Citrate Utilization

The citrate test determines whether an organism can use sodium citrate as a sole carbon source. Simmons citrate medium is frequently used to determine citrate utilization. In addition to citrate, the test medium contains ammonium salts as the sole nitrogen source.

Bacteria able to use citrate will use the ammonium salts releasing ammonia, The alkaline pH that results from use of the ammonium salts changes the pH indicator (bromthymol blue) in the medium from green to blue. It is important to keep the inoculum light because dead organisms can be a source of carbon, producing a false-positive reaction (Figure 9-8). Christensen’s citrate medium is an alternative test medium. This medium incorporates phenol red (as the pH indicator) and organic nitrogen. At an alkaline pH, the indicator turns from yellow to pink.



DNA is a polynucleotide composed of repeating purine and pyrimidine mononucleotide monomeric units. Most bacterial DNases are endonucleases cleaving internal phosphodiester bonds resulting in smaller subunits.

Extracellular DNAse can be produced by a number of bacteria such as Staphylococcus aureus and Serratia marcescens. DNase test medium usually contains 0.2% DNA. A heavy inoculum of bacteria is streaked onto the

surface of the medium in a straight line; several organisms can be tested at one time. The plate is incubated at 35°C for 18 to 24 hours, and then IN HCl is added to the surface of the plate. Unhydrolyzed DNA is insoluble in HCI and will form a precipitate. Oligonucleotides formed from the action of DNase will dissolve in the

acid, forming a clear zone (halo) around the inoculum.


Gelatin Liquefaction

Gelatin is a protein derived from animal collagen. A number of bacteria produce gelatinases-proteolytic enzymes that break down gelatin into amino acids.

Gelatinase activity is detected by loss of gelling Oiquefaction) of gelatin. Gelatinase activity is affected by several factors, including the size of the inoculum and incubation temperature. Some bacteria produce larger amounts of gelatinase at room temperature compared to 35° C. It may be necessary to incubate media several

weeks to detect a positive reaction. Several methods are available for detecting gelatinase.


Indole Production

Indole is one of the degradation products of the amino acid tryptophan. Organisms that possess the enzyme tryptophanase are capable of deaminating tryptophan, with formation of the intermediate degradation products of indole, pyruvic acid, and ammonia. Bacteria are inoculated into tryptophan or peptone broth. Most

commercial peptone broth contains enough tryptophan for a positive reaction; tryptophan can be added to obtain a final concentration of 1%. After inoculation, the broth should be incubated at 35° C for 48 hours.

After incubation, one of two methods can be used to detect indole. In the Ehrlich’s indole test, the indole is extracted from the broth culture by the addition of 1 ml of xylene. After the xylene is added, the tube is shaken well. After waiting a few minutes for the xylene to rise to the top, 0.5 mL of Ehrlich’s reagent, containing paradimethylaminobenzaldehyde (PDAB), is added. If indole is present, a red color develops after the addition of PDAB (Figure 9-9). Alternatively, Kovac’s reagent, which also contains PDAB, can be used. This method does not use a xylene extraction.

Approximately 5 drops of Kovac’s reagent is added directly to the broth culture. The tube is shaken, and if indole is present, a red color develops. Ehrlich’s method is more sensitive than Kovac’s reagent and is preferred with nonfermentative bacteria. If indolenitrate (trypticase nitrate) medium is used, the indole test can be performed from the same broth culture as a nitrate test. Before adding any reagents, the broth is divided in half, one aliquot for the indole test and the other for the nitrate test, see Malonate utilization.


Malonate Utilization

The malonate test determines whether the organism is capable of using sodium malonate as its sole carbon source. Malonate broth normally contains bromthymol blue as a pH indicator. Bacteria able to use malonate

as a sole carbon source will also use ammonium sulfate as a nitrogen source. A positive test results in increased alkalinity from utilization of the ammonium sulfate, changing the indicator from green to blue (Figure 9-10).



Motility can be determined by microscopic examination of bacteria or by observing growth in a semisolid medium. Motility test media have agar concentrations of 0.4%or less to allow for the free spread of organisms.

A single stab into the center of the medium is made. Best results are obtained if the stab is made as straight as possible. After incubation, movement away from the stab line or a hazy appearance throughout the medium indicates a motile organism. Incubation temperature is important. Some bacteria are motile only at room temperature, but this temperature may not be optimal for growth. It is suggested that two motility tubes be inoculated, one incubated at room temperature and the other at 35° C. Comparing inoculated to uninoculated tubes may help in interpreting results.


Nitrate and Nitrite Reduction

The nitrate reduction test determines whether the organism has the ability to reduce nitrate to nitrite and further reduce nitrite to nitrogen gas (N2). The organism is inoculated into a nutrient broth containing a nitrogen source. After 24 hours of incubation, N,Ndimethyl-a-naphthylamine and sulfanilic acid is added. A red color indicates the presence of nitrite.


Nitrate reduction test reaction

Nutrient broth with 0.1 % potassium nitrate –+ Nitrate reductase –+ Nitrite + Sulfanilic acid + N,N-Dimethyl-1-naphthylamine –+ Diazo red dye

If no color develops, this may indicate that nitrate has not been reduced or that nitrate has been further reduced to N2, nitric oxide (NO), or nitrous oxide CNP), which the reagents will notbe able to detect. Adding a small amount of zincdust will help to determine whether the test has produced a true negative result or whether the lack of color production was due to reduction beyond nitrite. Zinc

dust reduces nitrate to nitrite. Therefore development of a red color after the addition of zinc confirms a true-negative test result. Alternatively, a small glass tube, called a Durham tube, can be inserted into the broth upside down when the medium is aliquoted into test tubes. During incubation, if nitrogen gas is produced, it will be trapped in the inverted Durham tube.



The oxidase test determines the presence of the cytochrome oxidase system that oxidizes reduced cytochrome with molecular oxygen. The oxidase test is helpful in differentiating between the Enterobacteriaceae, oxidase negative, and the pseudomonads, which are oxidase positive. The oxidase test is also useful in identifying Neisseria organisms, which are oxidase positive. A modified oxidase test is used to distinguish Staphylococcus from Micrococcus. Several methods for performing an oxidase test are available.

Kovac’s oxidase test uses a 0.5 or 1%aqueous solution of tetramethyl-p-phenylenediamine dihydrochloride.

A drop of the reagent is added to filter paper, and a swab is used to rub a colony to the moistened filter paper. The development of a lavender color on the swab within 10 to 15 seconds is a positive reaction.

p-Aminodimethylaniline oxalate is less sensitive than tetramethyl-p-phenylenediamine dihydrochloride, but it is cheaper and more stable. Commercial forms of oxidase reagent are available in glass ampules and on filter paper disks.



The urease test determines whether a microorganism can hydrolyze urea, releasing a sufficient amount of ammonia to produce a color change by a pH indicator.

Urease hydrolyzes urea to form ammonia, water, and CO2,Different formulations of urea agar are available, but Christensen’s urea agar is generally preferred. The

medium is an agar slant inoculated on the slant surface but not stabbed. The medium contains phenol red as the pH indicator. The resulting alkaline pH from hydrolysis of urea is indicated by a bright pink color  (Figure 9-11).

Lysine Iron Agar Slant

The Lysine Iron Agar (LIA) test is a tubed agar slant. It contains the amino acid lysine, glucose, ferric ammonium citrate, and sodium thiosulfate. The Ph indicator is bromcresol purple. LIA is used primarily

to determine whether the bacterial species decarboxylates or deaminates lysine (see Figure 9-6). Hydrogen sulfide production is also detected in this medium. LIA is inoculated in the same manner as a TSI agar slant. LIA is most useful in conjunction with TSI in screening stool specimens for the presence of enteric

pathogens, differentiating Salmonella (lysine positive) from Citrobacter species (lysine negative). Decarboxylation only occurs anaerobically; the presence of a dark purple butt is positive for lysine decarboxylation.

The production of H2S can mask the purple color in the butt of the tube. Because HzS production in LIA occurs only in an alkaline environment, a black precipitate indicating H2Sis also a positive result for decarboxylation.

LlA is also useful in differentiating Proteus,Morganella, and Providencia species from most other members of Enterobacteriaceae. This group of enterics deaminates (attacks the NH2group instead of the carboxyl group) amino acids. In the LlA slant, deamination of lysine turns the original light purple color slant to a plum or reddish-purple color; the butt turns yellow due to glucose fermentation.


Motility-Indole-Ornithine Agar

Motility-indole-ornithine (Mia) is a semisolid agar medium used to detect motility and indole and ornithine decarboxylase production. Mia is useful in differentiating Klebsiella spp. from Enterobacter and Serratia spp. The medium is inoculated by making a straight stab down the center of the medium with an inoculating needle. Motility is shown by a clouding of the medium or spreading growth from the line of inoculation. Ornithine decarboxylation is indicated by a purple color throughout the medium. Because Mia is a semisolid medium, it does not have to be overlayed with mineral oil to provide anaerobic conditions.

Indole production is detected by the addition of Kovac’s reagent; a pink to red color is formed in the reagent area if the test result is positive. Ornithine decarboxylase and motility should be read first before addition of Kovac’s reagent.

Sulfide-Indo Ie-Motility Agar

Sulfide-indole-motility (SIM) medium is a semisolid agar helpful in differentiating gram-negative bacteria in the family Enterobacteriaceae. An inoculating needle is used to make a straight stab down the center of the medium. Cloudiness spreading from the inoculation line is positive for motility. The production of H2S is indicated by a black precipitate, and a pink to red color after the addition of Kovac’s reagent is positive for indole.

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