Biochemical Tests in Microbiology: Types, What They Serve and Importance

The biochemical tests in microbiology they are a set of chemical tests that are made to the microorganisms present in a sample in order to identify them; These microorganisms are usually bacteria. There is 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 characters, which allow the bacteria to be assigned to a particular category. The biochemical tests are based mainly on the metabolic properties of each type of bacteria.

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

Often less than 15 biochemical tests are required for the reliable identification of a bacterium up to the species level. Performing more biochemical tests can increase confidence in identification.

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

Classification

The 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 reliable identification.

Example

The catalase and oxidase test.

Differentials

These are the tests that are carried out to identify the microorganisms present in the sample up to the species level.

The identification is made based on the results of a combination of tests, since the individual results are not informative enough to make the 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 usually done to confirm or identify at the subspecies level. The individual tests are informative by 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 decomposing 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 bacterial species and to determine the ability of some microbes to break down hydrogen peroxide by producing the catalase enzyme.

If oxygen bubbles are observed it means that the bacterium possesses the catalase enzyme, because it catalyzes the decomposition of hydrogen peroxide into oxygen and water. It is then said that the organism is catalase positive (for example: 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 families Enterobacteriaceae and Pseudomadaceae.

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

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

Salted Mannitol Agar Test (MSA)

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 species of Staphylococcus in contrast to the species of Streptococcus , whose growth is inhibited in these conditions.

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

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

Coagulase test

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

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

The urease test

This test is used to identify bacteria capable of hydrolyzing urea, using the enzyme urease. It is commonly used to distinguish gender Proteus of 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 positive urease bacterium is Proteus mirabilis .

What is the use of biochemical tests in microbiology?

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

Importance

The structural differences with respect to the shape, size and disposition of the bacteria help very little in the identification process, because there are many species of bacteria that have a similar shape, size and arrangement.

For this reason, ultimately the identification of bacteria is based mainly on the 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, P. H., & Cowan, S. T. (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.