Microbiology: A
Crash Course in
Lab Basics
Microbiology: A
Crash Course in
Lab Basics
By: Azure Bradger
Table of contents
v
Table of contents
Table of contents ..................................................... v
Introduction ........................................................... vii
Understanding Aseptic Technique .......................... 3
Rules of Aseptic Technique ................................ 4
Creating Agars and Broths ...................................... 9
Creating Culture Media....................................... 9
Using the Autoclave .............................................. 15
Standard Settings .............................................. 15
Safety Guidelines .............................................. 15
Inoculation Technique and Incubation.................. 19
Broth Inoculation .............................................. 19
Slant Inoculation ............................................... 20
Plate Inoculation ............................................... 22
Incubation Conditions ....................................... 24
Analyzing Cultures ............................................... 27
Smear Preparation ............................................. 27
References ............................................................. 31
Index ..................................................................... 31
Introduction
vii
Introduction
In today’s world, science has become a field that
many people, young and old, have taken an interest
in. As young people begin their college education,
they spend increasing amounts of time in the
laboratory discovering the complexities of life on
Earth.
However, with these educational adventures come
many obstacles that students don’t realize they face.
Going into any biological lab means that a student
may come into contact with dangerous organisms
and chemicals. Doing so means they must prepare
themselves for this contact, to avoid harming
themselves with any sort of accidental contact.
In microbiology labs, specifically, there are many
different types of organisms that can grow inside
the human body, from bacteria to fungi, and
handling these organisms can be helpful if not vital
to a scientist’s work. Improper handling of these
organisms could result in negative consequences for
anyone exposed to them.
In addition, the chemicals used in the lab are often
riddled with many ingredients that, if even inhaled,
can cause severe damage to the human body. Using
these chemicals is a necessity, however, for many of
the tests run in the everyday biology lab. Many
students may not be aware of the issues they may
face in mishandling these products.
The purpose of this manual is to better prepare
students for their time in the microbiology labs, and
in their studies and research beyond. The use of the
manual could be the survival guide to the novice
viii
Microbiology: A Crash Course in Lab Basics
microbiologist, or a reference manual for the
forgetful professional. Either way, the use of this
manual will greatly benefit any person with an
interest in the techniques discussed here, because it
will break down the most complex of techniques to
a common language.
Chapter 1:
Understanding Aseptic
Technique
Understanding Aseptic
Technique
3
Understanding Aseptic Technique
Aseptic technique is the sterile techniques used by microbiologists
when working at all times in the laboratory. Its sole purpose is to
ensure chemical and biological safety, as many of the chemicals
and organisms used in labs can be harmful if they aren’t handled
properly. The biggest rule when entering a microbiology lab,
whether it is a college department lab or a research lab for the
CDC, is using caution and aseptic technique when handling
anything.
Of course, there are the common lab rules, such as:

No eating, drinking, smoking, chewing gum, etc. in the lab.

Never apply cosmetics or manipulate contact lenses in the
lab.

Wash hands before and after completing lab work.

No open-toed shoes in the lab.

Do not touch your face while working in the lab.
With microbiology, there are extra precautions to take when
working in the lab:

Do not store food in the microbiology lab.

Disinfect lab bench before and after working.

Dispose of bio-hazardous materials in the properly.

Do not move through the lab with open containers or
uncovered samples of chemicals or organisms.

Wear gloves when working with chemicals that can be
dangerous if absorbed through the skin, or if there is a rash
or cut on your hands.
4
Microbiology: A Crash Course in Lab Basics
Rules of Aseptic Technique
Among several others, these rules are necessary for the lab
students’ and instructors’ safety. The practice of aseptic technique,
along with all rules in the laboratory, can prove to be the difference
between a person finding their specimen on their slide or on their
hands!
1. Any materials used for biological inoculations or transfers
must be sterilized before and after use.
a. Loops, needles, and other tube/plate inoculating
tools can be sterilized with a flame.
b. Plates, broths, beakers, and other types of media
must be sterilized in the autoclave, which is a
machine used strictly to sterilize equipment and
supplies.
2. Covers, lids, caps, and anything placed on the biological
sample must never touch the lab bench. Use the hand
holding the inoculation tool (loop, needle, etc.) to hold tops
to tubes, and only uncover plates enough for the purpose
you need.
3. Always work near a flame. The flame creates an updraft
that can help aerosols and dust from settling in samples.
4. Unless you are holding it, all tubes must stay in a tube rack.
This ensures that the tube is safe and will not spill while not
in use.
5. All materials must be disposed of in the proper fashion.
a. Plastic plates and tools must be discarded in the
biohazard trash.
b. Small sampling tools (toothpicks, swabs, etc.) must
be discarded in the small biohazard containers on
the bench.
Understanding Aseptic
Technique
6. If a spill occurs, clean and decontaminate the area
following the safety codes for each biohazard level. For
microbiology student labs, the organisms are biohazard
level 1, which means they can be dangerous if inhaled or
ingested.
There are many other rules for aseptic technique, most of which
that are specific to the type of lab being used and the organisms
being observed. Overall, though, these rules apply to every
microbiology lab and are essential in proper and pure lab work.
5
Chapter 2:
Creating Agars
and Broths
Creating Agars and Broths
9
Creating Agars and Broths
All organisms need a source of nutrients to grow,
metabolize, and reproduce. With microorganisms,
scientists can use various ways to grow and
maintain a population of them. These ways, called
culture media, allow the scientist to grow
microorganisms any way they want. There are 3
main types of media:

Agars

Slants

Broths
Broths, the simplest of media, allows the scientist to
grow a large population of microorganisms in a
short amount of time (usually 18-24 hours). Agars,
a broad term to describe many things, are a gel-like
substance used to grow isolated cultures of
microorganisms. Slants are made from test tubes
filled with agar, and tilted, to give a plane to
inoculate with an organism. They are used most
commonly when analyzing growth with the naked
eye, and performing biochemical tests on an
organism.
Creating Culture Media
Below are the listed steps to follow when creating a
broth and agar. These use nutrient broth and agar,
but the only difference between making other types
is the broth powder used.
10
Microbiology: A Crash Course in Lab Basics
1. Dissolve 2.8 g of Nutrient Broth powder in
350 mL of distilled water using an
Erlenmeyer flask and a sterilized stir bar.
2. Pour into 10 test tubes 5 mL of the mixture.
These are completed broths.
3. Add 6 g of agar into the remaining 300 mL
of the solution.
4. Use medium heat and stir constantly until
the agar dissolves. Watch liquid carefully
until it is clear and reaches a boil.
Immediately remove it from heat. (Note: the
liquid normally bubbles and foams up when
it is ready to be removed.)
5. Pour into 10 screw-cap test tubes 5 mL of
agar mix. Cap loosely. These are what will
be the slants.
6. Cover the flask with the remaining 250 mL
of agar with foil. Place all test tubes and the
flask in the autoclave on standard
sterilization settings (see Chapter 3).
7. After autoclaving is complete, place all
screw-cap test tubes in a separate rack, and
place the longer side of the rack at a 30degree angle (2 inches of support under the
tubes should be about equal to the angle).
8. Broth tubes are ready to use, so place them
in a separate rack.
9. After cooling the flask to 50-60 degrees C,
use aseptic technique to pour 25 mL into 10
empty plastic Petri plates. Work quickly, as
the agar thickens as it cools.
Creating Agars and Broths
11
10. Allow plates to cool and solidify. All media
are ready for use within 2-4 hours.
Below you will find an image diagram that shows
the process. This will assist you in perfecting your
technique.
Creating Agars and Broths
13
Chapter 3: Using
the Autoclave
Using the Autoclave
15
Using the Autoclave
Standard Settings
Sterilization in a biology lab is a major concept for a student to
comprehend. Along with the many methods of sterilization
discussed, the use of the autoclave allows a student to sterilize
large amounts of materials in a relatively quick amount of time.
In standard sterilization, the autoclave must have certain settings to
reach the point where no microorganisms can grow. Pressure,
temperature, and time are essential in these settings. For the
purposes of this lab, we use standard sterilization settings, which
are:

15 minutes (takes about 20 minutes to heat up and cool
down, so it becomes about an hour)

15 PSI (or ATM, depending what the unit uses)

121 degrees Celsius
Safety Guidelines
When using the autoclave, remember that:

Anything that goes in will undergo these conditions for
some time, so they must be able to withstand them.

Organize tubes, plates, and other items flatly to allow them
to receive full sterility.

Always put smaller items onto tray or into a container, so
they can be inserted and removed in a timely manner.

Ensure that any screw-cap test tubes have been loosened, so
the contents inside can be sterilized.

Any broken glass getting sterilized will have to be in a
container or a tray.
16
Microbiology: A Crash Course in Lab Basics

Ensure the door is completely closed before changing
settings and starting cycle.
These are essential when it comes to safety in the lab, because
every person using the autoclave is potentially at risk for harm.
Use the autoclave in a good manner, though, and you will save a
lot of time.
Chapter 4: Inoculation
Technique and
Incubation
Inoculation Technique and
Incubation
Broth Inoculation
As mentioned before, broths can be used to grow certain
microorganisms quickly and safely. A broth is simply a liquid
medium solely used for the purpose of growing microorganisms.
Broths can be inoculated from anywhere, from samples in lab to
locations in the environment, as long as the sample has a
microorganism. To inoculate a broth:
1. Sterilize the inoculation loop by placing the wire and loop
in a burner flame for 15-20 seconds (metal will turn red
when sterilized).
2. Remove the lid of the culture with the hand holding the
loop, and hold it in your hand.
3. If your sample culture is environmental or a plate, bypass
this step. If your culture is a tube, flame the mouth of the
tube for 2-3 seconds.
4. Insert loop into the sample you will inoculate your broth
with, and remove it after you obtain a sample on the loop
(small enough to fit on the wire of the loop, so you may not
see the sample).
5. If you didn’t flame in step 3, bypass this step. Flame the
mouth of the tube again.
6. Close the lid of sample culture, and place on lab table (or in
a rack, if it is a test tube).
7. Use the hand holding the inoculating loop to remove the
cap of the nutrient broth.
8. Flame the mouth of the tube for 2-3 seconds.
9. Insert and inoculating loop into broth. Mix well to put the
specimens in suspension.
20
Microbiology: A Crash Course in Lab Basics
10. Flame the mouth of the tube and close tube. Sterilize loop
in the flame again until red-hot.
Underneath is a series of images with steps underneath to assist
you in the process.
Slant Inoculation
Slant inoculation is a bit easier than other inoculations, because it
takes the best parts of each of the other culture media for its
purposes. The purpose of a slant is to look at the morphology of a
cell with the naked eye, or for biochemical tests. To inoculate a
slant:
1. Sterilize the inoculation loop by placing the wire and loop
in a burner flame for 15-20 seconds (metal will turn red
when sterilized).
2. Remove the lid of the culture with the hand holding the
loop, and hold it in your hand.
3. (If your sample culture is environmental or a plate, bypass
this step.) If your culture is a tube, flame the mouth of the
tube for 2-3 seconds.
4. Insert loop into the sample you will inoculate your broth
with, and remove it after you obtain a sample on the loop
(small enough to fit on the wire of the loop, so you may not
see the sample).
5. (If you didn’t flame in step 3, bypass this step.) Flame the
mouth of the tube again.
6. Close the lid of sample culture, and place on lab table (or in
a rack, if it is a test tube).
7. Use the hand holding the loop to remove the screw-cap of
the slant.
8. Flame the mouth of the tube for 2-3 seconds.
9. Insert the inoculation loop at the very bottom of the plane
of agar in the slant.
10. Drag loop in a zig-zag motion to the top of the plane, until
the loop is at the top of the agar.
11. Flame the mouth of the tube for 2-3 seconds. Close tube
with screw cap.
12. Flame the inoculation loop to sterilize it (red-hot).
Underneath is an image with what a slant will look like after
inoculation.
(
)
22
Microbiology: A Crash Course in Lab Basics
Plate Inoculation
Inoculation of a plate can become difficult if the biologist is
careless and does not pay attention, but the key is to be patient
when working on the plates. The purpose of a plate is to isolate a
pure culture of the microorganism for analysis. Inoculation can be
done in two ways, but both are considered good for the purpose.
The novice 4-quadrant streak involves a plate being split into 4
quadrants by splitting the plate in half vertically and horizontally.
The professional 4-quadrant streak involves a plate being streaked
on its edges in thirds, with the final quadrant in the center of the
other 3 plates. To inoculate a plate:
1. Sterilize the inoculation loop by placing the wire and loop
in a burner flame for 15-20 seconds (metal will turn red
when sterilized).
2. Remove the lid of the culture with the hand holding the
loop, and hold it in your hand.
3. (If your sample culture is environmental or a plate, bypass
this step.) If your culture is a tube, flame the mouth of the
tube for 2-3 seconds.
4. Insert loop into the sample you will inoculate your broth
with, and remove it after you obtain a sample on the loop
(small enough to fit on the wire of the loop, so you may not
see the sample).
5. (If you didn’t flame in step 3, bypass this step.) Flame the
mouth of the tube again.
6. Close the lid of sample culture, and place on lab table (or in
a rack, if it is a test tube).
7. Working near a flame, turn the plate to be inoculated
upside-down (the lid should be on its flat side on the table).
With your empty hand, pick up the plate.
8. Place tip of the inoculation loop gently against the section
of agar, and drag across in a zig-zag motion until the 1st
quadrant is covered. Close the plate in its inverted position.
9. Flame the loop again. After cooling, drag the loop gently in
a zig-zag motion through the 1st quadrant 2-3 times while
streaking across the 2nd quadrant. Finish the 2nd quadrant
without streaking through the 1st.
10. Close the plate in its inverted position and flame the loop
again.
11. Repeat step 9 with quadrants 3 and 4, using the previous
quadrant for the streaking across.
12. After the 4th quadrant is done, flame the loop to remove any
residual culture. Close plate and incubate it in the inverted
position.
The best way to make this technique easier is to rotate the plate,
making the cross-quadrant streaking easier. Below is a figure with
images showing the complete process.
24
Microbiology: A Crash Course in Lab Basics
Incubation Conditions
When incubating culture media, it is important that the system
being used fits the growth the scientist wants to see in the media.
The most important part of incubation, however, is maintaining a
constant temperature. If the scientist wants a media with organisms
in full growth, the best way to grow them is to place them in the
temperature environment they are best suited in. Organisms are
classified by their preferred temperature growth:

Psychrophiles: grow in extremely low temperatures
(between -10 and 20 degrees C).

Mesophiles: grow in temperatures above the freezing point
of water to body temperature (between 10-40 degrees C).

Thermophiles: grow in temperatures above the body
temperature (between 40 and 70 degrees C).

Extreme thermophiles: grow in temperatures close to and
greater than the boiling point of water (between 80 and 110
degrees C)
Below is a table of various bacteria and their temperature growth
range. Most bacteria need 18-24 hours to show a good incubation
cycle, although at the reaches of their growth range, it may take
longer.
Chapter 5: Analyzing
Cultures
Analyzing Cultures
27
Analyzing Cultures
When analyzing microorganisms, it is important that the scientist
uses the proper technique for the smear and stain they need to
make. Each culture media has specific spear techniques, and each
type of microorganism has a stain technique. Understanding the
basic smears for broths and agars, and the basic types of stains, are
essential for any laboratory.
Smear Preparation
In order to properly stain a cell, the microorganism must be
properly smeared and fixed to the slide. Smears are heat-fixed,
meaning they are placed in a flame, in order to fix microorganisms
to the slide during staining. Agar smears are prepared from slants
and culture plates, and broth smears are prepared from broths.
Below are steps to take when inoculating from agar or broth
culture media.
Broth Smears:
1. Flame an inoculation loop and the mouth of the broth
culture being used. Insert and remove the loop, inoculating
it with the cultured microorganism.
2. Place the drop of broth on the center of a microscope slide.
Smear the drop around the slide to form a thin layer of the
broth.
3. Allow the slide to air dry near an open flame.
4. Run the slide through the flame 3 times for 1 second each.
This should be adequate to fix all of the microorganisms to
the slide.
5. Let the smear cool, and it is ready for staining.
Agar Smears:
28
Microbiology: A Crash Course in Lab Basics
1. Place 1-2 loops containing distilled water onto a clean
microscope slide (should be a small drop on the slide).
2. Flame an inoculation loop. Insert and remove the loop from
the agar culture, inoculating it with the microorganism.
3. Run the loop through the drop of water on the slide, and
smear around the slide to form a thin layer of the water and
sample.
4. Allow the slide to air dry near an open flame.
5. Run the slide through the flame 3 times for 1 second each.
This should be adequate to fix all of the microorganisms to
the slide.
6. Let the smear cool, and it is ready for staining.
Here is a visual of each step in the smear process.
Analyzing Cultures
29
Stain Preparation
After properly preparing a smear, analyzing the bacteria requires
adding color to them to see them under a microscope. Through
biochemical reactions, we use various chemicals to create the color
stain that we need.
There are two types of stains discussed here: the simple stain and
the gram stain. The simple stain is used to view a cell’s
morphology. The gram stain is used to identify the specific nature
of a cell’s membrane, and the chemical it attracts determines
whether it is considered gram positive or gram negative. Below are
instructions to prepare both types of stains.
Simple Stain:
1. After preparing a smear, cover the smear with methylene
blue. Leave for 1 minute.
2. Briefly rinse the slide with distilled water. Blot dry with
bibulous paper.
3. The slide is now ready for viewing under oil immersion.
The bacteria will appear indigo on a clear background.
Below is an image of the process and the complete microscope
slide.
30
Microbiology: A Crash Course in Lab Basics
Gram Stain:
1. After preparing a smear, cover the slide with Crystal
Violet. Leave for 30 seconds to 1 minute.
2. Rinse briefly with distilled water.
3. Cover the slide with Gram’s Iodine. Leave on for 1 minute.
4. Rinse slide with decolorizing alcohol (95% ethanol) for no
more than 10 seconds. Immediately rinse with distilled
water.
5. Cover the slide with safranin. Leave on for 1 minute.
6. Rinse the slide with water. Blot the slide dry with bibulous
paper.
7. The slide is now ready for viewing under oil immersion.
Gram positive bacteria will have a violet color, and gram
negative bacteria will be red-pink.
Below is an image of the process and the complete product.
(
References
Hughes, R., & Hughes, L. (2013). Microorganisms: A Laboratory
Manual (2nd ed.). Kendall Hunt.
Index
4-quadrant streak, 22
Agar Smears, 27
Agars, v, 7, 9
Aseptic technique, 3
autoclave, 4, 10, 15,
16
Broth Smears, 27
Broths, v, 7, 9, 19
culture media, 9, 20,
24, 27
Gram Stain, 30
incubation, 24
inoculation, 4, 19, 20,
21, 22, 27, 28
Simple Stain, 29
smears, 27
Sterilization, 15