Lecture 7 - Exam 2: Methods For Studying Soil Microorganisms

Question 1

What are the methods we use to study microorganisms?

Answer 1

Cell numbers, biomass, microbial activity, microbial diversity.

Question 2

For the method of cell numbers, what are the two ways we study this?

Answer 2

Direct microscopic counts and viable counts.

Question 3

Before we can use direct microscopic counts to look at cell numbers, we first must do what?

Answer 3

Dispersion of soil. Disperse the soil, separate out soil particles, and make sure bacterial cells are detached from soil particles.

Question 4

We have to disperse the soil in order to begin the direct microscopic counts to look at cell numbers. What are the ways we can disperse the soil?

Answer 4

Physical dispersion: shaking, blending, ultrasonication
Chemical dispersion: sodium hexametaphosphate & sodium pyrophosphate (deflocculating agents) and can use detergents and ion exchange resins
Combined physical and chemical dispersion: We can combine physical and chemical dispersion so that we can use lower concentrations of chemicals and more gentle shaking methods.

Question 5

What is one type of microscopy used in direct microscopic counts?

Answer 5

Light microscopy.

Question 6

Light microscopy has two submicroscopies. What are they?

Answer 6

Fluorescence microscopy: uses UV light. In fluorescence, molecules absorb light of one color and emit light of a different color.
Confocal laser scanning microscopy: More expensive and gives 3D images of the cells and can look at cultures with better resolution.

Question 7

Fluorescent stains can or cannot differentiate between live and dead cells?

Answer 7

Cannot

Question 8

In fluorescence, if we have double stranded DNA, we will stain with what color?
What about if we have single stranded DNA?

Answer 8

Green
Orange

Question 9

Which is the best stain to use for soil samples?
What are the other three stains?

Answer 9

DTAF
Acridine orange (DNA stain), DAPI (DNA stain, better used for water samples), and FDA (used for fungal samples)

Question 10

There is also dead/live staining. What colors represent live cells and what color represents dead cells?

Answer 10

Live cells: green
Dead cells: red

Question 11

What does FISH stand for? What is it?

Answer 11

Fluorescence in-situ hybridization.
Can design fluorescent probe targeting the 16s rRNA. The probe that has a fluorescent label allows you to select the region and can even mix different types of probes together.

Question 12

There is other microscopy used in direct microscopic counts. What are the two others used?

Answer 12

Electron microscopy and atomic force microscopy (AFM and can see atoms). We don’t typically use these to do numeration.

Question 13

Electron microscopy has two submicroscopies, what are they?

Answer 13

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

Question 14

The other method we use for cell numbers is viable counts. What are the two ways we can do viable counts?

Answer 14

1) Dilution plate counts: serial dilution and take out of a small sample from solution and spread onto agar plate - Spread plate. Pour plate is when we put the sample in the Petri dish before we pour the agar. The agar will slow down the diffusion of the oxygen.
2) Most probable number (MPN) method: Inoculate broth media and make dilutions. Observe growth depending on the experiment. Some can be measured based on turbidity. Some need special media and turn a certain color when a certain substance is present. Have to use a MPN table to figure out correct number and have to multiply by dilution factor and use the middle number in MPN table.

Question 15

What is selective media?

Answer 15

Media that enhance the growth of certain organisms while retarding the growth of others due to an added medium component.

Question 16

What is a drawback of plate counts?

Answer 16

Less to 1-5% of bacteria won’t show up on the agar plat and most won’t grow on the agar plate. It is not a good way to numerate fungi. It is typically better to use soil extract agar and we can extract straight from soil this way.

Question 17

Bacterial biomass calculation

Answer 17

No. of bacteria/g soil x avg. vol. of bacteria x bacterial cell density (1.1ng/mm^3) x solid content of bacteria (0.4 in soil) x carbon content of dry bacteria (50%)

Question 18

When we determine volume of a bacterial cell, what must we assume?

Answer 18

That they are all rod shaped - 4/3Pi (r^3) + Pi (r^2)(l)

Question 19

So for the biomass method, we have to use the biovolume- biomass calculation. What is another thing we have to use when looking at biomass?

Answer 19

Chloroform fumigation incubation or extraction. In this, we use chloroform in the soil to kill cells. The cell content is then leaked out so the dead cells provide new carbon in the soil. We can then quantify the new carbon released by measuring carbon dioxide involved in incubation period or we can extract the carbon directly and measure dissolved carbon content. The fumigation part is the same for both the incubation and extraction procedure.

Question 20

What are three assumptions for CFI (chloroform fumigation incubation or extraction)?

Answer 20

1) The soil fumigation kills the microorganisms and it does not affect the non-living organic matter ; therefore the new C flush exclusively derives from the microbial biomass.
2) The number of organisms killed in the unfumigated soil is negligible compared with that in the fumigated soil.
3) The fraction of dead microbial biomass carbon mineralized over a given time period does not differ in different soil.

Question 21

CFI: the conversion factor is not always _________, and depends on the ______.

Answer 21

constant ; pH

Question 22

As soil pH increases…?

Answer 22

So does the conversion factor and stabilizes around a pH of 4.5.

Question 23

The third thing we use when discussing biomass is?

Answer 23

Cell constituents: Extraction, detection and quantification.
When we measure cell constituents, we need to extract the cell constituents first and we have to have some way to quantify them by detecting them first and then calculating the amounts. There are several parameters.

Question 24

What are the parameters for the cell constituent part of using the biomass method?

Answer 24

ATP, Muramic acid, Ergosterol (fungi), phospholipid fatty acids (PLFA), total DNA, small subunit rRNA gene abundance.

Question 25

The third way we can study microorganisms is by microbial activity. What are the four ways we can look at microbial activity?

Answer 25

Respiration, enzyme activity, nitrogen mineralization, and functional genes.

Question 26

We can measure respiration to look at microbial activity. What are the two types of respirations we can look at?

Answer 26

Basal respiration (actual activity) and substrate-induced respiration (potential activity).
Substrate-induce respiration is looked at by measuring glucose and CO2 measurements.

Question 27

When we measure microbial activity, what is the general theme that we see and measure?

Answer 27

Disappearance of substrate and appearance of product.

Question 28

Enzyme activity is used to study microbial activity. Overall vs specific activities are used. What is an example for determining overall activity?
What is another way we look at enzyme activity?

Answer 28

the use of the enzyme dehydrogenase. TTC -> TPF
We also use FDA hydrolysis to look at enzyme activity.

Question 29

Nitrogen mineralization is also used to measure microbial activity. What is it?

Answer 29

The mineralization of organic nitrogen compound to inorganic nitrogen compound.

Question 30

Functional genes are also a way to measure microbial activity. What is it?

Answer 30

Protein or enzymes measure their abundance and expression.

Question 31

Microbial diversity is a method we use to study microorganisms. We can analyze microbial community structure in order to look at microbial diversity. What is microbial community structure?

Answer 31

The phylogenetic makeup (identity and number) of the microorganisms present in a community.

Question 32

What are the approaches we use in order to look at the microbial community structure?

Answer 32

Phenotypic (observe characteristics of microbes, usually growth based), chemotaxonomic (looking at biochemical markers), genotypic. All are culture independent approaches.

Question 33

What is a culture dependent approach that can be used to look at microbial community structure?

Answer 33

Carbon source utilization: Have wells and substrate and an indicator in each well. If the color turns purple then you know if the substrate has been used.

Question 34

What is a chemotaxonomic method we can use to look at microbial community structure/diversity?

Answer 34

Phospholipid fatty acid analysis. These are culture independent and look at living microorganisms. Broad microbial groups and get some idea of metabolic status. Gives us information on microbes because once they die, their membranes degrade quickly after death, so gives us info on living.

Question 35

What is the PLFA nomenclature?

Answer 35

(w=omega) A:BwC
A = the number of carbon atoms in the fatty acid
B = the number of double bonds in the fatty acid
C = the position of the 1st double bond from the w end

Question 36

When there is starvation/stress, we usually see what kind of isomer?

Answer 36

Trans isomers, and we don’t know why.

Question 37

What are the ways we can look at microbial diversity?

Answer 37

Carbon source utilization, phospholipid fatty acid analysis, nucleic acid analyses, linking functions to communities.

Question 38

When we look at nucleic acid analyses, what do we look at?

Answer 38

Background (analyze the genetic characteristics of microbes) and use molecular fingerprinting techniques.

Question 39

The molecular fingerprinting techniques we use for nucleic acid analyses use what?

Answer 39

Extraction of DNA from soil samples, polymerase chain reaction, separation of PCR products, interpretation of fingerprints.

Question 40

Linking functions to communities is used when looking at microbial diversity. What is involved with that?

Answer 40

Microautoradiography combined with fluorescence in situ hybridization, stable isotope probing (DNA, RNA, PFLA, and proteins), shotgun metagenomic sequencing.