166 LABORATORY EXERCISES IN BACTERIOLOGY. 



colonies calculated for one square centimeter, multiplied by the number of . square 

 centimeters in the film, furnishes the total number of bacteria in the material inocu- 

 lated. Or, if one wishes, the outer surface of the tube may be laid off in squares by 

 longitudinal and circumferential lines, a wax pencil being used to write on the glass, 

 these serving as the squares laid beneath an ordinary plate or dish culture. After 

 a proper count of the colonies in each culture of the several dilution inoculations has 

 been made, and this number multiplied by the coefficient of dilution of the original 

 substance, the mean of the results should be taken as most nearly representing the 

 correct number sought. However, it should be recalled that the figures obtained are 

 really only approximations, and are understatements rather than exaggerations, owing 

 to the failure of this or that bacterium to develop into a recognizable colony for some 

 reason or other, and owing to the fact that some colonies may represent, not isolated 

 organisms, but clumps of germs. 



Exercise 30. Under the eyes of the instructor let each student in turn 

 make an inoculation of tubes of solid medium by stroke (using plain 

 needle), smear (using loop), and stab inoculations; and of liquid medium 

 by diffusion inoculation (using loop) . In this it should be seen that the 

 student properly holds the tubes from which material is obtained and to 

 which inoculation is to be made ; that he removes the stoppers correctly 

 and completely, and disposes of them properly; that he flames the needle 

 properly both before and after inoculation, flames the mouth of tube and 

 stopper before readjusting the latter, and that the whole process is carried 

 out without undue exposure to contaminations. 



Exercise 31. Each student prepare a swab, sterilizing it for use in ob- 

 taining material from the throat of a diphtheritic patient. 



Exercise 32. Prepare from soft glass tubing three capillary pipettes, the 

 capillary portion of each of which shall be uniform for at least twelve 

 inches; graduate each into tenths of a cubic centimeter, properly prepar- 

 ing it for sterilization in the oven (rinsing in alcohol and ether, and 

 plugging end with cotton) ; thereafter sterilize in oven for future exercise. 



Exercise 33. Each student prepare one Sternberg bulb, at least one- 

 half inch in diameter of bulb and having a capillary tube of at least two 

 inches in length. 



Exercise 34. By means of sterile pipette measure one cubic centimeter 

 of milk and add it to nine cubic centimeters of sterile distilled water in a 

 small sterilized flask, mixing by gentle agitation. From the mixture, by 

 means of a sterilized capillary pipette, remove o.i cubic centimeter and 

 transfer to a tube of liquefied gelatine (kept liquid in warm water at 30 C.). 

 At once diffuse, and, following instructions given above, distribute the diffu- 

 sion upon a sterile glass plate and place it in a culture dish for development. 

 Remove from the remainder of the decimal dilution, by means of the sterile 

 pipette, one cubic centimeter of the dilute milk ; with assistance of a fellow 



