LABORATORY AND TEACHING METHODS 615 



putrefactive bacteria and +10 to +25 or even higher for fungi. There are some 

 bacterial organisms which prefer distinctly alkaline media (Fuller's scale), while 

 others prefer acid media. A good general practice to follow in the preparation of the 

 basic culture media to be kept in stock is to standardize to +10 of Fuller's scale and 

 vary the reaction according to the preference of the organisms under cultivation. 

 When other acids than HCl are used for acidifying the media, the kind should be 

 definitely specified, when the reaction is expressed. 



Titration of Media. — In outlining the method of preparation of bouillon for routine 

 work, directions were given for neutralization of the medium and the addition of the 

 requisite amount of acid. In accurate work, or in the prosecution of research, a 

 more carefvJ method of standardization is employed. The medium should be 

 neutralized by the titration method. The process is as follows: 



1. Add exactly 5 c.c. of the medium to 45 c.c. of distilled water in an evaporating 

 dish (use a s-c.c. Mohr pipette), boil for three minutes to drive off the CO2 and add 

 I c.c. of phenolphthalein solution. 



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2. Add NaOH drop by drop from a burette, stirring constantly until the 



20 



solution turns a faint, but permanent pink. Repeat the titration for two more 5-c.c. 

 samples, and determine the average of the three readings. 



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3. Calculate the amount of ^^r-^jj necessary to neutralize the medium (10 to 



IS c.c), add the amount determined to the medium, test reaction and if neutral, 

 proceed with preparation of the medium; if not, repeat the titration on neutralization. 



LESSON 19 



Germination Studies. — The examination of spore germination of various fungi can 

 be studied best by the hanging-drop method. Take a hanging-drop slide and sterilize 

 thoroughly in the hot-air oven at iio°C. after it has been wrapped in a crepe napkin 

 or piece of tissue paper. After sterilization plunge it into a beaker of absolute alcohol 

 (or such sterilized slides may be kept in stock in absolute alcohol) and then drain 

 off the greater part of the spirit, grasping the slide in a pair of sterile forceps. Bum 

 off the remainder of the alcohol in the flames. 



Place the hanging-drop slide on a piece of blotting paper moistened with a 2 

 per cent, lysol solution and cover it with a small bell glass that has been rinsed with 

 the same solution and not dried. 



Raise the bell glass slightly and smear sterile vaseline around the rim of the cell 

 by means of a sterile spatula of stout platinum wire. Remove a dean cover-slip 

 from the alcohol pot with sterile forceps and burn off the alcohol; again raise the 

 bell glass and place the sterile cover-slip on the blotting paper by the side of the 

 hanging-drop slide. 



Remove a drop of the culture medium selected for use (see below) and place the 

 drop on the center of the cover-slip. Sterilize the loop. 



Raise the bell glass sufficiently to. allow of the cover-slip being grasped with the 

 sterile forceps, invert it and place over the cell of the hanging-drop slide. Remove 

 the bell glass altogether and press the cover-slip firmly on the cell. 



