The staining technique you will most often use are simple staining, negative staining and Gram staining. Once a heat-fixed or air-dried smear of a sample is made, you can add a drop of methylene blue for one minute and then wash off the dye. Dry the slide again, then add a drop of immersion oil and observe the sample using an oil immersion objective lens. For negative staining, you could use a stain such as eosin or nigrosin. Indian ink is a good substitute. The negative charges on the molecules of the stain will not penetrate the cells of some microorganisms (e.g. bacteria), because these cells have negative charge on their surface. There is no need to need to heat fix these cells, so there is no risk of any distortion to their shape. The microorganisms will show up as stained against a dark background. This method is ideal for finding the true shape and size of bacterial cells. To perform negative staining of bacteria, place a loopful of bacteria in a spot of Indian ink, positioned at one end of a microscope slide. Use another microscope slide to spread the mixture out and form a thin smear. Allow the slide to dry in the air and then place a drop of immersion oil onto the dried slide. Then you can examine the bacteria using oil immersion.
Gram stain is the most widely used stain, because Gram staining is one of the first stages performed to identify a bacterium. Gram stain divides bacterial cells into two major groups; Gram- positive or Gram-negative. Gram stain is an example of a differential stain. To perform Gram staining, apply four different chemical reagents in sequence to a heat-fixed smear of bacteria. The first regent, crystal violet, is called the primary stain; this colours all the cells purple. The second reagent, Gram’s iodine, is a mordant; this intensifies the purple colour. In Gram-positive cells, the crystal violet-Gram’s iodine complex binds to the magnesium- RNA content of the thick bacterial walls made of peptidoglycan, forming a complex that is difficult to remove. The third reagent, 95% ethanol, is a de-colouriser. It removes the colour from the Gram-negative cells that do not have the same structure in their cell walls and do not bind to the crystal violet-Gram’s iodine complex. The Gram-negative bacterial cell walls also contain more lipids, which the ethanol dissolves; this leads to large pores, through which the crystal violet dye leaks out. The fourth reagent, safranin, is a counterstain; this adds a different colour, pink, to the de-colourised Gram-negative cells.