The below mentioned article provides a study on the Growth of Bacteria by the help of Turbidimetric Technique.
The Growth Theory of Bacteria:
The growth of bacteria in a suitable population can be divided into the following phases:
(i) The lag phase
(ii) The log phase
(iii) The stationary phase and
(iv) The phase of decline.
The duration of each of these phases depends on various factors such as growth condition, the number of initial inoculum and growth medium etc. The lag phase is supposed to be the phase preparatory to the most active phase of growth, log phase. During this log phase, bacterial number increases in an exponential rate and increase can be expressed by the relation N = No + α t …(1)
where α is the constant for growth.
The generation time of the population of bacteria is defined as the time in which the population doubles itself and is obtained from the relation (1). Thus at t = T where the population doubles, N = 2No and from the expression (1)
2 = eαT
or log e2 = α T
or T = 23 log102/α
= 0.69/α
So, if one plots the number of bacteria per ml. (N) against the time of growth (t), one obtains a graph, from the slope of the logarithmic part of which T, the generation time, can be obtained.
Requirements:
(1) Microscope;
(2) Haemocytometer;
(3) Diluting pipettes;
(4) Colorimeter;
(5) Colorimeter tubes;
(6) Nutrient Broth;
(7) Bacterial cultures.
Procedure:
A. O.D. (Optical Density) against number of cells:
(i) Counting of Bacteria:
The given bacterial culture usually contains cells of the order of 109 per ml., hence the suspension has to be diluted for haemocytometer counts. 50 times dilution is generally suitable. To stop further growth of bacteria during experimental procedures, a few drops of formalin is added to the original suspension. After dilution, the haemocytometer is charged with the diluted suspension, and the bacterial counting done under microscope.
In the haemocytometer there are 25 big squares, each of which contains 16 small squares. The counting of bacteria is done in different squares (positions 1 – 1, 1 – 5, 3 – 3, 5 – 1, 5 – 5) and the mean count is then determined. It is then multiplied by the factor 25 × 104 which gives the number of bacteria per ml. at 50 times dilution. Multiplication again by 50 gives the total number of bacteria per ml. of the original sample.
(ii) Determination of O.D. of Bacterial Solution:
The optical density should be between 0.3 to 0.4. It is determined at 640 nm against the sterilised growth medium containing a few drops of formalin. The sample is then diluted twice, five times, ten times and so on. The dilution is done with the growth medium having a few drops of formalin in it. The O.D. of these different suspensions is measured against bacteria-free medium.
From the haemocytometer reading, the number of bacteria per ml. of the original sample is determined. Then the concentration of bacteria in other diluted samples is also known from the dilution factor. Thus the graph of optical density against the cell number can be drawn.
(iii) Determination of growth curve:
About 6 ml. of the growth medium is taken in a standard tube and the medium is inoculated with the given bacteria. It is now placed in water-bath shaker at 37°C for the bacteria to grow.
The tube can be taken out of the shaker and optical density measured at suitable intervals of time against the sterilised growth medium. The optical density is measured at 0 time and then at intervals of half an hour till the beginning of the stationary phase (about 6 hrs.).
From the calibration curve the values of the concentrations corresponding to different O.D. can ‘be obtained. The concentrations of bacteria at various times are then plotted against corresponding time of growth.
Such a plot is known as growth curve. On semi-logarithmic plot of concentration against time, the growth curve is straight for the logarithmic phase of growth. The generation of time T of bacteria is then determined from the slope of the growth curve.
Calculation of the generation time:
(a) Using relation: α(from graph) = 2.3 ∆(log10N)/∆t
(b) Directly from graph: Generation of time (T) = 0.69