Enteric organism produces pyruvic acid from glucose metabolism. This test detects the ability of microorganism to ferment glucose and to produce acidic end products. The absence of red color is indicative of tryptophan hydrolysis due to the lack of tryptophanse enzyme(Fig 3). Indole will react with Kovac's reagent to produce a cherry red complex, which indicates a positive indole test. If the enzyme is present, it will degrade the aminoacid tryptophan in the media and will produce Indole, ammonia and pyruvic acid. Indole tests looks for the presence or absence of tryptophanase enzyme production of the bacteria. They are Indole test,Methyl red test, Voges proskauer test and Citrate test collectively known as IMViC series of reactions. Usually four tests are used for differentiation of the various members of Enterobactericeae. Gram negative family includes Shigella, Salmonella, Proteus, Klebsiella,Escherichia,Enterobacter etc. Hence they are the main causative agents of intestinal infection. Gram negative enteric bacilli play an important role in the contamination of food. Gram positive bacteriaįig 2: Bacteria identification from stains The study of morphological features and staining characteristics help in the preliminary identification of the isolate. Hence after the gram staining, the gram positive cells appear as purple and gram negative cells appear as pink (Fig 2). But in the case of gram negative, the thin peptidoglycan layer and more lipid contents in the cell wall will easily make them susceptible to the action of decolorizer and hence CVI complex is easily washed out and hence the gram negative cells will the colour of counter stain Safranin. Hence the counter stain Safranin has no action on gram positive cells. Gram positive cells due to their thick peptidoglycan layer will retain the CVI complex even after it is subjected to decolourization with acetone or alcohol. The primary stain Crystal violet and mordent Iodine form a strong CVI complex all bacteria. Usually it differentiates bacteria into two groups gram positive and gram negative. ![]() Gram staining is a differential staining technique that imparts different colours to different bacteria or bacterial structures. The strong negative charge of the bacterial cell will strongly bind with the positive charged basic dyes and will impart its colour to all bacteria. The simple stain uses the basic dyes such as Methylene blue or basic fuschin. Simple staining is used to study the morphology of all microorganisms (Fig 1). Staining is a simple basic technique that is used to identify microorganisms. In order to obtain the pure culture of organism, the isolated colonies are aseptically transferred on to different nutrient agar slant tubes and incubated overnight at 37 degree Celsius. Nature: discrete or confluent, filiform, spreading or rhizoid. Degree of growth : scanty, moderate or profuse. Structure: opaque, translucent or transparent. Colour: Yelow, green etc.( Note the colour of the colony). Edges: entire, undulate, crenated, fimbriate or curled. Surface: Smooth, wavy, rough, granular, papillate or glistening. Elevation: elevated, convex, concave, umbonate/umbilicate. Size: small, medium, large( or in millimetres). The characteristics features of the colonies on solid agar media are then noted. Upon incubation, individual colonies will arise from the biomass. This will make a dilution gradient across the agar plate. The inoculation of the culture is made on the agar surface by back and forth streaking with the inoculation loop over the solid agar surface. The streak plate is a qualitative isolation method quadrant streaking is mostly done to obtain pure colonies. The importance of this step is to isolate pure colonies of bacteria. Various steps involved in the identification of unknown bacteria are: Qualitative observations were made on the tests, which were compared to unknown bacteria identification key to aid with the identification process. ![]() Multiple tests were performed to provide the fermentation abilities, presence of certain enzymes, and certain biochemical reactions. ![]() It produces benefits for many aspects of the research of microorganisms and helps physicians correctly treat patients. The identification of bacteria is a careful and systematic process that uses many different techniques to narrow down the types of bacteria that are present in an unknown bacterial culture. Isolation and Identification of Two Bacterial Unknowns Principle:
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