76 A MANUAL OF BACTERIOLOGY 



Exercise 111. Determination of the Numbers of Bacteria in Milk by 

 Direct Microscopical Examination 



The difficulties of enumerating bacteria by the poured-plate 

 method which were mentioned above have been partially over- 

 come by the introduction of methods involving a direct micro- 

 scopical examination. Such methods are desirable if for no other 

 reason, because time is not lost in waiting for colonies to grow 

 upon the plates. 



The following procedure is substantially that outlined by 

 Breed. See 



WINSLOW. Journ. Infect. Dis., Suppl. No. 1., p. 209. 1905. 

 PRESCOTT and BREED. Journ. Infect. Dis. 7 : 632-640. 1910. 

 BREED. Centralbl. f. Bakt., 2te Abt., 30 : 337. 1911. 



1. Shake thoroughly the sample of milk to be examined. Draw 

 0.01 cc. by means of a capillary pipette. 



2. Spread the required quantity of milk over an area of 

 1 sq. cm. on an ordinary glass slide. 



This can be done by placing the slide on a paper or glass on 

 which an area of this size is ruled. Circular areas are better 

 than square areas. Make duplicate smears on each slide. 



3. Dry the milk with gentle heat ; treat the film with xylol or 



chloroform to dissolve out 



the fat ; when dry, immerse 

 the slide in 95 per cent 



alcohol for three to five min- 

 FIG. 31. Glass slide with etched circle 



. . utes, to fix the film on the 



The circle has an area of 1 sq. cm. 



slide. 'Dry the slide again 



and stain with methylene blue or other simple stain. Avoid 

 alkaline stains and all others which would attack the casein 

 and loosen the film. 



4. When the slides have dried from washing, they are ready 

 to examine. Place the slides under the oil-immersion lens. Move 

 the draw tube until the field of the immersion lens is equivalent 

 to 0.16 mm. ; then each field of the microscope covers approxi- 

 mately one five-thousandth (0.0002) of a square centimeter. 

 On this basis each bacterium seen in a field taken at random 



