SELECTIVE MEDIUM

HEKTOEN ENTERIC AGAR

is a selective and differential medium used for the isolation and

differentiation of gram-negative enteric pathogens.
SUMMARY

King and Metzger developed HE Agar in an effort to increase the

recovery of Salmonella and Shigella species over the previously formulated
Salmonella-Shigella (SS) Agar.(7) This medium is particularly useful in the
isolation of Shigella species.

The present formulation of HE Agar incorporates larger amounts of

peptone in order to offset the inhibitory effect of bile salts. Also, sodium
deoxycholate has been eliminated and the amount of bile salts reduced. Bile
salts allow for the selective nature of HE Agar by inhibiting gram-positive
organisms. Bile salts can also be toxic for some gram-negative strains.
Salicin, sucrose, and lactose are the fermentable carbohydrates present.
They provide optimal differentiation of enteric pathogens. Lactose and
sucrose, in increased concentration, aid in the differentiation of enteric
pathogens from slow lactose fermenters. Bromothymol blue and acid fuchsin
(Andrade's) are added as acid-base indicators. The addition of ferric
ammonium citrate and sodium thiosulfate enable the detection of H 2S, noted
by the production of black centered colonies. Sodium thiosulfate serves as
the sulfur source while ferric ammonium citrate serves as the indicator.

HE Agar is currently recommended as one of several plating media for

the culture of Enterobacteriaceae from stool specimens. This is due to its
moderately selective nature as well as for its differentiation property.

FORMULA

Ingredients per liter of deionized water:

Peptic Digest of Animal Tissue 12.0gm

Lactose 12.0gm
Sucrose 12.0gm
Bile Salts 9.0gm
Sodium Chloride 5.0gm
Sodium Thiosulfate 5.0gm
Yeast Extract 3.0gm
Salicin 2.0gm
Ferric Ammonium Citrate 1.5gm
Acid Fuchsin 0.1gm
Bromothymol Blue 0.064gm
Agar 13.5gm

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8C. Products should not be used if there are
any signs of contamination, deterioration, or if the expiration date has
passed. Product is light and temperature sensitive; protect from light,
excessive heat, moisture, and freezing.

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the

laboratory without delay and protected from excessive heat and cold. If there
is to be a delay in processing, the specimen should be inoculated onto an
appropriate transport medium and refrigerated until inoculation. Consult
listed references for information on specimen collection. (1-6)

Method of Use: Medium should be brought to room temperature prior to

inoculation. If material is being cultured directly from a swab, roll the swab
over a small area of the surface edge. Streak the inoculum to obtain isolated
colonies. A nonselective medium should also be inoculated. This increases
the chance of recovery when the population of gram-negative organisms is
low. It also provides indication of other organisms present in the specimen.
Incubate plates for 18-24 hours at 35C. protected from light. If negative
after 24 hours, reincubate for an additional 24 hours.

PHYSICAL APPEARANCE - Hektoen Enteric (HE) Agar should appear clear,

and green in color.

Shigella flexneri (ATCC 12022) colonies

growing on Hektoen Enteric Agar (Cat. no.
G63). Incubated aerobically for 24 hours
at 35C.

Salmonella enterica (ATCC 14028)

colonies growing on Hektoen Enteric Agar
(Cat no. G63). Incubated aerobically for
24 hours at 35C.
Enterococcus faecalis (ATCC 29212)
growth inhibited on Hektoen Enteric Agar
(Cat no. G63). Incubated aerobically for
BAIRD PARKER AGAR

is a selective medium for the detection and enumeration of coagulase-

positive staphylococci from food samples.
SUMMARY

Baird-Parker medium was developed by Baird-Parker to isolate and

enumerate coagulase-positive staphylococci from foods. The present
formulation, by Baird-Parker, is a modification from a previous formula
developed by Zebovitz, Evan, and Niven Sodium pyruvate was added as a
selective growth stimulant, and egg yolk emulsion as a differentiation agent.
The medium allows growth of Staphylococcus aureus and selectively inhibits
growth of most other bacteria.

Selective inhibition is thought to be caused by the combination of the

selective agents tellurite and lithium. Glycine and pyruvate are added to
enhance the growth of staphylococci.

After 24 to 48 hours of incubation at 35-37C., colonies of S. aureus will

appear black, convex, shiny, and 1-1.5mm in diameter. The very distinct
black colonies formed by S. aureus are a result of the reduction of tellurite in
the medium. These colonies will normally be surrounded by clear zones, a
result of proteolysis or lipolysis. Occasionally, opaque zones will form within
this clear zone, a result of lipase or lecithinase activity. Other bacteria which
may grow on this medium are easily distinguished from S. aureus , as they
do not form black colonies.

FORMULA

Ingredients per 940ml of deionized water:

Glycine 12.0gm
Pancreatic Digest of Casein 10.0gm
Sodium Pyruvate 10.0gm
Beef Extract 5.0gm
Lithium Chloride 5.0gm
Yeast Extract 1.0gm
Egg Yolk Tellurite Enrichment 60.0ml
Agar 20.0gm

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8C. away from direct light. Media should
not be used if there are any signs of deterioration (shrinking, cracking, or
discoloration), hemolysis, contamination, or if the expiration date has
passed. Product is light and temperature sensitive; protect from light,
excessive heat and freezing.

PROCEDURES

Specimen Collection: Infectious material should be submitted directly to the

laboratory without delay and protected from excessive heat and cold. If there is to
be a delay in processing, the specimen should be inoculated onto an appropriate
transport media and refrigerated until inoculation. Consult listed references for
information on specimen collection. (1-4)

Method of Use: The plates should be warmed to room temperature and the agar
surface should be dry before inoculating. Inoculate the specimen onto the media as
soon as possible after it is received in the laboratory. If the material is being
cultured from a swab, roll the swab over a small area of the agar surface and streak
for isolation. Incubate plates aerobically at 35-37C. for 24 to 48 hours. Observe
plates for characteristic colonial morphology and color at 24 hours. If negative for
staphylococci, reincubate for an additional 24 hours and read again.

PHYSICAL APPEARANCE - Baird-Parker Agar should appear opaque, and light yellow
in color with no precipitate, chips or debris.

Staphylococcus aureus (ATCC 25923)

colonies growing on Baird-Parker Agar
(Cat. no. G96). Incubated aerobically for
24 hours at 35C.

Proteus mirabilis (ATCC 12453) colonies

Escherichia coli (ATCC 25922) growth
growing on Baird-Parker Agar (Cat. inhibited
no. on Baird-Parker Agar (Cat. no.
G96). Incubated aerobically for 24 hours
G96). Incubated aerobically for 24 hours
EMB AGAR (EOSIN METHYLENE BLUE)

recommended for use as selective and differential media for the isolation of
gram-negative bacilli (including coliform organisms and enteric pathogens)
from clinical and nonclinical specimens.
SUMMARY
EMB (Eosin Methylene Blue) Agar (HHT) was developed by Holt-Harris and
Teague as an alternative to Endo's medium for the isolation of enteric bacilli. Eosin
dye and methylene blue were employed to inhibit the growth of gram-positive
bacteria; the dyes also serve as differential indicators for the products of
fermentation. Lactose and sucrose provide a carbohydrate source. The production of
acid from lactose- or sucrose-fermentation results in the eosin-methylene blue dye
complex being taken up by bacterial cells to produce a brown to blue-black colony
appearance. The HHT formulation provides a clear distinction between colonies of
lactose and non-lactose-fermenting microorganisms. However, this formulation does
not discriminate between carbohydrate utilization (lactose or sucrose). For example,
Yersinia enterocolitica ferments sucrose and not lactose, but will produce the same
blue-black colonies as lactose-fermenters.
In 1918, Levine simplified the Holt-Harris and Teague formulation by omitting
sucrose and doubling the quantity of lactose. The modification facilitated the
differentiation of Escherichia coli from Enterobacter aerogenes, and paralleled the
reactions of MacConkey Agar for better colony identification.
 Historically, EMB Agar, Levine has become the predominant formulation for
detecting fecal and non-fecal coliforms. The American Public Health Association
recommends its use in the microbiological examination of potable water, waste
water, dairy products and foods. (1,11) The USP recommends its use in the
performance of Microbial Limit Tests, and the U.S. Food and Drug Administration
(FDA) recommends the medium for enumerating Escherichia coli and coliform
bacteria.
FORMULA
Ingredients per liter of deionized water:

EMB Agar, Levine:

Pancreatic Digest of Gelatin 10.0gm
Lactose 10.0gm
Dipotassium Phosphate 2.0gm
Eosin Y 0.4gm
Methylene Blue 65.0mg
Agar 15.0gm

EMB Agar (HHT):

Pancreatic Digest of Gelatin 10.0gm
Lactose 5.0gm
Sucrose 5.0gm
Dipotassium Phosphate 2.0gm
Eosin Y 0.4gm
Methylene Blue 65.0mg
Agar 13.5gm

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8C away from direct light. Media should not be
used if there are any signs of deterioration (shrinking, cracking, or discoloration),
contamination, or if the expiration date has passed. Product is light and
temperature sensitive; protect from light, excessive heat, moisture, and freezing.
PROCEDURE
Specimen Collection: Consult listed references for information on specimen
collection. Infectious material should be submitted directly to the laboratory without
delay and protected from excessive heat and cold. If there is to be a delay in
processing, the specimen should be inoculated onto an appropriate transport media
and refrigerated until inoculation.
Method of Use: Allow plates to warm to room temperature. The agar surface should
be dry before inoculating. Inoculate and streak the specimen as soon as possible
after collection. If the specimen to be cultured is on a swab, roll the swab over a
small area of the agar surface and streak for isolation with a sterile loop. Incubate
plates aerobically at 35-37C for 18-24 hours and protect from light. Examine plates
for colonial morphology. If negative after 24 hours, reincubate an additional 24
hours.

PHYSICAL APPEARANCE - EMB Agar, Levine should appear clear, and purple with a
green-orange tinge in color. A slight precipitate is also apparent. EMB Agar (HHT) should
appear clear to slightly hazy, and dark red to bluish-purple in color.

Escherichia coli (ATCC 25922) colonies

Salmonella enterica (ATCC 14028)
growing on EMB Agar, Levine. Incubated
colonies growing on EMB Agar, Levine.
aerobically for 24 hours at 35C. Incubated
Shot at aerobically for 24 hours at
an angle to show green sheen. 35C.

PHENYLETHYL ALCOHOL AGAR (PEA AGAR)

recommended for use as an enriched and selective medium for the

cultivation and selective isolation of gram-positive and negative obligate
anaerobic bacteria. It is useful in isolating obligate anaerobes from mixed
flora, by inhibiting gram-negative facultative anaerobes and the control of
swarming organisms.
SUMMARY
PEA should be inoculated for purulent specimens and when mixed infections
are suspected. PEA inhibits facultative gram-negative rods, preventing
Enterobacteriaceae from overgrowing the anaerobes, and inhibits swarming of
Proteus. It also prevents certain clostridia, such as a C. septicum, from swarming,
which will facilitate the isolation of other colonies. Most gram-negative and gram-
positive anaerobes will grow on primary PEA medium, especially in mixed culture,
and the morphology of the colonies is similar to that on blood agar plates; however,
a longer incubation time may be necessary to detect the more slowly growing and
pigmented anaerobes. However, pigment develops more rapidly than on non-
selective Brucella Agar with H and K.
 The basal medium of Anaerobic PEA consists of pancreatic digest of casein
and enzymatic digest of soybean meal. Both provide amino acids, carbohydrates,
and vitamins. The medium is supplemented with yeast extract, sheep blood, vitamin
K, hemin, and L-cystine. Yeast extract enhances growth of fastidious
microorganisms; sheep blood demonstrates hemolysis; vitamin K promotes growth
and enhancement of pigmented Prevotella spp.; hemin and L-cystine improve the
growth of Clostridium haemolyticum, Fusobacterium necrophorum, certain strains of
Actinomyces israelii, Bacteroides thetaiotaomicron, and thiol-dependent
streptococci.The medium is made selective with the addition of phenylethyl alcohol
which inhibits facultative anaerobic gram-negative bacilli.
AnaeroGRO Anaerobic PEA is packaged under oxygen-free conditions to
prevent the formation of toxic oxidized products that may damage obligate
anaerobes and inhibit the growth of more fastidious species.
Culture media that is exposed to environmental oxygen leads to a build-up of
reactive oxygen species (ROS) that initiate damaging free radical reactions, which
inhibit the growth of anaerobic bacteria. Therefore, ingredients have been added to
the AnaeroGRO media to neutralize the growth inhibiting effects of peroxide and
other reactive oxygen species (ROS) that may develop during the medium's brief
exposure to oxygen after it is sterilized and before it is packaged in an oxygen-free
environment.

FORMULA
Ingredients per liter of deionized water:

Pancreatic and Enzymatic Digests of Casein 15.0gm

Papaic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Yeast Extract 5.0gm
Phenylethanol 2.5gm
Reducing Agents/Peroxide Inhibitors 1.5gm
Vitamin K 10.0mg
Hemin 5.0mg
L-Cystine 0.4mg
Sheep Blood 50.0ml
Agar 15.0gm

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 15-30C. away from direct light. Media should not be
used if there are any signs of deterioration (shrinking, cracking, or discoloration),
hemolysis, contamination, or if the expiration date has passed. Product is light and
temperature sensitive; protect from light, excessive heat and freezing.
 The plates must be inoculated immediately after opening the AnaeroGRO
pouch. After inoculation, the plates must be placed immediately into an anaerobic
atmosphere (pouch, jar, or chamber) to ensure optimal growth of anaerobic
bacteria.

PROCEDURE
Specimen Collection: Consult listed references for information on specimen
collection.(1,2,4,6,9,10) It is not recommended that a swab be used for specimen
collection. Swabs are prone to drying and may be easily exposed to ambient air. The
preferred means of anaerobic specimen collection is aspiration with needle and
syringe. The specimen should be transferred to an anaerobic transport system (Cat.
no. S120D) in order to protect it from oxygen exposure. Infectious material should
be submitted directly to the laboratory without delay and protected from excessive
heat and cold.
Method of Use: Open pouch and immediately inoculate and streak the specimen for
isolated colonies. A large inoculum should be used in order to minimize the toxic
effects of oxygen.(8) Incubate plates at 35-37C. for 18-48 hours in an anaerobic
atmosphere. Examine for typical colonial morphology and characteristics.
A non-selective medium such as Brucella with H and K (Cat. no. A30) should be
inoculated in parallel to the selective medium for enhanced recovery of anaerobic
microorganisms.
Aerotolerance Testing: Confirmation of obligate anaerobic microorganisms should be
performed. A Chocolate Agar plate (Cat. no. E14) incubated in 5-10% CO 2 is required
for aerotolerance testing to detect isolates that require CO 2, especially slow-
growing, fastidious, facultative or microaerophilic species that do not grow alone on
media containing blood (such as Haemophilus and Actinobacillus spp.). Use of
traditional blood agar media alone for CO2 incubation may yield false-negative
results. An additional Blood Agar plate (Cat. no. A10) incubated in air will further
detail the atmospheric requirements and hemolytic properties of facultatively
anaerobic microorganisms.

PHYSICAL APPEARANCE - AnaeroGRO Anaerobic PEA should appear opaque, and

dark red in color.

Bacteroides fragilis (ATCC 25285)

colonies growing on AnaeroGRO
Anaerobic PEA Agar. Incubated
anaerobically for 24 hours at 35C.

Proteus mirabilis (ATCC 12453) partial to Clostridium perfringens (ATCC 13124)

complete inhibition on AnaeroGRO colonies growing on AnaeroGRO
Anaerobic PEA Agar. Incubated Anaerobic PEA Agar (Cat. no. AG901).
XYLOSE LYSINE DESOXYCHOLATE (XLD)

used as a selective and differential medium for the isolation of gram-negative

enteric pathogens from fecal specimens or from food and other samples.

SUMMARY
Xylose Lysine Deoxycholate (XLD) Agar was developed by Taylor for the
differentiation, isolation, and identification of enteric pathogens, and to support the
growth of more fastidious enteric organisms. XLD Agar was especially designed to
allow the growth of Shigella species, and is a proven medium for the isolation of this
organism. It has also been found to be an excellent medium for isolating Salmonella
species as well.
The selective agent in XLD Agar is sodium deoxycholate, which inhibits the
growth of gram-positive organisms. The carbohydrate source is xylose which is
fermented by most enterics except for Shigella species, and these colonies appear
red on this medium as a result. A second differential mechanism for Salmonella is
employed by the addition of lysine. Lysine decarboxylation reverts the pH of the
medium to an alkaline condition. To avoid this reversal to a Shigella reaction, lactose
and sucrose are added in excess. The addition of sodium thiosulfate and ferric
ammonium citrate as a sulfur source and indicator, respectively, allows hydrogen
sulfide forming organisms to produce colonies with black centers, under alkaline
conditions. Organisms which ferment xylose, are lysine decarboxylase-negative, and
do not ferment lactose or sucrose cause an acid pH in the medium, and form yellow
colonies. Examples of such organisms are Citrobacter spp., Proteus spp., and
Escherichia coli.
XLD Agar with Novobiocin contains novobiocin, which is commonly used to
inhibit the growth of Proteus spp., and helps reduce the potential for false-positives
from this organism.

FORMULA
Ingredients per liter of deionized water:

Lactose 7.5gm
Sucrose 7.5gm
Sodium Thiosulfate 6.8gm
L-Lysine 5.0gm
Sodium Chloride 5.0gm
Xylose 3.75gm
Yeast Extract 3.0gm
Sodium Deoxycholate 2.5gm
Ferric Ammonium Citrate 0.8gm
Phenol Red 0.08gm
Agar 15.0gm
Novobiocin 10.0mg/mL

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8C. away from direct light. Media should not be
used if there are any signs of deterioration (shrinking, cracking, or discoloration),
contamination, or if the expiration date has passed. Product is light and
temperature sensitive; protect from light, excessive heat, moisture, and freezing.
PROCEDURE
Specimen Collection: Consult listed references for information on specimen
collection. Infectious material should be submitted directly to the laboratory without
delay and protected from excessive heat and cold. If there is to be a delay in
processing, the specimen should be inoculated onto an appropriate transport media
and refrigerated until inoculation.
Method of Use: Allow the plates to warm to room temperature, and the agar surface
to dry before inoculating. Inoculate and streak the specimen as soon as possible
after collection. If the specimen to be cultured is on a swab, roll the swab over a
small area of the agar surface. Streak for isolation with a sterile loop. Incubate
plates aerobically at 35-37C. for 18-24 hours. Examine colonial morphology,
characteristics, and hemolytic reactions.
It is recommended that selective enrichment broths, such as GN Broth or Selenite
Cystine Broth (Cat. no. K39, or K69, respectively), be used in conjunction with other
selective plating medias, such as HE Agar (Cat. no. G63). This recommendation is
made in order to maximize the recovery of enteric pathogens.

PHYSICAL APPEARANCE - XLD Agar should appear a clear, and red in color, and may
have a slight precipitate.

Salmonella enterica (ATCC 14028)

colonies growing on XLD Agar. Incubated
aerobically for 24 hours at 35C.

Shigella flexneri (ATCC 12022) colonies

growing on XLD Agar. Incubated
aerobically for 24 hours at 35C.