The tests described in this Section are of general usefulness in the characterization and identification of bacteria, Some organisms require the use of an isolation medium specially adapted to particular nutritional or osmotic requirements. In other cases, a selective medium maybe employed which not only allows the isolation of the organisms under investigation but also incorpotates one or more biochemical tests
1.CATALASE TEST
This test demonstrates the, presence of catalase,an enzyme that catalyze the release of oxygen from hydrogen peroxide. Catalase is an enzyme which catalyses the decomposition of toxic hydrogen peroxide into harmless oxygen.and water.
The enzyme catalase, is presents in most cytochrome containing aerobic and facultative anaerobic bacteria. The main exception is streptococcus, It,is .primarily;,used to differentiate the genus Sireptocgceus (-) fom. Micrococcus (+), Staphylococcus (+) and aerobic Bacillusispecies (+) from the closely related but anaerobic clostridium species(-).
Hydrogen peroxide is used as a potent antimicrobial agent when cells are infected with a pathogen, Pathogens that are catalase powbve such, ,asi) Mycobactarium, tuberculosis, Legionella pneumophila, Campylobacter jejuni, make catalase in order to deactivate the peroxide radicals, thus allowing them to survive unharmed within the host.
PRINCIPLE OF CATALASE TEST
Catalase acts like a catalyst in the breakdown of hydrogen peroxide to oxygen and water. An organism is tested for catalase production by bringing it into contact with hydrogen peroxide. Bubbles of oxygen are released if the organism is catalase producer.
2. COAGULASE TEST
Coagulase is a protein (enzyme) produced by several microorganisms that enables the conversion of fibrinogen to fibrin. In the laboratory, it is used to distinguish between different types of Staphylococcus isolates; Staphylococcus aureus (+ve) while S. epidermidis (-ve). Coagulase reacts with prothrombin in the blood.
The result is a complex-staphlothrombin, which enables the enzyme to convert fibrinogen to fibrin. This results in clotting of blood. Thus, coagulase induces clotting through an alternative pathway which is not dependent on calcium ions.
A pathogenic Staphylococcus, S. aureus has the power of clotting or coagulating blood plasma. This is due to the production by the pathogenic Staphylococcus of the enzyme coagulase. Since Staphylococcus aureus is defined as the species consisting of the coagulase positive strains of the staphylococci, the test for coagulase production is the conclusive identifying test for the species.
Two types of coagulase are produced by most strains of S. aureus.
- Free coagulase, which converts fibrinogen to fibrin by activating a coagulase-reacting factor present in plasma. Free coagulase is detected by the appearance of a fibrin clot in a test tube.
- Bound coagulase (also known as _ clumping factor) which converts fibrinogen directly to fibrin without requiring coagulase-reacting factor. It is detected by clumping of bacterial cells in the rapid slide test.
3. OXIDASE TEST
The oxidase test is used to determine the presence of oxidase enzyme in organisms. The oxidase reaction is due to the presence of cytochrome oxidase system which catalyses the oxidation of reduced cytochrome by molecular oxygen. This molecular oxygen acts as an electron acceptor in the terminal stage of the electron transport system. Oxidase positive organisms are aerobic or facultative anaerobic, because they contain the cytochrome system which makes them capable of utilizing oxygen as a final electron acceptor. They. are alsa able to reduce molecular hydrogen to hydrogen peroxide ” (H2O2), which if allowed, to accumulate can kill the organism. Thus, all oxidase ‘positive organisms are also catalase positive.
During the test, artificial substrate such as tetdiméthyl-para phenylene diamine are, used as electron’ acceptor. Upon addition of this substrate, (oxidase reagent),’a deep purple colour, is developed indicating the Presence of the oxidase enzyme., This test was originally deviced to identify all Neisseria spp but later came into use to separate Pseudomonas (+ve), Aeromonas (+ve) and “Alcaligenes (+ve) from the members of the Enterobacteriaceae which give negative reactions.
4. CITRATE TEST
The test is based on the ability of an organism to use Citrate as its only source of carbon and ammonia as its only source of nitrogen. This test is one of several techniques used to assist in the identification of Enterobacteria, klebsiella spp, i Citrobacter spp, and Enterobacter spp utilize citrate but Escherichia coli does not. Pseudomonas aeruginosa and Proteus mirabilis are also citrate utilizers.
PRINCIPLE OF CITRATE TEST
The test organism is cultured in a medium which contains sodium citrate, an ammonium salt, and the indicator bromothymol blue. Growth in the medium is shown by turbidity and a change in colour of the indicator from light green to blue, due to the alkaline reaction, following citrate utilization.
5. UREASE TEST
Urease is an enzyme that catalyses the hydrolysis of urea into carbon dioxide and ammonia. Many gastro intestinal and urinary tract pathogens produce urease, enabling the detection of urease to be used as a diagnostic tool to detect the presence of pathogens. Urease positive pathogens include Proteus vulgaris, Klebsiella spp, Morganella sp, Helicobacter pylori etc.
PRINCIPLE OF UREASE TEST
The test organism is cultured in a medium which contains urea and the indicator phenol red. If the strain is urease producing, the enzymes will breakdown the urea to give ammonia and carbon-dioxide. With the release of ammonia, the medium becomes alkaline as shown by a change in colour of the indicator to pink (red-pink).
6. INDOLE TEST
Some organisms will breakdown tryptophan (contained in peptone water) to indole within 48-96 h at.37°c. Indole-testis for the detection of the production of indole in peptone water. Tryptophan can be oxidized by certain bacteria to form three major indolic metabolites including;
1. Indole
2. Methylindole
3. Indole acetic acid (IAA)
Through the agency of intracellular enzymes known as tryptophanase. The tryptophan in the test medium is supplied by peptone. Tryptone is the peptone in the peptone water since it is rich in tryptophan. PRINCIPLE The test organism is cultured in a‘medium which contains tryptophan (PW). Indole production is detected by kovac’s Or Ehrlich’s reagent which contains 4-p-dimethyl aminobenzaldehyde. This reacts with the indole to produce a red-colored compound. The indole test is used in differentiation between the genera Edwardsiella (+ve) from Salmonella (-ve) and to differentiate E. coli (+ve) from Klebsiella Enterobacter group (-ve).
7. MR-VP TEST
This test is occasionally used to assist in the differentiation of Enterobacteria. Fermentation of glucose by organisms will ultimately yield pyruvic acid, CH3.CO.COOH.
8. METHYL RED TEST (MR)
This is used to test the ability of an organism to produce and maintain stable acid end products from glucose fermentation. The test aids in the differentiation between E. coli (+ve) from Enterobacter aerogenes (-ve); Yersinia sp (+ve) from other gram negative, non-enteric bacilli which are -ve. It also aids in the identification of Listeria monocytogenes (+ve).
The MR test is based on the use of a ph indicator-methyl red to determine the H+ concentration present when an organism ferments glucose. However the MR negative organisms, though they produce acid in the fermentation of glucose continue further to metabolize by decarboxylation producing neutral end products such as 2,3-butanediol and acetoin, otherwise known as acetyl methylcarbinol.
9. VOGES-PROSKAUER TEST (VP)
This is used to determine the ability of some organisms to produce a neutral end product, acetoin, from glucose fermentation. The V.P test is used primarily to separate E. coli (-ve) from Klebsiella (+ve) and Enterobacter group (+ve). Glucose is metabolized to pyruvic acid which is the key intermediate in glycolysis.
From pyruvic acid, there are many pathways – production of acetoin being one pathway for glucose degradation. Production of 2, 3 – butanediol, causes an increase in the production of carbon dioxide and the accumulation of acetoin.
In the presence of atmospheric oxygen and alkali, the neutral end products -acetoin and 2, 3-butanediol are oxidized to diacetyl which reacts with the VP reagent to produce the characteristic pink or red colour. Enterobacteria group is usually either MR +ve & VP -ve or MR -ve & VP +ve. Klebsiella oxytoca is the only organism that is both MR +ve & VP +ve.
10. VP TEST (FOR NEGATIVE MR TEST)
This test detects acetyl-methyl carbinol which, in the | presence of potassium hydroxide, KOH, oxidizes in the atmosphere to form diacetyl. This diacetyl forms a pink colour with alpha – naphthol.
11. HYDROGEN SULPHIDE PRODUCTION (H2S)
Some organisms decompose sulphur – containing aminoacids cystine to form hydrogen sulphide amongst the products.
The hydrogen sulphide liberated can be detected by incorporating a heavy metal salt into the medium. Hydrogen sulphide reacts with these compounds to form black metal sulphides. 10% lead acetate strips or the hydrogen sulphide medium, e.g Kligler Iron Agar (KIA) are used. The detection of hydrogen sulphide gas is used mainly to assist in the identification of enterobacteria.
12. NITRATE REDUCTION TEST
This test is used to differentiate members of the Enterobacteriaceae that produce the enzyme nitrate reductase from gram negative bacteria that do not produce the enzyme. It is also used in differentiating Mycobacterium species.
13. PRINCIPLE NITRATE REDUCTION TEST
A heavy inoculum of the test organism is incubated in a broth containing nitrate. After 4h, it is tested for the reduction of nitrate by adding sulphanilic acid reagent. If nitrite is present the acid reagent diazotized and forms a pink-red compound with alpha-naphthylamine.
When nitrite is not detected, it is necessary to test whether the organism has reduced the nitrate beyond nitrite. This is done indirectly by checking whether the broth still contains nitrate. Zinc dust is added which will convert any nitrate to nitrite. If no nitrite is detected when the zinc dust is added, it can be assumed that the entire nitrate has been reduced beyond nitrite to nitrogen gas or ammonia by a nitrate reducing organism.
14. LITMUS MILK TEST
Litmus milk medium is used in the litmus milk reduction test to identify Enterococci. It can also be used to demonstrate the “stormy clot” reaction of Clostridium perfringens, The reduction of Litmus milk is by an enzymatic action and shown by a decolourization of the Litmus. Litmus milk test indicates both saccharolytic and proteolytic properties of bacteria by detecting whether they ferment Lactose or digest casein. Lactose fermenter in litmus milk will form acid and cause it to become pink. Large amounts of acid will precipitate the casein as a clot and if gas is formed during coagulation, the clots will be disrupted by it (storming clot).
15. PRINCIPLE LITMUS MILK TEST
A heavy inoculum of the test organism is incubated for up to 4h in a tube containing litmus milk. Reduction of the litmus milk is indicated by change in colour of the medium from mauve to white or pale yellow.
16. HYDROLYSIS OF STARCH
Starch is a complex sugar consisting of two long chains of D-glucose. Some bacteria are able to secrete amylase (also called diastase) to breakdown starch into maltose and glucose.
17. FERMENTATION OF CARBOHYDRATES
A wide variety of carbohydrates is fermented by bacteria and the pattern of fermentation is characteristic of certain species, genera or other taxonomic groups of organisms.
Thus, the ability of different organisms to ferment specific carbohydrates is used in their identification and classification. It is essential that the medium used for this test shall be free from all carbohydrates except those particularly added. Nutrient broth is useless for this purpose as it contains small amount of muscle sugar.
The medium consists of peptone water. The selected carbohydrates and ‘ indicator is added to the medium and it is dispensed in tubes or bottles containing a small Durham’s tube. The Durham’s tube must be inverted and completely filled with the medium.
Since the usual end products of carbohydrates fermentation are acid and/or acid and gas, the indicator will reveal production of acid and the inverted Durham’s tube | will trap gas, if produced. For the fermentation reactions of more delicate organisms such as Streptococcus pneumonia and Corynebacterium diphtheria, the medium is enriched with serum.