312 Biological Stains 



Method of Analysis: Dissolve 100 mg. of dye in 100 ml. of 95% alcohol, add 100 

 ml. of distilled water and 15 g. sodium acid tartrate, heat to boiling and titrate 

 with 0,05 A'^ TiCls to a pale yellow endpoint. The following data are used in cal- 

 culating the percentage of anhydrous dye in the original sample: 



Molecular weight 482.618 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCla per gram of dye , 41.440 



For certification, samples of this stain must contain not less than 85% anhydrous 

 dye. 



Biological Tests: Brilliant green is tested as to its suitability for determining the 

 presence of the colon organism in drinking water. The object of the dye is to pre- 

 vent formation of gas by the bacillus of gas gangrene. Bacillus Welchii. For this 

 purpose standard methods prescribe a medium containing 2% dried oxgall with a 

 1/75,000 dilution of brilliant green after addition to the water to be tested. In test- 

 ing a dye sample, varying amounts of brilliant green are added to the bile medium 

 for one series of tests, using a sufficient variety of solutions to be certain of growth 

 in the most dilute and absence of growth in the most concentrated. A second series 

 of tests is set up with varying dilutions of pure cultures of the colon organism (Esch- 

 erichia coll) on the standard medium as above mentioned, while another comparison 

 is made in standard lactose broth. A satisfactory sample should allow 10% gas 

 production in 1 to 3 days by the colon organism, but not by Bacillus Welchii at the 

 dilution called for in the standard medium. The object of the long series of tests 

 is to check up on the delicacy of the medium as used, and on the efiFect of varying 

 the quantities of brilliant green. 



It is also tested as a bacteriostatic agent by the following technic: 

 Make three daily transfers at 37° C. of a spore-former (e.g. Bacillus cereus) and 

 of two members of the colon-typhoid group (including Escherichia coli) into a broth 

 containing 1% peptone and 1% lactose. As soon as distinct turbidity appears in 

 the third transfer (3-6 hours incubation), make a microscopic count of the organ- 

 isms, with the use of a hemocytometer; (it is desirable to have the count between 

 4 and 20 million per milliliter). Dilute with the above-mentioned broth so that 

 the concentration of bacteria is approximately 200 per milliliter. Meanwhile 

 make a 0.01% solution of the sample to be tested and of a check batch of 

 brilliant green (known to have the correct bacteriostatic titre) by boiling 10 mg. 

 dye in a 100 ml. volumetric flask not quite full of distilled water and bringing up to 

 the volume mark after cooling over night. From this flask prepare 0.001%,0.0001% 

 and 0.00001% dilutions. Sterilize the flasks containing these solutions and there- 

 after handle them aseptically. Fill a series of test tubes with 7.5 ml. of a broth 

 containing 1.33% peptone and 1.33% lactose, and divide into three lots, one 

 intended for each of the three test organisms; sterilize in the autoclave. To each of 

 these three series of tubes add varying quantities of the dye by introducing, with a 

 sterile pipet, 0.2-2.0 ml. of the four above-mentioned dilutions; then bring the total 

 volume in each tube up to 9.5 ml. by adding sterile distilled water aseptically, and 

 mixing thoroughly. (In this way final dilutions from 1:50,000 to 1:500,000,000 may 

 be obtained, although a partial set of them can be selected for each particular or- 

 ganism, from 1:5,000,000 to 1:100,000,000 for the spore-former and from 1:50,000 to 



