Lab #4
Review of Lab #3
• Oxygen requirements
• Obligate aerobes (B. subtilis)
• Obligate anaerobes (C. sporogenes)
• Facultative anaerobes (E. coli, K. pneumoniae, and S.
epidermidis)
• Microaerophilic
• Slant – only aerobic and facultative anaerobic
• Motility tall – only aerobic (top) and facultative
anaerobic (throughout)
• Thioglycolate broth – all organisms will grow but in a
different location in the broth
Review of Lab #3
• Oxygen requirements
Review of Lab #3
• Catalase test
• Organisms that utilize oxygen produce enzymes to help
protect against toxic oxygen derivatives (H2O2, O2-, etc.)
• Catalase enzyme
H2O2
H20 + ½ O2
(Production of O2 is seen as bubbles)
• E. coli
• B. subtilis
• K. pneumoniae
• S. epidermidis
Catalase
Positive
Review of Lab #3
• Endospore staining
• Positive result = spores are present (you see green
spores and pink vegetative cells)
• Negative result = no spores are present (you only see
pink vegetative cells)
• Vegetative cells will be present in both cases!
• B. cereus – spore positive
• B. subtilis – spore positive
• E. coli – spore negative
• K. pneumoniae – spore negative
• S. epidermidis – spore negative
Lab #4 – Streak plate method
(4D)
• Culture technique used to obtain isolated
colonies  obtain a pure culture
Lab #4 – Streak plate method
(4D)
• 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!!
Lab #4 – Streak plate method
• Each student streak 3 plates – lots of practice!
• Label plates properly
• Name
• Date
• Organism
• Incubate the plates INVERTED
• Why?
Lab #4 – Acid fast staining (9)
• Type of differential stain
• Used to identify members of the genus Mycobacteria
• Mycobacterium tuberculosis
• Mycobacterium leprae
• Mycobacteria cell walls have a high concentration of
waxy lipid substances (mycolic acid)
• Prevent dyes from staining the cells
• Heat is used to help stain cells
• Primary stain: Carbolfushin
• Counterstain: Methylene blue
Lab #4 – Acid fast staining
• Method (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
Lab #4 – Acid fast staining
• 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)
Lab #4 – Acid fast staining
• Each student prepare two stains:
• M. tuberculosis
• S. epidermidis
• Record results on pg. 47
Lab #4 – Hanging drop method (8A)
• 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 mix vaseline with 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
Lab #4 – Hanging drop method
• Microscope observation:
• 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!)
• Observe to distinguish
motility vs. Brownian motion
• Motility is distinct movement
from point A to point B
• Brownian motion = jiggling
in one spot/ gliding in direction
Lab #4 – Hanging drop method
• Each student prepare a hanging drop for:
• S. epidermidis
• E. coli
• Record motile or non-motile on pg. 44