Ultraviolet light has a mutagenic effect on microorganisms, mainly caused by changes in the molecular structure of DNA (formation of covalently bound thymine dimers between neighboring pyrimidines of the same strand of DNA), thus causing genetic variation in the organism. After cell counting, add a few drops of iodine solution to the plate with about 5-6 colonies, and a transparent circle will appear around the colony. Measure the diameter of the transparent circle and the diameter of the colony and calculate the ratio (HC value), and compare with the control plate, according to the results, to illustrate the mutagenic effect.
Operation method
ultraviolet mutagenesis
Principle
Ultraviolet light has a mutagenic effect on microorganisms, mainly caused by changes in the molecular structure of DNA (the formation of covalently bound thymine dimers between neighboring pyrimidines of the same strand of DNA), which leads to the genetic mutation of the organism.
Materials and Instruments
Bacillus subtilis Move I. Main instruments, equipment and materials for the experiment For more product details, please visit Aladdin Scientific website.
Saline Culture medium
Hematocrit plate Microscope UV lamp Electromagnetic stirrer Centrifuge
Instruments: blood counting plate, microscope, UV lamp (15W), electromagnetic stirrer, centrifuge.
Strain: Bacillus subtilis.
II. Experimental methodology, operational steps
1. Preparation of bacterial suspensions
(1) Take 4-5 slants of Bacillus subtilis that have been cultured for 48 hours, wash down the moss with sterile saline and mount in a small triangular flask containing glass beads and shake for 30 minutes to break up the clumps;
(2) Centrifuge the above bacterial solution (3 000 r/min, centrifugation for 15 minutes), discard the supernatant, wash the bacterial body with sterile saline 2-3 times, and finally make a bacterial suspension;
(3) Count by direct microscopic counting, adjusting the cell concentration to 108 cells per milliliter.
2. Flatbed production
Dissolve the starch agar medium and pour the plate when it is cold to about 55 ℃, then solidify it for use.
3. Ultraviolet treatment
(1) Turn the UV lamp switch on to preheat for about 20 minutes.
(2) Take 2 sets of 6 cm diameter sterile petri dishes, add 5 ml of the above bacterial suspension to each, and place a sterile stirring rod into the petri dishes.
(3) 2 sets of flat dishes containing bacterial suspensions were placed on a magnetic stirrer and irradiated by stirring under a UV lamp with a distance of 30 cm and a power of 15 W for 1 min and 3 min, respectively.
Under red light, the above mutagenized-treated bacterial suspensions were diluted to 10-1-10-6 by the 10-fold dilution method (specific dilutions can be made according to the estimated survival rate).
4. Coating plates
Three dilutions of 10-4, 10-5 and 10-6 were applied to the plates, 3 plates for each dilution, 0.1 ml of diluted bacterial solution was added to each plate, and the plates were coated well with a sterile glass spatula. In the same way, the plates were coated with the diluted bacterial solution without UV treatment as a control.
5. Cultivation
Wrap the above well-coated plates with black cloth (or black paper) and incubate at 37 ℃ for 48 hours. Note that the treatment time and dilution should be labeled on the back of each plate.
6. Counting
The plates after 48 hours of incubation were removed for bacterial counting and the number of viable bacteria per milliliter of bacterial solution was calculated based on the number of colonies on the control plate. Similarly the number of surviving cells and their lethality after 1 minute and 3 minutes of UV treatment were calculated.
7. Observation of mutagenic effect
The plates after cell counting, respectively, to the number of colonies in the 5-6 or so of the plate to add a few drops of iodine solution, in the colony will appear around the transparent circle. Measure the diameter of the transparent circle and the diameter of the colony and calculate the ratio (HC value), and compare with the control plate, according to the results, to illustrate the mutagenic effect. The colonies with a large HC ratio are transferred to a test tube slant for culture. This slant can be used for re-screening.
III. Experimental results
1. Enter the results of the experiment at in the table below.
