SOIL SAMPLE ANALYSIS
LAB 1
Soil Acquisition
1) Don’t use really fertile soil…dry is better, but not in an area where no plants are
growing.
2) It is best to get soil close to roots of nearby plants, and dig down about 2 inches
first.
3) Fill a 15 ml test tube with about 3 cm of soil.
4) Record the place of collection, temperature, and any other data you can collect
(vegetation around etc.).
5) Add enough DI water to fill the tube, close the lid and shake.
6) Place the tube on a vortex machine to shake more vigorously.
7) Label the BOTTOM side of the Petri dish, around the EDGE, with your name,
“soil”, date, and instructor name.
8) Use a micropipette to dispense 100-200 µL of water from the tube into the Petri
dish. Open the lid of the Petri dish at a 45° angle, keeping the lid over the agar.
Use a spreader tool to spread the liquid over the entire plate. Incubate upside
down at room temperature. Jeremy will put them in the refrigerator on Monday so
they do not overgrow.
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
LAB 2
Make a Master Plate (reserve stock) using the Pick and Patch method
1) See how many different looking colonies you got, especially those that have color
(like yellow).
2) Then take a paper grid (supplied in lab), and draw it onto the bottom of a new
TSA plate and number the squares.
3) Number each square
4) Inoculate each square with a different colony that has some color in it. This will
become your Master Plate, or the “reserve stock” that you can go back to in case
your future plates become contaminated.
5) Incubate the Master Plate upside down at room temperature. Jeremy will put
them in the refrigerator on Monday so they do not overgrow.
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
LAB 3
Find the colonies that secrete antibiotics
1) Take another paper grid (supplied in lab), and draw it onto the bottom of a new
TSA plate and number the squares. This will be your Test Plate.
2) Spread E. coli (or a different ESCAPE organism) on that new plate
3) Transfer colony #1 from your Master Plate to grid square #1 of your Test Plate,
and repeat for all the colonies.
4) Incubate upside down at room temperature. Jeremy will put them in the
refrigerator on Monday so they do not overgrow.
LAB 4
Choose the best antibiotic-secreting colony and make your working stock:
1) Check your E. coli plate for zones of inhibition.
2) Choose the colony with the biggest zone, and streak for isolation on a new TSA
plate (this Isolation Plate will be your working stock, with which you will perform
tests to identify its genus.
3) Take a picture of your Test Plate with the zones of inhibition. Make sure you get
a good photo of the zones on the plate. Then you can discard the Test Plate.
4) Keep your Master Plate as a reserve stock in case your working stock becomes
contaminated. If you got at least one colony with a good zone of inhibition, you
can also now discard your original plate from Lab 1.
5) Put the reserve stock (Master Plate) back in the refrigerator, upside down. The
new isolation plate (working stock) will stay upside down at room temperature for
the weekend before it goes into the refrigerator.
Total plates to keep per student for the duration of the experiment:
1) Master Plate (reserve stock) with grid lines
2) Isolation plate (working stock) of the one organism that has the best zone of
inhibition from the Test Plate...The Isolation Plate will be used for the morphological,
cultural, and physiology tests.
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
LAB 5
Use your working stock (isolation plate) to perform the following tests for
morphological characteristics
1) Gram stain to determine Gram +/- and shape (rods or cocci)
2) Negative stain to determine shape and arrangements (staph, strep, diplo, singles)
3) Motility stab
4) Hanging drop to observe if it runs or tumbles
5) Endospore stain
Use your working stock to perform the following tests for cultural characteristics
1) Inoculate a broth and plate to observe colony morphology. Use the terminology
from your lab manual to describe it.
A. NUTRIENT BROTH CHARACTERISTICS:
1. Is there turbidity (cloudiness)?
2. Is there growth only at the top (pellicle), only at the bottom (sediment), flakes
in the middle (flocculant), or growth throughout (uniform turbidity)?
3. What color is it?
B. PLATE CHARACTERISTICS:
1. MARGINS (look at the surface of the plate)
a. Filiform (thread-like edge)
b. Arborescent (tree-like branches)
c. Beaded (starts smooth, breaks into individual colonies at top)
d. Effuse (edges are not clearly defined)
e. Rhizoid (root-like branches)
f. Echinulate (serrated edge)
2) TEXTURE (look at surface of plate)
a. Flay, dry (Bacillus megatarium)
b. Spreading (proteus, pseudomonas)
c. Crusty, waxy (mycobacterium)
d. Transparent
3) PIGMENTATION (color of plate)
a. Yellow
b. White
c. Green
d. Etc
4) ELEVATION
a. Convex
b. Flat
c. Raised
d. Umbonate
2) Determine the optimal temperature by inoculating 3 TSB tubes, labeled with
three different temperatures you want to incubate at. Use the spectrophotometer
to calculate their optical density at the next lab period. The tube with the most
growth (highest OD) is the temperature they prefer.
3) Determine the oxygen requirements by inoculating a thioglycolate tube. Next lab
period, record if it is a strict aerobe, strict anaerobe, or facultative anaerobe.
4) Determine if your organism displays hemolysis by inoculating a blood agar plate
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
LAB 6-7
Check Bergey’s Manual to see which tests to run to determine the genus (and maybe
species) of your organism. Use your working stock (isolation plate) to perform
whichever of the following tests for physiological characteristics that Bergey’s Manual
says to perform.
1) Lactase (MacConkey’s agar)
2) Fermentation broths
3) IMVIC
4) Indole test
5) MR-VP test
6) Citrate
7) Amylase
8) Oxidation-fermentation (O-F) test for glucose
9) Urease broth or slant
10) Gelatinase
11) Phenylalanine deaminase test
12) Sim media
13) Decarboxylase broths
14) TSI (triple sugar iron agar)
15) Catalase
16) Oxidase
17) Lipase
18) Nitrate reduction test
LAB 8
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.
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
LAB 9-10
Write a report about your project