CHAPTER XVIII.
ISOLATION OF BACTERIA IN PURE CULTURE.

As has been stated, the thorough study of a bacterium depends on first getting it in pure culture. In the early days of bacteriology supposedly pure cultures were obtained by (1) dilution in liquid media. A series of tubes or flasks containing sterile liquid media was prepared. Number one was inoculated with the material to be examined and thoroughly mixed. A small portion of the mixture was transferred to number two, and mixed; from this to number three, and so on until a sufficient number were inoculated, the last three or four in the series receiving the same amounts of a very high dilution of the original material. If one or two of these latter showed a growth and the others not, it was assumed that the dilution had been carried so far that only a single organism was transferred and therefore the culture obtained was “pure.” The method in this crude form is too uncertain to be of value today and recourse is had to more exact means. The procedure most widely used is that of (2) “plating out” by means of gelatin or agar plates. The material to be plated out is diluted by transferring to three or more tubes of melted gelatin or agar as in the first method and then all the tubes are poured into Petri dishes and grown under suitable conditions. By proper mixing in the tubes the bacteria are well scattered through the medium which holds the individual organisms separate when it solidifies. On some of the plates a sufficient dilution will be reached so that the colonies developing from the bacteria will be so few that they are separate and pure cultures may be obtained by inoculating from one of these a tube of the appropriate medium (Figs. 131 to 134). The chief uncertainty with this method is that occasionally two kinds of bacteria stick together so closely that even the separate colonies contain both organisms. This is not common, however. The plate colonies frequently develop from groups of bacteria which were not separated, but as these are of the same kind the culture is essentially pure.


Fig. 131.—Dilution plates. × 3⁄10. 1, shows the first dilution, the colonies are so numerous and small that they are invisible (compare Fig. 132); 2, shows fewer and hence larger colonies, but too crowded to isolate (compare Fig. 133); 3, shows the colonies larger and well separated, so that it is easy to isolate from them (compare Fig. 134).

Fig. 132.—A portion of plate 1 in Fig. 131 as seen under the low-power objective. × 100. Very small, closely crowded colonies.

Another method which is frequently applicable with material from human or animal sources is to (3) rub the material over the surface of a slope tube or of medium solidified in a Petri dish with a sterile heavy platinum needle, glass rod, or cotton swab. If the bacteria are not too numerous, pure cultures may frequently be obtained. A modification of this method is to make a series of (4) parallel streaks on a slope tube or plate of medium with a needle inserted but once into the material to be plated. On the first streak most of the bacteria are rubbed off and a continuous growth results, but usually on the last of a series only isolated colonies appear, which are presumably pure. The ideal method for securing pure cultures is to be absolutely certain that the culture starts from a single organism. This may be accomplished by means of the (5) apparatus and pipettes devised by Professor Barber of the University of Kansas (Figs. 135 and 136). With this instrument a single organism is picked out under the microscope and isolated in a drop of culture medium and observed until it is seen to divide, thus proving its viability. Transfers are then made to the proper media. The method requires much practice to develop the necessary skill in the making of pipettes, determining the proper condition of the large cover-glasses used over the isolating box, and in manipulation, but the results fully compensate.


Fig. 133.—From the thinnest part of plate 2, Fig. 131 as seen under the low-power objective. × 100. Colonies much larger than on plate 1, but still crowded.

Fig. 134.—The smallest colony on plate 3, Fig. 131, as seen under the low-power objective. × 100. Large, single, isolated colony.

Professor W. A. Starin of the author’s department, a former student of Professor Barber, has done some excellent work with this apparatus.


Fig. 135.—Diagram of Barber’s isolation apparatus. b, moist chamber; ms, large cover-glass over moist chamber; p, small pipette drawn out to a fine point; k, r, g, pipette holder; f, screw for raising and lowering k, r, g; s, screw for lateral motion of k, r, g; n, screw for clamp on pipette which allows it to be moved in or out; m, mechanical stage of microscope; t, rubber tube held in the mouth and used to move the liquid culture medium in the pipette. (Journal of Infectious Diseases, October 20, 1908, vol. 5, No. 4, p. 381.)
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Fig. 136.—Photograph of microscope with Barber’s isolation apparatus set up to use.

A number of procedures may be used to greatly facilitate the above methods of isolation by taking advantage of the different physiological properties of different organisms in a mixture such as ability to form spores, different resistance to antiseptics, special food requirements, and pathogenic properties. (a) If material contains resistant spores, it may be heated to temperatures high enough to kill all of the organisms except the spores (80° for half an hour, for example) and then plated out. Or (b) an antiseptic which restrains the growth of some organisms and not others may be placed in the culture media (carbolic acid, various anilin dyes, (p. 162), excess acid, or alkali, ox bile, etc.), when the more resistant organisms grow on the final plates, the others not. (c) Special food substances (various carbohydrates) from which the organism desired forms special products (acids, aldehydes) that may be shown on the plates by various indicators, is one of the commonest means. Or media in which certain organisms thrive and others not, so that the former soon “crowd out” the latter (unsterilized milk for lactic acid bacteria, inorganic media in soil bacteriology) may be used. A combination of the general methods (b) and (c) is much used in the separation of the organisms of the “intestinal group” in human practice. (d) The inoculation of a susceptible animal with a mixture suspected to contain a given pathogenic bacterium frequently results in the development of the latter in pure culture in the body of an animal, from which it may be readily recovered. In all of the above methods (except Barber’s) the first “pure culture” obtained should be “purified” by replating in a series of dilution plates to make sure that it is pure.