1 88 LABORATORY GUIDE IN BACTERIOLOGY 



Experiment i. The dilution method 



1. Prepare a culture of a species of yeast in liquid 

 wort. 



2. Examine a large loopful under the microscope 

 and dilute the culture with sterile wort until there is 

 an average of about one cell to the loopful. 



3. Inoculate a series of ten tubes of liquid wort 

 with a loopful each of the diluted culture, or inoculate 

 with a drop from a sterile capillary pipette. 



4. Incubate. 



All those tubes in which growth has appeared con- 

 tain a culture originating from one cell. 



Experiment 2. Hansen's gelatin method 



1. Prepare a culture of a mixture of two or three 

 species of yeast. 



2. After growth has appeared inoculate a tube with 

 liquefied wort gelatin at a temperature of about 30 to 

 35 C. 



3. Examine under the microscope and dilute with 

 liquefied gelatin until there are only one or two cells 

 to each loopful. 



4. Prepare cover slips in the following manner: 

 Dip several cover slips into liquid paraffin. After the 

 paraffin has solidified draw lines through the paraffin 

 with a sharp steel needle, so as to form a number of 

 squares. Mark each square with numbers or letters 

 with the same needle. Dip the cover glass into hydro- 

 fluoric acid for a few seconds, wash in water, then 

 chloroform, ether, and alcohol until the glass is clean 

 and free from fat. The hydrofluoric acid will have 

 etched the glass so as to show the marks permanently. 



5. Cover the slip with a thin coat of the diluted 

 wort gelatin containing the culture. 



