Lab 3

Question 1

Functions of soil microbes

Answer 1

-Decomposition of organic materials and the release of carbon, nitrogen and other nutrients to support plant growth.

Question 2

Nitrogen is fixed by what bacteria?

Answer 2

Diazotrophic bacteria

Question 3

Diazotrophic bacteria

Answer 3

-Nitrogen is fixed by these bacteria that are either free living (Azotobacteria) or in symbiotic assosiation with plants (Rhizobium)

Question 4

Abundance of microorganisms

Answer 4

-2 basic approaches to enumarating bacteria that are grouped into direct or indirect methods

Question 5

Direct Methods

Answer 5

1. Direct Microscopic counting (Petroff-Hausser Counting Chaamber)
2. Electronic particle counters (Coulter counter)

Question 6

Indirect methods

Answer 6

1. Membrane filtration
2. Most probable number (MPN) Method
3. Viable plate counts (Standard plate count)
4. Other methods

Question 7

Direct microscopic counting

Answer 7

• Petroff-Hausser Counting Chamber
• A dilution of the cell suspension is placed on a special slide with a grid etched into its surface
• The grid has a SA of 0.1mm^2 and after a cover is placed over the suspension the volume of 0.1mm^3
• The number of microogranisms in this small volume is counted under a microscope and extrapolated to determine a total number in the population.

Question 8

Disadvantages of the direct microscopic counting

Answer 8

• Extra particles in the material (soil) makes it difficult to see microbes
• Dead and alive bacteria cannot be differentiated
• If the cell suspension contains less that 10^6/ml dilution the cells cannot be counted

Question 9

Electronic particle counters

Answer 9

• Coulter counter
• Based on the property of electricity
• Used to count cells in suspensions that are free of extraneous matters and is often used to count larger eukaryotic cells such as red and white blood cells

Question 10

Membrane filtration

Answer 10

https: //microbeonline.com/membrane-filter-technique/
- Go through the semi permeable membrane with an aid of a vacuum and the cells that are larger than the pore size of the membrane get trapped on the surface.
- Then the membrane is placed on the surface nutrient and incubated until colonies are formed on the membrane surface.
- Then they can be counted

Question 11

Most probably number (MPN) method

Answer 11

• statistical way of estimating the number of microorganisms in samples that cointain extraneous materials and it is used to count bacteria in water for POTABILITY testing.
• This method assumes bacteria are randomly distributed and if one or more cells are present they will elicit a positive response or grow.

Question 12

Viable plate counts (STANDARD PLATE COUNT)

Answer 12

0nly live cells are counted and the method is the ability of cells to grow on or in a solid medium to produce visible colonies that can be counted.

Question 13

two methods are of viable plate counts

Answer 13

1. spread plate count

2. Pour plate count

Question 14

Spread plate count

Answer 14

-smaller known volume is spread on the surface in the prepoured medium.

Question 15

Pour plate count

Answer 15

-slightly larger volume (1.0mL) of the sample is pipetted into a sterile petri plate, then a molten agar medium is mixed in with the sample and allowed to solidify.

Question 16

Colony unit

Answer 16

colonies forming units per mL (CFU/g or CFU/ml)

Question 17

CFU/ml

Answer 17

• descirbe the origin of the colonies more accurately then just cells.
• we need to dilute the original sample to have more isolated colonies.
• usually in the range of 30-300 to be easily counted.

Question 18

In our exercise we use

Answer 18

Pour plate method

Question 19

Other methods

Answer 19

-measurment by chemical assay of particular cell organells such as dna, protein and we need to find the biomass within a sample