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Laboratory 2:
Bio 4905 Lab #2
Growth is an orderly increase in the quantity of cellular components. In most bacteria, growth involves duplication of the bacterial chromosome, number of ribosomes , synthesis of new cell wall and plasma membrane, and eventual partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission.
For unicellular organisms such as the bacteria, growth can be measured in terms of two different parameters: changes in cell mass and changes in cell numbers.
Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. This method is simple and nondestructive. However, the sensitivity is limited to about 10^{7} cells per ml for most bacteria, which requires proper dilution of cell suspension before measurements can be taken.
In the laboratory, under unlimited (unrestricted) conditions, the number of bacteria doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations). This is called exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature where many growth restrict factors are present.
When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve
During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing.
Lab 2: Cell Culture and Measure Bacterial Growth Rate
Dilute over night culture 10X with LB medium
Measure o/n culture cell density with spectrometer at 600nm
Inoculate o/n culture to fresh LB medium (60ml in 250ml flask) to 0.05 OD600
Need to dilute 50X to get 0.05 OD
60ml/50 = 1.2ml (noculate 1.2ml of o/n culture to fresh 60ml medium)
o/n culture OD = ( _________ ) , then __________ /0.05= ( ________ )
Need to dilute 50X to get 0.05 OD
60ml/ ( ________) = ( _______ )ml
Incubate inoculated culture at 37oC, 250 rpm shaker (start timer)
Measure cell density with spectrometer at 600nm
(you have to blank out with blank medium before measure your sample)
*** when cell density reaches over 1.00 OD600, sample need to be diluted within 0.1 ~1.0 OD600
Ex) if OD600 without dilution is 1.2, need to dilute at least 2~3X with LB medium
(if 2X dilution, 1 part sample and 1 part fresh LB medium)
Plot the cell growth on semilog scale graph against time and cell density (OD600)
(xaxis time scale, yaxis cell density in logarithmic scale)
Predict cell growth on the semilog scale plot
Ex), OD= 0.5, 3/0.5= 6ml
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