Bacterial growth measurement

Bacterial growth measurement by different methods

1. Bacterial Growth Measurement Rajeshwari Jagadish Bangalore University

2. Growth is increase in number, mass, size of cells. Growth in bacterial usually refers to reproduction i.e increase in population size rather than enlargement of cells . Reproduction in bacteria occurs mainly through binary fission or transverse fission. The time taken by a population of bacterial cells to double its population is called “doubling time” or “generation time”. The length of generation time is a measure of the growth rate of an organism. The average time in bacteria is 30–60 mins under optimum conditions. Staphylococcus aereus and Salmonella enteriditis are example of pathogens having relatively short doubling time (20 mins). Mycobacterium leprae (14 days) has the largest generation time. Growth

3. Lag phase -During this phase the number of cells remains the same but considerable metabolic activity occurs . Log phase -In this stage bacteria begins dividing and therefore cell number increases exponentially, it is called exponential phase . Stationary phase -In this phase the population remains constant for a time since the reproduction rate is balanced by an equivalent death rate . Death phase -The bacteria die at a faster rate than the formation of new cells, therefore the number of viable cells decreases exponentially The bacterial growth curve shows 4 distinct phases of growth.

4. Turbidometric method 2) Cell count by haemocytometer 3) Standard plate count method. Bacterial growth can be measured by the following method.

5. Aim : To measure the growth of bacteria by Turbidometric method. Priciple : Scattering of light increases with increase in cell number. When light is passed through bacterial cell suspension, light is scattered by the cells. Therefore, transmission of light declines . At a particular wavelength absorbance of light is proportional to the cell concentration of bacteria present in the suspension. TUBIDOMETRIC METHOD

6. Thus cell growth of any bacterial suspension at a particular wavelength at different intervals can be measured in terms of absorbance and a standard graph (between absorbance and cell concentration) can be prepared . It is an indirect method of determining the microbial mass by light absorption. Optical density is directly proportional to the concentration of the cell .

7. REQUIREMENTS 10 -12hr old broth culture of the bacterium whose generation time is to be determined. Cuvetts and spectrophotometer /colorimeter. Pipette, conical flask, Nutrient broth PROCEDURE ; Prepare 100ml of nutrient broth and sterilise by autoclaving at 121 c for 15 mins at 151 lbs . 2. About 0.5 ml of 10 -12hr old broth culture of the test bacterium was added to the conical flask after bringing it to room temperature . 3. Contents were mixed well and immediately 0 min reading was noted against uninoculated media as blank at 620nm. Then incubate the conical flask at 37 C for a regular interval of 30 mins,60 min, 90,120, 150 till 12 hours . 4. The graph of the time v/s absorbance at 620nm was plotted.

8. Principle : Haemocytometer is instrument used to mainly count the number of blood cells and estimate their concentration. Dead cells cannot be distinguished from living ones. Cell growth is also measured by counting total cell number of the microbes present in that sample. Total cells (both live and dead) of liquid sample are counted by using a special microscope glass slide called Haemocytometer. In this slide a grid is marked on the surface of the glass slide with squares of known area. The whole grid has 25 large squares, a total area of 1 mm 2 and a total volume of 0.02 mm 3 (1/50 mm). All cells are counted in large square and total number per ml sample is measured. If 1 square contains 12 cells, the total number of cells per ml sample will be: 12 cells x 25 square x 50×103 = 1.5xl07 cells. CELL COUNT METHOD AIM : To measure the growth of bacteria by cell count method.

9. REQUIREMENTS: Nutrient broth media, bacterial culture , flasks PROCEDURE : Prepare 100ml of nutrient broth and sterilise by autoclaving at 121 c for 15 mins at 151 lbs . 2. About 0.5 ml of 10 -12hr old broth culture of the test bacterium was added to the conical flask after bringing it to room temperature . 3. Using a pipette, take 100 µL of cell suspension and apply to the  hemocytometer . Using a microscope, focus on the grid lines of the  hemocytometer  with a 10X objective. When counting, the cells are only counted when they are set within a square or on the right-hand or bottom boundary line.

10. 4.The number of cell was counted in at least in 5 small squared separately. The average in each square was calculated . 5. The slide should not be allowed for a long period to be counted because this will affect counting due to evaporation from the suspension . 6. The number of cells per ml of the suspension was calculated . Then incubate the conical flask at 37 C for 12 hours and regular interval of 30 mins,60 min, 90,120, 150 till 12 hours 100 µL of culture was removed and cells were counted. 7. The graph of the time v/s no. of cells was plotted.  

11. AIM : To measure the growth of bacteria by plate count method. Principle : Indirect viable cell counts, also called plate count method, It is the one of the most routinely used produce because of the possibility of enumerating viable cells. This method is based on the principle that when material containing bacteria is cultured, every viable bacterium develops into a viable colony on a nutrient agar medium. Therefore the number of colonies are same as the number of organism (individual cells) in the sample. The colonies developed on the NA media are counted. The number of organism is obtained by the number of colony obtained on the plate . Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. Disadvantages are only living cells develop colonies that are counted; clumps or chains of cells develop into a single colony; colonies develop only from those organisms for which the cultural conditions are suitable for growth. PLATE COUNT METHOD

12. ( i ) Spread Count Method: A volume of culture (0.1 ml) is spread over the surface of an agar plate by using a sterile glass spreader. The plate is incubated to develop colonies. Then the number of colonies is counted There are two ways of forming plate count. (ii) Pour Plate Method: In this method a known volume (0.1-1.0 ml) of the culture is poured into the sterile Petri dishes. Then melted agar medium is poured and mixed gently. The plate is incubated. Colonies growing on the surface of agar are counted.

13. PLATE COUNT METHOD In this method a known volume (0.1-1.0 ml) of the culture is poured into the sterile Petri dishes. Then melted agar medium is poured and mixed gently. The plate is incubated. Colonies growing on the surface of agar are counted. PROCDURE: Prepare 100ml of nutrient broth and sterilise by autoclaving at 121 o Cfor 15 mins at 151 lbs. 2. About 0.5 ml of 10-12hr old broth culture of the test bacterium was added to the conical flask after bringing it to room temperature. 3. Using a sterile pipette, take 0.1mL of cell suspension the agar surface and spread around using a sterile glass rod and label it as 0 min. 4. Then incubate the conical flask at 37 o C for 12 hours and at regular interval of 30 mins, 60 min, 90,120, 150 till 12 hours 0.1mL of cell suspension was removed and spread onto fresh NA plates and labelled accordingly. 4. The graph of the time v/s no. of colonies was plotted.

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