The purpose of lab is to provide a hands-on approach in understanding the concepts of
microbiology. Everything you see and do in lab are cornerstones of microbiology practiced
in the “real world.”
Lab Safety is Priority #1…
Be sure to review the Do’s & Don’ts of lab etiquette and Fire Safety.
DON’TS - eating or drinking or biting your nails or applying lip balm / no phones
DO’S – wear safety glasses and closed toe shoes wash hands / close gas jets after lab / tie
Where do you put it?
Contaminated media plates, plastic tubes, swabs, etc. go in the biohazards (pails on discard
carts in the back of lab).
Non-contaminated paper and towels soaked with Amphyl go in the crocks on the bench.
Uncontaminated broken glass or slides that have been heat-fixed goes in the
uncontaminated broken glass box at the back of the lab.
Contaminated slides that have not been heat-fixed, used pipettes, etc. go in the
contaminated bucket on the lab bench.
What are the three kinds of tape you are likely to use in the lab and why?
1. Autoclave indicator tape: tape shows diagonal slants once it has been autoclaved and is
2. Masking tape: to keep stacks of plates together
Don’t be afraid or embarrassed to tell your TA of mistakes and accidents. It is better to be
safe than sorry!
Exercise 1-2 – Media (TSB & TSA)
Just like us bacteria need nutrients to survive. These can be found in broth (liquid) and agar
(solid) forms. There are many different types of media since some organisms are very
“picky/ selective.” If we can’t grow the microorganisms we can’t study them!
We talk about media as being undefined and defined.
A defined media is one in which we know exactly all of the ingredients and amounts.
An undefined media is one in which the exact ingredients and amounts are not known.
To make media solid you have to have a “thickener.” In microbiology we use agar which
comes from seaweed. STERILITY is the single most important thing about making media. The most effective way of
sterilizing media is by autoclaving.
There are three parameters that are critical when autoclaving media to ensure sterility.
Why are these three things important? IT IS THE ONLY EFFECTIVE WAY TO KILL SPORES
When using an autoclave you also need to remember the more you put into it the MORE
time it takes to reach sterilization temperatures.
Steam is exhausted from an autoclave in two ways:
Fast-Exhaust which is used for “dry” things like glass, instruments, swabs, etc.
Slow-Exhaust which is used for “liquids” so that they don’t bubble over.
Things coming out of the autoclave are extremely hot and require you wear protection so
you are not burned!
How can you tell something has been autoclaved and is sterile? Autoclave indicator tape –
the stripes on the tape darken during the steam sterilization process.
Exercise 1-4 -- Aseptic Transfer & Innoculation
We “move” bacteria around in the lab using the methods of aseptic transfers and
inoculation for maintaining and testing.
What is the primary reason for using aseptic techniques to maintaining sterility of our
cultures/sample? Contamination of a culture with additional bacterial species can
confound results used in the identification of pathogenic bacterium and could lead to the
wrong diagnosis of an infectious disease.
When sterilizing your loop or needle in a flame which cone do you pass it through? Why?
You pass your loop through the inner flame to heat it sufficiently.
How do you know you have heated the loop or needle sufficiently?
You know you’ve heated your loop sufficiently when it turns a uniform orange color.
Why should you keep your tubes in the test-tube rack and other work items in a convenient
place? You keep your tubes in the test-rack so that they don’t leak, or roll of your bench,
or break. You keep everything in a convenient place to ensure safety and sterile
procedures. Leaning over a Bunsen burner for example, could cause you to get burned!
What are the steps to transfer from broth to broth? To slant?
From broth to broth:
When inoculating broth, gently hand-mix the broth culture prior to transfer by tapping the
tube several times with your finger. Make sure to flame the lip of the tube for sterility and
dip in your inoculating loop. Move it around in the broth and simply dip your loop into the
broth being inoculated. From broth to slant:
When inoculating an agar slant, begin at the bottom of the tube and drag the loop up in a
zig zag pattern taking care not to cut or rip the media. If you’re using culture tubes, pass
the mouth of the tube (cap off) several times in order to sterilize the lip of the tube.
Exercise 1-5 – Streak Plate
In all fields of microbiology it is absolutely critical to be able toisolate bacteria.
The goal of isolation is to obtain a pure culture from a mixed culture
containing two or more species.
Why is obtaining a pure culture so important?
If you have a sample with more than one bacterial species, any test you perform on that
culture will be unreliable because the test results may be from any one of the organisms
in that culture.
Streaking for isolation is a dilution technique. Meaning you start off with a
high concentration of mixed/non mixed bacteria and as you streak the
concentration of the bacteria lowers Finally the bacteria are at such low
concentration that individual cells on the agar surface can form a colony .
The goal of quadrant streaking is to get isolated colonies.
Exercise 2-1 – Ubiquity
What is a simple definition of ubiquity?
Ubiquity is existing or being everywhere
The three types of organisms we find in our world are:
1. Free-living Microorganisms are organisms that are not associated
with any specific organism and are non-pathogenic (NOT disease causing).
2. Pathogenic Microorganism are organisms that cause disease
3. Opportunistic Pathogens are organisms normally inhabit our bodies
without causing disease, but if introduced to a new area or if immune system
is suppressed, they can cause disease.
Understanding ubiquity is important for everyone (health-care workers and hospital
visitors ) to understand since one out of twenty patients will acquire an infection
in the hospital which could be life threatening.
Why do you streak environmental samples (zigzag) and clinical samples (quadrant/urine)
Unlike clinical samples, the amount of bacteria in an environmental sample is likely to be
much lower. If there is a low density of bacteria to begin with, quadrant streaking for
isolation would not be effective since the bacteria would likely all appear in the 1
quadrant and not be well separated. Quadrant streaking for isolation is difficult away
from a lab setting since there is generally not a flame source available. Making Dilutions
A 1:10 dilution means you have 1 part sample in a total volume of 10 parts.
If you want to reduce the volume of the dilution you are making you divide all the
volumes used in the dilution by the same number. To increase the volume of the
dilution you would multiply all the volumes by the same number.
When you are making dilutions there are 3 very important points to remember:
1. Always add the sample to the saline/broth
2. Always mix each dilution thoroughly after adding the sample.
3. Always, always change pipet tips after each sample transfer
Why is it important in a healthcare setting to know how to make dilutions?
It’s important in a healthcare setting to know how to make dilutions because you may be
required to dilute drugs prior to administering them to patients.
Exercise 6-1 – Standard Plate Counts (Viable Counts)
Why is it important to learn how to determine/quantify the number of bacterial colonies in
a sample? Learning how to determine / quantify the number of bacterial colonies in a
sample is especially important for food and water safety. Is the level of bacteria in our
drinking water acceptable by local, state or federal standards? Is the lettuce in the
processing plant contaminated and if so, what is the level of contamination?
Two techniques that are important in determining/quantifying bacteria:
1. Making Dilutions:
2. Making Spread Plates:
Label each tube the sample will be taken from (ex. 10^-4), put 0.1 ml of the bacterial
dilution onto the middle of the agar plate, put a metal hockey stick into a beaker of
alcohol, remove stick and let any excess alcohol drip off, pass metal hockey stick
through Bunsen burner so that the alcohol ignites and kills any bacteria on the stick,
let it cool, spread the sample over the entire surface of the plate, once bacterial
solution has been spread over the surface of the plate allow the solution to be
absorbed into the agar, after it’s absorbed, turn plate over and incubate over night.
Making Spread Plates
Once the number of colonies at a specific dilution is known, then it
is possible to calculate the number of colony forming units (CFU) in 1ml of
1 colony does NOT equal 1 bacterial cell. (One bacterium is NOT visible on a plate.)
However, 1 bacterial cell can form a colony. (A visible colony is hundreds of thousands
of cells that started from one bacterial cell that has divided into a group of identical
cells.) Can you figure the number of CFU/ml of a sample?
CFU/ml = __________(# of colonies)____________
(dilution factor) (volume plated)
What is the rule for using plates in your CFU/ml calculations?
Only use plates with between 20/30 to 200/300 colonies for calculations
Exercise 2-2 -- Colony Morphology
Why is Colony Morphology important?
Colony Morphology is important because it can give you an idea of how many different
bacterial species are in a sample. It provides you with the first clue as to the identity of
the bacterial species in a sample.
What are a few of the things to be considered when talking about morphology?
Things to consider when talking about morphology are form, elevation, margin,
pigmentation, texture and growth patterns.
Could you describe the colony morphology if you were given a plate with a sample? Supplemental: Sub-Culturing Bacterial Isolates
The purpose of sub-culturing is to obtain a pure culture of a bacterial species. Pure
cultures contain only one type of bacteria.
Without a pure culture you could not analyze morphology, physiology or biochemistry. You
could not identify the bacteria or determine antibiotic resistance.
Each colony type may indicate a different bacterial species. To know which colony is the
pathogenic bacterial species, you need to “pick” each different colony type and test that
particular bacterial species. Therefore, you need a pure culture of each microorganism. Each
colony needs to be well isolated
Exercise 6-3 – Urine Culture
Urinary Tract Infections (UTIs) are the 2 ndmost common type of infection.
It is the number one type of hospital acquired infection.
Why would UTIs be so much more common in the hospital environment?
The reason UTI’s are so common in the hospital environment is likely due to
catheterization, which is the insertion of a tube into the bladder.
The bacteria E. coli is most commonly associated with UTIs.
Urine MUST be culture within 1-2 hours after collection or if refrigerated within 24
Diagnosis of a UTI is based on the number of CFU/ml in the sample. Determining
CFU/ml by serial dilution and spread plates is NOT practical in a diagnostic lab. So we
use an alternate method.
First an inoculating loop is used to transfer a known volume of sample
to a Blood Agar Plate (BAP). This special plate contains sheep’s blood and is nutrient rich
so many different bacterial species can grow on it. Then the sample is spread out over the
entire surface of the plate using a specific streaking method called a semi -
Frist, streak in a straight line down the middle of the plate that fully extends from
one end of the plate to the other. Then perform a zigzag streak that runs back and
forth across the diameter of the plate and continually through the first streak line.
Loop is NOT flamed between streak #1 and #2. Using the plate counting rule ( 20/30 – 200/300) we can determine the original cell density
OCD = ______(# of colonies)_____ CFU/ml
General Guidelines for interpreting the results of a urine culture:
1. If there are more than 3 colony types on the plate, there is considered to be
contamination with normal flora and a new specimen needs to be taken
2. If there are 1-3 colony types on the plate, the number of each colony needs to be
determined. If colony has <10,000 CFU/ml, it is likely to be normal flora
3. If a colony type has >10,000 CFU/ml then the patient is diagnosed with a UTI
Exercise 8-11 – Soil Microbiology
What is soil composed of?
Inorganic material, organic matter, air, H20, and microorganisms
The number of microbes decreases as you go deeper into the soil layers.
What roles do microbes play in the soil?
Microbes play a role in fixing various elements such as nitrogen, carbon and sulfur. They
are also a key player in soil food web.
Chemoautotrophs oxidize inorganic compounds to yield energy and reduce CO 2.
Decomposers break down organic biomass/compounds. (dead plant material)
Actinobacteria are a class of bactieria that also produce antibiotics.
Mutualists form partnerships with plants.
Ex. Mychorrhizal fungi: colonize plant roots and bring nutrients such as nitrogen and
phosphorus to the plant in exchange for carbon from the plant.
Disease Suppressors are bacteria that suppress the occurrence of plant disease by
secreting anti-fungal or anti-insecticidal compounds.
Pathogens are bacterial species that can cause disease in plants/animals. Exercise 7-4 – Biofilms
A biofilm is a complex association that arises from a mixture of microorganisms
growing together on a surface of a habitat.
What are a few examples of biofilms?
Some examples of biofilms are scum on stagnant pond, scum on inside of water/sewage
pipes, scum on diseased fruit and vegetables, dental plaque on teeth.
Know the basics of the formation of a biofilm on a surface
1. Surface Conditioning: Production of a surface film composed of biomolecules and
2. Initial Attachment: Primary bacterial colonizers adhere
3. Slime Formation: Primary colonizers secrete slime that allows irreversible attachment
4. Secondary Colonization: More bacteria join
5. Maturation: Fully functioning biofilm is formed
6. Dispersal: Bacteria released and will go on to form biofilms on more distant surfaces
Biofilms are medically important and can lead to life threatening infections. List a few
“indwelling devices” that are susceptible to biofilm formation.
- contact lens
- prosthetic joints
The bacteria within a biofilm are generally more resistant to disinfectants and
Exercise 3-1 – Intro to Microscope
In lab we use a brightfield microscopy meaning it has two (2) sets of lenses.
Ocular Lenses located within the eye-piece .
Objective Lenses are on the nose-piece.
When carrying the microscope always use two (2