LABORATORY 8
SOIL BIOLOGY
I
Objectives
Learn procedure for enumerating soil microorganisms. Quantify effect of organic
amendments on soil microbial respiration.
II
Introduction
The soil is home to a wide range of plant and animal life. Roots are the largest form of
plant life and certain rodents, insects and earthworms, the largest animals. Though
higher plants are the primary producers of chemical energy that sustains the terrestrial
ecosystem, plants are dependent on the invisible (other than certain fungal structures)
community of soil microorganisms for continued supply of many essential nutrients.
Nutrient elements bound in organic combinations would be inaccessible to plants
without microbial decomposition of organic matter and mineralization of these elements.
Not only would nutrient cycling stop but also biological N-fixation. Soil microorganisms
are indispensable to life on earth.
Soil microorganisms live in thin films of water that surround soil particles. These tiny
organisms include microflora (bacteria, fungi and actinomycetes) and microfauna
(protozoa and nematodes). In terms of numbers and biological activity the microflora
are dominant. Bacteria are small (about 1 - 10 µm) and occur in three general shapes
Brod (bacillus), spherical (coccus) and spiral (spirilla). Bacilli and cocci are more
common in soil. Fungi are filamentous and much larger. The branched hyphae exhibit
cell divisions and fungal mycelia (hyphal mass) are often macroscopic. Actinomycetes
are also filamentous and branched but smaller.
The exercises included in this laboratory are intended as a survey of the numbers and
activity of soil microorganisms. Quantitative evidence of microbial activity is provided by
respiration data. Furthermore, by enriching the soil with one type of substrate or
another, the effect of C source on microbial growth is readily seen (for example,
vigorous growth with a N as well as C rich substrate but slower growth otherwise).
-1
A
Agar Plate Method for Microbial Count
In this method, soil is dispersed in an agar medium so that individual microbial cells,
spores or mycelial fragments develop into macroscopic colonies. The procedure
involves successive dilutions of soil. Depending upon extent of dilution, plates may be
filled with a huge number of colonies or very few. Enumeration of colony-forming units
initially present in the soil is from plates in between these extremes. This method
requires sterile technique to avoid introduction of extraneous microbes.
Any one of several different growth media can be used but no single growth medium is
optimal for all microorganisms that inhabit soil. Thus, growth of certain organisms is
favored in the chosen medium and growth of others is stymied. Antagonistic or
antibiotic effects may also affect colony development in the agar plate method.
B
Soil Respiration
Soil is teeming with organisms that release CO2 as organic matter is oxidized.
Respiration rate in soil can be determined by measuring the rate of CO2 evolution. A
straightforward method of determining CO2 takes advantage of its conversion to CO32in the presence of excess OH-. This reaction consumes OH- so that if the initial and
final amounts of OH- were known by titration, CO2 could be calculated. However, the
CO32- must somehow be removed before titrating OH-. Precipitation of CO32- by Ba2+ to
form the very insoluble BaCO3 takes care of this problem.
The below reactions summarize the foregoing.
CO2
+
H2O
H2CO3 + 2NaOH
Na2CO3 +
BaCl2
6
6
6
H2CO3
Na2CO3 + 2 H2O
BaCO3 9 + 2NaCl
If soil organisms are supplied with an oxidizable organic substrate, respiration rate
temporarily increases. This exercise measures the average respiration rate of moist soil
that has and has not been amended with organic matter.
-2
III
Procedures
A
Agar Plate Method for Microbial Count
You are provided with a homogenized, field-moist sample of topsoil. You are also
provided with bottles containing 90 mL of sterilized water. One has glass beads in it.
1.
Add a 10 g subsample of topsoil to the bottle of sterilized water that contains
glass beads (used to aid dispersion of soil). Tightly cap and shake vigorously for
10 minutes to disperse the soil. This is the 10-1 dilution.
2.
Transfer 10 mL of the 10-1 dilution to another bottle of sterilized water. Use a
sterile pipette. Take sample from the middle. Tightly cap and shake to uniformly
mix. This is the 10-2 dilution.
3.
Repeat step 2 using the 10-2 dilution to make a 10-3 dilution and proceed
similarly, making 10-4, 10-5, 10-6 and 10-7 dilutions.
4.
From the 10-7 dilution, transfer 1 mL to each of 2 sterile petri dishes using a
sterile 1 mL pipette. Make similar transfers from the 10-6, 10-5 and 10-4 dilutions.
5.
Into each seeded petri dish, pour enough sterile, melted agar to fill dish about 2
full. Immediately swirl it around to ensure good mixing of soil inoculant and agar.
6.
After the agar has solidified, invert plates and incubate at room temperature for 1
week.
7.
Next week, count the number of colonies on plates from the dilution that contains
from 30 to 300 colonies. Do not count from those plates that contain colonies
larger than 2 cm diameter. Record in Table 1. Multiply by dilution, take the
average and correct to oven-dry moisture content of the soil. This gives the
number of colony-forming units (CFUs) per gram of soil.
B
Soil Respiration
You are provided fresh, moist soil to which has been added a high C / N substrate, a
low C / N substrate or nothing. One-half g of each organic amendment was added per
25 g soil (oven-dry equivalent).
1.
Transfer 25 g oven-dry equivalent (moisture content given) of each amended and
unamended soil to a separate Mason jar. Place soil in jar to one side rather than
uniformly across bottom.
2.
Based on initial moisture content, add sufficient distilled water to bring moisture
-3
to 30 % by weight. Label each jar with amendment and lab group.
3.
Similarly, label each of three 50 mL centrifuge tubes with amendment and lab
group, then pipette exactly 20 mL of 1 N NaOH to each centrifuge tube.
4.
Carefully set a tube containing NaOH into each of the Mason jars. Do not spill
caustic NaOH on you or soil. It will chemically burn you and kill soil
microorganisms.
5.
Tightly cap jars to prevent any exchange of CO2 with the atmosphere and set
aside until next lab.
6.
Next week, carefully uncap and remove centrifuge tubes from the Mason jars.
7.
Add 10 mL of 2 N BaCl2 to each centrifuge tube.
8.
Spin BaCO3 precipitate down in centrifuge. Your lab instructor will help you.
9.
Carefully pipette 10 mL from the upper portion of supernatant (do not disturb
precipitate) to each of three 125 mL Erlenmeyer flasks.
10.
Add 40 mL distilled water and 4 drops of phenolphthalein indicator. Titrate with
0.25 N HCl. Endpoint is change from pink to colorless. Record mL (to nearest
0.1 mL) of HCl used in Table 2.
11.
Calculate milliequivalents of OH- consumed by reaction with CO2.
You are given the initial normality of NaOH. The meq of OH- consumed by
reaction with CO2 is given by difference with final meq of OH-.
meq OH- consumed = meq OH- initial - meq OH- final
where
meq OH- final
= 3 x (mL HCl x N HCl)
The factor 3 arises since only 1/3 of the NaOH solution is titrated (10 mL of 20
mL NaOH + 10 mL of BaCl2).
Each meq OH- consumed corresponds to 2 millimole of substrate C oxidixed
(see earlier equation) or 6.00 mg C.
IV
Worksheet
-4
A
Total Microbial Count
Table 1.
Plate No.
Dilution
No. of Colonies
No. CFU / g soil
Average CFU / g
Calculations
B
Soil Respiration
Table 2.
Treatment
mL HCl
meq OH- Final
None
Calculations
-5
meq OH- Consumed
mg CO2 / g soil