1. 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

2. Exercise 18: Motility Determination Motility Test Medium 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 (From the Media List in the Supplemental packet) Figure 18.4 Each pair makes wet mount slides (supplement directions) for: Proteus vulgaris (broth), Micrococcus luteus (broth), Bacillus subtilis (broth) (additional culture)Each organism on separate slide

3. Exercise 27: Effect of Oxygen on Growth Obligate aerobes: 20% O 2 Obligate anaerobes: O 2 toxic Facultative: w/ or w/o, better with O 2 Microaerophiles: 5-10% O 2 Aerotolerant: ignore O 2 Fig 27.5 Fig 27.1

4. Fluid Thioglycolate Medium (FTM) - tube Contains: rich medium with very low agar content (viscous) Sodium thioglycolate (removes oxygen) Resazurin oxygen indicator (pink when oxidized: O 2 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 faecalisEscherichia 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

5. 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 = H 2 + CO 2, H 2 combines with O 2 creating H 2 O, 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 growth on anaerobic agar only = obligate anaerobe even growth on both = aerotolerant heavy growth on aerobic, lighter growth on anaerobic = facultative anaerobe Fig 27.2 Fig 27.4 2 Anaerobic Agar plates (all 3 on one) (1 ANAEROBIC, 1 AEROBIC (control added))

6. 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

7. Potential Unknown List: Bacillus subtilis Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa 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, Put name on original provided slants and return for storage Salmonella typhimurium Shigella flexnari Staphylococcus aureus Staphylococcus epidermidis Enterococcus (Streptococcus) faecalis Streptococcus pyogenes

8. 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! Identification of Unknown Bacterium

9. 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! Unknown Project: Data Collection

10. Lab 6 Goals and Objectives: Exercise 18: Motility Determination 1. Each pair makes wet mount slides (supplement directions) for: 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): Staphylococcus aureus Bacillus subtilis Enterococcus faecalisEscherichia 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 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 4 3 2 1

11. Each pair needs for lab 6: Group 1 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 Group 2 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 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