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Direct and indirect bacterial number assessment in the soil

Lap report, Rhizosphere Ecology, WeberV, 24.12.2007

1 Practical Session No 2

1.1 Bacterial number assessment through cultivation
To the soil NaCl solution was added. Then different dilutions from 10-1 to 10-10 of soil suspension were prepared. 200 µl of each suspension was spread on an agar plate.

1.2 DNA extraction from soil
DNA extraction was followed after the instructions of kit’s manufactures. Then the DNA was diluted 1:100 for PCR. Then PCR with two primers was run to amplify DNA.

1.3 Extraction of lipids from the soil
Internal standard and chloroform-methanol were added to 10g soil samples. After centrifugation the liquid phase was transferred to a new vial. Then NaCl was added. After centrifugation the clear phase was taken out, filtered and evaporated.

1.4 Direct bacterial cell enumeration in soil
To 1-2g soil formaldehyde which fixes the cells was added. 0,2 ml of the soil suspension were transferred into a new vial. Afterwards DAPI was added which is a phenol that captures DNA. Then alternating water was added, centrifuged and then the supernatant was removed (all in all 3 times). 15 µl of the resulting suspension was masked on microscope slides and evaporated. Then this slide was put under the microscope. In the resulting pictures the bacteria colonies were counted and then the amount of bacteria in the soil was computed.

Expected observation: least bacteria in bulk soil but here most bacteria in bulk soil. The problem was that only 4 different areas where counted by 4 different people and the pictures where sometimes blurry

Questions
Comparison of cultivate and total bacterial counts

  • It could be 1,3 * 107 bacteria be cultivated
  • It could be 3 * 108 detected with DAPI straining

So as a result only 5% of the total bacteria colonies could be counted within the cultivated plates

Why there is usually a difference between estimated cell counts at different dilutions?
Bacteria inhibit themselves. That’s why there are fewer bacteria then expected

2 Practical Session No 3

2.1 Reading bacteria enumeration assay
The cultivable bacterial colonies on the agar plates were counted, which make only 1% of the total population in the soil.

2.2 RFLP patterns of 16S amplicon from planted, rhizosphere and bulk soils
With restriction enzymes the DNA was cut to fragments in a PCR. While the process was running an agarose gel was prepared. Then the gel was loaded with the restricted DNA and a standard. Then the electrophoresis was run. The result was observed under UV light and photographed.
First RFLP didn’t function because the Elution Buffer was used instead of PE-Buffer => DNA was washed out to early

Questions
Resolution of RFLP and lipid profiles? Can they be drawn back to species level?
The first gel was empty. The second one shows no clear difference of the DNA length between the three soils (rhizosphere, plant and bulk). So the amount of species can not be estimated out of the gel. The advantage of this gel is that within one band the species could be detected by taking the DNA in this band out and analyzing it.

2.3 Gas chromatography of fatty acids from soil

Idea behind
Membranes of organisms are composed of lipids. A high amount of lipids represents a high activity of organisms (lipid storage, turnover). Different organisms have a different lipid composition. This specification is visible in the FA profile.  Lipid chromatography helps to get general idea about organism community present or to detect an organism with special FAs (e.g. mycorrhiza). The approach depends on objective. If the investigation of activity / functional groups is of interest a lipid chromatography is the right method. If the detection of a specific organism is of interest molecular methods should be used. With molecular methods a differentiation between dead and living organisms is not possible. On the other hand is the resolution of molecular methods a lot higher than with lipid chromatography.

Method:
0,5 µl of the mixture was injected to the GC. The GC calculates the absolute contents of fatty acids by using the internal standard in the mixture. The fatty acids were burnt with H2 inside the chromatograph. Within this process ions are produced due to temperature. The amount of charged ions was then detected. The separation of the fatty acids is according to the size and the saturation of the lipid (the more chains and unsaturated the later). The Peaks (see graphic) represent the fatty acids. The area of the peak reflects the amount of the fatty acids. Due to the standard C11:0 the amount of lipids was computed. Towards the end the baseline is to low. With this the calculation of the peak area is too high. As a result the surplus area ahs to be subtracted in order to quantify fatty acids longer than C18:1.

lab-report_rhizosphere-ecology_ii_p1.jpg

The more lipids the more active organisms are in the soil. With the distance the lipid concentration should decrease (Plant > rhizosphere > bulk; but here bulk > plant > rhizosphere maybe because of the low internal standard)

Questions
Which of the molecular and lipid assays are more relevant for identifying living biomass? Why?
The Approach depends on objective. If the activity of micro organisms or functional groups of the lipids are the aim of a research the lipid chromatography should be used, because lipids are metabolic products which are only produced if the micro organism is alive. If a specific organism is to be detected than molecular methods should be used, because with these the resolution is higher. The Problem is that molecular methods don’t differentiate between dead and living organisms because they rely on DNA.

Imagine that you find a soil where PVC is quickly metabolized by unknown organisms. What would you undertake to identify and exploit those organisms? Do you think you get rich with patent protecting your rights to use such organisms.
To identify this organism the organisms has to be grown first on a specific agar. With the investigation a specific lipid structure could be researched and with this a lipid chromatography could be used. To use the microorganism for degradation PVC it has to be multiplied and then released on PVC. With this special bacterium you could earn a lot of money because PVC is nearly not degradable.

2.4 In-situ rhizosphere pH under different N fertilization
Agar was produced and then poured over the plant or put on the soil layer as agar plate. Then the coloring of the agar was observed. In the ammonium application the roots are alkaline whereas the soil is more acid. The reason for this is that the plant takes ammonium and exudates H+. In the case of nitrate the opposite is the case. The plant takes nitrate and H+. With this the soil around the roots is getting more acid.

lab-report_rhizosphere-ecology_ii_p2.jpg

Questions
How will microorganisms affect the pH in the rhizosphere? And how will they be affected by changes induced by the plant.
Mycorrhizas acidify for example the soil by nitrification (because they need lots of Ca to do it). I don’t know an additional example.
With KNO3 a pH of 7 was observed at the plat roots. The reason is, that the plant releases OH parallel to taking in NO3
With Ammonium a pH of 4 was observed. The reason is that the plant takes Ammonium in exchange of H+. With this the soil is acidified

December 24th, 2007
Topic: Crop Science, Plant nutrition Tags: None

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