TYPES OF AGAR CULTURE MEDIA

I. Blood Agar:
(Enriched, Differential, but NOT Selective)

Blood agar media are non-selective media, good

for culturing many bacterial species including Gram-
negative and Gram-positive species (such as Staphylo-
cocci and Streptococci). Blood agar media contain anti-
coagulated mammalian blood (usually that of sheep or
horse), typically at a concentration of 5-10%. These media
are enriched, differential media used to isolate fastidious
organisms and detect their hemolytic activity.

Hemolysis on Blood Agar:

Hemolysis on blood agar is used for the preliminary or confirmatory iden-

tification of many types of clinically important bacteria. While it is factored into the
differential diagnosis of a specific infectious agent, hemolysis type is not specific
enough to be a final diagnosis criterion. The three hemolysis conditions continue to be
described by terms that are somewhat confusing.

1) Alpha-hemolysis: This will only partially lyse hemoglobin and will appear
green. I.e. alpha-hemolysis appears as a greenish discoloration of the blood agar
surrounding a bacterial colony. It is a characteristic of Streptococcus viridans and
Streptococcus pneumoniae.

2) Beta-hemolysis: Beta-hemolytic
activity will show complete lysis of red blood
cells surround-ing colony. I.e. it appears as a
clear zone in the blood agar in the area
surrounding a bacterial colony. It is a charac-
teristic of Streptococcus haemolyticus, Strep-
tococcus pyogenes as well as some strains of
Staphylococcus aureus.

3) Gamma-hemolysis (or non-hemo-

lytic): Gamma-hemolysis is the term referring
to a lack of hemolytic activity in the area
surrounding a bacterial colony growing on blood agar. Gamma-hemolysis would
therefore describe bacterial growth that results in neither a greenish tinge to the
discoloration (alpha-hemolysis) nor a clear zone that the observer "could read a
newspaper through" (beta-hemolysis).

In fact, culture of bacteria on blood agar for the purpose of hemolysis clas-
sification is performed at 37oC in the presence of 5% CO2. This results in an overall
brownish discoloration of the blood agar, from its original blood-red hue.
 II. Chocolate agar (CHOC) - Boiled Blood Agar
(Enriched, but NOT Selective)

Chocolate agar is a type of blood agar in

which the blood cells have been lysed by heating
them to 56 °C. It is a non-selective enriched gro-
wth medium used for growing fastidious respire-
tory bacteria, such as Haemophilus influenzae.
These bacteria need growth factors, like NAD and
hematin, which are inside red blood cells; thus, a
prerequisite to growth is lysis of the red blood
cells. No chocolate is actually contained in the
chocolate agar plate; it is just named for its color-
ation, since chocolate agar plates usually appear as a light brown/milk chocolate
colored. Chocolate agar has the same composition as blood agar; except that the red
blood cells in chocolate agar have been heated until they are lysed, producing the cha-
racteristic brown color of this medium.

III. Thayer-Martin agar (TM)

(Only Selective)

Thayer-Martin agar (Thayer-Martin

medium) is a chocolate agar designated to
isolate Neisseria bacteria. TM agar is a
Mueller-Hinton agar with 5% chocolate (lysed)
sheep blood and antibiotics. It is used for
culturing and primarily isolating Neisseria
bacteria, including Neisseria gonorrhoeae and
Neisseria meningitisdis, as the medium inhibits
the growth of most other microorganisms. When
growing Neisseria meningitidis, one usually
starts with a normally sterile body fluid (blood
or CSF), so a plain chocolate agar is used.

The opposite figure shows Neisseria

gonorrhoeae colonies growing on Thayer
Martin Agar plate. N. gonorrhoeae appears as
small, grayish-white to colorless mucoid colo-
nies. N. meningitidis forms similar colonies to
N. gonorrhoeae, but larger and blue-gray.

- Components of TM: Thayer-Martin

agar usually contains the following combination
of antibiotics called VCN inhibitor: (1) Vancomycin, which is able to kill all Gram-
positive organisms, except Lactobacillus and Pediococcus which are intrinsically resi-
stant; (2) Colistin, which is added to kill all Gram-negative organisms except Neiss-
eria; and (3) Nystatin, which can kill all fungi.
- Chocolate Agar Vs Thayer-Martin Agar: (V. Important)

** Comparison of two culture media types used to grow

Neisseria gonorrhoeae bacteria:-

Known as overgrowth, note that the non-selective chocolate agar medium on

the left, due to its composition, allowed for the growth of bacterial colonies other than
those of Neisseria gonorrhoeae, while the selective Thayer-Martin medium on the
right, containing antimicrobials that inhibit the growth of organisms other than N.
gonorrhoeae, shows no overgrowth, but is positive for N. gonorrhoeae bacteria.

Culture Media Used in the

Diagnosis of Enterobacteriaceae
Enterobacteriaceae (enterics) are facultative Gram-negative rod-shaped bacte-
ria that inhabits the intestinal tracts of humans and many animals. Enterics that can
ferment lactose (lactose positive) are called coliforms and usually are considered to
nonpathogenic in the intestinal tract (e.g. Escherichia coli, Enterobacter aero-genes).
(Note that in recent years certain strains of E. coli such as O157:H7 have been
implicated in causing bloody diarrhea.) The enteric pathogens Salmonella and
Shigella are unable to ferment lactose (lactose negative).

Two types of selective and differential media are commonly employed for
isolating and identifying Enterobacteriaceae; MacConkey agar and Eosin Methylene
Blue (EMB) agar. Lactose positive colonies are visibly different from lactose
negative colonies on both types of media.
 IV. MacConkey Agar (MAC)
(Both Selective and Differential)

MacConkey agar is both selective and differe-

ntial. It contains bile salts and the dye crystal violet,
which inhibit the growth of gram-positive bacteria and
select for gram-negative bacteria. It also contains the
carbohydrate lactose, which allows differentiation of
gram-negative bacteria based on their ability to ferment
lactose. A MAC plate can be used in isolating and
differentiating lactose-fermenting from lactose non-
fermenting gram negative enteric bacilli. Organisms
which ferment lactose produce acidic end-products
which react with the pH indicator neutral red, and produce a pink color.

- MAC chemical composition:

 10g lactose
 20g peptone
 5g bile salts
 "pinch" or small amount of 1mg/mL Bromcresol purple or Crystal violet solution.
 15g agar
 1L distilled water

The opposite figure shows a MAC

plate divided into 4 quadrates, each
inoculated with a different organism:

- Quadrant 1: Growth on the plate

indicates the organism, Enterobacter ae-
rogenes, is not inhibited by bile salts and
crystal violet and is a gram-negative
bacterium. The pink color of the bacter-
ial growth indicates E. aerogenes is able
to ferment lactose.

- Quadrant 2: Growth on the plate

indicates the organism, Escherichia coli, is not inhibited by bile salts and crystal
violet and is a gram-negative bacterium. The pink color of the bacterial growth
indicates E. coli is able to ferment lactose.

- Quadrant 3: Absence of growth indicates the organism, Staphylococcus epi-

dermidis, is inhibited by bile salts and crystal violet and is a gram-positive bacterium.

- Quadrant 4: Growth on the plate indicates the organism, Salmonella typhimurium,

is not inhibited by bile salts and crystal violet and is a gram-negative bacterium. The
absence of color in the bacterial growth indicates S. typhimurium is unable to ferment
lactose (i.e. non-lactose fermentor).
 V. Eosin Methylene Blue Agar (EMB)
(Both Selective and Differential)

Eosin Methylene Blue (EMB) agar is both selective and differential. It con-
tains the dyes eosin and methylene blue, which inhibit the growth of gram-positive
bacteria and therefore select for gram-negative bacteria. It also contains the carbo-
hydrate lactose, which allows differentiation of gram-negative bacteria based on their
ability to ferment lactose.

The opposite figure shows an EMB plate

divided into 4 quadrates, each inoculated
with a different organism:

- Quadrant 1: Growth on the plate indica-

tes the organism, Escherichia coli, is not in-
hibited by eosin and methylene blue and is a
gram-negative bacterium. The green met-
allic sheen indicates E. coli is able to fer-
ment lactose to produce strong acid end pro-
ducts.

- Quadrant 2: Growth on the plate indica-

tes the organism, Pseudomonas aeruginosa,
is not inhibited by eosin and methylene blue
and is a gram-negative bacterium. The
absence of color in the bacterial growth
indicates P. aeruginosa is unable to ferment
lactose (non-lactose fermentor).

- Quadrant 3: Growth on the plate indica-

tes the organism, Enterobacter aerogenes,
is not inhibited by eosin and methylene blue
and is a gram-negative bacterium. The
dark purple color of the bacterial growth
indicates E. aerogenes is able to ferment
lactose to produce weak acid end-products.

- Quadrant 4: Absence of growth indicates

the organism, Staphylococcus aureus, is
inhibited by eosin and methylene blue and is
a gram-positive bacterium.

** As shown in figures: Only gram-negative bacteria

grow on EMB agar. (Gram-positive bacteria are
inhibited by the dyes eosin and methylene blue added
to the agar). Based on its rate of lactose fermentation,
E. coli produces dark, blue-black colonies with a
metallic green sheen on EMB agar. Weaker fermen-
tation of lactose results in colonies with a pinkish-
purple color. Colonies of nonlactose fermenters remain colorless, or at least are no darker than the
color of the media.
 VI. Salmonella-Shigella Agar (SS)
(Both Selective and Differential)

Salmonella-Shigella (SS) agar is another selective and differential medium

used for stool culture. It is mainly used for the isolation of Salmonella and Shigella
from faeces, foodstuffs and other materials. Like MacConkey, this medium inhibits
the growth of gram-positive bacteria and differentiates lactose-positive from lactose-
negative gram-negative rods, where:

1. Lactose-negative colonies appear to be colorless (as in the case of

Shigella and some Salmonella species).

2. Lactose-positive colonies appear pink to red (as in the case of

Escherichia coli and Klebsiella pneumoniae).

3. In addition to that, production of

hyd-rogen sulfide H2S can be detected
when black-centered colonies are
produced (as in the case of Proteus and
most Salm-onella species).

VII. Mannitol Salt Agar

(MSA)
(Both Selective and
Differential)

Mannitol Salt Agar (MSA) is a

selective and differential medium. The
high concentration of salt (7.5%) selects
for members of the genus Staphylococcus,
since they can tolerate high saline levels.
Organisms from other genera may grow,
but they typically grow very weakly.

MSA also contains the sugar man-

nitol and the pH indicator phenol red. If
an organism can ferment mannitol, an aci-
dic byproduct is formed that will cause the
phenol red in the agar to turn yellow. Most pathogenic staphylococci, such as Sta-
phylococcus aureus, will ferment mannitol. Most non-pathogenic staphylococci will
not ferment mannitol. And as shown in figure - The Staphylococcus aureus ferments
mannitol and turns the medium yellow. The Serratia marcescens does not grow bec-
ause of the high salt content.

VIII. Thiosulfate Citrate Bile salts Sucrose Agar (TCBS)

(Both Selective and Differential)

TCBS Agar (Thiosulfate-Citrate-Bile

salts-Sucrose agar) is both differential and
selective plating medium for Vibrio adjusted
to pH 8.6 (alkaline); Vibrio cholerae colonies
appear yellow (since it is sucrose fermentor);
other Vibrio spp. colonies appear green.
 MOST COMMON MICROBIOLOGICAL
TESTS

I. Catalase Test

Catalase is an enzyme found in most bacteria. It catalyzes the breakdown of

hydrogen peroxide to release free oxygen.

2 H2O2  2 H2O + O2

Procedure:

Add one drop of H2O2 to a glass slide with a loopful of growth from each
culture to be tested. The development of an immediate froth of bubbles is indicative
of a positive catalase test. The test must be performed on a BLOOD-FREE medium
(in order to avoid false positive results since RBCs also contain the catalase enzyme).
Staphylococci and Micrococci are catalase-positive, whereas Streptococci and enter-
ococci are catalase-negative.

II. Oxidase Test

A positive oxidase reaction reflects the ability of a microorganism to oxidize

certain aromatic amines, such as tetramethyl-p-phenylene diamine (TPD), producing
colored end products. This is due to the activity of cytochrome oxidase (a.k.a., indo-
phenol oxidase) in the presence of atmospheric oxygen. One use of the test is for the
preliminary identification of Neisseria and Moraxella species, which are both oxidase
positive gram-negative diplococci. Enterobacteriaceae, on the other hand, are oxidase
negative.

Procedure:

Using a sterile wooden stick, remove 2-3 colonies from each culture to be
tested and smear on a piece of filter paper. Add a drop of the spot test (TPD) reagent
to each spot. If the organism has oxidase activity, it will turn purple within 30 sec-
onds.

III. Coagulase Test

The coagulase test is used to differentiate the potentially pathogenic species

Staphylococcus aureus from the usually non-pathogenic species Staphylococcus
epidermidis. The presence of coagulase results in the formation of a clot in a tube of
citrated platelet-rich plasma (~ >150 x 106 platelets/cc plasma). The citrate is an
anticoagulant that is added to avoid autoclotting.

Procedure:
 Add a generous loopful of the organism to be tested to a tube of citrated rabbit
plasma. Thoroughly homogenize the inoculum with the loop and incubate the tube at
37o C for one to four hours. Examine the tube at 30 minute to hourly intervals for the
first couple of hours for the presence of a clot by tipping the tube gently on its side. A
test that shows any degree of clotting within 24 hours is considered coagulase posit-
ive.

Reincubate the tube overnight to see if the clot subsequently lyses. In strains
that produce fibrinolysin, the clot will be slowly digested. This illustrates the impor-
tance of reading the coagulase results within 24 hours. Thereafter, the lack of clotting
could be a false negative reaction with a coagulase-positive strain.