Transcript Lab 7
Lab 7 Goals and Objectives: Read the results: 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 (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 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 Not Non-motile Motile Motile Oxygen requirements 1. Fluid Thioglycolate Medium (FTM) 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 2. Brewer’s Anaerobic Agar 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 27.2 Fig 21.4 Which Groups Grow on Anaerobic Agar In the Anaerobic Jar? Which Groups Grow on Brewer’s in 20% Oxygen? Brewer’s No Oxygen Brewer’s 20% Oxygen Obligate aerobes: 20% O2 Obligate anaerobes: O2 toxic Facultative: w/ or w/o, better with O2 Microaerophiles: 5-10% O2 Aerotolerant: ignore 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 27.5 Fig 27.1 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: 1. 2 FTM tubes for O2 requirements (Ex. 38 pg. 267) **leave caps loose to incubate! 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) Nutrient Gelatin Stab Inoculation method: stab with needle Contains: beef extract, peptone, high gelatin concentration to gel (no agar) Must be set on ice 5-10 min before reading Discriminates organisms that can produce gelatinases to hydrolyze the gelatin into amino acids Results: liquid = gelatin hydrolysis, positive for gelatinase production solid = negative for gelatinase production Data to collect for Exercise 37 & 38 For Next Lab Transfer to Data Chart Gram result, size, shape, arrangement Motility Amount of growth Color Opacity Transfer to Data Chart Form Surface (broth) Subsurface (broth) Sediment (broth) Growth (broth) Temperature Oxygen requirements (FTM) Gelatin Transfer to Data Chart Transfer to Data Chart Transfer to Data Chart Identification of Unknown Bacterium For next lab: Read over formal lab report directions in packet. State a hypothesis “If the collective results of a profile of key biochemical assays are compared to the known results for limited subset of bacterial species, then a specific unknown culture from that subset of bacterial species can be correctly identified based on the results obtained from the biochemical assays.” Each pair needs for Lab 7 • • • • • 4 motility stabs 4 FTM tubes 4 gelatin stab 4 BHI broths 12 BHIA plates