82 



THE AMERICAN MONTHLY 



[May, 



cases, but it requires much labor and 

 apparatus. 



A method has been recently intro- 

 duced by Dr. Koch -which enables us 

 to secure pure cultures from mixtures 

 without much labor, and which fur- 

 nishes, in addition, the means of dis- 

 tinguishing different species with a 

 low power. The method, in brief, 

 consists in distributing individual mi- 

 crobes through a liquid medium which 

 rapidly gelatinizes. The microbes are 

 thus forced to remain isolated and to 

 multiply 171 situ. The steps of the 

 process are as follows : A minute por- 

 tion of tissue or liquid which contains, 

 among others, the pathogenic microbe 

 is thoroughly shaken up in about lo 

 c.c. of sterilized water. We prefer 

 sterilized broth, and thus obtain a 

 liquid culture of the various forms, 

 the examination of which may serve 

 as a check upon errors in the plate- 

 cultures to be described later. We 

 have at hand some nutritive gelatin 

 which is prepared according to Loff- 

 ler's formula : \o% gelatin, i''o pep- 

 tone, ^"o common salt dissolved in an 

 infusion of meat. This gelatin re- 

 mains solid at ordinary temperature, 

 but liquefies above 80° F. The gel- 

 atin is stored for this purpose in test- 

 tubes pi'ovided with a cotton plug, 

 each containing about 10 c.c. The 

 gelatin having been liquefied by gen- 

 tle warming or by placing in the ther- 

 mostat at 100° F. for a short time, the 

 protruding portion of the cotton plug 

 is singed aw^iy and the tube thorough- 

 ly heated in the flame around the plug 

 itself until the latter turns slightly 

 brown. This will insure the destruc- 

 tion of all bacteria that may have gath- 

 ered on or around the plug. A drop 

 of the fluid in which the bacteria have 

 been distributed is transferred to the 

 liquid gelatin, and this in turn is 

 thoroughly shaken to secure uniform 

 distribution. The gelatin is then 

 poured upon sterilized glass plates 

 which have been cooled in a refrig- 

 erator, and upon which it rapidly 

 solidifies, so that the plates may be 

 placed under a bell-jar in a moist at- 



mosphere within fifteen minutes. In 

 these manipulations the object sought 

 is to secure such a distribution of the 

 bacteria on the plates that they will 

 not interfere with each other as they 

 develop, and that they are far enough 

 apart to allow each centre of growth 

 to be touched with a needle without 

 touching adjacent centres. Since the 

 number of bacteria will vary with 

 their source, the amount of dilution 

 necessary in each case cannot be fore- 

 told. Judgment and experience must 

 be our guides. We have found glass 

 plates measuring about 8 by 10 cm. a 

 convenient size. It is more expedi- 

 tious in most cases to transfer fluid 

 with a* platinum loop than with a 

 pipette. The loop carries smaller 

 quantities, and is more easily steril- 

 ized. 



After a period of from 24 to 48 

 hours minute opaque points are per- 

 ceptible on the plates. Each of these 

 represents the progeny of a single mi- 

 crobe, which has multiplied until the 

 brood numbering thousands becomes 

 visible to the naked eye. Under a low 

 power these so-called colonies are ob- 

 served to vary greatly according to the 

 species of bacteria of which they are 

 made up. Some are spherical, with 

 shai-ply-defined outlines, others with 

 circumference not very distinct ; some 

 bear protuberances of various forms ; 

 some have an area of liquid gelatin 

 surrounding them, or are situated at 

 the bottom of steep, funnel-shaped 

 depressions ; some have peculiar 

 markings on their surface. They 

 may be made up of granules or ap- 

 pear homogeneous. Finally, they 

 may difter in refrangibility and in 

 color. It is needless to say that such 

 distinctions must be carefully noted. 

 By removing, with a sterilized needle 

 under a dissecting microscope, a col- 

 ony, as each centre of growth is called, 

 and mixing on a slide with a drop of 

 sterilized water, the microscopic char- 

 acters of the different forms of colonies 

 are readily determined. The plate- 

 culture has thus given us pure cul- 

 tures of all the bacteria in the origi- 



