Isolation experiments of drug-resistant mutant strains
Isolation experiments of drug-resistant mutant strains
Changes in the order of bases in a gene can lead to genetic variation in microbial cells. This variation can sometimes enable the cell to survive in a harmful environment, as exemplified by drug resistance mutations. Source: Laboratory Microbiology (3rd Edition)
Operation method
gradient plate method
Principle
Resistance mutations in organisms are caused by structural changes in a particular position of the DNA molecule, and have nothing to do with the presence of drugs. The presence of a certain drug is only used as a means to isolate a certain drug-resistant strain, not as an inducer to trigger the mutation, and thus, when a large number of cell populations are coated on a plate containing a certain concentration of a growth-inhibiting drug, a very small number of cells with resistance mutations will grow into colonies on the plate. These colonies will be picked and purified, and further resistance tests can be conducted to obtain the required resistant strains. Resistance mutations are often used as genetic markers, so it is essential to master the method of isolating resistant mutant strains. In order to facilitate the selection of appropriate drug concentration, the isolation of drug-resistant mutant strains commonly used gradient plate method. In this experiment, the gradient plate method was used to isolate streptomycin-resistant mutant strains of E. coli. The gradient plate was prepared as shown in the figure. First pour in the bottom medium without drug, put the plate diagonally (Fig. 1, A), put the plate flat after solidification, and then pour in the top medium containing streptomycin (Fig. 1, B), so that we can get the gradient plate with the concentration of streptomycin gradually decreasing from one side to the other. A large number of sensitive bacteria (or mutation-treated strains) are coated on this plate, and after incubation, streptomycin-resistant mutant strains can be isolated from colonies grown at the site where the streptomycin concentration is relatively high. Figure 1 Streptomycin concentration gradient plate
Materials and Instruments
Escherichia coli Move 1. Inoculate E. coli in a test tube containing 5 ml of liquid and incubate at 37°C for 24 h. 2. Lysogenize LB agar medium in a hot water bath. 2. Dissolve LB agar medium in a hot water bath. 3. 3. Pour 10 ml of dissolved drug-free LB agar medium into a sterile Petri dish and immediately raise one end of the dish so that the agar medium covers the entire bottom and the surface of the medium reaches the junction between the bottom and the sides of the dish at the raised end, and allow the medium to solidify at this inclined position (Fig. 1, A). 4. 4. Mark the bottom of the solidified plate away from the agar with "low" and return it to the horizontal position, then add 10 ml of LB agar medium containing 100 ug of streptomycin per ml on top of the bottom layer of the medium, and solidify it to make a plate with a gradient of streptomycin concentration from 0 ug/ml at one end to 100 ug/ml at the other end. 5. 0.2 ml of E. coli culture solution was pipetted into the gradient plate with a 1 ml sterile pipette, and the solution was spread over the entire surface of the plate with a sterile glass applicator. If using a glass applicator dipped in ethanol and flame-sterilized, the plate can be cooled slightly by the flame or by reaching into the plate and cooling it slightly on the plate cover to avoid scalding the cells. 6. Invert the plate and incubate at 37℃ for 48 h. 7. Select 1-2 growing cells. 7. Select 1 or 2 single colonies growing in the middle of the gradient plate, and use a sterile inoculating loop to touch the single colonies to draw a line in the direction of high drug concentration. 8. Invert the plate to 37 ℃ and incubate for 48 hours. For more product details, please visit Aladdin Scientific website.
Streptomycin LB liquid medium 10 ml agar medium tube
1 ml sterile pipette Beaker with 70% ethanol Glass pipette Water bath