Culture Media, Biochemical Tests, and TechniquesIntroduction: Culture media provide the chemical and physical environment necessary for bacterial growth.

Bacteria vary greatly in their nutritional requirements and often, different media are needed to enrich for specific bacteria. Most of the media used for this course is obtained commercially. Below is a description of a method used to produce broth and agar media.

General methodology for producing nutrient broth- and agar-based media: 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., 120oC, 15-20 min.) results in liquefaction of the agar; the mixture is then cooled to 41-42oC. 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 40oC, and cannot be reliquified without reheating to 100oC, hence the necessity for adding sterile, heat-labile ingredients at 41-42oC, 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, Biochemical Tests, and Techniques Used in this Laboratory Course (Note: descriptions and procedures may be more or less detail than those in the laboratory section)

 

Acid-fast Stain (AF): see common laboratory techniques (pp. viii-xviii).Aseptic Technique: see common laboratory techniques (pp. viii-xviii).

Bacitracin and SXT Susceptibility Tests: This bacitracin susceptibility test (with 95% accuracy) helps distinguish Group A Streptococcus from other beta-hemolytic streptococci that are much less susceptible to this antibiotic. In rare instances, beta-hemolytic strains of Groups B, C, D and G may show some degree of bacitracin susceptibility and, to avoid incorrect identification, this test is done in conjunction with sulfamethoxazole and trimethoprim (SXT) sensitivity test. Group A streptococci exhibit susceptibility to bacitracin and resistance to SXT. Procedure: Streak a single colony heavily onto BAP either over the whole/half plate. Using sterile forceps, aseptically place a bacitracin disc (0.04 units per disc) onto the densely streaked region of the plate. If performing SXT sensitivity, place SXT disc (contains 23.75 :g sulfamethoxazole and 1.25 :g of trimethoprim) approximately 2 cm from bacitracin disc on same dense region of plate. After 24 hours incubation at 35oC in 3-5% CO2, determine susceptibility to the antibiotic by examining growth inhibition around disc(s).

BACTEC 12B Medium: Because of the slow growth rate of mycobacteria on solid media, diagnostic laboratories in the United States must also inoculate a liquid medium such as BACTEC 12B media (for use in the BACTEC automated radiometric detection system) as a more rapid means of detecting mycobacteria. A portion of the treated specimen is inoculated into a vial containing the liquid medium consisting of glycerol, inorganic salts, albumin (provides added protein and protects against toxic components), glucose (energy source), catalase (protects against peroxides), and 14C-labeled palmitic acid. Additionally, the medium may be supplemented with antibiotics to make it more selective for mycobacteria. The vial is incubated under conditions similar to those on solid media. Utilization of the radiolabeled fatty acid results in evolution of 14CO2 that can be detected by the BACTEC system. A presumptive-positive detection of mycobacteria may occur within 4-14 days. Once mycobacteria have been detected in the BACTEC system, the positive culture from the BACTEC vial is stained to confirm the presence of AFB and subcultured for further testing.

BactiTM Staph (Remel) Latex Agglutination Test: see coagulase test

Beta-lactamase Production: A rapid test for detecting beta-lactamase activity from H. influenzae, N. gonorrhoeae, Moraxella catarrhalis, Enterococcus spp., anaerobic bacteria, and Staphylococcus species employs the Cefinase or Nitrocefin disc, which is impregnated with the chromogenic cephalosporin. This compound is yellow in its complete state but becomes red upon cleavage of the beta-lactam ring. Procedure: Moisten a Nitrocefin-impregnated disc with one drop of water. Smear growth of the isolated organism being tested for beta-lactamase production on the disc. Bacteria producing beta-lactamase in significant quantities will cleave the beta-lactam ring of the chromogenic cephalosporin changing the color of the disc from yellow to red. A negative result will show no color change on the disc. The reaction can occur as early as 30 seconds; however, the test is read after 15 minutes.

Bile-Esculin Slant: This agar medium is used for the presumptive identification of Group D streptococci and enterococci. These organisms hydrolyze esculin (6-$-glucoside-7-hydroxy coumarin) in the presence of 4% bile salts. The hydrolytic reaction yields glucose and esculetin. Esculetin then reacts with ferric iron in the medium to form a black diffusable complex that results in blackening of the bile-esculin agar slant within 24 to 48 hours. Other streptococci do not produce a color change. Growth on this medium is largely limited to streptococci, since most (except S. pneumoniae) tolerate the presence of bile salts. Procedure: Growth from

suspected streptococci isolates are streaked onto the surface of the slanted region of the bile esculin agar slant. The tubes are incubated at 35o C for 24 to 48 hours.

Bile Solubility Test: This test helps distinguish S. pneumoniae from viridans streptococci and other alpha-hemolytic streptococci. Bile salts, such as sodium deoxycholate, activate the pneumococcal autolytic enzymes and lead to dissolution of a colony or to loss of turbidity in a suspension. These agents rarely lyse other streptococci. Procedure: Place a drop of a 10% solution of sodium deoxycholate directly onto an isolated colony of S. pneumoniae. Pneumococcal colonies will disappear or flatten, while other streptococci colonies (e.g. viridans streptococci) will be unaffected.

Blood Agar Plate (BAP): Blood agar is a standard type of supportive medium used in the isolation and cultivation of most organisms except the most fastidious bacteria. The most common type used in clinical laboratories and in this laboratory is trypticase soy agar (soybean-casein digest agar base) containing 5% sheep defibrinated blood. The blood provides additional nutrients and serves as an indicator of hemolytic reaction, but it can also contain components that are toxic to fastidious bacteria such as Neisseria. Blood agar plates prepared with rabbit or horse blood tend to support the growth of the more fastidious bacteria.

Brain Heart Infusion (BHI) Broth: Brain heart infusion broth is a liquid medium widely used for the cultivation of a number of bacteria including pathogenic cocci and other organisms generally considered fastidious or difficult to grow. Virulence, antigenicity, and other serological characteristics are quite uniformly maintained when the organisms are grown on brain heart infusion.

BHI-6.5% NaCl Broth or Salt Broth: Enterococci are capable of growing in such a medium resulting in turbidity, whereas other streptococci including Group D streptococci cannot tolerate such a high salt concentration and will not grow at all. Therefore, this is a useful test to differentiate Group D Streptococcus from Enterococcus. Procedure: Transfer growth of an isolated colony to BHI-6.5% NaCl broth tube, incubate tube at 35oC, and examine tube for turbidity (growth) within 24 hours.

Carbohydrate Fermentation: The term fermentation is ordinarily applied to the anaerobic degradation of carbohydrates, although other products 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 or other pH indicator dyes are included to monitor the fermentation reaction (production of acidic endproducts). A change in the color of the dye within 24 to 48 hours usually indicates fermentation of the particular carbohydrate.

Catalase Test: The production of the enzyme catalase is an important diagnostic tool to differentiate members of the genus Staphylococcus from those of the genera Streptococcus and Enterococcus. Most aerobes and facultative anaerobes including Staphylococcus possess the enzyme, catalase, which enzymatically releases O2 from hydrogen peroxide (H2O2), the toxic endproduct of aerobic carbohydrate metabolism. Streptococcus and Enterococcus species, which are aerotolerant, do not possess this enzyme. Procedure: Smear growth of the organism to be identified onto a microscope slide and add a drop of a solution of 3% H2O2 onto the smear. If catalase is present, the H2O2 is broken down into water and oxygen, resulting in the immediate formation of gas bubbles. Remember that red blood cells (present in blood agar) also possess catalase activity.

Cefinase Test: See beta-lactamase production.

Chocolate Agar Plate (CAP): An enriched supportive medium that supports growth of most bacteria including the more fastidious bacteria such as Neisseria and Haemophilus. This medium is essentially the

same as BAP except that the agar medium containing the blood is heated to 70 oC for 10 min. to lyse the red blood cells (giving it a chocolate appearance). In heating the blood agar mixture, intracellular nutrients, such as hemin and NAD, are released from red blood cells and some components of BAP that are toxic to certain fastidious bacteria are neutralized or buffered.

Citrate Test: See Simmon's citrate agar.

Coagulase Test: The production of the enzyme coagulase by S. aureus is the most widely accepted criterion for differentiating this species from other staphylococci, which are categorized collectively as coagulase-negative staphylococci (CNS). Coagulase can either be detected in the extracellular environment (referred to as “free”) or bound to the cell surface (referred to a “clumping factor” or “fibrinogen receptor”) and both the free and bound type can bind to plasma fibrinogen causing a cascade of reactions that allow plasma to clot. Commercial latex agglutination kits to detect bound coagulase production (and in some kits, also Protein A, a cell wall component found in S. aureus but not in other staphylococci) are used by many clinical laboratories to identify suspected S. aureus isolates. As well, tube coagulase test with rabbit plasma (detects free coagulase) may also be performed if the latex agglutination reaction gives conflicting results with other findings.

Procedure for BactiTM Staph (Remel, Lenexa, KS) Latex Agglutination Test: Mix an isolated colony into one drop of the latex reagent on one circle of the reaction card, filling the whole circle. The latex reagent contains latex beads coated with human fibrinogen for detection of coagulase and IgG for detection of Protein A. The card is rocked in a circular motion for up to 60 sec. Aggregation of the black latex suspension with subsequent loss of black background represents a positive reaction for agglutination. For S. aureus, this usually occurs within 15 sec. A negative reaction is reported as little or no agglutination (without the loss of black background) within 60 sec.Procedure for tube coagulase test: Mix several colonies or 0.5 ml of broth culture into 0.5 ml of oxalated or citrated rabbit plasma in a tube. The tube is covered to prevent evaporation and incubated at 35 oC. The test is read by slowly tilting the tube. A positive test results in a highly viscous clot formation in the plasma. Once a coagulum, no matter how small, has formed the test is considered positive (usually within 4 hours). A negative test results in the plasma remaining free flowing with no evidence of a clot. The plasma should be incubated overnight before a test is called negative, but prolonged incubation (over 24 hours) may result in the dissolution of a formed clot.

Colony Isolation: see common laboratory techniques (pp. viii-xviii).

Columbia Colistin-Nalidixic Acid (CNA) Agar: CNA agar is a selective medium for the isolation of Gram-positive organisms especially staphylococci and streptococci from clinical specimens containing mixed flora. Not all Gram-positive bacteria grow on these plates (some Bacillus species are inhibited by CNA media). CNA agar contains meat peptones, casein, and yeast extract as the nutritional base, 5% defibrinated sheep blood, and antibiotics, colistin (C) and nalidixic acid (NA), which inhibit growth of most Gram-negative organisms. Colistin acts on the cytoplasmic membranes of these Gram-negative organisms and nalidixic acid, a quinoline, binds to DNA gyrase to stop DNA synthesis. While this medium may also be used to determine hemolytic properties of Gram-positive organisms, BAP is often the better choice for determining these properties because, for some bacteria, the hemolytic property observed on CNA agar may be an atypical reaction (i.e. some beta-hemolytic organisms appear as alpha-hemolytic on CNA agar).

Crystal Enteric Kit: see laboratory 6.

Cystine Trypticase Agar (CTA) Carbohydrate Test: 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%), 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). For CTA carbohydrate test, CTA is in a semi-solid form and contains 1% carbohydrate (glucose, maltose, sucrose, or fructose). This test is used to differentiate Neisseria species based on acid production from carbohydrate utilization reactions (although this test may not be as

sensitive in detecting oxidative carbohydrate utilization as in detecting carbohydrate fermentation). Procedure: The medium is heavily inoculated in the top 1/3 layer (5 mm) by repeatedly stabbing with a loopful of culture. The cultures are incubated at 35oC with tops of tubes tightened. A yellow color in the upper third of the stab indicates positive utilization of the carbohydrate. If an insufficient amount of inoculum is used, a false negative may occur. Because Neisseria species utilize sugars oxidatively, a tube exhibiting a yellow color the entire length of the tube indicates that the sugar is being fermented, suggesting that a contaminant is present. Table A1-1 (Appendix 1) shows the carbohydrates oxidized by various Neisseria species and Moraxella catarrhalis and the patterns of carbohydrate utilization that are used to differentiate these organisms.

Differential Medium: a medium that contains a factor or factors necessary to distinguish metabolic or cultural characteristics of specific bacterial isolates. These characteristics can be used to differentiate this group of bacteria from other strains growing on the agar plate. An example of differential medium is MacConkey agar that distinguishes lactose fermentation.

Elek Plate: This antigen-antibody immunodiffusion assay demonstrates the production of diphtheria toxin by lysogenic strains of C. diphtheriae carrying the temperate bacteriophage, corynephage $, which harbors the structural gene for the exotoxin. 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. Once the agar has solidified, then 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. The Elek test is subject to error (false negative results) due to small differences in media preparation and technical skills of personnel performing the test. For this reason, it is almost never performed outside a reference laboratory.

Esculin Hydrolysis: See Bile-esculin slant.

E-Test: The E-test, a quantitative test, has been developed that combines the convenience of disk diffusion with the ability to generate MIC data. The E-test (AB Biodisk, Solna, Sweden) utilizes plastic strips that are impregnated on one side with an antimicrobial agent concentration gradient. The other side contains a numeric scale that indicates the drug concentration. Mueller-Hinton plates are inoculated as for the Kirby-Bauer test and the strips are placed on the inoculum lawn. Multiple antimicrobials can be tested on a single isolate. Following overnight incubation, the plate is examined and the number present at the point where the border of growth inhibition intersects the E-strip is taken as the MIC. The same MIC criteria used for dilution methods are used with the E-test value to assign a category of susceptibility or resistance. The E-test (usually used for fastidious or slow-growing bacteria) is useful for producing MIC data in situations in which the level of resistance can be clinically important (e.g. penicillin or cephalosporins against S. pneumoniae).

Evaluation of Bacterial Growth: see common laboratory techniques (viii-xviii).

Flaming a Loop: see common laboratory techniques (pp. viii-xviii).

Gram Stain: see common laboratory techniques (pp. viii-xviii).

Haemophilus ID Quad Plates: Most commonly encountered clinical strains of Haemophilus spp. can be identified by hemolytic reactions on horse blood agar and growth requirements for X and V factors. These Quad plates are divided into 4 sectors and contain one quadrant each for X factor (hemin)-enriched medium, V factor (NAD)-enriched medium, both X and V factors-enriched medium and an enriched medium containing X and V factors with horse blood (to determine growth and hemolysis). Procedure: A single colony of a purified culture is streaked onto each of the quadrants and the plate is incubated at 35 oC in a candle jar for 24 to 48 hours.

Hemolysis on BAP: Certain bacteria exhibit hemolytic properties on BAP because of their ability to produce extracellular enzymes that lyse red blood cells in the agar. Hemolysis can only be determined around well-isolated single colonies and is accurately determined by holding a plate up to a light and observing the hemolysis with the light coming from behind. Hemolysis observed around areas of confluent growth may be

due to nonspecific lysis of cells rather than to the action of hemolysin. 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 upon microscopic examination. Alpha hemolytic colonies are surrounded by a narrower greenish zone of hemolysis that consists of unlysed red blood cells (or incomplete lysis of RBCs) and an unidentified reductant of hemoglobin that causes the greenish color. Many Gram-negative organisms will produce a greenish discoloration around colonies due to diffusion of growth by-products into the medium and not to a hemolysin. Gamma hemolysis actually refers to no observable hemolytic activity.

Surface colonies of beta hemolytic Group A streptococci may appear alpha or gamma hemolytic. Two hemolysins, streptolysins O and S, are responsible for b-hemolytic activity in these streptococci. These hemolysins are distinct antigenically and differ in susceptibility to oxidation. Streptolysin O is antigenic and oxygen-labile; it is enzymatically active under reduced oxygen tension. Thus, surface growth of streptococcal isolates, producing only Streptolysin O, exhibits alpha or gamma hemolysis on BAP and subsurface growth of these isolates exhibits beta-hemolysis in BAP. Streptolysin S is non-antigenic and oxygen-stable; it is enzymatically active in oxygen or reduced oxygen tension. Surface and subsurface growth of isolates producing only Streptolysin S exhibit b-hemolysis in BAP. To correctly identify beta hemolytic Group A streptococci, blood agar plates should be stabbed several times in the initial streak to ensure that the streptolysin O will be active in the subsurface colonies under the reduced oxygen environment.

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: This test is one (I) of four tests in the IMViC reaction and detects indole, a by-product of metabolic degradation of the amino acid tryptophan. Bacteria that are positive for indole production possess tryptophanase, the enzyme involved in hydrolyzing and deaminating tryptophan to indole. Typically, a 1% solution of tryptone is used in tests for indole production because it is rich in tryptophan. In practice, though, combination media such as Motility Indole Ornithine (MIO) deeps are also rich in tryptophan and may be used to detect indole production (see MIO agar deeps below). Procedure: Growth from an isolated colony or pure culture is inoculated into a 1% tryptone broth tube or into a MIO deep (semi-solid agar stabbed halfway down the center of the tube with an inoculating needle). The cultures are incubated at 35 oC in ambient air for 48-72 hours. To test for indole production, 6-8 drops of Kovac's reagent (p-dimethylamino-benzaldehyde in amyl alcohol) are added to the 48-72 hour culture tube. Indole reacts with the aldehyde group of the substrate to give a red complex in the reagent mixture (contains alcohol) that sits on top of the culture. The red color indicates that the isolate is positive for indole production. If the reagent mixture remains colorless or slightly yellow, then the isolate is negative for indole production.

JEMBEC Agar Plates: When delays in transport of suspected pathogenic Neisseria specimens are expected, the specimens should be inoculated to a plate containing selective medium with an incorporated method for generating an increased CO2 environment. The JEMBEC plate (an acronym from John E. Martin Biological Environmental Chamber) is one type of system in use today. It contains modified Thayer-Martin medium (see modified Thayer-Martin medium below), a molded inner well that holds a CO 2-generating tablet, and a bag in which to seal the plate. The plate should be incubated at 35oC.

Kirby-Bauer Method for Testing Antibiotic Susceptibility: The Kirby-Bauer method for determining antibiotic susceptibility is a qualitative test based on diffusion of antibiotics from antibiotic-impregnated paper discs into an inoculated agar culture medium in a Petri plate setting up a concentration gradient that radiates from the disc. 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. 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. Because of

the qualitative nature of this method, it is not widely used in larger diagnostic laboratories. Procedure: The Kirby-Bauer susceptibility test is performed using a pure culture of previously identified bacterial organism. 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 caliper. A zone size interpretive chart can be used to determine susceptibility or resistance of the organism to each antibiotic. At the University Hospital's Diagnostic Microbiology Laboratory, the caliper is electronically connected to a computer that contains the zone-size interpretive information, and when the technician presses a key, the interpretation appears on the screen as: "susceptible", "intermediate", or "resistant", according to data accumulated from many other tests. Thus, zone sizes are not reported to the clinician but only the three possible interpretations.

Latex Agglutination: For differentiation of Staphylococcus species, see coagulase test. For differentiation of Streptococcus species, see PathoDx Strep latex agglutination. Note: Latex agglutination is a way to rapidly visualize an antigen-antibody reaction or a substrate binding reaction by observing agglutination (clumping) of polystyrene latex beads coated with a specific antibody/substrate and the target antigen/enzyme. 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 including antibiotic susceptibility on the organism isolated from the patient. In IID, the BactiTM Staph (Remel, Lenexa, KS) latex agglutination test is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci. PathoDx Strep latex agglutination is used to differentiation Group B Streptococcus from other beta-hemolytic streptococci. Similar tests are also available for identifying Groups A, C, D, F, and G streptococci using latex beads coated with antibody to group-specific cell wall carbohydrates (Group A, B, C, F, or G) or lipoteichoic acid (Group D).

Loeffler's Serum Slant: Loeffler's is an enriched nutrient medium with the base medium containing heart infusion, peptones, and glucose. Egg and horse serum are added to the medium, providing additional nutrients and causing the medium to coagulate during the sterilization process. The slants are smooth and grayish white. These slants are used primarily for the cultivation of corynebacteria, especially Corynebacterium diphtheriae, from clinical specimens. Specimens taken from the throat and nasopharynx are inoculated promptly onto the surface of a Loeffler's slant. C. diphtheriae will frequently outgrow other organisms present in the specimen, especially within a short period of incubation (<18 hours). This medium also enhances characteristic morphologies of C. diphtheriae including metachromatic granule formation and pleomorphic cellular arrangements (i.e. palisade formation) which can be observed when stained with methylene blue.

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 and the presence of occasional particles of egg yolk can be characteristic. Specimens are usually cultured on 3 slants; One tube is incubated at room temperature to identify saprophytes; the other 2 tubes are incubated with and without light, respectively, to differentiate photochromogens, scotochromogens, and non-photochromogens. 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.

MacConkey (Mac) Agar: MacConkey (Mac) agar is used by many clinical laboratories as a primary selective and differential medium for Gram-negative bacteria, especially enteric organisms from stool specimens, urine and other specimens. This medium contains peptones, lactose, bile salts, crystal violet dye and pH indicator (neutral red). Gram-positive organisms and fungi are inhibited by the crystal violet dye and bile present in this medium; thus, Gram-negative organisms can selectively grow on this medium. Lactose and neutral red allow differential identification of lactose-fermenting Gram-negative organisms. The acidic endproducts of lactose fermentation by Gram-negative bacteria decrease the pH of the medium causing the dye to give the colonies a light pink to reddish color. A zone of precipitated bile may also be observed around Lac+ colonies. Non-lactose fermenters utilize peptones in the agar, yielding an alkaline reaction that changes the color of the medium to clear or yellow. These colonies are colorless and transparent.

Methyl Red Test: The Methyl Red (MR) test is one (M) of four IMViC tests and is always run in conjunction with the Voges-Proskauer (VP) test below. Several members of Enterobacteriaceae produce large quantities of mixed acids (formic, acetic, lactic, and succinic acids) from glucose fermentation. The Methyl Red test is a colorimetric pH indicator test that detects mixed acid producers and is based upon the final hydrogen ion concentration reached by a culture in Methyl Red-Voges-Proskauer (MR-VP) broth (a glucose broth) after prolonged incubation (48 to 72 hours) at 35oC. The pH at which methyl red detects the acidity of the medium is lower than that for other indicators; it ranges from pH 6.0 (yellow) to pH 4.4 (red). Procedure: The 48-72 hour MR-VP culture is used for both the MR and VP tests (one milliliter is used for the VP test and four milliliters is used for the MR test--see MR-VP broth below). To four milliliters of a MR-VP culture, 6-8 drops of methyl red indicator is added and the tube is mixed well. A methyl red positive reaction will be red (pH <4.5) and a negative reaction will be yellow (pH >4.5). Within a 24-hour incubation time period, most members of Enterobacteriaceae will produce a sufficient amount of acid end products that can be detected initially by methyl red (so all will be MR positive within 24 hours). A true methyl red positive organism, which produces larger quantities of stable mixed acid end products, is capable of maintaining a low pH over the prolonged incubation time period (48 to 72 hours), and thus, can overcome the buffering capacity of the medium.

Methyl Red-Voges Proskauer (MR-VP) Broth: MR-VP broth is recommended for the performance of the Methyl Red (MR) and Voges-Proskauer (VP) tests in differentiation of coliform organisms. Members of the family Enterobacteriaceae may be divided metabolically into two groups based on glucose fermentation: the mixed acid producers and the acetylmethylcarbinol producers. The MR test is used to detect the mixed acid production from the mixed acid pathway and the VP test is used to detect acetylmethylcarbinol production from the butylene glycol pathway. The MR-VP broth contains peptones, glucose, and salts. One culture can be used for both tests. Procedure: MR-VP broth is inoculated with growth from an isolated colony or pure culture. The culture tube is incubated at 35oC for 48 to 72 hours. On the day of the tests, one milliliter (ml) of culture will be used for the VP test (see Voges-Proskauer test below). The remaining 4 ml will be used for the MR test (see Methyl Red test above). The MR and VP tests are two (MV) of the four IMViC tests.

Middlebrook 7H10 or 7H11 Agar: This medium is an enriched medium used for cultivation of mycobacteria and contains glycerol, inorganic salts, oleic acid (fatty acid required by mycobacteria for metabolism), albumin (provides added protein and protects against toxic components), glucose (energy source), catalase (protects against peroxides), casein hydolysate (stimulates growth of drug-resistant Mycobacterium tuberculosis--in 7H11 medium only), and malachite green (inhibits faster growing bacterial contaminants). Culture plates are sealed and examined after 2-7 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.

Minimal Inhibitory Concentration (MIC) Method of Testing Bacterial Susceptibility to Antibiotics: This test determines quantitatively the lowest concentration of an antibiotic that prevents

growth of an organism. The technique is carried out with standardized antibiotic concentrations, inocula, and time and temperature of incubation, so that results from various laboratories will be comparable and related to the standards associated with the quality control strains. MIC tests are performed upon request and for all organisms isolated from normally sterile anatomical sites (e.g., blood, peritoneal fluid, pericardial fluid, spinal fluid, and fluid obtained by suprapubic tap). MICs can be determined from a broth tube dilution method or by automated commercial tests (i.e. Dade International Dade Microscan [West Sacramento, CA] or bioMerieux Vitek [Hazelwood, MO]). Procedure for broth tube dilution: 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 turbidity of the inoculum is based on comparison to a MacFarland standard for turbidity. 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). For commercial tests, the optical density of the culture well is monitored over a specific period of time and the results are sent directly to the computer where the MIC is determined automatically from these results.

Motility Medium. In this semi-solid agar deep, motile organisms spread throughout the agar, while non-motile organisms grow only in the inoculated site. Reduction by bacteria of tri-phenyl tetrazolium chloride (TTC) to a red color aids in visualizing the growth. Procedure: The motility-TTC medium is inoculated with an isolated colony or a liquid culture by stabbing the semi-solid agar with the inoculating loop straight down to about ½ to 1 inch below the surface. The tubes are incubated at 35oC in ambient air for 24 hours. If the isolate is motile, then the medium will look turbid and the red color will have diffused throughout the medium. If the isolate is non-motile, then the growth of the organism and the red color will be confined within the site of inoculation. Typically, if the test is negative, the tube is left at room temperature for another day because some organisms synthesize their flagellar proteins optimally at room temperature rather than at 35oC.

MIO (Motility Indole Ornithine) Agar Deep: MIO medium is a semi-solid purple-colored medium consisting of peptone, tryptone (rich in tryptophan), yeast extract, glucose, L-ornithine hydrochloride, and the pH indicator, brom cresol purple (detect ornithine decarboxylase activity). This medium is used to differentiate enterics on the basis of motility, production of indole, and ornithine decarboxylase. Kovac’s Reagent is added to the surface of the medium to detect indole production (see indole test). Motility is determined by growth of the organism from the inoculation site. If the isolate is motile, then growth of the organism will be observed away from the site of inoculation and the medium will look turbid. If the isolate is non-motile, then the growth of the organism is confined within the site of inoculation. Ornithine decarboxylase activity is determined by color changes of the pH indicator. During growth of the enteric organism, glucose is fermented producing acidic byproducts that decrease the pH, changing the pH indicator from purple to yellow. If the isolate is positive for ornithine decarboxylase activity (enzyme is active at the acidic pH), then ornithine is decarboxylated to putrescine, an alkaline byproduct that increases the pH, and the color of the medium or pH indicator changes back to a purple color. If the isolate is negative for ornithine decarboxylase activity, the pH remains acidic and the color of the medium remains yellow. Procedure: Using an inoculating needle, growth of an isolated colony or pure culture is stab halfway down the center of the tube. The cultures are incubated at 35oC in ambient air for 48-72 hours. The cultures are examined for motility and indole production (see respective tests). If required, the cultures may be examined for ornithine decarboxylase activity as described above. Usually for the ornithine decarboxylase test, a control culture is set up without L-ornithine hydrochloride (MI medium) to monitor glucose fermentation and validate that the purple color in the MIO medium is actually due to ornithine decarboxylase activity.

Modified Thayer Martin (MTM) Medium: Thayer-Martin agar is a selective chocolate agar-type medium for the isolation of pathogenic Neisseria, N. gonorrhoeae and N. meningitidis. The medium consists of peptones, cornstarch (neutralize fatty acids), phosphate buffer (control pH), hemoglobin (causes chocolate color of agar and provides hemin and NAD) and IsoVitaleX (provides NAD, vitamins, glucose,

and other nutrients). The medium is made selective by the addition of antimicrobial agents, vancomycin (inhibits Gram-positive bacteria), colistin (inhibits Gram-negative bacteria including nonpathogenic Neisseria), nystatin (inhibits fungi), and trimethoprim lactate (inhibits Proteus species).

Mueller-Hinton Agar: Mueller-Hinton agar is an enriched beef infusion medium recommended for determining antibiotic susceptibility using the Kirby-Bauer method or E-test. This medium shows good batch-to-batch uniformity and is low in tetracycline and sulfonamide inhibitors. The incorporated starch in the medium protects the organism from toxic material present in the medium. The medium used for susceptibility testing of Streptococcus pneumoniae is Mueller-Hinton agar with 5% sheep blood.

Nitrocefin Test: see beta-lactamase production.

Novobiocin Resistance Test: Staphylococcus saprophyticus is resistant to novobiocin (at a concentration of 5 :g) and this criterion is used to differentiate this organism from S. epidermidis, which is sensitive to novobiocin. Procedure: Streak a single colony onto BAP either over the whole/half plate. Aseptically place a novobiocin disc (C disc) onto the middle of the densely streaked region of the plate. Lightly press the disc onto the agar media using your forceps, so that the disc sits on top of the agar and not squashed into the agar. Incubate the plate inverted at 35oC overnight. A zone of inhibition that is less than 16 mm is usually indicative of a resistant strain. A zone of inhibition that is greater than 16 mm is indicative of a sensitive strain.

Nutrient Agar: This is a low-nutrient medium consisting of beef extract, peptone, and agar. 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: The Optochin disc is used in most laboratories for differentiation of S. pneumoniae from other alpha hemolytic streptococci or enterococci. Discs impregnated with optochin (ethylkhydrocupreine hydrochloride), when placed on a freshly streaked blood agar plate, will inhibit the growth of pneumococci but allow other a-hemolytic streptococci to grow normally. Procedure: Streak a single colony onto BAP either over the whole/half plate. Aseptically place an optichin disc (P disc) onto the densely streaked region of the plate. Lightly press the disc onto the agar media using your forceps, so that the disc sits on top of the agar and not squashed into the agar. Incubate the plate inverted at 35oC in 3-5% CO2 for 24-48 hours. A zone of inhibition 14 mm or greater is read as a positive test (strain is sensitive); a zone of 6 to 14 mm is a questionable result.

Ornithine Decarboxylase Activity: see MIO agar deep.

Oxidase Test: The oxidase reaction is based upon the production of an enzyme, indophenol oxidase (cytochrome oxidase), and is produced by strict aerobes, in particular Neisseria and Pseudomonas. The reaction is employed in most laboratories on suspected cultures of gonococcus or meningococcus and Pseudomonas aeruginosa. Procedure: On a slide, filter paper is impregnated with the oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride). An isolated colony is spread onto the saturated filter paper using an inoculating loop. If the color in the smear turns immediately from colorless to purple, then the isolate is positive for the production of cytochrome oxidase. If the color of the smear remains colorless or slowly changes to a light purple or rose color, then the isolate is negative for the production of cytochrome oxidase. As an alternative method, a drop of the oxidase reagent can be placed directly on the suspected colonies (especially for Neisseria species). The development of a deep purple color indicates a positive reaction. In clinical laboratories this test is performed with colonies from primary isolation plates; the results help determine which multi-test system to inoculate for further identification. If a colony is to be transferred to another medium, it may be done within 10 to 15 minutes after addition of the reagent because the organisms are not immediately killed.

PathoDx Strep Latex Agglutination Test (Remel, Lenexa, KS): This is one of several commercial latex agglutination kits available that identify Lancefield Groups A, B, C, D, F, and G streptococci. Group-specific cell wall carbohydrate (beta hemolytic streptococci Group A, B, C, F, and G) or lipoteichoic acid (Group D streptococci) antigens are extracted from the bacterial cell envelopes by treatment of the bacteria using a nitrous acid extraction. The extracted antigens are neutralized and mixed with latex beads coated with IgG antibodies against a particular Group-specific antigen (each latex reagent is for a specific Lancefield Group). When the Group-specific antibodies on the latex beads bind to their corresponding carbohydrate-specific or lipoteichoic acid antigen, then strong antigen-antibody reactions occur that crosslink the latex beads resulting in an observable agglutination reaction. According to the technical literature, cross-reactivity from other streptococci should be minimal due to the high specificity of IgG for each streptococcal group antigen and the nature of the nitrous acid extraction procedure. Not all the Lancefield antigens are specific for a single streptococcal species; however, the presence of Group B antigens from an isolated strain seems to be specific only for S. agalactiae. Procedure for detecting Group A and Group B streptococci: To extract the carbohydrate antigens from suspected streptococci colonies, two drops of Reagent 1 and 2 drops of Reagent 2 are added to a tube. Using an applicator stick, 1 to 4 isolated colonies are mixed into the extraction reagents. Four drops of Reagent 3 are added to the extraction mixture to neutralize the mixture. The extraction mixture is then mixed thoroughly by tapping the tube. One drop of the extraction mixture is placed on the end of the oval on a reaction card. The Group A or Group B latex reagent is resuspended by inverting the dropper. One drop of either Group A or Group B latex reagent is placed on the other end of the oval. The latex and extraction mixture are mixed together using a wooden stick or stirrer (cover entire oval) and the card is rocked back and forth for no more than one minute. The oval on the card is examined for agglutination. A positive reaction is agglutination of the blue latex with a loss of the blue background. A negative reaction remains as a uniform blue milky appearance.

Quantitation of Urinary Tract Infections: see laboratory 6.

Sabouraud's Agar: This agar medium is a supportive for the isolation and cultivation of both pathogenic and non-pathogenic fungi. This medium consists of peptone, glucose, and agar. 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.

Salt Broth: see BHI-6.5% NaCl broth.

Selective Medium: This type of medium contains inhibitory reagent(s) that restrict growth of some or most organisms and allow selective growth of other organisms.

Simmons Citrate Agar. This agar medium is used for the differentiation of Gram-negative enteric bacteria on the basis of citrate utilization and is one (C) of the IMViC test. Only those organisms capable of utilizing citrate (a TCA metabolite) as a sole source of carbon and ammonium salts as the sole nitrogen source 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. Additionally, the pH of the medium can rise due to utilization of ammonium salts for growth, resulting in the production of alkaline by-products (most notably, ammonia). Several coliforms either do not grow at all on this medium or grow so sparsely that no change is reaction is apparent. Procedure: Simmons Citrate agar slants are inoculated by streaking the slanted region with growth from an isolated colony or pure culture. These tubes are incubated at 35oC in ambient air for 24 hours. A change in color from green to blue in the slanted region is indicative of an alkaline change in pH and positive citrate utilization. No color change indicates negative citrate utilization.

Staph Latex Agglutination: see coagulase test.

Strep Latex Agglutination: see PathoDx strep latex agglutination test.

Streptolysin O and S: see hemolysis on BAP.

Sugar Reactions: see CTA carbohydrate test

Supportive medium: The type of medium contains enough nutrients to allow growth of all except the most fastidious organisms.

SXT susceptibility: see bacitracin and SXT susceptibility tests.

Tellurite medium: McLeod's tellurite agar or serum tellurite medium, a selective growth medium for corynebacteria, contains tellurite salt (K2TeO3; colorless) that inhibits growth of most of the throat flora. When this salt is reduced by the bacteria, a precipitate of metallic tellurium is produced and the colonies are gray-black in color.

Triple Sugar Iron (TSI) Agar: TSI agar slant is a screening medium used to identify the ability of gram-negative bacilli to ferment carbohydrates (glucose, sucrose and/or lactose) and/or to produce hydrogen sulfide. Procedure: The slant is inoculated by streaking the surface of the slanted agar with an isolated colony or a pure culture and then, without removing the inoculating loop, stabbing the butt of the agar slant from top to bottom. The tubes are incubated at 37oC in ambient air and the reactions are read within 24 h. After that time period, oxidative reactions of fermentative by-products may cause reversion of an acid reaction (yellow) to an alkaline reaction (red) and thus, produce false readings. The results shown in Table A2-1 are discussed below.

Table A2-1: Interpretation of reactions in TSI agar slants.

Reaction Interpretation

–Alkaline slant (red), acid butt (yellow) (K/A) Glucose fermented–Acid slant and butt (medium yellow throughout) (A/A) Lactose or sucrose (or both) in addition to glucose

fermented

– Alkaline slant and butt (medium entirely red) (K/K)None of the three sugars fermented

– Gas bubbles in butt, medium sometimes split (G) Gas (CO2) produced as a by-product of fermentation

–Blackening of the butt and/or stab (H2S) Hydrogen sulfide produced

The TSI agar medium contains 10 times as much lactose and sucrose as glucose and a phenol red indicator to detect acidification of the medium from fermentative by-products. Bacteria that ferment these carbohydrates (an anaerobic process) produce a variety of acid by-products that turn the color of the phenol red indicator in the slanted region and butt (or deep) region of the medium from red to yellow. If glucose is the only carbohydrate fermented by the isolate, then the results observed within 24 h will show that the butt of the medium will remain yellow. The slanted region of TSI, though, will revert back to alkaline (red/dark pink) due to the relatively low concentration of glucose in the medium and the increasing alkaline by-products produced from aerobic oxidation of the fermentative by-products. (The alkaline by-products are capable of neutralizing the small amounts of acids present in the slanted region but are unable to neutralize the larger amounts of acid present in the butt.) Bacteria that ferment lactose or sucrose (or both), in addition to glucose, produce such large amounts of acid by-products that the subsequent oxidative reactions do not yield enough alkaline by-products to cause a reversion of pH in the slanted region of the medium. Thus, these bacteria produce an acid slant and acid butt (yellow/yellow). Bacteria that do not ferment lactose, sucrose, or glucose (nonfermenters) do not produce any changes in the color of the medium (slant and butt remain a red color). The formation of gas (CO2 and H2) as a by-product of fermentation is detected by the presence of cracks or bubbles in the TSI agar medium. The presence of ferrous sulfate in TSI medium serves as an indicator for the detection of those isolates capable of producing hydrogen sulfide (H2S). H2S, a colorless gas, combines with the ferric ions to produce the insoluble black precipitate ferrous sulfide. H2S is produced

only in an acid environment and blackening of the agar usually occurs in the butt of the tube. Since the black precipitate may frequently obscure the color of the butt, then the assumption is that the color of the butt is yellow (i.e. the isolate ferments one, two, or all three sugars) because of the requirement for an acid environment for H2S production.

Trypticase Soy Agar (TSA): TSA (soybean-casein digest base) is a general purpose medium used for the cultivation of nonfastidious organisms. Some of more fastidious organisms, such as streptococci, will grow on TSA when blood is added to the agar medium (see Blood agar plate).

Tyrptone Broth: This broth is rich in tryptophan and a 1% solution of tryptone is used to grow bacteria to determine indole production (see Indole Test).

Urea Agar: Urea agar slants can be used to differentiate Proteus species and Yersinia enterocolitica from other Enterobacteriaceae by their ability to rapidly hydrolyze urea, a reaction that is catalyzed by the enzyme urease. Sufficient amount of ammonia is produced by these organisms in a short period of time (4 to 8 hours) to alkalinize the media to a pH >8.1. The shift in pH is detected by a color change of the phenol red indicator from light orange at pH 6.8 to magenta (or dark pink) at pH 8.1. In comparison to Proteus species and Y. enterocolitica, other enterics are slower at hydrolyzing urea, and thus, may be weakly positive after 48 hours of incubation. The reactions in the slants are normally read within 24 hours of incubation. Procedure: Christensen’s Urea agar slants are inoculated by streaking the slanted region with growth from an isolated colony or pure culture. These tubes are incubated at 35 oC in ambient air for 24 hours. A change in color from light orange to magenta in the slanted region is indicative of an alkaline change in pH and a strong positive urease activity. No color change indicates negative (or maybe weak) urease activity.

Voges-Proskauer Test: The Voges-Proskauer (VP) test is one (V) of four IMViC tests and is always run in conjunction with the Methyl Red (MR) test above. Several members of Enterobacteriaceae produce acetylmethylcarbinol (acetoin) as a major end product of glucose fermentation and smaller quantities of mixed acids. Acetoin is a neutral compound produced from pyruvate (the pivotal compound of glucose fermentation) via the butylene glycol pathway and this compound can be detected by the VP test. An alkaline reagent, potassium hydroxide, is added to a Methyl Red-Voges-Proskauer (MR-VP) culture that has been incubated for 48 to72 hours (should contain a build up acetoin end product) to oxidize acetoin to diacetyl. This in turn reacts with guanidine compounds in the broth and an added catalyst, alpha naphthol, to produce an intensified red-colored complex. This color reaction develops particularly in the top part of the medium exposed to the air because oxygen is required for the initial oxidation step. Procedure: The 48-72 hour MR-VP culture is used for both the MR and VP tests (one milliliter is used for the VP test and four milliliters is used for the MR test--see MR-VP broth above). To one milliliter of a MR-VP culture, 15 drops of 5% alpha naphthol (VP reagent A) is added, mixing the tube well, and then 10 drops of 40% KOH (VP reagent B) is added. The tube is mixed well and then placed in a tube rack for up to 30 minutes (it can take 15 minutes to see a positive reaction). Do not shake tube during the 30-minute time period. If the reaction is positive for acetoin production, then the culture will begin turning red at the surface or top layer of the liquid medium and the color will diffuse gradually throughout the tube over time. A negative reaction for acetoin production shows no evidence of color development (medium remains a yellow to yellow orange color) after 30 minutes.