Test suspension— Reconstitute the freeze-dried BCG Live as per the manufacturer's instructions for human use with the diluent recommended by the manufacturer,and dilute aseptically to about 2mg per mLwith sterile BCGdiluent.

Procedure— Randomly select not fewer than six guinea pigs of the same sex,each weighing 250to 300g.Inject each animal with a total of at least 4mg of the Test suspension intramuscularly or subcutaneously in the rear left internal thigh,and observe them for a period of 6weeks.Note the number of animals that survive at the end of the observation period,and then sacrifice them.Perform autopsies of all animals postmortem to examine them for evidence of tuberculous infections,particularly at the popliteal and inguinal lymph nodes,liver,spleen,pancreas,and lungs,as well as at the injection site.If any abnormalities are found,perform a histological examination using standard and Acid-Fast staining techniques to detect Acid-Fast organisms.The product complies with the test if none of the animals show signs of tuberculosis and not more than one-third of the animals die during the observation period.

Test suspensions— Using the same diluent and the Test suspension prepared as directed in the test for Virulent mycobacteria,further dilute aseptically by making three serial tenfold dilutions.

Procedure— Randomly select two guinea pigs (male or female),each weighing 250to 300g.Inject 0.1mLof each of the four suspensions intradermally at different sites on the back of each animal.After 4weeks,the animals are shaved so that the injection sites and any reactions are made clearly visible.The diameters of the reactions are measured,and the presence of necrosis or nodules are noted.The reaction for the largest dose is between 4and 10mm and the smallest dose induces a nodule less than or equal to 4mm.Each animal gains weight during the observation period.

Tuberculin solution— Use tuberculin,purified protein derivative,to prepare a solution containing 25U.S.Tuberculin Units per 0.1mL.Dilute aseptically,if necessary,with sterile 0.9%sodium chloride solution.

Procedure— Use the same animals on which the Skin reactivitytest is performed.After the Skin reactivitytest is completed,inject each animal intradermally on the back with 0.1mLof the Tuberculin solution,and observe after 18to 24hours.An erythematous reaction of not less than 10mm in diameter is measured on each animal.