Lab 7 Instructions: See lab manual
Summary
Record the results of the tubes from last week.
Use your unknown organism to inoculate new test media for carbohydrate and protein catabolism.
Inoculate an Enterotube.
Results from last week
1)
2)
3)
1)
2)
3)
4)
5)
MaConkey’s agar (yellow is positive for lactose fermentation)
Thioglycolate broth (to determine oxygen requirements)
Motility tube
Simmons Citrate agar slant (blue is positive)
Dextrose fermentation tube (yellow is positive; record any gas present)
Lactose fermentation tube (yellow is positive; record any gas present)
Sucrose fermentation tube (yellow is positive; record any gas present)
MRVP broth (add reagents to finish this test)
Controls
Groups 1 and 2 inoculated one Thio Broth with Pseudomonas aeruginosa (aerobic)
Group 3 and 4 inoculated MRVP tube with E. coli (+/ -)
Group 5 and 6 inoculated MRVP tube with Enterobacter aerogenes (-/+)
MRVP broths
Use a pipette to remove half the liquid from each MRVP tube and place it in an empty test tube.
In the test tube, perform the methyl red test. In the original MRVP tube, perform the VP test.
Methyl Red Test: add 5 drops of methyl red to the tube. If it turns red, it is positive.
VP Test: See Ch 14 for instructions. Add 12 drops of Barrits’s A reagent (alpha napthol, a carcinogen!) and 3 drops of
Barrett’s B reagent (potassium hydroxide; KOH, a very caustic base, found in draino). Put the test tube cap back on
and shake. TAKE OFF CAP and allow to stand exposed to air for 15 minutes. A rust color (brownish red) is
positive.
Record all these results in your chart.
TODAY’S LAB WORK
Use your unknown organism to inoculate all these tests
Ch 13 (Carbohydrate Catabolism)
Starch agar plate (loop)
Two OF-glucose deeps with and without mineral oil (needle)
Controls
Group 1 and 2 inoculate the 2 mediums above with E. coli (O/F, G)
Group 3 and 4 inoculate the 2 mediums above with Pseudomonas aeruginosa (O)
Ch 15 (Protein Catabolism)
Urea tubes (use loop, but inoculate zig-zag on top)
Gelatin tubes (needle)
Controls
Group 5 and 6 inoculate the 2 mediums above with Pseudomonas aeruginosa (positive)
1
Ch 16 (Protein Catabolism)
Phenylalanine (Phe) slants (use loop, but inoculate zig-zag on top)
SIM media (needle); We will use this media instead of peptone iron and MIO, as stated in the
book.
Controls
Group 7 inoculate the 2 mediums above with E. coli (H2S and indole +)
Group 8 inoculate the 2 mediums above with Enterobacter aerogenes (H2S and indole neg)
Ch 51 (Rapid ID methods)
4 Enterotubes
Groups 1 and 2 get one tube to inoculate with a pure culture of E. coli
Groups 3 and 4 get one tube to inoculate with a pure culture of Pseudomonas aeruginosa
Groups 5 and 6 get one tube to inoculate with a pure culture of Enterobacter aerogenes
Groups 7 and 8 get one tube to inoculate with a pure culture of your choice!
Take off the caps at both ends of the tube that cover up the ends of a wire that runs through the
whole tube. The straight wire will be dipped into the pure organism tube (tilt the tube), and the bent
side of the wire is the handle. Dip the wire into the inoculum and pull it out of the Enterotube in a
twisting motion, then twist it again as you push the wire back into the Enterotube. That will
inoculate all the wells in the tube at once. Then look for a tiny notch in the wire of the handle. Bend
the handle wire back and forth to break it off at the notch. Put both caps back on the tube. Use the
broken piece of wire to poke a hole in the cellophane covering the flat side of the Enterotube, on the
8 wells on the right side of the tube. Leave the cellophane intact on the first 4 wells on the left. Put a
piece of green tape on the tube and label it with “Magrann”, the date, and the name of the organism
you used.
SOIL PROJECT
1) Record the results of the previous tests.
2) Run a PCR on your isolated organism to determine the 16sRNA.
3) Send it to the San Diego lab for sequencing for Genus.
4) Use Bergey’s Manual to determine Genus and possibly species.
5) Write a report about your project
2