THE following metabolic products, present in the metabolism solution of Penicillium griseo-fulvum Dierckx when this mould is grown under different cultural conditions and on different culture solutions, have been isolated in this laboratory: 6-hydroxy-2-methylbenzoic acid [Anslow & Raistrick, 1931], 2:5-dihydroxybenzoic acid (gentisic acid), fumaric acid and mannitol [Raistrick & Simonart, 1933] and fulvic acid, C14H1208, a yellow crystalline substance of at present undetermined molecular constitution [Oxford et al. 1935]. The purpose of the present communication is to record observations on a hitherto undescribed chlorine-containing metabolic product of P. griseo-fulvum which has been isolated from the mycelium of this mould and for which the name griseofulvin is proposed.Griseofulvin, C17H.706C, M.P. 218-219', is a colourless, crystalline, neutral compound giving no colour with FeCl3 and containing no free hydroxyl or carboxyl groups. It is highly dextrorotatory, [ac],61+ 417g.One oxygen atom is present as >CO since a crystalline mono-oxime, C,7H1806NC1, was readily obtained; derivatives are also formed with phenylhydrazine and with 2:4-dinitrophenylhydrazine, but these could not be obtained crystalline. The function of three other oxygen atoms is obvious since griseofulvin contains three methoxyl groups. A study of the products obtained on acid and alkaline hydrolysis showed that griseofulvin must be the methyl ester of a carboxylic acid, and hence the function of a fifth oxygen atom is accounted for. The function of the sixth oxygen atom has not been definitely settled though evidence given later indicates that it is present as an oxygen bridge.Griseofulvin, when hydrolysed with boiling N aqueous-alcoholic H2SO4, yields griseofulvic acid, C16H1506C1, [x]5461+ 5080, a monobasic acid containing two methoxyl groups and giving only a feeble colour with FeCl3, but these facts in themselves are not sufficient to establish the presence of the grouping -COO. CH3. However, hydrolysis of griseofulvin, or further hydrolysis of griseofulvic acid, with boiling aqueous N/2 NaOH yields norgriseofulvic acid, C15H130O6C1, [a]05M+609', a dibasic acid containing only one methoxyl group, together with decarboxygriseofulvic acid, C,5H1504C1, [x]e1 -310, an insoluble neutral compound containing two methoxyl groups, giving no colour with FeCl3, and derived from griseofulvic acid by the loss of 1 mol. of CO2. Decarboxygriseofulvic acid is stable to acid hydrolysis and hence it seems certain that griseofulvin contains only one -COO.COH3 group and that the second acidic ( 240 )

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SUMMARY:In a suitable buffer a t 39" the life of all three species of holotrich ciliates of the sheep's rumen can be extended for one or more days by addition of glucose, fructose, galactose, sucrose, cellobiose, raffinose, inulin, bacterial levan, salicin or nielibiose (the least effective). Mannose, glucosamine or galactosamine are definitely toxic in that they greatly shorten protozoan life. This toxic effect is observed even in the presence of glucose. Isotrichw prostoma and I . intestinalis, but not Dasytricha ruminantium, will ingest vegetable starch of small grain size, e.g. rice starch, thereby prolonging protozoan life.The nitrogenous requirements of these protozoa are best met by whole grass juice, which extends the life of the culture for several weeks; and even the ethanolic precipitate from boiled and cleared grass juice is a better nitrogenous supplement than cleared rumen liquor. The ash from this alcoholic precipitate will definitely extend protozoan life in absence of nitrogenous supplement. The effect is probably due to a 'trace' metal, which is not Zn, Fe, Sn, Sr, Mn, Cu or Ni, but may be Ti, Mo, Cr, co or v.The holotrich ciliates here studied do not ingest lactobacilli, either when starved or when in an actively motile state after addition of glucose to the medium.

SUMMARY: When holotrich ciliates of hay-fed sheep's rumen acted upon watersoluble carbohydrates under conditions comparable with those in the m e n save for the absence of appreciable bacterial competition, only glucose, fructose, sucrose, inulin, bacterial levan (from Bacillus megatherium) and to a lesser extent cellobiose were utilized for rapid and extensive storage of iodophilic polysaccharide granules (cf. Oxford, 1951). No other soluble carbohydrate tried (including maltose) was so utilized. When polysaccharide storage did take place the product always gave a purple colour with iodine, and had the properties of a starch rather than glycogen in that it gave an insoluble iodine complex under the conditions of Pucher, Leavenworth & Vickery (1948). Storage of starch by oligotrich ciliates was much slower than with holotrichs, and did not take place with cellobiose as substrate. Holotrich ciliates continued to replace themselves in the sheep's rumen even when large volumes of m e n contents were periodically withdrawn over a long period. The protozoan starch could be isolated in about 1 % yield by application of the Pucher et al. (1948) method direct to the dry matter of strained m e n liquor taken from sheep which had grazed on starch-free Spring grass.The ciliate literature contains numerous references to the storage of polysaccharides variously termed glycogen, starch, paraglycogen, paramylum, etc. (cf. Doyle, 1943; Kudo, 1947), but these iodine-staining inclusions have never been isolated in sufficient quantity to allow proper chemical investigation of them. The observation made previously (Oxford, 1951) that the holotrich ciliates of sheep fed on a starch-free diet (hay) stored relatively enormous numbers of tiny iodophilic polysaccharide granules when glucose, for example, was fermented in vitro by muslin-strained lumen liquor, has now been utilized in the preparation of a large specimen (more than 50 g.) of this polysaccharide in a relatively pure state in order that such a chemical investigation might be made, beginning with a decision between the starch and glycogen possibilities.In these in vitro fermentations the protozoa were, of course, competing with bacteria for the available sugar, and the yield of protozoan starch was never more than 5-10 % of the total sugar fermented. Attempts were, therefore, made to use an inoculum consisting mainly of protozoa, not only to minimize bacterial interference in order to obtain larger overall yields of protozoan starch, but also to find out which carbohydrates other than glucose, fructose and sucrose can be converted into starch by the protozoa acting alone. Owing to the difficulties of large-scale isolation of protozoa from lumen liquor without impairment of their fermentative powers, the first objective (greater overall yield) has not been attained, but the second, which obviously does not depend on the isolation of the greater part of the protozoa