Download 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
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4 motility stabs
4 FTM tubes
4 gelatin stab
4 BHI broths
12 BHIA plates