Stain Technology
ISSN: 0038-9153 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ibih19
Some Observations on Glass-Knife Making Robert T. Ward To cite this article: Robert T. Ward (1977) Some Observations on Glass-Knife Making, Stain Technology, 52:6, 305-309, DOI: 10.3109/10520297709116803 To link to this article: http://dx.doi.org/10.3109/10520297709116803
Published online: 12 Jul 2009.
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Date: 19 April 2016, At: 22:57
STAIN TECHNOLOGY VOLUME 52
NOVEMBER1977
NUMBER6
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
SOME OBSERVATIONS ON GLASS-KNIFE MAKING ROBERTT. WARD,Department of Anatomy, Downstate Medical Center, Brooklyn, New York 11203 ABSTRACT.The yield of usabie knife edge per M e (for thin sectioning) wss markedly increased when glass knives were made at an included angle of 55"rother than the customary 45". A large number of measurements of edge check marks made with a routine light scattering method as well as observations made on a smaller number of test sections with the electron microscope indicated the superiority of 55" knives. Knives were made with both taped pliers and an LKB Knifemaker. Knives were graded by methods easily applied in any biological electron microscope laboratory. Depending on the mode of fracture, the yield of lmives having more than 33%of their edges free of check marks was M to 100 times greater at 55" than 45".
Since the introduction of the glass knife to ultramicrotomy by Latta and Hartmann (1950), there have been few important advances in the process of making glass knives. Certainly the most important contribution to the successful ultramicrotomy of epoxies with glass knives was that of Andre (1962), who called attention to the fact that only a small portion of the average glass knife was sufficiently sharp €or obtaining good epoxy sections. Though Cameron's method of fracture by heat (1956) enjoyed a brief span of popularity, the use of taped glazier's pliers for glass fracture as suggested by Weiner (1959) quickly became the method of choice in most laboratories. In recent years several mechanical devices for breaking knives have been developed and have come into wide use. It has been customary to make glass knives with an included angle of 45', but knives scored at 55" have been used in this department for about seven years. Many thousands of such knives have been used for thin sectioning. Those who have tried them for an extended period of time have always preferred them to knives made at 45" because of their longer usable edges. The present study is an attempt to quantify with some objectivity this preference for knives broken at a nominal included angle of 55". Whether made with taped pliers or an LKB Knifemaker, 55" knives have in fact been found to be significantly superior. MATERIALSA N D METHODS
Knives were broken by either of two methods: (1) Glazier's pliers taped according to the method of Weiner (1959). A single wholesaler, who was aware- of our requirements, supplied Belgian plate glass in sheets, usually 12 inches square. In general, the procedure 305
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
so6
STAIN TECHNOLOGY
outlined by Porter (1964) was followed. The glass sheet was repeatedly divided in half until small squares were obtained. After the first break, which followed a score, short score marks were made to start otherwise free breaks. For the final (diagonal) break, a score was drawn toward the corner at which the knife edge was to be formed. The score was directed slightly toward the smoothest (most defect free) of the two surfaces and as close to the corner as possible without running the risk of cross-over breaks. For 55" knives, the final score was made following a carefully measured angle drawn on cardboard placed beneath the glass, and the included angle formed was checked after the break was made. All scores were made with Millers Falls #66 carbide wheel glass cutters. (2) A carefully adjusted LKB Knifemaker. Knives were made with glass obtained from the LKB Company according to instructions supplied with the instrument. Using a Bausch & Lomb Stereozoom microscope (45 X magnification) with a Bausch & Lomb Nicholas illuminator, knife edges were examined as follows: the bottom of the knife was pressed into modelling clay which served as a mount for orientation. The light was directed so that a bright reflection was obtained on the surface where the trough would be attached. The knife was then rotated toward the observer so that the bright patch of light was gradually narrowed to a thin line confined to the cutting edge. Such a line is seen in Figure 1. This light scattering image makes the detection of minute imperfections possible. A reticle in one ocular was used for measurement. Two hundred and seventy-five knives were made in each of four ways: with pliers at 45" and 55" and with the Knifemaker at 45" and 55". The entire 1100 knives were graded by measuring the fraction of total length of edge completely free of flaws observable under the conditions described .l To determine the relation of such quality assessments of knife edges to their capacity to produce thin sections, a single block of rat liver embedded in Epon (Luft 1961) was sectioned on a Reichert ultramicrotome using knives made with the Knifemaker. The sections obtained were scored for interference color and chatter. Using a similar block, other knives made with the Knifemaker were used to study the relation between the light microscopic assessment o f a knife edge and the quality of the sections actually obtained with it, as seen under the electron microscope. Thin sections taken across the measured flaw-free edge were observed in the electron microscope. They were judged "acceptable" if free from chatter and/or numerous fine scratches and with no more than four serious scratches. The ratio of good edge as determined by electron microscopy to that observed with the light microscope was determined. Ratios less than 0.6 were classified as unsatisfactory. The block used On 55" knives, unlike 45" knives, check marks are sometimes present in the region of the stress mark, so one must examine the entire edge of such knives with particular care. If the damaged area is slight, the adjacent flaw-free area is suitable for thin sectioning.
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
GLASSKNIFE MAKING
FIG. 1. Image of knife edge seen with light scattering. The irnperfecriom arc Seen to the right as irregular spots of light. The flaw-free section of the edge appears to the left a a thin, even, light line.
measured 0.5 x 0.75 mm, with the longer dimension parallel to the knife edge.
RESULTS In general, it was found that as the included angle was increased above 45", one obtained more knives with edges free of check marks over greater
lengths. Knives made at angles above 60" did not cut satisfactory sections. Knives made at 55" were found to be the equal of those made at 45" as far as freedom from compression or other sectioning difficulties were concerned. Most important, however, is the finding that the yield of knives with over 33% of their edge flawless is markedly increased when they are made with the 55" included angle (Table 1). Forty knives made at 55" with the Knifemaker were tested on a block of Epon-embedded rat liver to determine if their capacity to obtain optimal sections was in any way inferior to that of knives made at 45'. Twenty-nine of these had 40% or more flawless edge. All but one yielded silver or grey sections from the entire flawless edge. The thinnest secGons cut by these knives were as follows: 18 knives, gray; 10 knives, siIver. One knife had 40% flawless edge but would not cut better than gold sections. The remaining eleven knives had less than 40% flawless edge. However, tests showed that all would cut gold sections or better and some were capable of producing gray sections. Knives from the same batch of glass made at 45" at the same time proved to be in no way superior to those made at 55" and had markedly shorter flaw-free edges. Thirty-two knives broken at 55" with the Knifemaker and thirty broken at
308
STAIN TECHNOLOGY
45" were used to produce thin sections for electron microscopic study (Table 2). About two-thirds of the knives judged good at the light microscopic level yielded satisfactory sections as seen with the electron microscope. The ratio of good edge (EM) to good edge (LM) is somewhat better for 55" than 45" knives. A total of 372 thin sections were scored in this study.
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
DISCUSSION Our results do not agree with the suggestion first made by Andr6 (1962) that knives with included angles in excess of 50" will not produce satisfactory thin sections of Epon. Those of our Epon blocks which can be sectioned with glass edges will section well with knives made at either angle. However, many blocks can only be sectioned well with a good diamond knife and still others cannot be sectioned well with either glass or diamond edges. Our grading of knives made use of a procedure which is routine is most biological laboratories. We did not attempt more elaborate, time-consuming methods. Ultimately the only completely reliable test is cutting thin sections and observing them in the electron microscope. Examination of the light microscopic assessment of knives made at either 45" or 55" using either pliers or the Knifemaker (Table 1) shows that with either method a great many more knives with more than 33% of their edge flaw-free are produced at 55". With the Knifemaker this gain is without increase in the number of unusable knives (less than 26% flaw-free edge) produced as compared with results at 45". With pliers the breaking of usable knives at 55" is evidently more difficult than at 45", but since the poor knives are readily detected and discarded, the greatly increased proportion of superior knives that remain constitute a great improvement over the results obtained at 45". Considerable loss of time results when knives must be TABLE 1. PERCENT OF FLAW-FREE EDGEON KNIVESMADE AT T W O DIFFERENT ANGLES BY Two DIFFERENT.MEANS Knives made with pliers (%)
Percent of edge free of visible defects
(26 2633 >33
Knives made with Knifemaker (%)
45"
55"
45"
55"
26.6 72.7 0.7
38.6 40.0 21.5
29.5 70.2 0.4
29.1 28.7 42.2
A total of 1100 knives were scored to obtain the data above; 275 knives were scored for each of the modes tested. Copies of the tables of raw data are available from the author on request. TABLE 2. CORRESPONDENCE BETWEEN LIGHTMICROSCOPIC ASSESSMENT OF KNIFEEDGESAND THEIR ACTUAL SECTION PERFORMANCE AS JUDGED IN THE ELECTRONMICROSCOPE Ratio Good edge (EM)/Good edge (LM) Knives
1 .o
0.9
(Included Angle) 55" 45"
14 6
3 3
0.8
0.7
(Numbers of knives) 1 2 6
4
o.6
ISSN: 0038-9153 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ibih19
Some Observations on Glass-Knife Making Robert T. Ward To cite this article: Robert T. Ward (1977) Some Observations on Glass-Knife Making, Stain Technology, 52:6, 305-309, DOI: 10.3109/10520297709116803 To link to this article: http://dx.doi.org/10.3109/10520297709116803
Published online: 12 Jul 2009.
Submit your article to this journal
Article views: 5
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=ibih19 Download by: [University of Lethbridge]
Date: 19 April 2016, At: 22:57
STAIN TECHNOLOGY VOLUME 52
NOVEMBER1977
NUMBER6
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
SOME OBSERVATIONS ON GLASS-KNIFE MAKING ROBERTT. WARD,Department of Anatomy, Downstate Medical Center, Brooklyn, New York 11203 ABSTRACT.The yield of usabie knife edge per M e (for thin sectioning) wss markedly increased when glass knives were made at an included angle of 55"rother than the customary 45". A large number of measurements of edge check marks made with a routine light scattering method as well as observations made on a smaller number of test sections with the electron microscope indicated the superiority of 55" knives. Knives were made with both taped pliers and an LKB Knifemaker. Knives were graded by methods easily applied in any biological electron microscope laboratory. Depending on the mode of fracture, the yield of lmives having more than 33%of their edges free of check marks was M to 100 times greater at 55" than 45".
Since the introduction of the glass knife to ultramicrotomy by Latta and Hartmann (1950), there have been few important advances in the process of making glass knives. Certainly the most important contribution to the successful ultramicrotomy of epoxies with glass knives was that of Andre (1962), who called attention to the fact that only a small portion of the average glass knife was sufficiently sharp €or obtaining good epoxy sections. Though Cameron's method of fracture by heat (1956) enjoyed a brief span of popularity, the use of taped glazier's pliers for glass fracture as suggested by Weiner (1959) quickly became the method of choice in most laboratories. In recent years several mechanical devices for breaking knives have been developed and have come into wide use. It has been customary to make glass knives with an included angle of 45', but knives scored at 55" have been used in this department for about seven years. Many thousands of such knives have been used for thin sectioning. Those who have tried them for an extended period of time have always preferred them to knives made at 45" because of their longer usable edges. The present study is an attempt to quantify with some objectivity this preference for knives broken at a nominal included angle of 55". Whether made with taped pliers or an LKB Knifemaker, 55" knives have in fact been found to be significantly superior. MATERIALSA N D METHODS
Knives were broken by either of two methods: (1) Glazier's pliers taped according to the method of Weiner (1959). A single wholesaler, who was aware- of our requirements, supplied Belgian plate glass in sheets, usually 12 inches square. In general, the procedure 305
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
so6
STAIN TECHNOLOGY
outlined by Porter (1964) was followed. The glass sheet was repeatedly divided in half until small squares were obtained. After the first break, which followed a score, short score marks were made to start otherwise free breaks. For the final (diagonal) break, a score was drawn toward the corner at which the knife edge was to be formed. The score was directed slightly toward the smoothest (most defect free) of the two surfaces and as close to the corner as possible without running the risk of cross-over breaks. For 55" knives, the final score was made following a carefully measured angle drawn on cardboard placed beneath the glass, and the included angle formed was checked after the break was made. All scores were made with Millers Falls #66 carbide wheel glass cutters. (2) A carefully adjusted LKB Knifemaker. Knives were made with glass obtained from the LKB Company according to instructions supplied with the instrument. Using a Bausch & Lomb Stereozoom microscope (45 X magnification) with a Bausch & Lomb Nicholas illuminator, knife edges were examined as follows: the bottom of the knife was pressed into modelling clay which served as a mount for orientation. The light was directed so that a bright reflection was obtained on the surface where the trough would be attached. The knife was then rotated toward the observer so that the bright patch of light was gradually narrowed to a thin line confined to the cutting edge. Such a line is seen in Figure 1. This light scattering image makes the detection of minute imperfections possible. A reticle in one ocular was used for measurement. Two hundred and seventy-five knives were made in each of four ways: with pliers at 45" and 55" and with the Knifemaker at 45" and 55". The entire 1100 knives were graded by measuring the fraction of total length of edge completely free of flaws observable under the conditions described .l To determine the relation of such quality assessments of knife edges to their capacity to produce thin sections, a single block of rat liver embedded in Epon (Luft 1961) was sectioned on a Reichert ultramicrotome using knives made with the Knifemaker. The sections obtained were scored for interference color and chatter. Using a similar block, other knives made with the Knifemaker were used to study the relation between the light microscopic assessment o f a knife edge and the quality of the sections actually obtained with it, as seen under the electron microscope. Thin sections taken across the measured flaw-free edge were observed in the electron microscope. They were judged "acceptable" if free from chatter and/or numerous fine scratches and with no more than four serious scratches. The ratio of good edge as determined by electron microscopy to that observed with the light microscope was determined. Ratios less than 0.6 were classified as unsatisfactory. The block used On 55" knives, unlike 45" knives, check marks are sometimes present in the region of the stress mark, so one must examine the entire edge of such knives with particular care. If the damaged area is slight, the adjacent flaw-free area is suitable for thin sectioning.
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
GLASSKNIFE MAKING
FIG. 1. Image of knife edge seen with light scattering. The irnperfecriom arc Seen to the right as irregular spots of light. The flaw-free section of the edge appears to the left a a thin, even, light line.
measured 0.5 x 0.75 mm, with the longer dimension parallel to the knife edge.
RESULTS In general, it was found that as the included angle was increased above 45", one obtained more knives with edges free of check marks over greater
lengths. Knives made at angles above 60" did not cut satisfactory sections. Knives made at 55" were found to be the equal of those made at 45" as far as freedom from compression or other sectioning difficulties were concerned. Most important, however, is the finding that the yield of knives with over 33% of their edge flawless is markedly increased when they are made with the 55" included angle (Table 1). Forty knives made at 55" with the Knifemaker were tested on a block of Epon-embedded rat liver to determine if their capacity to obtain optimal sections was in any way inferior to that of knives made at 45'. Twenty-nine of these had 40% or more flawless edge. All but one yielded silver or grey sections from the entire flawless edge. The thinnest secGons cut by these knives were as follows: 18 knives, gray; 10 knives, siIver. One knife had 40% flawless edge but would not cut better than gold sections. The remaining eleven knives had less than 40% flawless edge. However, tests showed that all would cut gold sections or better and some were capable of producing gray sections. Knives from the same batch of glass made at 45" at the same time proved to be in no way superior to those made at 55" and had markedly shorter flaw-free edges. Thirty-two knives broken at 55" with the Knifemaker and thirty broken at
308
STAIN TECHNOLOGY
45" were used to produce thin sections for electron microscopic study (Table 2). About two-thirds of the knives judged good at the light microscopic level yielded satisfactory sections as seen with the electron microscope. The ratio of good edge (EM) to good edge (LM) is somewhat better for 55" than 45" knives. A total of 372 thin sections were scored in this study.
Downloaded by [University of Lethbridge] at 22:57 19 April 2016
DISCUSSION Our results do not agree with the suggestion first made by Andr6 (1962) that knives with included angles in excess of 50" will not produce satisfactory thin sections of Epon. Those of our Epon blocks which can be sectioned with glass edges will section well with knives made at either angle. However, many blocks can only be sectioned well with a good diamond knife and still others cannot be sectioned well with either glass or diamond edges. Our grading of knives made use of a procedure which is routine is most biological laboratories. We did not attempt more elaborate, time-consuming methods. Ultimately the only completely reliable test is cutting thin sections and observing them in the electron microscope. Examination of the light microscopic assessment of knives made at either 45" or 55" using either pliers or the Knifemaker (Table 1) shows that with either method a great many more knives with more than 33% of their edge flaw-free are produced at 55". With the Knifemaker this gain is without increase in the number of unusable knives (less than 26% flaw-free edge) produced as compared with results at 45". With pliers the breaking of usable knives at 55" is evidently more difficult than at 45", but since the poor knives are readily detected and discarded, the greatly increased proportion of superior knives that remain constitute a great improvement over the results obtained at 45". Considerable loss of time results when knives must be TABLE 1. PERCENT OF FLAW-FREE EDGEON KNIVESMADE AT T W O DIFFERENT ANGLES BY Two DIFFERENT.MEANS Knives made with pliers (%)
Percent of edge free of visible defects
(26 2633 >33
Knives made with Knifemaker (%)
45"
55"
45"
55"
26.6 72.7 0.7
38.6 40.0 21.5
29.5 70.2 0.4
29.1 28.7 42.2
A total of 1100 knives were scored to obtain the data above; 275 knives were scored for each of the modes tested. Copies of the tables of raw data are available from the author on request. TABLE 2. CORRESPONDENCE BETWEEN LIGHTMICROSCOPIC ASSESSMENT OF KNIFEEDGESAND THEIR ACTUAL SECTION PERFORMANCE AS JUDGED IN THE ELECTRONMICROSCOPE Ratio Good edge (EM)/Good edge (LM) Knives
1 .o
0.9
(Included Angle) 55" 45"
14 6
3 3
0.8
0.7
(Numbers of knives) 1 2 6
4
o.6
