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Microbiology Lab Procedure Notes

Microbiology Lab Procedure Notes By ©2018 Shirley S. Chung, Green River Community College

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Smear

  1. Clean slide
  2. Label: my name, organism, date, medium

Broth Smear

  1. Agitate broth unless otherwise stated
  2. FLAME, 1 loopful in center of slide spread to dime-size.
  3. FLAME, 2nd loopful in center of slide spread to dime-size.
  4. FLAME

Solid Smear

  1. FLAME, 1 loopful water (or drop)
  2. FLAME, 2nd loopful water
  3. FLAME 1 loopful specimen and mix well in water.
  4. FLAME 2nd loopful specimen if needed and mix well in water.
  5. FLAME
  6. Air Dry
  7. Heat Fix
  8. Use clothespin.
  9. Pass slide’s underside over the flame (coolest part of flame)
  10. 3 passes

Simple Stain (Methylene blue, Crystal Violet, Carbol Fuchsin)

  1. Smear and heat fix.
  2. Flood stain

Carbol Fuchsin, 15-30 sec

Crystal Violet, 20-60 sec

Meth. Blue, 1-2 min

  1. Rinse with Di Water
  2. Bibulous blot, Do not wipe

Gram Stain

  1. Flood with Crystal Violet
  2. Wait 1 min
  3. Flush with Di water
  4. Flood with Gram’s Iodine mordant
  5. Wait 1 min
  6. Flush with Di water
  7. Decolorize
  8. Flush immediately
  9. Counterstain Safranin
  10. Wait 45 sec
  11. Flush with Di water
  12. Bibulous Blot

Acid-Fast Staining

  1. Smear and heat fix.
  2. Flood smears with carbol fuchsin
  3. Place over a beaker of water on a warm hot plate
  4. Steam 5 minutes (do NOT allow stain to evaporate, replenish as needed)

OR

Flood smear with carbol fuchsin WITH TERGITOL for 5-10 minutes

  1. If slides are heated, allow to cool.
  2. Flush with Di water
  3. Decolorize with acid-alcohol
  4. Flush with Di water
  5. Counterstain with Meth. Blue for 2 min
  6. Flush with Di water
  7. Bibulous blot.

Spore Staining

  1. Smear and heat fix
  2. Flood with malachite green
  3. Place on beaker of water sitting on hot plate
  4. Steam for 2-3 minutes (do NOT allow stain to evaporate, replenish as needed)
  5. Remove from hot plate
  6. Cool
  7. Flush with Di water (this is the decolorizing step)
  8. Counterstain Safranin for 30 seconds
  9. Flush with Di water
  10. 10. Bibulous blot

 

Microbiology Lab Procedure Notes By ©2018 Shirley S. Chung, Green River Community College