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IDENTIFICATION OF MICROORGANISMS (Methods for identifying microbes).pptx
IDENTIFICATION OF MICROORGANISMS **Methods for identifying microbes 1. Macroscopic Features 2. Staining and Microscopy (Gram staining, Endospore staining, Ziehl-Neelsen Staining, Stains for Fungi and Yeast) 3. Simple Biochemical Tests (Catalase Testing, Oxidase Testing) 4. Identifying Microbes Using PCR 5. Immunological Identification
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IDENTIFICATION OF MICROORGANISMS (Methods for identifying microbes).pptx
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- 2. Species-level identificationis necessary to discriminate between two species from the same genus, which is often essential in the treatment of infectious diseases. For example, the bacterial genus Yersinia contains approximately 15 species, some of which form part of the normal human microflora, but others of which are serious pathogens and require medical treatment (e.g. Yersinia pestis is the causative agent of bubonic plague).
- 3. Methods for IdentifyingMicrobes 1. Macroscopic Features Macroscopic features include the overall appearance of a microorganism, including its shape, size, color, and smell (i.e. the features that you can see with the naked eye). The type of microorganism can be determined by examining the morphological/macroscopic features on an agar culture.
- 4. Filamentous fungior molds appear as ‘hairy’ irregularly shaped colonies and often produce visible spores that may look powdery or dusty. Fungal colonies can contain more than one color, usually with a darker color in the center and a lighter color radiating from it.
- 5. Bacteria oftenform distinct colonies, which are sometimes smaller than fungal colonies and can be anything from slimy to very dry in texture. They range in color from white to bright red. Bacteria often have a strong odor while filamentous fungi can be odorless or have an earthy smell.
- 6. 2. Staining andMicroscopy Stains enable easier visualization under a microscope. Following are the most popular microbiological stains. Gram Staining Gram staining is often the go-to test in bacterial identification. This purple stain, based on the crystal violet dye, is named after the Danish bacteriologist Hans Christian Gram, who developed it. Typical Gram-positive bacteria include Bacillus, Staphylococcus, Streptococcus, and Clostridium spp., while Escherichia, Helicobacter, and Salmonella spp. are Gram-negative.
- 7. When thestain combines with bacteria in a sample, the bacteria will either stay purple or turn pink or red. If the bacteria stays purple, they are Gram-positive. If the bacteria turns pink or red, they are Gram-negative.
- 8. Endospore Staining Thisinvolves applying a stain to a bacterial sample to check for the presence of spores. Because not all bacteria produce spores, this information can be useful in identification. Several spore stains are available, but malachite green is probably the most popular.
- 9. Ziehl-Neelsen Staining This isan important tool for the staining of Mycobacterium tuberculosis (TB), which can’t be Gram-stained. The red stain carbol fuchsin is used first, followed by a counterstain such as methylene blue. M. tuberculosis stain red while other bacteria stain blue.
- 10. Stains for Fungiand Yeast Several fungal stains exist, although they are generally non-specific. They help visualize fungal elements for identification rather than discriminate between fungal species. Examples of fungal stains include: Lactophenol cottonblue, Periodic-acid Schiff stain (PAS stain), Trypan blue, and calcofluor white.
- 11. 3. Simple BiochemicalTests Catalase Testing If the unidentified bacterial species has catalase activity (enzyme that reduce Hydrogen peroxide to water and oxygen), bubbles of oxygen appear when hydrogen peroxide is added to a bacteria scraping on a microscope slide. Staphylococci, Micrococci, E. coli, Salmonella spp. are catalase-positive bacteria, while Streptococcus and Enterococcus bacteria are catalase-negative.
- 12. Oxidase Testing Theoxidase test identifies bacteria with cytochrome c oxidase (CCO) activity. This enzyme forms part of the bacterial electron transport chain. When present, CCO oxidizes the reagent (tetramethyl-p-phenylenediamine) to a purple- colored product. When the enzyme is not present, the reagent remains in the reduced state and is colorless. Bacteria with oxidase-positive include: Actinobacillus, Aeromonas spp. Vibrio spp. Oxidase- negative, on the other hand, includes E. coli, Serratia spp. and Acinetobacter spp.
- 13. 4. Identifying MicrobesUsing PCR PCR is probably the most widely used molecular technique for identifying microbes. Using PCR, one can rapidly detect and identify microbial species directly from clinical samples, thus speeding up diagnostic procedures.
- 14. 5. Immunological Identification ELISA-based methods can be set up for microbial detection on a species-by-species basis. All microbial species have unique antigen(s), which is helpful in the identification of the specific microorganism.