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The alveoli of the mammalian lung are lined by a film of surface-active material (surfactant) which is commonly believed to be a phospholipid (1,5,8,11) . A considerable body of evidence has accumulated which suggests that surfactant is synthesized by the granular pneumonocytes (type II cells) of the alveolar epithelium (1, 9, 10) . Although a number of acid hydrolases have been demonstrated in the inclusion bodies of granular pneumonocytes (4, 6), no attempt has been made to localize enzymes specifically concerned with phospholipid metabolism . Phosphatidic acid phosphatase is of particular interest because of its key role in the biosynthesis of phosphatides . Unfortunately, this enzyme cannot be demonstrated by conventional histochemical methods because of the insolubility of the substrate (phosphatidic acid) in water . Stable emulsions of phosphatidic acid can be prepared but these do not diffuse into cells . One method of overcoming this difficulty is to inflict minor localized damage on cells and expose the organelles to an emulsion of phosphatidic acid in a suitable medium .A method of producing minor trauma by osmotic forces has been developed to enable the localiza-THE JOURNAL OF CELL BIOLOGY . VOLUME 53, 1972 . pages 249-252 tion of phosphatidic acid phosphatase to be studied in granular pneumonocytes . MATERIALS AND METHODSYoung adult hamsters of 150-200 g body weight were killed by cervical dislocation, and small blocks of lung tissue were excised and fixed for 2 hr in ice-cold 3% glutaraldehyde in 0 .1 M cacodylate buffer (pH 7 .2) containing 7 .5% sucrose . After fixation the blocks were washed for 20 min in 0 .1 M cacodylate buffer (pH 7 .2) containing 7 .5% sucrose, and then 0 .5 mm slices were cut freehand. The tissue slices were incubated at 37°C for 15-90 min in a freshly prepared medium containing phosphatidic acid as substrate . The medium consisted of 3 ml of 0.1 M acetate buffer (pH 5 .1) containing 3% sucrose, 5 ml of distilled water, 1 ml of 5% lead nitrate solution, and 1 ml of 2% phosphatidic acid (Sigma London Chemical Co ., London, England) emulsion prepared by the method of Clark and Hiibscher (2) . The medium was preheated to 37°C before use . Control sections were incubated in substrate-free media or in complete media containing 0.01 M sodium fluoride . After incubation the tissues were washed briefly in cacodylate buffer, postfixed in cacodylate bufferOs04 (1%), dehydrated in a series of graded alcohols, and processed through propylene oxide to Araldite CY212 . Ultrathin sections were examined

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The respiratory epithelium in the lungs of the newt Triturus cristatus has been studied by electron microscopy. T h e entire pulmonary gas-exchange area is covered by a continuous epithelium, the cells of which are all of the same type and are termed "pneumonocytes". Typically each pneumonocyte is squamous and has attenuated sheets of cytoplasm which cover the pulmonary capillaries. Its free surface bears microvilli while mitochondria, multivesticular bodies and small inclusions are prominent in its cytoplasm. Many pneuomonocytes send cytoplasmic processes deep into the substance of the lung wall. I t is postulated that these processes may help to anchor the epithelium.

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