Microbiology Lab Procedure Notes By ©2018 Shirley S. Chung, Green River Community College
Smear
- Clean slide
- Label: my name, organism, date, medium
Broth Smear
- Agitate broth unless otherwise stated
- FLAME, 1 loopful in center of slide spread to dime-size.
- FLAME, 2nd loopful in center of slide spread to dime-size.
- FLAME
Solid Smear
- FLAME, 1 loopful water (or drop)
- FLAME, 2nd loopful water
- FLAME 1 loopful specimen and mix well in water.
- FLAME 2nd loopful specimen if needed and mix well in water.
- FLAME
- Air Dry
- Heat Fix
- Use clothespin.
- Pass slide’s underside over the flame (coolest part of flame)
- 3 passes
Simple Stain (Methylene blue, Crystal Violet, Carbol Fuchsin)
- Smear and heat fix.
- Flood stain
Carbol Fuchsin, 15-30 sec
Crystal Violet, 20-60 sec
Meth. Blue, 1-2 min
- Rinse with Di Water
- Bibulous blot, Do not wipe
Gram Stain
- Flood with Crystal Violet
- Wait 1 min
- Flush with Di water
- Flood with Gram’s Iodine mordant
- Wait 1 min
- Flush with Di water
- Decolorize
- Flush immediately
- Counterstain Safranin
- Wait 45 sec
- Flush with Di water
- Bibulous Blot
Acid-Fast Staining
- Smear and heat fix.
- Flood smears with carbol fuchsin
- Place over a beaker of water on a warm hot plate
- Steam 5 minutes (do NOT allow stain to evaporate, replenish as needed)
OR
Flood smear with carbol fuchsin WITH TERGITOL for 5-10 minutes
- If slides are heated, allow to cool.
- Flush with Di water
- Decolorize with acid-alcohol
- Flush with Di water
- Counterstain with Meth. Blue for 2 min
- Flush with Di water
- Bibulous blot.
Spore Staining
- Smear and heat fix
- Flood with malachite green
- Place on beaker of water sitting on hot plate
- Steam for 2-3 minutes (do NOT allow stain to evaporate, replenish as needed)
- Remove from hot plate
- Cool
- Flush with Di water (this is the decolorizing step)
- Counterstain Safranin for 30 seconds
- Flush with Di water
- 10. Bibulous blot
Microbiology Lab Procedure Notes By ©2018 Shirley S. Chung, Green River Community College