255.

A PHOTOGRAPHIC TECHNIQUE FUR THE D E T f R M I N A T I O N OF PERCENT AREA OF E L A ' S T I C F IBERS A N D COLLAGENOUS BUNDLES IN H U S C I E 1 * 2 ~ 3

Various chemical methods have been u t i l i zed -Go determine the amount of collagen and/or e l a s t i n in muscle. been made t o d i f fe ren t ia te the various tissue6 by staining procedures. Fewer attempts have been made t o quantitatively determine the amount of collagen o r e l a s t in u t i l i z i n g photographic techniques.

However, few attempts have

Previous work by the authors R l a t i v e t o var ia t ion i n muscle and

After several attempts, a one-inch borer was used t o excise sample location caused some concern i n obtaining a sample representative of t h e muscle. sample cores. thoracic vertebra. Care was taken t o assure that the bore was sharpened t o a keen edge p r io r t o i ts use. This provided a smoth cut and reduced fiber d is tor t ion resul t ing f r o m undue pressure during sampling. Three sample cores were removed fmm each longissimus dors i muscle by cutt ing parallel t o the long axis of the muscle; one near the vertebra (dorsal), one close t o the l a t e r a l margin of the muscle ( l a t e ra l ) and one i n the middle of the muscle (middle). semitendinosus muscle parallel t o i t s long axis. a pre-chilled screw-cap bo t t l e and kept refrigerated during the brief delay pr ior t o preparing them f o r the f ixat ive. thick were cut transversely and placed i n a f ixat ive.

The longissimus dorsi muscle w88 sampled opws i t e the 13th

A single core was removed f r o m the center of the Each core was placed i n

Sl ices one-fourth of an inch

The f ixa t ive solution consisted of 1 part formalin t o 9 par t s d i s t i l l e d water with the addition of 20 gms. per l i t e r sodium acetate and 9 gms. per l i t e r sodium chloride. The sodium chloride served no usef'ul purApse and could have been excluded. The sodium acetate, however, acted a8 a buffer.

The one-quarter inch th ick s l i ce s from each core were fixed a t 36OF. Fresh f ixa t ive was added after 24 hours t o res tore the formalin con- centration t o near 10 percent and provide the storage medium. minimum storage of 48 hours, the thiclmess of the s l i c e s was halved t o one- eighth of an inch and pmcessed f o r paraffin embedding by dehydration i n graded solutions of isopmpanol and clearing i n acetone.

After a

lJourna1 Series Paper No. 802.

'Presented before the 15th Annual Reciprocal Meat Conference, Columbus,

3Financed i n part by the U. S, Dept. Agri. under the Research and Market-

Approved by the Director of the OUahoma Agriculture Experiment Station.

Ohio, June 13-15, 1962.

ing Act, Contract No. 12-25-010-576.

256.

The embedding pmce6s'too'k place i n a la rge thick-walled glass container, under reduced pressure and y i t h a slow continuous a i r wash over the surface of the molten paraffin. temperature of 54OC. during a four-hour process period.

The container was held i u an oven a t a

The acetone i n the t i s sues sh i f ted t o the vapor phase and sassed out through the vacuum tube of the container with the a i r of the wash. Ehbedding temperatures above 56.5OC. were found t o increase the b r i t t l eness of muscle proteins . t i on and block formation. f i n blocks.

Paper molds held molten paraff in f o r t i s sue or ienta- Ice water was used t o rapidly so l id i fy the paraf-

Before sectioning, the paraff in blocks were trirmped t o expose one surface of the t i s sue , and soaked in i ce The i c e water rehydrated the muscle proteins and helped t o minimize t i s sue shat ter ing. Sections 6 microns th ick were cut on a rotary microtome, and f loa ted i n water held a t a temperature of 48 C. Thirty-two milliliters of a 1 percent ge la t in solut ion were added t o the 1850 m i l l i l i t e r s of water i n the bath t o provide a weak adhesive between s l i d e and section. slide was permitted t o dry f o r 24 hours i n a 54OC. oven and then stored a t room temperature u n t i l stained.

water f o r four or'more hours.

Each

The s t a i n f o r elastin wasGomri'6 Aldehyde Fuchsin, modified by exchanging isopropanol f o r ethanol. t he s t a i n caused them t o become a deep purple color.

The a f f i n i t y of e l a s t i c f i b e r s f o r

Collagen was stained by u t i l i z i n g the masking properties of p i c r i c acid, on mst t i ssues , and the s ta ining propert ies of acid fuchsin on collagen (Modification of t he Van Gieson Technique).

The deparaffinized and rehydrated t i s sue aections were placed i n a saturated aqueous solut ion o f p i c r i c acid f o r approximately 30 minutes. The s l ides were then placed d i r ec t ly in to a O , l $ aqueous solution of acid fuchsin saturated with p i c r i c acid and l e f t f o r one hour. collagen fibers adsorbed a limited amount of fuchsin while the p i c r i c acid masked the o ther t i s sues . aqueous solution of ac id f'uchsin containing p i c r i c acid (90 percent satur- ated) f o r 5-10 minutes. solut ion and in tens i f ied the s ta ining of collagen and permitted the muscle t o absorb some f'uchsin. any required d i f fe ren t ia t ion since p i c r i c acid and acid fuchsin a re re- moved f r o m a l l t i s sues a t about the saw ra t e . higher concentrations of isopropanol in to xylene lessened f'urther undesir- able d i f fe ren t ia t ion .

I n t h i s solution

The s l ides were then t ransferred t o a 0.1%

Picr ic acid diffused from the t i s sues i n t h i s

Seventy percent isopropanol provided a means f o r

Rapid passage through the

The s ta ined balsam mounted t i s sue sections were photographed using Kadalith Super Ortho film i n a Bausch and Lomb Model H, Camera equipped with a Zeiss Tessar 49 mm. lens . proximately 1 . 2 x .95 cm. a 4 x 5 inch negative. (3 min,) with Super Line developer and dried. negative was placed i n an enlarger, and the amount of l i g h t transmitted t o a phototube mounted i n a "search uni t" was recorded on the indicator of a Photovolt Electronic Photometer, Model 514-14. The amount of l i g h t t r a n s - mitted through the negative was expressed aa a percent of the are8 composed of collagen o r e l a s t in .

The area of t i s sue photographed was ap- This was enlarged approximately eleven times t o

The exposed f i l m (24 sec.) was then developed An 8 x 11 cm. section of the

257.

To evaluate the e r ror i n the photometric determination f o r e l a s t in and collagen, each of four balsam mounted sections were photographed several times. with the photometer,

The negatives were developed separately and multiple readings made

The data representing the percent area of the negative occupied by e l a s t i c fibers are shown i n table I.

Table I. Repeatability Studies f o r Determining Percent Area of Elast ic Fibers i n b s c l e

I Neg . 1st Reading 2nd Reading 3rd Reading 1 ,380 .360 .360 2 ,350 .355 .355 3 ,340 ,345 .342 4 .340 ,350 .355

Similar data representing the percent area of the negative occu- pied by collagenous bundles may be seen i n table 11,

Table 11. Repeatability Studies f o r Determining Percent Area of Collagenous Bundles i n Muscle

Neg . 1st Reading 2nd Reading 3rd Reading 1 .049 .053 ,043 2 .041 ,042 ,036 3 .a0 ,042 .041 4 .035 .036 ,045 5 . a 0 .041 ,052

Mean .042 2.006$

The greater percent e r ro r f o r collagen is due to the thinness of the collagen fibers. Variation i n exposure and development time have their e f fec ts primarily a t the junction of opaque and c lear areas of the negative rather than uniform changes throughout the image area. smaller the image, the greater the e r ro r .

Therefore, the

Pin holes o r other a r t i f a c t s in the negative a re responsible f o r less than opt ica l density of the oN.que background of the negative resulted i n varia- t i on of 2 .005 percent. sul ted i n ?: .015 percent.

.0003$ variat ion i n the area determined. Variation i n t h e

Variation i n the distinctness of the image re-

This technique, applied t o t issues f r o m animals differ ing i n age, gave data as presented i n tables I11 and IV.

258.

Table 111. Percent Area of Collagenous Bundles i n Two Bovine Muscles f r o m Animals Varying i n Age

6 18 42 90

- Left Side LD ST

0.141 0 211 0.151 0.214 0.146. 0.086 0.177 . 0,350

Right Side LD ST

0.141 0.092 0.206 0.147 0 185 0.100 0.119 0.098

Table IV. Present Area of Clastic Fibers i n Two Bovine Muscles f r o m Animals Varying i n Age

1 I Left Side

LD ST Right Side

LD ST

0.790 1 8 0.072 1.006 0.024 1.286 42 0.007 0.340 0.036 0.584

0.470 90 0.023 0.288 0.42

6 0,019 0,653 0.013

Some variation i n percent collagen o r e l a s t in was evident from side t o side. same muscle section, as w e l l as, differences within a 6 micron section f r o m the same core. presence o r absence o r a r t e r i e s . may be responsible f o r i t s variation.

It is evident f r o m t h i s work tha t the technique may be useful i n accessing the quantity of the various t i s sues i n muscle. However, fur ther work w i l l be necessary t o provide la rger samples f o r analysis. scanning device tha t could u t i l i z e the en t i r e stained preparation would be preferable t o the photographic technique with i ts ever present errors .

Furthermore, differences were noted amng cores from the

Part of the var ia t ion i n e l a s t in may.be at t r ibuted t o the The minute s ize of the collagen f ibe r s

An automatic

summary:

One inch diameter sections of muscle t i s sue have been success- f u l l y prepared and stained f o r collagen and e las t in . The stained balsam mounted t i s sue sections were photographed and the area of t i s sue enlarged approximately eleven times. The precent area of collagenous bundles and e l a s t i c f ibe r s w&s determined using a Photovolt Electronic Fhotometer t o measure the quansity of l i g h t t ha t passed through the exposed negative. Refined methods f o r measuring la rger sections of t i s sue would be useful.

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Breslau, A . M. 1953. Photographic and Photometric Processes i n Cytochem-

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Lewis, Barnadine, D. L. Harrison and D. S. Folse. 1958, A Photo-micro- graphic Method f o r Estimating the Quantity and Distribution of Fat i n Muscle Tissue. Food Res. 23:l-6.

Pol l i s te r , A. W. and L. Omstein. 1959. The Photometric Chemical Analysis of Cells. Analytical Cytology McGmw-Hill Book Co., Inc., New York.

Rasch, E. and H. Se i f t . 1960. Microphotometric Analysis of Cytochemical Millon Reaction. Jour. Histochem. and Cytochern. 8:4-17.

Rudkin, G. and S. Corlettee. 1957. Ultraviolet Radiations Scattered f r o m Microscopic Objects. Biophy . Biochem. Cytol., 3 :821-825.

A Photographic Method f o r Measuring Jour.

Swift, H. and E. Rasch. 1956. Microphotometry with Visible Light. Physical Techniques i n Biological Research Academic Press, Inc., New York, N. Y.

Wang, H. 1949. A Histo-photoelectric Method of Collagen Determination In k s c l e s . Anat. Rec. 105:537.

Wang, H., E. Rasch, V. Bates, F. J. Beard, J. D. Pierce and 0. G. Hankins. 1943. Cooked Beef. Food Res. 19:314.

Histological Observation on Fat Loci and Distribution i n

(Applause )

MR. BRISKEX: In Relationship of Connective Tissue t o Meat Quality, we'll ask M i l t Bailey t o make h i s presentation f o r us.

l l e t l l move r ight on before we c a l l f o r questions.

Milt.

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