Bacterial Identification| 8 Methods & Tests In Microbiology
Bacteria are single-celled micro-organisms and are invisible to naked eye.
They are classified under plants due to similar resemblances.
But many bacteria infect humans and animals causing deadly diseases.
They can act as parasites in almost all the organs of human body.
Further they easily spread from one person to another by means of water, air, food, insects, sex etc.
So bacterial identification is a necessary part of disease diagnosis and cure.
Without the identification of causative bacteria, it is tough to provide effective treatment with available antibiotics.
Identification of bacteria methods:
Bacteria as mentioned before or so tiny that one cannot watch without a microscope.
So either we identify them using a microscope or by enhancing their population to a huge bulk or by biochemical methods.
Hence in identification of bacteria, we use microscopical, medical, biochemical, & serological methods.
Identification by morphology:
This method is to determine the individual bacterial physical appearance.
1. Based on size: Here bacteria are identified based on their physical size. Like small size or big one expressed in mircorns. Here bacteria are viewed under a microscope to view the size. One can apply some stain to the culture use methods like glass slit or hanging drop method. In glass slit method a layer of bacterial culture is applied, stained and viewed by microscope above 40x magnification. In hanging drop method a drop of culture is made to hang between glass slide and slit and viewed under a microscope. The glass slide has a concave aperture on one side at the center where the glass slit with culture drop is placed over such that drop suspends in the concave aperture. The advantage of hanging drop method is we can identify motile bacteria.
2. Based on shape. The structure of bacteria varies like spherical (cocus), bar or stick like (bacilli), chain like (strepto), comma shaped like cholera bacteria etc. Even the shape of the bacteria colony grown on the nutrition medium also differ for a different strain of bacteria. Hence under microscope bacterial colony radius, the shape can be viewed and strain of bacteria be identified.
3. By extra-cellular characters: Here some extra-cellular characters are used in the identification of bacteria. Some bacteria have flagella for motion. So they can be identified based on the number of flagella and the arrangement of flagella on the bacterial surface. Ex: Atrichous bacteria have no flagella, Monotrichous have one flagellum, Polytrichous have many flagella. Even some bacteria have two flagella one on each side of the cell.
By Cultural characteristics: Here bacteria are identified as group or culture as a whole and not individual bacteria. Since most bacteria grow in colonies and also divide fast, they can be easily grown into a culture in suitable nutrition media. Based on the characteristics of the culture they can be identified as
a. The shape of culture: Circular, irregular, rhizoid etc.
b. Size of culture or colonies in millimeters.
c. Type of elevation of culture like effuse, convex, concave etc.
d. Margins of the colony like dentate, rough, smooth etc.
e. The surface of the colony: Like smooth, wavy, papillate etc.
f: Color of culture.
Based on resistance:
Antibiotics are specific to certain bacteria. Hence few bacteria develop resistace to certain type of antibiotics. By adding the antibiotics to culture and measuring the resistance of microbe to the said antibiotic, the type of bacteria can be identified.
Ex: TB bacteria i.e. Mycobacterium tuberculosis is not affected by penicillin but killed by ciproflaxain. So this differentiation can be used to identify few bacteria.
Few bacteria are so specific for metabolic requirements. Based on this specificity, they can be identified.
Ex: Aerobic bacteria require oxygen for survival.
Anaerobic bacteria get killed when exposed to oxygen. Similarly some require carbon dioxide, blood or other pigments for metabolism.
Identification of bacteria by Biochemical tests.
This is one of the widely used methods and more important.
a. Sugar fermentation test: Bacteria is grown in a sugar media.
b. Litmus milk test: When bacteria is grown in this medium, there may be the production of acids or alkali or even no change in pH.
c. Indole production test: Bacteria is grown in the peptone water culture. After 48 to 96hrs, incubation at 37°C, it is checked for the presence of red color. Red color indicates the production of indole from the amino acid typtophane. The reagent used is called as kovac’s reagent.
d. Methyl Red test: Bacteria is grown in glucose phosphate medium at 30°C for five days. Then few drops of 0.04% of methyl red are added and mixed to observe for color change. Red color indicates positive while yellow color indicates a negative test for glucose fermenting bacteria.
e. Citrate utilization test: This test helps identify mostly intestinal gram negative bacteria. The citrate medium also called as Koser’s medium is used here. Since the only carbon source is citrate here, its utilization is seen in terms of turbidity. If positive indicates the above-said microbes.
6. By differential staining: The identification depends on staining of bacteria. And most bacteria can be stained by specific stain ex: Gram +ve bacteria are stained by Gram satin while Gram -ve bacteria don’t take up gram stain. Tuberculous bacteria can be stained by acid-fast stain specifically and other strains don’t take up this stain.
7. Serological methods. here the identification of bacteria is done by use of antibodies and antigens which are specific against the suspected bacteria. Antigens and antibodies are very specific and bind to a single type of bacteria.
8. By protein and nucleotide analysis. the cellular constituents like protein content, nucleotide sequence in DNA are used for identification of bacteria. This requires methods like Polymerase chain reaction (PCR), gel electrophoresis, radio immuno asssay etc.
Identification of bacteria is necessary because
1. Identify the disease: It helps to know the type of infection and the disease caused by the individual. Ex. Tuberculosis can be identified by an acid-fast stain test for mycobacterium. So bacterial identification is important in health care.
2. Select suitable drug: Not all drugs (antibiotics) are active against all the bacteria. So identification helps to target that specific bacteria with suitable choice of drugs. Example rifamycin is best suited for tuberculosis and cannot be controlled by other antibiotics.
3. Evaluation of treatment progress: Identification of bacteria is also necessary to know how far the drug is effective and if the patient is freed from the said bacteria by the treatment.
During the treatment, the patient body samples are repeatedly checked during the course to see if there is a recovery in the patient. This is ascertained by performing identification test for the causative bacteria. An indication of a negative result in the test indicates, he is free from infection.
4. For industrial purposes: It also helps isolate a specific strain of bacteria needed for research or other industrial productions like fermentation etc. Many vitamins (B12), hormones (insulin) and supplements are produced by rDNA technology by using the principles of biotechnology.
5. For storage: Some bacteria are specifically stored with an intention of future requirement. Identification is also necessary to store the bacteria in pure form without foreign contamination. These stored bacteria are supplied when required for research or industrial manufacture.
Because of above advantages, identification of bacteria is of most importance in microbiology.
Unlike virus, bacteria are easily traceable by simple staining methods. Further by use of common microscope one can even see if they are moving (motile) or not moving, grouping etc. Identification of bacteria is a part of diagnosis of the many diseases and hence is a good demand for microbiologists.