Simple and Differential Stains

• Methylene blue stains chromatin in the nucleus a bright blue, differentiating cell types based on their nuclear morphology.
• Simple staining depends on the fact that bacteria differ chemically from their surroundings and thus can be stained in contrast with their environment.
• Bacteria also differ from one another chemically and physically and may react differently to a given staining procedure.
• Differential staining can distinguish between bacterial types
• The Gram stain is an example of differential staining
• Gram-positive cells retain crystal violet dye while gram-negative cells lose it during decolorization
• Crystal Violet (CV) - primary stain
• Crystal Violet (CV) - primary stain
• bacteria are divided into two group gram negative and gram positive.
• A bacterial smear is a dried preparation of bacterial cells on a glass slide.
• In a bacterial smear that has been properly processed, (1) the bacteria are evenly spread out on the slide in such a concentration that they are adequately separated from one another, (2) the bacteria are not washed off the slide during staining, and(3) bacterial form is not distorted.
• making a smear, bacteria from either a broth culture or an agar slant or plate may be used.
• If a slant or plate is used, a small amount of bacterial growth is transferred to a drop of water on a glass slide and mixed, and the mixture is spread out evenly over a large area on the slide.
• Common error in smear preparation from agar cultures: using too large an inoculum results in large aggregates of bacteria piled on top of one another
• If the medium is liquid, place one or two loops of the medium directly on the slide and spread the bacteria over a large area
• Allow the slide to air dry at room temperature after spreading the bacteria
• Next step after the smear is dry: attach the bacteria to the slide by heat-fixing
• Heat-fixing is accomplished by gentle heating, passing the slide several times through the hot portion of the flame of a Bunsen burner
• Most bacteria can be fixed to the slide and killed through heat-fixing without serious distortion of cell structure
• Simple staining is the use of a single stain or dye to create contrast between the bacteria and the background
• Chief value of simple staining lies in its simplicity and ease of use
• Simple staining is often employed when information about cell shape, size, and arrangement is desired
• Procedure for simple staining:

  • Place the heat-fixed slide on a staining rack
  • Cover the smear with a small amount of the desired stain for the proper amount of time
  • Wash the stain off with water for a few seconds
  • Blot it dry
• Basic dyes used in simple staining:

  • Crystal violet (20 to 30 seconds staining time)
  • Carbolfuchsin (5 to 10 seconds staining time)
  • Methylene blue (1 minute staining time)
• Once bacteria have been properly stained, it is usually easy to discern their overall shape
• Bacterial morphology is usually uncomplicated and limited to one of a few variations
• Smear Preparation Steps:
• Use a wax pencil to mark the name of the bacterial culture in the far left corner on each of three slides
• For the broth culture:

  • Shake the culture tube
  • Aseptically transfer 1 to 2 loopfuls of bacteria to the center of the slide
  • Spread this out to about a 1/2 inch area
  • When preparing a smear from a slant or plate, place a loopful of water in the center of the slide
  • With the inoculating needle, aseptically pick up a very small amount of culture and mix into the drop of water
  • Spread this out as above
  • Prepare three slides: one each of B.subtilis or C. pseudodiphtheriticum, M. luteus, and S. volutans
• Allow the slide to air dry or place it on a slide warmer
• Pass the slide through a Bunsen burner flame 3 times to heat-fix and kill the bacteria
• Staining procedure for simple staining:

  • Stain one slide with alkaline methylene blue for 1 to 1.5 minutes
  • Stain one slide with carbolfuchsin for 5 to 10 seconds
  • Stain one slide with crystal violet for 20 to 30 seconds
• After staining, wash the stain off the slide with water for a few seconds
• Carefully blot the slide dry with bibulous paper to avoid rubbing the smear and removing the stained bacteria
• Examine the slide under the oil immersion lens and complete the report
• It is recommended to treat smears of the same bacterium with all three stains for direct comparison
• Cover bacterial smears for varying lengths of time with a given stain to understand reactivity and the effects of over-staining or under-staining
• Refer to figure 8.5a-c for examples of bacteria stained with crystal violet
• Gram Staining Steps:
• Prepare heat-fixed smears of E. coli, S. aureus, and a mixture of E. coli and S. aureus