BIOL 206

LAB MANUAL
 Table of Contents
Microbiology Lab Safety​ 3
Ubiquity of Microorganisms and Blood Agar Media​ 6
Ubiquity of Microorganisms Experimental Procedure​ 8
Growth Morphology​ 10
Microbiological Culture Media and Sterilization​ 12
Aseptic Inoculation Methods​ 13
Aseptic Inoculation Methods Experimental Procedure​ 17
Quadrant Streak Plate​ 21
Quadrant Streak Plate Experimental Procedure​ 22
Spread Plate Method of Isolation​ 24
Spread Plate Experimental Procedure​ 28
Pour Plate Method of Isolation​ 30
Pour Plate Method of Isolation Experimental Procedure​ 31
Introduction to the Light Microscope​ 32
Bacterial Cell Morphology and Arrangement​ 34
Smear Preparation and Simple Stain​ 36
Smear Preparation and Simple Stain Experimental Procedure​ 38
Gram Stain​ 40
Gram Stain Experimental Procedure​ 44
Negative Stain​ 46
Negative Stain Experimental Procedure​ 47
Antimicrobial Susceptibility Test​ 48
Antimicrobial Susceptibility Test Experimental Procedure​ 50
Bacteriophage Sensitivity​ 51
Bacteriophage Sensitivity Experimental Procedure​ 52
ABO-Rh Agglutination - Transfusion​ 54
ABO-Rh Agglutination (Transfusion) Experimental Procedure​ 55
Transfusion Worksheet​ 56
Selective and Differential Media and Biochemical Tests​ 57
Triple Sugar Iron Agar​ 58
Triple Sugar Iron Agar Experimental Procedure​ 60
Catalase Test​ 62
Catalase Test Experimental Procedure​ 63
Oxidase Test​ 64
Oxidase Test Experimental Procedure​ 65

1
Citrate Utilization​ 66
Citrate Utilization Experimental Procedure​ 68
Malonate Utilization​ 69
Malonate Utilization Experimental Procedure​ 71
Lysine Iron Agar​ 72
Lysine Iron Agar Experimental Procedure​ 74
MIO (Motility, Indole, Ornithine)​ 76
MIO (Motility, Indole, Ornithine) Experimental Procedure​ 77
Carbohydrate Fermentation (Phenol Red Broth)​ 78
Carbohydrate Fermentation Experimental Procedure​ 81
(Phenol Red Broth)​ 81
Urea Hydrolysis​ 82
Urea Hydrolysis Experimental Procedure​ 84
MacConkey Agar​ 85
Unknown Identification Worksheet Page 1​ 86
Unknown Identification Worksheet Page 2​ 87
Unknowns Project Day 1 Experimental Procedure​ 88
Unknowns Project Day 2 Experimental Procedure​ 89
Unknowns Project Day 3 Experimental Procedure​ 90
Unknowns Project Day 4 Experimental Procedure​ 91
Unknowns Project Final Day Experimental Procedure​ 92
Enteric Chart​ 93

2
 Microbiology Lab Safety
SAFETY IS A PRIORITY. We expect you to actively assist us in preventing accidents in the lab,
and in responding appropriately to any accidents which might occur. You should be familiar
with the location and operation of safety equipment. For your protection, and for the
protection of your fellow students, we require that the following safety guidelines be followed
in the Microbiology laboratory. Failure to follow these safety guidelines will result in
disciplinary action and dismissal from this laboratory.
______________________________________________________________________________
General Laboratory Procedures

●​ All students must follow the lab safety procedures.

●​ No electronic devices (i.e. cell phone, ipad, ear buds, computer, etc.) in the lab room.
●​ Personal belongings such as extra clothing, unnecessary books, purses, backpacks, and
paraphernalia should be stored in designated areas away from the work area.
Benchtops should be free from clutter and personal items.
●​ Horseplay and other inappropriate, disruptive or unsafe behaviors are prohibited.
●​ Eating, drinking, applying makeup or lotions, and tobacco products of any kind are
forbidden at all times in the laboratory. Do not place anything in your mouth or eyes
while in laboratory.
●​ Clean your work area with disinfectant before and after each laboratory period. Return
all reagents, cultures, and glassware to their appropriate places.
●​ Wash your hands after all experiments, after removing gloves, and immediately
following contact with potentially hazardous materials.
●​ Lab access is not allowed unless an instructor is present.
●​ Label all experimental material with your: Name, date, class & lab section,
specimen/organism
●​ Note that incinerators are very hot and retain heat after being turned off and Bunsen
burner flames may appear invisible. Treat both with caution.
●​ Read and be familiar with the current lab procedures before you come to lab each
week.
●​ Open sores or wounds must be bandaged before entering the laboratory.
●​ Only write with provided pens, pencils, and sharpies.
______________________________________________________________________________
Personal Protection

●​ Wear a laboratory apron, smock, or lab coat when working in the laboratory. A lab
apron will be provided for you.
●​ Clear lens safety glasses must be worn when working in the laboratory. You will be
dismissed from the lab for failing to wear protective eyewear when instructed.
●​ Avoid touching your face and eyes while working in the laboratory.
●​ When working with microorganisms, use provided gloves. When lab is complete,
dispose of gloves in the biohazard waste and then wash hands thoroughly.

3
 ●​ Wear appropriate clothing. Closed-toe shoes must be worn in lab at all times. You will
be dismissed from the lab for wearing open-toed shoes or sandals. Pants and skirts
must reach the ankles and no shirts with dangling sleeves. Tie back long hair and
remove dangling jewelry. No loose hats or large hair accessories. Shirts must cover your
shoulders and torso.
●​ Any student not wearing proper safety attire will be required to leave the lab – this is a
University safety requirement and will result in an unexcused absence.
______________________________________________________________________________
Chemical Safety

●​ Do not sniff or ingest chemicals.

●​ All solutions must be clearly labeled. Do not remove information labels from bottles.
Notify your instructor if a label is missing.
●​ Report spills immediately! Clean spills promptly. Alert your instructor of all chemical
spills and follow guidelines for cleanup from the MSDS.
●​ Keep flammable chemicals away from heat sources.
______________________________________________________________________________
Sharps and Broken Glass

●​ Use extreme caution when handling sharps.

●​ Notify your instructor of any broken glass. It must be swept up and disposed of
immediately.
●​ Dispose of sharps waste properly: broken glass in a glass waste container and metal
waste in a metal sharps container. Sharps must not be placed in the regular garbage.
______________________________________________________________________________
Infectious or Transgenic Materials

* You have enrolled in BIOL 351 where you will be working with various microorganisms
during the upcoming semester. Some of the microorganisms (bacteria) used in this class have
the potential of causing disease in healthy adult humans. You will be provided with training
and protective equipment, including lab aprons and gloves, in order to safely work with and
handle these microorganisms. However, if you have an underlying health condition which is
known to weaken your immune system, or if you are taking certain medications which cause
immunosuppression or weakening of the immune system, or if you are pregnant, HIV+, etc.
you may be at increased risk of infection with some of the bacteria in use in this class. If you
have any concerns about your health, please request an appointment with the Occupational
Health Care Provider contracted by the University by calling 979-845-6649. Alternatively, you
may choose to discuss your concerns with your personal physician. A list of the organisms in
use in this teaching lab will be provided to you upon your request. If you have any
questions related to biosafety or your health in relation to this course, please call
979-845-6649.

4
 ●​ Biohazard containers will be provided for infectious materials. When instructed, place
used materials (including culture tubes and gloves) into these receptacles for
autoclaving.
●​ When infectious material is spilled, cover it immediately with a disinfectant and notify
your instructor at once.
●​ Sterilize wire loops before and immediately after transfer of cultures.
______________________________________________________________________________
First Aid and Emergency Procedures

●​ Be familiar with the location and operation of safety equipment: eyewash (side room of
the lab), emergency shower (hallway outside of the lab), first aid kit (hanging on front
wall), fire extinguisher (wall by door).
●​ In case of an emergency, telephone numbers are posted on the laboratory door.
●​ Immediately report any injuries to your TA.
●​ If you observe what you consider to be unsafe conditions or are concerned for your
safety in the lab, please report safety concerns to your TA or Lacy Basile as soon as
possible.

By signing the online safety agreement on Canvas, I verify that I have read, understood, and
agree to follow the safety regulations required for this course as established by the Department
of Biology and Texas A&M University. I have located all emergency equipment and know how to
use it. While in the laboratory, improper conduct and horseplay of any kind that may endanger
others or myself will not be tolerated and appropriate disciplinary action will be taken. I
understand that I may be dismissed from this laboratory for failure to comply with the stated
safety regulations. I also understand that it is my responsibility to follow the safety guidelines
and I will receive an unexcused absence or safety violation due to the violation of any of the
safety guidelines. These guidelines include, but are not limited to:

●​ Cell phones, headphones, and any other personal electronic devices must be turned off
and put away before entering the lab room and cannot be brought back out until
washing hands and fully exiting the lab room.

●​ Cell phones and electronic devices are not allowed out in the lab room under any
circumstances. This is true even if the experiment is complete or there is no experiment
being performed that day.

●​ Closed-toe shoes must be worn at all times in the lab room and you must wear long
pants or long skirts - no shorts. Shirts must cover your shoulders and torso. You are
required to leave lab if you aren't dressed according to these guidelines and you will
receive an unexcused absence.

●​ Safety glasses must be worn at all times in the lab until all students have completed the
lab experiment.

5
 Ubiquity of Microorganisms and Blood Agar Media
Background: Ubiquitous means present or found everywhere, and microorganisms are
ubiquitous. In most environments many types of organisms can be found including viruses,
bacteria, and fungi. Some environments will also have algae, protozoans, and helminths
(worms).

One of the places you will be testing in this laboratory exercise is the air. Dust in the air carries
bacteria and fungi. How many and which type of fungi are found in dust are largely dependent
on climate and region, whereas bacteria in dust depend on who occupies an indoor space-
people, pets, and insects all contribute. (1)

Therefore, though microorganisms are ubiquitous, the number and type of organisms often
depend on the environment. The human body also has different environments that affect which
microorganisms can be found, bacteria that grow on the skin are often limited to those that are
tolerant to salt, since sweat is salty. However, since hands touch a lot of surfaces there may also
be transient bacteria that are temporarily on the hands, but that will not grow and thrive on
skin.

Different microbes will have different nutritional requirements, so you will be using a variety of
media throughout the semester. One media you will be using is an undefined media used to
grow a multitude of microorganisms; the tryptic soy agar plate (TSA plate). The other media is
an enriched media, where blood is the enriched nutrient that will encourage fastidious
(nutritionally picky) organisms to grow. The Blood Agar plate is also a differential plate.
Differential means you should be able to detect the difference between some microorganisms
based on their biochemical/physiological characteristics. In the case of the Blood Agar plates,
you can detect between two colonies that may look alike by their ability to lyse the Red Blood
Cells (RBC) and this test is often used to distinguish amongst different species of Streptococci.

Organisms used: The organisms in this exercise will be from the natural environment
surrounding you. Most of these organisms are not pathogenic, meaning they do not cause
disease. The percentage of microorganisms that are pathogenic are small. Most microorganisms
are of benefit to humans because they are saprophytic, which means they play an important
role in decomposing dead organic matter. However, caution should be used after the organisms
have been incubated on the plates and you are observing them in the next laboratory period, as
a high concentration of bacteria/yeast that normally don’t cause you problems may still be
irritating at high concentration.

6
 α hemolysis β hemolysis ϒ hemolysis

Partial hemolysis of RBC’s Complete hemolysis RBC No hemolysis

hemoglobin to and breakdown of
methemoglobin hemoglobin

Greenish color in media Clear/yellow zone of color

surrounding bacteria around the bacteria

Photos by Christy Longcrier

Expected Outcome/Tips for keeping your notebook:

You should be able to answer the following questions: Since microorganisms are ubiquitous, do
you expect growth on all of the plates? What about the control plate? Would you expect the
same number and type of organisms to be obtained from different environments? Why is this
experiment being done (how might this be used in a restaurant or hospital for example)?

Morphology is the shape or physical characteristics of a microbe. This can include relative size
(small, large), color, whether the colony looks smooth or fuzzy, etc. Note: Many bacteria look
smooth, whereas fungi look fuzzy usually

1. Barberán Albert, Dunn Robert R., Reich Brian J., Pacifici Krishna, Laber Eric B., Menninger
Holly L., Morton James M., Henley Jessica B., Leff Jonathan W., Miller Shelly L. and Fierer Noah
2015 The ecology of microscopic life in household dustProc. R. Soc. B.2822015113920151139
[Link]

7
Ubiquity of Microorganisms Experimental Procedure
●​ Work in groups of 4
●​ Supplies
o​ 6 Tryptic Soy Agar plates (TSA)
o​ 1 Blood Agar plate (BAP)
o​ 2 sterile cotton swabs
●​ Procedure
1.​ Label each plate with your group number, section number, the date, and number them 1
through 7. The blood agar plate will be labeled plate #3.
2.​ Plate #1: Remove the lid from plate #1 and lay it on your tabletop, so that it is exposed
to the air for at least 20 minutes. After 20 minutes have passed, place the lid back on the
plate.
3.​ Plate #2 and #3: Use a sterile cotton swab to sample the bottom of a group member’s
shoe. Once sampled, gently press the contaminated part of the swab to the agar surface
and swipe back and forth, making a zigzag pattern across the agar (see Figure 1.1). Be
sure to press lightly so that the agar surface is not broken. Repeat the zigzag pattern of
inoculation on plate #3 (BAP).
4.​ Plate #4: Use a second sterile cotton swab to sample any surface in the lab area.
Inoculate plate #4 using the same zigzag pattern used in the previous step. Label the
plate with the surface your group sampled.
5.​ Plate #5: Pick one group member to remove one of their gloves. That group member will
then gently touch the surface of the agar with their fingertips. Replace glove that was
removed.
6.​ Plate #6: Using a scoopula, sprinkle a small soil sample from the beaker provided onto
the agar surface.
7.​ Plate #7: Do not uncover or inoculate plate #7. This will serve as your control plate.
8.​ Invert and incubate your plates in the 25° incubator until the next lab period. Plates are
inverted to keep condensation from dripping off the lids and onto the agar surface and
distorting microbial growth. Place your plates on the shelf in the incubator labeled with
your section number.

8
●​ Clean up
○​ Dispose of cotton swabs and its packing into the tabletop biohazard bags.
○​ Dispose of gloves in the large biohazard bin at the front of the lab room.
○​ Spray benchtops with lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

9
 Growth Morphology
Usually when a sample of bacteria or fungi has been placed on a media plate, you cannot see it
with the naked eye because it is microscopic. If you let the plate incubate under the right
conditions individual cells will start to divide logarithmically, doubling in number with each
division. Once the number is high enough this “pile” of cells that are genetically identical may
be seen with the naked eye, and is termed a colony. This can happen quite quickly, often within
24 to 48 hours. These colonies are typically consistent in appearance for a species of bacteria on
a particular media and therefore can be used to help you identify an organism, along with other
tests/factors. You will be counting the approximate number of these colonies from your plates
made in the previous laboratory period and can use this number to get a rough estimation of
which environments harbored the most microorganisms!

Another thing you will do is try to determine the approximate number of DIFFERENT types of
colonies on one plate. A plate can have multiple colonies of all of the same organism, in which
case the colonies will all look similar as seen in box A below, or your plate may have a handful of
colonies, but they look different—suggesting there are numerous species on this plate as seen
in box B.

A B

Photos by Christy Longcrier

For plate B, you might want to consider how you would describe some of the colonies. Here is
an example of some of the different aspects you can look for below. These are Colony
Description Helpers for your notebook.

1) Differences in color. Some bacteria might be white (opaque) while others are more clear
(translucent) and others are yellow/red/black etc…

2) Differences in size

10
3) Differences in texture- does the colony look dry and dull, or does it look mucoid (sticky
looking),

4) Differences in shape/edges- does your colony look perfectly round, or like tiny dots
(punctiform), or are the edges irregular? Are there branches like roots sticking out (rhizoid)?

5) Elevation- Is it flat to the plate, or does it stand up all over(raised)? If it stands up, is it
convex? If it is raised just in the center it is called umbonate.

11
 Microbiological Culture Media and Sterilization

Microbiology Culture Media and Sterilization: In order to work with microorganisms you will
need to be able to create a clean environment, make media that is not contaminated, and make
sure your benchtop is safe for the next person. The following 3 terms are frequently used when
describing the level of “clean” needed in micro laboratories, and are used in the medical field as
well:

1) Decontamination- this refers to a low level of control of microorganisms. You will be using
decontamination when you use cleansers/ soap and water/ alcohol to clean your benchtop
before you work there.

2) Disinfection- This term is a more rigorous cleaning and kills a large number of
microorganisms. There will be some microorganisms that are resistant to this level of cleaning
due to their ability to make endospores or other protective structures. Common ways to
disinfect are using chemical cleaners or by use of UV radiation.

3) Sterilization- This is the highest level of pathogen control and what you need to do to
bacterial growth media. It will eliminate all microorganisms, including those that make
protective structures like endospores. This can include dry heat and moist heat, but one of the
most common sterilization methods is a combination of heat and pressure. A machine called an
autoclave is used in this process

12
 Aseptic Inoculation Methods
Types of Media:
​
You will be working with different categories of media during the semester and you need to
know what each category is used for. Below is a picture of each type, followed by a description.

Pictures taken by Christy Longcrier

From Left to Right:

A small slant or a stab: The first tube is a small slant or a stab. This type of media can be used
when you want to grow an organism in anaerobic (low/no oxygen) conditions. You can inoculate
it by stabbing the media with an inoculating loop or needle. In this lab, you will only use the
slanted portion of the media (aerobic conditions) by streaking across the top.

A liquid (or broth) culture: The second tube shows a liquid culture. These are used when you
want to increase the number of cells of a particular microorganism. You inoculate this with an
inoculating loop and cells will grow exponentially, resulting in millions of cells in a typical
overnight culture grown under ideal conditions.

A slant: Slants can have several advantages. You can use them if you would like to try and
simultaneously grow the organism in anaerobic conditions (by stabbing the media) and also
aerobic conditions (by streaking across the top of the slanted media).

Another use of a slant is when you would like to incubate some cells, and then need to add a
liquid in order to read the outcome of the test. Having the cells in a tube rather than a plate
keeps the liquid contained over the top of the cells.

13
An agar plate: The last object shown is an agar plate. Agar plates have the advantage over
liquid media in that you can take a mixed culture and spread it thinner and thinner in a process
called “streaking a plate” so that individual bacteria can be separated from one another. If you
then incubate the plates under ideal growth conditions overnight, colonies arise from those
individual bacteria. This would allow you to then harvest that colony and place it in a
liquid/broth to grow a pure culture!
​
Agar plates also have many other uses depending on the type of media on the plate. There are
many biochemical tests that can be done on a plate that are used to identify an unknown
organism.

Aseptic Technique
​
Doing a procedure aseptically means doing that procedure without contaminating anything.
Learning aseptic technique is one of the most important skills you will learn in the microbiology
laboratory, and is also a skill set that directly applies to the medical field when keeping a
clean/sterile field. In this lab you are going to be learning how to aseptically transfer a culture
from one tube or plate into another tube or onto a plate.
​
So what is a culture? Any medium that contains living microorganisms is called a culture. If it
only contains one species, then we would call that a pure culture. If you have a pure culture and
would like to keep it that way when transferring it, here are a few important tips:

1) Start with clean hands and a clean surface. Wash your hands before the lab and dry them
before putting on gloves. Make sure you are working at a clean benchtop (we ALWAYS clean our
benchtops when we are done so they should be clean!), and one that doesn’t contain excess
books and papers.

2) Take your time and be organized. A few minutes of organization saves you from potentially
having to redo the whole experiment. You will want to have read the protocol, and pre-label any
plates or tubes. Always write on the bottom of a plate—in other words, the outside of the dish
with the agar in it, since the lid might come off and also because the lid can spin around but the
bottom is “fixed”.

3) If you are going to be using liquid cultures, make sure you have a test tube rack to set the
tubes in so they cannot leak. Usually the caps on cultures are put on loosely, so that the culture
can have oxygen. If you lay it down on the counter, you will make a mess! Since the caps are
loose, never pick up a tube by the cap.

4) You need to remember to use the incinerator (or flame if using a Bunsen burner) on your
inoculating loop both before entering a culture AND after you have transferred the culture. You
flame before, so that you don’t contaminate the culture. After you transfer the culture to a plate
or tube, there will still be bacteria on the loop, so you flame it again so that you don’t spread
bacteria around the lab from a contaminated loop.

14
4) Once you start the protocol, try not to talk or be distracted. The actual transfer only takes
about a minute and you want to work in a calm, relaxed manner and concentrate on what you
are doing. You will hold the inoculating loop like a pencil, with your dominant hand. You will
hold the tube cap with your pinky finger in your dominant hand, and the tube or plate lid with
your non-dominant hand. Nothing should be set down on the counter top, and your TA will
demonstrate this technique for you.

Here are some examples of how you will hold equipment:

I. Holding the inoculating loop like a pencil in your dominant hand, as well as the test tube cap
(seen in red) being held by the dominant hand pinkie finger. The test tube is held at a slant in
the non-dominant hand.

II. Holding the inoculating loop like a pencil in your dominant hand while using your
non-dominant hand to tilt up a plate lid. Notice that the lid is NOT taken off the plate and set on
the counter! Also notice that the liquid/broth culture that the inoculum came from was placed
back into a test tube rack before dealing with the plate. Next to that is the same thing done with
a micropipette.

15
III. WHAT NOT TO DO

DO NOT take the lid off the agar plate and

leave it on the counter when inoculating.

DO NOT forget to slant the tube when you

are working with it. If the opening faces
directly up like in this picture—dust, with
germs on it, can fall in and contaminate your
culture.

16
 Aseptic Inoculation Methods Experimental Procedure
●​ Work in groups of 4
●​ Supplies
o​ 3 Tryptic Soy Broths (TSB)
o​ 3 TSA slants
o​ 1 TSA slant inoculated with Staphylococcus cohnii
o​ 1 TSB inoculated with Staphylococcus cohnii
o​ 1 TSA plate inoculated with Staphylococcus cohnii
o​ Metal inoculating loop
o​ Incinerator
●​ Procedure
o​ Label all tubes with your group number, section number, date, and the organism name.
Label the tube directly on the glass, NOT the cap. Caps are removed and can easily be
mixed up.
●​ Broth to Broth
1.​ Gently vortex your TSB tube inoculated with S. cohnii. Be careful not to vortex too
vigorously because the broth can spill out of the tube, even with the cap on.
2.​ Sterilize your inoculating loop by placing the loop end inside of your incinerator for 5-7
seconds. Allow your loop to cool for a few seconds without touching anything. Do NOT
wave your loop around in an attempt to cool your loop.
3.​ Holding your S. cohnii tube in one hand and the loop in the other, remove the cap with
the pinky of your loop hand.
4.​ While holding the S. cohnii tube at an angle, carefully insert your sterile loop into the
broth and then remove the loop from the tube. There should be a film of broth on the
inside of the loop.
5.​ Replace the cap of the S. cohnii tube, put the tube back in a rack, and pick up a sterile
TSB tube.
6.​ Remove the cap of the sterile TSB tube with the pinky of your loop hand, carefully insert
your loop into the broth, and gently mix the broth with your loop.
7.​ Replace cap on the tube, place your tube in a rack, and re-sterilize your inoculating loop
by placing it inside the incinerator for 5-7 seconds.
●​ Broth to Slant
1.​ Gently vortex your TSB tube inoculated with S. cohnii. Be careful not to vortex too
vigorously because the broth can spill out of the tube, even with the cap on.
2.​ Sterilize your inoculating loop by placing the loop end inside of your incinerator for 5-7
seconds. Allow your loop to cool for a few seconds without touching anything. Do NOT
wave your loop around in an attempt to cool your loop.
3.​ Holding your S. cohnii tube in one hand and the loop in the other, remove the cap with
the pinky of your loop hand.

17
 4.​ While holding the S. cohnii tube at an angle, carefully insert your sterile loop into the
broth and then remove the loop from the tube. There should be a film of broth on the
inside of the loop.
5.​ Replace the cap of the S. cohnii tube, put the tube back in a rack, and pick up a sterile
TSA slant.
6.​ Remove the cap of the sterile TSA slant with the pinky of your loop hand, gently insert
your loop into the tube and lightly touch the base of the agar slant.
7.​ Gently move your loop in a zigzag pattern up the slant, using care to not cut the agar
surface with the loop.
8.​ Replace cap on the tube, place your tube in a rack, and re-sterilize your inoculating loop
by placing it inside the incinerator for 5-7 seconds.
●​ Slant to Broth
1.​ Sterilize your inoculating loop by placing the loop end inside of your incinerator for 5-7
seconds. Allow your loop to cool for a few seconds without touching anything. Do NOT
wave your loop around in an attempt to cool your loop.
2.​ Holding your TSA slant inoculated with S. cohnii in your other hand, remove the cap with
the pinky of your loop hand.
3.​ Gently insert your loop into the tube and lightly touch the bacterial growth along the
agar surface. You do not need a large amount of growth on your loop, just a small
amount that you can still see present on the loop.
4.​ Replace the cap onto the agar slant, place the tube in a rack, and pick up a sterile TSB
tube.
5.​ Remove the cap with the pinky of your loop hand, carefully insert the loop into the
broth, and gently mix the broth with your loop.
6.​ Replace cap on the tube, place your tube in a rack, and re-sterilize your inoculating loop
by placing it inside the incinerator for 5-7 seconds.
●​ Slant to Slant
1.​ Sterilize your inoculating loop by placing the loop end inside of your incinerator for 5-7
seconds. Allow your loop to cool for a few seconds without touching anything. Do NOT
wave your loop around in an attempt to cool your loop.
2.​ Holding your TSA slant inoculated with S. cohnii in your other hand, remove the cap with
the pinky of your loop hand.
3.​ Gently insert your loop into the tube and lightly touch the bacterial growth along the
agar surface. You do not need a large amount of growth on your loop, just a small
amount that you can still see present on the loop.
4.​ Replace the cap of the S. cohnii tube, put the tube back in a rack, and pick up a sterile
TSA slant.
5.​ Remove the cap of the sterile TSA slant with the pinky of your loop hand, gently insert
your loop into the tube and lightly touch the base of the agar slant.
6.​ Gently move your loop in a zigzag pattern up the slant, using care not to cut the agar
surface with the loop.

18
 7.​ Replace cap on the tube, place your tube in a rack, and re-sterilize your inoculating loop
by placing it inside the incinerator for 5-7 seconds.
●​ Plate to Broth
1.​ Sterilize your inoculating loop by placing the loop end inside of your incinerator for 5-7
seconds.
2.​ Lift the lid of the petri dish and gently touch the loop to the agar near the edge of the
TSA plate, where there is no bacterial growth, to cool your loop.
3.​ Touch the loop to bacterial growth and pick up a small amount that you can still see on
your loop. Do not scoop up a large amount or cut into the agar surface with your loop.
Replace lid onto petri dish and pick up a sterile TSB tube.
4.​ Remove the cap with the pinky of your loop hand, carefully insert the loop into the
broth, and gently mix the broth with your loop.
5.​ Replace cap on the tube, place your tube in a rack, and re-sterilize your inoculating loop
by placing it inside the incinerator for 5-7 seconds.
●​ Plate to Slant
1.​ Sterilize your inoculating loop by placing the loop end inside of your incinerator for 5-7
seconds.
2.​ Lift the lid of the petri dish and gently touch the loop to the agar near the edge of the
TSA plate, where there is no bacterial growth, to cool your loop.
3.​ Touch the loop to bacterial growth and pick up a small amount that you can still see on
your loop. Do not scoop up a large amount or cut into the agar surface with your loop.
Replace lid onto petri dish and pick up a sterile TSA slant.
4.​ Remove the cap of the sterile TSA slant with the pinky of your loop hand, gently insert
your loop into the tube and lightly touch the base of the agar slant.
5.​ Gently move your loop in a zigzag pattern up the slant, using care not to cut the agar
surface with the loop.
6.​ Replace cap on the tube, place your tube in a rack, and re-sterilize your inoculating loop
by placing it inside the incinerator for 5-7 seconds.

19
●​ Clean up
○​ Dispose of original S. cohnii broths and slants in the large biohazard bin at the front of
the lab room. Remove the caps and put them in the appropriate cap receptacle at the
front of the room and then put the tubes in the biohazard.
○​ Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

20
 Quadrant Streak Plate

A quadrant streak plate is used when a person wants to isolate cells away from one another in
order to get a pure culture. Many cultures are obtained from the environment or from a patient,
and these are commonly a mixture of bacteria and fungi. It is often necessary to isolate a single
bacterium in research or medicine, and an example of this is when a pathogen needs to be
identified.

The idea is to begin in one quadrant (1/4th of the plate) and streak the cells from the original
culture across a quadrant of the plate to thin them out. Once this is done the inoculating loop is
sterilized and allowed to cool so that it is free of cells and then a portion of the cells that are
now in that first quadrant are dragged across and thinned out into a second quadrant. This
sterilization of the loop and further thinning out of the cells will be repeated for quadrants
three and four (the pattern of the streaking is seen in the protocol pictures below, but your lab
TA will demonstrate the sterilizing and cooling process that happens between the streaking for
you). In the end, what you would like to have is a single species of bacteria isolated away all the
others.

If this plate is then incubated overnight, each bacterium will divide until a lump of bacteria that
can be seen with the naked eye will appear. This lump of bacteria that are basically genetic
clones of the original bacteria are called colonies. If the protocol is done well, the result should
be isolated colonies of bacteria in the third or fourth quadrant that are not touching each other.
It won’t be done in this protocol, as we are just practicing our streak technique, but you could
put that colony into a new media with a great deal of certainty that you were growing a “pure
culture” of a single species.

Note: A quadrant streak plate is often just called a streak plate in a laboratory setting. This can
get confusing when you are told to “streak a plate” and the TA only needs a single line of
bacteria on a plate for a biochemical test. Once you have experience you will know which to do
by the context, but in early micro labs we will often say “streak a plate for isolation” if we mean
a quadrant streak plate in order to help newer microbiologists like you!

21
 Quadrant Streak Plate Experimental Procedure
●​ Work individually
●​ Supplies
○​ 1 TSA plate
○​ 1 TSA slant inoculated with Escherichia coli and Staphylococcus cohnii mixed
○​ Metal inoculating loop
○​ Incinerator
●​ Procedure
○​ Label the plate with your initials, section number, date, and the organism(s) name. Label
along the edge of the bottom of the plate. The lid is removed and rotated.
○​ Draw three lines on the bottom of your plate as illustrated in Figure 3.1. The lines will
help you visualize your quadrants while you are streaking. Label your quadrants 1-4, and
leave your plate upside down (lid down) on the benchtop.
○​ Sterilize your inoculating loop in the incinerator, let cool for a few seconds in the air or
gently touch the loop to the surface of the agar. DO NOT wave around in the air to cool.
○​ Inoculate your loop with the mixed culture bacteria from your slant by lightly touching
the bacterial growth on the slant with your loop. You should see a little of the growth on
your loop.
○​ Pick up the bottom part of the plate, leaving the lid on the table, and hold your plate at
an angle where light reflects off the surface of the agar.
○​ Gently touch the loop to the edge of the plate in the quadrant labeled 1 and drag across
the surface, back and forth, a few times. This is illustrated in Figure 3.2. Be careful to not
cut into the agar with the loop. Place your plate back down onto the lid.
○​ Sterilize your loop in the incinerator and let cool for a few seconds as stated in Step 3.
DO NOT get more inoculum from the slant.
○​ Rotate your plate about 90 degrees, and repeat the streaking pattern from quadrant 1 in
quadrant 2, starting your pattern at the end of quadrant 1 so that the patterns slightly
overlap. The second streak should only intersect the first streak 2-3 times. This is
illustrated in Figure 3.3. Place your plate back down on the lid.
○​ Sterilize your loop and cool.
○​ Rotate the plate 90 degrees and repeat the same process for a third streak into quadrant
3. Remember that your third streak pattern should only intersect the second pattern 2-3
times. See Figure 3.4. Place your plate back down.
○​ Sterilize loop and cool.
○​ Rotate your plate 90 degrees and repeat the last streak pattern for quadrant 4. Be
careful not to intersect your fourth streak into your first streak! See Figure 3.5. Place
your plate back down and sterilize the loop.
○​ Incubate plate in the appropriate incubator with the lid down (inverted) until the next
class.
●​ Clean up
○​ Dispose of the mixed starter culture in the large biohazard bin. Remove the cap and put
them in the appropriate cap receptacle at the front of the room and then put the tubes
in the biohazard. Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

22
23
 Spread Plate Method of Isolation

Uses of the Spread Plate Technique

​
The spread plate technique can be used in two different ways. The first way in which you can
utilize this technique to solve a problem in the laboratory is when you have a sample with more
than one microbe in it and you would like to have the cells spread out on the plate such that
each individual cell on the plate is not touching other cells. The best way to accomplish this is by
spreading out a very dilute culture. After this is done, the plate can be incubated overnight, and
colonies of bacteria that can be seen on the plate the next day should all be genetically identical
if they arose from a single cell.
​
The group of genetically identical cells derived from the original single cell is called a colony.
When we discuss cell concentration on these plates we use the term Colony Forming Units
(CFU) because we cannot absolutely say 2 cells have not overlapped (though we are fairly
certain!).
​
The second way the spread technique can be used is if you would like a lawn of bacteria on a
plate. To make a lawn, you would inoculate the plate with a small amount of a dense culture
before spreading the cells around. These lawns of bacteria that arise after incubation can then
be used to test many different things such as antibiotic efficiency and the ability of a virus to kill
that type of bacteria. This use of the spread technique will be used in another lab later in the
semester.
​
This week you will be using the spread plate technique to isolate genetically identical cells of a
single species. Therefore, you will need to know how to do a serial dilution in order to place a
dilute sample on your plate.

Serial Dilution
​
It is often important to know how many microorganisms are in a sample. Therefore, techniques
have been developed to count these organisms. Whether one is counting viruses or counting
bacteria, a shared concept is to first make a serial dilution. Serial dilutions take the original
concentrated tube of microorganisms and then transfers a portion of this into a tube containing
a known amount of media/solution. This will be done in “series” repeatedly and is usually set up
so that the dilution factor remains the same with each transfer.

Look at the diagram below and we will use this set up as an example. Let’s say we have an
original stock culture that has been growing overnight in the incubator, such that the culture is
very thick. (If you tapped it gently on the side you could see a swirling pattern in the
tube—which means you have millions of bacteria in that tube!). You now want to make a much
more dilute culture. The dilution scheme you chose depends on just how dilute you want to go,
but step downs by 1/10th and by 1/100th are pretty common.

24
For this example, we are going to want each tube in the series to be 1/10th of the concentration
of the tube before, so we will dilute this in a series (which is why it is called serial dilution). If we
want to transfer samples in 1ml amounts between tubes, we need the final volume of tubes 1-4
below to be 10 so that it would be a 1 in 10 dilution (1 ml in 10ml total). If we start with 9mls of
media in tubes 1-4 and transfer 1 ml into it, that works!

Note: To find the dilution factor in your serial dilution of given tubes with
known liquid in them just use the amount you are transferring between tubes
on the top of the fraction, and the amount you are transferring PLUS the
amount in the tube as the bottom of the fraction. So, in this example you have
1ml/ 1ml+9ml = 1ml/10ml or a 1:10 dilution

If Tubes 1 to 4 have 9 mls, what you do is take 1 ml from the original stock culture and put it in
tube 1. You then would gently mix tube 1 and you would have a culture that was 1/10th of the
original culture in tube 1. If you then take 1 ml from tube 1 and place it in tube 2 and mix it you
would then have a 1/100th culture (you multiply the 1/10th from the first tube by the 1/10th of
the second tube). If you then went from tube 2 to tube 3 with 1 ml, what would the dilution
be? You can take the 1/100 form tube 2 and multiply it by the 1/10th dilution you did for tube
3…. And you get 1/1000 dilution.
​
What would you have in tube 4 if you transferred 1 ml from tube 3 into tube 4? (answer at end
of intro so you can check yourself).

Things to remember about working in the lab this week:

25
REMEMBER to tilt the tube at a roughly 45 degree angle. Below is a picture of someone failing
to do this such that the mouth of the tube will allow dust, and therefore bacteria, into the tube.

DON’T DO THIS, tilt the tube:

You will then pipette the liquid onto the plate. This part is done correctly, as the lid is kept
hovered over the plate to prevent contamination instead of being set on the counter!

26
You will then spread the liquid on the plate. The lid is again in the correct position in this photo.

Photos by Christy Longcrier

Answer to dilution question: 1/10,000.

27
 Spread Plate Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 4 TSA plates
o​ 1 5mL sterile water
o​ 1 TSB inoculated with Micrococcus luteus
o​ 4 eppi tubes
o​ Disposable spreaders
o​ 1 P1000 Pipette
o​ 1 P200 Pipette
o​ 1 P20 Pipette
o​ Eppitube rack
o​ Blue and yellow tips
●​ Procedure
1.​ Label the bottom of the 4 TSA plates each with A, B, C, or D. Also label your plates with
your normal identifying information (initials, section number, date, and organism name).
2.​ Label the 4 eppitubes with A, B, C, or D and place in the eppitube rack.
3.​ Using a P1000 pipette, transfer 990 µL of sterile water to eppitube A and 900 µL of
sterile water to eppitubes B, C, and D.
4.​ Gently vortex the TSB tube inoculated with M. luteus.
5.​ Using a P20 pipette, transfer 10 µL of the M. luteus broth to the eppitube labeled A.
Eject the used tip into the tabletop biohazard bag. Close the eppitube and vortex.
6.​ Using a P200 pipette, transfer 100 µL from eppitube A to eppitube B. Eject the used tip
in the tabletop biohazard bag. Close the eppitube and vortex.
7.​ Repeat step 6 from eppitube B to eppitube C.
8.​ Repeat step 6 from eppitube C to eppitube D.
9.​ Using a P200 pipette, transfer 100 µL from eppitube D to the middle of TSA plate D. Eject
tip into the tabletop biohazard bag.
10.​Using a disposable spreader, spread the inoculum around on top of the agar as shown in
figure 4.2. Use gentle pressure and do not cut into the agar surface.
11.​Repeat steps 9 and 10 for transferring and spreading for eppitube C to plate C, eppitube
B to plate B, and eppitube A to plate A.
12.​Invert and incubate the plates until the next class.
●​ Clean up
o​ Dispose of starter culture, spreaders, and water tubes into the large biohazard bin.
Remember to remove caps and place them in the bins at the front.
o​ Dispose of gloves in the large biohazard bin.
o​ Return eppitube racks, pipettes, and tip boxes to where you got them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

28
29
 Pour Plate Method of Isolation

The pour plate procedure is one of many techniques used to obtain isolated colonies of bacteria and
fungi. The original sample is diluted several times to sufficiently reduce the microbial population in
order to yield separate colonies upon plating. Small volumes of each dilution are mixed with molten
tryptic soy agar (48o-50oC) and then poured into sterile petri plates. Most bacteria and fungi will not be
killed by the brief exposure to the warm agar. After the agar has solidified, each isolated cell will
proliferate and form a colony. Isolated colonies will result when the sample is diluted to a point that the
cells are separated enough in the agar to make individual colonies. Assuming no chaining or cell clusters,
the total number of colonies is equivalent to the number of viable microorganisms in the diluted sample.
This is recorded as cfu/mL (colony forming units per milliliter of media). To prepare pure cultures for
experimentation, a single, isolated colony is used to inoculate sterile broth or agar. Plates are incubated
in the inverted position (lid down) to keep condensation from forming and falling onto the agar surface
and interfering with individual colony formation. One disadvantage of this technique is that some of the
colonies will be embedded within the solidified agar. These subsurface colonies will be smaller and thus
you must take extra care to include them when performing total colony counts.

30
 Pour Plate Method of Isolation Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 3 Sterile Petri Plates
o​ 3 9.9mL H2O Tubes
o​ 3 TSA Moltens (Do not get these until ready to use - found in water bath)
o​ p200 Micropipette
o​ p1000 Micropipette
o​ Yellow Tips
o​ Blue Tips
o​ 1 TSB inoculated with Micrococcus luteus

●​ Procedure
1.​ Label the bottom of all plates with your group number, section number, date, dilution
(10-2, 10-4, 10-6) and the organism name. Label H2O tubes with the dilution (10-2, 10-4,
10-6). Label the tube directly on the glass, NOT the cap. Caps are removed and can easily
be mixed up.
2.​ Gently vortex your TSB tube inoculated with M. luteus. Be careful not to vortex too
vigorously because the broth can spill out of the tube, even with the cap on.
3.​ To make dilutions of your M. luteus culture, add 100uL of M. luteus culture to the tube
labeled 10-2 using the p200 micropipette and a sterile yellow tip. Gently vortex your 10-2
tube.
4.​ Using a new sterile yellow tip, add 100uL of the 10-2 dilution to the tube labeled 10-4.
Gently vortex your 10-4 tube.
5.​ Using a new sterile yellow tip, add 100uL of the 10-4 dilution to the tube labeled 10-6.
Gently vortex your 10-6 tube.
6.​ Using the p1000 micropipette and sterile blue tip, add 1mL of the 10-2 dilution to the
plate labeled 10-2.
7.​ Get one TSA molten from the water bath, pour it into the 10-2 plate containing 1mL of
the dilution, gently swirl, and let solidify. Get only one molten at a time.
8.​ Repeat steps 6 and 7 with the 10-4 and 10-6 dilutions.
9.​ Allow plates to cool and solidify. Solidified agar will become opaque in appearance.
Once solidified, invert and incubate until the next lab period.
●​ Clean up
o​ Dispose of starter culture and H2O tubes into the large biohazard bin. Remember to
remove caps and place them in the bins at the front.
o​ Dispose of plates and/or tubes from previous experiments after you have read results.
o​ Dispose of gloves in the large biohazard bin.
o​ Return eppitube racks, pipettes, and tip boxes to where you got them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

31
 Introduction to the Light Microscope

Using the Light Microscope:

PARTS OF THE MICROSCOPE: Below is a picture of a light microscope. Make sure you know all of
the parts of the microscope below from the slides.
​
There are two lenses in the light microscope for magnification. The first lens you look through is
where you put your eyes (the ocular-see picture). The ocular lens can come out of the
microscope, so we always carry the microscope in an upright position with both hands! A very
common magnification for an ocular lens in a light microscope is 10X (the image is 10 times
bigger than the actual size).
​
The second lens is called an objective (see picture). Most decent light microscopes will have 4
different objective lenses of various strengths that can be rotated into position when needed.
Typical magnifications for the 4 lenses is 4X, 10X, 40X and 100X. The objective lens you will use
will depend on the type of organism you are observing.

32
​
In order to figure out the total magnification of an object, you will use the following formula.
(which is really common sense, since it is one lens magnifying after the other you just multiply
the magnifications together). Try filling out the table below with the correct total
magnifications. The first example has been done for you.

Magnification of the ocular X Magnification of the objective = Total Magnification

Ocular 4X 10X 40X 100X

Objective Objective Objective Objective
10X 10 X 4= 40X total
magnification

33
 Bacterial Cell Morphology and Arrangement

Identifying a bacterial species is important in the field of microbiology and also in medicine. Often the
first steps are going to be to determine the morphology and arrangement of the species cells and the
composition of the cell wall and cell membrane system. Before getting into the different techniques
used to do that, we will review some of the most common morphologies in the table below:

Cocci- these are round spheres Bacilli- these are rod shapes Other- includes corkscrew
shaped organisms or those
without a consistent shape

Diplococci- cocci in pairs Bacilli – normal length rods, Spirillum- rigid corkscrew
these can sometimes be shape
Tetrad- cocci in groupings of attached end to end
four Spirochete- corkscrew shape
that isn’t rigid
Streptococci – cocci in chains
Coccobacilli- short rods Pleomorphic – shape of the
Staphylococci – cocci in grape organism can be inconsistent
clusters Vibrio- slightly curved rods

Bacilli Images

34
Cocci Images

35
 Smear Preparation and Simple Stain

The first step in any slide staining protocol is preparing the smear. The bacteria must first be distributed
on the slide in such a way that the cells are not too dense, because if they are you may not be able to see
the individual morphology of the cells. Conversely, if the cells are spread too thin, it may be difficult to
find them under the microscope, or you might misinterpret the arrangement. You will get a chance to
make smears in the next few laboratory assignments.

After making a good smear, you will need to decide whether to “heat fix” the slide or not. Heat fixing is
part of many traditional staining methods, such as the Gram stain. Heat fixing consists of passing a slide
with a smear on it over a heat source. You will be told what to do in the protocol, but should understand
the implications. The majority of staining techniques require heat fixing, and you can see the advantages
in the chart below.

No Heat Fixing Heat Fixing

Cons: Cons:

●​ Bacteria are still alive, which can be ●​ For some more fragile bacteria, the
dangerous if they are pathogenic morphology is deformed during heat
●​ Bacteria are still alive, and it will be fixing
impossible to focus on them if they are
motile Pros:
●​ Bacteria are not fixed to the slide and
can be spilled off when liquid is added ●​ Any pathogenic bacteria will be killed
by the heat fixing process
Pros: ●​ Because bacteria are dead, they will be
non-motile
●​ The morphology of the bacteria is not ●​ Bacteria will be stuck to the slide
potentially changed

Simple stains are stains that are used on heat fixed slides to tell the basic morphology and
arrangement of cells. Gram staining (you will learn this later) is used much more often, but it
takes a bit more skill to do, so simple staining is a good introduction to staining techniques, and
can give you some basic information about morphology if that is all you need.

How does simple staining work? Bacterial cells are made out of molecules that give them a
negative charge on their surfaces. We can utilize this fact by using a solution containing a
colored molecule (a chromogen) that is positively charged. The positively charged chromogen
will then make an ionic bond to the negatively charged bacterial surface, allowing for easier
visualization under a microscope. Simple stains therefore work by bonding to the surface of a

36
cell. Three of the most common stains are crystal violet, methylene blue and safranin. These
basic stains will pick up a hydrogen ion in the solution, becoming positively charged, which then
bonds to the bacterial cell wall.

Collecting data:

You will want to record the shape and density of your cells, as well as the color. It is good to
write any notes you might have for improving your staining technique the next time you do it
such as changing the amount of culture you place on the slide, improvements you could make
to the heat fixing protocol, and staining time.

37
Smear Preparation and Simple Stain Experimental
Procedure
●​ Work Individually
●​ Supplies
o​ 1 glass microscope slide
o​ Clothespin
o​ Metal inoculation loop
o​ Incinerator
o​ Water bottle
o​ Simple Stains – Carbolfuchsin, Methylene blue, Crystal violet, Safranin (each person will
pick one stain)
o​ Paper towels
o​ Microscope
o​ Immersion oil
o​ Lens paper
o​ Lens cleaner
o​ 1 TSA slant inoculated with Bacillus megaterium
o​ 1 TSA slant inoculated with Micrococcus luteus
o​ 1 TSA slant inoculated with Staphylococcus cohnii
●​ Procedure
o​ Smear Preparation
1.​ Label your microscope slide with the three organisms you will be staining as
illustrated in Figure 5.1.
2.​ Using the water bottle, place a very small drop of DI water underneath each
organism name (Fig 5.1).
3.​ Sterilize your loop and cool for a few seconds. Then inoculate your loop with one
of the three organisms by lightly touching your loop to the surface of the slant.
You do not need to have a lot of bacteria on your loop. Less is more when it
comes to staining procedures.
4.​ With your inoculated loop, smear the bacteria into the water droplet by moving
your loop back and forth and in circles. Keep in mind that you will be staining all
three organisms on the same slide, so leave space between each so that you can
differentiate the smears. Sterilize your loop after smearing.
5.​ Repeat step 4 with the other two organisms. Make sure to sterilize your loop in
between.
6.​ Leave the slide on the benchtop and let the water droplets dry.
7.​ After the slide is completely dry, attach a clothespin to one end of the
microscope slide. While holding the clothespin, hold the slide, with the smear
facing away, in front of your incinerator for 5 to 10 seconds to heat fix the
bacteria on the slide.
8.​ Allow to cool.
o​ Simple Staining
1.​ With the clothespin attached, lay your slide on the staining tray.
2.​ Using the dropper bottles of stain, cover your smear completely with the liquid
stain.
3.​ Allow the stain to sit on the smear for one minute.

38
 4.​ Rinse the slide with DI water from the water bottles into the staining tray.
5.​ Lay your slide onto a paper towel and gently dab the top of the slide with
another paper towel. Do not wipe the slide with the paper towel – this will ruin
your smear. Just dab the slide to remove excess water.
6.​ After the slide is dry, you will observe your slide under immersion oil on the
microscope.
●​ Clean up
o​ Dispose of starter cultures into the large biohazard bin.
o​ Dispose of used microscope slides into the GLASS DISPOSAL.
o​ Dispose of gloves in the large biohazard bin.
o​ Throw away any old results from previous experiments.
o​ Return water bottles and stain bottles to the cabinet where you got them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

39
 Gram Stain

The Gram stain is a staining technique which allows the visualization and differentiation of
bacteria based on morphological characteristics, which in turn, can aid in the identification of
bacteria. It was developed by a Danish physician named Hans Christian Gram while studying
bacteria that caused pneumonia (1).
​
Most bacterial cells lack color under the microscope making them difficult to see, so one
advantage of Gram staining is that it allows you to visualize the cells and determine basic
morphology such as cocci, bacilli, spirillum, etc. When a media or stain allows us to categorize
organisms based on their biochemical or morphological characteristics, we say it is a differential
media or a differential stain. In this case, the Gram stain is a differential stain because it not only
allows us to physically see the cells, it also allows us to differentiate bacteria as either Gram
positive or Gram negative based on their cell wall or cell membrane structures.
​
The Gram stain utilizes crystal violet, iodine, and safranin that stain bacteria either purple or
pinkish red depending on their cell wall or membrane. Gram positive bacteria (Gram+) appear
purple after the Gram stain technique because they possess a cell wall that has a thick layer of
peptidoglycan. After application of crystal violet to the bacterial cells, iodine is applied as a
mordant. The crystal violet and iodine form a complex that’s molecular structure is too large to
bypass the thick peptidoglycan layer and the cross linked teichoic acids. So when the decolorizer
is applied, the crystal violet-iodine complex remains trapped in the cell walls making them
appear purple under the microscope. Gram negative bacteria appear pinkish red after the Gram
stain technique due to their cell wall only containing a thin layer of peptidoglycan and
possessing an outer cell membrane rich with lipopolysaccharides (LPS). After the crystal violet
and iodine are added, a decolorizer, such as ethanol, is applied which dissolves the LPS. Since
Gram negative bacteria do not possess a thick layer of peptidoglycan and the outer membrane
is gone, the crystal violet-iodine complex is easily washed out of the cells making them appear
colorless. Safranin is then applied as a counter stain, so that we can visualize the cells and they
appear pinkish red under the microscope. The next page has a very basic drawing of the
differences in the cell wall/cell membrane system (it lacks some details):

40
 Gram +* Gram-*

Stains purple Stains pink or red

Created with [Link] Created with [Link]

*the + and – do not hold any further meaning in Gram staining, so do NOT confuse this
with the charge on the outer layer of the bacteria. The charge on the outer layer of
bacteria is negative for both Gram+ and Gram- bacteria.

It is important to be able to identify bacteria, and many fields of biology and medicine rely on
the ability to quickly group or classify cells by differential staining. For example, the Gram stain
technique can help to determine what group of organisms are found in a patient with an
infection which will allow physicians to select the appropriate antibiotic for treatment. Different
antibiotics will kill some groups of bacteria but not others since these drugs often work by
binding to part of the structure of the cell, so being able to differentiate and classify cells is an
integral part of choosing the correct pharmacological agent.

41
Gram Stain examples:

Gram + organism example: Gram – organism example:

Micrococcus nishinomiyaensis Escherichia coli

This bacteria is Gram+ cocci, found in tetrads This bacteria is Gram- bacilli
(groups of 4)

Basic Gram Stain Theory:

Step 1) Prepare a smear of bacteria and heat fix the slide. – Helpful hint: always use cultures less
than 24 hours old. You may get some Gram+ that stain pink like a Gram- if the cells are old and
the thick cell wall has thinned out due to having an “elderly” culture.

Step 2) Primary Stain (1o stain) is Crystal violet, a dark purple color. This stain is used to stain the
cell wall/membrane system a dark purple. —Helpful hint: As the name suggests, if this stain is
old or unfiltered you can mistakenly get crystals on your slide. You don’t want this.

Step 3) Add a mordant. A mordant is a chemical that will allow a dye to stick better. This term is
used in biology but also in art when dying fabric! In this case the mordant is called Gram’s
Iodine and will help the Crystal Violet to be trapped by the cell wall.

Step 4) Decolorize. This step adds ethanol to the slide. The outer membrane in Gram negative
cells are made of lipopolysaccharides (LPS), and these dissolve. Because the outer membrane is
gone and the cell wall is thinner in a Gram negative cell, it doesn’t hold onto the dark purple
Crystal Violet. The Gram + does retain the stain and will appear purple on a slide. – Helpful hint:
Decolorizing is tricky and you want to follow the timing that your TA gives you exactly here or
you may get incorrect results.

42
Step 4) Secondary Stain (2o stain) is the counter stain, Safranin. This stain will stain the
de-stained Gram negative cells a pinkish red color.

These are the basic steps. Some protocols will rinse the slide between steps with distilled water,
and the time you leave each stain on the slide will vary between protocols. Things like
temperature, humidity, freshness of stain, and the age of the culture can affect the Gram stain
outcome. So there is an art to how well it turns out and each lab has typically worked out a
protocol that works best for them, though they are all extremely similar.

Written by Christy Longcrier and Donna Janes. Photos by Christy Longcrier, illustrations by Donna Janes

References

1. Tripathi N, Sapra A. Gram Staining. 2023 Aug 14. In: StatPearls [Internet]. Treasure Island (FL):
StatPearls Publishing; 2024 Jan–. PMID: 32965827.

43
 Gram Stain Experimental Procedure
●​ Work Individually
●​ Supplies
o​ 1 glass microscope slide
o​ Clothespin
o​ Metal inoculation loop
o​ Incinerator
o​ Water bottle
o​ Ethanol bottle
o​ Crystal violet
o​ Safranin
o​ Paper towels
o​ Microscope
o​ Immersion oil
o​ Lens paper
o​ Lens cleaner
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Bacillus megaterium
●​ Procedure
o​ Smear Preparation
1.​ Using the water bottle, place a very small drop of DI water in the center of your
slide.
2.​ Sterilize your loop and cool for a few seconds. Then inoculate your loop with E.
coli by lightly touching your loop to the surface of the slant. You do not need to
have a lot of bacteria on your loop. Less is more when it comes to staining
procedures.
3.​ With your inoculated loop, smear the bacteria into the water droplet by moving
your loop back and forth and in circles. Sterilize your loop after smearing.
4.​ Repeat steps 2 and 3 using B. megaterium, mixing both the organisms together
into the water droplet.
5.​ Leave the slide on the benchtop and let the smear dry.
6.​ After the slide is completely dry, attach a clothespin to one end of the
microscope slide. While holding the clothespin, hold the slide, with the smear
facing away, in front of your incinerator for 5 to 10 seconds to heat fix the
bacteria on the slide.
7.​ Allow to cool.
o​ Gram Stain
1.​ With the clothespin attached, lay your slide on the staining tray.
2.​ Using the dropper bottles of stain, cover your smear completely with crystal
violet.
3.​ Allow the stain to sit on the smear for one minute.
4.​ Rinse the slide with DI water from the water bottles into the staining tray.
5.​ Lay your slide on the staining tray and cover with iodine.
6.​ Allow the stain to sit on the smear for one minute.
7.​ Rinse the slide with DI water from the water bottles into the staining tray.

44
 8.​ Holding your slide at an angle over the staining tray, gently decolorize with
ethanol by letting the ethanol run down the smear until it runs clear. Be careful
that you don’t decolorize for too long.
9.​ Immediately rinse the slide with DI water from the water bottles into the
staining tray.
10.​ Lay your slide on the staining tray and cover with safranin.
11.​ Allow the stain to sit on the smear for one minute.
12.​ Rinse the slide with DI water from the water bottles into the staining tray.
13.​Lay your slide onto a paper towel and gently dab the top of the slide with
another paper towel. Do not wipe the slide with the paper towel – this will ruin
your smear. Just dab the slide to remove excess water.
14.​After the slide is dry, you will observe your slide under immersion oil on the
microscope.
●​ Clean up
o​ Dispose of starter cultures into the large biohazard bin.
o​ Dispose of used microscope slides into the GLASS DISPOSAL.
o​ Dispose of gloves in the large biohazard bin.
o​ Throw away any old results from previous experiments.
o​ Return water bottles, ethanol bottles, and stain bottles to the cabinet where you got
them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

45
 Negative Stain

Many types of staining depend on charge attractions or repulsions. The teichoic acid found in in the
Gram+ cell wall causes it to have an external net negative charge. The outer membrane made of
lipopolysaccharides on a Gram – cell causes it to also have a net negative charge. Negative staining is a
technique that utilizes the net negative charge of bacterial cells to stain the surroundings of the bacteria,
allowing us to visualize their morphology, size, and arrangement.

The technique uses an acidic stain, such as Nigrosin or India ink, that has a net negative charge. When it
is applied to bacteria that also have a net negative charge, the stain is repelled by the cells causing a halo
to form around the bacteria. The stain will not enter the bacteria and stay on the outside. All of the
slides will be colored dark by the negative stain except where the bacteria are, which will allow you to
see the outline of the bacteria and you can then determine the morphology. The negative stain is
advantageous because it does not require heat fixing the bacteria to the slide, which can cause cells to
shrink or be too harsh for more fragile cells. One of the disadvantageous of negative staining is it does
not allow us to differentiate between types of bacteria like the Gram stain, so it provides less
information.

Written by Christy Longcrier and Donna Janes. Photos by Christy Longcrier

46
 Negative Stain Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 glass microscope slides
o​ Metal inoculation loop
o​ Negative stain - Nigrosin
o​ Microscope
o​ Immersion oil
o​ Lens paper
o​ Lens cleaner
o​ 1 TSA slant inoculated with Micrococcus luteus
o​ 1 TSA slant inoculated with Bacillus megaterium
●​ Procedure
1.​ Lay one microscope slide on the benchtop.
2.​ Using the dropper bottle of nigrosin stain, add one small drop of stain to the left side of
your slide.
3.​ Sterilize your loop and cool for a few seconds. Then inoculate your loop with one of the
two organisms by lightly touching your loop to the surface of the slant. You do not need
to have a lot of bacteria on your loop. Less is more when it comes to staining
procedures.
4.​ With your inoculated loop, gently smear the bacteria into the nigrosin stain by moving
your loop back and forth and in circles. Sterilize your loop after smearing and then rinse
your loop to remove the nigrosin stain.
5.​ Sterilize your loop and cool for a few seconds. Then inoculate your loop with the second
of the two organisms by lightly touching your loop to the surface of the slant.
6.​ With your inoculated loop, gently smear the bacteria into the nigrosin stain that already
contains your first organism by moving your loop back and forth and in circles. This will
mix your cultures so you’ll see both organisms on the same stain. Sterilize your loop
after smearing and then rinse your loop to remove the nigrosin stain.
7.​ Place the edge of your second microscope slide in the middle of your first slide at a 45
degree angle and push the edge into the stain and bacteria mixture. The stain and
bacteria mixture should spread to the width of the slide.
8.​ Gently pull the second slide across the top of the first slide, spreading the stain and
bacteria mixture to the end of the first slide.
9.​ Leave the slide on the benchtop and let the stain dry.
10.​After the slide is dry, you will observe your slide under immersion oil on the microscope.
●​ Clean up
o​ Dispose of starter cultures into the large biohazard bin.
o​ Dispose of used microscope slides into the GLASS DISPOSAL.
o​ Dispose of gloves in the large biohazard bin.
o​ Throw away any old results from previous experiments.
o​ Return stain bottles to the cabinet where you got them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

47
 Antimicrobial Susceptibility Test
Why do we need to be able to test how sensitive a strain of bacteria is to a particular antibiotic? One of
the reasons is that not all antibiotics naturally work against all types of bacterial infections. For example,
some drugs like vancomycin have trouble crossing the outer membrane of Gram negative bacteria.
Because of their outer membrane, Gram negative bacteria are harder to kill and some classes of
antibiotics are used exclusively on Gram positive bacteria.

Furthermore, an increase in antibiotic resistance of both Gram positive and Gram negative bacteria has
led to an increase in infections that are very difficult to treat. One way to combat an increase in
antibiotic resistance of a wide number of bacteria is to always select the appropriate antibiotic for
treatment, particularly if a narrow spectrum antibiotic can be chosen that is specific for that pathogen.
How do we determine what the appropriate antibiotic to use is? We do this by testing how well a
particular antibiotic works against a particular bacteria.

The test for how well an antibiotic will work to inhibit or kill a microorganism is called the Kirby-Bauer
Disk Diffusion Method. The protocol and the result interpretations for this method have been
standardized by the U.S. Food and Drug Administration (USDA) and the Clinical Laboratory Standards
Institute (CLSI). The CSLI will determine the Minimum Inhibitory Concentration (MIC) of a drug. The
standardization of both the protocol and the result interpretation allows for both clinical and research
laboratories to share information with certainty that the results can be repeated.

The basic protocol spreads bacteria out on an agar plate so that it will grow in a lawn to cover the whole
plate upon incubation. Prior to incubation, little disks of paper infused with the antibiotic to be tested
are placed on the plate. It is common to test several antibiotics on one plate. During the incubation, the
antibiotic in the disk will diffuse out into the surrounding media. If the disk has a clear ring around it
instead of bacterial growth after incubation, that is an indication that the antibiotic is having a
bacteriostatic (inhibition from growth) or a bactericidal (killing) effect on the bacteria on the plate. These
clear rings are called Zones of Inhibition.

In order to make sure the diffusion of the antibiotic from the disk is standardized every laboratory uses
the same media at the same pH, Mueller-Hinton Agar. The Mueller-Hinton Agar (MH Agar) is also poured
to the same depth in each plate, 4mm. The amount of bacteria used to make the lawn is also
standardized. A tube with a turbidity (cloudiness) equal to 1 X 10 8 CFU/ml is held next to the tube of
bacteria, and dilutions are made if necessary to bring them to the same visual turbidity. Another option
to determine the right amount of cells for the original inoculation is to measure the absorbance of the
culture in a 1cm cuvette with a spectrophotometer set at 625 nm. Newly inoculated cultures generally
need to incubate 4 to 6 hours if you want to fall within the right range without having to dilute your
sample.

Once a plate has been incubated, you will measure the Zones of Inhibition and compare to the standard
charts that are provided by the CLSI. There are 3 possible outcomes. If the bacteria are resistant to that
antibiotic the measurement in mm will be the number in the Resistant column on the chart or smaller.
This means that would not be an appropriate antibiotic to use for an infection with that organism. There
is sometimes an intermediate range, in which the measured zone in mm is such that the CSLI cannot say
that the organism is susceptible to the drug. The third column on the chart is Susceptible. If the Zone of
Inhibition you measure in mm is as big as this number, or larger, then you know that this antibiotic will

48
work well to kill/inhibit the organism on the plate! You will want to take careful data on your
measurements for each drug, and you will also want to be able to interpret your data or any data given
to you from a chart. You won’t need to memorize the numbers on a chart like this for an exam, everyone
looks up those numbers, but it will be important for you to know how to use the chart.

References:
Clinical Laboratory Standards Institute (CSLI). Performance Standards for Antimicrobial Disk

Susceptibility Tests; Approved Standard, 11 th edition. CSLI document M02-A11. Mayne, PA: CLSI, 2012.
Bauman, Robert. Chapter 10 in Microbiology With Diseases by Body System, 5 th ed. Pearson, 2018.

49
 Antimicrobial Susceptibility Test Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 Meuller Hinton plates
o​ 1 TSB inoculated with Escherichia coli
o​ 1 TSB inoculated with Staphylococcus cohnii
o​ Antibiotic discs (1 antibiotic per group of four - assigned by TA)
o​ Disposable spreaders
o​ 1 P200 Pipette
o​ Yellow tips
o​ Alcohol
o​ Bunsen Burner
o​ Forceps
●​ Procedure Day 1
1.​ Label the bottom of the 2 Mueller Hinton (MH) plates with organism name (1 plate per
organism) and name of antibiotic. Also label your plates with your normal identifying
information (initials, section number, date, and organism name).
2.​ Gently vortex the TSB tube inoculated with E. coli.
3.​ Using a P200 pipette, transfer 100 µL of culture to the appropriate MH plate.
4.​ Using a disposable spreader, spread the inoculum around on top of the agar as you
learned during the Spread Plate Method of Isolation. Use gentle pressure and do not cut
into the agar surface.
5.​ Repeat steps 2-4 for S. cohnii.
6.​ Take both plates to the side of the room where the Bunsen burner is located.
7.​ Dip the forceps in alcohol and sterilize using the Bunsen burner. The alcohol will catch
on fire and quickly burn away. If the jar of ethanol catches on fire, carefully but quickly
put the lid on top of the jar to extinguish the fire.
8.​ Using the sterilized forceps, gently place and press down your assigned antibiotic disc
into the center of the plate.
9.​ Repeat steps 7 and 8 with your second plate.
10.​DO NOT invert - place in the incubator upright and incubate the plates until the next
class.
●​ Procedure Day 2
1.​ Using the provided rulers, measure the zone of inhibition on each plate. Compile results
as a class.
2.​ Use the Antibiotic Inhibition Chart to determine if the organism is resistant,
intermediate, or susceptible to each antibiotic.
●​ Clean up
o​ Dispose of starter culture and spreaders into the large biohazard bin. Remember to
remove caps and place them in the bins at the front.
o​ Dispose of gloves in the large biohazard bin.
o​ Return pipettes and tip boxes to where you got them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

50
 Bacteriophage Sensitivity

Bacteriophages are very specific for the bacterial host they infect and eventually lyse.
Bacteriophage T4r is a double stranded DNA phage and ΦX174 is a single stranded DNA phage
and they both infect Escherichia coli.. T4r is one of the largest phages and can only undergo a
lytic lifestyle. This is the main method of viral replication and involves the use of the host cell’s
machinery to produce large quantities of viral components. After these components are
assembled into virus particles, the cell wall of the host is broken down and the cell is lysed,
releasing the newly formed viruses. The sensitivity or resistance of bacteria to these phages is
helpful as a tool for strain differentiation.

Certain strains of E. coli show either sensitivity or resistance to different phages, as you will see
in this experiment. When reading your results, note that some strains are resistant to one
phage, neither phages, or both phages. Based on bacteriophage sensitivities, researchers can
distinguish between two different strains of E. coli due to different patterns of plaque formation
on a bacterial lawn when drops of various bacteriophages are tested. The same E. coli strains
should have the same sensitivity patterns and so the same plaque (clearing) patterns. For
instance, in a food poisoning outbreak due to E. coli, E. coli isolates are obtained from the sick
individuals and from environmental and food samples. Comparing the bacteriophage
sensitivities (or phage typing) of the samples can determine the source of the E. coli causing the
disease.

51
 Bacteriophage Sensitivity Experimental Procedure
●​ Work in groups of 4
●​ Supplies
o​ 2 TSA plates
o​ 1 TSA slant inoculated with Escherichia coli C
o​ 1 TSA slant inoculated with Escherichia coli B
o​ 1 TSA slant inoculated with Escherichia coli K12
o​ 1 TSA slant inoculated with Escherichia coli HB101
o​ Phage T4r
o​ Phage ΦX174
o​ Incinerator
o​ 1 P20 Pipette
o​ Yellow tips
●​ Procedure
1.​ Label the bottom of the 2 TSA plates with your initials, section number, and date. You’ll
also label each plate as shown below with all four E. coli strains and one phage per plate.
2.​ Using a P20 pipette, transfer 20 µL of phage T4r to the top of the appropriate plate.
Sterilize your loop, aseptically cool it, and use it to draw the phage in a line down the
plate as shown in the figure below (perpendicular to the E. coli labels). You MUST add
the phage before the bacteria.
3.​ Aseptically get a loopful of E. coli C and streak from left to right on the plate, crossing the
line of phage only one time as shown in the figure below. Use gentle pressure and do
not cut into the agar surface.
4.​ Repeat step 3 with E. coli B, E. coli K12, and E. coli HB101.
5.​ Repeat steps 2-4 with phage ΦX174.
6.​ Invert plates after they have dried and incubate until the next class.

52
●​ Clean up
o​ Dispose of starter culture into the large biohazard bin. Remember to remove caps and
place them in the bins at the front.
o​ Dispose of gloves in the large biohazard bin.
o​ Return pipettes and tip boxes to where you got them.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

53
 ABO-Rh Agglutination - Transfusion
Blood transfusions are used routinely during various medical procedures. However, if the blood
transfused (donor blood) is not properly matched with the patient’s blood a transfusion reaction can
occur. The chief danger from incompatible transfusions lies in the fate of the injected cells (1). The
patient may suffer a transfusion reaction if there is sufficient anti-A and/or anti-B antibodies in his
circulation to cause agglutination or hemolysis of the donor’s red blood cells (RBCs). Despite the
presence of anti-A and anti-B antibodies in the sera of group O individuals, their blood can often be given
to persons of any group. The titer of normal anti-ABO antibodies is normally low, and they are diluted by
the blood of the recipient to such an extent that they do not agglutinate or hemolyze the patient’s cells
(1). Therefore, members of group O are called universal donors-they have no blood antigens that can be
recognized by the recipient. Group AB individuals are likewise called universal recipients because they
have no Abs in their serum. Of course homologous blood is always the best due to other minor blood
groups (1).

Transfusion of blood into a recipient possessing antibodies to one of the donor blood group antigens can
result in a transfusion reaction. Transfusion reactions may trigger an immediate hemolytic reaction
against the donor RBCs, resulting in both intravascular lysis of RBCs and extensive phagocytosis of
antibody coated RBCs by macrophages of the liver and spleen (1). Lysis of the donor RBCs lead to free
hemoglobin that can be detected in the plasma and it is filtered through the kidneys. Hemoglobin can be
present in quantities that may be toxic for kidney cells, producing acute tubular cell necrosis and renal
failure. High fevers, chills, nausea, pain in the lower back, shock, hemoglobin in the urine and
disseminated intravascular coagulation may also develop (1). Some of the hemoglobin gets converted to
bilirubin which at high levels is toxic.

Treatment involves:
▪​ Prompt termination of the transfusion and
▪​ Maintenance of urine flow with a diuretic because the accumulation of hemoglobin in the
kidney can cause acute tubular necrosis.
The disseminated intravascular coagulation consumes clotting factors faster than they can be
synthesized and the patient may paradoxically die of bleeding (1).

In this exercise, you are a clinical blood technician of a hospital and you’re called to the
emergency room where you find an injured woman. Her left arm has been ripped open and the
emergency room medical team is trying to stop the flow of blood from a major artery. You know that the
hospital’s blood bank was depleted when victims from a local bus accident were airlifted to the hospital.
A nurse quickly explains that the woman has five friends in the emergency waiting room who are eager
to donate blood. You collect a blood sample from the injured woman (Rita) and her five friends (Alex,
Ben, Daniel, Lucy, and Christy). You must now determine which, if any, of these people can donate blood
to save their friend’s life (2).

1.​ Carpenter PL. 1975. In Vitro Antigen-Antibody Reactions: Precipitation and Agglutination.
p.220-221. In Immunology and Serology, 3rd ed., Saunders College Publishing., Philadelphia, PA
2.​ Carolina Biological Supply Company. 2003. Transfusion Matching with Synthetic Blood Kit
#700104. Carolina Biological Supply Company, Burlington, SC.
3.​ Moyes R. 2005. ABO System. Texas A&M University, College Station, TX. http://
[Link]/COURSES/microbio/notes/ABO%[Link].

54
 ABO-Rh Agglutination (Transfusion) Experimental
Procedure
●​ Work individually
●​ Supplies
○​ 1 Blood Typing Slide
○​ Synthetic Blood (Each group of four will be assigned a different person by their TA)
○​ Synthetic A Antiserum (Blue)
○​ Synthetic B Antiserum (Yellow)
○​ Synthetic D Antiserum (clear)
○​ Mixing sticks
●​ Procedure
○​ Place a small drop of the accident victim’s blood sample in each well of the blood typing
slide. Replace the cap on the dropper vial. Always replace the cap on one vial before
opening the next vial to prevent cross contamination.
○​ Add a drop of synthetic A Antiserum to the well labeled A. Replace the cap.
○​ Add a drop of synthetic B Antiserum to the well labeled B. Replace the cap.
○​ Add a drop of synthetic D Antiserum (Rh) to the well labeled Rh. Replace the cap.
○​ Using a mixing stick, gently stir the synthetic blood and anti-serum drops until
completely mixed - approximately 30 seconds. Remember to discard each mixing stick
after a single use to avoid contamination of your samples.
○​ Carefully examine the wells for agglutination. Compile class results to get results for all 6
samples.
○​ Complete the worksheet on the next page - this will be used for your online assignment.
●​ Clean up
○​ Dispose of the blood typing slide and mixing sticks in the large biohazard bin.
○​ Return blood and antiserum to TA
○​ Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

55
 Transfusion Worksheet

Complete the table below. Answer yes or no as to whether agglutination occurred in each sample. A
positive agglutination reaction indicates the blood type.

Rita (victim) Alex Ben (donor) Daniel Lucy (donor) Christy

(donor) (donor) (donor)
A Antiserum
B Antiserum
D Antiserum (Rh)
Blood Type

You must use a donor whose blood will not agglutinate when mixed with that of the accident victim.
Which of Rita’s friends can donate blood to her?

Which of Rita’s friends is your first choice to donate blood? Why?

Rita’s first friend has donated all the blood that he/she can but more blood is needed. Does Rita have a
friend who, although not a preferred donor, can be used in this emergency? Who is it and why can this
person be used as a donor even though the blood types are not the same?

56
 Selective and Differential Media and Biochemical Tests

In your work in the laboratory so far you have used agar plates to grow cell cultures and to isolate cells.
Agar plates can additionally be utilized in identification of a microorganism.

One strategy is to use plates containing selective media. Selective media contains added chemicals that
will inhibit or kill some species of bacteria while letting others grow. These selective plates limit what will
grow on the plate and therefore select for a particular type of organism.

Another strategy is to use plates containing differential media. Differential media will allow a wide range
of bacteria to grow but the visual appearance of the colonies will be different between species based on
their biochemical properties. This is typically dependent on which enzymes a species produces based on
genetics.

Some media can utilize both strategies and are therefore selective and differential at the same time. For
example, MacConkey Agar will be used in the unknown process and is both selective and differential.

57
 Triple Sugar Iron Agar

The Triple Sugar Iron Agar test is most often used to differentiate Gram- enteric (gut) bacteria,
though it can be used to differentiate other types of Gram- bacteria as well. The Triple Sugar
Iron Agar (TSIA) test has 3 basic components to it.

First, it tests for the ability of the microorganism to ferment 3 sugars: glucose, sucrose, and/or
lactose. The TSIA media comes as a solid slant and is a dark fuchsia color. (This fact will help to
keep you from accidentally picking up and inoculating a TSA media by mistake, since that media
is tan!) The media is a fuchsia color because it contains phenol red, a weak organic acid that
goes from fuchsia to yellow when the pH drops below 6.8. Since it is a slant, it also has the
advantage that you can observe what happens when cells grow anaerobically (you stab the butt
of the slant and distribute your microorganism deep in the agar) and aerobically (you do a
second inoculation on the surface of the tube).

The end product of sugar fermentation is acidic, so one will expect to see a change to yellow if
the microorganism has the enzymes to ferment that sugar. Because this media puts in 0.1%
glucose but 1% of both sucrose and lactose, any microorganisms that ferment ONLY glucose will
initially turn the media yellow within 12 hours. However, you want to incubate to 24 hours since
once it runs out of glucose it will start to break down amino acids near the surface for energy,
which produces basic ammonia (NH3) and the faster growing aerobic slant will revert back to
the original color. At 24 hours then, any glucose only fermentation tube will have a yellow butt
and because of the reversion to alkaline, a red slant. If a microorganism can ferment glucose
and either lactose or sucrose (or both) it will turn the media yellow throughout. Since there is
so much more of the other sugars, the media will stay yellow longer without having to break
down proteins. (This is way you don’t want to read the color changes after 24 hours—eventually
they will run out of sugars). Note that you will know if it can ferment glucose after this test, and
you will be able to say it ferments either lactose or sucrose—but you won’t be able to say which
one or if it ferments both.

If it doesn’t ferment any of the sugars, the microbe might still be able to grow if it has the
enzymes that allow it to use amino acids or peptones for energy. Using these compounds will
turn the fuchsia tube to red. If it is an obligate aerobe, only the slant will turn red. This is a more
subtle color change than turning to yellow, so it might be helpful to compare the tube next to a
control tube that has not been inoculated.

You will want to record if your tubes produced gas or not upon fermentation. Since the bacteria
that can grow anaerobically will grow in the butt of the slant, if they produce gas they will either
crack the media, form bubbles, or if they produce enough, even push all of the media up away
from the bottom of the tube. The third thing you will be able to test for is if the microbe is
capable of hydrogen sulfide (H2S) production from the breakdown of the amino acid cysteine or
reduction of thiosulfate in acidic conditions. When a bacteria can breakdown these compounds
and ferment sugar they will interact with the ferrous sulfate in the media turn black.

58
Pictures of possible TSIA tests:

How to understand the picture (front and side view shown for each tube):
❖ ​ Slant / Butt/Gas/H2S ex. K/A/-/+
❖ ​ K- alkaline
❖ ​ A-acidic
❖ ​ Bubbles or cracks in the tube indicate gas production
❖ ​ Black precipitate indicates H2S production
❖ ​ You should never get an A / K reaction. If you do, you did not inoculate correctly.
You must stab AND streak.

59
 Triple Sugar Iron Agar Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 3 TSIA slants
o​ Inoculating loop
o​ Incinerator
o​ 1 TSA slant inoculated with E. coli
o​ 1 TSA slant inoculated with C. freundii
o​ 1 TSA slant inoculated with P. rettgeri
●​ Procedure
1.​ Label each TSIA tube with the appropriate identifying information and organism name
that you will be inoculating with.
2.​ Sterilize your inoculating loop in the incinerator and allow to cool.
3.​ Aseptically inoculate your loop with bacteria from the starting E. coli culture slant.
4.​ You will be performing the “stab and streak” method. Touch your inoculated loop to the
surface of the agar slant and gently push your loop through. You will push your loop all
the way down to the bottom of the tube, while trying to keep the loop roughly in the
center of the agar slant. This is called the “stab”.
5.​ Remove your loop from the agar, and then perform a zig zag streak along the surface of
the agar slant. See the figure below for an illustration of this method.
6.​ Repeat steps 4 and 5 for C. freundii and P. rettgeri.
7.​ Incubate at 37 until the next lab period.

60
●​ Clean up
○​ Dispose of gloves in the large biohazard bin at the front of the lab room.
○​ Spray benchtops with Lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

61
 Catalase Test
The catalase test determines the presence or absence of the enzyme catalase. This test is
typically used to differentiate between catalase positive cocci organisms such as Staphylococci
(positive) and Streptococcus (negative). It can also assist in differentiating other types of
bacteria, including types of bacilli such as aero tolerant Clostridium (negative) verses Bacillus
(positive).

Catalase is an enzyme found in many organisms that use aerobic respiration to produce ATP.
Normally during aerobic respiration, electrons are passed down the electron transport chain
(ETC) until they use oxygen as the final electron acceptor. If this pathway is not taken with
complete efficiency, it can result in reactive oxygen species (ROS).

ROS like superoxide radicals, hydrogen peroxide, and hydroxyl radicals can damage cell
membranes, proteins, and nucleic acids. In order to protect itself, any organism that is an
obligate or facultative aerobe must have enzymes that can convert these toxic ROS into less
toxic molecules. The most common enzymes are superoxide dismutase, peroxidase, and
catalase. The reactions for these 3 enzymes can be seen below. Once superoxide dismutase
makes the hydrogen peroxide, then either peroxidase or catalase will convert it into less toxic
molecules. In this laboratory exercise we will be testing for catalase specifically.

As you can see in the catalase catalyzed reaction above, the end products are water and oxygen
gas. Because these are the products, if you grow bacteria in a tube overnight and then add
hydrogen peroxide, if it makes catalase the hydrogen peroxide solution will start to bubble due
to making oxygen gas! You can also grow bacteria overnight and put a clump onto a slide, which
is what you see when you observe the reaction in the pictures below:

62
 Catalase Test Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 1 glass microscope slide
o​ Metal inoculation loop
o​ Incinerator
o​ Hydrogen peroxide
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Streptococcus lactis
●​ Procedure
1.​ Label your microscope slide with both organisms - E. coli on the left side of the slide, S.
lactis on the right side of the slide.
2.​ Sterilize your inoculating loop in the incinerator and allow to cool.
3.​ Aseptically inoculate your loop with bacteria from the E. coli culture slant.
4.​ With your inoculated loop, smear the bacteria in a small area on the left side of the
slide. Keep in mind that you will be testing both organisms on the same slide, so leave
space between each so that you can differentiate the results. Sterilize your loop after
smearing.
5.​ Repeat steps 3 and 4 with S. lactis on the right side of your slide.
6.​ Without touching the bacterial smear, carefully add one drop of hydrogen peroxide to
each bacterial smear.
7.​ Observe each smear for bubble formation. Bubbles indicate the presence of catalase
which is a positive result.
●​ Clean up
o​ Dispose of starter cultures into the large biohazard bin.
o​ Dispose of used microscope slides into the GLASS DISPOSAL.
o​ Dispose of gloves in the large biohazard bin.
o​ Throw away any old results from previous experiments.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

63
 Oxidase Test
During the breakdown of glucose during aerobic respiration, the glucose molecule will be
oxidized until eventually all of the carbons will be CO2. In any chemical reaction when one
molecule is oxidized since it loses an electron, then another molecule must be reduced and gain
that electron. (A helpful mnemonic is OIL RIG, where oxidation is loss and reduction is gain—of
an electron). Many enzymes involved in the breakdown of glucose therefore have coenzymes
that are capable of gaining an electron! Some common coenzymes are NAD, nicotinamide
adenine dinucleotide, and FAD, Flavin adenine dinucleotide. These molecules gain hydrogen,
along with its electron, in the metabolic reactions involved.

One of the jobs of the Electron Transport Chain (ETC) is to take the electron off the hydrogen in
NADH and FADH2 and recycle it back into NAD and FAD so that the reactions in glycolysis and
the Krebs cycle can continue to move forward. (The H+ then moves into the periplasmic space in
bacteria, or the inner mitochondrial space in eukaryotes). If a bacteria has an ETC similar to
those in Eukaryotes, it will have a series of four complexes that carry the electron until it gets
joined with oxygen and protons in a reaction that creates water. The last of these complexes
contains the enzyme cytochrome c oxidase.

The oxidase test will test for the presence of this cytochrome c oxidase. A chromogenic
compound is one that changes color when its oxidation/reduction status changes. The
chromogenic reducing agent tetramethyl-p-phenylenediamine is used and when added to
bacterial growth that contains the enzyme it will result in a positive test. You will see a color
change to a purple/dark blue color if the reducing agent (tetramethyl-p-phenylenediamine) gets
oxidized in the process, indicating the presence of cytochrome c oxidase.

Picture of the Oxidase Test

64
 Oxidase Test Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 pieces of sterile filter paper
o​ Metal inoculation loop
o​ Incinerator
o​ Oxidase reagent dropper bottle
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Pseudomonas fluorescens
●​ Procedure
1.​ Sterilize your inoculating loop in the incinerator and allow to cool.
2.​ Aseptically inoculate your loop with bacteria from the E. coli culture slant.
3.​ With your inoculated loop, smear the bacteria in a small area on one piece of filter
paper. Sterilize your loop after smearing.
4.​ Repeat steps 2 and 3 with P. fluorescens on the second piece of filter paper.
5.​ Have your TA place 1-2 drops of fresh oxidase reagent on each piece of filter paper.
6.​ Observe for blue color within 20 seconds. A positive test will turn the paper blue in less
than 20 seconds. A negative test may turn blue but it will take much longer.
●​ Clean up
o​ Dispose of starter cultures into the large biohazard bin.
o​ Dispose of gloves in the large biohazard bin.
o​ Throw away any old results from previous experiments.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

65
 Citrate Utilization
The citrate test is used to differentiate between bacteria in the family Enterobacteriacae. This
family contains bacilli that are Gram (-) facultative anaerobes. This test determines if an
organism has the enzyme citrate permease and citrate lyase, allowing you to narrow down what
organism you have based on whether the enzymes are present or absent.

The media for the citrate test can do this since the only source of carbon for the bacteria comes
from sodium citrate. Citrate cannot be broken down in glycolysis to make pyruvate for the citric
acid cycle, but if the enzyme citrate permease is present citrate may be transported into the
cell:

Once the citrate is inside the cell, it can enter the citric acid cycle and be used under aerobic
conditions (Also called the TCA or Kreb’s cycle). The citric acid will also be used in fermentation
processes. If the cell also contains citrate lyase, this enzyme can convert citrate to oxaloacetate
and acetate, and the oxaloacetate can then be converted into pyruvate under basic pH
conditions. This reaction directly makes ATP for the cell during fermentation (no aerobic
respiration needed). In a side reaction, it can also convert NAD+ to NADH.

How are these reactions visualized on this media? Besides the citrate, this media also contains
ammonium phosphate. Organisms that use citrate as a sole carbon source also will convert
ammonium phosphate into ammonia and ammonium hydroxide, which will make the media
become more alkaline. There is a color indicator called bromophenol blue which is green at a pH
of 6.9, but becomes blue above the more alkaline pH of 7.6. The original media is slightly acidic
and should come as a nice dark green color. If the organism can use citrate as its carbon source,
it will also convert the ammonium phosphate. The resulting metabolism will make the media
more alkaline and modify boromophenol blue so that it turns from green to blue. Both the
green and the blue are a really beautiful color, as seen below, so this is a fun test to run!

66
67
 Citrate Utilization Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 Citrate slants
o​ Metal inoculating loop
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Enterobacter aerogenes

●​ Procedure
1.​ Label all tubes with your group number, section number, date, and the organism name.
Label the tube directly on the glass, NOT the cap. Caps are removed and can easily be
mixed up.
2.​ Using aseptic technique, inoculate the citrate slant with E. coli by fishtail streaking the
slant.
3.​ Repeat step 2 using E. aerogenes. Incubate both tubes at 37C until the next lab period.
●​ Clean up
o​ Dispose of starter culture into the large biohazard bin. Remember to remove caps and
place them in the bins at the front.
o​ Dispose of plates and/or tubes from previous experiments after you have read results.
o​ Dispose of gloves in the large biohazard bin.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

Result Interpretation

Some or all of the slant is blue Positive for Citrate Utilization

Slant is completely green Negative for Citrate Utilization

68
 Malonate Utilization
The Malonate Utilization Test can determine if a microorganism has the enzymes needed to use
malonate as a sole carbon source. This test is typically done to distinguish between some
Enterobaceriaceae.
Malonate can act as an enzyme inhibitor. For bacteria that do aerobic respiration and therefore
use the Krebs cycle (also called Citric Acid Cycle or TCA cycle), the molecule succinate must get
converted to fumarate, and this reaction is carried out by the enzyme succinate
dehydrogenase, as seen in step 6 of the Krebs Cycle below.

The molecule malonate is similar in shape to succinate and therefore can act as a competitive
inhibitor to the enzyme succinate dehydrogenase. If this enzyme is inhibited, no succinate can
be converted to fumarate and succinate builds up in the cell. The Krebs cycle is backed up and
cells will die unless they can use the malonate as their sole carbon source.

If the cells can use the malonate, it gets oxidized and in this case that makes the media more
ALKALINE (higher pH). The media for the test has a pH sensitive color indicator molecule,
bromothymol blue, which is green at more acidic pH and blue at more alkaline pH,7.6. Since
malonate utilization results in more alkaline conditions, an inoculated test that remains green
would be considered negative for malonate utilization and a positive result would be when the
tube is blue after incubation of the culture for up to 48 hours.

[Link]

69
Examples of Malonate Utilization Outcomes:

70
 Malonate Utilization Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 Malonate broths
o​ Metal inoculating loop
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Enterobacter aerogenes

●​ Procedure
1.​ Label all tubes with your group number, section number, date, and the organism name.
Label the tube directly on the glass, NOT the cap. Caps are removed and can easily be
mixed up.
2.​ Using aseptic technique, inoculate the malonate broth with E. coli by mixing your
inoculated loop in the broth.
3.​ Repeat step 2 using E. aerogenes. Incubate both tubes at 37C until the next lab period.
●​ Clean up
o​ Dispose of starter culture into the large biohazard bin. Remember to remove caps and
place them in the bins at the front.
o​ Dispose of plates and/or tubes from previous experiments after you have read results.
o​ Dispose of gloves in the large biohazard bin.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

Result Interpretation

Broth is blue Positive for Malonate Utilization

Broth is green Negative for Malonate Utilization

71
 Lysine Iron Agar
The Lysine Iron Agar test is most often used to differentiate enteric bacteria. Like the TSIA test,
the Lysine Iron Agar (LIA) test comes as a slant for both aerobic and anaerobic growth, and tests
for multiple things. It has a small amount of sugars and peptones and yeast extract in the media
that allows for hardy cell growth, but it also contains a color indicator that is pH dependent. The
color indicator is Bromocresol purple, which turns purple above pH 6.8 and is yellow at or below
pH 5.2.

You will be testing for the ability of the microorganism to produce hydrogen sulfide (H2S) gas by
the anaerobic reduction of thiosulfate. Ferric ions in the media react with the H2S to form black
articles in the butt of the tube.

You will also be testing for the presence of two different enzymes. The enzyme lysine
decarboxylase is made by some microorganisms to combat the stress induced when media gets
too acidic. Therefore, when fermentation of glucose starts to take place this media will originally
turn yellow as it gets more acidic. This will cause the organism to make the enzyme lysine
decarboxylase as a way to make the media more alkaline (k) again by converting lysine to an
amine called cadaverine. This will make the whole tube go back to purple. If the butt is yellow
and there is a purple slant, it means it fermented the glucose, but didn’t have the genes to code
for the enzyme lysine decarboxylase.

The second enzyme you will be testing for is lysine deaminase, an enzyme that works in the
presence of oxygen. The media contains ferric ammonium citrate, and deamination of lysine
produced compounds that interact with ferric ammonium citrate to turn red. Because this
reaction requires oxygen, you will only see this color at the surface/top of the slant.

72
73
 Lysine Iron Agar Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 3 LIA slants
o​ Inoculating loop
o​ Incinerator
o​ 1 TSA slant inoculated with E. coli
o​ 1 TSA slant inoculated with E. aerogenes
o​ 1 TSA slant inoculated with P. rettgeri
●​ Procedure
1.​ Label each LIA tube with the appropriate identifying information and organism name
that you will be inoculating with.
2.​ Sterilize your inoculating loop in the incinerator and allow to cool.
3.​ Aseptically inoculate your loop with bacteria from the starting E. coli culture slant.
4.​ You will be performing the “stab and streak” method. Touch your inoculated loop to the
surface of the agar slant and gently push your loop through. You will push your loop all
the way down to the bottom of the tube, while trying to keep the loop roughly in the
center of the agar slant. This is called the “stab”.
5.​ Remove your loop from the agar, and then perform a zig zag streak along the surface of
the agar slant. See the figure below for an illustration of this method.
6.​ Repeat steps 4 and 5 for E. aerogenes and P. rettgeri.
7.​ Incubate at 37oC until the next lab period.

74
●​ Clean up
○​ Dispose of gloves in the large biohazard bin at the front of the lab room.
○​ Spray benchtops with Lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

75
 MIO (Motility, Indole, Ornithine)
MIO is a semi-solid medium that tests for motility, indole production, and ornithine decarboxylation.
Motility can be determined due to the semi-solid consistency of the medium. If an organism is motile,
the tube will appear cloudy. If an organism is non-motile, the original stab line will be visible.

Organisms that possess the enzyme “tryptophanase” cleave the amino acid tryptophan with the
resulting production of indole, pyruvic acid, and ammonia. Upon addition of Kovac’s reagent
(p-dimethylaminobenzaldehyde), indole reacts with the aldehyde group of
p-dimethylaminobenzaldehyde yielding the formation of a red complex. If indole is present, a cherry red
layer will form at the top of the medium.

Decarboxylase enzymes attack the carboxyl (COOH) group of amino acids resulting in the formation of
alkaline-reacting amines. The ability of organisms to produce arginine decarboxylase, lysine
decarboxylase, or ornithine decarboxylase is tested using three different but very similar media. In all
decarboxylation reactions, the microbe must first ferment glucose. Upon glucose fermentation, acidic
byproducts are formed turning the media yellow due to a drop in pH. The presence of acid from glucose
fermentation will activate decarboxylase enzymes. Ornithine decarboxylation yields putrescine which
causes a rise in the pH and corresponding color change of the bromcresol purple from yellow to purple.
In this case, if the inoculated medium remains yellow, the organism is decarboxylase-negative for
ornithine.

76
MIO (Motility, Indole, Ornithine) Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 MIO tubes
o​ Metal inoculating loop
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Klebsiella ozaenae

●​ Procedure Day 1
1.​ Label all tubes with your group number, section number, date, and the organism name.
Label the tube directly on the glass, NOT the cap. Caps are removed and can easily be
mixed up.
2.​ Using aseptic technique, inoculate the MIO with E. coli by stabbing straight down and
pulling the loop straight back out of the medium. Remember - don’t wiggle the loop
when inoculating because this can skew the motility results.
3.​ Repeat step 2 using K. ozaenae. Incubate both tubes at 37C until the next lab period.
●​ Procedure Day 2
1.​ Read the motility and ornithine decarboxylation tests.
Result Interpretation

Uniform cloudiness Positive Motility

Stab line is clearly visible Negative Motility

Purple Positive for Ornithine Decarboxylation

Some or all of the medium is yellow Negative for Ornithine Decarboxylation

2.​ Add 3-5 drops of Kovac’s reagent to the tube. Read the indole production test.
Result Interpretation

Cherry Red/Pink Ring Positive for Indole Production

No color change/Yellow Ring Negative for Indole Production

●​ Clean up
o​ Dispose of starter culture into the large biohazard bin. Remember to remove caps and
place them in the bins at the front.
o​ Return reagents to the refrigerator.
o​ Dispose of plates and/or tubes from previous experiments after you have read results.
o​ Dispose of gloves in the large biohazard bin.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

77
 Carbohydrate Fermentation (Phenol Red Broth)
There are a series of biochemical tests based on carbohydrate fermentation that can be used to
determine the identity of a bacterium. These tests determine what enzymes and metabolic pathways are
present that allow for the fermentation of carbohydrates in that particular species. These tests are
frequently used to tell the members of Enterobacteriaceae from one another, as well as to differentiate
between a few important Gram+ species.

Louis Pasteur discovered that fermentation of carbohydrates is due to the presence of microorganisms in
1857(1). Fermentation of carbohydrates results in the breakdown of the carbohydrate under anaerobic
conditions in order to produce energy for the cell. The carbohydrate is typically broken down into
monosaccharides, and then these simple sugars are further broken down, resulting in the loss of
electrons (oxidation). Oxidation is always accompanied with a reduction reaction, because that electron
needs to go somewhere! The electron will be gained by another organic molecule that acts as the final
electron acceptor. You might already be familiar with some of these electron acceptors from freshman
biology; such as pyruvate which will be converted to lactic acid or ethanol when it gains the electron
from NADH, depending on which enzymes the organism produces.

One of these fermentation tests utilizes Phenol Red Broth. This test consists of a series of tubes, each
with a different carbohydrate to be tested. The media in all of the tubes also contains a color indicator
that is pH sensitive (Phenol red), which will be pink above pH 7.4, red from pH 6.8 to pH 7.4, and yellow
below pH 6.8. The end product of carbohydrate fermentation is acidic and will turn the media yellow
because of the drop in pH below 6.8. The media also has the protein casein in it, and deamination of the
amino acids found in casein will produce ammonia and make the media become more alkaline, turning it
pink. Below is a picture of two Phenol Red Broth tubes containing the carbohydrate lactose. The picture
on the left shows a positive yellow test, indicating that the pH of the media has dropped below 6.8 due
to fermentation of the sugar. In the tube on the right, the organism lacked the enzymes available to
break down lactose and the pH of the solution remained between 6.8 and 7.4 and therefore there was
no color change and the tube remained red.

78
Some enzymes that ferment carbohydrates also release gas in the process. This data can be easily
collected and utilized to help identify an unknown organism. When using Phenol Red broth to detect
fermentation one can simply insert a smaller tube, called a Durham tube, upside down in the broth and
it is filled with media. The tube is then incubated overnight. If the organism produces gas during
fermentation, the gas bubbles will rise to the top of the Durham tube and push the liquid out the
bottom, leaving a gas bubble at the top of the upside down tube. Below is a picture of Phenol Red Broth
tubes with the carbohydrate glucose in the media. From left to right you see in the first tube a positive
test of yellow with a gas filled Durham tube, in the second tube a positive test for fermentation of
glucose but negative for gas production as the Durham tube is still full of liquid, and in the last tube a
negative test for glucose fermentation as the media stays red.

79
Pictures by Christy Longcrier

(1) Stanier RY, Doudoroff M, Adelberg EA. 1963. The microbial world, 2nd ed. Prentice-

Hall, Englewood Cliffs, NJ

80
Carbohydrate Fermentation Experimental Procedure

(Phenol Red Broth)

●​ Work in pairs
●​ Supplies
o​ 1 Lactose broth
o​ 1 Sucrose broth
o​ 1 Glucose broth with durham tube
o​ Metal inoculating loop
o​ 1 TSA slant inoculated with Escherichia coli, Providencia rettgeri, OR Pseudomonas
fluorescens (each pair will use only one organism)

●​ Procedure
1.​ Label all tubes with your group number, section number, date, and the organism name.
Label the tube directly on the glass, NOT the cap. Caps are removed and can easily be
mixed up.
2.​ Using aseptic technique, inoculate the lactose broth with your designated organism by
mixing your inoculated loop in the broth.
3.​ Repeat step 2 using sucrose broth.
4.​ Release step 2 using glucose broth. Incubate tubes at 37C until the next lab period.
●​ Clean up
o​ Dispose of starter culture into the large biohazard bin. Remember to remove caps and
place them in the bins at the front.
o​ Dispose of plates and/or tubes from previous experiments after you have read results.
o​ Dispose of gloves in the large biohazard bin.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

Result Interpretation

Broth is yellow Positive for Carbohydrate Fermentation

Broth is red/pink Negative for Carbohydrate Fermentation

81
 Urea Hydrolysis
Some bacteria can break down urea in a hydrolysis reaction using the enzyme urease. The urea test,
which tests for the presence of the enzyme urease, can help you differentiate between organisms that
are rapid-urease positive (Proteus, Morganella morganii, and some strains of Providencia stuartii) and
slower urease-positive bacteria or urease-negative bacteria. The overall reaction can be seen below. (1)

If a bacteria contains the enzyme urease, It is capable of breaking down urea into carbon dioxide and
ammonia. Since ammonia is basic, this particular reaction results in a higher, more alkaline, pH in the
solution. The media for this test is an orange color because it contains the pH sensitive indicator phenol
red, which is orange at a more neutral pH.

If a bacteria contains the enzyme urease, ammonia is produced and the pH raises above pH 8.2. At this
pH, the phenol red turns hot pink which is a positive test for the presence of urease. Proteus, Morganella
morganii, and some strains of Providencia stuartii is good at utilizing urease and will turn the media hot
pink overnight (rapid positive), but some of the organisms that are urease positive with weaker/delayed
reactions might take up to 24 to 48 hours on solid media or not react at all in the liquid media since it is
more buffered (2). Since you check your tubes after 48 hours of incubation you can be confident in your
test results.

If a bacteria does not contain the enzyme urease it might still ferment the available carbohydrates in the
media, and if the media drops below 6.8 due to the acids produced during fermentation of
carbohydrates the phenol red will turn yellow. Therefore, a negative test will be either the original
orange color or yellow. See photo below:

1. [Link]

2. [Link]
[Link]

82
83
 Urea Hydrolysis Experimental Procedure
●​ Work in pairs
●​ Supplies
o​ 2 Urea broths
o​ Metal inoculating loop
o​ 1 TSA slant inoculated with Escherichia coli
o​ 1 TSA slant inoculated with Providencia rettgeri

●​ Procedure
1.​ Label all tubes with your group number, section number, date, and the organism name.
Label the tube directly on the glass, NOT the cap. Caps are removed and can easily be
mixed up.
2.​ Using aseptic technique, inoculate the urea broth with E. coli by mixing your inoculated
loop in the broth.
3.​ Repeat step 2 using P. rettgeri. Incubate both tubes at 37C until the next lab period.
●​ Clean up
o​ Dispose of starter culture into the large biohazard bin. Remember to remove caps and
place them in the bins at the front.
o​ Dispose of gloves in the large biohazard bin.
o​ Spray benchtops with Lysol and wipe down with the provided sponges.
o​ Wash your hands before leaving the lab.

Result Interpretation

Broth is pink Positive for Urea Hydrolysis

Broth is orange/peach Negative for Urea Hydrolysis

84
 MacConkey Agar

As stated previously, some media are selective and differential at the same time. The agar you are using
today, MacConkey Agar, is both selective and differential. It is typically used to isolate bacteria in the
family Enterobacteriaceae and then differentiate them based on the ability of the organism to ferment
lactose. In a clinical setting MacConkey agar is often used to determine the causative agent of UTIs
(urinary tract infections) or gastroenteritis. Examples of clinically important members of
Enterobacteriaceae are the lactose fermenter Escherichia coli and Salmonella, a non-lactose fermenter
(1). Most coliforms, a sub group of Enterobacteriaceae that grow in the gut, are lactose fermenters.
MacConkey agar can therefore also be used to determine if water might potentially be unsafe due to
sewage contamination.
​
The components of MacConkey Agar that make it selective are bile salts and crystal violet, both of which
inhibit Gram+ bacteria from growing but allow the growth of Gram- bacteria. The components of the
media that make it differential are lactose and the pH sensitive dye Neutral Red. If an organism cannot
ferment lactose, the colony will stay white/tan. If the organism does ferment lactose, then the end
product of that fermentation will usually be lactic acid. The lactic acid will lower the pH and the Neutral
Red dye will turn pink/red when the pH gets below 6.8 (1).

Photos by Christy Longcrier

In your notebook you should write the answers to the following questions: Why would you use
MacConkey Agar? What would a positive test for lactose fermentation look like and what would a
negative test look like? Can you tell if your sample ferments lactose or not if what you had in your culture
was a Gram+ organism? The answer should be no, but you should be able to explain why the answer is
no. You should be able to explain which components of the media make the plate selective and which
make the plate differential.

1.​ [Link]

85
 Unknown Identification Worksheet Page 1

Name:____________________________ Section:_______
Unknown Number:_______

TSA
Appearance of colonies

MacConkey
Amount of growth

Appearance of colonies

Lactose fermentation

Gram Stain
Color Shape/Morphology

Catalase
Result (+/-) Color/Reaction

Oxidase
Result (+/-) Color/Reaction

Unknown Group Number

86
 Unknown Identification Worksheet Page 2
TSIA
Slant Butt H2S Gas
Result (+/-)
Color/Reaction

Biochemical Test Result (+/-) Color / Reaction

Unknown Identified as:

87
 Unknowns Project Day 1 Experimental Procedure

●​ Work individually
●​ Supplies
○​ 1 TSA plate
○​ 1 MacConkey
○​ 1 TSA slant inoculated with your unknown (assigned by your TA)
○​ Metal inoculating loop
○​ Incinerator
●​ Procedure
1.​ Label both plates with your initials, section number, date, and the Unknown number.
2.​ Fill out the top of your unknown worksheet with your name, section number, and
unknown number.
3.​ Using aseptic technique and the quadrant streak plate technique, streak your unknown
for isolation on your TSA plate.
4.​ Repeat step 3 with your MacConkey plate.
5.​ Invert and incubate your plates until the next class.
6.​ Return your unknown slant to your TA.
●​ Clean up
○​ Dispose of plates and/or tubes from previous experiments after you have read results.
○​ Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with lysol and wipe down with the provided sponges.

88
 Unknowns Project Day 2 Experimental Procedure

●​ Work individually
●​ Supplies
○​ Unknown culture from TSA plate or slant
○​ Metal inoculating loop
○​ Incinerator
○​ Microscope slide
○​ Hydrogen peroxide
○​ Sterile filter paper
○​ Oxidase reagent
●​ Procedure
1.​ Record results from your TSA and MacConkey plates on your Unknown worksheet. Be
sure to record how your unknowns grow and what reactions occur on TSA and
MacConkey plates (lactose fermentation, size, pigmented colony, etc.). Keep your
plates until your unknowns project is complete.
2.​ Using aseptic technique, complete the catalase test for your unknown. Make sure you
are familiar with the procedure before you begin. Record this result on your Unknown
worksheet.
3.​ Using aseptic technique, complete the oxidase test for your unknown. Your TA will add
the oxidase to your filter paper. Make sure you are familiar with the procedure before
you begin. Record this result on your Unknown worksheet.
●​ Clean up
○​ Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with lysol and wipe down with the provided sponges.

89
 Unknowns Project Day 3 Experimental Procedure

●​ Work individually
●​ Supplies
○​ Unknown culture from TSA plate or slant
○​ 1 glass microscope slide
○​ Clothespin
○​ Metal inoculation loop
○​ Incinerator
○​ Water bottle
○​ Ethanol bottle
○​ Crystal violet
○​ Safranin
○​ Paper towels
○​ Microscope
○​ Immersion oil
○​ Lens paper
○​ Lens cleaner
●​ Procedure
1.​ Perform a Gram stain on your unknown organism. Make sure you are familiar with the
procedure before you begin. Record your results on the Unknown worksheet.
●​ Clean up
○​ Dispose of used microscope slides into the GLASS DISPOSAL.
○​ Dispose of gloves in the large biohazard bin.
○​ Return water bottles, ethanol bottles, and stain bottles to the cabinet where you got
them.
○​ Spray benchtops with Lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

90
Unknowns Project Day 4 Experimental Procedure

●​ Work individually
●​ Supplies
○​ Unknown culture from TSA plate or slant
○​ Enteric battery (TSIA, MIO, urea, citrate, LIA, malonate, arabinose, inositol, sorbitol,
raffinose, rhamnose and adonitol)
●​ Procedure
1.​ Label all tubes with your initials, section number, date, and unknown number. Write the
type of media on each tube. The order of the tubes is listed above. Arabinose, inositol,
sorbitol, raffinose, rhamnose, and adonitol are all phenol red carbohydrate broths.
2.​ Aseptically inoculate the enteric battery using your unknown culture.
3.​ Incubate at 37oC until the next lab period.
●​ Clean up
○​ Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with Lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

91
 Unknowns Project Final Day Experimental
Procedure

●​ Work individually
●​ Supplies
○​ Kovak’s reagent
●​ Procedure
1.​ Read all enteric battery results and record data on your Unknown worksheet.
2.​ Identify your unknown based on your test results using only the enteric chart below. Do
not use outside sources to identify your organism. Organisms can be variable and these
charts are based on the strains that are used in this lab.
●​ Clean up
○​ Dispose of plates and/or tubes from previous experiments after you have read results.
○​ Dispose of gloves in the large biohazard bin.
○​ Spray benchtops with Lysol and wipe down with the provided sponges.
○​ Wash your hands before leaving the lab.

92
Enteric Chart

93