Exercise VII 



STUDIES IN MICROBIOLOGY (2) 39 



or streptomycin. Some of you may find strains 

 that are resistant to both antibiotics. Such 

 strains do not develop by the reorganization of 

 cells of the original parent stock. On the con- 

 trary, the antibiotic eliminates these, and per- 

 mits only one or a few antibiotic-resistant mutant 

 forms that happen to be present to multiply to 

 form a new antibiotic-resistant population. 

 What we observe, therefore, is not the inherit- 

 ance of an acquired character, but, as always, 

 the selection of those individuals which through 

 mutation already possess that character. 



As you must know very well by now from 

 discussions of atom bombs and fallout, it is 

 possible to increase the rate of mutation far 

 above that which occurs naturally. One way is 

 by exposing cells to high-energy radiation. 

 Ultraviolet light of wavelengths near 260 m/i 

 has this effect. It is high in energy, correspond- 

 ing with its short wavelength (£ = hcj\, in 

 which E is the energy per quantum, /; is Planck's 

 constant, c is the velocity of light, and X is 

 the wavelength). The organic bases in the nucleic 

 acid chains strongly absorb these wavelengths 

 of ultraviolet light. Indeed, such ultraviolet 

 light in large enough doses kills all living cells 

 through its destructive effects upon their nucleic 

 acids. If one subjects a population of cells to a 

 large enough dose of ultraviolet light to kill 

 many of them but not all, the survivors usually 

 display an extraordinarily high incidence of 

 mutation. 



Today you will perform such an experiment 

 upon Serratia marcescens and look for induced 

 mutants among the surviving cells. The brilliant 

 red pigmentation of this bacterium makes it 

 particularly suitable for such studies, since 

 mutants that lack the normal pigmentation and 



hence look pink, white, or speckled are easily 

 recognized. 



Isolation of antibiotic-resistant strains 



Prepare your own agar plate containing an 

 antibiotic concentration gradient by the follow- 

 ing procedure (see diagram): Pour enough 

 melted nutrient agar (about 15 ml) into a slanted 

 petri dish (place a stirring rod under one end) 

 so that you have just covered the bottom of the 

 dish. Let the agar harden. Place the plate in a 

 horizontal position and add enough additional 

 agar containing penicillin (P) or streptomycin 

 (S) (whichever one you choose, your neighbor 

 should use the other) to just cover the already 

 solidified agar. (Don't fill the dish to the top.) 

 The antibiotic will establish a linear concentra- 

 tion gradient during subsequent incubation by 

 diffusing into the nutrient agar below it. Before 

 inoculation, dry the surface of the agar by open- 

 ing the dish slightly by propping up one edge of 

 the lid and incubating the dish for one hour at 

 37°C. Mark with an arrow on the bottom of 

 the dish the direction of the gradient of anti- 

 biotic concentration. 



The plate is inoculated with either S. mar- 

 cescens or Escherichia coli bacterial suspension, 

 parallel to the gradient. Give the labeled plates 

 to your instructor to store until next week. 



Radiation effects 



With sterile technique pour about 1 ml of the 

 diluted saline suspension of S. marcescens (10* 

 cells/ml) into a sterile test tube. Obtain an agar 

 plate containing synthetic medium and divide it 

 into four quadrants marked on the bottom of 

 the glass with wax pencil. Label the quadrants 

 150, 120, 90 (standing for seconds of radiation), 



