1902.] the Bactericidal Paver of small Samples of Blood. 57 



A ten-fold dilution of the culture — the dilution which is perhaps 

 most often required — is made by taking first 25 cm. of the culture, 

 and then, after the interposition of an air bubble, filling up to the 

 250-cm. mark with sterile broth. It can also, and this avoids any 

 contamination of the sterile diluting fluid, be made by filling up first 

 with 225 cm. of the broth, and completing up to the 250-cm. mark with 

 the culture. 



A six-fold dilution, should such be required, would be obtained by 

 filling in to the 250-cm. mark with sterile broth, and then completing 

 with two volumes of culture, these last being isolated as before by 

 intervening air bubbles. 



A five-fold dilution is obtained by filling in with two separate 25-cm. 

 volumes of the culture, and completing up to the 250-cm. mark with 

 sterile broth. 



A two and a half-fold dilution would be obtained by filling in with 

 four separate volumes of 25 cm., and completing up to the 250-cm. 

 mark with sterile broth. 



Dilutions of a different order can be obtained by filling in the 

 pipette as occasions may require with 2*5 or 5 cm. of culture, and then 

 completing to 250 cm. with sterile broth. By this means dilutions of 

 1 in 100 and 1 in 50 respectively can be obtained uno saltu. 



By a series of successive dilutions, made in each case after washing 

 out the pipette with boiling sterile water, any desired attenuation of 

 the culture can be quickly arrived at. The dilution of 1 in 1,000,000 

 ordinarily required for the purposes of enumeration will be obtained 

 by three successive dilutions of 1 in 100. 



(3.) Method of Eliciting the Number of Micro-organisms contained in the 



Diluted Culture. 



The required dilution of, let us say, 1 in 1,000,000 having been 

 piepared, the pipette would, after sterilisation in boiling sterile water, 

 be filled in with, say, three successive 10 cm. volumes of the diluted 

 •culture. A corresponding number of agar tubes having been taken in 

 hand, the three 10 cm. volumes of diluted culture would be separately 

 transferred to the surface of the nutrient medium, care being taken in 

 •each case to spread out the fluid over as large an area of surface as 

 possible. 



After incubation, the number of bacteria in each 10 cm. volumes of 

 diluted culture would be deduced from the number of colonies which 

 develop on the corresponding agar tube. 



After averaging the number of bacteria contained in the three tubes, 

 the number of living bacteria contained in 1 c.c. of the original culture 

 would be found by a simple arithmetical process. 



