SOIL SAMPLE ANALYSIS
LAB 1
1) Don’t use really fertile soil…dry is better, but not in an area where no plants are
growing. It is best to get soil close to roots of nearby plants, and dig down about 2
inches first.
2) Fill a 15 ml test tube all the way up with soil.
3) Record the place of collection, temperature, and any of the below data you can collect
LOCATION
Location
Latitude
Longitude
Date and Time Collected *
Date
Time
Sample Site Descriptors *
SOIL SAMPLE
Air Temperature (°C)
Humidity (%)
Depth (In.)
Type of Soil
Soil Temperature (°C)
pH of Soil
Water Content (%)
4) Pour the whole 15 ml tube of soil into a 250 ml Erlenmeyer flask and fill it to the 100 ml
mark with sterile water in the large plastic jar by the sink. That gives a 15% soil to water
ratio. Shake it up to make it muddy.
5) EACH STUDENT will obtain and label the following three plates PER SOIL SAMPLE: NA
(nutrient agar), TSA (tryptic soy agar), PDA (potato dextrose agar)
6) Label the BOTTOM side of the Petri dishes, around the EDGE, with your name, “soil”,
date, and instructor name.
7) Use a pipette to drop 0.5 ml of the solution (half a stem full of the pipette) onto a Petri
dish by opening the lid of the Petri dish at a 45° angle, keeping the lid over the agar.
8) Use a sterile spreader, but you can use the same spreader for each of the three plates
from the same soil sample. Spread across the whole plate surface while rotating the
plate to be sure the whole surface is covered.
9) Wrap your three plates with green tape with your name on the tape. Make sure the
agar is upside down. Leave these at room temperature. Do NOT put these in the
incubator. The Tech 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. The Tech will put
them in the refrigerator on Monday so they do not overgrow.
6) Record the following data, which you will need to input into the Yale website at
the end of the semester”
Media
Temperature (°C)
Oxygen
Length of Incubation (hr)
CFU/g (colony forming units per gram)
Total Number of Isolates
Frequency of Antibiotic Producers (%)
Antibiotic Resistance Frequency (%)
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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 ESKAPE 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. The Tech will put them in the
refrigerator on Monday so they do not overgrow.
5) Write down which of the following ESKAPE organism(s) you used
Enterococcus raffinosus
Escherichia coli
Enterobacter aerogenes
Acinetobacter baylyi
Psuedomonas putida
Staphylococcus epidermidis
Staphylococcus cohnii
Bacillus subtilis
Erwinia carotovura
Pythium ultimum
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.
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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
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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 7
1) Record the results of the previous tests.
2) Run a PCR on your isolated organism to determine the 16sRNA.
3) Run a gel electrophoresis on part of your PCR sample.
4) Send the rest of the PCR sample to the San Diego lab for sequencing for Genus.
5) After 24 hours, go to the BLAST website and find out what genus your organism
is. Look at the list of possible species they give you.
6) Use Bergey’s Manual to compare the possible species with your physiological
tests that you ran. See if you can determine the species.
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LAB 8-10
Write a report or a poster about your project