Lab #3
Review of Lab #2
• Gram staining  record results on pg. 35
• Gram positive – purple
• Gram negative – pink
• Bacillus subtilis –
• Escherichia coli –
• Klebsiella pneumoniae –
• Staphylococcus epidermidis –
Review of Lab #2
• Motility tall (pg. 43 – 44)
• Semisolid media (1/3 the normal amount of agar)
• Motile bacteria are able to move around in the media
• Observe motility for:
• E. coli –
• B. subtilis –
• K. pneumoniae –
• S. epidermidis –
• What bacterial structure
provides motility?
• Flagella
Review of Lab #2
• Aseptic technique
• Observe growth on slant, broth (pg. 20), and motility tall (pg. 43 - 44)
Bacterial
growth
Slant
Review of Lab #2
• Aseptic technique
• Observe growth on slant, broth (pg. 20), and motility tall (pg. 43 - 44)
Lab #3 – Oxygen requirements
• Oxygen requirement varies for microbes
• 4 major groups
• Obligate Aerobes
• Grow only in presence of oxygen
• Use O2 as final electron acceptor in electron transport chain
• Obligate Anaerobes:
• Grow only in the absence of oxygen – O2 is toxic to them
• Use other molecules (nitrates, sulfates, etc) as final electron
acceptor
• Microaerophilic
• Require oxygen to be lower than atmospheric level for growth
• Facultative anaerobes
• Use oxygen when available but can grow in its absence
Removal of oxygen in lab
• Reducing agent
• Thioglycollate broth (inoculated in lab #2)
• Reduces 02 to H20
• Produces a range of oxygen levels
• Mechanical removal
• Vacuum to remove O2 and replace with other gas
• Chemical Reaction
• Reaction of O2 and other chemicals results in loss of O2
Oxygen requirements
(thioglycollate broth)
Oxygen requirements (pg. 109-111)
• Use the slant, motility tall, and the thioglycolate broth to
determine the oxygen requirements
Oxygen requirements (pg. 109-111)
• Use the slant, motility tall, and the thioglycolate broth to
determine the oxygen requirements of the following
organisms:
• E. coli –
• B. subtilis –
• K. pneumoniae –
• S. epidermidis –
• C. sporogenes –
• Record results on pg. 110 and 111
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
Lab #3 – Catalase test
• Test for enzyme catalase (pg. 85-86):
• Aseptically transfer a visible amount of bacteria into the
depression well of a concave slide
• Spread bacteria
• Add a drop of 3% or 10% H2O2 to the bacteria on the
slide
• Observe for bubbles
(positive result)
• NOTE: Do not touch
H2O2 with loop
Catalase test result
• Test all four organisms:
• E. coli
• B. subtilis
• K. pneumoniae
• S. epidermidis
• Record results on pg. 86
Lab #3 – Endospore staining
• Type of structural staining
• Endospores
• Round or oval in shape
• Resistant structures produced for
survival of some bacteria under
stressful conditions
• Resistant to heat, drying, UV
radiation, acids/bases,
disinfectants and even Gram
staining
• Usually found in Bacillus and
Clostridium species
Endospore cycle
Endospore staining procedure
• Procedure on 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
• Do this step 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
Endospore staining results
• 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 –
• B. subtilis –
• E. coli –
• K. pneumoniae –
• S. epidermidis –
• Record results on pg. 39