Summary
Tuesday night 5:30-7pm
SOIL PROJECT:
1) Run a PCR on each of your isolated organisms that had a zone of clearing to determine the 16sRNA.
2) Run a gel electrophoresis on part of your PCR sample or observe the demo.
3) Send the rest of the PCR sample to the San Diego lab for DNA sequencing of the 16S rRNA gene to determine what the Genus is of your soil organism.
FERMENTATION TUBES
Inoculate three sugar tubes with your unknown organism.
Inoculate another three sugar tubes with your soild organism that showed clearing
Thursday 5:30 -7 pm
Add the reagent to the last well on the right of the API strips and add the other reagents to the two MRVP broths from last week and r ecord the colors of your other media from last week as well.
Use your unknown organism to inoculate new test media for carbohydrate and protein catabolism: starch, O-F, urea, gelatin, Phe, and enterotubes.
Use your microscope to observe the worm slides: Enterobius, Trichinella, Taenia, and Echinococcus.
After dinner, we will finish the lab lecture on how to ID an unknown organism, take a break and then stay in the lab room to finish the lecture material.
Results from last week
In the VP well, add 1 drop of VP reagent II (KOH) and then add 1 drop of VP reagent I (alpha napthol). A positive reaction produces a red color after 10 minutes.
In the NIT well, add 2 drops of nitrate reagent A (dimethyl-alpha-napthylamine) and 2 drops of nitrate reagent
B (sulfanilic acid). A positive (red) may take 3 minutes to develop. If it is negative, add zinc dust/ If it then turns red, it is negative. Make a + sign under each well that is positive (use this paper) and make a negative (-) sign under each well that is negative.
This is what all positive tests look like on the API Staph strip
This is what all negative tests look like

ID your Staph organism.
Next, start with the first three wells. For all the wells that were POSITIVE, add the numbers (printed on each well below) and put that number under the blue circle. For instance, if all three wells were positive, 1+2+4 =7, so you would put the number 7 in the blue circle below. Then add the numbers of all the positive wells in the next group of three. If only the first two wells in this group were positive, it would be 1+2=3, so put 3 in the second blue circle. If all wells are negative in a group, put 0. Ignore the last well called LSTR on the document below. Once you have a 7-digit number, your instructor will login to this website, enter that number, and it will tell you what your organism is.
For the API Staph results you will need to use this website to access the key: https://apiweb.biomerieux.com/servlet/Authenticate?action=prepareLogin
Other Results from last week: Record these results on your unknown chart
1) MaConkey’s agar plate (growth means Gram neg, yellow agar instead of pink is positive for lactose fermentation)
2) Thioglycolate broth (to determine oxygen requirements). Growth at top means aerobic. Growth throughout means facultative.
3) Motility tube: First look to see if your stab in the middle of the tube is wispy, indicating motility (see picture below). Then see if the tube is yellow, which means is positive for the enzyme that degrades ornithine; add 4-5 drops of Kovac’s reagent to the tube and mix. If it turns red, it is positive for indole, which means it uses the 2,
3, butanediole fermentation pathway.
4) Simmons Citrate agar slant (blue is positive; organism can use citrate as its sole carbon source )
5) MRVP broth (add reagents to finish this test; instructions below)
6) Dextrose fermentation tube (yellow is positive; record any gas present)
7) Lactose fermentation tube (yellow is positive; record any gas present)
8) Sucrose fermentation tube (yellow is positive; record any gas present)
9) TSB tubes which were incubated at different temperatures. Put a pipette full of each in a cuvette and place in the spectrophotometer at 600 nm and record the transmission. The one with the lowest transmission is the one with the most growth. That tells you the organism’s preferred temperature.
Controls
Groups 1 and 2 inoculated one Thio Broth with Pseudomonas aeruginosa (aerobic)
Group 3 and 4 inoculated 2 MRVP tubes with E. coli (+/ -)
Group 5 and 6 inoculated 2 MRVP tubes with Enterobacter aerogenes (-/+)
Group 7 and 8 inoculated 2 MRVP tubes with Proteus vulgaris (+/-)
MRVP broths
In one MRVP tube, perform the methyl red test. In the other 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 in your one-page unknown chart I gave you.
You will be able to finish your chart next week and identify your unknown organism.

TODAY’S LAB WORK
Use your unknown organism to inoculate all these tests (6 tubes total)
Ch 13 (Carbohydrate Catabolism)
Two OF-glucose deeps with and without mineral oil (stab straight and back out with a needle). Add about ½ inch of mineral oil to ONE of them AFTER you inoculate.
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)
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. Inoculate your soil bug too.
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)
OBSERVE GEL ELECTROPHORESIS DEMO UNDER THE HOOD BEFORE DINNER
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. Make sure you do not touch the straight wire with your gloves, just touch the handle. Put both of the caps back on the tube, making sure the blue cap is on the left, closest to the well with glucose (see the label on the tube). Use a toothpick 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
(glucose, lysine, ornithine, H2S). Put a piece of tape on the tube and label it with “Magrann”, the date, your lab group number (GROUP #) and the number of the organism (ORG #) you used, and place it in the incubator.

Ch 36 (Invertebrates; p.278 lab manual)
Preserved parasitic worm slides
Enterobius vermicularis (roundworm/pinworm; identify mouth, pharynx, eggs)
Trichinella spiralis (roundworm/threadworm; identify cyst in muscle)
Taenia saginata (flatworm/tapeworm; identify scolex with hooks/suckers, proglottids w/uterine branches, eggs)
Echinococcus (flatworm/tapeworm; id scolex with hooks/suckers, proglottids with uterine branches and eggs)