Traditional biochemical tests for microbial identification
Current tests for microbial identification can be split into traditional methods and modern methods. Simple biochemical tests such as catalase testing, oxidase testing, and substrate utilization tests fit under the category of traditional tests, alongside staining and microscopy methods such as gram staining, endospore staining, and Ziehl-Neelsen staining.
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Catalase activity is specific to certain bacterial strains such as Staphylococci, Micrococci, E. coli and the other Enterobacteriacaea, and Salmonella spp. Others are known not to cause this activity, such as Streptococcus and Enterococcus bacteria. Catalase testing involves inducing catalase activity by adding hydrogen peroxide to a bacterial scraping placed on a microscope slide. Bubbles that appear on the slide indicates catalase activity and suggests the presence of catalase-positive bacteria.
Bacteria with cytochrome c oxidase activity (CCO) can be identified with oxidase testing. When present, the COO enzyme, which forms part of the bacteria's electron transport chain, oxidizes tetramethyl-p-phenylenediamine (used as a reagent). This oxidation reaction causes the reagent to turn purple in color, therefore, the presence of this enzyme can be confirmed visually, as when the enzyme is not present, the reagent does not change color.
The limitation of oxidation tests is that they are vulnerable to inaccurate results given that while oxidase-positive bacteria are aerobic, some are capable of anaerobic respiration also. Additionally, false-negative results can be induced if the bacterial species under investigation possesses an oxidase that does not react with the reagent.
Finally, a range of substrate utilization tests is commercially available for microbial identification. These tests involve the addition of unknown bacterial species to a panel of substrates. Scientists can then identify the bacteria based on the color changes induced in the separate panels, demonstrating unique patterns associated with specific bacterial species. These tests are often conducted alongside catalase and/or oxidase tests in order to increase accuracy and reliability.
Modern biochemical tests for microbial identification
In addition to traditional methods, recent years have seen new, modern methods emerge that have been adapted for microbial identification. Polymerase chain reaction (PCR), and immunological tests such as ELISA are among these modern methods. Newer biochemical tests that have emerged include fatty acid profiling and metabolic/chemo profiling.
Fatty acids are vital to the construction of the cell membranes of bacterial cells. As with enzymes, fatty acid profiles are unique to specific bacterial species. Therefore, fatty acids obtained from a species of unknown bacteria can be used to identify it. The methods of gas chromatography and mass spectrometry are most commonly used to conduct fatty acid profiling.
Finally, metabolic/chemo profiling is used to detect the unique secondary metabolic profiles of bacteria. All microbes produce metabolites, primary metabolites such as ATP that are associated with basic functioning, and secondary metabolites, such as antibiotics and immunosuppressive compounds that are more specific to the species of bacteria.
These secondary profiles can be measured by methods such as high-performance liquid chromatography (HPLC) and mass spectrometry to identify the species of bacteria that they are associated with.