Experiments on fixation for electron microscopy 2. Alkalized osmium tetroxide. By S. K. MALHOTRA

Transcription

1 28? Experiments on fixation for electron microscopy 2. Alkalized osmium tetroxide By S. K. MALHOTRA (From the Cytological Laboratory, Department of Zoology, University Museum, Oxford) With 6 plates (figs. I to 6) Summary The effect of fixation with osmium tetroxide solution, made alkaline by the addition of potassium acetate, was studied by electron microscopy. Exocrine cells of the pancreas and the cells of the first ('proximal') convoluted tubules of the kidney of the mouse were used as test-objects. Partially prepolymerized methacrylate was used for embedding. The preservation of the various cell inclusions was similar to what is generally produced after fixation with Palade's buffered osmium tetroxide. Introduction OSMIUM tetroxide has come to take a predominant place as a fixative for studies of biological materials by electron microscopy. It is considered as the most suitable substance for preservation of the whole of the cellular constituents. Potassium permanganate, which is nowadays rather widely used for studies of membranous structures (Robertson, 1959, i960; Mollenhauer, 1959; Zebrun and Mollenhauer, i960), removes ribonucleic acid and histones from the tissue (Luft, 1956; Bradbury and Meek, i960). Osmium tetroxide is mostly used in buffered solutions, because it has been suggested that the quality of fixation by osmium tetroxide is largely dependent on the concentration of hydrogen ions. It is believed that when tissues are fixed in an unbuffered solution of osmium tetroxide, a wave of acidity precedes the osmium into the tissue, and that the structure of cells is best stabilized if the fixing solution is buffered slightly on the alkaline side of neutrality (Palade, 1952a). Palade recommended the use of Michaelis's (1931) sodium veronal/ sodium acetate buffer (without sodium chloride) for this purpose, probably because it is not harmful to most cells. Palade experimented with phosphate buffer at the same ph as the veronal/acetate buffer, but found it less satisfactory. Dalton (1955) used a chromate buffer (K 2 Cr /K0H) in osmium tetroxide solution for fixation at ph 7-2. He considered that his fixing solution had some advantages over Palade's fluid. However, there is some evidence that the quality of preservation is not necessarily determined by the concentration of the hydrogen ions in fixing solutions (Malhotra, 1962). Sjostrand and his associates (see Sjostrand, 1956) have obtained comparable preservation of a wide variety of animal cells by [Quart. J. micr. Sci., Vol. 103, pt. 3, pp , 1962.] X

2 288 Malhotra Experiments on fixation for electron microscopy using unbuffered osmium tetroxide made isotonic by the addition of indifferent salts. They regard the tonicity of the fixing fluids as of vital importance for ideal preservation of cell structures. It may be remarked that Palade's fluid is greatly hypotonic to the body-fluids of mammals. I have elsewhere stated (1962) that I am studying the effect on fixation of the osmotic pressure due to those non-fixative ions that are commonly added to fixing solutions for electron microscopy; and it is intended to complete this work in a series of papers. In the first paper of this series I have described the results of fixation with a simple solution of osmium tetroxide in distilled water. In the present paper, the effect on fixation by an alkalized solution of osmium tetroxide is described. Material and method The exocrine cell of the pancreas and the cells of the first ('proximal') convoluted tubule of the kidney of the mouse have been used as test-objects for this series of experiments on fixation. The reasons for the selection of these cells have been given in the first paper of the series. A 1% solution of osmium tetroxide in 'deionized' water (see Malhotra, 1962), made alkaline by addition of potassium acetate (ph 7-3 to 7*5), was used as fixative. Potassium acetate is hygroscopic, and therefore a small amount from the stock supplied by the manufacturer was dried for several days over phosphorus pentoxide in a vacuum desiccator before use. The ph of a 0-22% solution of the dried salt varies between 7-3 and 7-5, probably because of impurities in the salt; 'Analar' grade salt is not available. A stock solution of the salt was prepared at 0-44% and mixed with an equal volume of a 2% solution of osmium tetroxide. The mixture was used for fixation. The depression of the freezing-point of this fixing solution, as determined by the use of a Beckman thermometer, varies between and C. The depression of the freezing-point due to osmium tetroxide alone in this fixing solution varies between an( i 0-06 C. The material was also fixed in a 1 % solution of osmium tetroxide that had been made alkaline by the addition of potassium acetate at ph 7-2 to 7-3, but at lower concentration than 0-22%. This was done by considerably diluting the stock solution of potassium acetate and noting its ph. The diluted salt solution was mixed with an equal volume of 2% osmium tetroxide. The depression of the freezing-point of this solution was 0-08 C. The tissue was fixed, dehydrated, and embedded according to the procedure standardized for fixation with the unbuffered solution of osmium tetroxide, so that the results could be closely compared. The tissue was FIG. 1 (plate). Micrograph showing the general appearance of the exocrine cells of the pancreas fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate at ph 73 to 7"S)- Parts of three cells appear in the micrograph, cm, limiting cell membrane; er, endoplasmic reticulum; v, vacuoles found in association with the non-granular membranes (y-cytomembranes); m, mitochondria; n, nucleus; z, zymogen granules.

3 FIG. I S. K. MALHOTRA

4 FlC. 2 S. K. MAI,HOTRA

5 Malhotra Experiments on fixation for electron microscopy 289 fixed for a short time (less than an hour) in a solution previously cooled to just above freezing-point. It was then dehydrated through a graded series of ethanols and embedded in partially prepolymerized w-butyl methacrylate. A reference may be made to the first paper of this series (Malhotra, 1962) for details of the preparative technique. Sections were cut on the Huxley ultramicrotome manufactured by the Cambridge Instrument Co. Ultrathin sections were mounted on grids coated with formvar, and examined in the Akashi 'tronscope' (model TRS 50 Ei) operated at 50 kv, with anode aperture of 100 fx and objective aperture of 50 or 100 fx. Results It is the purpose of this paper simply to record the general appearance of the various cell structures seen after fixation with alkalized osmium tetroxide, according to the experimental technique, and to compare this appearance with the results produced by the unbuffered and unbalanced solution of osmium tetroxide (Malhotra, 1962). It is also intended to compare these results with what is already known about the exocrine cell of the pancreas and the cells of the first convoluted tubules of the kidney. The appearance of the various cell inclusions is essentially similar with lower or higher concentrations of potassium acetate used to alkalize the fixative, and it will therefore not be necessary to give separate accounts of the results. Exocrine cell of the pancreas A general survey of the various inclusions of this cell fixed with alkalized osmium tetroxide solution shows that the addition of potassium acetate has not produced any gross alterations suggestive of poor preservation. Fig. 1 shows the general quality of fixation. It would appear that the cell inclusions are satisfactorily preserved, and there is no difficulty in recognizing their identity. The nuclear contents are more or less evenly dispersed and appear finely granular. The nuclear membrane does not seem to be disrupted and is sharply delimited. The two nuclear membranes are closely apposed to each other (fig. 2, A). The maximum thickness of the nuclear membrane is about FIG. 2 (plate). A, from exocrine cell of the pancreas fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate at ph 7-3 to 7-5); showing the endoplasmic reticulum and ribonucleoprotein granules in the basal region. The arrow indicates a pore in the nuclear membrane (nm). A mitochondrion is also seen in the middle of the field of view. B, from exocrine cell of the pancreas fixed by osmium tetroxide (alkalized with lower concentration of potassium acetate at ph 7-2 to 7 P 3); showing a part of the apical region. er, endoplasmic reticulum with its associated ribonucleoprotein particles; g, non-granular paired membranes (y-cytomembranes) in association with large vacuoles and small vesicles; 2 X and^2 are probably different stages of condensation of protein in the vacuoles leading to the formation of zymogen granules (z).

6 290 Malhotra Experiments on fixation for electron microscopy the same as after fixation with unbuffered osmium tetroxide, about 60 m/z. The small dense particles, of the size of the ribonucleoprotein granules, often seen on the outer nuclear membrane, are easily seen in the micrographs (fig. 2, A). The nuclear pores (fig. 2, A) are quite evident and similar to those seen after fixation with buffered (Watson, 1955, 1959) or unbuffered (Malhotra, 1962) osmium tetroxide. The characteristic ultrastructure of the mitochondria seems to be remarkably well preserved, and there is no evidence of any marked swelling due to the hypotonic nature of the fixing solution used. The limiting double membrane and the cristae are clearly seen. The mitochondrial matrix appears almost structureless in the micrographs (figs. 2, A; 3). The endoplasmic reticulum is also very well shown. Its form and distribution throughout the greater part of the cytoplasm do not suggest any noticeable disorganization (figs. 1; 2, A). The membranes bounding the elements of the endoplasmic reticulum are well defined. The ribonucleoprotein particles are well preserved (figs. 2; 3). Their distribution on the surfaces of the cisternae of the endoplasmic reticulum at the base of the cell is clearly depicted in the micrographs (fig. 2, A). The highly organized membranous complex, consisting of y-cytomembranes (paired non-granular membranes), vacuoles, and small vesicles (or granules), generally present in cells that actively secrete protein, is clearly seen (figs. 2, B; 3). This complex is commonly referred to as the Golgi apparatus, after its first description by Dal ton and Felix (1954) in the epithelial cells of the mammalian epididymis (see Dalton, 1961, for details). The structure and appearance of this complex, and of the zymogen granules, is essentially similar to that described after fixation with unbuffered osmium tetroxide (Malhotra, 1962), and no additional remarks seem to be necessary. Some of the vacuoles associated with the non-granular membranous complex appear more or less empty, while others have contents of higher electron density (fig. 2, B). This appearance has been interpreted as suggestive of a gradual condensation of protein in these vacuoles, leading to the formation of zymogen granules (see Palay, 1958; Hirsch, 1961; Palade, 1961; Malhotra, 1962). The vacuoles associated with the non-granular membranous complex have sometimes been seen in close proximity to the elements of the endoplasmic reticulum (fig. 3). This appearance has been interpreted as suggestive of an organic association of the endoplasmic reticulum with the non-granular membranous complex (Palade and Siekevitz, 1956). The boundaries between each cell and the next are easily recognized. They are represented by thin, sharply demarcated, dense lines in the micrographs (fig- 1). (alk FIG. 3 (plate). Apical region of exocrine ceil of the pancreas fixed by osmium tetroxide ^alkalized with higher concentration of potassium acetate at ph 7-3 to 7-5). Endoplasmic reticulum (er) and ribonucleoprotein particles are seen throughout the cytoplasm. The arrow indicates a close contact between the endoplasmic reticulum (er) and a vacuole belonging to the non-granular membranous complex (g), m, mitochondria; z, zymogen granules.

7 FIG. 3 S. K. MALHOTRA

8 Fie. 4 S. K. MALHOTRA

9 Malhotra Experiments on fixation for electron microscopy 291 Cells of the convoluted tubules of the kidney The general preservation of the various structures of these cells is essentially similar to what is seen after fixation by unbuffered (Malhotra, 1962) or buffered (Sjostrand and Rhodin, 1953; Rhodin, 1954; Pease, 1955 a, b) osmium tetroxide. The micrographs clearly show this resemblance. The nuclear contents are fairly and uniformly granular (figs. 4; 6, B). The nuclear membrane is sharply delimited and seems to be well preserved. Its double nature is clearly retained, and the two membranes are not pulled apart. The inner nuclear membrane often appears thicker than the outer (fig. 4), as described by Porter (1961). The characteristic infoldings of the cell membrane into the cytoplasm at the basal end of the cell are very well preserved (figs. 4; 5, A). The membranes of the infoldings are clearly demarcated and appear as thin, dense lines. The two membranes of an infolding (j3-cytomembranes of Sjostrand, 1956, 1959) are regularly disposed, parallel to each other, and more or less parallel to the major axis of the cell. They are also closely apposed to each other, about 15 m/x to 20 m/u. apart (see Rhodin, 1954; Porter, 1959; Forster, 1961). In micrographs at high resolution, each of the thin, dense lines is seen to be composed of two very thin, dark lines, separated by a thin, pale line (fig. 5, B). Rough estimates of the total thickness of this tripartite membranous complex give a value between 7 mju. and somewhat less than 9 m/x; that is to say, about the thickness of the 'unit membrane' of Robertson (1959, i960). Porter (1959) has given 8 ntyi as the thickness of this tripartite complex of the infoldings of the tubules of the kidney, as compared to 6 m/x given by Rhodin (1954). The tripartite complex is not so well seen after fixation in osmium tetroxide as after potassium permanganate (Robertson, 1959, i960). The double membranes of the infoldings are formed by invaginations of the cell membrane (Rhodin, 1954; Pease, 19556; see Porter, 1959; Sjostrand, 1959). The cell membrane at the base of the cell is seen as a single dark line in fig. 6, A. The mitochondria do not show any obvious defects of preservation (figs. 4; 5, A; 6, c): their characteristic cristae and outer double membrane are clearly shown, and the cristae are densely packed and arranged in a regular fashion, especially in the mitochondria towards the base of the cell. In this situation the mitochondria are known to be disposed more or less perpendicularly to the base of the cell (Sjostrand and Rhodin, 1953; Rhodin, 1954). This arrangement is quite evident in the micrographs (fig. 4). In the micrographs at high resolution, each of the outer double membranes and also the membranes of the cristae have been seen to be tripartite, but the images are not quite clear enough for reproduction in print. These observations, and the FIG. 4 (plate). Micrograph showing basal region of a cell from the first convoluted tubule of the kidney fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate at ph 7-3 to 7-5). cm, infoldings of the cell membrane; er, endoplasmic reticulum; I, dense bodies, which may be lipid globules; m, mitochondria; n, nucleus. Small particles of the size of ribonucleoprotein granules are scattered about in the cytoplasm.

10 292 Malhotra Experiments on fixation for electron microscopy demonstration of the tripartite nature of the membranes of the infoldings, suggest that alkalized osmium tetroxide reveals essentially the same structure as is discernible after fixation with buffered osmium tetroxide. The cylindrical processes of the cytoplasm, forming the brush border at the apical end of the cell (Rhodin, 1954; Pease, 1955 a, b), are of fairly uniform diameter. Rhodin gives 50 mju, to 70 m/x for the diameter of these processes. In my micrographs they are mostly closely packed (fig. 6, c, D). It has been pointed out that this close packing is quite characteristic of the material that has been removed from the animal in small pieces and subsequently immersed in the fixing solution. In tissue fixed in situ the cytoplasmic processes are wide apart (Pease, 19556). Pease thinks that the apical region of this cell is particularly sensitive to osmotic changes and becomes swollen during fixation. At the base of the brush border, vacuoles of various sizes appear scattered about in the cytoplasm. They are delimited by a membrane, which is seen as a thin, dense line in the micrographs (fig. 6, c). Pease (1955 a, b) has demonstrated that these vacuoles are connected with small ducts (fig. 6, c), situated between the cytoplasmic processes of the brush border. Novikoff (1961) has also described similar ducts as canalicular structures. Small, dense granules of the size of ribonucleoprotein granules (Pease, 19556) are scattered throughout the cytoplasm. Some of these granules are associated with elements of the endoplasmic reticulum, as in figs. 4 and 5. Fig. 6, B shows what appears to be a continuation of the endoplasmic reticulum into the outer nuclear membrane (see Porter, 1959,1961 for references). y-cytomembranes (Golgi apparatus of Rhodin, 1954; Forster, 1961) have very seldom been seen in my micrographs of the cells of the first convoluted tubules. They appear to be confined to the cytoplasm in the vicinity of the nucleus (fig. 6, c). It would appear that their preservation is satisfactory. The large vacuoles and granules (or small vesicles) that are usually associated with y-cytomembranes in a wide variety of cells have not been encountered in my micrographs of these cells of the kidney. In addition to the inclusions described above, there are nearly spherical bodies, about 0-14 /x to 0-2 p in diameter, dispersed in the cytoplasm at the basal end of the cell. In the micrographs they appear as homogeneously dense structures (fig. 4). They may perhaps be minute lipid globules. The basement membrane that surrounds the tubules appears to be rather uniformly dense in the micrographs (fig. 6, A). The appearance is the same as after fixation by unbuffered osmium tetroxide. FIG. 5 (plate). From a cell of the first convoluted tubule of the kidney fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate at ph 7'3 to 7"s). A, showing infoldings [cm) of the cell membrane into the cytoplasm disposed more or less parallel to the mitochondria (m). er, endoplasmic reticulum in association with ribonucleoprotein particles. B, magnified view of a part of fig. 5, A, showing tripartite nature of the double membranes of the infoldings. Arrows indicate where the tripartite appearance is easily seen, m, mitochondrion.

11 FIG. S S. K. MALHOTRA

12 FIG. 6 S. K. MALHOTRA

13 Malhotra Experiments on fixation for electron microscopy 293 Discussion Solutions of osmium tetroxide used in fixation for electron microscopy are usually held at a particular ph by an elaborate buffer (see Palade, 1952a; Pease, i960; Glauert, 1961). The exocrine cell of the pancreas and the cells of the first convoluted tubules of the kidney look the same in electron micrographs whether a carefully buffered solution is used (see Palade, 1952a, 1961; Palade and Siekevitz, 1956; Sjostrand, 1956; Sjostrand and Hanzon, 1954 a, b; Bradbury and Meek, i960 for the pancreatic cell; and Sjostrand and Rhodin, 1953; Rhodin, 1954; Pease, 1955 a, b for the kidney cell) or whether, on the contrary, the osmium tetroxide solution is simply slightly alkalized by a small addition of potassium acetate. It therefore seems that a solution alkalized in this way might well be substituted for buffered solutions in the study of other cells. However, the various inclusions of the cells discussed in this paper, when fixed by alkalized osmium tetroxide, are very similar in their appearance to cells fixed by unbuffered osmium tetroxide, without any addition of salts (Malhotra, 1962). It therefore seems doubtful whether it is necessarily advantageous to use buffered or alkalized solutions of osmium tetroxide in fixation for electron microscopy. Sjostrand and Rhodin (see Sjostrand, 1956) have achieved comparable preservation of the cell structures by fixing in unbuffered osmium tetroxide solutions that had been made isotonic by the addition of indifferent salts. Rhodin (1954) fixed first convoluted tubules of the kidney of the mouse with solutions of osmium tetroxide varying in ph from 4-5 to 7-9. He did not observe any marked difference in the general appearance of the cells, but he demonstrated slight swelling of the double membranes (especially of mitochondria) in the acidic fixing solutions. It is noteworthy that Mollenhauer (1959) obtained 'particularly good preservation' of the cells of the root tips of Zea mays after fixation with unbuffered potassium permanganate (at 2 to 5 %) and embedding in epoxy resin. Zebrun and Mollenhauer (i960) used also unbuffered (in addition to buffered) potassium permanganate and embedding in araldite for study of rat testes by electron microscopy. FIG. 6 (plate). From cells of the first convoluted tubules of the kidney. A, fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate at ph 7-3 to vs). bm, basement membrane; cm, cell membrane represented by a single dense line at the base of the cell. B, fixed by osmium tetroxide (alkalized with lower concentration of potassium acetate at ph 7-2 to 7'3). The arrow indicates what appears to be a continuity of the endoplasmic reticulum (er) into the outer nuclear membrane (nm), m, mitochondrion; n, nucleus. C, fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate at ph 7"3 to 7'S); showing the general appearance of the apical zone of the cell, bb, brush border; d, ducts which Pease (1955a) has shown to be connected with the vacuoles (v); g, non-granular membranes (y-cytomembranes); m, mitochondria. D, fixed in osmium tetroxide (alkalized with lower concentration of potassium acetate at ph 7-2 to 7'3); showing the microvilli of the brush border (bb) sectioned longitudinally (on the left) and transversely (on the right).

14 294 Malhotra Experiments on fixation for electron microscopy Tissue fixed in potassium permanganate (Luft, 1956) 'seems unduly sensitive' to damage during polymerization of methacrylates (Pease, i960). Tissue fixed in formaldehyde solution also is often badly damaged when methacrylates polymerize, but if fixation in formaldehyde is followed by fixation in buffered osmium tetroxide the damage is much less (see Glauert, 1961). It has also been reported that the preservation of the ground substance of the cytoplasm is better if fixation with permanganate is followed by fixation by formaldehyde (see Glauert, 1961). Tissue fixed in Dalton's (1955) chrome-osmium fixative has been found to suffer less polymerization damage than after fixation in Palade's buffered osmium tetroxide fluid. It seems possible that tissue fixed in an unbuffered and unbalanced solution of osmium tetroxide may undergo disorganization of the cell inclusions during polymerization of monomer methacrylates, but the damage may be much less if a buffered solution of osmium tetroxide is used. Tissues do not suffer this damage if partially prepolymerized methacrylate is used. Hypotonic solutions may cause swelling of cell inclusions. Sjostrand and his associates (see Rhodin, 1954; Sjostrand, 1956; Pease, i960) have therefore emphasized the importance of using isotonic fixing solutions. Both the alkalized and the simple unalkalized solutions of osmium tetroxide that I have so far used for experiments on fixation are greatly hypotonic to the bodyfluids of mammals, but they do not appear to show any obviously deleterious effect on preservation. Palade's fluid, which is so widely used in its original form (Palade, 1952a) in electron microscopy, is also hypotonic to the bodyfluids of mammals. Palade (1952&) added enough sucrose to his buffered osmium tetroxide solution to bring the total osmotic pressure up to that of the blood-plasma, and found that this fixing solution gave better preservation in cells that had high water-content in the cytoplasm, such as fibroblasts, leucocytes, and the centro-acinar cells of the pancreas. In other kinds of cells he did not notice any differences in results produced by hypotonic or isotonic buffered fixative. Sager and Palade (1957) did not find any 'appreciable improvement' in preservation of cell inclusions in Chlamydomonas by the addition of sucrose to buffered osmium tetroxide. Rangan (i960) found that it was better to have hypotonic rather than isotonic fixing fluids for the preservation of the nucleoplasm of malignant tumour cells. Though it may sometimes be desirable to add indifferent salts to solutions of osmium tetroxide (Sjostrand, 1953; Zetterqvist, 1956; Caulfield, 1957), it does not always seem to serve a useful purpose in fixation of the minute pieces of tissues that are generally required for electron microscopy. Robbins (1961) has found that the quality of fixation of metaphase chromosomes is not determined by the tonicity and ph of the solutions of osmium tetroxide. He fixed cultured cells in 'several isotonic solutions' that had been brought to the same ph and noticed 'significantly different' qualities of fixation. Baker (1958) investigated the effect on fixation for light microscopy by adding indifferent salts to unmixed fixatives, and he found that, with osmium tetroxide, the addition of salts showed no demonstrable improvement in the quality

15 Malhotra Experiments on fixation for electron microscopy 295 of preservation. However, I have myself found some evidence that with marine invertebrates, the addition of salts to osmium tetroxide solution in fixation for electron microscopy greatly improves the quality of preservation (unpublished observations). This supports the findings of Berthold (1882) and many subsequent investigators (see especially Young, 1935) that certain fixatives should be made up in salt solutions for the study of marine invertebrates and marine algae. I am grateful to Dr. J. R. Baker, F.R.S., who suggested the trial of potassium acetate as a weak alkalizing agent, for his invaluable help and advice, and to Mrs. B. M. Luke for her skilled assistance. Mrs. Luke made all the photographic enlargements except fig. 5, B (which was made by Dr. J. T. Y. Chou). My election to the Grocers' Company Fellowship at New College, Oxford, is greatly appreciated, and so also is the award of a Senior Studentship of the 1851 Exhibition. The Akashi electron microscope used in this investigation was provided by the Wellcome Trustees for research carried out under the direction of Dr. Baker. The Huxley ultramicrotome was provided by the Parliamentary Grants Committee of the Royal Society for the same purpose. References BAKER, J. R., Principles of biological microtechnique. London (Methuen). BERTHOLD, G., Jahrb. wiss. Bot. (Pringsheim), 13, 569. BRADBURY, S., and MEEK, G. A., i960. Quart. J. micr. Sci., 101, 241. CAULFIELD, J. B., J. biophys. biochem. Cytol., 3, 827. DALTON, A. J., Anat. Rec, 121, In The cell, 2, edited by J. Brachet and A. E. Mirsky. New York (Academic Press). and FELIX, M. D., Amer. J. Anat., 94, 171. FORSTER, R. P., In The cell, 5, edited by J. Brachet and A. E. Mirsky. New York (Academic Press). GLAUERT, A. M., In Techniques for electron microscopy, edited by D. Kay. Oxford (Blackwell). HIRSCH, G. C, In Biological structure and function, edited by T. W. Goodwin and O. Lindberg. London (Academic Press). LUFT, J. H., J. biophys. biochem. Cytol., a, 799. MALHOTRA, S. K., Quart. J. micr. Sci., 103, 5. MICHAELIS, L., Biochem. Zeit., 234, 139. MOLLENHAUER, H. H., J. biophys. biochem Cytol., 6, 431. NOVIKOFF, A. B., In The cell, 2, edited by J. Brachet and A. E. Mirsky. New York (Academic Press). PALADE, G. E., 1952a. J. exp. Med., 95, *. Anat. Rec, 114, In Electron microscopy in anatomy, edited by J. D. Boyd and others. London (Arnold). and SIEKEVITZ, P., J. biophys. biochem. Cytol., 2, 671. PALAY, S. L., In Frontiers in cytology, edited by S. L. Palay. New Haven (Yale Univ. Press). PEASE, D. C, 1955a. J. Histochem. Cytochem., 3, Anat. Rec, 121, 723. i960. Histological technique for electron microscopy. New York (Academic Press).

16 296 Malhotra Experiments on fixation for electron microscopy PORTER, K. R., In The biology of myelin, edited by S. R. Korey. New York (Hoeber- Harper) In X/ie ctf/z, 2, edited by J. BrachetandA. E. Mirsky. New York (Academic Press). RANGAN, S. R. S., i960. J. Ult. Res., 3, 392. RHODIN, J., 'Correlation of ultrastructural organization and function in normal and experimentally changed proximal convoluted tubule cells of the mouse kidney.' Thesis, Stockholm. ROBBINS, E., J. biophys. biochem. Cytol., 11, 449. ROBERTSON, J. D., Biochem. Soc. Symp., 16, 3. i960. Progress in Biophys. biochem. Chem., 10, 343. SAGER, R., and PALADE, G. E., J. biophys. biochem. Cytol., 3, 463. SJOSTRAND, F. S., J. cell. comp. Physiol., 42, Intemat. Rev. Cytol., 5, In Biophysical science a study program, edited by J. L. Oncley and others. New York (Wiley). and RHODIN, J., Exp. Cell Res., 4, 426. and HANZON, V., 1954a. Ibid., 7, Ibid., 7, 415. WATSON, M. L., J- biophys. biochem. Cytol., 1, Ibid., 6, 147. YOUNG, J. Z., Nature, 135, 823. ZEBRUN, W., and MOLLENHAUER, H. H., i960. J. biophys. biochem. Cytol., 7, 311. ZETTERQVIST, H., Thesis. Stockholm. Quoted from Pease, i960.