Biochemical tests are the tests that are used for the identification or confirmation of different bacterial species or different micro organisms based on the biochemical activities that the microorganisms are performing.
In order to classify different bacteria as unique or different from each other we need to identify them as unique and to do their structural and chemical differences are identified. Identification based on morphology in case of bacteria, because structural differences, which may differentiate one species from the other, are not visible even under a microscope.
The structural differences such as shape, size and morphology of bacteria do not help much if we want to identify the different species of the bacteria, because there are various species of bacteria that have similar shape, size and arrangement. Hence, the identification of bacteria should majorly be based on the differences in the bacterial biochemical.
Some bacterial species perform a metabolic process which is known as fermentation, where they utilize sugars and produce ATP or also known as the energy source of the bacteria. The utilization of the sugar depends upon the specific enzymes that the bacteria shall possess. Bacteria use the nutrients that are present in their environment (for example, the media in which they are inoculated) to produce ATP (energy source) for their biological processes such as growth (i.e. cell division) and reproduction. Many bacteria possess the enzyme system required for the oxidation and utilization of the simple sugar, glucose. Some bacteria have the ability to break down complex carbohydrates like lactose, sucrose or even polysaccharides, into simpler sugars that they can then utilise to produce energy required by other metabolic processes. Such bacteria shall have the required enzyme that is able to cleave the bonds between the sugar units so the resulting simple carbohydrate can be transported into the cell.
Glucose is the sugar that we get from almost every food that we eat and the body uses it to produce energy for our metabolism. It is the simplest form of carbohydrate.
Sucrose is the common sugar that we use in day to day life. It is comprised of glucose and fructose. The enzyme sucrase breaks down the sucrose (also known table sugar) into its constituent units i.e., glucose and fructose.
Lactose is a disaccharide consisting of the glucose and galactose bonded by glycosidic bond. The bacteria which produce the enzyme lactase has the ability to break this bond and thus release free glucose that can easily be utilized by the bacteria, hence, lactase breaks lactose (or milk sugar) into glucose and galactose.
Mannitol is a sugar alcohol most commonly used as a medication. It is given to diabetic patients as it is poorly absorbed in intestine.
This ability of the bacteria to produce such enzymes enables them to use various carbohydrates which in turn help in the identification of unknown bacteria.
Carbohydrate utilisation tests (Glucose, Lactose, Sucrose, Mannitol)
The carbohydrate fermentation test is used to determine whether or not a bacteria has the ability to ferment (or utilise) a specific carbohydrate (or the sugar). Carbohydrate utilisation test’s results are useful in differentiating different bacterial species. The results tell us which carbohydrate can be utilised by the bacteria for fermentation. It tests for the presence of acid and/or gas production from carbohydrate fermentation.
Carbohydrate utilisation broth:
Phenol red broth is a media that is generally used as a fermentation media, it comprises of sodium chloride, trypticase, phenol red and any carbohydrate.
The trypticase serves as a supplier of amino acids, minerals, vitamins, and other nitrogenous substances so that it can provide nutrition to the microorganisms.
Sodium chloride provides a balance to the osmotic pressure and provides the essential electrolytes for the transportation purpose (into the cell).
Carbohydrate serves as the energy source for the bacteria.
Phenol red acts as the pH indicator which is initially at neutral (pH 7). Once the fermentation is completed by the bacteria which will lead to the ph change of the medium the colour of the pH will accordingly change indicating the acidity of the medium.
The phenol red broth supports growth of most of the bacteria whether they are able to ferment sugar or not. But here when we want to find out if the bacteria is able to ferment the carbohydrate that is provided to the medium, the production of acid should be observed. The production of acid is confirmed with the change in the colour of the phenol red that was provided to the medium.
Glucose acts as an electron donor. The other carbohydrates are first converted into glucose and then the fermentation reaction will occur.
When the fermentation is completed, the reaction will result in the end products like acid, ethanol, Carbon dioxide, Hydrogen and other compounds, which is depended on the species of bacteria that is performing fermentation. The production of gas is confirmed by placing inverted Durham’s tubes into the test tubes containing the phenol red medium. If gas production will be there, it will be visible as a bubble at the top of the Durham’s tube. Acid production is indicator of the production of lactic acid, acetic acid or formic acid which lowers the pH of the medium and may turn the colour of the medium to yellow. The pink colour at the top of the medium containing tube indicates the rise in pH or the production of alkaline products.
In some bacteria oxygen (O2) is reduced to toxic substances called hydrogen peroxide (H2O2) and superoxide (O2-) due to the presence of flavoproteins that catalyse this reaction. These substances are powerful oxidizing agents and have the ability to destroy cellular components very frequently. These toxic substances ultimately will result in the death of the bacteria if they are not reduced to their previous form. Hence, in order to protect itself it produces enzymes that catalyse the reduction of these toxic substances.
The reduction of the hydrogen peroxide is as follows:
2H2O2 à 2H2O + O2 (Catalysed by Catalase enzyme)
Catalase activity (or the catalase production) is a very useful test in differentiating between groups of bacteria which cannot be distinguished on the basis of their morphology. For example, the species that are morphologically similar like: Enterococcus (catalase negative) and Staphylococcus (catalase positive), can be differentiated using this test.
In this test one or two drops of the 24 hour culture broth of test bacteria is mixed with two or three drops of freshly 3% hydrogen peroxide. After about 15 seconds, the presence of gas bubbles is observed for the presence of the catalase enzyme in the bacteria.
Coagulases are the enzymes that help in the blood plasma clot by a mechanism that is quite similar to normal clotting. Coagulases enzyme cross-links α and β chain of fibrinogen that is present in the blood plasma to form fibrin, which is a clot that deposits on the cell wall. As a result, individual coccus stick to each other and clump is observed.
The coagulase test is used to identify whether an organism is capable to produce this enzyme. This enzyme clots the plasma component of blood. The only significant disease causing bacteria of humans that produce coagulase enzyme are Staphylococcus aureus. Hence this test is performed to check the pathogenic potential of Staphylococcus aureus.
The action of coagulase enzyme in human host causes clotting of plasma by converting fibrinogen to fibrin.
Most strains of S.aureus produce one or two types of coagulase:
1) Free coagulase.
2) Bound coagulase.
Free coagulase is an extracellular enzyme which reacts with prothrombin. It is heat labile. It has seven antigenic types. It is secreted extracellular. It can be detected in tube coagulase test.
Bound coagulase is localized on the surface of the cell wall. It is heat stable. It has only one antigenic type. It is a cell wall associated protein. It can be detected in slide coagulase test. Slide coagulase test is used to screen isolates of S.aureus, whereas, tube coagulase is used for further confirmation.
In the coagulase test, the sample is added to rabbit plasma and is kept at 37° C for a specified period of time. Clot formation occurs within 4 hours and is interpreted as a positive result and is an indicative of the presence of a virulent Staphylococcus aureus strain. The absence of coagulation after 24 hours of incubation is a negative result, which is an indicative of presence of an avirulent strain of Staphylococcus aureus.