Lab 6 Goals and Objectives:
Exercise 18: Motility Determination
Each pair makes wet mount slides (supplement directions) for:
Proteus vulgaris (broth), Staphylococcus aureus replaces M. luteus (broth),
Bacillus subtilis (broth) (additional culture)Each organism on separate slide
Each pair makes Motility Test Media tube for P. vulgaris and S. aureus only
No plates, no hanging drop
Exercise 27: Effect of Oxygen on Growth
Team up with another pair and split the work (group of 4):
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):
3 FTM tubes (one tube each)
2 Anaerobic Agar plates (all 3 on one) (1 ANAEROBIC, 1 AEROBIC
(control added)), No TGYA shakes
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
Play BacterialMotility.mpg
(From the Media List in the Supplemental packet)
Motility Test Medium
Inoculation method: vertical single stab with straight needle
Contains: nutrient medium with low (0.5%) agar concentration
(semisolid) and TTC which changes from colorless to dark pink in
the presence of bacterial growth (enhances visualization)
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
Figure 18.4
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)
Inoculation method: loop transfer, careful mixing, screw cap must be loose
Contains: rich medium with very low agar content (viscous)
Sodium thioglycolate (removes oxygen)
Resazurin oxygen indicator (pink when oxidized: O2 present)
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
growth only in middle = microaerophile
Fig 27.1
Brewer’s Anaerobic Agar
Inoculation method: surface streak with loop, must be 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.
Contains: Nutrient agar with sodium thioglycolate and resazurin (see FTM)
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
growth on anaerobic agar only = obligate anaerobe
even growth on both = aerotolerant
heavy growth on aerobic, lighter growth on anaerobic = facultative
Fig 27.4
Fig 27.2
Possible Unknowns:
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
•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
4
Each pair makes wet mount slides (supplement directions) for:
Proteus vulgaris (broth), Staphylococcus aureus replaces M. luteus (broth),
Bacillus subtilis (broth) (additional culture)Each organism on separate slide
Each pair makes Motility Test Media tube for P. vulgaris and S. aureus only
No plates, no hanging drop
Exercise 27: Effect of Oxygen on Growth
Team up with another pair and split the work (group of 4):
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):
2
3 FTM tubes (one tube each)
2 Anaerobic Agar plates (all 3 on one) (1 ANAEROBIC, 1 AEROBIC
(control added)), No TGYA shakes
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