Lab 7 Goals and Objectives:

1
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 39 and 40 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. 40 pg. 267) leave caps loose to
incubate! 2. 2 BHI broth tubes for growth in
liquid characteristics (Ex. 40 pg. 266 fig.
40.2) and for Gram stain (Ex. 39 pg. 260) and wet
mount for motility if desired (Ex. 39 pg.
262) 3. 2 Gelatin stab cultures for gelatin
hydrolysis ability (Ex. 40 pg. 267) 4. 3 BHI
plates each unknown (6 total), streak for
isolation, to grow at 25C, 30C, 37C to
determine optimal temp. (Ex. 39 had you
compare 2 slants to broths in Ex. 40 for this
but it is better to compare the same media
plates let us check for contamination) 5. 2
Motility test media stabs (Ex. 39 pg. 262) No
endospore, acid fast, other structures (Ex. 39
pg. 262)
2
(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
3
Motility Test Media Results
Not
Motile
4
Brewers Anaerobic Agar Inoculation method
surface streak with loop, must be incubated in
Brewers 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 21.2
Fig 21.4
5
Which Groups Grow on Brewers Anaerobic Agar In
the Anaerobic Jar? Which Groups Grow on Brewers
in 20 Oxygen?
Brewers No Oxygen
Brewers 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
6
Obligate aerobes 20 O2 Obligate anaerobes O2
toxic Facultative w/ or w/o, better with
O2 Microaerophiles 5-10 O2 Aerotolerant ignore
O2
Fig 21.1
7
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 21.1
Fig 21.5
8
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!

9
Example Data Chart for Exercise 40
10
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 39 and 40 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. 40 pg. 267) leave caps loose to
incubate! 2. 2 BHI broth tubes for growth in
liquid characteristics (Ex. 40 pg. 266 fig.
40.2) and for Gram stain (Ex. 39 pg. 260) and wet
mount for motility if desired (Ex. 39 pg.
262) 3. 2 Gelatin stab cultures for gelatin
hydrolysis ability (Ex. 40 pg. 267) 4. 3 BHI
plates each unknown (6 total), streak for
isolation, to grow at 25C, 30C, 37C to
determine optimal temp. (Ex. 39 had you
compare 2 slants to broths in Ex. 40 for this
but it is better to compare the same media
plates let us check for contamination) 5. 2
Motility test media stabs (Ex. 39 pg. 262) No
endospore, acid fast, other structures (Ex. 39
pg. 262)