PREPARATION FOR MICROBIOLOGICAL EXPERIMENTS 137 



e. Bacteriological loops 



These should be made from nichrome wire, 

 No. 23 gauge. The loop should be about \" in 

 diameter; it is convenient to form such loops by 

 bending the wire around two nails of appropriate 

 size imbedded in a wood block, as shown in the 

 diagram. 



The wire is either inserted into a commercially 

 available loop holder or fused into the end of a 

 4" length of thick-walled capillary tubing to 

 serve as a handle. Alternatively loops may be 

 purchased already assembled. 



B. WASHING OF GLASSWARE 



All glassware should be thoroughly washed 

 with detergent and rinsed at least five times in 

 order to remove traces of detergent which might 

 be toxic for the bacteria. It is not necessary to 

 rinse this glassware with distilled water, though 

 the latter should be used for making up all 

 media and solutions. It is advisable that the 

 students be taught to do as much of the washing 

 and plugging of pipets and aeration tubes as 

 possible. 



C. STERILIZATION 



In general, vessels containing liquids, or 

 assemblies containing rubber parts, should be 

 autoclaved at 120°C (15-lb pressure) for 15 min. 

 Large volumes (greater than 200 ml), particu- 

 larly of viscous liquids such as agar, should be 

 autoclaved for longer periods (30 min to 1 hr). 



Empty glassware should be dry-sterilized in 

 an oven at 160°C for at least 90 min. 



Serological pipets, culture tubes, flasks, and 

 aerators must be plugged with nonabsorbent 

 cotton before sterilization. If proper cans are 

 not available, serological pipets, dropper pipets, 

 and aerators may be wrapped in bunches with 

 aluminum foil so that sterility is preserved on 

 removal. 



Sugar solutions are best sterilized by heating 

 for 20 min in a boiling water bath or for one 

 hour in an inspissator (steam box). However, 

 concentrated glucose solutions (10%) may be 

 wet-autoclaved the same as media and then 

 measured out after cooling. 



Solutions of labile materials must be sterilized 

 by filtration. 



D. SOURCES OF CULTURES 



Cultures of Bacillus megatherium, Serratia 

 marcescens, and Escherichia coli B may be ob- 

 tained from the American Type Culture Collec- 

 tion, 212 M Street, N.W., Washington, D.C. 

 They should be propagated every two months 

 by streaking a sample of the old culture onto 

 the surface of a fresh nutrient agar slant and 

 incubating until growth is completed, after 

 which the cuhures should be conserved in the 

 cold. Agar slants are prepared by adding about 

 5 ml of liquid nutrient agar to a sterile screw- 

 cap vial, about 16 mm X 150 mm in dimen- 

 sions. Tilting the tube before solidification 

 results in a slant surface of considerable area, 

 in a tube of small cross section, and hence rela- 

 tively little risk of contamination. 



Strains of Pneumococcus which are nonencap- 

 sulated or rough, and therefore nonpathogenic, 

 must be used. A normal strain sensitive to 

 streptomycin, as well as a streptomycin-resistant 

 strain, can be obtained from universities or insti- 

 tutes carrying on research on the transformation 

 of Pneumococcus. Among such institutions are 

 The Rockefeller Institute, New York City; 

 University of Colorado Medical Center, Denver, 

 Colorado; Laboratoire de Ge'netique Physiolo- 

 gique, C.N.R.S., Gif-sur-Yvette (Seine-et-Oise), 

 France; and Brookhaven National Laboratories, 

 Upton, New York. 



