MICROBIAL DISEASES OF INSECTS 915 



tally in the direction of the head to a depth of about 3 mm. for an adult insect, a 

 little less for a nymph. The point of the needle should enter the abdominal cavity, 

 not merely pierce the tegument as in the latter case the effect would be nil. If 

 the needle is inserted too deep the internal organs will be injured. A very fine- 

 pointed bent pipette could be employed equally well. One or two drops of the emul- 

 sion of the old culture are injected. 



As soon as the locusts in the first series become sick or preferably are nearly 

 dead, press the abdomen between the fingers and collect in a watch glass the blackish 

 liquid which issues from the anus. Inject a drop of this liquid into the abdominal 

 cavity of the second series of locusts, following the same technic and observing the 

 same precautions as for those of the first series. These insects will die in a shorter 

 period of time. Obtain as previously, in a watch glass the intestinal liquid of three 

 or four of the first dead locusts of the second series, dilute half with water and sterile 

 broth and inoculate the third series. To inoculate the fourth series, use the intes- 

 tinal liquid of the first dead of the third series diluted to a third; a fifth series with 

 the liquid diluted to a fourth and continue with the series in this way. It is excep- 

 tional that it will be necessary to proceed further than the twelve series. The viru- 

 lence of B. acridiorum is increased sufficiently if death occurs eight hours after injec- 

 tion. One-hundredth of a cubic centimeter of virus at its maximum virulence in- 

 iected into a locust will cause the characteristic diarrhosa in two hours and death 

 an hour later. This method of increasing the virulence takes five to six days and 

 this period of time has to be taken into consideration when it is necessary to employ 

 the culture on a practical scale. 



When the acridian to be infected belongs to a different species than that for which 

 the virulence of B. acridiorum has been previously augmented, a large number of 

 passages may be necessary as a culture virulent for one species may not be able to 

 infect another species. In one case fifty-two passages were necessary in order to 

 kill Stauronautus maroccanas (Algeria) in eight hours while for the same insect at 

 Cyprus only twelve passages were necessary. It is also desirable that the first 

 few series consist of a large number of insects, as there will be apt to be some 

 which will be more sensitive to the virus. Their natural resistance can be weakened 

 by fasting for several days before inoculation. The intestinal contents should not 

 be diluted until the virus will kill within fifteen hours. 



When the virulence is sufficiently increased, the specific bacillus is isolated by 

 means of an agar slant or plate and cultivated for twenty-four to thirty-six hours 

 at room temperature; it may then be isolated if the virus is to be conserved ; if 

 desired for direct infection experiments, it may be placed dfrectly in broth. The 

 broths used is made as follows: 



Water 1,000 c.c. 



Peptone 40 gr. 



Salt S gr- 



Gelatin 30 gr. 



Dextrose S gr. 



Boil, alkalinize slightly and filter; place in bottles, plug with cotton, 

 cover mouth and neck with parchment paper cap, and sterilize at 

 120 for thirty minutes. 



The gelatin serves to fix the organisms in place when the culture is sprayed on the 

 vegetation, and on account of the dextrose the plants are greedily devoured by the 

 locusts. 



