CULTURE MEDIA
- contains ingredients that promote or
Liquid Media
- Commonly called broth in the
microbiology laboratory
- Media does not contain agar, but
nutrients in the liquid
- The enrichment properties of the media
enable microorganisms to be recovered in
higher numbers
- Is used for detecting the presence of a
low number of microorganisms that may
be missed if plated on solid agar only
- Has specialized formulas that may be
used for biochemical testing and other
uses
Solid Media
• A solidifying agent, such as agar, is added in
the preparation of solid culture media
• Does not inhibit bacteria or liquefy at room
temperature
• Is used for developing surface colony growth
of microorganisms
• Is critical for obtaining isolated colonies
Semi-Solid Media
• Has the consistency between a liquid and a
solid
• Is often equated to jelly consistency
• Has a lower concentration of agar than solid
media
• Is commonly used in determining organism
motility
CULTURE MEDIA TYPE
There are three main types of culture media:
enrichment, selective, and differential.
Enrichment media 
- has specific nutrients added to the
basic culture media to encourage the
growth of specific types of
microorganisms.
Selective media 
inhibit the growth of particular types
of microorganisms. Growth inhibiting
agents can include items such as dyes,
acids, alcohols, and/or antibiotics.
 
Differential media 
- Differentiates microorganisms based
on major metabolic or biochemical
reactions such as the production of
acid from certain carbohydrate
fermentation.
Culture media is useful to isolate and identify
microorganisms from various sites in the
human body.
(Culture Media TABLE)
(Specimen TYPE)
Some microorganisms grow best in the
presence of oxygen and are called AEROBES.
ANAEROBES, on the other hand, cannot grow
in the presence of oxygen.
There are specialized media to grow
anaerobes. Also, additional conditions must
be established in the laboratory, such as
anaerobic chambers or jars, to grow these
microorganisms.
For a sputum specimen, the media of choice
is: Blood Agar Plate (BAP) or Sheep Blood
Agar (SBA), Chocolate Agar (CHOC), and
MacConkey Agar (MAC).
BAP/SBA
• a base media
• an enriched and differential media
• Commonly used for culturing many bacterial
species including Gram negative (such as
Escherichia coli) and Gram positive species
(such as Streptococci).
Interpreting SBA/BAP
When reading or interpreting a specimen that
has been inoculated to SBA/BAP and
incubated for 18-24 hours, you may see a
clearing around some of the isolated colonies.
This is ß (Beta) hemolysis.
This is a result of hemolysins that the
microorganisms possess that causes the
complete or partial lysis of blood cells
(hemolysis).
There are two other types of hemolysis that
can be exhibited around colonies: 
α (Alpha) hemolysis results in a greening
around a colony and γ (Gamma) hemolysis in
which you will not see hemolysis at all.
Chocolate Agar (CHOC)
Characteristics
• an enriched media
•Prepared by heating whole blood to 80-90 ºC
• when heated, the media turns a chocolate
brown color
•when heating of the blood occurs, the X and
V factors are released from red blood cells,
which is needed by some microorganisms for
growth
•supports the growth of fastidious
microorganisms, such as Haemophilus spp.
MacConkey Agar (MAC)
Characteristics
• a common selective and differential media
• used for processing various specimen types
where Gram negative rods are a suspected
potential pathogen.
• selectively inhibits the growth of Gram
positive microorganisms resulting in the
“selection” of Gram negative microorganisms
for growth
• lactose and neutral phenol red (pH
indicator) allow the “differentiation” of Gram
negative rods based on their ability to
ferment lactose and the resulting color
appearance of the colonies.
• as a differential media, MAC differentiates
between Gram negative microorganisms that
are able to ferment lactose (lactose
fermenters) and those that cannot (non-
lactose fermenters)
• lactose fermenters will appear as pink
colonies on the media while non-lactose
fermenters will appear as clear or colorless
colonies
For a tissue specimen, you would inoculate
the following culture media:
•Anaerobic blood agar (AnaBAP)
•Blood agar (BAP)
•Chocolate agar (CHOC)
•Columbia Colistin-Nalidixic Acid agar (C-CNA)
•MacConkey agar (MAC)
•Phenylethyl alcohol agar (PEA)
•Thioglycollate broth (THIO)
Anaerobe Blood Agar (AnaBAP)
•is a non-selective media
• allows the growth of the facultative and
obligate anaerobic microorganisms when
incubated anaerobically. Anaerobic
microorganisms will die in the presence of
oxygen over a short amount of time.
Thioglycollate Broth (THIO)
Characteristics
•is a broth media
•allows the growth of anaerobes and aerobes
•growth patterns within the broth tube may
be useful in helping with microorganism
classification of anaerobic or aerobic
Note:  If there is growth in this media after
incubation, subculturing to appropriate
culture media as indicated by Gram stain will
be necessary.
Columbia Colistin Nalidixic Agar (C-CNA)
• enriched and selective media that contains
two antibiotics, colistin and nalidixic acid,
used to encourage the growth of Gram
will grow bottom of
Anaerobic
tube
will grow throughout
Facultative
tube
Aerobe will grow top of tube
positive microorganisms while inhibiting the
growth of Gram negative microorganisms.
Note: Some laboratories will use either C-CNA
or PEA in their laboratory set-ups for wound
or tissue specimens.
Phenylethyl Alcohol Agar (PEA)
• a selective culture media
• inhibits the growth of facultative or
anaerobic Gram negative microorganisms
• specifically formulated to allow the growth
of Gram positive microorganisms and some
anaerobic Gram negative microorganisms
Strep throat is an infection caused by the
microorganism Streptococcus pyogenes. There
will be a clearing around colonies grown on
BAP due to the hemolysis of red blood cells (ß
hemolysis) in the media.
The microorganism that causes strep throat,
Streptococcus pyogenes, will grow on BLOOD
AGAR. Blood agar meets its growth
requirements and allows you to see a type of
hemolysis typical of this bacterium.
“If the culture grew Streptococcus
pyogenes after overnight incubation, would
you expect to see hemolysis?”
- YES, If the microorganism is S.
pyogenes you will expect to see Beta
hemolysis.
The culture media used to isolate
microorganisms from stool specimens can be
both selective and differential. Stools are
routinely inoculated onto BAP and the
following media:
•MAC
•SMAC
•CAMPY
•GN Broth/SF Broth
•HE/XLD
•SS
•CHROMagar for Enteroccoccus or
CHROMagar for E. coli O157
Note:  TCBS may be used for testing
gastrointestinal specimens, however, it was
not an option in this case scenario
as Vibrio spp. was not suspected.
MacConkey Agar (MAC)
• a common selective and differential media
•used for processing various specimen types
where Gram negative rods are a suspected
potential pathogen.
In a stool culture, there will be normal flora
present, but potential pathogens as well. You
will need to rule out normal flora from
pathogens.
Mac-Sorbitol for O157 (SMAC)
Xylose Lysine Deoxycholate Agar (XLD)
• a selective and differential media
•is a selective and differential media
• a modification of MacConkey agar where
the carbohydrate lactose is replaced by •is used for the isolation of Salmonella and
sorbitol Shigella species from clinical and food
samples
• one of several media commonly used to
•Gram positive microorganisms are inhibited
isolate E.coli O157 from stool
by the sodium deoxycholate in the media

•allows colonies of lactose fermenters such as

Campylobacter Agar (CAMPY) E.coli to be yellow

•one of several culture media used to isolate •non-lactose fermenters such as, Salmonella
Campylobacter spp. from stool specimens and Shigella, will be clear

•Campylobacter spp. are microaerophilic •those species of Salmonella that produce

H2S, the colony will be black
•Campylobacter spp. colonies will appear as
gray, glistening colonies
Salmonella-Shigella Agar (SS)

Hektoen Enteric Agar (HE) • is a selective media for Salmonella and

Shigella
• is a selective and differential culture media
• other microorganisms are inhibited by
• uses bile salts and dyes (e.g. bromthymol brilliant green and bile salts
blue or acid fuschin) to allow Gram negative
• lactose fermenting microorganisms will have
enteric microorganisms to grow
pink or red colonies
•is used to isolate Salmonella and Shigella • non-lactose fermenting microorganisms
•allows lactose fermenting enteric
such as Salmonella or Shigella will be colorless
microorganisms to grow and produce
salmon to yellow-orange colored colonies •some species of Salmonella produce H 2S and
will turn the colony black in the center
• Salmonella and Shigella are non-lactose
fermenters and the colonies can appear
colorless or green from retaining the color of CHROMagar
the culture media or dye
• Differential and selective media agar
• if the Salmonella sp. is a hydrogen sulfide
(H2S) producer, the colonies will appear clear • designed to identify certain/specific
or green with black centers. pathogens easier and faster when culturing
specimens.
Note: some laboratories will use either HE or
•Composition/ingredients of the media are
XLD in their laboratory set-ups for stool
relevant to the suspected microorganism
specimens.
being isolated.

•Some microorganisms will produce a

characteristic color on CHROMagar with
different colors being positive dependent on
the media company used.
The media to isolate MRSA is CHROMagar
Gram Negative (GN) Broth for MRSA

•is a selective liquid media used for the
isolation of Gram negative enteric
microorganisms
•contains sodium citrate as an inhibitory
agent for other microorganisms
Some microorganisms will produce a
characteristic color (mauve, pink, blue) on
CHROMagar with different colors being
positive dependent on the media company
used.
Examples:

Selenite F Broth CHROMagar for yeast (Candida)

CHROMagar for STEC

- Selenite F broth is another broth, similiar
to GN broth, that can be used to isolate
suspicious pathogens from a stool
sample.
Thiosulfate Citrate Bile Salts Sucrose
(TCBS)
TCBS may be used as a selective media to
isolate the stool pathogen, Vibrio spp. when
requested.
•Vibrio requires high salt content for growth.
CHROMagar for KPC
“You receive a C-CNA agar plate containing an
isolate with a positive Gram stain result for
Gram positive microorganisms.  No other
information is given.  What  culture media
should you use?”
- Blood agar (BAP) is an enrichment and
differential agar and will tell you if the
microorganism produces any hemolysis.

•if a species of Vibrio is able to ferment

sucrose, they will produce yellow colonies
•if a particular species does not ferment
sucrose, the colonies will be green
“Your GN broth is showing turbid growth and
you need to subculture it to see what is
growing.  What culture media should you use
to isolate the microorganism(s)?”

- MacConkey, XLD, SS, and HE culture

To subculture an isolate from an STD clinic media’s should be used to isolate enteric
you would use Blood agar (BAP),in addition microorganisms
to Thayer Martin (TM) or Martin Lewis agar.

Characteristics

•are enriched and selective media

•are used to isolate Neisseria gonorrhoeae

from clinical specimens known to contain
normal flora

•contains bovine hemoglobin as a source of

X Factor. V Factor is added as a supplement
(e.g. IsoVitaleX)

Culture Media Quality Control (QC)
To ensure proper performance, all culture
media must pass quality control (QC)
measures prior to use. Inspect all media upon
receipt and before use for the following:
•Evidence of inappropriate storage
conditions, such as freezing or desiccation
•Problems in transport such as cracks in the
media or plastic
•Evidence of poor quality, such as bubbles,
uneven fill or contamination
If any issues are observed, report them
immediately to the manufacturer and do not
use. Document date of receipt or preparation
(if made in-house), lot numbers, and
expiration dates for your records. Refer to
appropriate Clinical Laboratory Improvement
Amendments (CLIA) regulations and Clinical
and Laboratory Standards Institute (CLSI)
M22-A3 guidelines to determine if user QC is
required as well as any in-house laboratory
guidelines.
Good Laboratory Practices
When working in a microbiology laboratory, it
is important to use good laboratory practices.
Good laboratory practices include:
•Clean your workspace with the appropriate
disinfectant before and after each laboratory
session.
•Use sterile or disposable loops, needles and
pipettes.
•Keep cultures closed when possible and
replace caps and lids on cultures as soon as
possible.
•Do not lay caps or lids on the benchtop.
•Minimize risk of creating aerosols.
•When opening specimens or cultures, keep
the sample away from your face.
•Wear gloves to minimize contamination.
Note: Proficiency in demonstration of your
laboratory’s specific practices should be a part
of your laboratory’s guidelines or safety
manuals.
“In performing quality control on media, what
important information must be documented
for your records?”
- You should record the date the media
was prepared or received, the lot
numbers, and the expiration dates just in
case you have problems later on.
“When applying aseptic technique when
working with tube media, you should?”
- Clean workspace with disinfectant before
and after work, a clean workspace and
the use of a BSL-3 safety cabinet will help
to prevent contamination.
Inoculation of Media
Inoculation of culture media is an important
basic laboratory skill to possess as a
microbiologist.
When you receive a specimen or isolate in
the laboratory, you choose the media on
which to plate the specimen or isolate. The
next step is to inoculate the appropriate
culture media to isolate colonies.
Streaking for isolation is the ability to obtain
individual colonies on a culture medium plate
after inoculating the medium with a
specimen. This must be completed before
you can perform the necessary tests to
identify the infectious microorganisms.
Note: sometimes it may be necessary to
subculture growing broth media or isolated
colonies. This will be discussed further in this
module.
Pre-Inoculation Steps
Before you inoculate tube media or plate
media, there are certain steps that must be
followed:
1. Organize and label the media with the
appropriate identifiers such as culture
number, isolate number, and date.
2. Check to make sure the media is within
the expiration date and does not appear to
be contaminated.
Tube Media Inoculation Procedure
To inoculate a specimen to a tube of liquid
media (e.g.GN broth), you will find the steps
for inoculating tube media below:
1. Pre-loosen the cap(s).
2. Hold the specimen (e.g., swab, loop or
pipette) in one hand.
3. Pick up the tube to inoculate with the
other hand.
4. Use your little finger of the hand holding
the inoculum/specimen to remove the cap of
the tube.
5. Place the inoculum/specimen into the
tube according to the protocol for that media
and replace the cap.
6. Incubate according to protocol.
Streaking Culture Media
The purpose of streaking a agar plate is to
obtain isolated colonies from a specimen.
Each isolated colony represents several
thousand microorganisms which were
derived from a single mother cell.
When inoculating an agar plate from a single
colony, the colonies that grow are clones
from that single cell.
Quadrant Streaking
There are several techniques available for
streaking a plate. The most commonly used
method is quadrant streaking. Obtaining
isolated colonies can be achieved by
performing a four quadrant streak that
results in a dilution of the inoculum from one
quadrant to the next (with quadrant 1 being
the heaviest inoculum and quadrant 4 being
the lightest).
Quadrant Streaking Procedure
1. Inoculate the plate by placing a specimen,
on the first quadrant. If you have a swab
sample, touch the swab to the first quadrant.
2. Inoculate the first quadrant of the new
plate by making a one inch streak down the
first quadrant from the outside edge, then
streak back and forth across this inoculum.
3. Turn the plate a quarter turn.
4. Use a sterile loop to streak the second
quadrant by going through the edge of the
first quadrant approximately four times and
then continue streaking the second quadrant
without going back into the first quadrant
again.
5. Turn the plate another quarter turn.
6. Use a sterile loop and repeat the process
above but only go into the second quadrant
two or three times and complete the third
quadrant streak.
7. Turn the plate another quarter turn.
8. Use a sterile loop and streak the fourth
quadrant the same as the third but let the
streaks get smaller and trail off.
9. Replace the lid on the plate and incubate
the plates upside down (to decrease
condensation) according to your laboratory’s
testing protocol.
Subculturing from an Isolated Colony
If you need to obtain more growth of a
particular colony type, you will need to
subculture that colony. Subculturing is
performed in the same manner as quadrant
streaking, however, your specimen is an
isolated colony from the primary media
culture plate.
Incubation of Culture Media Plates
After culture media plates have been streaked
for isolation, the plates are incubated based
on environmental conditions that
microorganisms grow best in/on. Incubation
time for most organisms is 18 to 24 hours.
Incubation Notes:
•Special conditions must be established in
the laboratory, such as anaerobic chambers
or jars, to grow anaerobic microorganisms.
•There is a group of anaerobes that can grow
in the presence of oxygen, but grow best in
anaerobic conditions. These microorganisms
are called facultative anaerobes.
Staphylococcus spp. are facultative
anaerobes.
•Capnophiles are microorganisms that must
have 5-10% CO2 to survive, such as
Haemophilus influenzae.
•Campylobacter species are microaerophillic
bacteria that require a microaerobic
environment for isolation from clinical
specimens.
Problems Associated with Streaking
Plates
There may be problems that occur with
culture media and the testing procedure.  
Let’s work through some potential common
problems or issues.
Troubleshooting Problem 1
You remove blood agar plates from the
refrigerator and immediately inoculated
them. After incubation, this photo shows what
you observed. What has happened?
Ans: Allow the plates to come to room
temperature before inoculation.
Troubleshooting Problem 2
You begin streaking a plate, but forget to
flame the metal loop beforehand
Ans: If you have some of the specimen left,
reinoculate using new plates.
If you do not have any specimen left, you will
have to request another specimen and report
that a laboratory error has occurred.
Troubleshooting Problem 3
You flame the metal loop, but forgot to wait
to start streaking the plate. The colonies
burn.
Ans: If you burned the colonies in one area of
the streak, find another area to streak from.
Do this only after you have flamed the loop
and allowed the loop to cool.
Note: not allowing the loop to cool
completely and touching the media is a
frequent cause of aerosol creation.
 - Let them come to room temperature
before you use them

Troubleshooting Problem 4 “You streaked and incubated a blood agar

Streaking half of a blood agar plate resulting
in less surface area for a four quadrant
streaking.
Or
First and second quadrants were not streaked
closely together so there is not enough space
to allow for appropriate dilution.
Ans: Ensure appropriate amount of inoculum
is added to the plate and each quadrant is
used fully.
Troubleshooting Problem 5
Not using a sterile loop for each quadrant can
result in heavy carryover from one quadrant
to the next.
Ans: Ensure use of sterile loops for each
quadrant. Do not rush the streaking process. If
using a metal loop and incinerator, sterilize
between quadrants or use a new sterile
disposable loop until you have mastered
streaking for isolation with one or two
disposable loops.
plate. When you remove the plate from the
incubator, you observe heavy carryover from
one quadrant to another.  Could this be due
to not replacing or flaming the loop between
quadrants?”
- Yes, not using a sterile loop for each
quadrant can result in heavy carryover
from one quadrant to the next.
Summary
Culture media comes in three forms: solid,
liquid, and semisolid. Media is also classified
into three main types: enrichment, selective,
and differential.
The correct culture media to use for isolation
is based on the specimen type and/or Gram
stain results.
Streaking a culture media plate for isolation,
whether it is a primary culture or from a
subculture, is a critical microbiology technique
that should be mastered.

Troubleshooting Problem 6

When streaking the fourth quadrant, one of

the streaks runs into the first quadrant which
brings microorganisms from quadrant one
into quadrant four.

Ans: Ensure accuracy by staying within the

boundaries of each quadrant.

“You have taken a bag of MacConkey plates

out of the refrigerator and after you open
them you notice moisture on the surface of
the plate.  What do you do?”