Exercise VIII 



STUDIES IN MICROBIOLOGY (3) 45 



the transfer.) Tube No. 1 will be the control. 

 Tube No. 2 should receive 2 drops of your DNA 

 solution. Label the tubes so that you can iden- 

 tify them as your own. Incubate them for 30 

 minutes in a 30°C water bath. When the 30 

 minutes are up, transfer the tubes to the 37°C 

 water bath and incubate them for 90 minutes 

 more. 



So that you can count the bacteria which are 

 resistant to streptomycin, a blood agar plate 

 containing streptomycin will be provided. Di- 

 vide the plate into three equal sectors. On one 

 sector spread two loopfuls of culture from the 

 control tube; on another spread two loopfuls of 

 the transformed culture; on the third spread 

 two loopfuls of your DNA solution. Label the 

 sectors appropriately and put your name on the 

 plate. Place it, upside-down, in the bin that is 

 provided. It will be incubated at 37° for two 

 days, and then refrigerated until the next 

 laboratory session. 



Antibiotic-resistance experiment 

 (continuation) 



Today you will demonstrate that the bac- 

 terial colonies which grew on your antibiotic 

 gradient plates are, indeed, mutations. You 

 will test this by transferring mutants from your 

 antibiotic gradient to fresh antibiotic plates. 

 Obtain a nutrient agar plate containing the 

 antibiotic you used last week. The antibiotic 

 concentration is set so that the original strain 

 of E. coli will not grow but the resistant mutant 

 will. Divide the plate into four quadrants and 

 label them Hi-P, Lo-P, Hi-S, Lo-S. 



Pick a mutant from the end of your gradient 

 plate containing a low antibiotic concentration, 

 and with a wire loop transfer it to a small test 

 tube containing sterile saline and stir. If your 

 antibiotic is penicillin (or streptomycin), transfer 

 a loop of this saline suspension to the "Lo-P" 

 ("Lo-S") quadrant of your new penicillin 

 (streptomycin) plate. Transfer another loopful 

 to the "Lo-P" ("Lo-S") quadrant of your 

 neighbor's streptomycin (penicillin) plate. If 

 any of your bacteria survive when transferred 



to your neighbor's plate, they should represent 

 double mutants, that is, mutants able to resist 

 both antibiotics. Repeat the operation with a 

 mutant from the high-antibiotic-concentration 

 end of your gradient plate. This mutant should 

 have a better chance of growing on your new 

 plate than the mutant from the low end of 

 the gradient. Should a penicillin mutant 

 have an increased chance of surviving on 

 streptomycin? 



Take your plates home and keep them in a 

 warm place for three days. If you find any 

 colonies which you believe to be double mutants, 

 and wish to check them, place your plates in a 

 refrigerator until the next laboratory session 

 (there may be space in the laboratory refrigera- 

 tor). 



Irradiation of S. marcescens (continuation) 



You can now examine the results of last 

 week's irradiation experiment. Make note of 

 two things : survival, and the presence of color 

 mutants. The latter will be propagated on fresh 

 medium to see if they breed true. 



Obtain your irradiated 5. marcescens plate 

 and pick out a colony that appears clearly to 

 represent a color mutation. With a loop, trans- 

 fer this colony to a small test tube containing 

 sterile saline. Next, transfer a loop of this saline 

 suspension of bacteria to one quadrant of a new 

 plate (synthetic medium). Repeat this process 

 with three additional mutant colonies. Take 

 your plates home and incubate them at room 

 temperature. A genuine mutation will breed 

 true: the new colonies that result should be 

 identical in color with the original mutant. 



Microorganisms in the air (continuation) 



While your cultures are incubating, examine 

 the growth on the agar plates you exposed last 

 week to the air. Make a list of the different 

 colonies on the plates; describe them as you see 

 them. Can you distinguish molds from bac- 

 teria? Do any two colonies appear to corre- 

 spond to the same organism? 



