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.