Determination of bacterial proliferation curve experiment
Determination of bacterial proliferation curve experiment
A small amount of bacteria is inoculated into a certain volume of suitable fresh medium and cultivated under suitable conditions, and the amount of bacteria in the culture solution is measured at regular intervals, with the logarithm of the amount of bacteria as the vertical coordinate and the growth time as the horizontal coordinate, and the curve drawn is called a growth curve.
Operation method
turbidimetric method
Principle
A small amount of bacteria will be inoculated into a certain volume of fresh medium suitable for cultivation under suitable conditions, the amount of bacteria in the culture medium will be measured regularly, and the curve drawn with the logarithm of the amount of bacteria as the vertical coordinate and the growth time as the horizontal coordinate is called growth curve. It reflects the group growth pattern of single-cell microorganisms in liquid culture under certain environmental conditions. According to the different growth rates, the growth curve can be divided into delayed period, logarithmic period, stabilization period and decline period. The length of these four periods varies depending on the genetics of the strain, the amount of inoculum and the culture conditions. Therefore, by determining the growth curve of microorganisms, we can understand the growth pattern of each bacterium, which is of great significance for scientific research and production. There are many different methods to determine the number of microorganisms, which can be chosen according to the requirements and laboratory conditions. In this experiment, the turbidimetric method is used, because the concentration of the bacterial suspension is directly proportional to the optical density (OD value), so we can use a spectrophotometer to determine the optical density of the bacterial suspension to infer the concentration of the bacterial solution, and the measured OD value and its corresponding incubation time graphs, you can draw the growth curve of the bacteria under certain conditions.
Materials and Instruments
E. coli Move I. Experimental main instruments, equipment and materials For more product details, please visit Aladdin Scientific website.
Beef paste Peptone Sodium chloride
Balance Spectrophotometer Colorimetric Cup Constant Temperature Shaker Sterilizer, Pipettes Test Tubes Tripods
Balance, 721 spectrophotometer, colorimetric cups, thermostatic shaker, sterilizing pot, sterile pipettes, test tubes, triangular flasks.
Strain: Escherichia coli.Medium: beef paste, peptone, sodium chloride liquid medium
II. Experimental methodology, operational steps
1. Seed solution preparation
Take 1 branch of Escherichia coli slant strain, under aseptic conditions, pick 1 ring of bacterial moss, access to the sterile beef paste peptone culture solution, stationary culture for 18 h for seed solution.
2. Inoculation culture
2 mL of seed solution was accurately sucked up with a sterile pipette and added into a 250 mL triangular flask containing 50 mL of sterile beef paste peptone culture solution, and incubated at 37 ℃ with oscillation (oscillation frequency 250 r/min).
3. Growth curve measurement
At 0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h, respectively, the triangular flask was removed, 1 ml of culture solution was aspirated, and the uninoculated culture solution was used as a blank control, the above culture solution was colorimetrically measured at a wavelength of 600 nm (before the determination of the OD value, the culture solution to be measured was oscillated so as to make the cells uniformly distributed), but it was required that the extinction degree was in the range of 0.10 to 0.65, and the uninoculated beef paste peptone was used as a blank control for the bacterial suspension with a large concentration. For large concentration of bacterial suspension with uninoculated beef paste peptone liquid medium diluted appropriately after the determination of the OD value measured after dilution should be multiplied by the number of times of dilution, is the actual OD value of the culture fluid.
4. Experimental results
(1) Fill in the following table with the OD values measured at different time points.
(2) Plot the growth curve of E. coli using the time in the above table as the horizontal coordinate and the OD600 value as the vertical coordinate.