The methyl green-pyronin technique has become a popular method in histochemistrysince Brachet's application for differential stain of desoxyriboncleic and ribonucleic acidsin 1940. Due to variation in concentration of different brands of pyronin and variationin stainability of different tissues, one often has to spend considerable dyes before theirright proportion is obtained. In the experiment under discussion the results were unsatis-factory when stained in the stock solutions of methyl green (1% E. Merk, in distilledwater containing 0.25% phenol or in 0.1 M sodium acetate buffer, pH 4.1) and pyronin(1%, National Aniline Dye Co., in the same medium as for methyl green) either separatelyor in a mixture in various proportions. On the other hand, it was found to be much easierto handle in a diluted mixture. To each 40 cc. of distilled water or 0.1 M acetate buffersolution, pH 4.1, was added 20-25 drops of the methyl green and pyronin stock solutionsrespectively, stain overnight, blot and destain in pure acetone for 15-30 seconds. The timeshould be shortened when sections were thinner than 10 ?. The destaining could bemuch faster when the acetone absorbed atmosphoric moisture. To overcome the difficulty of obtaining complete dehydration, it was found thatimmersing sections either in dioxan or castor oil before in xylol gave good results. Therate of destaining in acetont could be lowered by adding 1/3 of either dioxan or castoroil into it.

The limb buds of mouse embryos from 10-17 days were used for the study on the distribution of acid and alkaline phosphatases according to MacDonald's modification of lead nitrate and Danielli's modification of calcium cobalt methods respectively. McManus' modification of Hotchkiss' PAS was used for the demonstration of glycogen with control sections stained after diastase digestion. A modified diluted methyl green-pyronine method was used for the stain of nuclei acids. The reaction of both alkaline and acid phosphatases was weak in the limb buds of 2-3 mm embryos when they began to bulge out from the somatopleure. The reaction became stronger when the apical ectodermal thickening process in the limb buds of 3-4 mm embryos began. It increased further when the thickening reached its full development in 5-7 mm embryos. Since then the enzymes were weakened together with the thinning of the thickening. The ectoderm, particularly that in the shoulder region, had more glycogen granules than that of the apical ectodermal thickening. The ribonucleic acid took deeper stain in the thickening than that of the general ectodermal covering due to vacuolization of the latter. There were pyknotic nuclear fragments of different sizes in the thickening, more frequently in the regressional ones. They were considered to be in the process of autolysis. The staining reaction of phosphatases and ribonucleic acids was increased in the mesodermal cells around the cartilage primordia and in the premuscle masses. It was even more prominent when the perichondrial fibroblasts and myoblasts were in process of elongation. During chondrification, the phos- phatases and ribonucleic acid began to increase in precartilage stage, reached their maximum in the chondrocytes before hypertrophy and became decreas- sed in the hypertrophied cells. The glycogen granules in the fully developed muscle fibers were about the same as that of the myoblasts, but the phos- phatases and nucleic acid were decreased in the former. In general the changes were prominent in phosphatases than in ribonucleic acid and gly- cogen. The above phenomena lead one to correlate the increase of phosphatases and nucleic acid with embryonic differentiation. The increase of glycogen content may or may not follow the increase of either the phosphatases or ribonucleic acid. Their significance in embryonic development was discussed.

The apical ectodermal thickening of the limb buds of mouse embryos, 11-day vaginalplug age, and epithelial cells of the villi of small intestine, 19-day mouse fetus, wereused for this observation. The limb bud ectoderm was fixed in 1% osmic acid andstained in 1% Pb(OH)_3, while the small intestine was fixed in 1.2% potassium per-magnate. There were transitional stages from minute dense vesicles of 0.01--0.07? with afew vague striations, gradually growing up into vesicles of 0.07--0.10? with definitemitochondrial cristae, and finally into organelles that could be definitely identified assmall mitochondria of 0.13?0.20? in size, They were neither fragments of degenera-ting mitochondria nor lysosomes, since their structures became more and more similar tothat of the mitochondria with their increase in size. These minute dense vesicles werefound among clusters of ribosomes, at first vaguely outlined and then with definite mem-branes and dense substance. The significance of ribosomes in relation to synthesis of themembranous protein and the matrix of the mitochondria was discussed. In the course of the formation of granulated endoplasmic reticulum, diffuse cyto-plasmic ribosomes at first arranged themselves into circular or tubular clusters. Theseclusters then transformed themselves into membrane bounded tubules and vesicles. Byfurther fusion and extension, definite granulated endoplasmic reticula were formed.Close relationship between mitochondrium, granulated endoplasmic reticulum and Golgibody has been observed but there showed no evidence of developmental significance.The granulated endoplasmic reticulum was thus considered to be formed by ribosomes.

The duodenal absorptive cells were studied in 20-day mouse fetuses as well as innew born mice after fasting for 8 hours or after resuckling following 12-hour fasting.Specimens were mainly fixed in veronal buffered 1% osmic acid, pH 7.4, at 0-4℃,sectioned in Niklowitz microtome and photographed under SEM Ⅲ electron microscope.Potassium permagnate was occasionally used as an alternative fixative. In the epithelial cells of the fetal intestine, the microvilli of the striated borderwere shorter and looser than those of the new born animal. Invaginations between thebases of the microvilli as well as the pinocytic vesicles in the terminal web had appeared.There were occasional connections between these vesicles. Below the terminal web, therewere oval mitochondria with radiating cristae. After birth they changed to rod-shape withoblique cristae. A small yet typical Golgi complex consisting of tubules and vesicles ofsmall and medium sizes but without lipoid droplet, could be located in the supranuclerregion. The fetal cells contained more ribosomes and granulated endoplasmic reticulathan those in the newborn. The invaginations and pinocytic vesicles in the terminal web of the newborn micewere found to be somewhat more prominent than those in the fetus. Interconnectionsbetween invagination and vesicles and coalescence of vesicles were present. Besides the pinocytic vesicles there were smooth surfaced tubular structures both in the terminal weband endoplasm. No lipoid droplet was present in or between the microvilli nor in thevesicles of the terminal web and endoplasmic reticulum. On the other hand there werecloudy patches in some of the vesicles. In some of the larger vesicles, particularly inthose near the inner region of the apical endoplasm, the cloudy patches changed into fatdroplets of medial density. There were a kind of small granules in the groud substanceof the terminal web and endoplasm between the endoplasmic reticula and mitochondria,where they were arranged in linear groups, looking as if they were moving inward. In animals fasted for 8 hours, the absorptive epithelial cell failed to show muchchange in apical invaginations and the pinocytic vesicles. The smooth-surfaced endo-plasmic reticulum contained cloudy patches. The Golgi complex and intercellular spacewere filled with large fat droplets. In certain specimens, there appeared one kind oflipoid substance, much smaller and denser than fat droplets, in the apical smooth surfacedendoplasmic reticulum and Golgi complex. In resuckling specimens the pinocytic and apical endoplasmic vesicles were in generalvoid of dense substance, but with occasional cloudy patches. There were also specimenscontaining lipoid droplets in their vesicles, Golgi complex and intercellular space. It isinteresting to note that some cells might have their apical vesicles filled with homogenoussubstance of medium density which gradually rounded up into small lipoid masses in theinner region. This change together with the presence of the above-mentioned cloudy sub-stance in the outer and fat droplet in the inner vesicles of the cytoplasm were con-sidered to be the evidences of gradual formation of fat droplet by resynthesizing thehydrolyzed fat in the vesicles. The fat drops were then transferred into the inter-cellular space by the Golgi complex and smooth surfaced endoplasmic reticulum. Thecytoplasm of the resuckling intestinal was epithelium particularly rich in smooth endo-plasmic vesicles. Part of them contained fat droplets. Ribosomes and granulated endoplasmic reticulum were of common occurence. Lyso-somes were occasionally found both in the fetal and new born intestine. The structureof the terminal bar and the interdigitation of the lateral cell membranes were described.