6 LABORATORY MANUAL OF MICROBIOLOGY 



of certain kinds of culture media, it has been found that the 

 removal of these inorganic salts prevents the formation of a 

 precipitate during sterilization and hence gives a clearer medium. 



Preparation of Silica Gel — Prepare a 25 per cent solution of 

 equal parts of sodium and potassium silicate (C.P.). Adjust 

 the specific gravity of the solution to 1.06. Five cubic centi- 

 meters of this solution is then poured into a Petri dish, and a 

 drop of phenolphthalein added. An approximately 5 per cent 

 solution of HCl is added to this silicate, from a graduated pipette, 

 until the color is just discharged. The acid is then adjusted so 

 that 5 cubic centimeters of HCl solution will just neutralize 5 

 cubic centimeters of the sihcate. If care is taken not to overrun 

 the acid, the gel will set in a few minutes. 



To make the Petri plates, add a definite volume of the silicate, 

 solution to an equivalent amount of acid, placed in a large flask. 

 Shake the mixture vigorously and immediately pour 20- to 50- 

 cubic centimeter portions into Petri dishes. Allow the plates 

 to harden upon a flat surface. They are then placed in deep flat 

 vessels and dialyzed in running tap water until free from chlorides. 

 About 24 hours is required for this purpose. The dishes are then 

 removed and transferred to a sterile vessel containing boiled 

 distilled water. This is replaced several times. After they have 

 been properly washed, the dishes are drained and treated with 

 the nutrient medium. 



Sterilization. — Unless otherwise stated, it is recommended 

 that all media be sterilized in the autoclave. In the case of 

 liquid and agar media, about 120°C. for 20 minutes will be found 

 sufficient for complete destruction of all microorganisms. In 

 sterilization, it must be remembered that the time required to 

 kill bacteria depends upon the degree of heat at the center of the 

 vessel and the nature of the medium. This degree of heat is 

 determined by the size of the container, the original temperature 

 and viscosity of the contents, and also by the free access of the 

 steam to the surface of the container. All sterilizers should be 

 equipped with temperature controls and with air outlets at the 

 bottom. To secure the best results, place the medium .n small 

 containers and space in the autoclave in such a way as to give 

 free access of the steam to the surfaces. The time of steriliza- 

 tion must be determined for the various types of media. Agar 



