388 GASOMETRIC-MANOMETRIC METHODS 



Placing IF, POi, and P. With the diver attached to the rubber 

 block of the pipette, the biological sample in its aqueous medium 

 W is drawn in, followed at once by the short seal of oil POi. The 

 filling is carried out under the microscope. The liquids should be in 

 small narrow cylinders which prevent the rubber block from coming 

 into contact with them. Speed is essential in bringing in the oil seal 

 after W has been taken in, since Zeuthen has found that if W is 

 exposed to evaporation for not more than 10 sec. at about 50% 

 humidity a loss of less than 10% of W will be incurred. It is advis- 

 able to work in a moist chamber in order to reduce evaporation 

 losses. 



The paraffin seal (P) is next placed by pushing the end of the 

 diver through a layer of paraffin 0.2-0.3 mm. thick. The paraffin 

 (m.p. 58-60°) must be absolutely clear and white; the layer is 

 formed by kneading the material between the fingers. It is then 

 placed on a piece of rubber to minimize damage to the diver when 

 piercing the paraffin. The diver is held with the forceps and wiped 

 off to remove any paraffin or oil from the outside. A small blunt 

 glass rod which can go into the diver is used to push in the P, 

 POx, W combination about 1.2-1.5 mm. to leave space for Mx. 

 This must be done under the microscope and very slowly. Caution 

 is required to prevent the sample in W from coming into too inti- 

 mate contact with the air-water interface or particularly the oil- 

 water interface. Now the brake is clamped vertically and the length 

 of the diver is measured, if this has not been done previously. The 

 transparency of the rubber block permits the end of the diver to 

 be seen. 



Placing PO2' A rubber stopper with dimensions approximately 

 the same as those in B, Figure 135, is fastened on its wide end to 

 a glass rod by which it may be clamped. A slit, 2-3 mm. deep, is 

 cut into the surface of the small end of the stopper and the end of 

 the diver is placed in this slit. With the diver mounted vertically in 

 this fashion, the ball-tipped pipette is clamped directly over it, and 

 the clamp holding the rubber stopper is raised so that the tip of the 

 pipette enters the diver. The ball is brought to the point where PO2 

 is to be placed and the oil is carefully blown out of the pipette to 

 form the seal. A slight jerk is used to disengage the ball from the 

 seal and then the pipette is withdrawn from the diver entirely. The 

 flexibility of the pipette stem makes strict alignment between the 



