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Methods of Enumeration and Activity of
Microorganisms
Cultural Methods
The cultural methods are selected depending upon the type of microbes
These methods vary with different types of microbes
Some specific microbes need a selective method
- Ex
...
Standard Plate Count
2
...
Streak plate method
b
...
Pour plate method
d
...
Choose appropriate nutrient agar plate
2
...
Dip glass “hockey stick” in 70% ethanol
...
Do NOT put back into alcohol
4
...
With one hand rotate the dish
...


Serial Dilution Method
This method is used to obtain pure culture of those microorganisms that have not yet
successfully cultivated on solid medium and grow only in liquid media
Series of dilutions are made from a distinct colony in the test tubes
From each dilution 100ul inoculum is spread on solid medium
Incubation at optimum temperature
Further isolation by streak plate method
It is better to make dilutions of
1:10
10-1

1:100
10-2

1:1000
10-3

1:10,000
10-4

After a few days bacterial colonies will appear which can be counted on colony counter
Number of colonies are multiplied by dilution factor to find out the total number of cells per ml of
original sample
Example:
-

Number of bacterial colonies/plate: 15
Dilution Factor: 1:1000
Total number of cells in 1 ml sample
- 15x 1000 = 15,000

Characterization of Isolates (Criteria)
-

Ensure you that you start with pure culture

-

Determine whether gram positive or gram negative

-

Establish the relationship with oxygen (anaerobic or aerobic)

-

Whether the cells are capable of motility

-

Observe morphological characteristics (cell shape, flagellum)

-

Colony morphology on agar medium

-

Colony size

Use different test for identification

Bacteria from Water
Two techniques are adopted:
a
...
Plating method
Membrane filter technique
- In this method water sample is concentrated rather than dilution because they contain
low number of bacteria
-

The bacteria are trapped on a membrane which are then place on agar medium and are
allowed to grow at proper temperature

Plating Method: for example
a
...
Streak plate method
c
...
General purpose media:
- For common heterotrophic bacteria
b
...
Nutrient rich media (ex
...
Nutrient poor media (ex
...
Elective enriched medium

-

It allows the growth of a single or limited type of bacteria based on unique
combination of nutritional or physical attributes

2
...
g
...
Specialized isolation medium
- This meets the requirements of specific groups of microbes
- Ex
...
Initial enrichment
b
...
Serial dilution method
b
...
Pour plate method

Cultural Methods for Algae and Cyanobacteria
Dilution and plating method are used
MPN (Most probable number) is also used
Filamentous algae need grinding of soil in mortar and pestle to homogenize the soil
Antibiotics and fungicides are added to check the bacterial and fungal growth
Incubation for 1-3 weeks at 20-25 C with photoperiod of 12 hours and 16 hours darkness
Very specific medium is used for cyanobacteria

Detection of Viruses by Cell Culture
The main cell cultures are used in virology
a
...
Continuous cell culture
Primary cell culture
- In this case cells are removed directly from an animal and can be used to subculture
viral cells for only a limited number of times
Continuous cell culture
- In this case cell lines are derived from animal or human and are sub-cultured indefinitely

Detection and Quantification of Viruses
Two methods are used for this purpose:
1
...
Plaque forming unit (PFU)
Cytopathic effect
- In this case the changes in cells are observed due to viral replication
-

The infected cells become lysed

-

Different viruses produce different CPE
- Ex
...
Serial dilution end point (TCID50 Method)
2
...
g
...
Substrate disappearance
b
...
Cell mass increase
d
...
g

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