Lab 6 Goals and Objectives:
Exercise 18: Motility Determination – presence of flagella
Exercise 27: Effect of Oxygen on Growth
Exercise 37: Morphological Study of Unknown Bacterium
Exercise 18: Motility Determination
Motility Test Medium (From the Media List in the Supplemental packet)
• Contains: nutrient medium with low (0.5%) agar concentration (semisolid) and
TTC which changes from colorless to dark pink or maroon in the presence of
bacterial growth when it is reduced by cellular enzymes involved in redox
reaction that are common to all cell types.
• Inoculation method: vertical single stab with straight needle
• Discriminates motility (presence of flagella), ability to “swim” through media
• Results:
– organism growing only in line of inoculation = non-motile
– organism appears as haze beyond line of inoculation = motile
Each pair makes Motility Test
Media tube for
P. vulgaris and S. aureus only Figure 18.4
No plates, no hanging drop
Each pair makes wet mount slides (supplement directions) for:
Proteus vulgaris (broth), Staphylococcus aureus (replaces Micrococcus luteus)
(broth), Bacillus subtilis (broth) (additional culture)Each organism on separate slide
Exercise 27: Effect of Oxygen on Growth
Obligate aerobes: 20% O2
Obligate anaerobes: O2 toxic
Facultative: w/ or w/o, better with O2
Microaerophiles: 5-10% O2
Aerotolerant: ignore O2
Fig 27.1
Fluid Thioglycolate Medium (FTM) - tube
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Contains: rich medium with very low agar content (viscous) Sodium thioglycolate (removes
oxygen) Resazurin oxygen indicator (pink when oxidized: O2 present)
Inoculation method: loop transfer, careful mixing, screw cap must be loose
Discriminates oxygen requirements: obligate aerobes, obligate anaerobes, facultative
anaerobes, microaerophiles, aerotolerant
Results:
– growth only at top = obligate aerobe
– growth only at bottom = obligate anaerobe
– even growth throughout = aerotolerant
– heavy growth at top, lighter growth at bottom = facultative anaerobe
– growth only in middle = microaerophile
One pair use (broth):
Other pair use (broth):
Staphylococcus aureus
Bacillus subtilis
Enterococcus faecalis
Escherichia coli
Clostridium sporogenes
Clostridium rubrum (replaces C. beijerinckii)
For your pair’s set of 3 organisms, make (per pair):
1. 3 FTM tubes (one tube each) **leave cap loose to incubate!
2. 2 Anaerobic Agar plates (all 3 on one) (1 ANAEROBIC, 1 AEROBIC
(control added)), No TGYA shakes
FTM Agar - Brewer’s Anaerobic
Contains: Nutrient agar with sodium thioglycolate and resazurin (see FTM above)
Inoculation method: surface streak with loop,
-incubated in Brewer’s anaerobic jar (water+ gas pack = H2 + CO2, H2 combines with
O2 creating H2O, sealed jar is oxygen free).
(Inoculate in conjunction with BHIA in aerobic 20% oxygen atmosphere)
Discriminates oxygen requirements if read in conjunction with normal incubated
BHIA:obligate aerobes, obligate anaerobes, facultative anaerobes, aerotolerant
Results: growth on aerobic agar only = obligate aerobe
Fig 27.2
growth on anaerobic agar only = obligate anaerobe
even growth on both = aerotolerant
heavy growth on aerobic, lighter growth on
anaerobic = facultative anaerobe
Fig 27.4
2 Anaerobic Agar plates (all 3 on one) (1
ANAEROBIC, 1 AEROBIC
(control added))
Exercise 37: Morphological Study of Unknown Bacterium
Unknown Selection: each person choose 1 pair unknowns and sign out numbers
Make new BHIA slant of each (2 total)
Make BHIA streak for isolation plate of each (2 total)
Label original tube with your name and return to stock rack
Potential Unknown List:
• Bacillus subtilis
• Enterobacter cloacae
• Escherichia coli
• Klebsiella pneumoniae
• Proteus vulgaris
• Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexnari
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus (Streptococcus) faecalis
Streptococcus pyogenes
• Unknowns should be provided as a pair with one Gram positive and
one Gram negative
• Students select and sign out a pair of unknowns (preset pair, see
below).
1. Make new slants (to serve as reserve stock: can be “bought” later for
10 points off unknown report grade), one slant per culture, 37°C
2. Make streak for isolation plates to work with next time, one plate
per culture,
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Put name on original provided slants and return for storage
Identification of Unknown Bacterium
•Record all data for both unknowns: fill in on blank data report in lab
•Type in data for each category on the data report on the day you collect
it: do not wait until the end of the project!
Unknown Project: Data Collection
1. Get the data charts formatted on the computer: one for each
unknown.
2. Use the return key to make spaces in all the boxes.
3. Print blank charts to write in data as you collect it in lab.
4. Type new data into the chart the day you collect it in lab:
fill in all the required information for each assay.
5. On the last day of data collection, print the nearly complete
charts and bring them to class to compare information with
classmates. Catch the silly mistakes before they cost you
points!
Lab 6 Goals and Objectives:
Exercise 18: Motility Determination
1. Each pair makes wet mount slides (supplement directions) for: 4
Proteus vulgaris (broth), Micrococcus luteus (broth),
Bacillus subtilis (broth) (additional culture)Each organism on separate slide
2. Each pair makes Motility Test Media tube for P. vulgaris and M. luteus
only
No plates, no hanging drop
Exercise 27: Effect of Oxygen on Growth
One pair use (broth):
Other pair use (broth):
3
Staphylococcus aureus
Bacillus subtilis
Enterococcus faecalis
Escherichia coli
Clostridium sporogenes
Clostridium rubrum(replaces C. beijerinckii)
For your pair’s set of 3 organisms, make (per pair):
1. 3 FTM tubes (one tube each) **leave cap loose to incubate!
2
2. 2 Anaerobic Agar plates (all 3 on one) (1 ANAEROBIC, 1 AEROBIC
(control added)), No TGYA shakes
Exercise 37: Morphological Study of Unknown Bacterium
Unknown Selection: each person choose 1 pair unknowns and sign out numbers
Make new BHIA slant of each (2 total)
1
Make BHIA streak for isolation plate of each (2 total)
Label original tube with your name and return to stock rack
Each pair needs for lab 6:
Exercise 37: Morphological Study of Unknown Bacterium
Sign out a pair of unknowns
4 BHIA plates
4 BHIA slants
2 motility test media stabs
3 Fluid Thioglycolate Media (FTM) tubes
1 Brewer’s Anaerobic Agar plate
1 BHIA plates
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Group 1
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Group 2
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1 broth culture of Proteus vulgaris
1 broth culture of Micrococcus luteus
1 broth culture of Bacillus subtilis
1 broth culture of Staphylococcus aureus
1 broth culture Enterococcus faecalis,
1 broth culture Clostridium sporogenes
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1 broth culture of Proteus vulgaris
1 broth culture of Micrococcus luteus
1 broth culture of Bacillus subtilis
1 broth Escherichia coli,
1 broth culture Clostridium rubrum