.
5. Wipe one side clean with a paper towel so that the biofilm is on the other side.
6. Place in another slide holder to bring it to the lab.
7. When in the lab, air-dry the slide (probably already dried during transport) and
heat-fix it.
8. Save the slide in your slide holder.
9. Repeat steps 4-8 for lab sessions three and four.
10. Observe your biofilm during lab session four by
a. Comparing the appearance of the slides
b. Staining the slides with methylene blue and then observing under oil.
Summary – Human Biofilm
You will make a smear from the bacteria resident on your gum line. You will then collect
a sample of your saliva and mix it with molten Snyder agar. Analyzing the color change
of the agar at 2 days and 1 week will suggest how predisposed one is to getting dental
caries.
1. Clean a slide and place a small drop of sterile water to make a smear.
2. Gently scrape around your gum line with toothpick.
3. Tap the toothpick into the drop and make a smear.
4. Stain with methylene blue and view under oil.
Page | 158 5. Collect non-bubbly saliva in your saliva cup.
6. Using the sterile transfer pipette, pipette 0.25 ml of saliva into the Snyder agar
tube. Work quickly since Snyder agar solidifies quickly!
7. Vortex about 10 seconds to mix.
8. Inoculate one loop of Lactobacillus acidophilus into the control Snyder agar tube.
Vortex to mix. Add 0.25 ml of sterile water to another Snyder agar tube for
another control.
9. Label your tube with Class ID, Team number and first name/last initial.
10. Place in designated incubation area. The tubes will be incubated at 35-37˚C.
11. Record your results after 2 days and one week of incubation.
Results:
Environmental Biofilm
The slides were placed ____________________________________________
Slide 1
Slide 2/3
Slide 4/5
Length of
incubation
Visual Slide
appearance
Draw your most “populated” slide:
Page | 159 Types of microorganisms identified (check all applicable):
___ Bacteria
___ Algae
___ Fungi
___ Protozoa
___ Helminths
___ Archaea
Most abundant types of microorganisms were: ________________
Human Biofilm
Draw and color your bacterial sample as it appears in the simple stain from your gum
line.
Using the positive and negative controls, determine a color scale from 1-4 where 1 is
dark green, 2 is light green, 3 is light yellow and 4 is bright yellow.
Incubation time
Tube color value
Page | 160 Conclusion:
Based on my results, I
AM
AM NOT
predisposed to dental caries.
Does this result match whether you have actually had cavities in the past?
YES
NO
Questions:
1. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
2. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
3. _______________________________________________________
_______________________________________________________
Answer: _________________________________________________
_______________________________________________________
Page | 161 Exercise 29: Getting information from PubMed
Goal: To use PubMed to find the most recent medically related articles on
specific subjects.
Introduction:
In 1988, PubMed was developed through a national effort to compile all biomedical
related information in one location. This national center is called the National Center
for Biotechnology Information (NCBI), and is located at the National Library of
Medicine (NLM) at the National Institutes of Health (NIH). The purpose of PubMed
was to provide citations (references) of all biomedical related literature. Each citation
link usually includes the abstract of the article, citation information, links to the journal
that published the article and other articles that reference the searched article.
One excellent resource are the reviews on PubMed. Reviews are relatively short articles
that give a great summary of the topic and the latest research that pertains to it. This is
another great source for your disease projects!
To access PubMed:
1. Enter NCBI into Google and the following will appear:
Page | 162 2. Click on the “National Center for Biotechnology Information” link. The following
homepage will appear.
3. From the “Popular Resources” bar on the right select “PubMed”.
4. Enter “Super oxide dismutase” in the search box. Be sure to include the quotes!
5. How many citations appear (under “All”)? How many of these citations are
Reviews?
All:______________
Reviews:_____________
6. What is the difference between a journal article and a review?
_______________________________________________________
7. What is the Month and Year of the most current title that appears?
Month:____________
Year:_______________
8. What are the authors, article title and name of the journal for this latest citation?
_______________________________________________________
Page | 163 9. Now enter “Superoxide dismutase”. How many citations appear? After this,
enter Super oxide dismutase without quotes. How might these results change
your search strategy?
_______________________________________________________
_______________________________________________________
_______________________________________________________
_______________________________________________________
10. Now enter your disease from your disease project.
11. How many citations appear (under “All”)? How many of these citations are
Reviews?
All:______________
Reviews:_____________
12. On the right hand side click “Free full text”.
13. A plus sign will appear next to the highlighted phrase:
Click on the “+” sign.
14. The search box will include the “Free full text” filter like this:
Now click on the blue search button again.
Page | 164 15. Click on the Review link and all the free reviews will be listed.
16. Download a review that you will use as the starting point for your new disease
development. Write down the author, date and name of the review below:
_______________________________________________________
Appendix A: Microbes used in the lab
Alcaligenes faecalis
Bacillus cereus
Bacillus subtilis
Branhamella catarrhalis
Citrobacter freundii
Clostridium sporogenes
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Geobacillus stereothermophilus
Klebsiella pneumoniae
Lactobacillus acidophilus
Lactococcus lactis
Micrococcus luteus
Mycobacterium smegmatis
Proteus vulgaris
Pseudomonas aeruginosa
Pseudomonas fluorescens
Salmonella typhimurium
Serratia marcescens
Sporosarcina ureae
Staphylococcus aureus
Staphylococcus epidermidis
Page | 165