Staining (Microbiology)

COLONY CHARACTERS The appearence of bacterial colonies when cultured on semisolid media are also useful for identification. When inoculated on to a solid medium, bacteria develop into colonies of characteristic size, shape, colour, sheen, elevation and margin, which aid in identification. Common colony features are listed below and are illustrated in Fig. 13. nse Punceate Small Spreading (pimbread) (a) Surface —___ am a A. Flat Raised Convex Umbonste (b) Elevation Circular CS C ; (entire) Undatated Rhivoid Lobae fc) Margin Fig. 13° Morphological features of bacterial colonies Morphology and Classification of Bocisria vu * Colour Translucent, white, creamy, yellow, red. * Elevation Fiat, raised, convex, urmbonate, * Sheen Smooth and shining, dull. © Margin Circular, undulated, rhizoid, lobate. * Size Punctate (pinhead colonies), small, large, spreading,Staining Procedures As they exist in nature, bacteria are colorless, transparent, and difficult to see. Therefore, various staining methods have been devised to enable scientists to examine bacteria. In preparation for staining, the bacteria are smeared onto a glass microscope slide (resulting in what is known as a “smear”), air-dried, and then “fixed.” (Methods for prepar- ing and fixing smears are further described in CD-ROM Appendix 5: “Clinical Microbiology Laboratory Proce- dures.) The two most common methods of fixation are heat-fixation and methanol-fixation. Heat-fixation is usu- ally accomplished by passing the smear through a Bunsen: burner flame. If not performed properly, excess heat can distort the morphology of the cells. Methanol fixation, which is accomplished by flooding the smear with absolute methanol for 30 seconds, is a more satisfactory fixation technique, Fixation serves three purposes: © it kills the organisms © it preserves their morphology (shape) * itanchors the smear to the slide AA. Sear book ot BAe © dig rnanot mmusebes ent Shao aa on om =f Flood wide F. Examine wth win sain vi00 ory (odiennersion) FIGURE 6-12. Simple bacterial staining technique. (A) Wh a flamed loop, smear a looptl of bacteria saspended in broth or water onto a slide (8) Al ‘ow side to air-dry. (C) Facthe smear with absolute (100%) methanol (D) Flood the side with the stain. (F) Rinse wath water and blot dry with bibulous paper or pape tome. (F} Examine the sie with the * 100 microscope objective, using a drop a immersion ol directly onthe smear.Specific stains and staining techniques are used to observe bacterial cell morphology (eat, size, shape, marpho- logic arrangement, composition of cell wall, capsules, flagella, and endospores). ‘A.simple stains sufficient to determine bacterial shape and morphologic arrangement (c.g. pairs. chains, clusters). The Origin of the Gram Stain While working in a laboratory in the morgue of = Bertin hospital in the 1880s, a Danish physician ‘named Hans Christian Gram developed what yas to become the most important of all bacterial staining. procedures. He was developing a staining technigue Ai ese es eee a Hea sues of patients who had died of sl eg ana el ‘stain—demonstrated that two general categories of bacteria cause prcumonia: some of them stained blue and some of them stained red. The blue ones came to be known as Gram-positive bacteria, and the red ones came to be known as Gram-negative bacte- fia. It was not until 1968 that the mechanism of ‘Gram differentiation was explained by M.RJ, Salton. For this method, shown in Figure 4-12, a dye (such as meth- ‘ylene blue) is applied to the fixed smear, rinsed, dried, and ‘examined using the oil immersion lens of the microscope. The procedures used to observe bactevial capsules, spores, and flagella are collectively referred to as structural statn- ing In 1883, Dr. Hans Christian Gram developed a staining technique that bears his name—the Gram stain or Gram staining procedure. (The details ofthis staining procedure are found in CD-ROM Appendix 5: “Clinical Microbiology Laboratory Procedures.”) The Gram stain has become the most important staining procedure in the bacteriology laboratory, because it differentiates between “Gram-positive” and “Gram-negative” bacteria (these terms will be explained shortly). The organism's Gram reaction serves as an extremely important “clue” when attempting to leam ‘the identity (species) of a particular bacterium. There are nine steps in the Gram staining procedure, as described in ‘CD-ROM Appendix 5: “Clinical Microbiology Laboratory Procedures.” ‘The color of the bacteria at the end of the Gram staining ‘procedure depends on the chemical composition of their cell wall (Table 4-5), Ifthe bacteria were not decolorized during the decolorization step, they will be blue-to-purple at the conclusion of the Gram stai procedure; such ‘bacteria are said to be “Gram-positive.” The thick layer of ‘peptidoglyean in the eel! walls of Gram-positive bacteria makes it dificult to remove the crystal violet~fodine com- plex during the decolorization step. Figures 4-13 through (4-17 depict various Gram-positive bacteria. 1f, on the other hand, the erystal violet was removed ‘rom the cells during the decolorization step. and the cells ‘were subsequently stained by the safranin fa red dye), they

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