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BACKGROUND
Growth is the increase in the cell mass and the cell size of an organism and is one of the unique traits of each and every organism. There is need of certain basic parameters by the organism for generating energy and cell biosynthesis. Every organism growth is affected by physical factors like pH, temperature, moisture content and nutritional factors like nitrogen, Sulphur, phosphorus and many other trace elements. As we know bacteria are unicellular organism and when they reach a particular size they start dividing by binary fission. The time taken by the bacterial population to double its number is called generation time. The basic objective of this experiment is to calculate the generation time and specific growth rate of bacteria from the graph plotted with a given set of data.
Principle: The bacteria are allowed to grow under specific set of conditions. The bacterial growth dynamics is studied by plotting a graph of the cell growth against the incubation time and the resultant curve is called standard growth curve. Basically, this growth curve has four distinct phases that is the lag phase, where the cell metabolism is increases, there is increase in the size of the cells but there is no replication of bacteria and thus no enhancement in the cell mass. Then comes the exponential or the log phase where the cells grows rapidly and divides. The time taken here by the bacteria to double its number is called the generation time. Then comes the stationary phase where the nutrients in the medium are used up completely, thereby creating a very unfavorable condition for the bacteria to grow and it stops completely. Then comes the decline or the death phase where the deposition of the waste products and other toxic materials prohibits the bacteria from growing and the bacteria moves to the death phase.
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REQUIREMENTS
Sample: Overnight culture of the bacteria
Media: Nutrient broth
Apparatus: Sterile Petri plates
Micropipettes
Cuvette
Conical flask
Colorimeter
Sterile tips
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PROCEDURE
Take an isolated colony of the organism and inoculate it in 15 ml of nutrient broth and incubate it overnight. Then after that day measure the OD of the culture.1 Adjust the OD of the inoculum to the standard value by using the dilution formula.2
OD1V1 = OD2V2
OD1 = OD of the broth culture, inoculated the previous day.
V1 = volume of this broth culture to be added to the inoculums
OD2 = OD of the inoculum
V2 = volume of the inoculums
Now accordingly substitute the values and calculate V1. The more amount of inoculum that is V1 was removed before adding equal amount of the broth to it, so the total volume remains same. Then the OD was checked in every 30 minutes. By using this OD a standard growth curve was plotted. From this Generation time was calculated by using the following formula.
The exactly doubled points from the absorbance readings were taken and, the points were extrapolated to meet the respective time axis.3
Generation Time = (Time in minutes to obtain the absorbance 0.4) – (Time in minutes to obtain the absorbance 0.2)4
Let No = the initial population number
Nt = population at time t
N = the number of generations in time t
Therefore,
The growth rate can be expressed in terms of mean growth rate constant (k), the number of generations per unit time.
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CONCLUSION
The bacterial growth rate of the sample is studied and recorded.
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REFERENCES
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Herbert, d., Elsworth, r. & Telling, r. C. (1956). The continuous culture of bacteria: a theoretical and experimental study. J. Gen. Microbial. 14, 601.
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Hughes, w. H. (1955). The inheritance of differences in growth rate in Escherichia coli. J. Gen. Microbiol. 12, 265.
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Johnson, L. (1949). Bivariate distributions based on simple translation systems. Biomtrika, 36, 297.
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Kendall, d. G. (1948). On the role of variable generation time in the development of a stochastic birth process. Bimnetrika, 35, 316.
