354 METHODS OF OBTAINING PURE CULTURES 



plug of glass wool, whicli supports a layer of sand. Broth is poured into both 

 tubes till it reaches the same level. The mixed culture is seeded into the broth 

 of the outer tube, and the apparatus is put in the incubator. The organisms 

 that are motile will pass through the holes into the inner tube, grow up through 

 the wool and sand, and produce a turbidity in the broth of the inner tube ; from 

 this they may be recovered in pure culture. The non-motile organisms remain 

 confined to the broth of the outer tube. An alternative method described by 

 Craigie (1931) is now extensively used for the separation of motile from non- 

 motile organisms of the Salmonella group. It consists of an ordinary test-tube, 

 6 X f in., containing a piece of glass tubing 4 in. long, having the bottom cut 

 off obliquely. About 6-8 ml. of melted semi-solid (0-25 per cent.) nutrient agar 

 are poured into the outer tube. After autoclaving, the agar is cooled to 45° C, 

 and the inner tube, which projects above the level of the agar, is inoculated with 

 the organism under test. If motile organisms are present, or appear as the result 

 of incubation, they grow down the inner tube and up the outer tube, from the 

 top of which they may be subcultured in a day or two's time (see also Tulloch 1939). 



E. Optimum Temperature. — It is sometimes possible to make use of the optimum 

 temperature of growth of an organism when it is desired to obtain it in pure culture, 

 as for instance when one wants it to multiply freely in a mixed culture. The 

 thermophiUc bacteria may be separated from other organisms by incubating the 

 medium at about 60° C. ; none of the ordinary bacteria will grow at this tem- 

 perature, so a pure growth of the thermophiUc organisms is obtained. Again, 

 certain bacteria will not grow at 22° C, whereas others will. If a mixture of the 

 two is incubated at 22° C, only one type will develop ; this may then be picked 

 off pure. In this way N. catarrhalis may be separated from the meningococcus. 

 In water analysis, when it is desired to know the numbers of potentially pathogenic 

 bacteria in a given sample, the cultures are incubated at 37° C. ; many of the 

 saprophytic forms fail to grow at this temperature, and the resulting growth con- 

 sists largely of potential parasites. 



F. Aerobic and Anaerobic Incubation. — This is a simple method of separating 

 aerobes from anaerobes. Incubated aerobically, the strict anaerobes will not 

 grow ; incubated anaerobically, the strict aerobes will not grow. And as most 

 facultative anaerobes seldom grow as well under anaerobic as under aerobic condi- 

 tions, anaerobic incubation favours the strict anaerobes more than the facultative 

 ones. 



G. Heating. — Heating a mixed culture at 80° C. for 10 minutes will destroy all 

 the vegetative non-sporing bacteria, while leaving the spores unaffected. This 

 method is largely used in the prehminary purification of the anaerobes. In the 

 body, most of the organisms with which the anaerobes are likely to be contaminated 

 are non-sporing cocci and bacilli ; all of these are destroyed at 80° C, and conse- 

 quently the anaerobic organisms alone develop. 



H. Selective Bactericidal Substances. — Certain substances with a germicidal 

 action are useful in destroying susceptible organisms, while leaving the more 

 resistant unaffected. One of the best examples of this method is the isolation of 

 the tubercle bacillus from sputum by the use of antiformin. Tubercle bacilU are 

 very resistant to chemical disinfectants, even though they are easily killed by heat. 

 If the sputum, which generally contains numerous other organisms, is treated with 

 15 per cent, antiformin (equal parts of 15 per cent. NaOH and Liq. sodae chlorinatae 



