44 STUDIES IN MICROBIOLOGY (3) 



Exercise VIII 



Certain species, such as S. marcescens and E. 

 coli, are very versatile. They can grow on a 

 simple mixture of a sugar and the salts potas- 

 sium phosphate, ammonium sulfate, calcium 

 chloride, magnesium chloride, and ferric chlo- 

 ride. They do not require vitamins and amino 

 acids, which they can synthesize for them- 

 selves. In our experiments, however, we want 

 them to grow more rapidly since we have only 

 a short time in which to work, so we provide 

 them with a richer medium. The nutrient broth 

 we use is a mixture of a protein hydrolysate 

 (such as you made from yeast proteins) and a 

 beef extract, which like whole beef contains 

 salts, vitamins, and sugars as well as amino 

 acids. By adding agar, we obtain a solid growth 

 medium, the surface of which can be used for 

 bacterial counts. 



Pneumococcus is a more fastidious organism, 

 and requires many preformed vitamins and 

 amino acids. The growth of Pneumococcus can 

 be supported on a medium composed of potas- 

 sium phosphate, calcium chloride, a protein 

 hydrolysate fortified by the addition of the amino 

 acids cysteine and glutamine, and a tiny trace 

 of yeast extract which acts as a source of vita- 

 mins. In order for the cells to become able to 

 incorporate DNA and be transformed, it is 

 necessary to supplement the medium. In par- 

 ticular serum albumin, a protein found in 

 blood, must be added. 



In preparing a solid medium on whose surface 

 Pneumococcus can grow, whole blood must be 

 added. Pneumococcal cells not only do not use 

 air, but are inhibited by its presence. They are 

 so-called obligate anaerobes, as opposed to the 

 aerobic bacteria which tolerate the presence of 

 air. What probably happens is that in the pres- 

 ence of oxygen, bacteria produce hydrogen 

 peroxide, which poisons Pneumococcus since it 

 lacks catalase. The addition of blood, which 

 contains an active catalase, repairs this de- 

 ficiency. 



The strain of Pneumococcus which we use, 

 though not pathogenic, that is, capable of 

 causing disease, is closely related to the strain 

 which causes pneumonia. The chief difference 



between the two is that the pathogenic strain is 

 covered by a capsule of polysaccharide which 

 protects it in the body. We see that the nutri- 

 tional and environmental requirements of 

 Pneumococcus stem from its parasitic mode of 

 life and the nature of its habitat in body tissues. 

 Another bacterium of wide distribution and 

 interest, E. coli, does not grow within the body 

 tissues, but normally is found in the large intes- 

 tine, where it thrives on the organic material 

 passed along by the digestive apparatus. For 

 this reason it occurs also in sewage and polluted 

 waters. 



EXPERIMENTS 



Cells of a streptomycin-resistant strain of 

 Pneumococcus were grown overnight, collected 

 by centrifugation, and resuspended in the flasks 

 marked SR. Take 5 ml (about | inch) of this 

 culture in a wide test tube. Add 5 drops of 

 deoxycholate solution. Mix. Incubate at 37°C 

 for 5 minutes. Do the cells lyse? Does the 

 solution become viscous? (Viscosity of the 

 solution can be estimated by swirling the con- 

 tents and observing the rate with which bubbles 

 rise.) The increased viscosity is caused by re- 

 lease of the long chains of DNA. 



Pour an equal volume of alcohol slowly down 

 the side of the test tube so that it does not mix, 

 but forms a layer over the solution. Gently 

 insert a glass rod into the center of the tube 

 and by rotating the rod wind up on it the fibers 

 of DNA which form at the interface of the 

 alcohol and water. Keep turning the rod until 

 the two layers have mixed. Withdraw the rod 

 with the fibers wound on its end, dip into 2 ml 

 of sterile sodium chloride solution in a small 

 test tube, and stir to remove the DNA. Plug 

 the tube immediately. Swirl until the fibers 

 have dissolved. 



Prepare two small test tubes for the trans- 

 formation experiment by adding 1 ml of medium 

 to each with a sterile pipet. Add to each tube 

 2 drops of the streptomycin-sensitive cells which 

 are to be transformed. (These will be found in 

 the ice baths. Use a sterile dropper pipet for 



