Fermentation of sugar is one of the characteristic property of some bacteria this can be tested easily and can be used to identify that particular bacteria from the mixed culture of bacteria. Beside, fermentation other biochemical properties are also noted for the identification of bacterial colonies. Following are some common biochemical properties used to identify bacteria.
This fermentation property is basically tested in sugar media. Acid production is shown by change in the color of the medium to pink or red, and the gas produced collected in Durham’s tube.
This test is done when there may be no change in the medium, or acid or alkali may produced; clothing of milk, pretonisation or saponification may occur. The clot may be disrupted by the gas produced (stormy fermentation).
Indole production test can be done to identify bacterial colonies. This is tested in a peptone water culture after 48 or 96 hours incubation at 37 C. this test demonstrates the production of indole from tryptophan. Add 0.5 ml Kovac’s reagent and shake gently. Red color indicates a positive reaction.
Kovac’s Reagent Is Cosists Of
• Paradimethylaminobenzaldehyde 10 g
• Amyl or isoamyl alcohol 150 ml
• Concentrated HCl 50 ml
This reagent is prepared in small quantities and stored in the refrigerator.
Methyl Red Test (MR)
This test is about to detection of acid production during the fermentation of glucose and the maintenance of a pH below 4.5 in an old culture. Five drops of 0.04% solution of methyl red are added to the culture in glucose phosphate medium which had been incubated at 30 C for i’ve days, mixed well and read at once. Red color is positive while yellow signifies a negative test.
Voges-Prokauer (VP) Test
This test is done to identify the production of acetyl methylcarbinol from pyruvic acid, as an intermediate stage in its conversion to 2 -3 butylene glycol. In the presence of alkali and atmospheric oxygen, the small amount of acetyl methyl carbinol present in the medium is oxidized to diacetyl which reacts with the peptone of the broth to give a red color.
VP test is performed by adding 0.6 ml of 5% solution of alpha-naphthol in ethanol and 0.2 ml of 40% KOH to one ml of a glucose phosphate medium culture of the organism incubated at 30 C for five days or 37 C for 48 hours. In a positive reaction, pink color appears in 2 -5 minutes, deepening to magenta or crimson in half an hour. In a negative reaction, it remains colorless for half an hour. Traces of pink coloration should be ignored.
Loser’s citrate medium has citrate as the sole source of carbon. Ability to use this substance is indicated by the production of turbidity in the medium.
Indole, MR, VP and citrate tests are very useful in the identification and classification of enteric Gram negative bacteria. These test are commonly referred to by he sigla “IMViC” test.
This is a test for the presence of the enzyme nitrate re-educates which reduces nitrate to nitrite. Nitrate reduction is tested from a culture of five days at 37 C in a broth containing 1% KNO3. The test reagent consist of a mixture of equal volume of solutions of sulphanilic acid and alpha-naphthylamic in 57 N alpha acetic acid mixed just before use. 0.1 ml of the test reagent is added to the culture. A red color developing within a few minuted signifies a positive reaction, while absence of color indicates a negative reaction.
Production Of Ammonia
Production of ammonia is tested to a peptone water culture grown for five days at 37 C, Nessler’s reagent is added. Brown color is positive and faint yellow color negative.
This test is done in Christensen’s urease medium. Inoculate the slope heavily and incubate at 37 C. examine after four hours and after overnight incubation. The test should not be considered negative till after four days of incubation. Urease positive cultures produce a purple pink color. Urease producing bacteria reduce urea to ammonia which is responsible for the color.
Hydrogen Sulfide Production
Some organisms decompose sulphur – containing amino acids producing H2S among the products. When cultured in media containing leas acetate, they turn them black or brown. Instead of lead acetate, ferric ammonium citrate or ferrous acetate can be used. The organisms can be grown in culture pure tubes. Between the cotton plug and the tube insert a filter paper strip soaked in 10% lead acetate solution and dried. Browning of the paper indicated H2S production.
Methylene Blue Reduction
One drop of 1% aqueous methylene blue is added to the broth culture, and incubated at 37 C. complete decolonization is strongly positive, while green color is weakly positive.
Place a loopful H2O2 on colonies on nutrient agar. Prompt effervescence indicates catalase production. Culture media containing blood are unsuitable for the test as blood contains catalase.
Egg Yolk Reaction
Organisms producing lecithin are (for example, Cl. Perfringens) when grown on a solid egg yolk medium, from colonies surrounded by a zone o clearing.
Growth In Presence Of KCN
Buffered liquid medium containing KCN in a final concentration o about 1/13,000 is used to identify some KCN tolerant enteric bacilli.
Composite media are being used increasingly for the identification of isolates. These are convenient and economical, as a single composite medium indicates different properties of the bacterium which otherwise would have required the use of many separate media.
A popular composite medium is the Triple Sugar Iron (TSI) medium which indicates whether a bacterium ferment glucose only, or lactose and sucrose also, with or without gas formation, besides indicating H2S production as well. The medium is distributed in tubes, with a butt and slant.
After inoculation if the slant remains red and the butt becomes yellow, all the sugars – glucose, lactose and sucrose – are fermented. Bubbles in the medium shows formation of H2S. the TSI medium facilitates preliminary identification of Gram negative bacilli.
Other tests such as fermentation of organic acids, oxidation of gluconate, amino acid decarboxylation, and hydrolysis of sodium hippurate and sometimes employed. With increasing knowledge of the metabolic processes in the growth of various bacteria, the number of tests too is on the increase. Special manuals have to consulted for the details and utility of these test.
Rapid Identification Methods
While classical phenotypic characterization of isolates takes days, automated methods are now available which only take hours. Identification is simplified by the detection of specific enzymes, toxins, antigens or metabolic end products of the isolates. For example, many obligate anaerobes can be identified rapidly by gas liquid chromatography of the short chain fatty acids produced by them during glucose fermentation. Molecular methods such as polymerase chain reaction and other amplification procedures coupled with nuclei acid probes carrying specific DNA or RNA base sequences are now widely used for identifying microbes.