9SO CONTROL OF BIOLOGICAL PRODUCTS 



fit being well mixed with the melted agar for each plate. Incubation is at 37° C. for forty- 

 eight hours. The glycerinated lymph intended for a single vaccination should show not to 

 exceed fifty organisms when this technique is followed. In practice it is rare to find glycerin- 

 ated lymph which carries more than a dozen contaminating organisms, and those usually 

 clearly of a harmless nature. Many tubes are entirely free from contaminants. 



The tests for tetanus are required to be carried out on the harvest from each calf sepa- 

 rately. The glycerinated pulp must be tested before any other preservative has been added, 

 and the test must be made after the bacterial count has been reduced to less than fifty per 

 vaccination quantity or after storage for at least seven days at a temperature of 10 °C. Two 

 CO. of the glycerinated vaccine are planted in each of several fermentation tubes, each tube 

 containing not less than 25 cc. of meat-infusion broth which, immediately preceding plant- 

 ing, shall have been heated to 100° C. for thirty minutes, any air bubbles being removed by 

 tipping the tubes. The tubes are incubated at 37° C. and examined daily. The presence of gas 

 or clouding in the closed arm shall be taken as indication for the inoculation of animals with 

 the culture. Either mice or guinea pigs may be used. The inoculation should be made upon 

 the first observation of growth in the closed arm and again at the end of nine days after the 

 planting. The animals should be kept under observation for at least six days and closely 

 observed for the development of symptoms of tetanus. 



There are a number of procedures for testing the potency of smallpox vaccine, all 

 of which are described by Force and Leake.' These authors suggest a method of their 

 own, which is a modification of that proposed by Calmette and Guerin. The modifi- 

 cation may be described as follows: 



Albino rabbits are employed, preferably mature animals. The hair is removed by clip- 

 ping, followed by shaving or by plucking. The area utilized extends from the scapular to the 

 iliac region and about 12 cm. on each side of the vertebral column. By means of a sterile plate 

 made from thin galvanized iron the area on each side is marked off into four rectangles of 

 2.5X5 cm. 



The dilutions to be tested are i : 1,000, 1 13,000, i : 10,000, 1 130,000. These dilutions are 

 employed for the vaccine under test and for one of known satisfactory potency which is used 

 as a control. 



The inoculations are made by means of glass pipettes, 3-mm. inside diameter, which are 

 broken off squarely. With these pipettes charged with about 0.4 cc. of diluted vaccine the 

 tightly stretched skin within the rectangle is inoculated. Corresponding areas on the right 

 and left sides of the rabbit are used for the same dilutions of the control vaccine and the 

 vaccine under test. 



The readings are made at the end of seven days and a comparison made between cor- 

 responding dilutions of the vaccine being tested and the vaccine used for control purposes. 



The authors suggest the following criterion of potency: 



A smallpox vaccine of high potency, when diluted i : i ,000 should produce a confluent 

 eruption on from 90 per cent to 100 per cent of the vaccinated area on the back of a rabbit, 

 and when diluted i : 3,000 the decrease in confluence should not be over 20 per cent. A vaccine 

 satisfying this criterion should produce in all previously unvaccinated human subjects a 

 circular vesicle measuring at least 7 mm. in diameter on the seventh day when applied, un- 

 diluted, to a circle of the exposed derma measuring 2 mm. in diameter. 



' Force, J. N., and Leake, J. P.: Hygienic Laboratory Bull. i4g. 1927. 



