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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

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Lab 7 goals and objectives 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:

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)


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

(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


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

Motility Test Media Results

Non-motile

Motile

Motile

Non


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

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


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

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


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

Obligate aerobes: 20% O Anaerobic Jar?2

Obligate anaerobes: O2 toxic

Facultative: w/ or w/o, better with O2

Microaerophiles: 5-10% O2

Aerotolerant: ignore O2

Fig 27.1


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

Fluid Thioglycolate Medium Anaerobic Jar? (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


Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

Unknowns Anaerobic Jar?

  • 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!



Lab 7 goals and objectives gather all plates and tubes so we can discuss results together

Lab 7 Goals and Objectives: Anaerobic Jar?

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:

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)