India ink staining whole blood9/1/2023 In Gram staining bacteria fixed to a slide are treated with a basic dye that binds electrostatically to the negativelyĬharged cells. Overheat the smear, melting cell walls and possibly breaking the slide.Heat the smear before letting it air dry, boiling the bacteria instead of attaching them.Use so much liquid that it takes forever to dry.Use too much material - before air drying the smear the liquid should be somewhat cloudy and without particulate matter.After cooling the slide, conduct the staining procedure.Each pass through the flame should take about a second. Hold the slide horizontally with a clothes pin and pass it through a flame three times with the smear up, to kill the bacteria and cause them to adhere. Light a bunsen or Fisher burner and adjust to produce a vigorous flame.Allow the drop to air dry completely (usually a couple of minutes for a single loopful).To prepare a smear from a broth culture, aseptically remove several loopfuls of culture and spread over the circled area.Try to disperse the culture material completely, so that there are no visible chunks of material. Aseptically remove a barely visible amount of material from a culture with a loop or stick and use the loop or stick to mix it with the drop. To prepare a smear from a colony, place a loopful (~25 µl) of deionized water over the circledĪrea.When the slide de-fogs immediately after breathing Vigorously with a Kimwipe or paper towel to remove the fog. A good way to clean a slide is to repeatedly breathe on it, followed by rubbing The circles help you locate smears that are hard to see with the naked eye and help you locate the surface of the slide on which you have made the smear. Use a glass etching tool to mark a circle on the under side of a slide at each positioin at which you plan to make a smear.Too few, and they cannot be located on the slide. Giving false positives or crowding each other to make a mess. Is to learn to recognize the correct density of bacteria to place on the slide. It also kills them, rendering pathogenic bacteria safe to handle. It causes bacteria to adhere to a slide so that they can be stainedĪnd observed. Preparing a smearĪ properly prepared smear accomplishes two things. Shapes, patterns of association, and motility. You will observe living bacteria to become familiar with features that can be seen without staining, including cell You will also carry out spore staining on appropriate cultures. To clearly distinguish shapes of bacteria. You will learn the technique of negative staining with nigrosine dye in order In the laboratory you will practice the Gram stain technique on a variety of Gram positive and Gram negative bacteria As with the Gram stain, a spore stain distinguishes spores Stains or by phase contrast microscopy of living cells, however differential staining methods may be necessary toĬonfirm or reject the presence of spores in a culture. Some species produce spores, which are dormant cells with thickened cell walls. Cells remain unstained against a dark background. Acidic dyes have a negative chargeĪnd are repelled by the negatively charged cell walls. In indirect, or negative, staining, smearsĪre produced by mixing material with India ink or acidic dyes such as nigrosine. The Gram stain is a direct method, since the cells themselves retain dye. Since two dyes are used to distinguish types ofīacteria, Gram staining is called a differential staining method. Gram negative bacteria do not retain the dark blue color, but can be counterstainedĪ light red so that they can be seen in bright field microscopy. A species can be classified as Gram positive, Gram negative, or Gram variable depending on the ability ifĬells to retain the blue dye. The Gram stain is routinely used as an initial procedure in the identification of an unknown bacterial species.īacteria bear a slight net negative charge and usually bind positively charged dyes such as methylene blue and crystal Slide and air dried, then fixed to the surface by passing the slide quickly through a flame, melting the complexĬarbohydrates of the cell walls to the glass and killing the cells. The rigid cell walls of bacteria prevent distortion of morphology upon drying, samples can be spread onto a glass The staining methods we will use kill the bacteria, reducing the risk of infection by pathogenic organisms. Of stains have been developed to distinguish spores, nuclear bodies, capsules, and characteristics of the cell wall. To reveal different properties and to enhance contrast for viewing with conventional bright field microscopy. However bacteria are routinely stained with different dyes in order The phase contrast or dark field microscope. Important information such as shape and degree of motility can be obtained by observation of living bacteria with
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