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0 5m-phosphate buffer (pH 6 0), 1 ml. 1% (v/v) H202 and 15 ml. water were warmed to 200 in a beaker. An aqueous suspension (1 ml.) of the ciliate, which had been twice frozen and thawed to disrupt the cells, was added, and the mix:ture incubated at 200 for 5 min.; the reaction was stoppedbytheadditionofl ml.2 N-H2SO4. As much protein was present, and interfered with the extraction of purpurogallin, 1 ml. 100 % (w/v) trichloroacetic acid was added, and the mixture centrifuged. The supernatant was extracted twice with ether, and the extract diluted to 25 ml, with ether. Purpurogallin was estimated photoelectrically in the 31-2 483

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A number of Protozoa synthesize intracellular carbohydrate, described as glycogen on account of the brown colour given with iodine; this polysaccharide may be diffusely distributed throughout the cell, or localized in 'glycogen vacuoles'. There do not seem to have been any investigations of the chemical nature of protozoal glycogen, nor of its physiological function. Recently, however, an opportunity for such an investigation arose from the observation, briefly reported in the preceding paper of this series (Ryley, 1951), that the protozoan Glaucoma piriformis may have an intracellular glycogen content as high as 22 %. This protozoan will be referred to in the present study as Tetrahymena pyriformis in accordance with the new nomenclature advocated by Corliss (1952). The protozoal glycogen has been isolated in pure form and subjected to a detailed analysis. MATERIALS AND METHODS Ciliate preparations. Tetrahymena pyriformi8 was cultivated in a peptone medium as described in the following paper (Ryley, 1952). It was found that the peptone used in the preparation of the medium contained 0.44 % of a KOHresistant, ethanol-precipitable, glycogen-like material. Cells were harvested after 6 or 12 days growth, using a small angle centrifuge, or, in the case of larger preparations, the Sharples supercentrifuge. Analytical methods. Total nitrogen was determined by digestion with H2SO4, using SeO2 as catalyst. After digestion, ammonia was distilled in vacuo in the Parnas-Heller apparatus (Parnas & Heller, 1924), and estimated colorimetrically with Nessler's reagent. Phosphorus was estimated by the method of Fiske & Subbarow (1925), after digestion of a sample with 60 % HCl04 and a little HNO3. Glycogen was estimated by the method of Good, Kramer & Somogyi (1933). Reducing sugar was estimated iodometrically by the method of Somogyi (1945), while glucose was determined as the loss in reducing value of a sample after treatment with glucose oxidase and catalase (Mann, 1946). The glycogen end-group assay was carried out by the method of KIb4 oxidation developed by Halsall, Hirst & Jones (1947), as modified by Bell & Manners (1952). fi-Amylolysis ofglycogen was effected by treating the glycogen * Present address: Imperial Chemical (Pharmaceuticals) Ltd., Biological Laboratories, Morley, Wilmslow, Manchester.