MINIMAL LETHAL DOSE 317 



5. Similarly, o.i c.c. of the mixture is transferred 

 from capsule II to capsule III (i c.c. of bouillon in 

 capsule III contains o.ooooi loop), and then from 

 capsule III to capsule IV (i c.c. of bouillon in capsule 

 IV contains o.ooooooi loop). 



The dilutions thus prepared may be summarised in 

 a table ; 



Capsule I=i loopful+io c.c. water .'. i c.c. = o.i loop. 



Capsule II = o.i c.c. capsule I +9.9 c.c. water .'. i c.c. = o.ooi loop. 



Capsule III = o.i c.c. capsule II +9.9 c.c. water .'. i c.c. = 0.00001 loop. 



Capsule IV = o.i c.c. capsule III + 9.9 c.c. water .'. i c.c. = 0.0000001 loop. 



6. With sterile graduated pipettes remove the neces- 

 sary quantity of bouillon corresponding to the various 

 divisors of ten of the loop from the respective capsules, 

 and transfer each "dose" to a separate sterile capsule 

 and label; and to such doses as are small in bulk, add 

 the necessary quantity of sterile bouillon to make up 

 to i c.c. 



7. Multiples of the loop are prepared by emulsifying 

 i, 2, 5, or 10 loops each with i c.c. sterile bouillon in 

 separate sterile capsules. 



8. Inoculate a series of animals with these measured 

 doses, filling the syringe first from that capsule con- 

 taining the smallest dose, then from the capsule con- 

 taining the next smallest, and so on. If care is taken, 

 it will not be found necessary to sterilise the syringe 

 during the series of inoculations. 



9. Plant tubes of gelatine or agar, liquefied by heat, 

 from each of the higher dilutions, say from o.ooooooi 

 loop to o.oi loop; pour plates and incubate. When 

 growth is visible enumerate the number of organisms 

 present in each, average up and calculate the number 

 of bacteria present in one loopful of the inoculum. 



10. The smallest dose which causes the infection and 

 death of the inoculated animal is noted as the minimal 

 lethal dose. 



