Simple staining



Principle

 In the simple staining bacterial smear is stained with a single reagent, which produces distinctive contrast between an organism and its background. Basic stains with a positively charged chromogen are preferred because bacterial nuclei and cell wall are negatively charged. That charge will ultimately result in a strong binding with the positively charged chromogen. The purpose of staining is to visualize the morphology and arrangement of the bacterial cells.

Materials

24h nutrient agar culture of Staphylococcus or E. coli, Methylene blue, crystal violet, carbol fuchsin, Bunsen burner, glass slides, inoculation loop, staining tray, bibulous/blotting paper, and microscope. 

Procedure

  1. Prepare the bacterial smear as previously described.
  2. After heat fixation, place the slide over the staining tray and flood with any of the simple stains so that it can cover the whole area.
  3. Wait for the proper exposure time for the respective stain: carbol fuchsin; 15-30 sec, Crystal violet; 2060 second, and methylene blue; 1-2 min.
  4. Gently wash the smear with tap water to remove excess stain. During washing, hold the slide parallel to the water stream to avoid the loss of bacteria by the water stream.
  5. Dry the slide by using blotting paper, never wipe the slide.
  6. Examine under the microscope oil immersion lens.
Various shapes are: cocci (spheres), bacilli (Rods/cylinders), spiral (helical), Curved rods (vibrio)
 Various arrangements: diplococci (pairs), streptococci (cocci chains), staphylococci (cocci clusters), streptobacillus (bacilli chains), sarcina/ tetrads (cluster of four), palisades (stacks) 
Spores locations: Terminal spores (non bulging), terminal spores (bulging), subterminal spores, central spores.

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