Laboratory Identification Of Microbes

Biochemical Reactions

Biochemical tests demonstrate the presence of enzyme systems within the microbial cell, such as those responsible for the fermentation of carbohydrates or the decomposition of proteins and are done so that individual species of microbes may be identified. By noting the presence of specific enzymes in a pure culture of bacteria being studied under a microscope, one can usually make its identification.

Miniaturization of microbiologic technique refers to the use of the commercially prepackaged test units wherein basic reagents for a given biochemical reaction are pre-measured, standardized, and compacted into a tablet or onto a paper disk or filter paper strip. The test unit is applied to cultures in liquid or solid media and is chemically designed so that enzymatic action of the test organism usually results in a color change, or end point, a quick and easy observation under a microscope. There are many varieties of such tests available.

Fermentation of Sugars

Microbes ferment many organic compounds, including carbohydrates, generating in the process energy-rich -chemical bonds. Through fermentation simple sugars can serve as the main source of energy for many different- kinds of microbes. The end products of fermentation depend on the substrate, enzymes present, and conditions under which the reaction proceeds. For instance, yeast enzymes that break down glucose into carbon dioxide and alcohol have no effect on sucrose (cane sugar), whereas enzymes produced by pneumococci and other streptococci ferment glucose to lactic acid. Products of bacterial fermentation are lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, and the gases carbon dioxide and hydrogen.

Fermentation reactions, which vary among species of microorganisms,, are of great value in differentiating species with the use of a microscope. From their specific action on a given sugar in the laboratory, bacteria (and other microbes) are classified as (1) those that do not ferment the sugar, (2) those that do ferment it with the production of acid only, and (3) those that ferment it with the production of both acid and gas. Sugar-containing media are inoculated with the bacteria (or other test microbes) and observed for gas and acid formation. Gas production in liquid media is detected by the accumulation of gas that displaces the fluid medium contained in the closed arm of a special tube, as in a Smith fermentation tube, and then examined under a microscope. Gas may be detected in a small tube (one end of which is sealed off) placed in an inverted position within a larger tube of liquid medium. As gas is formed in the inoculated liquid, it collects in the small tube within the depths of the culture and rises toward the sealed end of the smaller tube where it is trapped.

One inoculates solid media for fermentation studies by plunging a straight needle carrying some of the bacteria (or other test microbes) deep into the medium, a maneuver known as “stabbing”, and then examined under a microscope. The solid medium may also be melted, cooled to 42° C., and then inoculated.

In solid media, gas production is seen as bubbles of gas disrupting the medium. Acid formation in both liquid and solid media is measured by color changes in pH indicators incorporated therein. Many carbohydrates are used in routine fermentation studies. Important sugars are glucose, lactose, maltose, sucrose, xylose, mannitol, and salicin. The fermentation of lactose is crucial in the identification of bacteria recovered from the intestinal tract, since it makes a division between the nonpathogenic coliform organisms, which rapidly ferment lactose, and the pathogenic species, such as Salmonella and Shigella, which cannot ferment lactose (with a few exceptions).

Hydrolysis of starch

When microbes, seen under a microscope, are grown on starch agar, a clear zone develops around the colonies of those that digest starch. If the agar is covered with a weak solution of iodine, the undigested starch assumes a blue color.

Liquefaction of Gelatin

A gelatin medium is stabbed with an inoculating wire having bacteria on it, incubated at 20° C, and observed for liquefaction. It may be incubated at 37° C. (gelatin is liquefied at this temperature) and then placed in the refrigerator, where the unliquefied gelatin solidifies. Incubation should be continued for at least 2 weeks unless liquefaction occurs before that time, and a tube of uninoculated gelatin should be incubated as a control.

Citrate Utilization

Utilization of citrate by bacteria is determined by inoculation of Simmons’ citrate agar, a commercially prepared test medium, colored green that contains ammonium phosphate as a nitrogen source and sodium citrate as the sole source of carbon. If the test microorganism possesses the enzyme permease to transport citrate into the cell, the medium becomes alkaline, turning from its original green to a deep Prussian blue. The test is useful in the differentiation of certain coliforms, as Enterobacter and Klebsiella species, which can use citrate as their sole carbon source, from Escherichia coli, which cannot.

Indole Production

Production of indole (from the amino acid tryptophan) is determined by culture of the test organisms in a medium containing tryptophan, with the use of a microscope. The reaction is mediated by.”tryptophanase” a term for the complete system of enzymes responsible. A strip of filter paper soaked with a saturated solution of oxalic acid may be hung over the culture and held in place either by the cotton plug or by the screw cap of the culture tube. A pink color on the paper indicates indole production. When Kovacs reagent (paradimethyl aminobenzaldehyde dissolved in amyl alcohol and concentrated hydrochloric acid) is added to the broth, a red color indicates indole production. Important indole producers are Escherichia coli and Proteus, which avidly decompose proteins.

The proteolytic enzymes (protein-splitting enzymes, proteinases) produced by bacteria split complex proteins into proteases, peptones, polypeptides, amino acids, ammonia, and free nitrogen. Protein decomposition is known as putrefaction. Some authorities restrict the term putrefaction to the decomposition of proteins by anaerobic bacteria, which results in the formation of hydrogen sulfide and other foulsmelling decomposition products, and they use the term decay for the decomposition of proteins by aerobic bacteria. The latter does not result in the formation of malodorous decomposition products:

Nitrate Reduction

Nitrate reduction means the removal of oxygen from the nitrate radical to convert it into nitrite (NO2). The reaction is mediated by the enzyme nitratase produced by a wide variety of microorganisms. The test organisms are incubated in broth containing 0.1% potassium nitrate, and the broth is tested for nitrite with sulfanilic acid and alpha-naphthyfamine reagents. A red color is a positive test.

Deoxyribonuclease Elaboration

Production of deoxyribonuclease (DNase) is demonstrated by culture of test microbes on the surface of agar to which deoxyribonucleic acid (DNA) has been added. After 24 hours of incubation, the surface is flooded with either 1N hydrochloric acid to highlight the clear zones about the DNase-positive growth or with 0.1% toluidine blue, in which case a bright rose pink color is the end point, that is, indicates the presence of DNase. Staphylococci produce this enzyme.

Hydrogen Sulfide Production

A stab culture in an agar that contains basic lead acetate or iron acetate is incubated for 1 to 4 days and then examined under a microscope. The production of hydrogen sulfide from the sulfur-containing amino acids of the medium is indicated by the appearance of a black compound, lead sulfide (or iron sulfide) formed from the combination of the hydrogen sulfide with the lead (or iron) acetate. Instead of being incorporated into the medium, the basic lead acetate may be impregnated on sterile filter paper strips suspended over the medium as in the test for indole production. The production of hydrogen sulfide facilitates the identification of Brucella species and enteric bacilli.

Splitting of Urea

Certain bacteria (for example, Proteus) convert urea to ammonia by producing urease. With phenol red as the indicator, the presence of urease is seen by the appearance of the` red color. This test helps to separate species of the genus Proteus from the urease-negative Salmonella and Shigella species.

Digestion of Milk

Bacterial growth in sterile milk may be alkaline or acid and with or without curdling. Curdling may or may not be followed by liquefaction of the casein curd. Excessive gas production in milk produced by Clostridium perfringens is called stormy fermentation.

Oxidase Reaction

The enzyme oxidase is produced by Neisseria species, and its detection is of great value in the identification of Neisseria gonorrhoeae. The oxidase reagent (N,N-dimethyl-p-phenyl-enediamine manohydrochloride) colors the positive colonies pink to red to black.

Niacin Test

The niacin test is useful in distinguishing Mycobacterium tuberculosis from other species of mycobacteria. A 4% alcoholic aniline solution and a 10% aqueous solution of cyanogen bromide (Note toxicity to humans) are added to 1 or 2 ml. of emulsified bacterial growth. A complex yellow compound is formed when niacin or nicotinic acid, formed by the tubercle bacillus, but not by other mycobacteria, reacts with the cyanogen bromide and a primary amine.

Optochin Growth-Inhibition Test

The Optochin growth-inhibition test is performed by placement of a 6 mm absorbent paper disk containing Optochin ethylhydrocupreine hydrochloride) in contact with a heavy inoculum of test streptococci in pure culture on a blood agar plate, which is incubated overnight. A zone of inhibition greater than 18 mm. identifies the growth as that of pneumococci, whereas zones about other streptococci, if present, are much smaller. Currently this is the most widely used test for differentiating pneumococci from other alpha-hemolytic streptococci.

Catalase Test

The catalase test is performed by pouring of hydrogen peroxide over the growth of a heavily inoculated, 18- to 24-hour agar slant and then placing the tube in an inclined position to demonstrate nicely the rapid evolution of oxygen gas bubbles, the positive reaction. In microbes, the transfer of hydrogen to oxygen in respiration may result in the production of hydrogen peroxide, which is toxic to the microbial cell. Most aerobic, but not anaerobic, bacteria produce an enzyme, catalase that oxidizes the hydrogen peroxide to water and oxygen. Staphylococci are catalase positive; streptococci are catalase negative; Acid-fast bacilli produce catalase, and the detection of catalase activity is useful in the laboratory evaluation of mycobacteria.