Starch Hydrolysis Test for Bacteria and more

Nitrate Reduction Broth:

Bacterial species may be classified into different groups depends on  their ability to reduce nitrate to nitrite or nitrogenous gases provided in the growth medium. The reduction in nitrate can be coupled  to anaerobic respiration in some bacterial species.  Nitrate, present in the broth, is reduced to nitrite which  is  then reduced to nitric oxide, nitrous oxide, or nitrogen. The basis of nitrate reduction test the detection of nitrite and its ability to form a red colored  compound when it reacts with reagent A which is 

sulfanilic acid  and to form a complex (nitrite-sulfanilic acid) which then reacts with Reagent B which is α-naphthylamine to give a red precipitate (prontosil). Zinc powder act as a catalyst and that will favours the reduction of nitrate to nitrite. Nitrate reaction occurs only under anaerobic conditions (Fig 8). The medium is then transferred in tubes to make a low surface area to depth ratio that will limit the diffusion of oxygen into the growth medium. Most bacteria utilize the available oxygen in the medium for their growth and will rapidly produce anaerobic conditions for the further reactions.

Catalase Test

The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase may explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the toxic concentration of H₂O₂ cannot be degraded when these organisms are cultivated in the presence of oxygen. Organisms capable of producing catalase rapidly degrade hydrogen peroxide which is a tetramer  containing four polypeptide chains, which are usually  500 amino acids long. It also contains four porphyrin heme groups(ie., iron groups) that will allow the enzyme to react with the hydrogen peroxide.

The enzyme catalase is present in most cytochrome containing aerobic and facultative anaerobic bacteria. Catalase has one of the highest turnover numbers of all enzymes such that one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen in a second.

Catalase production and activity can be detected by adding the substrate H₂O₂ to an appropriately incubated (18- to 24-hour) tryptic soy agar slant culture. Organisms which produce the enzyme break down the hydrogen peroxide, and the resulting O₂ production produces bubbles in the reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the catalase enzyme and are unable to break down hydrogen peroxide, into O₂ and water and are catalase negative.  

Coagulase Test:

Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting. The coagulase test identifies whether an organism produces this exoenzyme. This enzyme clots the plasma component of blood. The only significant disease-causing bacteria of humans that produce coagulase are Staphylococcus aureus. Thus this enzyme is a good indicator of the pathogenic potential of S. aureus. In the test, the sample is added to rabbit plasma and held at 37° C for a specified period of time. Formation of clot within 4 hours is indicated as a positive result and indicative of a virulent Staphylococcus aureus strain. The absence of coagulation after 24 hours of incubation is a negative result, indicative of an avirulent strain.

 Oxidase Test:

Oxidase test is an important differential procedure that should be performed on all gram-negative bacteria for their rapid identification. The test depends on the ability of certain bacteria to produce indophenol blue from the oxidation of dimethyl-p-phenylenediamine and α-naphthol. This method uses N,N-dimethyl-p-phenylenediamine oxalate in which all Staphylococci were oxidase negative. In presence of the enzyme cytochrome oxidase (gram-negative bacteria) the N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol react to indophenol blue. Pseudomonas aeruginosa is an oxidase positive organism.

Starch Hydrolysis Test:

Amylases are a class of enzymes that are capable of digesting these glycosidic linkages found in starches. Amylases can be derived from a variety of sources. Amylases are present in all living organisms, but the enzymes vary in activity, specificity and requirements from species to species and even from tissue to tissue in the same organism. Alpha-amylase (1,4 alpha D-Glucan-glucanohydrolase) acts upon large polymers of starch at internal bonds and cleaves them to short glucose polymers. Alpha-amylase catalyzes the hydrolysis of internal Alpha-1-4 glucan bonds in polysaccharides containing 3 or more alpha 1-4 linkages; it results in a mixture of maltose and glucose. Amyloglucosidase works on the shorter polymers and splits off single glucose sugars. Bacterial alpha-amylase is particularly suited for industrial usage since it is inexpensive and isothermally stable.
Starch agar is an example of differential medium which tests the ability of an organism to produce certain alpha-amylase and oligo-1, 6-glucosidase that hydrolyze starch. Starch molecules are too large to enter into the bacterial cells, so some bacteria will secrete exoenzymes that will degrade starch into subunits that can be then easily utilized by the organism.
Starch agar is a simple nutritive medium with starch added.  Since no colour change occurs in the medium when organisms hydrolyze starch, iodine solution is added  to the plate after incubation. Iodine turns blue, purple, or black (the colour depends on the concentration of the iodine used) in the presence of starch. A clearing around the bacterial growth shows that the organism has hydrolyzed starch.

Lipid Hydrolysis:

Trybutyrene agar is used for the detection and enumeration of lipolytic microorganisms in food and other material (Fig 9).

                                   Fig 9:  Lipid hydrolysis: Left side;positive for lipid hydrolysis;right side;negative for lipid hydrolysis

Growth on selective and differential media:

 Selective media allows only the growth of certain types of organisms, while inhibiting the growth of other organisms.

Eg: Mannitol salt agar, Hektoen enteric agar (HE), Phenylethyl alcohol agar.

Differential media are employed to differentiate certain closely related organisms or groups of organisms. Depending on the presence of specific dyes or chemicals in the growth media, the organisms will tend to produce certain specific characteristic changes or growth patterns that can be used for further identification or differentiation steps.
 

Eg: MacConkey (MCK)agar, Eosin Methylene Blue (EMB) agar .

Enriched media are media that have been supplemented with highly nutritious materials such as blood, serum or yeast extract for the purpose of cultivating fastidious organisms.
Eg: Blood agar, Chocolate agar
 

Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from mixed cultures.

 Eosin methylene blue agar is both a selective and differential medium used for the detecting and isolating Gram-negative pathogens residing in the intestine.

MacConkey’s Agar is both a selective & differential media that is selective for Gram negative bacteria and can differentiate those bacteria that are able to ferment lactose.

Different streptococci produce different effects on the red blood cells in blood agar. Those that produce incomplete hemolysis and only partial destruction of the cells around colonies are called alpha-hemolytic Streptococci. Characteristically, this type of hemolysis is seen as a distinct greening of the agar in the hemolytic zone, and thus this group of Streptococci has also been referred to as the viridans group.

Species whose hemolysins cause complete destruction of red cells in the agar zones surrounding their colonies are said to be beta-hemolytic. When growing on blood agar, beta-hemolytic Streptococci are small opaque or semi translucent colonies surrounded by clear zones in a red opaque medium.

Some species of Streptococci do not produce hemolysins. Therefore, when their colonies grow on blood agar, no change is seen in the red blood cells around them. These species are referred to as nonhemolytic or gamma hemolytic Streptococci.

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