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Simple Staining Procedure
Preparation of a smearUsing a sterilized inoculating loop, carry loopful of liquid suspension containing bacteria to a slide (clean grease complimentary microscopic slide) or deliver an isolated swarm from a culture plate to a slide through a water drop.Disperse the bacteria on the loop in the drop of water on the slide and also spreview the drop over a room the size of a dime. It should be a thin, even smear.Allow the smear to dry thoapproximately.Heat-solve the smear cautiously by passing the underside of the slide through the burner flame 2 or 3 times. It fixes the cell in the slide. Do not overwarm the slide as it will certainly distort the bacterial cells.
StainingCover the smear via methylene blue and permit the dye to remajor in the smear for about one minute (Staining time is not critical here; somewbelow between 30 secs to 2 minutes should offer you an acceptable stain, the longer you leave the dye in it, the darker will certainly be the stain).Using distilled water wash bottle, gently wash off the excess methylene blue from the slide by directing a gentle stream of water over the surchallenge of the slide.Wash off any type of stain that got on the bottom of the slide as well.Saturate the smear aget yet this time through Iodine. Iodine will certainly collection the stainWash of any type of excess iodine with gently running tap water. Rinse thoaround. (You might not acquire a point out of step 4 and 5 in some textbooks)Wipe the back of the slide and also blot the stained surface via bibulous paper or via a paper towel.Place the stained smear on the microscope stage smear side up and emphasis the smear using the 10X objective.Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be stupassed away, use immersion oil straight to the smear, and also emphasis the smear under oil with the 100X objective.
Left: Cocci in Cluster; Right: Bacilli (Image source: microrao.com)
ResultsThe bacterial cells typically stain uniformly and also the color of the cell counts on the kind of dye supplied. If methyene blue is provided, some granules in the internal of the cells of some bacteria may show up even more deeply stained than the remainder of the cell, which is due to visibility of different chemical substances.
UsesDiagnostic microbiology laboratory mostly does not perdevelop basic staining approaches. Differential staining such as Gram Staining and AFB Staining are typically supplied to identify and also differentiate the bacterial isolates. Simple staining have the right to be useful in the following circumstances.To differentiate bacteria from yeastern cells: When endocervical swab society is done in blood agar both Staphylococcus spp and also yeast cells may offer similar-looking colonies in Blood agar (a common error for a new technologist or microbiologist through less experience). Performing the wet mount strategy or easy staining from the isolate deserve to be valuable.
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To presumptively recognize the bacterial isolateDue to their common visibility, Bacillus spp might present as a contaminant in the culture plates. In some situations (e.g. expansion in Blood Agar but no growth in MacConessential Agar), identifying the shape of the bacteria (rod or cocci) might help to get rid of the isolate as feasible contaminants (e.g., Bacillus spp) or further procedure as a potential pathogen (cocci).