168 LABORATORY EXERCISES IN BACTERIOLOGY. 



student at once rinse out the flask with sterile distilled water, placing 

 thereafter in it nine cubic centimeters of sterile distilled water. To this 

 add the one cubic centimeter of the decimal dilution, thus preparing a one 

 per cent, dilution, diffusing thoroughly by agitation. From this second 

 dilution remove with sterilized capillary pipette o. i cubic centimeter and 

 transfer to a tube of liquefied agar (kept liquid in water -bath at 45 C.) ; 

 diffuse by agitation, and transfer the contents to a Petri dish, in which it 

 should be quickly cooled so as to harm as little as possible the bacteria by 

 the temperature of the agar, placing the dish after solidification in the in- 

 cubator. From the one per cent, dilution remove with a sterile pipette 

 one cubic centimeter of the dilute milk; again quickly rinse out the flask 

 with sterile distilled water, and place nine cubic centimeters of the sterile 

 distilled water in the flask. Add the one per cent, dilution to the latter, 

 thereby making a one in a thousand dilution. Of this, with sterile capillary 

 pipette inoculate o. i cubic centimeter to a second tube of liquefied gelatine 

 and roll over ice to make an Ksmarch tube. Put aside in a dark place at 

 room temperature for further development. Observe each culture at 

 close of the first, second, and third days, and at the close of the last period 

 count the number of bacteria obtained in each dilution. Calculate there- 

 from the number in one cubic centimeter of the undiluted milk. (Should 

 liquefaction of medium endanger the definition of the colonies before the 

 seventy-second hour shall have been passed, count at an earlier period as 

 conditions require.) 



Note the differences shown by the colonies on the agar in the incubator 

 and those of the gelatine plate and tube at room temperature. 



(As far as possible let the instructor witness the various steps taken by each 

 student in the processes of dilution, inoculation, and the different modes of plating 

 the inoculated media, to insure all necessary precautions against contamination and 

 to correct errors of manipulation.) 



Exercise 35. Make an inoculation of one cubic centimeter of water 

 from the tap or from a cistern into a tube of liquefied agar ; diffuse by agita- 

 tion; transfer to a Petri dish; grow in incubator. Make a similar inocula- 

 tion in a tube of liquefied gelatine ; transfer to Petri dish ; place in a dark 

 place at room temperature for development. Compare the rate of devel- 

 opment of these psychrophilic bacteria at the two temperatures. Are 

 those on the agar different in gross appearance of colonies from those on the 

 gelatine ? 



Exercise 36. Inoculate two tubes of solid blood-serum by smears from 

 a known culture of Mycobacterium diphtheria. Let one remain at room 

 temperature; grow the second in the incubator at body temperature. 



