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Comparison of methods for measuring sarcomere length in beef semitendinosus muscle
Meat Science 5 (1980-81) 261-266
C O M P A R / S O N OF M E T H O D S F O R M E A S U R I N G S A R C O M E R E L E N G T H IN BEEF S E M I T E N D I N O S U S M U S C L E H.R. CROSS
Meat Science Research Laboratory, USDA, AR Beltsville, M D 20705, USA R. L. WEST
Animal Science Department, University oJ Florida, Gainesville, USA
T.R. DUTSON
Animal Science Department, Texas A & M University, College Station USA (Received: 4 February, 1980)
S UMMAR Y
The objective of the study was to compare the precision of the laser method for measuring sarcomere length with the precision of two oil-immersion microscope methods. Eighteen semitendinosus muscles were assigned to one of three postmortem treatments to provide a wide range in sarcomere length. Two 5.0cm sections were removed from the centre of each muscle. Each section was subdivided into six equal-size pieces and randomly allotted to each of three participating institutions. Analysis of variance revealed that sarcomere length measurement was not significantly affected by the method of measurement or by the technician. For 99% precision, the laser method required 34 measurements, whereas the two microscope methods required 45 and 66 measurements, respectively.
INTRODUCTION
Since Locker (•960) reported on the relationship of muscle shortening (cold shortening) to meat tenderness, several workers have conducted further investigations on this relationship (Locker & Hagyard, 1963; Marsh & L e e t , 1966; Herring et aL, 1965; and Hostetler et aL, 1970). These workers and others have clearly demonstrated that when muscles are treated to alter sarcomere length, there is a high, positive correlation between sarcomere length and tenderness. Since a direct Any reference to a brand or firm name within this paper, does not constitute endorsement by the US Department of Agriculture.
261 Meat Science 0309-1740/81/0005-0261/$02-50 © Applied Science Publishers Ltd, England, 1981 Printed in Great Britain
262
H . R . CROSS, R. L. WEST, T. R. DUTSON
correlation between state of muscle contraction and ultimate meat quality does exist, it would be desirable to know whether the different methods of measuring sarcomere length are similar and which method is the simplest and most reliable one available. The most commonly used method of sarcomere length measurement is the oilimmersion, phase-contrast microscopy of unstained tissue. With this method, the space between adjacent Z-discs is measured with various calibrated eye-piece measuring devices. The type of measuring device is usually some type of eye-piece micrometer; however, more sophisticated and costly devices, such as the particle counter/size analyser, have been used. A different method which has been used (Voyle, 1971; Rome, 1967; and Ruddick & Richards, 1975) was derived initially from early observations that a striated muscle acts as a transmission grating when placed in a beam of light. With this method, diffraction patterns are formed on a screen by transmitting monochromatic light through muscle fibres, and the separation of the orders of diffraction are determined by the contractile state of the muscle. Rome (1967) and Cleworth & Edman (1969) described developments in which a gas laser was used as a source of coherent monochromatic light. Ruddick & Richards (1975) compared sarcomere length measurement of cooked chicken peetoralis muscle by laser diiiraction and oilimmersion microscopy. Their results indicated that the laser method was equal, or superior to, the oil-immersion methods. The primary objective of this study was to compare the precision of the laser technique with the precision of two oil-immersion techniques for measuring sarcomere length in bovine muscle. An additional objective was to evaluate the human error within each procedure and to determine the minimum number of measurements needed to obtain a given degree of precision with each procedure.
EXPERIMENTAL
Sample selection and preparation Semitendinosus muscles from four beef carcasses were excised at about lh postmortem, divided lengthwise into three equal parts, and one part of each muscle was assigned to one of three post-mortem treatments. Muscle strips in treatment 1 were stretched maximally and held in that position by clamps on each end of the muscle strip. Muscle strips in treatment 2 were immobilised in a slightly stretched condition, with clamps at each end of the muscle. Muscle strips in treatment 3 were not stretched or immobilised. These three treatments were designed to provide a wide range in sarcomere length. Two 5.0cm sections were removed from the centre of each muscle strip, and each section was subdivided into six equal-size pieces. Then two pieces from each section (four pieces from each muscle strip) were randomly allotted to each of the three participating institutions. Each piece was double wrapped in aluminium foil and
SARCOMERE LENGTH MEASUREMENT
263
stored in liquid nitrogen until shipped to the respective institutions. Samples were shipped in dry ice via air freight. After the samples reached the respective institutions, one piece from each section (two pieces from each muscle strip) was allotted to each of two highly trained technicians for measurement of sarcomere length by one of the three methods described below.
Laser diffraction method Twoborate-KC1 buffer solutions containing glutaraldehyde were used as fixatives. Solution A consisted of 0.1M KC1, 0.039M boric acid, and 5mM E D T A in 2-5% glutaraldehyde. Solution B consisted of 0.25M KC1, 0.29M boric acid, and 5raM E D T A in 2.5% glutaraldehyde. The muscle tissue was cut into 3.0 × 3.0 × 2.0cm pieces and placed in a vial and covered with solution A for 2h. The sample was then transferred to a fresh vial containing solution B for 17-19h. Sarcomere length was measured t h e following day. Sarcomere length by laser diffraction was determined by procedures similar to those described by Ruddick and Richards (1975) except that the calculations differed due to the use of a flat surface (screen) instead of a convex surface (as in this instance). The apparatus consisted of a helium-neon laser (with a wavelength of 632-8nm) which was mounted on an optics bench with a specimen-holding dbvice and a screen (Fig. 1). Individual fibres were teased from the muscle bundle and placed on a microscope slide with a drop of solution B. The slide was then placed horizontally in the path of a vertically orientated laser beam to give an array of diffraction bands
I
i
,: ! ',
Fig. 1. Diagram of laser technique.
264
H. R. CROSS, R. L. WEST, T. R. DUTSON
on the screen. These bands were perpendicular to the long axis of the fibres (Fig. 1). Sarcomere lengths were calculated by use of the following formula: 632.8 x 10-3 x D x N/(-~TD)2 + 1 Sarcomere length (microns)= T where D equals the distance (mm) from the specimen-holding device to the screen (throughout this experiment, D had a constant value of 100mm) and T equals the separation (mm) between the zero and the first maximum band. Twenty-five fibres were measured by each individual on each sample piece.
Filar micrometer method A 5g muscle sample was cut into small pieces and homogenised at low speed (approximately 5000rpm) in 30ml of chilled 0-25M sucrose solution, in a Virtis '23' homogeniser, for 60sec. A drop of homogenate was transferred to a slide and covered with a cover slip. The slide was examined under the phase microscope (100 X objective, 10 X eye-piece) with oil-immersion for the number and length of myofibrils. I f fibres were not sufficiently broken apart, the sample was homogenised an additional 20-30sec. This procedure was repeated as necessary up to three times, taking care that the myofibrils were not homogenised to less than 10 sarcomeres in length. The length of 10 sarcomeres on each of the first 25 myofibrils, located by starting at one edge of the slide and moving straight across, were measured with a filar micrometer containing a Vernier scale calibrated against a stage micrometer. Values were converted using the calculated calibration factor (the filar micrometer was adjusted so that 0 . 0 1 m m = 100~tm thereby eliminating the conversion of each measurement). Shearieon size analyser method A 3g sample of muscle was cut into small pieces and allowed to thaw. The sample was placed in a 50ml beaker with 35ml of 0.25 M surcrose and blended at high speed for 30sec in a Virtis '33' macro-homogeniser. After blending, a small drop of the sucrose solution was placed on a slide, covered with a cover slip, and placed under the oil-immersion lens on a Zeiss W L phase-contrast microscope equipped with a Timbrell Coulter Shearicon particle counter/size analyser. The Shearicon was calibrated with a stage micrometer to give measurements to the nearest 0.01mm. The mean sarcomere length of each muscle was determined by measuring 25 myofibrils containing four sarcomeres each. Statistical analysis Data were treated by analysis of variance procedures as outlined by Snedecor & Cochran (1967). The power of the test was calculated according to Guenther (1964).
SARCOMERE LENGTH MEASUREMENT RESULTS
AND
265
DISCUSSION
Results o f analysis o f variance revealed that s a r c o m e r e length m e a s u r e m e n t did not differ significantly with different m e t h o d s of m e a s u r e m e n t or with different technicians. A s expected from the experimental design, effects of different muscle t r e a t m e n t s were all highly significant. M e a n sarcomere lengths are segregated by m e t h o d o f m e a s u r e m e n t , technician and treatment in T a b l e 1. A s predicted, restraint t r e a t m e n t significantly altered s a r c o m e r e length. W i t h i n each muscle treatment, s a r c o m e r e length was n o t affected b y the m e t h o d o r the technician. M e a n s and s t a n d a r d deviations within each t r e a t m e n t are r e m a r k a b l y similar. One possibility when the m i c r o s c o p e techniques are used to m e a s u r e s a r c o m e r e length is t h a t fatigue m a y a c c o m p a n y the method. F a t i g u e can result in error, increased variability, or both. H o w e v e r , the results o f this s t u d y indicated t h a t all six technicians obtained c o m p a r a b l e results, regardless o f m e t h o d ; and fatigue, which might affect the results, was n o t observed. TABLE 1 MEAN SARCOMERE LENGTH ( ~ m ) AND STANDARD DEVIATION BY MUSCLE TREATMENT AND TECHNICIAN WITHIN METHOD
Method of measurement
Muscle treatment Maximum stretch
LaseF
Technician 1 Technician 2 Fi!ar micrometer Technician 1 Technician 2 Shear±con/size analyser Technician 1 Technician 2
Slight stretch
Unrestrained
3.35 + 0-27~ 3-37 _+0.24 a
2.77 + 0.25 ~ 2-72 ± 0.27 ~
1-89 + 0-0Y 1-93 + 0-06c
3.39 ± 0-17 a 3-39 + 0-18a
2-70 + 0-14 ~ 2.73 + 0-22 b
1.98 + 0-08c 1-99 ± 0-09c
3.19 + 0-22 a 3-31 + 0.15 a
2-71 _+0-18~ 2.67 + 0.18 ~
1-97 + 0. I(F 1-97 _ 0-10e
~.n.cMeans in the same row and column with different superscripts are significantly different (P < 0"001). The numbers o f sarcomeres per sample m e a s u r e d and reported in the literature varies considerably. T o determine the m i n i m u m n u m b e r of m e a s u r e m e n t s n e c e s s a r y to o b t a i n a p a r t i c u l a r level o f precision, we calculated the p o w e r of the test using the coefficient o f variation f r o m each method. Since technicians h a d no significant effect on sm.comere length, technicians within e a c h m e t h o d were c o m b i n e d (Table 2) for calculating the coefficient o f variation. F o r 99% precision, the laser method required 34 m e a s u r e m e n t s whereas the two oil-immersion m e t h o d s required 45 and 66, respectively. A precision o f 95% required one, two a n d three m e a s u r e m e n t s for the laser a n d the two m i c r o s c o p e m e t h o d s , respectively. Differences in coefficients of variation m a y have been due, in part, to the n u m b e r o f s a r c o m e r e s m e a s u r e d per observation. T h e laser m e t h o d m e a s u r e s m a n y s a r c o m e r e s within the b e a m ' s path, but the Film" m i c r o m e t e r and Shear±con/size a n a l y s e r techniques measure only ten
266
H . R . CROSS, R. L. WEST, T. R. DUTSON TABLE 2 MINIMUM NUMBER OF MEASUREMENT UNITS REQUIRED TO ASSURE A
PRECISION OF 95 OR 99 PER CENTa
Method of measurement
Number oJ units required ~ Coefficient of variation
95% precision
99% precision
5.8 6- 7 8.1
1 2 3
34 45 66
(%)
Laser Filar micrometer Shearicon/size analyser
"Measurement units differ for each method as follows: Laser--fibres; Filar micrometer--myofibrils with 10 sarcomere units; and, Shearicon/size analyser--myofibrils with four sarcomere units. bPower of the test was calculated by: n ~ (coefficient of variation)2/p2, where P is the proportion of desired accuracy.
and four sarcomeres per myofibril per measurement unit, respectively. For high precision (99%) measurement, 34 fibres would have to be dissected and measured by the laser method. Measurement of 40 to 60 units by the microscopic methods could be accomplished by the preparation of three to four slides. Based on the performance of the technicians participating in this study, we estimate that one technician could measure up to 500 samples per week with the laser (95% precision) and up to 65 samples per week with the microscope. High precision (99%) would reduce the number of samples measured by the laser and microscope to less than 30 per week. However, if the 95% level of precision is used, technicians using the laser method would measure only one muscle fibre per animal, whereas use of the microscope methods would provide the possibility of measuring sarcomeres from two or three fibres, respectively. This may be of significance in samples having fibres of the same muscle with different amounts of shortening, in that one, two or three fibres may not be representative of the remainder of the muscle. Therefore, it is suggested that a greater number of observations be made (four or five) to alleviate this possibility. REFERENCES
CLEWORTH,D. & EDMAN,K. A. P. (1969). Science, 163,296. GUEWrHER, W. C. (1964). In Analysis oJ variance. Prentice-Hall, Inc., Englewood Cliffs, N J, USA. HERRING,H. K., CASSENS,R. G. & BRISKEY,E. J. (1965). J. Food Sci., 30, 1049. HOSTETLER, R. L., LANDMANN,W. A., LINK, B. A. & FITZHUGH, H. A. JR. (1970). J. Anita. Sci., 31, 47. LOCKER, R. H. (1960). FoodRes., 25, 304. LOCKER, R. H. • HAGYARD, C. J. (1963). J. Sci. FdAgric., 14, 787. MARSH, B. B. t% LEET, N. G. (1966). J. Food Sci., 31,450. ROME, E. (1967). J. MoL Biol., 27, 591, RUDOICK, J. E. & RICHARDS, J. F. (1975).,/. Food Sci., 40, 500. SNEDECOR, G. W. & COCHRAN, W. G. (1967). Statistical methods (6th edn.). Iowa State University Press, Ames. VOYLE, C. A. (1971). Proc. 17th Europ. Meeting of Meat Res. Workers, Bristol, England, p. 95.
C O M P A R / S O N OF M E T H O D S F O R M E A S U R I N G S A R C O M E R E L E N G T H IN BEEF S E M I T E N D I N O S U S M U S C L E H.R. CROSS
Meat Science Research Laboratory, USDA, AR Beltsville, M D 20705, USA R. L. WEST
Animal Science Department, University oJ Florida, Gainesville, USA
T.R. DUTSON
Animal Science Department, Texas A & M University, College Station USA (Received: 4 February, 1980)
S UMMAR Y
The objective of the study was to compare the precision of the laser method for measuring sarcomere length with the precision of two oil-immersion microscope methods. Eighteen semitendinosus muscles were assigned to one of three postmortem treatments to provide a wide range in sarcomere length. Two 5.0cm sections were removed from the centre of each muscle. Each section was subdivided into six equal-size pieces and randomly allotted to each of three participating institutions. Analysis of variance revealed that sarcomere length measurement was not significantly affected by the method of measurement or by the technician. For 99% precision, the laser method required 34 measurements, whereas the two microscope methods required 45 and 66 measurements, respectively.
INTRODUCTION
Since Locker (•960) reported on the relationship of muscle shortening (cold shortening) to meat tenderness, several workers have conducted further investigations on this relationship (Locker & Hagyard, 1963; Marsh & L e e t , 1966; Herring et aL, 1965; and Hostetler et aL, 1970). These workers and others have clearly demonstrated that when muscles are treated to alter sarcomere length, there is a high, positive correlation between sarcomere length and tenderness. Since a direct Any reference to a brand or firm name within this paper, does not constitute endorsement by the US Department of Agriculture.
261 Meat Science 0309-1740/81/0005-0261/$02-50 © Applied Science Publishers Ltd, England, 1981 Printed in Great Britain
262
H . R . CROSS, R. L. WEST, T. R. DUTSON
correlation between state of muscle contraction and ultimate meat quality does exist, it would be desirable to know whether the different methods of measuring sarcomere length are similar and which method is the simplest and most reliable one available. The most commonly used method of sarcomere length measurement is the oilimmersion, phase-contrast microscopy of unstained tissue. With this method, the space between adjacent Z-discs is measured with various calibrated eye-piece measuring devices. The type of measuring device is usually some type of eye-piece micrometer; however, more sophisticated and costly devices, such as the particle counter/size analyser, have been used. A different method which has been used (Voyle, 1971; Rome, 1967; and Ruddick & Richards, 1975) was derived initially from early observations that a striated muscle acts as a transmission grating when placed in a beam of light. With this method, diffraction patterns are formed on a screen by transmitting monochromatic light through muscle fibres, and the separation of the orders of diffraction are determined by the contractile state of the muscle. Rome (1967) and Cleworth & Edman (1969) described developments in which a gas laser was used as a source of coherent monochromatic light. Ruddick & Richards (1975) compared sarcomere length measurement of cooked chicken peetoralis muscle by laser diiiraction and oilimmersion microscopy. Their results indicated that the laser method was equal, or superior to, the oil-immersion methods. The primary objective of this study was to compare the precision of the laser technique with the precision of two oil-immersion techniques for measuring sarcomere length in bovine muscle. An additional objective was to evaluate the human error within each procedure and to determine the minimum number of measurements needed to obtain a given degree of precision with each procedure.
EXPERIMENTAL
Sample selection and preparation Semitendinosus muscles from four beef carcasses were excised at about lh postmortem, divided lengthwise into three equal parts, and one part of each muscle was assigned to one of three post-mortem treatments. Muscle strips in treatment 1 were stretched maximally and held in that position by clamps on each end of the muscle strip. Muscle strips in treatment 2 were immobilised in a slightly stretched condition, with clamps at each end of the muscle. Muscle strips in treatment 3 were not stretched or immobilised. These three treatments were designed to provide a wide range in sarcomere length. Two 5.0cm sections were removed from the centre of each muscle strip, and each section was subdivided into six equal-size pieces. Then two pieces from each section (four pieces from each muscle strip) were randomly allotted to each of the three participating institutions. Each piece was double wrapped in aluminium foil and
SARCOMERE LENGTH MEASUREMENT
263
stored in liquid nitrogen until shipped to the respective institutions. Samples were shipped in dry ice via air freight. After the samples reached the respective institutions, one piece from each section (two pieces from each muscle strip) was allotted to each of two highly trained technicians for measurement of sarcomere length by one of the three methods described below.
Laser diffraction method Twoborate-KC1 buffer solutions containing glutaraldehyde were used as fixatives. Solution A consisted of 0.1M KC1, 0.039M boric acid, and 5mM E D T A in 2-5% glutaraldehyde. Solution B consisted of 0.25M KC1, 0.29M boric acid, and 5raM E D T A in 2.5% glutaraldehyde. The muscle tissue was cut into 3.0 × 3.0 × 2.0cm pieces and placed in a vial and covered with solution A for 2h. The sample was then transferred to a fresh vial containing solution B for 17-19h. Sarcomere length was measured t h e following day. Sarcomere length by laser diffraction was determined by procedures similar to those described by Ruddick and Richards (1975) except that the calculations differed due to the use of a flat surface (screen) instead of a convex surface (as in this instance). The apparatus consisted of a helium-neon laser (with a wavelength of 632-8nm) which was mounted on an optics bench with a specimen-holding dbvice and a screen (Fig. 1). Individual fibres were teased from the muscle bundle and placed on a microscope slide with a drop of solution B. The slide was then placed horizontally in the path of a vertically orientated laser beam to give an array of diffraction bands
I
i
,: ! ',
Fig. 1. Diagram of laser technique.
264
H. R. CROSS, R. L. WEST, T. R. DUTSON
on the screen. These bands were perpendicular to the long axis of the fibres (Fig. 1). Sarcomere lengths were calculated by use of the following formula: 632.8 x 10-3 x D x N/(-~TD)2 + 1 Sarcomere length (microns)= T where D equals the distance (mm) from the specimen-holding device to the screen (throughout this experiment, D had a constant value of 100mm) and T equals the separation (mm) between the zero and the first maximum band. Twenty-five fibres were measured by each individual on each sample piece.
Filar micrometer method A 5g muscle sample was cut into small pieces and homogenised at low speed (approximately 5000rpm) in 30ml of chilled 0-25M sucrose solution, in a Virtis '23' homogeniser, for 60sec. A drop of homogenate was transferred to a slide and covered with a cover slip. The slide was examined under the phase microscope (100 X objective, 10 X eye-piece) with oil-immersion for the number and length of myofibrils. I f fibres were not sufficiently broken apart, the sample was homogenised an additional 20-30sec. This procedure was repeated as necessary up to three times, taking care that the myofibrils were not homogenised to less than 10 sarcomeres in length. The length of 10 sarcomeres on each of the first 25 myofibrils, located by starting at one edge of the slide and moving straight across, were measured with a filar micrometer containing a Vernier scale calibrated against a stage micrometer. Values were converted using the calculated calibration factor (the filar micrometer was adjusted so that 0 . 0 1 m m = 100~tm thereby eliminating the conversion of each measurement). Shearieon size analyser method A 3g sample of muscle was cut into small pieces and allowed to thaw. The sample was placed in a 50ml beaker with 35ml of 0.25 M surcrose and blended at high speed for 30sec in a Virtis '33' macro-homogeniser. After blending, a small drop of the sucrose solution was placed on a slide, covered with a cover slip, and placed under the oil-immersion lens on a Zeiss W L phase-contrast microscope equipped with a Timbrell Coulter Shearicon particle counter/size analyser. The Shearicon was calibrated with a stage micrometer to give measurements to the nearest 0.01mm. The mean sarcomere length of each muscle was determined by measuring 25 myofibrils containing four sarcomeres each. Statistical analysis Data were treated by analysis of variance procedures as outlined by Snedecor & Cochran (1967). The power of the test was calculated according to Guenther (1964).
SARCOMERE LENGTH MEASUREMENT RESULTS
AND
265
DISCUSSION
Results o f analysis o f variance revealed that s a r c o m e r e length m e a s u r e m e n t did not differ significantly with different m e t h o d s of m e a s u r e m e n t or with different technicians. A s expected from the experimental design, effects of different muscle t r e a t m e n t s were all highly significant. M e a n sarcomere lengths are segregated by m e t h o d o f m e a s u r e m e n t , technician and treatment in T a b l e 1. A s predicted, restraint t r e a t m e n t significantly altered s a r c o m e r e length. W i t h i n each muscle treatment, s a r c o m e r e length was n o t affected b y the m e t h o d o r the technician. M e a n s and s t a n d a r d deviations within each t r e a t m e n t are r e m a r k a b l y similar. One possibility when the m i c r o s c o p e techniques are used to m e a s u r e s a r c o m e r e length is t h a t fatigue m a y a c c o m p a n y the method. F a t i g u e can result in error, increased variability, or both. H o w e v e r , the results o f this s t u d y indicated t h a t all six technicians obtained c o m p a r a b l e results, regardless o f m e t h o d ; and fatigue, which might affect the results, was n o t observed. TABLE 1 MEAN SARCOMERE LENGTH ( ~ m ) AND STANDARD DEVIATION BY MUSCLE TREATMENT AND TECHNICIAN WITHIN METHOD
Method of measurement
Muscle treatment Maximum stretch
LaseF
Technician 1 Technician 2 Fi!ar micrometer Technician 1 Technician 2 Shear±con/size analyser Technician 1 Technician 2
Slight stretch
Unrestrained
3.35 + 0-27~ 3-37 _+0.24 a
2.77 + 0.25 ~ 2-72 ± 0.27 ~
1-89 + 0-0Y 1-93 + 0-06c
3.39 ± 0-17 a 3-39 + 0-18a
2-70 + 0-14 ~ 2.73 + 0-22 b
1.98 + 0-08c 1-99 ± 0-09c
3.19 + 0-22 a 3-31 + 0.15 a
2-71 _+0-18~ 2.67 + 0.18 ~
1-97 + 0. I(F 1-97 _ 0-10e
~.n.cMeans in the same row and column with different superscripts are significantly different (P < 0"001). The numbers o f sarcomeres per sample m e a s u r e d and reported in the literature varies considerably. T o determine the m i n i m u m n u m b e r of m e a s u r e m e n t s n e c e s s a r y to o b t a i n a p a r t i c u l a r level o f precision, we calculated the p o w e r of the test using the coefficient o f variation f r o m each method. Since technicians h a d no significant effect on sm.comere length, technicians within e a c h m e t h o d were c o m b i n e d (Table 2) for calculating the coefficient o f variation. F o r 99% precision, the laser method required 34 m e a s u r e m e n t s whereas the two oil-immersion m e t h o d s required 45 and 66, respectively. A precision o f 95% required one, two a n d three m e a s u r e m e n t s for the laser a n d the two m i c r o s c o p e m e t h o d s , respectively. Differences in coefficients of variation m a y have been due, in part, to the n u m b e r o f s a r c o m e r e s m e a s u r e d per observation. T h e laser m e t h o d m e a s u r e s m a n y s a r c o m e r e s within the b e a m ' s path, but the Film" m i c r o m e t e r and Shear±con/size a n a l y s e r techniques measure only ten
266
H . R . CROSS, R. L. WEST, T. R. DUTSON TABLE 2 MINIMUM NUMBER OF MEASUREMENT UNITS REQUIRED TO ASSURE A
PRECISION OF 95 OR 99 PER CENTa
Method of measurement
Number oJ units required ~ Coefficient of variation
95% precision
99% precision
5.8 6- 7 8.1
1 2 3
34 45 66
(%)
Laser Filar micrometer Shearicon/size analyser
"Measurement units differ for each method as follows: Laser--fibres; Filar micrometer--myofibrils with 10 sarcomere units; and, Shearicon/size analyser--myofibrils with four sarcomere units. bPower of the test was calculated by: n ~ (coefficient of variation)2/p2, where P is the proportion of desired accuracy.
and four sarcomeres per myofibril per measurement unit, respectively. For high precision (99%) measurement, 34 fibres would have to be dissected and measured by the laser method. Measurement of 40 to 60 units by the microscopic methods could be accomplished by the preparation of three to four slides. Based on the performance of the technicians participating in this study, we estimate that one technician could measure up to 500 samples per week with the laser (95% precision) and up to 65 samples per week with the microscope. High precision (99%) would reduce the number of samples measured by the laser and microscope to less than 30 per week. However, if the 95% level of precision is used, technicians using the laser method would measure only one muscle fibre per animal, whereas use of the microscope methods would provide the possibility of measuring sarcomeres from two or three fibres, respectively. This may be of significance in samples having fibres of the same muscle with different amounts of shortening, in that one, two or three fibres may not be representative of the remainder of the muscle. Therefore, it is suggested that a greater number of observations be made (four or five) to alleviate this possibility. REFERENCES
CLEWORTH,D. & EDMAN,K. A. P. (1969). Science, 163,296. GUEWrHER, W. C. (1964). In Analysis oJ variance. Prentice-Hall, Inc., Englewood Cliffs, N J, USA. HERRING,H. K., CASSENS,R. G. & BRISKEY,E. J. (1965). J. Food Sci., 30, 1049. HOSTETLER, R. L., LANDMANN,W. A., LINK, B. A. & FITZHUGH, H. A. JR. (1970). J. Anita. Sci., 31, 47. LOCKER, R. H. (1960). FoodRes., 25, 304. LOCKER, R. H. • HAGYARD, C. J. (1963). J. Sci. FdAgric., 14, 787. MARSH, B. B. t% LEET, N. G. (1966). J. Food Sci., 31,450. ROME, E. (1967). J. MoL Biol., 27, 591, RUDOICK, J. E. & RICHARDS, J. F. (1975).,/. Food Sci., 40, 500. SNEDECOR, G. W. & COCHRAN, W. G. (1967). Statistical methods (6th edn.). Iowa State University Press, Ames. VOYLE, C. A. (1971). Proc. 17th Europ. Meeting of Meat Res. Workers, Bristol, England, p. 95.