![]() This eliminates the parameter: smear thickness and focuses on reagent quality and staining and destaining times. Note : It is recommended to carry out, with the same preparation of diluted stools, one or more dozens of slides which are dried, fixed and wrapped separately with aluminum foil. There should appear many Gram positive and negative bacteria. Spread a thin layer of stool on a slide and color it. Decolorizing agent left too long or poorly rinsed.Lugol's solution left for too little time,.In any population of Gram + we see Gram - (sometimes very numerous): these are corpses of Gram + bacteria,.Some bacteria are even called “low gram” or “variable gram” (ex: corynebacteria).īe careful, poor staining can lead to bacterial identification errors or poor diagnostic orientation. It should be noted, however, that Gram staining can be variable for the same species (for example, pneumococcus discolors easily, likewise, certain Gram-positive bacteria when the colonies are old). Gram-negative bacteria will stain pink/red and Gram-positive bacteria will stain blue/purple. One may use the intermediate acting solution of 95% ethanol with acetone, which decolorizes in ∼7 to 10 sec ( acetone can be used as the decolorizer with a 1- to 2-sec reaction time). Note : 95% ethanol is the slowest acting solution (10 to 20 sec) and, thus, the most forgiving, it is suggested for beginners. Examine under the microscope, x100 objective Observe the results of the staining procedure under oil immersion.Be sure the bottom of the slide is clean. Wash the slide in a soft, indirect stream of tap water until no color appears in the effluent, then dry with paper towel.Flood the slide with counterstain, 'safranin' and wait 30 seconds to 1 minute.Flood the blade with decolorizing agent and wait 15 seconds or add drop by drop to release decolorizing agent.Flood with the mordant: iodine or lugol and wait 1 minute.Wash the blade in a gentle, indirect stream of tap water for 2 seconds.Please note that the quality of the smear (too heavy or too light cell concentration) will affect the staining results. ![]()
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