Transcript RCC Lab 5 S14

Lab #5
Review for Practical #1
• Colony morphology (pg. 19)
Review for Practical #1
• Preparing a smear (pg. 31-32)
Same procedure
for all staining
methods except
Acid fast staining
(1% albumin)
Review for Practical #1
• Gram staining (pg. 34)
5-10 sec
1 min
30-45 sec
Review for Practical #1
Gram positive cocci
Where Are Microorganisms Found and How Do We Study Them?
Lab 2
• Bacterial shape and
arrangement
Coccus (round)
Bacteria - 1000x
Rod (bacillus)
Spirochete
Single
Pair
Single
Gram negative bacillus
Tetrad
Chain
Chain
Cluster
(masses)
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Review for Practical #1
• Endospore staining (pg. 37 – 38)
• Prepare and heat fix a smear
• Flood slide with Malachite green for 1 min
• Steam for 3 minutes
• Use Bunsen burner like a blowtorch, intermittently
• Make sure the stain doesn’t dry up
• Rinse with water for ½ minute
• Cover smear with 0.5% Safranin stain
• Rinse and blot dry
• Observe under the microscope
(+) Green = spores (inside cells and outside)
Pink = vegetative bacterial cell
Review for Practical #1
• Acid fast staining (Pg. 45-46):
• Prepare smear
• Add 2 loopfuls of 1% albumin (helps hold bacteria onto slide)
• Mix in bacteria aseptically
• Air dry and heat fix
• Flood slide with Carbolfushin
• Blow torch for 5 minutes, intermittently  DON’T LET STAIN
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DRY
Rinse with water
Decolorize with Acid Alcohol until color stops to run
Rinse with water
Cover smear with Methylene blue for 1 minute
Rinse with water
Blot dry and observe under microscope
Review for Practical #1
• Acid fast positive = Red cells
• Acid fast negative = Blue cells
• Only Mycobacterium species will be Acid fast positive 
resist de-colorization and retain primary stain
(Carbolfushin)
Review for Practical #1
• Hanging drop method to determine motility (pg. 41 - 42)
• Use a broth culture
• Need a depression slide, coverslip, and Vaseline
• Smear Vaseline on palm  scrape onto the four edges of
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coverslip (Don’t get vaseline mixed w/ culture)
Transfer loopful of bacterial culture onto coverslip
Place depression slide, concave side down, onto
coverslip  DON’T PRESS
CAREFULLY invert slide  see the hanging drop
Observe under microscope for motility
Review for Practical #1
• Microscope observation of hanging drop:
• Start at 10X objective  look for the edge of the drop
• Center the edge of drop in field of view
• Switch to 40X objective  FINE focus
• Bacteria will appear “ghost like” within the drop
(no stain!)
• Motility is distinct
movement from point A
to point B
Review for Practical #1
• Streak plate method (pg. 26 – 29):
• Sample is only take once – only for quadrant 1
• Streak from top to bottom – don’t go up and down
• Flame loop in between each quadrant!!
• Work aseptically!
Capsule staining
• Some bacteria produce this external structure
• Capsules composed of polysaccharides
• Encapsulated bacteria is more virulent
• Procedure
• Negative staining (stain the background and the bacteria)
• Capsule appears as a “halo” around the bacterial cell
Capsule staining