Download SOIL 4400 Soil Ecology

SOIL 4400 Soil Ecology: Laboratory 3 pg 1
SOIL 4400 Soil Ecology
Laboratory 3 : Enumerating from Dilution Plates, Streaking, and Viewing
Soil Organisms
You will in this laboratory:
a) Report your findings of cfu per gram of soil
b) streak out growth of boiled nutrient agar tubes onto agar medium
c) Learn how to adjust a microscope for optimal use
d) view fungal morphologies of prepared slides using the compound
microscopes
e) view prepared slides of various soil organisms
A) Follow laboratory exercise #1 to determine cfu/g soil for each treatment
and medium
B) Streaking Bacteria From Plates Derived From Boiling-soil Tubes:
1.
2.
3.
4.
5.
Sterilize an inoculating loop by placing it at an angle over a flame.
Cool the inoculating loop by stabbing it into the agar.
Remove the lid from the test tube.
Shake the soil tube and dip the loop into the slurry.
Streak the loop containing the bacteria at the top end of the agar plate
moving in a zig-zag horizontal pattern until 1/3 of the plate is covered.
6. Sterilize the loop again in the flame and cool it at the edge of the agar
away from the bacteria in the plate that you just streaked.
7. Rotate the plate about 60 degrees and spread the bacteria from the first
streak into a second area using the same motion in step 6.
8. Sterilize the loop again using the procedure in step 7.
9. Rotate the plate about 60 degrees and spread the bacteria from the
second streak into a new area in the same pattern.
10. Sterilize the loop again.
11. Replace the lid and invert the plate. Incubate the plate at 24 degrees
Celsius.
12. Next week you should see bacterial cells growing in streaks and in
isolated areas.
Tips:
1. When sterilizing the inoculating loop, make sure that the entire loop turns
orange before using on the agar plates.
2. When streaking the agar with the loop, be sure to keep the loop horizontal
and only streak the surface of the agar.
SOIL 4400 Soil Ecology: Laboratory 3 pg 2
C) Preparation of Fungal Wet Mounts
You will prepare wet mounts using water and cotton blue to view three different
colonies of fungi (seem to be different fungi) isolated previously.
1. Place a small drop of mounting medium on a microscope slide (water and
then on a different slide cotton blue)
2. Using a sterilized dissecting or inoculating needle remove a small (no
more than 2 mm square) portion of the colony near the margin, taking with
it a very thin layer of the agar surface. If the colony is thick and woolly, it
may not be necessary to take the agar, but in the more appressed type it
is essential.
3. Place the piece of colony in the mounting medium, and, with a second
needle, tease it out so that the filaments are well spread. A mount that has
not been teased out will appear as an opaque lump yielding little
information.
4. Place a cover-slip over the mount, lowering one edge to the slide before
the other so that air bubbles can escape.
5. If the stain to too heavy on the slide draw the stain out of the slide by
touching paper to the edge of the cover slip
6. Seal the edge of the cover slip with nail polish and view your slide
7. Can you see the following
a. Clamp connections
b. Septa
c. Spores
d. Irregular or tubular hyphal shape
SOIL 4400 Soil Ecology: Laboratory 3 pg 3
D) Prepared Slides
Observe one slide of the following prepared slide groups:
1. Gram +
a. 90 W 0533
b. 90 W 0131
c. 90 W 2021
2. Spiral bacteria
a. 90 W 0560
b. 90 W 3005
3. Gram –
a. 90 W 2070
b. 90 W 2073
c. 90 W 0565
4. Blue Green Algae
a. 91 W 0050
b. 91 W 0052
SOIL 4400 Soil Ecology: Laboratory 3 pg 4
Parts of A Microscope and Setting Kohler Illumination
Setting up Kohler Illumination
Having light focused through the condenser onto the specimen is necessary for optimum
illumination and contrast of the item being viewed on the slide. Kohler illumination is the
conditions of the microscope to optimum lighting of the specimen. To set up for Kohler,
do the following:
a) Place slide with specimen on the stage
b) Set objective to 10x magnification
c) Focus specimen
d) Move the polarizer lever on the condenser to the right (remove polarization)
e) Close iris diaphragm at illumination base (should see a small spot of light in the
ocular)
f) Move the condenser thumb screws to centre the spot of light
g) Adjust the condenser knob up and down to focus edge of iris diaphragm (should
see sharp edges, and blue colour edge, not red)
h) Open iris to just beyond field of view in ocular
i) Congratulations, you have reached Kohler status, yeh and enjoy.