BIOCHEMICAL TESTS: TYPES, WHAT THEY ARE FOR AND IMPORTANCE - BIOLOGY - 2024

The biochemical tests in microbiology are a set of test chemicals are carried to the microorganisms present in a sample in order to identify them; These microorganisms are usually bacteria. There are a large number of biochemical tests available to a microbiologist.

However, the choice of these tests is based on preliminary findings, such as the Gram stain pattern and growth traits, which allow the bacteria to be assigned to a particular category. Biochemical tests are mainly based on the metabolic properties of each type of bacteria.

Not all bacteria have the same properties, so it is investigated if they possess any particular enzyme by adding the substrate and waiting for the reaction to occur. Commonly this determination is given by a change in color or pH in the culture medium.

Less than 15 biochemical tests are often required for reliable identification of a bacterium down to the species level. Performing more biochemical tests can increase confidence in the identification.

Most of these biochemical tests are carried out on serum or blood plasma. However, they can also be performed on other biological secretions such as: urine, cerebrospinal fluid, pleural fluid and feces, among others.

Classification

Biochemical tests can be classified into 3 groups:

Universal

They are the tests that can be performed on any sample and that guide the microbiologist on the following biochemical tests that must be performed to obtain a reliable identification.

Example

The catalase and oxidase test.

Differentials

They are the tests carried out to identify the microorganisms present in the sample down to the species level.

Identification is made based on the results of a combination of tests, as individual results are not informative enough to make identification.

Example

IMViC tests and sugar utilization tests.

Specific

They are specific tests for a particular set of species or to subtype a species. These tests are generally done to confirm or identify at the subspecies level. The individual tests are informative in themselves.

Example

The γ-Glutamyl aminopeptidase test.

Types of biochemical tests

The catalase test

The catalase test is a test to demonstrate the presence of catalase enzyme by breaking down hydrogen peroxide into oxygen and water. A small amount of bacteria is added to a drop of hydrogen peroxide (3%) on the slide.

The catalase test is a simple test used by microbiologists to help identify species of bacteria and to determine the ability of some microbes to break down hydrogen peroxide by producing the enzyme catalase.

If bubbles of oxygen are observed, it means that the bacterium has the enzyme catalase, because it catalyzes the decomposition of hydrogen peroxide into oxygen and water. The organism is then said to be catalase positive (eg Staphylococcus aureus).

The oxidase test

This test is used to identify microorganisms that contain the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between the Enterobacteriaceae and Pseudomadaceae families.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. Artificial electron donor and acceptor molecules are provided in the oxidase test.

When the electron donor is oxidized by the action of cytochrome oxidase, the medium turns dark purple and is considered a positive result. The microorganism Pseudomonas aeruginosa is an example of an oxidase positive bacterium.

Salted Mannitol Agar (MSA) Test

This type of test is both selective and differential. The MSA will select organisms capable of living in environments with high salt concentrations, such as Staphylococcus species in contrast to Streptococcus species, whose growth is inhibited under these conditions.

The differential component in this test is mannitol sugar. Organisms capable of using mannitol as a food source will produce by-products of fermentation, which are acidic and thus lower the pH of the medium.

The acidity of the medium causes the pH indicator, phenol red, to turn yellow. Examples of species of bacteria that can be differentiated by this method are: Staphylococcus aureus (positive because it ferments mannitol) and Staphylococcus epidermidis (negative because it does not ferment mannitol).

Coagulase test

Coagulase is an enzyme that helps blood plasma clot. This test is performed on Gram positive and catalase positive bacteria species to identify Staphylococcus aureus (coagulase positive). In fact, coagulase is a virulence factor of this bacterial species.

Clot formation around an infection caused by this bacterium probably protects it from phagocytosis. This test is very useful when you want to differentiate Staphylococcus aureus from other Staphylococcus species that are coagulase negative.

The urease test

This test is used to identify bacteria capable of hydrolyzing urea, using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria.

The hydrolysis of urea produces ammonia as one of its products. This weak base increases the pH of the medium above 8.4 and the pH indicator (phenol red) changes from yellow to pink. An example of a urease positive bacteria is Proteus mirabilis.

What are biochemical tests for?

Biochemical tests in microbiology are used to diagnose diseases caused by microbes and to monitor treatments administered to combat them. Additionally, they are used for the screening of infectious diseases and for their prognosis.

The biochemical identification of microorganisms offers an idea of ​​what these microorganisms are capable of doing, being possible the discrimination of different strains of the same species by specific biochemical profiles.

Differences in specific enzyme activities inform the ecology, physiology or natural habitat of the microorganism, which in some cases can be considered important information.

Importance

Structural differences in the shape, size, and arrangement of bacteria help little in the identification process, because there are many species of bacteria that are similar in shape, size, and arrangement.

For this reason, the identification of bacteria is ultimately based primarily on differences in their biochemical activities.

Each species of bacteria has a well-defined set of metabolic activities different from all other species. These biochemical "fingerprints" are properties controlled by bacterial enzymes.

Thus, biochemical tests are important because they help the researcher to correctly identify the pathogens present in a sample and, in this way, to be able to recommend the appropriate treatment to the patient.

References

1. Beckett, G., Walker, S. & Rae, P. (2010). Clinical Biochemistry (8th ed.). Wiley-Blackwell.
2. Clarke, PH, & Cowan, ST (1952). Biochemical methods for bacteriology. Journal of General Microbiology, 6 (1952), 187-197.
3. Gaw, A., Murphy, M., Srivastava, R., Cowan, R., St, D. & O'Reilly, J. (2013). Clinical Biochemistry (5th ed.). Elsevier Health Sciences.
4. Goldman, E. & Green, L. (2008). Practical Handbook of Microbiology (2nd ed.). CRC Press.
5. Harrigan, W. (1998). Laboratory Methods in Food Microbiology (3rd ed.). Academic Press.
6. Vasanthakumari, R. (2009). Practical Microbiology. BI Publications Pvt Ltd.