9/28/2023 0 Comments India ink negative stain![]() ![]() DO NOT RINSE WITH WATER so you do not dissolve the capsules that are present.Ĩ. Allow the dye to remain on the slide for 2 minutes.ħ. Heat fixing will shrink the organisms and either produce artifacts that look like capsules or the heat will destroy the capsules that are present.Ħ. Sterilize the loop, allow it to cool and add another loopful of inoculum onto the slide. Gently place the inoculum on the slide.Ĥ. Remove the cap of the Klebsiella in the litmus milk, insert the inoculating loop and obtain a loopful of inoculum. Sterilize an inoculating loop and allow it to cool.ģ. Dispose of the cotton swab in the regular trash.Ģ. Clean a clean glass slide with lens cleaner and a cotton swab. PROCEDURE Prepare a bacterial smear for Capsule Stain (Anthony Methodįor this exercise – Use Klebsiella aerogenes in litmus milk brothġ. Since the capsules do NOT pick up any stain, the results produce a blue background, clear outline of the capsules, and dark blue microorganisms inside the capsule. The Anthony’s method for staining capsules uses litmus milk to provide a proteinaceous background for contrast. Bacterial capsules are soluble in water, so we do not rinse with water in capsule staining. Heat damages certain cellular features including bacterial glycocalyx (capsules and slime layers), therefore we do not heat fix when negative staining. Nigrosin and India ink are both examples of negative stains commonly used in microbiology, although any negatively charged stain maybe used. Therefore, in negative staining the background (rather than the microorganism) is stained. Negative stains are repelled by the negatively charged bacterial cells preventing the cells from taking up the stain. We need to utilize negative staining techniques to view the outline of the capsule. Streptococcus pneumoniae, for example, can be found as part of the normal microbiome of mucous membranes, yet it is pathogenic when it produces a capsule.Ĭapsules are non-ionic (no net charge) meaning capsules will not stain with a positively charged stain or a negatively charged stain. The capsule contributes to the virulence of various microbes. It is a source of nutrition, and some bacteria may find the need to metabolize their capsules as an energy source if food in their environment runs out. The capsule is sticky and helps bacteria attach to skin and mucous membranes. The capsule aids the bacteria in invading the human body by helping it to resist phagocytosis by white blood cells. Capsules may be made of polysaccharides, glycoprotein, or polypeptides. Capsules are generally easier to visualize than slime layers. If the structure is quite tightly bound, highly organized, and generally round or oval, it is called a capsule. If this structure is very loosely bound and somewhat irregularly shaped, it is called a slime layer. ![]() Many bacteria secrete a rather sticky substance that adheres to the cell and forms a coating around it called a glycocalyx. In a positive stain the microorganisms are stained and the background is not stained. Most staining procedures in the microbiology laboratory (simple, Gram, endospore, and acid fast stains) use a positive stain technique. ![]()
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