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LABORATORY EXERCISES IN BACTERIOLOGY. 



performed, if there be no further need for the material, the swab is burned and the 

 tube disinfected and washed in the usual manner (Fig. 35). 



Pipettes. Ordinary graduated pipettes of small caliber are often used for making 

 inoculations with definite quantities of liquid containing infectious germs, and simple 

 pipettes drawn in the laboratory from narrow glass tubing are likewise used for the 

 transfer of liquids to the nutrient media. The liquid thus added is usually diffused 

 through the nutrient which has been previously liquefied by heat, and the contained 

 microorganisms are thereby scattered throughout the nutrient mass. The resultant 

 culture thus grows either diffusely or in separate colonies (each representing at least 

 one bacterium) throughout the medium; such a culture is spoken of as a "diffusion 

 culture." In practice, the pipette, the upper end having been provided with a cotton 

 stopper, is sterilized in the usual manner in the oven or autoclave. If desired, a piece 

 of rubber tube may be attached to the upper end to serve as a mouthpiece. The 



small end is passed several times through 

 the flame as a precautionary measure and 

 then plunged into the infected liquid, which 

 is drawn into the interior to the desired 

 graduation by suction by the mouth. A tube 

 of liquid medium is at hand or a gelatine or 

 agar tube is liquefied in a water-bath (either 

 one extemporized or of a form especially de- 

 signed for the purpose, as shown in Fig. 36). 

 The stopper is removed from the culture 

 tube and the measured quantity of the in- 

 fected liquid passed into it. The pipette is 

 at once laid aside (into a pan of disinfectant 

 solution, if of no more consequence), the lip 

 of the tube flamed, and the stopper flamed 

 and readjusted. Then, with slight agitation, 

 care being exercised that the stopper is not 

 soiled by the contents of the tube, the 

 material is thoroughly diffused through the 

 medium. The tube may now be set aside 



and the contents allowed to set, or the latter may be poured into a Petri dish or 

 upon a plate for a dish or plate culture, if so desired. 



Should the liquid to be inoculated upon the nutrient medium contain but few 

 organisms, the usual amount employed is one cubic centimeter, which amount is gener- 

 ally diffused in nine cubic centimeters of the medium. If there be numerous organisms, 

 however, so that in the resulting culture the colonies are likely to be so numerous 

 and grow so close to each other as to interfere with proper observation and enumera- 

 tion, known dilutions with sterilized water may be made to reduce the number of 

 bacteria in the volume employed as one part of the suspected fluid to nine, or ninety- 

 nine, or nine hundred and ninety-nine parts of sterilized water. When very small 

 fractions of a cubic centimeter of the original liquid or of the known dilutions are de- 

 sired, they are best obtained by means of long capillary pipettes drawn from narrow, 

 soft glass tubing as needed. (To draw such a pipette select an eight- or ten-inch 

 piece of a soft glass tube of small caliber and heat a length of several inches near the 

 middle in the spread flame until it has become ductile ; then at once remove from the 

 flame, and with even, fairly rapid traction from each end draw the tube into a uniform 



FIG. 36. LABORATORY WATER-BATH 

 FOR MELTING SOLID MEDIA IN 

 TUBES. 



