Bacterial Stains. Overview. In our laboratory, bacterial morphology (form and structure) may be examined in two ways: by observing living unstained organisms (wet mount). by observing killed stained organisms.
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1: see greater contrast between the organism and the background,
2: differentiate various morphological types (by shape, arrangement, gram reaction, etc.),
3: observe certain structures (flagella, capsules, endospores, etc.).
Example of a dye:
Methylene Blue Chloride
MBCl MB+ + Cl-
1. Simple stain
2. Differential Stain
3. Special stain
1. Obtain broth cultures of the bacteria.
2. Using an inoculating loop, remove a loopful of suspension from one of the tubes. Remember to use sterile technique.
3. Smear the bacteria across the center of the slide with the loop. If the bacterial suspension is very thick, add a drop of water and mix the bacteria and the water on the slide.
4. Allow the smear to completely air dry.
5. Heat-fix the smear by quickly passing the slide through a Bunsen burner flame three times. This causes partial melting of the cell walls and membranes of the bacteria, and makes them stick to the slide. Do not overheat the slide as this will destroy the bacteria.
6. Cover the smear with a few drops of one of the stains. Allow the stain to remain for the following periods of time:
Carbolfuchsin- 15-30 seconds.
Methylene blue- 1-2 minutes.
Nigrosin- 20-60 seconds.
8. Gently blot the excess water from the slide with bibulous paper. Do not wipe the slide. Allow the slide to air dry.
Observe the slide under the microscope with air and oil lenses.
A coverslip is not required. Repeat this process with the other bacteria and stains. Note the differences between the various types of stains and their appearances
Crystal violet precipitate on epithelial cell:
May be confused with Gram positive cocci
Crystal violet precipitate crystal on gram stain
Acid Fast bacilli (red)
Non Acid Fast bacilli (blue)
Acid Fast bacilli (red) mixed with non acid fast (blue cocci
Acidic dye as India Ink and Nigrosen use to stain the background of the slide but basic dye as methylene blue and crystal violet use to stain the cell
Capsules appear as clear zones (halos) around the refractile organism.
Bacteria with capsules: Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas .