INDUSTRIAL A N D ENGINEERING CHEMISTRY
130
shown, thus, that some biotin is present in the basal medium although none is added to the tube. To discover whether or not synthesis of biotin had taken place in tubes containing no added pure biotin but showing maximum acid production under stimulation of lipoid substances the following flasks were pre ared: (1) basal medium alone, (2) basal medium lus rice oifin quantity comparable to that resent in rice polisph extract, and (3) basal medium plus crystalEne biotin comparable to that in standard tubes containing 800 mieromicrograms of biotin. After inoculation and 72 hours' incubation, the flasks containing the rice oil and the pure biotin greatly exceeded the blank control flask in both acidity and turbidity. The contents of all three flasks were made 4 N with concentrated sulfuric acid and autoclaved at 15 pounds' pressure for 2 hours. Vi orous hydrolysis was carried out to ensure the liberation of ced contents After hydrolysis the pH was increased to 6.0 by the addition of hot barium hydroxide octahydrate suspension. The precipitate was digested, filtered, and washed. The filtrate and washings were concentrated by eva ration, then made up to one fifth of the original volume. concentrate was sssayed a t the 2-ml. and 5-ml. levels. In the case of all three concentrates, the 5-ml. level proved to be too high in dissolved materials to permit satisfactory growth of the organisms. The 2-ml. levels, however, showed the following sharp differentiation in milliliters of 0.1 N sodium hydroxide required for titration: (1) basal medium alone, 1.80 ml. of alkali; rice oil 1.30 ml. of alkali: and (3) basal (2) basal medium medium ure biotin, 9,lb ml. of alkali. The results were checked by &plicate determinations.
Assay tubes showing high cell and acid production in the presence of stimulants and in the absence of added pure biotin were tested for evidences of biotin synthesis on the part of the organism. No synthesis could be demonstrated. LITERATURE CITED (1) Andrews, J. S., Boyd, H. M., and Terry, D. E., IND.ENO. CHEM.,ANAL.ED., 14, 271 (1942). (2) Bauernfeind, J. G., Sotier, A. L., and Boruff, C. S., Ibid., 666 (1942). (3) Brown, E. A , , Thomas, J. M., and Bina, A. F., Cereal Chem., 20, 201 (1943). (4) Cheldelin, V. H., Eppright, M. A., Snell, E. E., and Guirard, B. M., Univ. of Texas, Pubt. 4237, 26 (1942).
Clarke, M. F., Lechycka, M., and Light, A. E., J . Biol. Chem., 142, 957 (1942).
Cohen, S., Snyder, J. C., and Mueller, J. H., J. Bad.,41, 581 ( 1941).
l%i
+
Eckhsrdt. R. E., Gyorgy, P., and Johnson, L. V . , Proc. SOC. Ezptl. Biol. Med., 46, 405 (1941).
Feeney, R. E., and Strong, F. M., J. Bwl. Chem., 133, x u i (1940).
Ibid., Proc. xxxviii (1941).
Feeney. R. E., and Strong, F. M., J. B i d . Chem., 142, 961 (1942).
+
Light, A. E., and Clarke, M. F., Ibid., 147, 739 (1943). Neal, A. L., and Strong, F. M., IND.ENQ.CEIEM., ANAL.ED., 15, 654 (1943).
Pennington, D., SneIl, E. E., and Williams, R. J., J . B i d . Chem.,
It was concluded that no synthesis of biotin had taken place in the various tubes and flasks showing maximum acid production in the presence of rice oil and no added pure biotin. I t is possible that the bacteria had somehow adapted themselves to an environmeDt either biotin-free or containing ordinarily insufficient quantities of biotin. Work is being continued on this phase of the problem.
135, 213 (1940).
Scott, M. L., Randall, F. E., and Hesael, F. H., Ibid., 141, 325 (1941).
Shull, G. M . , Hutohings, B. L., and Peterson, W. H., Ibid., 142, 913 (1942).
Shull, G. M., and Peterson, W.H., Zbid., 151, 201 (1943). Snell. E. E.. and Strona. - F. M.. IND.ENO.CHEM..ANAL.ED.. 11, 346 (1939).
Snell, E. E., and Wright, L. D., J . Biol. Chem., 139, 675 (1941). Strong, F. M., and Carpenter, L. E., IND. ENQ.CHEM.,ANAL. ED., 14, 909 (1942). Strong, F. M., Feeney, R. E., and Earle, A., Ibid., 13,566 (1941). Wegner, M. I., Kernmerer, A. R., and Fraps, G. S., J . Biol.
SUMMARY
Growth stimulants in the microbiological biotin assay have
been investigated and methods for their removal devised. Various supplements for the basal medium were studied, but
Chem., 144, 731 (1942).
Williams, V. R., and Fieger, E. A., Cereal Chem., 21, 540
none has been found worthy of recommendation.
Color Test for
Oils and
Vol. 17, No. 2
(1944).
Resins, Using Hirschsohn Reagent for Cholesterol
HOWARD C. BRINKER,
Minnesota Mining and Manufacturing Company, St. Paul, Minn.
H E Hirschsohn reagent for cholesterol is useful &S a simple T c o i o r test for oil types. I t also gives color reactions with some resins. when using this reagent in making tests, it is good practice to have authentic samples for comparison with the unknown. Hirschsohn reagent for cholesterol (I), 9 grams of trichloroacetic acid and 1 cc. of distilled water.
PROCEDURE. Place 1 or 2 drops of an oil or a corr-ponding amount of resinous material on a porcelain spot plate, add 3 or 4 drops of the reagent, and wait a short time for the color to develop. As the color develops rather slowly at 20" c. or l-, the test may be speeded up by warming the spot plate to 35' C. LITERATURECITED (1)
Merck, Index, 5th ed.,p. 764, test 1835, 1940.
Table I. Color Test lor Oils Material Oils Tung Byntnenol Isoline Linseed Heavy-bodied linseed Corn (maiae) Neat's-foot SOY Cold pressed cantor Cottonseed Blown castor Fish Perilla Oiticica Tung 3, linseed 1 Tung 1 , iinawd 3 Tung 1, linseed 9
Color, 1 to 2 Minutes Gels, gel turns red Yellow Yellow Blue Dark brown Light blue Light ink Dirty.glue Practically no color Light purple Orange Dirty red Mauve Gels gel turns red Gels' gel turns brown red Gela: gel turns light red Red
Color, 5 Minutes
...
Brown Slight pink Blue purple
... Purple ...
Purplu
Medium purple Brown Blue Violet
... ...
...
Brown red
Reains -.
Material
Rosin
Ester gum Cyclohexanone formaldehyde Cumar Staybellite (hydrogenated rwin)
COlO?,
1 to 2 Minutes
Blue green Red B n h t red Lig%tred Light blue green
Varnish Ester gum, tun oil Eater gum, perilla
Dark red Brown
Dried varnish film Linseed type Dehydrated castor, type Tung type
Brown Light yellow Practically no color
Color. 5 Minutes Dark blue Purple
... . . ..
...
Purple
B r o w red
...
...
