The Fundamentals of Bacteriology

by Charles Bradfield Morrey

Sterile graduated pipettes varying in capacity from 1 cc graduated in hundredths, upward, permit the transfer of definite amounts of liquids. Large quantities are conveniently transferred by means of Pasteur flasks ([Fig. 130]). The details of inoculation are best derived from laboratory practice.

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.