120 LABORATORY EXERCISES IN BACTERIOLOGY. 



times, when a comparatively pure water is to be examined, the bacteria from a large 

 amount are concentrated by filtration through sterile powder, and particles of the 

 powder are transferred to the culture tube by means of the forceps, carrying with 

 them a greater or smaller number of the germs. Short lengths of capillarv tube (sterile), 

 into which a suspected liquid has been drawn, may be broken off and thrown into 

 a tube of liquefied material and the inclosed liquid diffused through the medium by 

 agitation ; the known length of the tube affording opportunity of approximate quan- 

 titative estimation of the organisms developing into colonies in the medium if desired. 

 Another and somewhat similar method of approximate quantitative estimation of 

 bacteria in a liquid is to take equal lengths of thread and place one in a liquid of the 

 same general nature as that to be examined but not infected (water or bouillon), 

 and a second into the fluid to be investigated. The first is weighed when wet and 

 placed in the oven to dry, after which it is again weighed, the difference in weight 

 representing the fluid it was capable of absorbing. The second piece of thread is 

 taken from the infected fluid with a sterile pair of forceps, and cut with sterile scissors 

 at a definite length (one-half, third, or fourth, as desired), and the bit thus cut off 

 transferred to the tube of liquefied medium, and the tube agitated so as to scatter 

 the bacteria from it throughout the medium. Thereafter the tube is set aside for 

 growth as a diffusion culture or its contents transferred to a Petri dish or plate 

 as best suited. Sterile granulated sugar is often used in filtration of air and then 

 placed in liquefied medium so that the sugar may dissolve and leave the bacteria which 

 had collected in the filter upon the grains scattered through the medium. 



Fractional Inoculation. When the substance to be inoculated into a nutrient 

 material is especially rich in microorganisms, the resulting cultures are apt to be so 

 crowded with colonies, of single or mixed type, that it is impossible to recognize the 

 characteristic appearances of a single colony or to separate it from the mass. To 

 prevent this, dilution inoculations are generally made. A series of three culture tubes 

 is usually used, the first to be inoculated from the original infected matter, the second 

 from the first tube, and the third from the second. Thus, for example, in the clinical 

 cultivation of the diphtheritic organism, Mycobacterium diphtheria, three tubes of blood - 

 serum are usually inoculated. The first is inoculated by smearing the swab, infected 

 from the patient's throat, over the surface of the solidified serum. The platinum loop 

 is then taken up and sterilized and drawn over this inoculated surface, and then rubbed 

 over the surface of the serum in the second tube. The loop is again flamed and drawn 

 over the surface of the medium in the second tube and rubbed over that in the third 

 tube, probably carrying to this last but few of the organisms which were originally 

 deposited upon the serum of the first inoculation. On comparison of the three tubes 

 after growth of the infections the value of the procedure will at once be appreciated, 

 the scattered colonies in the third tube being much the most easily studied and the 

 most characteristic in appearance. 



The same principle is followed in diffusion inoculations of material rich in bacteria. 

 The first tube having been inoculated and the material diffused in the liquefied medium, 

 a given small amount (as one, two, or three loopfuls, or more definite amounts, as a 

 fraction of a cubic centimeter, if desired) of the mixture is carried into the second tube 

 and similarly diffused. The procedure is repeated from the second to a third tube, 

 in which the organisms of this final dilution are likely to be few and scattered and the 

 colonies resulting from their growth distinct and characteristic. 



