Download LAB 6B

PHT 381
Lab # 6
Bacterial population count
Many bacteriological studies require that we are
able to determine the number of m.o per unit
volume of a given sample.
This measurement is needed for:1.
Standardization of inocula in
microbiological assay [e.g; evaluation of
antimicrobial agents, assay of vitamins]
2. Industrial fermentation.
3. Evaluation of sterilization technique
Several different methods can be used for
determination of either
total counts.
(both dead and living bacterial cells )
or
viable counts.
(living bacterial cells only)
Determination of Total Bacterial
Counts:
1. Direct microscopic count.
2. Turbidimetry determinations.
3. Dry weight and nitrogen content
determinations
1. Direct microscopic count :
Diluting out the organisms and counting
the organisms in a number of
microscopic fields on a slide.
(homocytometer).
2. Turbidimetric determination:

Increased turbidity in a culture is
another index of bacterial growth and
cell numbers.
☂ ↑ the # of cells during growth ↑ the
turbidity.


There is a liner relationship between
turbidity and cell number of a given
bacterial culture.
By using a spectrophotometer, the
amount of transmitted light decreases as
the cell population increases.
USE OF THE SPECTROPHOTOMETER:

A clear solution will allow almost all of the light through
(BLANK).

Light entering a cloudy solution will be absorbed.

The amount of absorbance is determined by measuring
what fraction of the light passes through a given solution
and compared to that absorbed by a clear solution.
SPECTROPHOTOMETER


The amount of cells
in the solution is
directly proportional
to the absorbance
reading.
A graph of
absorbance vs.
concentration will
give a straight line.
Turbidimetric determination
3. Dry weight and nitrogen content
determinations:



In this method, the bacterial cell are collected by
centrifugation, then dried in an oven overnight
at 85℃.
The dry weight of bacterial mass will be
proportional to their number.
Also the nitrogen content of the dry sample can
be determined by micro-kjeldahl method.
Determination of Viable Bacterial Count:
1. Measurement of microbial activity
2. Pour plate method.
3. Spread plate method.
1.
Measurement of microbial activity:
Many microbial activity measured
quantitatively and used as a measure of
microbial growth.
e.g; the growth of acid forming bacteria may
be measured by simple titration of the culture
using standard alkali.
 in this method only viable cells which are
capable for reproduction are counted.

2. Pour plate method:
Principle:
Based on the fact that if the viable cell are
allowed to grow apart from each other on a
solid medium, each cell develops into one
visible colony. The number of colonies
obtained is equal to the number of viable
cells.
In this method only viable cells which are
capable for reproduction are counted.
Pour Plate Method
Materials:
 Culture of C. albicans.
 Melted nutrient agar.
 Ringer solution.
 3 Test tubes.
 3 Petri dishes.
 Sterile 1ml pipette.
 Sterile 10ml pipette.

 Drawer 
9 ml
1ml
1ml
C
1ml
1
2
3
1:10
1:100
1:1000
R.S
Melted NA
1 ml
1
1 ml
2
1 ml
3
dilution
Result
Dilution factor
mean
X*Y
(X)
# of colonies /plate
1
2
3
(Y)
10
10²
10³
No of cell / 1ml of original culture (cfu/ml)= (X₁*Y₁)+ (X₂*Y₂)+(X₃*Y₃)
3
Result

Count the # of colonies on each plate
which are in the range of 30-300.

Over 300 reported as TNTC.

Under 30 reported as TLTC.