![]() To prepare a smear from a broth culture, instead of carrying out steps 2 and 3, aseptically remove several loopfuls of culture and spread over the circled area.Įxpect the smear to be far less dense than smears prepared from colonies on agar.The hottest part of the flame is just above the tip of the inner cone. To obtain a sufficiently hot flame, adjust the air-gas mixture so that the gas burns with a pale blue flame (inner cone) with a nearly colorless large outer cone.Make sure that the liquid is completely dry before passing the slide through the flame.Heating the slide briefly partially melts the cell walls, causing the cells to adhere to the glass surface.Let the slide cool for 30 sec or so before starting the staining procedure.Each smear should pass through the hottest part of the flame (see the third note below) and each pass through the flame should take about a second. Hold one end of the slide horizontally with a clothes pin and pass it through a flame three times with the smears up.Allow the drop to air dry completely (usually a couple of minutes for a single loopful).Try to disperse the culture material completely, so that there are no visible chunks of material, and spread the liquid into a thin layer within the circle. Use a loop or stick to aseptically remove a barely visible amount of material from a culture and stir it into the drop.To prepare a smear from a colony, place a loopful (~25 µl) of deionized water over the circled.Use a glass etching tool to mark one or more dime-sized circles on the surface of a slide, leaving room on one end for handling the slide.When the slide de-fogs immediately after breathing With a Kimwipe or paper towel to remove the fog. One way to clean a slide is to repeatedly breathe on it, followed by rubbing If water tends to bead up on your slides you may need to clean them before starting. ![]() ![]() Here is a description of our procedure, to be accompanied by a video demonstration. The objective is to obtain a single layer of cells so that all cells are exposed to the staining reagents. We start by preparing a smear on a clean microscope slide, then we heat-fix the cells to the surface and follow with the staining procedure. Species with a single plasma membrane surrounded by a thick peptidoglycan cell wall stain positive and species with a thin peptidoglycan wall sandwiched between two lipid membranes stain negative. We start there because the Gram stain differentiates nearly all isolates into one of two major groups, namely Gram-positive or Gram-negative (a small proportion of species stain unpredictably or not at all). Nearly every attempt at identifying an unknown bacterial culture begins with a Gram stain.
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