ANDRE LWOFF AND ANTOINETTE GUTMANN 



parallel to that of the stage of the microscope. The pipette is then displaced 

 towards the left, out of the microscope field. 3. A Pasteur pipette whose tip has 

 been bent at a right angle about two to three mm. from the end is introduced into 

 the right needle carrier of the Peterfi micromanipulator. The pipette contains 

 dilution medium for a length of about 2 cm. It is so oriented that the angle 

 formed by the terminal bend is in a plane parallel to the plane of the stage. 

 4. The tip of this pipette is brought into the center of the microscope field. The 

 micropipette is then moved towards the right and its level regulated in such a 

 manner that its point is opposite the liquid meniscus of the Pasteur pipette. One 

 introduces the micropipette into the liquid. One inoculates the content of the 

 micropipette into the liquid column of the Pasteur pipette. If one manipulates 

 the bacterium, it is possible to see that bacterium pass from micropipette to 

 Pasteur pipette. In any case, one knows that the operation is finished when a 

 drop of oil appears at the point of the micropipette. All these manipulations are 

 carried out in the incubator box. 5. The Pasteur pipette is then detached from 

 its support, removed from the box and its contents transferred either into a tube 

 or onto a petri plate. After a little practice, these operations can be performed 

 very quickly and with great assurance. All of these manipulations are naturally 

 carried out aseptically. According to our experience, which includes almost 1000 

 samplings, the chances of contamination are practically nil, since one manipulates 

 liquid quantities of the order of 10~^ to 10~^ mm^. 



III. Bacterial Multiplication 

 WITHOUT Liberation of Bacteriophage 



a. A diplo-bacillus, after five washings, is inoculated into a drop of peptone 

 water. After each division, one of the daughter cells is removed with a minimum 

 of the liquid. Twenty-two daughter cells are thus successively removed. The 

 entire drop is then removed: no bacteriophages. 



b. In another experiment of the same type, one removes successively 42 

 daughter cells: the result is identical. Furthermore, assays of four samplings in 

 the course of the experiment and of the total sample at the end show no bacterio- 

 phages. 



c. The washed filament of three bacteria is observed. One removes successively 

 14 daughter cells and makes 5 samplings. After the last division, the entire fluid 

 is assayed: no bacteriophages. 



d. Three diplo-bacilli are washed and inoculated separately into drops of 

 peptone water. When the number of bacteria has attained respectively 24, 47, 

 and 54, the entire fluid is sampled. There are no bacteriophages in the fluid nor 

 in any of the samples previously taken. 



e. Three diplo-bacilli are washed and transferred into drops of peptone water 

 of 72 to 100 mil diameter. Multiplication stops spontaneously when the number 

 of bacteria reaches respectively 8, 12 and 16. All of the fluid is removed and 

 sampled: no bacteriophages. 



f. A filament of 7 bacteria is washed and inoculated into a synthetic medium. 



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