PHT 381PHT 381Lab # 6Lab # 6

Bacterial population count Bacterial population count

Many bacteriological studies require that Many bacteriological studies require that we arewe are able to determine the number of able to determine the number of m.o per unitm.o per unit volume of a given sample. volume of a given sample.

This measurement is needed This measurement is needed for:-for:-

1.1.Standardization of inocula in Standardization of inocula in microbiological assaymicrobiological assay [e.g; [e.g; evaluation of antimicrobial agents, evaluation of antimicrobial agents, assay of vitamins]assay of vitamins]

2. 2. Industrial fermentation.Industrial fermentation.

3. 3. Evaluation of sterilization techniqueEvaluation of sterilization technique

Several different methodsSeveral different methods can be can be used for determination of either used for determination of either

total total counts.counts.

(both dead and living bacterial cells )(both dead and living bacterial cells ) oror

viable viable counts.counts.

(living bacterial cells only)(living bacterial cells only)

Determination of Determination of Total Total Bacterial Counts:Bacterial Counts:

1. Direct microscopic count. 1. Direct microscopic count.

2. Turbidimetry determinations. 2. Turbidimetry determinations.

3. Dry weight and nitrogen 3. Dry weight and nitrogen content determinationscontent determinations

1. Direct microscopic 1. Direct microscopic count :count :

Diluting out the organisms and Diluting out the organisms and countingcounting thethe organisms in a organisms in a number of microscopic fields on a number of microscopic fields on a slide.slide.

(homocytometer).(homocytometer).

2. Turbidimetric 2. Turbidimetric determination:determination:

Increased turbidity in a Increased turbidity in a culture is another index of culture is another index of bacterial growth and cell bacterial growth and cell numbers. numbers.

☂ ☂ ↑ ↑ the # of cells during growth ↑ the # of cells during growth ↑ the turbidity.the turbidity.

There isThere is a liner relationshipa liner relationship betweenbetween turbidityturbidity andand cell numbercell number of a given bacterial culture.of a given bacterial culture.

By using By using a spectrophotometer,a spectrophotometer, the amount of transmitted light the amount of transmitted light decreases as the cell population decreases as the cell population increases. increases.

USE OF THE USE OF THE SPECTROPHOTOMETER:SPECTROPHOTOMETER:

A clear solution will allow almost all of the light A clear solution will allow almost all of the light through (BLANK).  through (BLANK). 

Light entering a cloudy solution will be Light entering a cloudy solution will be absorbed. absorbed. 

The amount of absorbance is determined by The amount of absorbance is determined by measuring what fraction of the light passes measuring what fraction of the light passes through a given solution and compared to that through a given solution and compared to that absorbed by a clear solutionabsorbed by a clear solution. .

SPECTROPHOTOMETERSPECTROPHOTOMETER

The amount of The amount of cells in the cells in the solution is directly solution is directly proportional to the proportional to the absorbance absorbance reading. reading. 

A graph of A graph of absorbance vs. absorbance vs. concentration will concentration will give a straight line.give a straight line.

Turbidimetric determinationTurbidimetric determination

33. Dry weight and nitrogen . Dry weight and nitrogen content determinations:content determinations:

In this method, the bacterial cell are In this method, the bacterial cell are collected by centrifugation, then collected by centrifugation, then dried in an oven overnight at 85dried in an oven overnight at 85℃. ℃.

The dry weight of bacterial mass The dry weight of bacterial mass will be proportional to their number. will be proportional to their number.

Also the nitrogen content of the dry Also the nitrogen content of the dry sample can be determined by micro-sample can be determined by micro-kjeldahl method.kjeldahl method.

Determination of Determination of ViableViable Bacterial Bacterial Count:Count:

1. Measurement of 1. Measurement of microbial activitymicrobial activity

2. Pour plate method.2. Pour plate method.

3. Spread plate method.3. Spread plate method.

1.1. Measurement of microbial Measurement of microbial activity:activity:

Many microbial activity measured Many microbial activity measured quantitatively and used as a measure of quantitatively and used as a measure of microbial growth. microbial growth.

e.g; the growth of acid forming bacteria e.g; the growth of acid forming bacteria may be measured by simple titration of may be measured by simple titration of the culture using standard alkali.the culture using standard alkali.

in this method in this method only viable cellsonly viable cells which which are capable for reproduction are are capable for reproduction are counted.counted.

2.2. Pour plate method:Pour plate method: Principle:Principle: Based on the fact that if the viable Based on the fact that if the viable

cell are allowed to grow apart from cell are allowed to grow apart from each other on a solid medium, each each other on a solid medium, each cell develops into one visible colony. cell develops into one visible colony. The number of colonies obtained is The number of colonies obtained is equal to the number of viable cells. equal to the number of viable cells.

In this method In this method only viable cellsonly viable cells which are capable for reproduction which are capable for reproduction are counted.are counted.

Pour Plate MethodPour Plate Method

Materials:Materials: Culture of Culture of C. albicansC. albicans.. Melted nutrient agar.Melted nutrient agar. Ringer solution.Ringer solution. 3 Test tubes.3 Test tubes. 3 Petri dishes.3 Petri dishes. Sterile 1ml pipette.Sterile 1ml pipette. Sterile 10ml pipette.Sterile 10ml pipette.

Drawer Drawer

1 2 3

9 ml

C

1ml

1:10

1ml

1:100

1ml

1:1000 dilution

R.S

1 ml 1 ml 1 mlMelted NA

1 2 3

ResultResult

Dilution factor Dilution factor # of colonies# of colonies/ / plateplate mean X*Y mean X*Y

(X) 1 2 3 (X) 1 2 3 (Y) (Y)

1010

10²10²

10³10³No of cell / 1ml of original culture (cfu/ml)= (X₁*Y₁)+ (X₂*Y₂)+(X₃*Y₃) 3

ResultResult

Count the Count the ## of colonies on each of colonies on each plate which are in the range of plate which are in the range of 30-300.30-300.

Over 300 reported as TNTC.Over 300 reported as TNTC.

Under 30 reported as TLTC.Under 30 reported as TLTC.