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Introduction:

Culture media provide the chemical and physical environment necessary for bacterial growth. Different species of bacteria vary greatly in their nutritional requirements.

The basis for the media used in the study of the common bacteria is meat infusion broth, often referred to as nutrient broth. To an aqueous extract of meat, proteoses and amino acids are added in the form of commercial peptone (N source). The electrolyte content and buffer capacity of the medium may be adjusted by the addition of inorganic salts, particularly sodium chloride and phosphates. Media may be enriched by the addition of carbohydrates (C source), serum, whole blood, accessory growth factors (vitamins) and other substances. When solid media are desired, agar is added. Sterilization of the mixture when autoclaved (pressure of 15 lbs./sq. in., 120^oC, 15-20 min.) results in liquefaction of the agar; the mixture is then cooled to 41-42^oC. Heat labile constituents, such as serum, whole blood, vitamins, etc., may be added in sterile form as needed. The final mixture is poured into plates or tubes and allowed to solidify. Agar solidifies at temperatures below 40^oC, and cannot be reliquified without reheating to 100^oC, hence the necessity for adding sterile, heat-labile ingredients at 41-42^oC, the lowest temperature at which agar remains liquified.

Hydrogen Ion Concentration. It is essential that the hydrogen ion concentration of culture media be properly adjusted. The electrometric method is used for precise determination of pH but in many cases sufficiently accurate results may be obtained by colorimetric methods. The students will observe that indicators are added to many types of bacteriological media so that gross changes in pH, which accompany the growth of certain microorganisms, may be readily observed.

Alphabetical Listing of Culture Media and Biochemical Tests

Acid-fast stain. (AF) A complex waxy wall containing mycolic acid covalently linked to peptidoglycan limits accessibility of Gram stains, but carbol fuchsin (pink-red) stain, once infused into the wall won't come out upon treatment with ethyl alcohol containing 3% HCl. The "acid-alcohol" rinse decolorizes all bacteria readily except Mycobacterium and some Nocardia. Hence these organisms are termed "acid-fast". Acid-fast bacilli (AFB) are red, and other structures in the specimen are blue due to the blue counterstain employed. This property alone is sufficient to distinguish Mycobacterium and Nocardia from most other bacteria and is very useful for diagnosis: direct examination of sputum or exudates can demonstrate the presence of AFB and indicate the potential infectiousness of the patient. Procedure (Kinyoun carbolfuchsin method):

a. Place the fixed slide on a staining rack and flood the smear with the carbolfuchsin stain for 5 minutes.

 

b. Wash the slide under running water.

 

c. Decolorize to a faint pink with acid alcohol while continuously agitating the slide until no more stain comes off in the washings. Add more acid alcohol and leave the slide flat for approximately 30 seconds. Pour off and add more acid alcohol for approximately 30 seconds. Thoroughness in decolorization is essential in order to prevent the possibility of a false positive reading.

 

d. Wash the slide with water. Counterstain with methylene blue for 1 minute. Wash with water, blot dry, and examine under oil. Acid-fast bacteria are red; other organisms and most of the background are blue.

Bacitracin Susceptibility. This test helps distinguish Group A Streptococcus from other streptococci which are much less susceptible to this antibiotic. Procedure: Streak heavily a sector of a BAP (1/4-1/3) with the culture to be tested in order to obtain confluent growth. With sterile forceps lay a bacitracin disc (each containing 0.2 units) in the center of the inoculated sector. After 24 hours incubation "read the plate" to ascertain whether there is inhibition of growth around the disc.

Beta-lactamase production. A rapid test for beta-lactamase employs the Cefinase disc, which is impregnated with the chromogenic cephalosporin, Nitrocefin. This compound is yellow in its complete state but becomes red on cleavage of the beta-lactam ring. Therefore, when a bacterium produces a beta-lactamase in significant quantities, the yellow colored disc turns red on the area where the isolate is smeared within 5-10 minutes. A negative result will show no color change on the disc. Procedure: Moisten a Cefinase discs with 1 drop of tap water per disc. Then smear some of the organism being tested for beta-lactamase production on the disc.

Bile Solubility. This test helps distinguish S. pneumoniae from viridans streptococci. Only pneumococci are lysed when treated with bile or various other detergents, such as deoxycholate or dodecyl sulfate.

Bile-esculin slant. This medium is used for the presumptive identification of Group D streptococci. Group D enterococci yield a dark brown color at the slant within 24 hours, while non-enterococcus Group D streptococci produce this reaction only within 41 hours. Other streptococci grow but do not produce a color change. Only the surface is inoculated. Growth on this medium is largely limited to streptococci, since only they tolerate the bile present. The dark brown color is due to hydrolysis of esculin, which is 6,7-dihydroxycoumarin-6-glucoside.

Blood Agar. (Abb. = BAP) Blood agar is a standard type of medium used in the isolation and cultivation of most organisms including many fastidious pathogenic microorganisms. It consists of a beef-heart infusion, NaCl, a peptic digest of animal tissue, 1.5% agar, and defibrinated blood of sheep, horse, rabbit, or human. This consists of a nutrient agar base to which is added 5% sterile sheep blood. The blood not only provides additional nutrients but serves as an indicator of hemolytic reaction. In preparing this medium all the ingredients except the blood are autoclaved together. The sterile mixture is then cooled to 50^o C before adding sterile blood to a final concentration of 5% (V/V). Most bacteriology labs use sheep or sometimes human blood, but both are toxic to Haemophilus. However, blood agar plates prepared with rabbit or horse blood do support the growth of these fastidious organisms.

There are three types of hemolysis that occur on blood agar - alpha, beta, and gamma. Beta hemolytic organisms produce a wide clear zone of complete hemolysis in which no red cells are visible on microscopic examination. Alpha hemolytic colonies are surrounded by a narrower greenish zone of hemolysis with partially lysed red cells present. Gamma hemolysis actually constitutes no hemolysis at all. Surface colonies of beta hemolytic streptococci may appear alpha or gamma hemolytic. Two hemolysins are responsible for beta hemolytic activity in streptococci. These are differentiated on the basis of antigenicity and susceptibility to inactivation by oxidation. The antigen, oxygen-labile hemolysis is designated streptolysin O and nonantigenic, oxygen-stable hemolysin is designated streptolysin S. Therefore, surface colonies of the beta hemolytic streptococci may appear as alpha or nonhemolytic due to inactivation of the streptolysin O. To insure that the beta hemolytic streptococci are correctly identified, inoculated blood plates should be stabbed several times in the initial streak. The subsurface colonies will be under a reduced oxygen tension and therefore the streptolysin O will not be inactivated. Many gram-negative organisms will produce a greenish discoloration around colonies due to diffusion of growth by-products into the medium and not to an hemolysin. Hemolysis due to a specific hemolysin results in a definite zone of lysis around a colony.

Bordet-Gengou Agar. This medium is used for the isolation of Bordetella pertussis, the cause of pertussis (whooping cough). It contains 15-20% blood (3-4 times the amount in BAP) and an infusion from potatoes (the potato starch binds toxic fatty acids in the blood).

Brain Heart Infusion. Brain heart infusion is a liquid medium widely used for the cultivation of pathogenic cocci and other organisms generally considered difficult to grow. Virulence, antigenicity, and other serological characteristics are quite uniformly maintained when the organisms are grown on brain heart infusion. This medium is especially adapted for blood culture work. The addition of a small amount of agar (0.1-0.2%) to BHI is particularly recommended for the growth and isolation of pathogenic microorganisms, especially their primary isolation from blood and other specimen material. The flocculent agar develops variable degrees of anaerobiosis making the medium suitable for the growth of either aerobes or anaerobes.

Campylobacter Agar. Campylobacter (Campy) agar is a selective medium used to aid in the isolation of Campylobacter jejuni, coli and laridis from fecal specimens. The medium consists of Brucella agar base to which is added vancomycin, trimethoprim, polymyxin B, amphotericin B, and cephalothin. These agents inhibit the growth of normal stool flora. Sheep red blood cells (5%) are added for enrichment. Campy agar also contains sodium bisulfite which lowers the oxidation-reduction potential of the medium to enhance recovery of the microaerophilic Campylobacter organisms. The plates should be incubated at 42^oC, which allows good growth of Campylobacter but inhibits normal stool flora. Incubation in an anaerobic jar with active catalyst and BBL's CAMPY-PAK envelopes produces an atmosphere of 5-12% CO₂ and 5-15% O₂, which falls within the range of the ideal atmosphere of 10% CO₂ and 5% O₂. Campylobacter spp. grow as small, gray, nonhemolytic, flat, occasionally mucoid colonies.

Carbohydrate Fermentations. The term fermentation is ordinarily applied to the anaerobic degradiation of carbohydrates, although other produces can be fermented. The purpose of fermentation is to make energy available to a living cell, since carbohydrate molecules are rich in stored energy. Whether or not a carbohydrate is fermented depends upon the enzyme contained by an organism. The end products of fermentation vary from one organism to another. The fermentative properties of bacteria are valuable criteria and may be determined by culturing the organism in a suitable medium containing the appropriate fermentable substance. A satisfactory basic medium for determining the fermentation reactions of microorganisms must be capable of supporting growth of the organisms under study. To this broth base various carbohydrates, such as lactose and glucose, may be added at a concentration of 0.5-1.0%. Phenol red is included as an indicator of change in reaction. The pH at which phenol red changes color is about 6.8-6.9. Therefore, only a small amount of acid production changes the indicator to a yellow color. A test is not positive unless the medium is bright yellow. Once the carbohydrate has been depleted, reversal of the reaction can occur. Gas may be evolved during the process of carbohydrate fermentation caused by certain organisms. This is collected in the inverted vials, known as Durham tubes, in the broth.

Catalase Test. The production of the enzyme catalase is used to differentiate between the gram-positive Staphylococcus and Streptococcus and between the gram-negative Neisseria and Hemophilus. Procedure: Transfer a colony of the organism to be identified to a drop of 3% H₂O₂ on a microscope slide. If catalase is present, the H₂O₂ is broken down into water and oxygen which results in the immediate formation of gas bubbles. All members of the genus Staphylococcus are positive for the production of catalase, and all members of the genus Streptococcus are negative for the production of catalase. Neisseria is catalase-positive, whereas most Hemophilus isolates are catalase-negative. Remember that red blood cells (present in blood agar) also possess catalase activity.

Cefinase. See Beta-lactamase.

Chocolate Agar. (Abb. = CAP): One commonly used formula includes beef infusion, 1.5% agar, peptone, casein digest and corn starch. This mixture is autoclaved and cooled before adding sterile blood which has previously been heated to 80 C for 15 min. This treatment of the blood turns it a chocolate-brown color; hence the name of the plate. The advantages of a CAP are (1) release of erythrocyte contents such as hemin and NAD into the medium and (2) inactivation of certain erythrocyte enzymes which destroy various nutrient factors (e.g., NAD). Chocolate agar is not selective for particular organisms. Most organisms, including staphylococci, streptococci and gram-negative enterics, will grow very well on chocolate agar.

Citrate Test. See Simmon's Citrate Agar.

Coagulase Test. The test for production of the enzyme coagulase is used to distinguish S. aureus from S. epidermidis. The conventional version of the test is done with either human or rabbit plasma. Procedure: Several colonies of Staphylococcus are transferred with a loop to a tube containing 0.5 ml of plasma. The tube is covered to prevent evaporation and incubated at 37^oC overnight. The test is read by tilting the tube and observing for clot formation in the plasma. A negative test results in the plasma remaining free-flowing with no evidence of a clot.

Columbia Colistin-Nalidixic Acid (CNA) Agar. CNA agar is a selective medium for the isolation of gram-positive organisms from clinical specimens containing mixed species of bacteria. Gram-negative organisms are inhibited by the presence of colistin and nalidixic acid. Colistin is an antibiotic which acts on the cytoplasmic membranes of these organisms. Nalidixic acid is a quinoline which binds to DNA gyrase to stop DNA synthesis. When both gram-positive and gram-negative organisms are found upon microscopic examination of a specimen, a CNA plate should be used in conjunction with a plate such as MacConkey agar for isolation of gram-negative organisms. The CNA agar should not be depended upon for determination of hemolytic reactions since atypical reactions may be observed.

Corn Meal (Tween 80) Agar. Corn meal agar is simply a medium consisting of corn meal plus agar. This medium has been used particularly for morphological studies of Candida and is a recommended medium for production of chlamydospores. Incorporation of 1% Tween 80 reportedly stimulates more rapid and abundant chlamydospore formation than occurs on the plain medium by reducing surface tension.

Cystine Trypticase Agar. (Abb. = CTA) Cystine trypticase agar is designed as a simple and convenient basic medium for maintenance, detection of motility, and, with added carbohydrates (in a concentration of 1%) for determination of carbohydrate utilization reactions of fastidious organisms. CTA medium contains a pancreatic digest of casein, agar, cystine, and phenol red (yellow at pH < 6.8 and red at pH > 8.4; plate is normally red initially). This medium is particularly recommended as a basic medium for differentiation of Neisseria by means of carbohydrate utilization reactions. The medium is used in a semi-solid form in the Neisseria sugar test. Procedure: The medium is heavily inoculated in the top layer (5 mm) by repeated stabbing with a loopful of culture. Incubation is at 37^oC in a regular incubator.

Elek Plate. This plate demonstrates the production of diphtheria toxin by lysogenic strains of C. diphtheriae carrying the beta-prophage, which bears the tox gene. The medium consists of peptone, NaCl, and agar supplemented with amino acids, Tween 80, and glycerol. Immediately after the plate is poured and before the agar solidifies, a filter paper strip saturated with diphtheria antitoxin is submerged in it. The organisms being tested are heavily streaked at right angles to the filter paper strip. Plates must be incubated for 2-4 days before fine precipitin lines become visible.

Eosin Methylene Blue (EMB) Agar. EMB agar is used for differentiation and isolation of gram-negative bacilli from mixed populations of bacteria. EMB agar employs a combination of the dyes eosin and methylene blue as an indicator which gives a distinct difference between colonies of lactose fermenting organisms and those which do not ferment lactose. Lactose fermenting colonies are either dark or possess dark centers with transparent colorless peripheries, while organisms which do not ferment lactose remain uncolored. This purple color is due to the absorption of the esoin-methylene blue complex which forms in the presence of acid. (However, EMB should not be used as the sole indicator of lactose fermentation. It should be used in conjunction with MacConkey agar or lactose fermentation broth). Certain members of the coliform group, especially Escherichia coli, exhibit a greenish metallic sheen by reflected light. Enterobacter-Klebsiella colonies are larger, usually mucoid, occasionally confluent, and with dark blue centers. Gram-positive organisms are inhibited by the dye content of the medium making this medium selective for gram-negative bacteria.

Esculin hydrolysis. See Bile-esculin slant.

Gram Negative (GN) Broth. GN broth is a selective liquid enrichment which contains, among other things, sodium citrate and sodium deoxycholate to kill gram-positive organisms and inhibit the development of normal enteric flora. The increased concentration of mannitol over that of glucose favors and enhances the growth of mannitol-fermenting, gram-negative rods, such as Shigella and Salmonella, over that of nonfermenting species, particularly during the first six hours. The Salmonella and Shigella organisms are usually outnumbered by aerobic normal flora in fecal specimens, and the enrichment allows these organisms to increase in number so that they may be recovered on a selective medium such as Hektoen Enteric agar.

Hektoen (HE) Enteric Agar. HE agar is a selective and differential medium recommended for direct isolation of enteric pathogens from feces or indirectly after enrichment in selective broth. HE agar is used in many clinical laboratories to increase the yield of Salmonella and Shigella from a fecal specimen containing large numbers of normal enteric flora. Gram-negative enteric pathogens and nonpathogens are differentiated according to their ability to ferment lactose and sucrose and to produce hydrogen sulfide. Rapid lactose or sucrose fermenters produce bright orange to salmon pink colonies because of the combination of the yellow color from the bromthymol blue indicator and the red color from the acid fuchsin when acid is produced. Most of the nonpathogens ferment at least one of these carbohydrates. Salmonella and Shigella colonies are green to blue-green in color. Hydrogen sulfide-producing species generate hydrogen sulfide gas from sodium thiosulfate. The gas reacts with ferric ammonium citrate to yield a black precipitate that accumulates within colonies. The selectivity of the medium is due to the presence of bile salts that inhibit the growth of gram-positive bacteria.

Hippurate Hydrolysis. Sodium hippurate broth is made by adding sodium hippurate to brain heart infusion. This medium is used for differentiating Group B streptococci from other streptococci. All Group B streptococci possess the enzyme hippuricase, which is responsible for hydrolyzing sodium hippurate to benzoic acid. A few Group D streptococci are positive. However, these few Group D strains are also bile-esculin positive and thus can be differentiated from the Group B streptococci. Benzoic acid is detected by adding ferric chloride to cell-free supernatants and observing for formation of an insoluble precipitate. Both sodium hippurate and benzoic acid are initially precipitated from solution as ferric salts. However, on addition of excess ferric chloride, ferric hippurate redissolves, leaving ferric benzoate as the sole precipitate. Formation of a precipitate that does not redissolve after resting for 10 minutes is considered a positive finding for hydrolysis of sodium hippurate, that is, presence of benzoic acid in the test broth. Procedure. Inoculate the organisms to sodium hippurate broth and incubate at 35^oC for 48 to 72 hours. After incubation, centrifuge the culture and remove 0.8 ml of the cell-free supernatant to a clean tube. Add 0.2 ml of 12% ferric chloride (hippurate reagent) to the supernatant aliquot. Allow the mixture to stand for 10 minutes and observe for a cloudy precipitate.

IMViC Test.. The mnemonic IMViC was coined to designate the combined results of four different tests: Indole, Methyl Red, Voges-Proskauer, and Simmon's Citrate. The "i" was inserted for easier pronunciation. The media and tests are discussed separately under Indole Test, Methyl Red-Voges-Proskauer Broth, and Simmon's Citrate Agar.

Indole Test. Tests for the presence or absence of indole as a by-product of bacterial metabolism are of definite value in the identification of bacteria. Indole production is dependent upon the presence of tryptophan in the culture medium. A 1% solution of tryptone is generally used in tests for indole production because it is rich in tryptophan. Test for indole production by adding 6-8 drops of Kovac's Reagent (p-dimethylaminobenzaldehyde in amyl alcohol) to a 48-72 hour tryptone broth culture. Indole reacts with the aldehyde to give a red product in the alcoholic layer of the broth-reagent mixture. A yellow color is a negative test. This is one of four tests in the IMViC reaction.

Kirby-Bauer method for determining antibiotic resistance spectrum. The Kirby-Bauer method for this purpose is based on the diffusion of antibiotics from antibiotic-impregnated paper discs into an inoculated culture medium in a petri plate. The diameter of the area of growth inhibition around the antibiotic disc will give information that is useful for the effective treatment of the patient. Procedure: The Kirby-Bauer susceptibility test is performed using a pure culture of previously identified bacterial organism. The technique is carried out with standardized media, inocula, time and temperature of incubation, and antibiotic discs so that results from various laboratories will be comparable and related to the standards associated with the method. The inoculum to be used in this test is prepared by adding growth from 5 isolated colonies grown on a blood agar plate to 5 ml of broth. Several colonies are used to avoid clone differences in susceptibility. This culture is then incubated for 2 hours to produce a bacterial suspension of moderate turbidity. The turbidity is adjusted with sterile medium to match a standard turbidity tube of 1% barium chloride in 1% sulfuric acid (0.36 N). A sterile swab is used to obtain an inoculum from the standardized culture. This inoculum is then streaked evenly in three directions on a Mueller-Hinton plate so that confluent growth of the organism will be obtained.

The antibiotic discs are placed on the surface of the medium at evenly spaced intervals with flamed forceps or a disc applicator. Depending on the organism, different antibiotic-containing discs are used; thus, Gram positive and Gram negative organisms are tested against different panels of antibiotics. Antibiotics which produce smaller inhibition zones are best located in the center ring. Incubation is usually overnight with an optimal time being 14 hours. The diameters of the inhibition zones (including the 6 mm disc) are measured using a calipers. Zone size interpretive information is used to determine susceptibility or resistance of the organism to each antibiotic according to data accumulated from many other tests. The interpretation is given as "susceptible", "intermediate", or "resistant".

Latex agglutination. Latex agglutination is a way to rapidly visualize an antigen-antibody reaction through the easlily observed clumping that occurs between polystyrene latex beads coated with a specific antibody and the target antigen for the antibody. Latex agglutination assays are available for the rapid presumptive identification of several bacterial pathogens of humans and are easily performed in a physician's office. The speed of the test allows the physician to initiate therapy immediately; changes in therapy might need to be made later based on a more thorough series of diagnostic tests on the patient's isolate and on findings from antibiotic susceptibility testing for the isolate.

In IID, we used the Streptex latex agglutination test for the presumptive identification of the Group B Streptococcus. The Streptex test identifies Groups A, B, C, D, F, and G streptococci by antibodies specific for the Group carbohydrates. These Group-specific carbohydrate antigens can be released from the bacterial surface by treatment of the bacteria with a proteolytic enzyme. In the Streptex test, group-specific antibodies are coated onto polystyrene latex beads. When the latex beads are incubated with an extract containing the released corresponding group-specific carbohydrate antigen, a strong antigen-antibody reaction occurs, that crosslinks the beads in a clearly observable agglutination reaction.

Procedure:

Loeffler's Blood Serum Slant. Loeffler's is a minimal nutrient medium composed of a coagulated egg protein, beef serum, and inorganic salt and is used primarily for the recovery of Corynebacterium diphtheriae from clinical specimens. The slants are smooth and grayish white. Specimens taken from the throat and nasopharynx are inoculate promptly to the surface of a Loeffler's slant. On a slant that has been incubated for 2-8 hours. C. diphtheriae will frequently outgrow other organisms present and will exhibit a characteristic morphology (metachromatic granules and palisade formation) when stained with methylene blue. This morphology is more evident in cultures grown on Loeffler's medium than on some other type of medium. Slants should be examined in less than 18 hours to obtain C. diphtheriae before the other organisms have reached their full growth.

Lowenstein-Jensen Medium. Lowenstein-Jensen is an egg-based medium used for the cultivation of mycobacteria. The potato flour, glycerol, and egg components in this medium aid detoxification of components that would inhibit mycobacterial growth and supply some nutrients essential for mycobacteria. The malachite green is particularly inhibitory to contaminating bacteria. The surface of the medium should be light green in color. The presence of occasional particles of egg yolk is characteristic. Specimens are usually cultured on 3 slants. One tube is incubated at room temperature to identify saprophytes. The other 2 tubes are incubated one with and one without light to differentiate photochromogens from scotochromogens. Cultures should be examined after 4-7 days of incubation to detect rapidly growing mycobacteria, and weekly thereafter to identify Mycobacterium tuberculosis and slow-growing species. Cultures are observed 6-8 weeks before being reported as negative.

Lysine Decarboxylase (LDC) Medium. A positive (purple) reaction results from an alkaline pH generated by utilizing lysine. A yellow (negative) color occurs when organisms generate acid conditions by utilizing the limiting glucose present but not lysine. Incubation should continue for 4 days before a negative reaction can be confirmed. Note that the uninoculated medium is purple. Use an isolated colony from your EMB or Mac plate to inoculate the LD tube and overlayer the tube with -1 cm of mineral oil. DO NOT use the impure mineral oil with your microscope in place of the immersion oil.

MacConkey (Mac) Agar. MacConkey agar is a selective and differential plating medium recommended for use in the detection and isolation of Salmonella and Shigella from stool specimens, urine and other materials. This medium contains peptone, lactose, bile salts, neutral red, and crystal violet. Gram-negative bacteria are differentiated by their ability to ferment lactose. Isolated colonies of lactose fermenters are brick red and may be surrounded by a zone of precipitated bile. This reaction is due to the action of acids, produced by fermentation of lactose, upon the bile salts and subsequent absorption of the indicator neutral red. Non-lactose fermenters utilize the peptone thus yielding an alkaline reaction which changes the color of the medium to clear or yellow. These colonies are uncolored and transparent. Gram-positive organisms are inhibited by the dye crystal violet and bile salts.

Mannitol Salt Agar. This plate is used to help distinguish S. aureus from S. epidermidis. The former readily ferments the mannitol present in the medium (turning it bright yellow), while the latter only occasionally does. The medium also contains 7.5% sodium chloride. Although staphylococci are not inhibited by this concentration of salt, other organisms generally are.

Methyl Red-Voges Proskauer (MR-VP) Broth. MR-VP broth is recommended for the performance of the Methyl Red and Voges-Proskauer tests in differentiation of coliform organisms. One culture can be used for both tests. Transfer, using a Pasteur or serological pipet and rubber bulb, approximately one ml of a 48-hour MR-VP broth culture to a clean test tube to use for the VP test. Use the remaining five ml of culture for the MR test. The MR and VP tests are two of the four IMViC tests.

Members of the family Enterobacteriaceae may be divided metabolically into two groups: the mixed acid producers and the butylene glycol producers. The methyl red test is based upon the final hydrogen ion concentration reached by a culture in glucose broth after prolonged incubation (48 to 72 hours) at 37^oC. This change in pH is caused by mixed acid fermentation (i.e., the formation of formic, acetic, lactic and succinic acid) based upon a colorimetric procedure using the indicator methyl red. When 6-8 drops of methyl red (pH range 4.4-6.4) is added to the culture, the indicator will be orange-red (positive) if the pH is less than 4.5 and yellow (negative) if the pH is higher than 4.5.

The Voges-Proskauer reaction depends upon the ability of certain bacteria to produce acetylmethylcarbinol from the metabolism of glucose. When the alkaline reagent potassium hydroxide is added to a culture, acetylmethylcarbinol is oxidized to diacetyl. This in turn reacts with guanidine compounds in the broth to produce a red-colored complex. This color reaction develops particularly in the top part of the medium exposed to the air. Alpha naphthol serves as a catalyst and greatly intensifies the color. The test is performed on one ml of MR-VP broth culture by adding 15 drops of 5% alpha naphthol (reagent A) followed by 10 drops of 40% KOH (reagent B). Shake well and allow to stand for up to 30 minutes before calling a reaction negative. Do not shake again. If positive, the culture will begin turning red at the surface of the liquid, and the color will spread gradually throughout the tube.

MIC (Minimum Inhibitory Concentration of an antibiotic). This test determines the lowest concentration of an antibiotic that prevents growth of an organism. Procedure: In the broth tube dilution method, specific amounts of the antibiotic are prepared in decreasing concentrations. This dilution series of the antibiotic is then inoculated with a culture of the organism to be tested. The susceptibility of the organism is determined after suitable incubation by macroscopic observation for the presence or absence of growth in the varying concentrations of the antimicrobial agent. This bacteristatic end-point value is known as the Minimal Inhibitory Concentration (MIC).

Motility medium. In this semi-solid agar, motile organisms spread throughout the agar, while non-motile organisms grow only in the inoculated site. Reduction by bacteria of tri-phenyl tetrazolium chloride to a red color aids in visualizing the growth. With a straight inoculating wire (not a loop) inoculate the motility medium from colony or a liquid culture by stabbing the semi-solid agar to about 1/2 inch below the surface. Incubate at 37oC for 24 hours and observe for motility. If the test is negative, leave the tube at room temperature for another day. Some organisms synthesize their flagellar proteins optimally not at 37^oC but at room temperature.

Mueller-Hinton Agar

Mueller-Hinton agar is an enriched beef infusion medium recommended for determining antibiotic susceptibility using the Kirby-Bauer method. This medium shows good batch-to-batch uniformity and is low in tetracycline and sulfonamide inhibitors. The incorporated starch in the medium probably acts as a "protective colloid" against toxic material present in the medium.

Nutrient Agar

This is a low-nutrient medium consisting of beef extract, peptone, agar and water. It is recommended as a general culture medium for the cultivation of the majority of the less fastidious microorganisms. It also serves as a base to which a variety of materials (dyes, salts, carbohydrates, tissues, blood or serous fluid) may be added to give selective, differential or enriched media.

Optochin Sensitivity. This is a disc test to distinguish pneumococci from other alpha-hemolytic streptococci (Such as members of the viridans group). Procedure: A segment of a BAP is heavily inoculated with a single colony from a plate or with a pure broth culture. An optochin disc is laid on top of the inoculated area. Inhibition is assessed after 18-24 hours at 37^oC. Pneumococcal growth is inhibited by optochin (ethylhydrocupreine HCl).

Oxidase Test. The oxidase reaction is based upon the production of an enzyme, indophenol oxidase, by members of the genera Neisseria and Pseudomonas. Procedure: A disc impregnated with the oxidase reagent (p-aminodimethylaniline) is placed on a slide and moistened with several loopfuls of water. In an alternative method, break a vial of Oxidase Spot-Test and saturate a piece of filter paper in a petri dish. A colony suspected of being Neisseria or Pseudomonas is spread over the disc or onto the filter paper. If the color around the smear immediately turns rose to purple, the organism does produce indophenol oxidase. If the organism is negative for the production of oxidase, there is no color change (or a slow change to a rose color).

Sabouraud's Agar. This is a basic peptone, glucose, and agar medium used for the isolation and cultivation of both pathogenic and non-pathogenic fungi. Antibiotics such as cycloheximide and penicillin may be added to the medium to increase the inhibition of contaminating bacteria. The low pH (pH 5.6) of the medium also contributes to inhibition of contaminants.

Salmonella-Shigella (SS) Agar. The selective action of SS agar is attributed to brilliant green dye, which is inhibitory to most species of intestinal bacteria other than Salmonella, and bile salts and sodium citrate, which are inhibitory to most species of gram-positive bacteria and some of the lactose-fermenting members of the normal intestinal flora. Sodium thiosulfate is reduced by certain species of enteric bacteria to sulfite and hydrogen sulfide gas. Production of hydrogen sulfide is detected as an insoluble black precipitate, ferrous sulfide, formed upon reaction of hydrogen sulfide with the ferric ions of ferric citrate. When lactose is fermented by the few lactose-fermenting species of normal intestinal flora that can grow on SS agar, acid is produced and the pH indicator neutral red changes color from yellow to red. Thus, these organisms grow as pigmented colonies. Non-lactose-fermenting organisms grow as translucent colorless colonies with (Salmonella) or without (Shigella) black centers.

Salt Broth. This medium consists of Brain Heart Infusion broth containing 6.5% sodium chloride. Enterococci are capable of growing in such a medium resulting in turbidity, whereas other streptococci cannot tolerate such a high salt concentration and will not grow at all. Therefore, this is a useful biochemical test to be employed in conjunction with other tests in identifying a Group D Streptococcus.

Selenite-F Broth. This is an enrichment broth containing sodium hydrogen selenite. The action of this medium depends on maintenance of a certain pH at which sodium selenite is moderately toxic toward enterococci and normal intestinal flora while promoting growth of enteric pathogens. The critical pH is maintained by lactose. As enteric bacteria grow, they reduce selenite resulting in an alkaline environment. However, the acids produced from lactose fermentation neutralize the alkaline products produced during the reduction of selenite and thus promote the toxic effects of selenite. Because the medium loses its ability to suppress normal intestinal flora after 12 hours incubation, subculture between 8 and 12 hours incubation is recommended.

Simmon's Citrate Agar. This is used for the differentiation of gram-negative enteric bacteria on the basis of citrate utilization and is part of the IMViC test. Only those organisms capable of utilizing citrate as a sole source of carbon will grow on Simmon's citrate agar. These organisms are able to cleave citrate to oxaloacetate and acetate via the citritase enzyme. Another enzyme, oxaloacetate decarboxylase, then converts oxaloacetate to pyruvate and carbon dioxide. Carbon dioxide combines with water to form sodium carbonate, an alkaline compound. As a result, the pH of the medium rises and the indicator (bromthymol blue) changes from green to blue. Coliforms either do not grow at all on this medium or grow so sparsely that no change is reaction is apparent. Organisms are streaked onto a slant of Simmon's citrate agar and the reaction read after 24h of incubation.

Sodium Hippurate Broth. This is made by adding sodium hippurate to brain heart infusion. This medium is used for differentiating Group B streptococci from other streptococci. All Group B streptococci possess the enzyme hippuricase which is responsible for hydrolyzing sodium hippurate to benzoic acid. A few Group D streptococci are positive. However, these few Group D strains are also bile-esculin positive and thus can be differentiated from the Group B streptococci. Benzoic acid is detected by adding ferric chloride to cell-free supernatants and observing for formation of an insoluble precipitate. Both sodium hippurate and benzoic acid are initially precipitated from solution as ferric salts. However, on addition of excess ferric chloride, ferric hippurate redissolves, leaving ferric benzoate as the sole precipitate. Formation of a precipitate that does not redissolve after resting for 10 minutes is considered a positive finding for hydrolysis of sodium hippurate, that is, presence of benzoic acid in the test broth. Procedure. Inoculate the organisms to sodium hippurate broth and incubate at 35^oC for 48 to 72 hours. After incubation, centrifuge the culture and remove 0.8 ml of the cell-free supernatant to a clean tube. Add 0.2 ml of 12% ferric chloride (hippurate reagent) to the supernatant aliquot. Allow.

+6 the mixture to stand for 10 minutes and observe for a cloudy precipitate.

Streptex. See Latex agglutination.

Staphyloslide. There are several rapid diagnostic methods for identifying S. aureus in the modern diagnostic microbiology lab, including rapid tests for coagulase. In general, the rapid tests detect the presence of one or more proteins that are secreted by S. aureus or that are on the surface of the S. aureus bacteria. One that is used at UKMC is called the Staphyloslide. This test detects the presence of a fibrinogen receptor on the S. aureus surface. The fibrinogen receptor, also known as clumping factor, can bind to soluble fibrinogen (or to a fibrin clot). Fibrinogen is one of several serum proteins that coat our own cell surfaces and also deposit onto implanted devices (e.g., catheters). The ability of S. aureus to adhere to fibrinogen is thought to promote staphylococcal colonization of tissues and implanted foreign bodies. The presence of the fibrinogen receptor distinguishes S. aureus from other staphylococci and is thought to contribute to the virulence of S. aureus. The Staphyloslide test uses sheep red cells that have been coated with fibrinogen. Because fibrinogen is a dimer, one molecule can bind two bacterial cells; individual bacterial cells have many copies of the fibrinogen receptor on their surface and can bind to many fibrinogen molecules. Hence, if S. aureus is mixed with red cells coated with fibrinogen, a clumping reaction will occur as the bacteria and red cells bind to each other and adhere together. This clumping will not be seen if the isolate is S. epidermidis.

- Tap the tubes containing each of these reagents gently several times to resuspend the red cells.

- Label one end of a microscope slide: + and the other half: -.

- Using a Pasteur pipette and bulb, dispense a drop from the + reagent onto the + side of the slide and a drop from the - reagent onto the - side.

- A positive test for S. aureus will show clumping with the + reagent within15seconds; the - reagent drop will remain homogeneous. A negative reaction will show no clumping within 15 seconds for either reagent.

Sugar Reactions. Different sets of "sugar media" are commonly employed for distinguishing among Neisseria species and among various enteric organisms. Apart from the several sugars used, these sets differ from each other in certain basic ingredients required by the organisms being tested. All media in a set must be inoculated from the same colony or from a subculture of a single colony. The CTA (Cystine Trypticase agar) medium used for Neisseria is recommended for fastidious organisms which would not grow in ordinary carbohydrate fermentation broth. The tubes must be heavily inoculated in the top layer (5 mm) of the medium with the organism being tested. If an insufficient amount of inoculum is used, a false negative may occur. A positive result is indicated by turbidity and a yellow color change in the indicator in the upper layer of medium only due to acid production.

Neisseria Sugar Reactions
	Glucose	Maltose	Sucrose	Lactose
N. gonorrhoeae	+	0	0	0
N. lactamica	+	+	0	+
N. meningitidis	+	+	0	0
N. sicca	+	+	+	0
N. flavescens	0	0	0	0
Moraxella catarrhalis	0	0	0	0

N. lactamica can be differentiated from N. meningitidis through the former's oxidation of lactose. (Associate lactamica with lactose.) M. catarrhalis is included in the table because it is a GNDC that resembles Neisseria.

SXT susceptibility. Although the bacitracin susceptibility test is 95% accurate for recognizing Group A streptococci, rare strains of Groups B, C, D and G may show some degree of bacitracin susceptibility. To avoid incorrect identification of beta-hemolytic streptococcus as Group A, a disc containing the sulfonamide sulfamethoxazole and trimethoprim (SXT) is used in conjunction with a bacitracin disc. If the beta-hemolytic organism being tested is susceptible to both bacitracin and SXT, it is not Group A.

Tellurite medium. Good for permitting selective growth of corynebacteria. Tellurite salt (K₂TeO₃; colorless) is reduced by the bacteria, producing a precipitate of metallic tellurium, giving grey-black colors. Tellurite salts also reduce the number of contaminants: this is why tellurite medium is a selective medium.

Thayer-Martin Agar. Thayer-Martin agar is made selective for Neisseria gonorrhoeae and N. meningitidis by the addition of V-C-N antibiotic inhibitor to chocolate agar containing supplements, such as IsoVitale X. The V-C-N inhibitor is a mixture of vancomycin (which inhibits gram-positive bacteria by interfering with cell wall synthesis), colistin (which disrupts the cytoplasmic membrane of gram-negative bacteria), and nystatin (which inhibits yeasts by altering the permeability of their cell walls). This selective inhibition of contaminating flora permits the isolation of gonococci and meningococci. The modified formulation (modified Thayer-Martin or MTM) has an increased agar content (2% as opposed to 1%) to reduce spreading of Proteus species. The addition of dextrose provides a more nutritional medium for growth of gonococci and meningococci.

Triple Sugar Iron (TSI) Agar. TSI agar is a screening medium used to identify gram-negative bacilli based on the ability to ferment the carbohydrates glucose, sucrose and lactose, and to produce hydrogen sulfide gas. It is inoculated by stabbing the butt and streaking the surface of the agar with an inoculating loop. The medium contains 10 times as much lactose and sucrose as glucose. Bacteria that ferment glucose produce a wide variety of acids, turning the color of the medium from red to yellow. When any sugar is metabolized under anaerobic conditions (fermentation), there is increased acid production; thus the butt of the tube remains acid for a longer period of time. When glucose only is fermented, acid is produced throughout the medium but due to the relatively low concentration of glucose in the medium and the aerobic metabolism that is present on the slant, nitrogenous metabolic end products soon change the reaction of the slant to alkaline. These alkaline products neutralize the small amounts of acids present in the slant but are unable to neutralize the large amounts acid present in the butt. Thus, the appearance of an alkaline (red) slant and an acid (yellow) butt after 24 hours incubation indicates that the organism is a glucose fermenter but is unable to ferment lactose and sucrose. Bacteria that ferment lactose or sucrose (or both), in addition to glucose, produce such large amounts of acid that the oxidative deamination of protein that may occur in the slant does not yield enough alkaline products to cause a reversion of pH in that region. Thus, these bacteria produce an acid slant and acid butt. The inclusion of sucrose permits separation of certain Proteus organisms which ferment sucrose from the salmonellae since both of these groups of organisms are lactose-negative and both may produce hydrogen sulfide. The formation of gas (CO₂ and H₂) as a by-product of fermentation is detected by the presence of cracks or bubbles in the medium. Hydrogen sulfide (H₂S) gas is produced as the result of the reduction of thiosulfate. H₂S is a colorless gas and can be detected only in the presence of an indicator, in this case ferric ammonium sulfate. H₂S combines with the ferric ions to produce the insoluble black precipitate ferrous sulfide. Reduction of thiosulfate proceeds only in an acid environment and blackening usually occurs in the butt of the tube. Although the black precipitate may frequently obscure the color of the butt it can be assumed that the organism is a glucose fermenter because of the requirement for an acid environment.

Reaction Interpretation

Alkaline slant (red), acid butt (yellow) (KA) Glucose fermented

Acid (yellow) throughout the medium, butt Lactose or sucrose or both fermented

and slant acid (yellow) (A/A)

Alkaline slant and butt (medium None of the three sugars fermented

entirely red)

Gas bubbles in butt, medium Gas (CO₂) produced as a by-product of

sometimes split fermentation

Blackening of the butt and/or stab Hydrogen sulfide produced

Trypticase Soy Agar. TSA is a general purpose solid medium for isolation of nonfastidious organisms. If blood is added, some fastidious organisms, such as streptococci, will grow and type of hemolysis can be determined.

Trypticase Soy Broth. In TSB, as in TSA, certain aerobic and facultative microorganisms will grow without blood or serum. It can also be used as an enrichment medium for blood culture procedures.

Tyrptone Broth. Because it is rich in tryptophan, a 1% solution of tryptone is used to grow bacteria to test for indole production (see Indole Test). This is part of the IMViC test.

Urea Agar. Urea agar slants are recommended for the identification of the genus Proteus. The decomposition of urea by member of the Proteus group has been especially helpful to differentiate these organisms from other gram-negative intestinal bacteria. Using this strongly phosphate buffered medium, Proteus can be differentiated from most other members of the enteric group by its ability to produce sufficient ammonia to give a reaction more alkaline than pH 8.1 after 4-8 hours of incubation. This occurs as a result of hydrolysis of urea by the enzyme urease. Phenol red is included as a pH indicator, and a positive test is indicated by a change of color in the medium to dark pink or red. Yersinia enterocolitica also gives a strong positive reaction and some other organisms may be weakly positive after 48 hours of incubation. Slants are streaked and normally incubated for 24 hours before reading the reaction.

Voges Proskauer Test. See Methyl Red-Voges Proskauer (MR-VP) Broth.

X and V factors. Different species of Hemophilus require one or both of two factors found in blood. These factors are designated X and V. The X factor is heat stable and may be either hematin (hydroxyhemin) or hemin (chlorohemin). The X factor is not available from the whole blood in sheep blood agar plates but is liberated when sheep blood is used to make chocolate agar plates. The V factor is NAD/NADP; it is destroyed by autoclaving. It also is destroyed by enzymes which are present in sheep blood. These enzymes are inactivated when sheep blood is heated to 80^oC for 15 minutes, as in the preparation of chocolate agar.

Xylose Lysine Deoxycholate (XLD) Agar

XLD agar is recommended for isolation of enteric pathogens, especially Shigella species. This medium is made selective for these organisms by the addition of sodium deoxycholate which inhibits most gram-positive bacteria and partially inhibits the growth of normal intestinal flora. The differential action of XLD agar is due to fermentation of xylose, lactose, and sucrose, decarboxylation of lysine and production of H₂S. Most of the normal intestinal flora ferment xylose, lactose, sucrose, or all of these, producing organic acids and, as a result, yellow colonies. Species of Shigella do not produce acid from these carbohydrates and thus produce red or clear colonies. Hydrogen sulfide production is noted by the formation of black centers in the colonies due to reaction with ferric ammonium citrate.

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