Biochemical Test: 13 Principles That Are Easy To Grasp

1. DNAse Test

Deoxyribonuclease test is a biochemical test that investigates the ability of bacteria to produce DNA cleaving enzyme deoxyribonuclease. The reagents for the test include 2 gms/l DNA, hydrochloric acid, and Tyrosine Agar. Unhydrolyzed DNA precipitates in the presence of hydrochloric acid, and bacteria that produce DNAse utilize DNA for carbon and energy production. and therefore the expected observation for a positive test includes a clear zone around the colony of bacteria producing DNAse.

It is a test for the identification of Staphylococcus aureus.

2. Bacitracin Test

This is a test that distinguishes Group A streptococci (Streptococcus pyogenes) from the other streptococci. a bacitracin disk contains an antibacterial that inhibits the growth of Streptococcus pyogenes, which shows a clear zone of inhibition on an agar plate if present, thus constituting a positive test

3. Optochin Test

The optochin test is a biochemical test that involve the use of a filter paper impregnated with Ethyl Hydrocuprein Hydrochloride. It inhibits the growth of Streptococcus pneumoniae while allowing all the other bacteria present in the sample to grow. This, therefore, shows a clear zone of inhibition.

4. Urease Test

Urease is an enzyme that breaks down urea into ammonia and carbon (IV) oxide gas. the ammonia formed dissolve in the broth thus changing the pH to basic (8.1 from 6.8) resulting in phenol red color change to pinkish, thus constituting a positive result. This test has sometimes been referred to as campylobacter like organism test in the detection of Helicobacter pylori because of its ability to produce urease enzyme.

Of important to note, this test puts into consideration time factor because there are organisms that produce a lot of urease in a short time while others produce little urease enzyme over a long time. As well the person setting up the test should ensure that the apparatus does not contain Nessler’s reagent as it degrades Urease.

5. Aesculin Test

Aesculin or Esculin is a coumarin glycoside that is found in trees (Aesculus sp.) and in coffee (dandelion coffee). Bile Aesculin agar is composed of bile salts to inhibit other gram-positive bacteria, ferric citrate to confirm hydrolysis of aesculin, sodium azide to inhibit the growth of gram-negative organisms and aesculin which acts as a substrate for the enzyme aesculinase produced by the target organism in culture.

In the absence of ferric citrate, the culture media can be observed under fluorescent light as its hydrolysis results in loss of fluorescence. Otherwise, the aesculetin that results from its hydrolysis reacts with ferric ions to form a brown to black precipitate, which appears like a black or brown hollow around the colony.

6. Citrate Utilization Test

This is a biochemical test that examines the bacteria’s ability to utilize citrate as the only source of carbon in its energy production cycles. its major purpose is to differentiate members of Enterobacteriaceae family based on their metabolic by-products or density of bacterial growth.

This test can be done on two types of media namely Koser’s citrate media and Simon’s citrate agar. in the former turbidity is observed while in the later, color change from green to blue is observed if the test is positive.

7. Indole Test

Indole test is used to identify bacteria in the family Enterobacteriaceae with the ability to produce tryptophanase wich converts tryptophan into indole through reductive deamination. The reagents required are P3 broth and Kovacs or Ehrlich reagent. A positive reaction is shown by the formation of a brick-red ring in the Kovac’s reagent.

8. Methyl Red Test

Methyl red test identifies bacteria which are able to produce a large amount of acid when supplied with glucose. this test is reliant on the fact that methyl red only detects acid at very low pH, usually below 4.4. a positive test is confirmed if the media turns from yellow to red. organism such as E. Coli is methyl red positive and Enterobacter Aerogenes is methyl red negative.

9. Vogey’s Proskauer Test

Organisms such as Klebsiella sp., Enterobacter sp., Hafnia sp. and Serratia sp. produce Acetyl Methyl Carbinol (Acetoin) as the main final product with small amounts of mixed acids after glucose metabolism. The presence of acetoin is detected by reaction with 40% potassium hydroxide, in which it is converted to diacetyl, a reaction catalyzed by alpha naphthol. a red complex result which confirms a positive result.

10. Germ Tube Test

Candida albicans yeast forms are converted into mycelia when incubated in human plasma or serum at 37 degrees Celcius in a partially anaerobic state. these pseudohyphae are only observed in Candida albicans species thus differentiating it from other Candida species

11. Oxidase Test

Cytochrome oxidase or indophenol oxidase catalyzes the transport of electrons from NADH to oxygen (usually). Bacteria which are oxidase-positive possess these enzymes. In an oxidase test, they are detected through subjection to a substrate which changes color when acted upon by the enzymes such as TMPD or DMPD. the substrates act as artificial electron donors to the enzyme oxidase resulting in a purple or blue colored oxidized product, which confirms a positive reaction.

12. Coagulase Test

This is a biochemical test used to differentiate Staphylococcus aureus from the other coagulase-negative staphylococci (CONS). the former produces the coagulase enzyme which converts the soluble fibrinogen toa fibrin clot. there are however two forms of coagulase; one which is bound and one which is free tested by slide method and tube method respectively.

13. Catalase Test

Catalase is an enzyme produced by aerobic organisms to neutralize toxic products formed when oxygen is used in their metabolism such as hydrogen peroxide which has bactericidal effects.

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