Gather all plates and tubes so we can discuss results together: Lab 7 Goals and Objectives: Gather all plates and tubes so we can discuss results together: ***Do NOT shake the FTM tubes!!!*** Your Unknowns: Exercises 37 and 38 Check plates, record appearance, check for contamination Check slants for growth and place in my “reserve” rack Make new inoculations for both unknowns from the plates: 2 FTM tubes for O2 requirements (Ex. 38 pg. 267) 2. 2 BHI broth tubes for growth in liquid characteristics (Ex. 38 pg. 266 fig. 38.2) and for Gram stain (Ex. 37 pg. 260) 3. 2 Gelatin stab cultures for gelatin hydrolysis ability (Ex. 38 pg. 259) 4. 3 BHI plates each unknown (6 total), streak for isolation, to grow at 25°C, 30°C, 37°C to determine optimal temp. (Ex. 37&38 had you using slants and broths; side by side plates is easier) 5. 2 Motility test media stabs (Ex. 37 pg. 262) No: KOH, endospore, acid fast, other structures (Ex. 37 pg. 261-262)

(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 (reduced) 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 Fig 18.4

Motility Test Media Results Non-motile Motile Non Motile

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 plate 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 plate: 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

Which Groups Grow on Brewer’s Anaerobic Agar In the Anaerobic Jar? Which Groups Grow on Brewer’s in 20% Oxygen? Brewer’s No Oxygen Brewer’s 20% Oxygen Obligate anaerobes: O2 toxic Obligate aerobes: 20% O2 Aerotolerant: ignore O2 Facultative: w/ or w/o, better with O2 Microaerophiles: 5-10% O2

Obligate anaerobes: O2 toxic Facultative: w/ or w/o, better with O2 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

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!

Data to collect for Exercise 37 & 38 For Next Lab Gram result, size, shape, arrangement Transfer to Data Chart Motility Amount of growth Color Opacity Form Surface (broth) Subsurface (broth) Sediment (broth) Growth (broth) Temperature Oxygen requirements (FTM) Gelatin Colonies

Gather all plates and tubes so we can discuss results together: Lab 7 Goals and Objectives: Gather all plates and tubes so we can discuss results together: ***Do NOT shake the FTM tubes!!!*** Your Unknowns: Exercises 37 and 38 Check plates, record appearance, check for contamination Check slants for growth and place in my “reserve” rack Make new inoculations for both unknowns from the plates: 2 FTM tubes for O2 requirements (Ex. 38 pg. 267) 2. 2 BHI broth tubes for growth in liquid characteristics (Ex. 38 pg. 266 fig. 38.2) and for Gram stain (Ex. 37 pg. 260) 3. 2 Gelatin stab cultures for gelatin hydrolysis ability (Ex. 38 pg. 259) 4. 3 BHI plates each unknown (6 total), streak for isolation, to grow at 25°C, 30°C, 37°C to determine optimal temp. (Ex. 37&38 had you using slants and broths; side by side plates is easier) 5. 2 Motility test media stabs (Ex. 37 pg. 262) No: KOH, endospore, acid fast, other structures (Ex. 37 pg. 261-262)