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. 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. total counts. (both dead and living bacterial cells ) or or viable counts. 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. Diluting out the organisms and counting the organisms in a number of microscopic fields on a slide. (homocytometer). (homocytometer).

2. Turbidimetric determination: Increased turbidity in a culture is another index of bacterial growth and cell numbers. 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. 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. 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). A clear solution will allow almost all of the light through (BLANK). Light entering a cloudy solution will be absorbed. 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. 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. 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. 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 ℃. 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. 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. 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. 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.  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. 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. 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. In this method only viable cells which are capable for reproduction are counted.

Pour Plate Method Materials: Materials: Culture of C. albicans. Culture of C. 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

ml C 1ml 1:10 1ml 1:100 1ml 1:1000 dilution R.S 1 ml 1 ml1 ml Melted NA 1 23

Result Dilution factor # of colonies/ plate mean X*Y (X) (Y) (X) (Y) ² 10² 10³ 10³ No of cell / 1ml of original culture (cfu/ml)= (X ₁*Y₁ )+ (X ₂ *Y ₂ )+(X ₃ *Y ₃ ) 3

Result Count the # of colonies on each plate which are in the range of Count the # of colonies on each plate which are in the range of Over 300 reported as TNTC. Over 300 reported as TNTC. Under 30 reported as TLTC. Under 30 reported as TLTC.