Phenotypic Identification: Gram Staining PDF

PHENOTYPIC IDENTIFICATION: MICROBIOAL TAXONOMY PREPARED BY: MA. CRISTINA GRAM STAINING BARNES, RMT MICROSCOPY Microscopy is the most common method used both for the detection of microorganisms directly in clinical specimens and for the characterization...

PHENOTYPIC IDENTIFICATION: MICROBIOAL TAXONOMY PREPARED BY: MA. CRISTINA GRAM STAINING BARNES, RMT MICROSCOPY Microscopy is the most common method used both for the detection of microorganisms directly in clinical specimens and for the characterization of organisms grown in culture. DIRECT AND INDIRECT SMEAR Staining methods are either used directly with patient specimens or are applied to preparations made from microorganism. DIRECT SMEAR A direct smear is a preparation of the primary clinical sample received in the laboratory for processing. A direct smear provides a mechanism to identify the number and type of cells present in a specimen, including white blood cells, epithelial cells, and predominant organism type. GRAM STAINS OF DIRECT SMEARS SHOWING SQUAMOUS CELLS AND BACTERIA POLYMORPHONUCLEAR LEUKOCYTES AND BACTERIA DIRECT SMEAR Occasionally an organism may grow in culture that was not seen in the direct smear. Potential reasons: a slow-growing organism was present the patient was receiving antibiotic treatment to prevent growth of the organism the specimen was not processed appropriately and the organisms are no longer viable the organism requires special media for growth INDIRECT SMEAR Preparation of an indirect smear indicates that the primary sample has been processed in culture and the smear contains organisms obtained after purification or growth on artificial media. INDIRECT SMEAR Indirect smears may include preparation from solid or semisolid media or broth. ensure that the smear is not too thick when preparing the slide from solid media. smear from a liquid broth should not be diluted SOLID MEDIA LIQIUD MEDIA SEMI-SOLID MEDIA DIRECT SMEAR Generally, specimen samples are placed on the slide using a swab or a direct smear that contains patient material or by using a pipette into which liquid specimen has been aspirated. DIRECT SMEAR Material to be stained is dropped (if liquid) or rolled (if on a swab) onto the surface of a clean, dry, glass slide. To prevent contamination of culture media, once a swab has touched the surface of a nonsterile slide, it should not be used for subsequently inoculating media. INDIRECT SMEAR For staining microorganisms grown in culture or an indirect smear, a sterile loop or needle may be used to transfer a small amount of growth from a solid medium to the surface of the slide. This material is emulsified in a drop of sterile water or saline on the slide. INDIRECT SMEAR Smears should be air-dried completely before heat fixing to prevent the distortion of cell shapes before staining. To examine organisms grown in liquid medium, an aspirated sample of the broth culture is applied to the slide, air-dried, and fixed before staining. GRAM STAIN Gram stain is the principal stain used for microscopic examination of bacteria and is one of the most important bacteriologic techniques within the microbiology laboratory. Gram staining provides a mechanism for the rapid presumptive identification of pathogens. GRAM STAIN Nearly all clinically important bacteria can be detected using this method, the only exceptions are: organisms that exist almost exclusively within host cells (e.g., chlamydia) those that lack a cell wall (e.g., mycoplasma and ureaplasma) and those of insufficient dimension to be resolved by light microscopy (e.g., spirochetes). GRAM STAIN First devised by Hans Christian Gram during the late nineteenth century, the Gram stain can be used to divide most bacterial species into two large groups: those that take up the basic dye, crystal violet (i.e., gram-positive bacteria) those that allow the crystal violet dye to wash out easily with the decolorizer alcohol or acetone (i.e., gram-negative bacteria). GRAM STAIN Therefore the Gram stain is considered a differential stain, based on the chemical differentiation of organisms as a result of the structural chemical components of the organism’s cell wall. GRAM STAIN Gram stain procedure entails fixing clinical material to the surface of the microscope slide, either by heating or by using methanol. Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimens.