Summary
Highlights
The endospore stain is used to identify endospores produced by genera like Bacillus and Clostridium. Begin with a fixed smear of Bacillus. Cover the smear with a small piece of paper towel and saturate it with malachite green. Steam the slide on a boiling water bath for five minutes, adding more stain if needed to keep the paper towel wet. Remove the slide, let it cool for one minute, then wash with distilled water until clear. Counterstain with safranin for 30 seconds, then wash again with distilled water. Gently blot the slide dry. Under the microscope, endospores will appear as green structures within red cells, or as free green structures of uniform size and shape.
Capsules are visualized using negative staining. Place a loopful of Congo red on a slide and create a thick smear of the test bacterium in the dye. Allow the smear to air dry without heat-fixing. Fix the smear with acid alcohol for approximately 15 seconds; the Congo red will change color. Wash the smear with distilled water until clear. Cover the smear with acid fuchsin for one minute to stain the cells, then wash and gently blot dry. Examine under the microscope. The negatively charged Congo red is repelled by the bacterial surface, staining the background dark. The capsules appear as a clear, white halo around the red-stained cells, as the acid fuchsin stains the cell but not the capsule.
Bacterial motility is best observed in a wet mount. For a broth culture, place a drop of the culture on a slide, flaming the loop and tube mouth appropriately. Gently lower a coverslip onto the broth. For a solid medium, place two to three loopfuls of water on a slide, then add a loopful of bacteria from the plate to make a thick suspension. Gently lower a coverslip. Examine under the microscope, noting that Brownian movement (vibration of cells without movement relative to each other) is not true motility. Discard slides in disinfectant after viewing.
Flagella are too thin to see with a light microscope but can be viewed with special staining. Handle cells carefully to prevent flagella breakage. Sterilize a scalpel and forceps by dipping in alcohol and flaming. Use the sterile scalpel and forceps to cut a piece of agar with the test bacterium growing on it. Gently place the agar culture side down on a slide, then carefully remove the agar, returning it to the Petri plate. Replace the cover. Sterilize the instruments again. Let the slide air dry (do not fix). Cover the slide with flagella mordant for 10 minutes to thicken the flagella. Gently rinse the slide with distilled water and allow it to air dry (do not blot). Examine under the microscope; cells and flagella will appear brown. Observe the arrangement of the flagella on the cells. After viewing, blot oil from the slide and store.