352 METHODS OF OBTAINING PURE CULTURES 



every alternate tube. This method affords one no certainty that the cultures 

 obtained are pure ; further study must be undertaken to ascertain this. The 

 objections to the method are that too much guess-work is involved in judging the 

 correct dilution, and that several tubes of medium are inevitably wasted. It is 

 useful, however, in a modified form in conjunction with the plating method. That 

 is to say, when the culture is thick, it is wise to dilute it considerably before plating 

 out ; in this way there is more likelihood of obtaining single colonies from single 

 organisms. 



B. Koch's Plating Method. — Originally the solid medium used was spread out 

 in the melted state on microscopic slides and allowed to set ; these were then 

 streaked with a needle dipped in the culture, and incubated in a moist bell-jar. 

 Later, large glass plates were used ; these had to be specially levelled by means of 

 adjustable screw supports, and covered with a bell- jar. The method now employed 

 is to pour the melted medium into Petri dishes ; each of these is provided with a 

 cover, which protects the medium from contamination. The plating method may 

 be employed in one of two ways. Either the culture material may be streaked 

 on the surface of the solid medium, or it may be mixed with the medium in the 

 melted state, poured out into Petri dishes, and allowed to set. The former method 

 results in a surface growth, the latter in a growth throughout the whole thickness 

 of the medium. As a rule the former method is the more useful. In streaking 

 the surface of the medium, a drop of the fluid culture may be placed in the centre 

 of the dish, and spread out in all directions by means of a sterile glass or metal rod 

 bent at a right angle. This results in an even distribution of organisms over the 

 plate. If, however, single colonies are particularly desired — and this is usual — 

 it is best to make a series of streaks across the plate with a platinum loop dipped 

 in the culture ; the streaks should be about 10 mm. apart, and may be crossed at 

 right angles ; the platinum loop should not be re-charged with culture during the 

 process. If preliminary dilution has not been performed, it is often advisable to 

 continue the streaking over a second or even a third plate without re-charging the 

 loop. On the first plate the growth may be entirely confluent ; but on the second 

 and third, single colonies will generally be obtained. These single colonies can 

 then be examined with a hand lens, and picked off with a platinum needle into 

 broth. Except with certain organisms, single colonies obtained in this way, 

 especially if the culture has been previously diluted, are generally derived from 

 single organisms, and are hence pure. The purity of colonies picked from over- 

 crowded plates is less certain. In using the surface streak method it is important 

 that the plates should be fairly dry ; if there is a film of moisture on the medium — 

 resulting partly from condensationand partly from expression — organisms, partic- 

 ularly if motile, are apt to form a confluent growth over the whole surface. This 

 point must be borne in mind, especially when attempting to isolate anaerobic bacteria. 

 These organisms, instead of growing up from the medium, frequently spread in a 

 thin layer over it ; the edges of the colonies are difficult to define, and if there is 

 a film of moisture over the medium, they coalesce, thus rendering their isolation 

 impossible. The pour-plate method is, in general, of less value ; the deep colonies 

 are not usually as characteristic as the surface colonies, and to pick them off involves 

 stabbing the medium with a platinum wire — a process that takes longer than the 

 simple one of surface picking. In carrying out this method of plating, a tube con- 

 taining 15 ml. of the solid medium is heated in water till the medium is melted ; 

 for gelatin a temperature of 30° C. will suffice ; for agar the water must be boiled. 



