INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM 



practiced, can only furnish partial solutions to the problems of lysogenic bacteria. 

 Only observations carried out on individual bacteria, or on microcultures 

 containing a small number of individuals, are capable of leading to definite 

 conclusions. 



Strain. Our experiments were carried out on a lysogenic strain of Bacillus 

 megaterium. We have used the classic strain "899" of den Dooren and de Jong 

 and an asporogenic sensitive mutilat strain. Lysogenic and mutilat strains were 

 subcultured by passages on peptone broth agar. 



Technique of Micromanipulation. One utilizes an incubator box made from 

 transparent plastic material. An electric heater is placed under the bottom of 

 this box, into which 20 or so holes have been drilled. The temperature is thus the 

 same in all parts of the incubator box and a thermoregulator in one corner insures 

 a constant temperature of 37°. The microscope and the receptor for the de 

 Fonbrune micromanipulator are fixed on the base of the Zeiss-Peterfi micro- 

 manipulator. We have utilized the oil chamber of de Fonbrune. It is unnecessary 

 to go into details concerning this device, of which one can find a description in the 

 monograph of de Fonbrune (1949). A knowledge of these technical details, 

 however, can prevent considerable loss of time. 



Two pieces of glass of 1.5 X 12 X 7 mm. are glued to either side of the depres- 

 sion slide. Their separation is so calculated that it exceeds the length of the 

 coverslips by about .8 mm. The latter, made from neutral glass, are washed in 

 nitric acid, rinsed in double distilled water, wiped with fine cloth and sterilized 

 (as well as the slide) at 150°. 



For the preparation of the experiment: 1. one sets a cover slip on the slide and 

 places several drops of paraffin oil on the cover slip, so that it is covered by a layer 

 of oil. 2. One draws out the fine end of a Pyrex glass Pasteur pipette so that the 

 diameter of the terminal capillary allows passage of culture medium and that 

 when the tip of the pipette filled with liquid comes in contact with the cover slip 

 under the oil, one drop of liquid comes out and spreads out on the glass. This 

 drop must have a diameter of about 500 to 800 m- Nine rows of 9 drops can thus 

 be regularly placed. The second and third rows are interrupted at the point of 

 the fourth, fifth, and sixth drop. It is of course possible to place several different 

 media on the cover slip. The cover slip is then turned upside down and oil is 

 poured between it and the slide. On the bottom of the oil chamber one places 

 drops of distilled water in a manner so as not to disturb the observations; this 

 will prevent any significant evaporation of drops of less than 100 m diameter. 

 The oil chamber is then placed on the stage of the microscope. 



Technique of Sampling. It is necessary that one can transfer one bacterium, or 

 a minimum quantity of liquid of the order of 10"^ mm.^ into a tube or onto a 

 petri dish. The dimensions of the liquid column sampled can be measured with 

 the micrometer objective. In order to transfer the content of a pipette into the 

 tube, one can proceed in the following manner : 1 . The field of the microscope is 

 disengaged by placing the oil chamber towards the rear. 2. The needle and the 

 needle carrier are turned by 90°, in such a manner that the point of the needle 

 points towards the right. The angle formed by the micropipette is then in a plane 



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