British Poultry Science
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
The use of osmolality as an aid to establishing consistent fixation quality: Studies on the kidney of the domestic fowl B. Rothwell To cite this article: B. Rothwell (1978) The use of osmolality as an aid to establishing consistent fixation quality: Studies on the kidney of the domestic fowl, British Poultry Science, 19:2, 213-218, DOI: 10.1080/00071667808416466 To link to this article: http://dx.doi.org/10.1080/00071667808416466
Published online: 08 Nov 2007.
Submit your article to this journal
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Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [University of Florida]
Date: 12 November 2015, At: 01:32
Br. Poult. Sei., 19: 213-218. 1978
Longman: printed in Great Britain
THE USE OF OSMOLALITY AS AN AID TO ESTABLISHING CONSISTENT FIXATION QUALITY: STUDIES ON THE KIDNEY OF THE DOMESTIC FOWL
Downloaded by [University of Florida] at 01:32 12 November 2015
B. ROTHWELL Agricultural Research Council's Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh EH9 3JS, Scotland Received for publication 12th May 1977
1. Using the proximal convoluted tubule of the fowl kidney as indicator tissue the immersion application of fixatives of differing composition and osmolality was studied. 2. The best results were obtained with Dalton's buffered osmium tetroxide and with sodium cacodylate buffered glutaraldehyde followed by osmium post-fixation. 3. The most satisfactory component concentrations in both cases were those which most nearly reproduced a total osmolality close to that of fowl plasma. With both types of fixation the ultrastructural image of the kidney tubule was sensitive to changes in fixative osmolality and differences were clearly identified over a 30 to 50 mOsm range. 4. With glutaraldehyde/osmium double fixation it was found that it was the total osmolality of the glutaraldehyde buffer combination that was to be considered in a study of osmolotic effects, not the osmolality of the buffer vehicle alone. 5. The 30 to 50 mOsm difference in fixative osmolality that was able to be detected by studying the image of the kidney tubule was of the order of magnitude commonly experienced as experimental error in the laboratory as a result of changes in reagent batches or error in making up the solutions. The measurement of osmolality therefore provides an ideal quality control to the laboratory make-up of fixative solutions. INTRODUCTION
A list of standard fixative solutions and of buffers suitable for use with them can easily be drawn up. However, little information is available regarding osmolality, i.e. the physical measurement of particle concentration, in the buffers or fixative-buffer formulations used in the fixation process. This is despite a general appreciation that tissues vary in their degree of response to the difference in ion 213
Downloaded by [University of Florida] at 01:32 12 November 2015
214
B. ROTHWELL
balance between the solution and the cells' normal environment, more compact tissues being less sensitive than less compact and delicate ones (Hayat, 1970). Fahimi and Drochmans (1965) studied quality changes on compact tissues such as live muscle and noted changes with tonicity. Maser et al. (1967) quote work on newborn rat epidermis where a 50 mOsm difference in fixative concentration could be recognised. Gil and Weibel (1968) noted adverse changes in lung tissue when fixative osmolality was not equivalent to that of blood plasma. Others (Schultz and Karlsson, 1965; Maunsbach, 1966; Bone and Denton, 1971; BussonMabillot, 1971; Iqbal and Weakley, 1974) have studied osmolality in the more general sense of how variation on osmotic concentration can effect the morphological and morphometric features of cells and tissues and have identified procedures and preferences accordingly. Much of the information is confusing, however, in terms of trying to establish any general pattern on which other studies might be based as different tissues react differently to the various fixative formulations. This study was made to demonstrate the response of fowl kidney tissue to a consideration of osmolality in any fixation regime, and to try to establish a method of using osmolality measurements as a means of more reliably replicating experimental procedure. MATERIALS AND METHODS
Animals from a 6- to 8-week-old flock of a lightweight laying strain of domestic fowl (Shavers) were killed by a lethal dose of sodium pentobarbitone (Nembutal, Abbot Laboratories) and tissue was rapidly excised from the anterior kidney division and cut into 1 mm blocks prior to immersion in fixative solution. The fixation time was standardised to 1 h, followed, where applicable, by a compatible wash in buffer and post-fixation whose osmolality was as near to that of the fixative as it was possible to achieve. The tissue was then dehydrated in an ascending series of ethanol prior to embedding in Araldite. Ultrathin sections were cut after a fixed number of trimming sections had been taken from the block face thus establishing a level of comparability between blocks; these were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Phillips 300 electron microscope. The osmolality of all fixative and buffer solutions was measured using a Schuco Osmette S freezing-point depression osmometer. The criteria for the assessment of fixation quality were the same as those used in a previous study on fowl kidney fixation (Rothwell, 1974). Maunsbach (1966) noted that the ultrastructure of, for example, cytoplasmic bodies is little affected by the cellular alterations which occur immediately following the interruption of the blood supply to the kidney. The use of an immersion fixed image to evaluate fixation quality when it is known that a perfusion fixation technique is superior is therefore not unacceptable on ultrastructural criteria and was used here on the grounds of financial expediency and the minimisation of animal experimentation. RESULTS
Although the results are presented here as a single series of individual observations the information indicated is derived from a system whereby each experi-
OSMOLALITY AND FIXATION QUALITY
215
mental procedure was duplicated at least within and between animals and the amount of replication was further increased at the more critical observation stages. The immersion application to the kidney of variously buffered osmium fixatives was first studied and Dalton's buffered fixative clearly gave the most acceptable image. By varying the concentration of the buffer constituents a series of Dalton's buffered osmium fixatives of differing osmolalities was produced and applied to the kidney tissue. Their effect on image quality was noted first across a broad osmolality TABLE 1 The effect of a broad osmolality range of Dalton's fixative on kidney proximal tubule morphology
Downloaded by [University of Florida] at 01:32 12 November 2015
Dalton's OsO 4 fixative Potassium dichromate (g/1) 20 30 40 80 1
Sodium chloride (g/1) 17 26 34 68
Osmolality mOsm 230 334 390 674
Image quality1 0 0
Scale of assessment: 0, Very poor; + , Moderate; + + , Good; + + + > Very good; + + + + , Excellent.
TABLE 2 A more narrowly defined osmolality range of Dalton's fixative around the optimum indicated in Table 1 and its effect on kidney proximal tubule morphology
Dalton's OsO 4 fixative Potassium dichromate (g/1) 25 30 30 30 40
Sodium chloride (g/1) 21 26 30 30-3 34 1
Osmolality mOsm 294 325 357 374 406
Image quality1
See footnote to Table 1.
range (Table 1) and then in a much more narrowly defined range (Table 2) around the indicated optimum from Table 1. The preferred image was achieved by a fixative whose osmolality was very close to that recorded for fowl plasma—300 to 320 mOsm. It was possible for the microscopist experienced in studying avian kidney morphology and without prior knowledge of which material was being observed to perceive easily a difference in image quality produced by fixatives whose only variance was a small osmolality difference of around 30 mOsm. Plate-Fig. 1 shows the image change across a slightly broader osmolality range as this more clearly illustrates the effect to the person not used to viewing avian kidney material. Thus the fowl kidney proximal tubules show a distinct osmolotic sensitivity in their fixation with Dalton's osmium tetroxide fixation.
216
B. ROTHWELL
Downloaded by [University of Florida] at 01:32 12 November 2015
A glutaraldehyde-buffer combination followed by osmium post-fixation was now sought to establish what degree of osmolotic sensitivity existed with this typical double fixation procedure. Maunsbach (1966) observed on rat kidney proximal tubules that the osmolality of the medium in which the fixative was dispersed was more important than the total osmolality of the fixative, an observation which has been further substantiated by other workers (Bone and Ryan, 1972; Glauert, 1975). Fahimi and Drochmans (1965) further showed that the effect on tissue of hypertonicity created by the buffer component was worse than when created by glutaraldehyde. Thus the first experimental double fixation series chosen used only moderate glutaraldehyde concentrations (1 to 2-5%) with buffers approximately equiosmolotic with fowl plasma (300 to 320 mOsm). Negative results were obtained. A series was therefore devised in which the total osmolality of the fixative-buffer combination TABLE 3 The effect of a closely defined osmolality series of sodium cacodylate buffered glutaraldehyde (30 g/l) on kidney proximal tubule morphology
Molarity of sodium cacodylate buffer 0-075 0-1 0-125 1
Osmolality mOsm 330 362 392
Image quality1 + + ++ + + +
See footnote to Table 1.
was considered rather than the osmolality of the individual components. The buffers chosen were those recommended by Sabatini et al. (1963) for use with glutaraldehyde fixative and in all cases the osmolality of the buffer wash and postfixative solutions were equivalent to the fixative osmolality. A sodium cacodylate buffered glutaraldehyde combination produced the most acceptable image in this series and in all cases the preferential image was given by the fixative-buffer combination whose total osmolality was about 350 mOsm. A more closely identified osmolality range around the preferred 360 mOsm value was now prepared for sodium cacodylate buffered glutaraldehyde (Table 3). Once again the fowl kidney proximal convoluted tubule was shown to be osmolotically sensitive, and the preferred image was achieved with a fixative-buffer combination whose total osmolality was close to that recorded for fowl plasma. As with osmium fixation a 30 mOsm difference in fixative osmolality was able to be perceived by the experienced microscopist. Having identified that the fowl kidney proximal tubule was sensitive to the osmolality of the fixative solution and particularly to the formulation of the fixative and buffer components a test was made to identify any further limits to this fixativebuffer combination. Table 4 summarises an experiment in which the effect of varying the fixative to buffer ratio, whilst maintaining the osmolality around that already found adequate, is assessed. It will be seen that the relative proportions of the fixative and buffer components are vital in establishing the correct fixative formulation. In these studies the osmolality of every solution was checked before its use and adjusted or remade if found incorrect. In the course of this procedure it became
Downloaded by [University of Florida] at 01:32 12 November 2015 PLATE-FIG.
l(a).
PLATE-FIG.
l(b).
[facing p. 216.
Downloaded by [University of Florida] at 01:32 12 November 2015
tk*. PLATE-FIG. 1(C).
PLATE-FIG. 1.—(a) Normal image of the fowl kidney proximal tubule cell following fixation with 330 mOsm Dalton's osmium. (4) Fowl kidney proximal tubule cell fixed by 290 mOsm Dalton's osmium. Note the mitochondrial swelling and the tendency for cytoplasmic organelles to vesiculate. (c) Fowl kidney proximal tubule cell fixed by 390 mOsm Dalton's osmium. Note the shrunken dense mitochondria and the leached cytoplasm with few organelles evident. The scale marker represents 3 ftm.
OSMOLALITY AND FIXATION QUALITY
217
TABLE 4 The effect on kidney proximal tubule morphology of varying the fixative/buffer ratio whilst retaining a similar osmolality
Glutaraldehyde concentration 2 3 1
Molarity of sodium cacodylate buffer 0-1 0-025 0-025 1
Osmolality mOsm 368 362 344
Image quality1 ++ + + + +
See footnote to Table 1.
Downloaded by [University of Florida] at 01:32 12 November 2015
clear that different batches of reagent and different technical operators could very easily account for errors of 30 mOsm and errors of up to 100 mOsm were not uncommon. DISCUSSION
In the process of fixation the plasma membrane and the membranes of the cell organelles do not behave as " ideal " membranes in the sense that the term is used when assessing colligative properties as they are changed by the fixation process itself. Fixative osmolality therefore can only be considered as an arbitary factor and not as an absolute value having cytophysiological significance. Nevertheless, the value of using it as a measure around which a more standard laboratory procedure can be effected is well demonstrated in the foregoing experiments. Whilst accepting Hayat's (1970) contention that " due to relative lack of information on the molecular basis of fixation it is difficult to evaluate the exact quality of tissue fixation ", nevertheless it is possible to establish a mixture of objective and subjective criteria for the assessment of fixation quality. The proximal convoluted tubule was used as a sensitive indicator tissue in these experiments because, as Maunsbach (1966) observed, this tissue is influenced by variation in fixation and preparation techniques. The 30 mOsm variation from the ideal fixative osmolality value capable of identifying the osmolotic sensitivity of the fowl kidney tubule image quality and detail was well inside the experimental error obtained in the laboratory as a result of reagent batch changes and technical variations on preparation of solutions. By using osmolality measurements on all the fixative components to be used in a fixative regime, any small changes that would significantly affect fixation quality can be discovered and their effect avoided. Maunsbach (1966) studying the rat kidney proximal tubule concluded that the osmolality of the medium in which the fixative was dispersed was more important than the total osmolality of the fixative and that the type of buffer used as dispersion medium was not significant. These present studies on the fowl do not support this observation, total osmolality of the fixative buffer formulation was found to be the more important criterion. REFERENCES
BONE, Q,. AND DENTON, E. J. (1971). The osmotic effect of electron microscope fixatives. Journal of Cell Biology, 4 9 : 571-581.
19/2—F
Downloaded by [University of Florida] at 01:32 12 November 2015
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BONE, Q,. AND RYAN, K. P. (1972). Osmolality of osmium tetroxide and glutaraldehyde fixatives. Histochemical Journal, 4: 331. BUSSON-MABILLOT, S. (1971). Influence de la fixation chimique sur les ultrastructures. I. Étude sur les organites d'un follicle ovarian d'un poisson téléostéen. Journal de Microscopie, 12: 317-348. FAHIMI, H. D. AND DROCHMANS, P. (1965). Essais de standardisation de la fixation au glutaraldéhyde, II. Influence des concentrations en aldéhyde et de Posmolatité. Journal de Microscopie, 4 : 737-748. GIL, J . AND WEIBEL, E. R. (1968). The role of buffers in lung fixation with glutaraldehyde and osmium tetroxide. Journal of Ultrastructural Research, 25: 221-348. GLAUERT, A. M. (1975). Fixation, dehydration and embedding of biological specimens. I n : Practical Methods in Electron Microscopy, p . 9. Edit. GLAUERT, A. M. Amsterdam, North Holland Publishing Co. HAYAT, M. A. (1970). Principles and Techniques of Electron Microscopy, vol. 1, Biological Applications New York, Van Nostrand Reinhold Co. IQBAL, S. J . AND WEAKLEY, B. S. (1974). The effects of different preparative procedures on the ultrastructure of the hamster ovary. 1. Effects of various fixative solutions on ovarian oocytes and their granulosa cells. Histiochemistry, 38: 95-122. MASER, M. D., POWELL, T. E. AND PHILPOTT, C. W. (1967). Relationships among pH, osmolality
and concentration of fixative solutions. Stain Technology, 42: 175-182. MAUNSBACH, A. B. (1966). The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. II. Effects of varying osmolality, ionic strength, buffer system and fixative concentration of glutaraldehyde solutions. Journal of Ultrastructural Research, 15: 283-309. REYNOLDS, E. S. (1963). The use of lead citrate at high p H as an electron opaque stain in electron microscopy. Journal of Cell Biology, 17: 208. ROTHWELL, B. (1974). Perfusion fixation of the kidney of the domestic fowl. Journal of Microscopy, 100: 99-104. SABATINI, D. D., BENSCH, K. AND BARRNETT, R. J . (1963). Cytochemistry and electron microscopy.
The preservation of cellular ultrastructure and enzymic activity by aldehyde fixation. Journal of Cell Biology, 17: 19-58. SCHULTZ, R. L. AND KARLSSON, U. (1965). Fixation of the central nervous system for electron microscopy by aldehyde perfusion. II. Effect of osmolarity, p H of perfusate and fixative concentration. Journal of Ultrastructural Research, 12: 187-206. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. Journal of Biophysical and Biochemical Cytology, 4: 475-478.
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
The use of osmolality as an aid to establishing consistent fixation quality: Studies on the kidney of the domestic fowl B. Rothwell To cite this article: B. Rothwell (1978) The use of osmolality as an aid to establishing consistent fixation quality: Studies on the kidney of the domestic fowl, British Poultry Science, 19:2, 213-218, DOI: 10.1080/00071667808416466 To link to this article: http://dx.doi.org/10.1080/00071667808416466
Published online: 08 Nov 2007.
Submit your article to this journal
Article views: 8
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [University of Florida]
Date: 12 November 2015, At: 01:32
Br. Poult. Sei., 19: 213-218. 1978
Longman: printed in Great Britain
THE USE OF OSMOLALITY AS AN AID TO ESTABLISHING CONSISTENT FIXATION QUALITY: STUDIES ON THE KIDNEY OF THE DOMESTIC FOWL
Downloaded by [University of Florida] at 01:32 12 November 2015
B. ROTHWELL Agricultural Research Council's Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh EH9 3JS, Scotland Received for publication 12th May 1977
1. Using the proximal convoluted tubule of the fowl kidney as indicator tissue the immersion application of fixatives of differing composition and osmolality was studied. 2. The best results were obtained with Dalton's buffered osmium tetroxide and with sodium cacodylate buffered glutaraldehyde followed by osmium post-fixation. 3. The most satisfactory component concentrations in both cases were those which most nearly reproduced a total osmolality close to that of fowl plasma. With both types of fixation the ultrastructural image of the kidney tubule was sensitive to changes in fixative osmolality and differences were clearly identified over a 30 to 50 mOsm range. 4. With glutaraldehyde/osmium double fixation it was found that it was the total osmolality of the glutaraldehyde buffer combination that was to be considered in a study of osmolotic effects, not the osmolality of the buffer vehicle alone. 5. The 30 to 50 mOsm difference in fixative osmolality that was able to be detected by studying the image of the kidney tubule was of the order of magnitude commonly experienced as experimental error in the laboratory as a result of changes in reagent batches or error in making up the solutions. The measurement of osmolality therefore provides an ideal quality control to the laboratory make-up of fixative solutions. INTRODUCTION
A list of standard fixative solutions and of buffers suitable for use with them can easily be drawn up. However, little information is available regarding osmolality, i.e. the physical measurement of particle concentration, in the buffers or fixative-buffer formulations used in the fixation process. This is despite a general appreciation that tissues vary in their degree of response to the difference in ion 213
Downloaded by [University of Florida] at 01:32 12 November 2015
214
B. ROTHWELL
balance between the solution and the cells' normal environment, more compact tissues being less sensitive than less compact and delicate ones (Hayat, 1970). Fahimi and Drochmans (1965) studied quality changes on compact tissues such as live muscle and noted changes with tonicity. Maser et al. (1967) quote work on newborn rat epidermis where a 50 mOsm difference in fixative concentration could be recognised. Gil and Weibel (1968) noted adverse changes in lung tissue when fixative osmolality was not equivalent to that of blood plasma. Others (Schultz and Karlsson, 1965; Maunsbach, 1966; Bone and Denton, 1971; BussonMabillot, 1971; Iqbal and Weakley, 1974) have studied osmolality in the more general sense of how variation on osmotic concentration can effect the morphological and morphometric features of cells and tissues and have identified procedures and preferences accordingly. Much of the information is confusing, however, in terms of trying to establish any general pattern on which other studies might be based as different tissues react differently to the various fixative formulations. This study was made to demonstrate the response of fowl kidney tissue to a consideration of osmolality in any fixation regime, and to try to establish a method of using osmolality measurements as a means of more reliably replicating experimental procedure. MATERIALS AND METHODS
Animals from a 6- to 8-week-old flock of a lightweight laying strain of domestic fowl (Shavers) were killed by a lethal dose of sodium pentobarbitone (Nembutal, Abbot Laboratories) and tissue was rapidly excised from the anterior kidney division and cut into 1 mm blocks prior to immersion in fixative solution. The fixation time was standardised to 1 h, followed, where applicable, by a compatible wash in buffer and post-fixation whose osmolality was as near to that of the fixative as it was possible to achieve. The tissue was then dehydrated in an ascending series of ethanol prior to embedding in Araldite. Ultrathin sections were cut after a fixed number of trimming sections had been taken from the block face thus establishing a level of comparability between blocks; these were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Phillips 300 electron microscope. The osmolality of all fixative and buffer solutions was measured using a Schuco Osmette S freezing-point depression osmometer. The criteria for the assessment of fixation quality were the same as those used in a previous study on fowl kidney fixation (Rothwell, 1974). Maunsbach (1966) noted that the ultrastructure of, for example, cytoplasmic bodies is little affected by the cellular alterations which occur immediately following the interruption of the blood supply to the kidney. The use of an immersion fixed image to evaluate fixation quality when it is known that a perfusion fixation technique is superior is therefore not unacceptable on ultrastructural criteria and was used here on the grounds of financial expediency and the minimisation of animal experimentation. RESULTS
Although the results are presented here as a single series of individual observations the information indicated is derived from a system whereby each experi-
OSMOLALITY AND FIXATION QUALITY
215
mental procedure was duplicated at least within and between animals and the amount of replication was further increased at the more critical observation stages. The immersion application to the kidney of variously buffered osmium fixatives was first studied and Dalton's buffered fixative clearly gave the most acceptable image. By varying the concentration of the buffer constituents a series of Dalton's buffered osmium fixatives of differing osmolalities was produced and applied to the kidney tissue. Their effect on image quality was noted first across a broad osmolality TABLE 1 The effect of a broad osmolality range of Dalton's fixative on kidney proximal tubule morphology
Downloaded by [University of Florida] at 01:32 12 November 2015
Dalton's OsO 4 fixative Potassium dichromate (g/1) 20 30 40 80 1
Sodium chloride (g/1) 17 26 34 68
Osmolality mOsm 230 334 390 674
Image quality1 0 0
Scale of assessment: 0, Very poor; + , Moderate; + + , Good; + + + > Very good; + + + + , Excellent.
TABLE 2 A more narrowly defined osmolality range of Dalton's fixative around the optimum indicated in Table 1 and its effect on kidney proximal tubule morphology
Dalton's OsO 4 fixative Potassium dichromate (g/1) 25 30 30 30 40
Sodium chloride (g/1) 21 26 30 30-3 34 1
Osmolality mOsm 294 325 357 374 406
Image quality1
See footnote to Table 1.
range (Table 1) and then in a much more narrowly defined range (Table 2) around the indicated optimum from Table 1. The preferred image was achieved by a fixative whose osmolality was very close to that recorded for fowl plasma—300 to 320 mOsm. It was possible for the microscopist experienced in studying avian kidney morphology and without prior knowledge of which material was being observed to perceive easily a difference in image quality produced by fixatives whose only variance was a small osmolality difference of around 30 mOsm. Plate-Fig. 1 shows the image change across a slightly broader osmolality range as this more clearly illustrates the effect to the person not used to viewing avian kidney material. Thus the fowl kidney proximal tubules show a distinct osmolotic sensitivity in their fixation with Dalton's osmium tetroxide fixation.
216
B. ROTHWELL
Downloaded by [University of Florida] at 01:32 12 November 2015
A glutaraldehyde-buffer combination followed by osmium post-fixation was now sought to establish what degree of osmolotic sensitivity existed with this typical double fixation procedure. Maunsbach (1966) observed on rat kidney proximal tubules that the osmolality of the medium in which the fixative was dispersed was more important than the total osmolality of the fixative, an observation which has been further substantiated by other workers (Bone and Ryan, 1972; Glauert, 1975). Fahimi and Drochmans (1965) further showed that the effect on tissue of hypertonicity created by the buffer component was worse than when created by glutaraldehyde. Thus the first experimental double fixation series chosen used only moderate glutaraldehyde concentrations (1 to 2-5%) with buffers approximately equiosmolotic with fowl plasma (300 to 320 mOsm). Negative results were obtained. A series was therefore devised in which the total osmolality of the fixative-buffer combination TABLE 3 The effect of a closely defined osmolality series of sodium cacodylate buffered glutaraldehyde (30 g/l) on kidney proximal tubule morphology
Molarity of sodium cacodylate buffer 0-075 0-1 0-125 1
Osmolality mOsm 330 362 392
Image quality1 + + ++ + + +
See footnote to Table 1.
was considered rather than the osmolality of the individual components. The buffers chosen were those recommended by Sabatini et al. (1963) for use with glutaraldehyde fixative and in all cases the osmolality of the buffer wash and postfixative solutions were equivalent to the fixative osmolality. A sodium cacodylate buffered glutaraldehyde combination produced the most acceptable image in this series and in all cases the preferential image was given by the fixative-buffer combination whose total osmolality was about 350 mOsm. A more closely identified osmolality range around the preferred 360 mOsm value was now prepared for sodium cacodylate buffered glutaraldehyde (Table 3). Once again the fowl kidney proximal convoluted tubule was shown to be osmolotically sensitive, and the preferred image was achieved with a fixative-buffer combination whose total osmolality was close to that recorded for fowl plasma. As with osmium fixation a 30 mOsm difference in fixative osmolality was able to be perceived by the experienced microscopist. Having identified that the fowl kidney proximal tubule was sensitive to the osmolality of the fixative solution and particularly to the formulation of the fixative and buffer components a test was made to identify any further limits to this fixativebuffer combination. Table 4 summarises an experiment in which the effect of varying the fixative to buffer ratio, whilst maintaining the osmolality around that already found adequate, is assessed. It will be seen that the relative proportions of the fixative and buffer components are vital in establishing the correct fixative formulation. In these studies the osmolality of every solution was checked before its use and adjusted or remade if found incorrect. In the course of this procedure it became
Downloaded by [University of Florida] at 01:32 12 November 2015 PLATE-FIG.
l(a).
PLATE-FIG.
l(b).
[facing p. 216.
Downloaded by [University of Florida] at 01:32 12 November 2015
tk*. PLATE-FIG. 1(C).
PLATE-FIG. 1.—(a) Normal image of the fowl kidney proximal tubule cell following fixation with 330 mOsm Dalton's osmium. (4) Fowl kidney proximal tubule cell fixed by 290 mOsm Dalton's osmium. Note the mitochondrial swelling and the tendency for cytoplasmic organelles to vesiculate. (c) Fowl kidney proximal tubule cell fixed by 390 mOsm Dalton's osmium. Note the shrunken dense mitochondria and the leached cytoplasm with few organelles evident. The scale marker represents 3 ftm.
OSMOLALITY AND FIXATION QUALITY
217
TABLE 4 The effect on kidney proximal tubule morphology of varying the fixative/buffer ratio whilst retaining a similar osmolality
Glutaraldehyde concentration 2 3 1
Molarity of sodium cacodylate buffer 0-1 0-025 0-025 1
Osmolality mOsm 368 362 344
Image quality1 ++ + + + +
See footnote to Table 1.
Downloaded by [University of Florida] at 01:32 12 November 2015
clear that different batches of reagent and different technical operators could very easily account for errors of 30 mOsm and errors of up to 100 mOsm were not uncommon. DISCUSSION
In the process of fixation the plasma membrane and the membranes of the cell organelles do not behave as " ideal " membranes in the sense that the term is used when assessing colligative properties as they are changed by the fixation process itself. Fixative osmolality therefore can only be considered as an arbitary factor and not as an absolute value having cytophysiological significance. Nevertheless, the value of using it as a measure around which a more standard laboratory procedure can be effected is well demonstrated in the foregoing experiments. Whilst accepting Hayat's (1970) contention that " due to relative lack of information on the molecular basis of fixation it is difficult to evaluate the exact quality of tissue fixation ", nevertheless it is possible to establish a mixture of objective and subjective criteria for the assessment of fixation quality. The proximal convoluted tubule was used as a sensitive indicator tissue in these experiments because, as Maunsbach (1966) observed, this tissue is influenced by variation in fixation and preparation techniques. The 30 mOsm variation from the ideal fixative osmolality value capable of identifying the osmolotic sensitivity of the fowl kidney tubule image quality and detail was well inside the experimental error obtained in the laboratory as a result of reagent batch changes and technical variations on preparation of solutions. By using osmolality measurements on all the fixative components to be used in a fixative regime, any small changes that would significantly affect fixation quality can be discovered and their effect avoided. Maunsbach (1966) studying the rat kidney proximal tubule concluded that the osmolality of the medium in which the fixative was dispersed was more important than the total osmolality of the fixative and that the type of buffer used as dispersion medium was not significant. These present studies on the fowl do not support this observation, total osmolality of the fixative buffer formulation was found to be the more important criterion. REFERENCES
BONE, Q,. AND DENTON, E. J. (1971). The osmotic effect of electron microscope fixatives. Journal of Cell Biology, 4 9 : 571-581.
19/2—F
Downloaded by [University of Florida] at 01:32 12 November 2015
218
B. ROTHWELL
BONE, Q,. AND RYAN, K. P. (1972). Osmolality of osmium tetroxide and glutaraldehyde fixatives. Histochemical Journal, 4: 331. BUSSON-MABILLOT, S. (1971). Influence de la fixation chimique sur les ultrastructures. I. Étude sur les organites d'un follicle ovarian d'un poisson téléostéen. Journal de Microscopie, 12: 317-348. FAHIMI, H. D. AND DROCHMANS, P. (1965). Essais de standardisation de la fixation au glutaraldéhyde, II. Influence des concentrations en aldéhyde et de Posmolatité. Journal de Microscopie, 4 : 737-748. GIL, J . AND WEIBEL, E. R. (1968). The role of buffers in lung fixation with glutaraldehyde and osmium tetroxide. Journal of Ultrastructural Research, 25: 221-348. GLAUERT, A. M. (1975). Fixation, dehydration and embedding of biological specimens. I n : Practical Methods in Electron Microscopy, p . 9. Edit. GLAUERT, A. M. Amsterdam, North Holland Publishing Co. HAYAT, M. A. (1970). Principles and Techniques of Electron Microscopy, vol. 1, Biological Applications New York, Van Nostrand Reinhold Co. IQBAL, S. J . AND WEAKLEY, B. S. (1974). The effects of different preparative procedures on the ultrastructure of the hamster ovary. 1. Effects of various fixative solutions on ovarian oocytes and their granulosa cells. Histiochemistry, 38: 95-122. MASER, M. D., POWELL, T. E. AND PHILPOTT, C. W. (1967). Relationships among pH, osmolality
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