32 THE BACTERIOPHAGE AND ITS BEHAVIOR 



tilled water. Place in a water-bath at SO^C. for 45 minutes. Next 

 wash the sac, outside and in, with a 10 per cent solution of ammonia, 

 taking considerable care for at this stage the sac is very fragile. Finally- 

 wash with distnied water. 



It is then necessary to completely dry the membrane, maintaining 

 a positive air pressure upon the inside to prevent the sac from becoming 

 deformed. The following has been found to be the most practical 

 method. A rubber stopper (with one hole) which will fit the opening 

 in the supporting tube is provided with a short glass tube, to the 

 latter being attached a piece of rubber tubing provided with a good 

 screw clamp (fig. 4). 



Pour a few cubic centimeters of water into the sac and insert the 

 stopper equipped with its tube and clamp. Blow through the rubber 

 tube to distend the sac, taking care not to rupture the still fragile 

 membrane. Close the clamp. When attached to a support, the sac 

 uppermost, the few cubic centimeters of water placed in the sac will 

 make an hydrauUc seal and prevent the escape of air. 



When dried, a denitrified sac will keep indefinitely. For use, it is 

 placed in a large tube* in distilled water, after the manner indicated 

 for sacs which have not been denitrified. 



Sterilization 



Such is the description of the preparation and assembhng of sacs for 

 ultrafiltration as given in texts designed for physical chemists. For 

 us, as microbiologists, the procedure can not stop here, for we must 

 work in an aseptic manner, that is, the ultrafilters must be sterilized. 

 But, it is practically impossible to steriUze a collodion ultrafilter by 

 moist heat in an autoclave. f The sac becomes deformed, the mem- 

 brane is rendered opaque, and porosity is modified. 



It is possible, as Duclaux has shown, to effect denitrification and 

 once this is accompHshed an ultrafilter can be sterilized by steam in 

 the autoclave. Nevertheless, the deformation of the membrane, 

 although very much less than with the original collodion membrane, 

 is still very considerable. 



After many trials I have adopted the following method, which has 



* A more minute description of the details of ultrafiltration will be found in 

 text-books dealing specifically with colloidal chemistry. 



t It is easy to see the enormous errors attending the use of unsterilized or 

 poorly sterilized ultrafilters,^" yet of all the proposed types of ultrafilters, the 

 only ones that can be effectively sterilized are those designed by Malfitano, the 

 preparation of which is here described. 



