506 GENETICS OF SOMATIC CELLS 



gauge before joining, by means of a Y-shaped connecting tube, to the reduced air. The/>H 

 of media is 7.0-7.2, which is excellent for Chinese-hamster cells. The C0 2 can be reduced 

 to 0.1 liters per minute to adjust the pH to approximately 7.3-7.4 for lines of human cells. 

 As the interior of the incubator becomes moist less C0 2 is needed and the incoming gas 

 mixture (about 2 per cent) is utilized more efficiently in the saturated environment. 



In the event it becomes necessary to check the actual concentration of C0 2 in the in- 

 cubator, especially when optimal levels are to be attained for cultures of a particular 

 species, the Burrell Kwik-Chek gas analyzer for C0 2 may be helpful. (Burrell Corporation, 

 Pittsburgh 19, Pa.) 



Filter assemblies for sterilization of media 



The following filter assemblies are listed in the order of preference based on experience 

 in this laboratory. 



Selas VFA-86-02 vacuum filtration assembly with the substitution of a heavy-wall 

 filtration flask (Pyrex) having two tubulations (G8847, Emil Greiner Company). The 

 upper tubulation is cotton stoppered, and this is held tightly in place with a rubber band 

 and linked to a cotton-filled, sterile, air-filter tube (14-2340, Bellco Glass, Inc.) by a sturdy 

 suction hose. The suction line is then linked to an intermediate catch vessel (500-1,000 c.c.) 

 that acts as a back-suction stop when the faucet or pump is turned off. The lower tubula- 

 tion is linked by means of a convenient length (11-12 inches) of latex tubing to a filling 

 attachment (3960, Corning Glass Works, or 14-23330, Bellco Glass Co.) having an inside 

 diameter of 20 mm. The lower end of tubing of the filling bell is cotton stoppered, the bell 

 covered with Kraft paper, and held in position with a rubber band prior to autoclaving. 

 The porcelain neck of the Selas-filter element is placed in the rubber stopper with the aid 

 of Kel-F stopper grease. Extreme care must be taken to prevent accidents when assembling 

 the glass chimney. 



After autoclaving, a clamp is placed onto the latex tube and the assembly is com- 

 pleted. When the media has passed through the filter cone, the entire assembly is raised 

 and held by a sturdy clamp about the upper tubulation of the filter flask to stand some 

 12-13 inches above the surface of the bench to facilitate filling of milk-dilution bottles with 

 the aid of the filling bell. Repeated flaming of the base of the filling bell and necks of the 

 milk-dilution bottle is adequate to insure continued sterility during the filling process. This 

 filter assembly will facilitate larger volumes of media to be sterilized because of the con- 

 venience and opportunity to remove the initial liter of solution via the lower tubulation 

 before continuing with added volumes. Larger capacity filter elements (1^ inches wide) 

 provide adequate precautions for the sterilization of 2 liters of media with a 1 -liter flask 

 adapted to this scheme. The final 10 ml. of medium should be tested on surplus cultures 

 and the batch frozen or stored in the refrigerator. On numerous occasions, the initial and 

 final milliliter of media are added to Difco thioglycollate medium to help disclose con- 

 taminations. However, the use of surplus cultures for testing of sterility has proved to be 

 the more sensitive system. 



Fritted glass, Millipore, and Seitz filters can be substituted for the Selas-filter assembly 

 in the order of listing, in the event adequate distilled water rinsing is not feasible. The 

 need for a furnace to incinerate organic particles remaining in the Selas filter element may 

 also be an initial drawback. However, every effort should be made to start with the Selas 

 filter when genetic lines are to be maintained. Conventional stock cultures that can be 

 replaced do not require these precautions. 



Milk-dilution bottles (Corning Glass) are excellent for the storage of solutions and 

 media. Their necks are long enough to permit setting of the bottle slant-wise in a half-pint 

 container, along with a stainless steel graduated medicine cup (8492, Vollrath Co.) covering 

 the opened neck. In this manner the cup is raised slightly and the media pipetted from a 

 convenient angle to deliver to culture vessels. The stainless steel medicine cups may be 

 comparatively expensive, but the breakage of beakers generally used for this purpose is 

 eliminated, and the purchase price is fully warranted. 



