MICROBIOLOGY OF DISEASES OF INSECTS 64 1 



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 directly in broth. The 

 broth used is made as follows: 



Water 1,000 c.c. ] 



Peptone 40 gr. J Boil, alkaHnize slightly and Hlter; place in bottles, plug with cotton. 



Salt 5 gr. r cover mouth and neck with parchment paper cap, and sterilize at 



Gelatin 30 gr. I I30° for thirty minutes. > 



Dextrose 5 gf . J 



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. 



It is necessary to remember that the virulence of B. acridiorum lowers very rapidly 

 in culture and is attenuated likewise by re-transplantations, so that a broth culture 

 so prepared should be used within two or three days at the utmost. If the campaign 

 against the locusts lasts for several months or the regions invaded are extensive, 

 it will be necessary to continue the series of passages during the campaign in order 

 to have on hand a virus of maximum efficiency. It is best to make two or three more 

 passages than necessary, rather than too few, for in the latter case the results of a 

 whole campaign may be nullified. 



The material necessary for a campaign against the locusts consists of a new 

 Spray pump, preferably tinned inside, such as is used in spraying fruit trees, and the 

 bottles containing the pure broth culture of B. acridiorum at a maximum virulence. 

 A used spray pump should never be employed as it is practically impossible to free 

 it from the antiseptic contained. The pure culture should be used as soon as it 

 shows turbidity. Broth cultures should never be used which have a putrefactive 

 odor. 



In practice it is generally necessary to infect the greatest number of locusts in 

 the largest possible number of bands in order to exterminate them with certainty 

 in as short a time as possible. The quantity of broth culture to be sprayed varies 

 with the area covered by the nymphs or locusts. One liter per hectare is sufficient 

 in all cases. For large areas, e.g., 100-200 hectares, spray over twenty dif- 

 ferent places, using one-half liter each time, taking in all ten liters. It is better 

 to spray over a large area rather than all in one place, choosing places where the 

 nymphs or locusts are in largest numbers, and always spraying the type of plants 

 preferred and in advance of where they are eating. Spraying should be done in the 

 early morning or preferably in the evening towards sunset. The heat and especially 

 the bright light of day rapidly attenuate the virulence of the bacillus. If necessary 

 to spray in the middle of the day, shady spots should be chosen. 



The virulence and vitality of B. acridiorum has been conserved in the dejecta 

 and dried cadavers for seven months while in culture the virulence especially, is 

 lost rapidly. 



A very hard rain will inhibit the progress of an epizootic for several days. The 

 rain washes the dejecta of the locusts from the contaminated vegetation, hindering 



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