Exercise 18: Motility Determination 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

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

Fig 27.4 Fig 27.2 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

Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa 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

Unknowns 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 Get the data charts formatted on the computer: one for each unknown. Use the return key to make spaces in all the boxes. Print blank charts to write in data as you collect it in lab. Type new data into the chart the day you collect it in lab: fill in all the required information for each assay. 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!

4 3 2 1 Exercise 18: Motility Determination 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 4 3 2 1