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N E T H O D S U S E D IN T H E S T U D Y O F H N O L O G ~ C A L
S T R U C T U R E O F H E A T
D R. P A U L I N E PAUL
M I C H I G A N STATE C O L L E G E
Histology deals with the study of the rniscroscopic appearance of c e l l s and tissues. So the study of the histological structure of meat involves prep- aration of sections thin enough t o be studied microscopically, and the observa- t i o n and interpretation of the r e s u l t s obtained.
Histological technics have been i n use f o r many years, but it i s only
The e a r l i e r work dealt principal-
within the past 10 o r 15 years that these technics have been applied t o obeer- vation of material from the food standpoint. l y with anatomical d e t a i l s. technics, and discuss b r i e f l y the types of r e s u l t s which can be obtained.
I propose t o review f o r you some o f t h e basic
The principal steps i n histological study consist of k i l l i n g and fix-
i n g the tissue, solidifying it i n eome way t o permit cutting it into very t h i n slices, cutting the sections, staining and mounting on slides, and studying the
r e s u l t s. Fixing t h e t i s s u e i s usually necessary i n order t o stop the a c t i v i t y
of the c e l l a and preserve the material f o r f'uture study. T e n percent formalin
(4$ formaldehyde i n water) i s frequently used f o r k i l l i n g and fixing.
sue blocks can be preserved i n formalin f o r several months i f ' time does not per- mit i m e d i a t e preparation of the slides. However, f o r some studies the m a t e r i d cannot be h a d l e d i n t h i s manner. t h e distribution of mobile fat droplets (not f a t deposits) within and around t h e c e l l s , he found it necessary t o work with unfixed material and prepare the sections immediately, If one wishes t o study glycogen distribution i n t h e
muscle t i s s u e , it i s necessary t o f i x the blocks i n 9545 alcohol, and the t i s s u e s
must be sectioned within a short time as prolonged exposure t o higher concentra- t i o n s o f alcohol w i l l shrink and harden the t i s s u e so that it i s extremely d i f - f i c u l t t o section.
The tis-
I n D r. Beard's work, where he was studying
There a r e three common technics f o r embedding o r hardening the t i s s u e so that it may be sectioned, When you r e a l i z e t h a t f o r most problems it i s necessary t o cut the sections 10 microns (1/2500 inch) or l e s s i n thickness, you will understand t h a t ordinary s l i c i n g with a sharp knife w i l l not do.
Paraffin embedding consists of dehydrating the tissue, i n f i l t r a t i n g with paraffin, and embedding i n a block of paraffin. Dehydration may be car- r i e d out i n a number of ways. The most common method8 a r e t o use (1)a graded series of alcohols of increasing concentration, (2) dioxane, or (3) chloroform. The point is that the water must be removed from the t i s s u e , and replaced with a solvent miscible w i t h paraffin, BO t h a t when t h e t i s s u e i s placed i n paraffin, t h e paraffin w i l l penetrate d l the way through, not j u s t form a framework around the t i s s u e. the paraffin, and w i l l not cut properly on the microtome.
If this happens, the t i s s u e w i l l d i f f e r i n hardness from
The microtome i s a device for cutting very t h i n sections and consists principally of a very sharp knife coupled t o a device f o r advancing t h e block any desired distance after each section i s cut. sections of only a few microne thicknesa.
This enables one t o cut uniform
Paraffin blocks are usually cut on a rotary microtome. I n t h i s de- vice, the block moves while the knife i s stationary. Since the paraffin sec- t i o n s w i l l adhere t o each other, it i s possible t o obtain t h e sections i n r i b - bons. This makes it possible t o make s e r i a l sections through a whole block of t i s s u e i f one wishes t o observe changes with depth. Another advantage i s t h a t t h e paraffin blocks can be preserved almost i n d e f i n i t e l y , sections can be mounted on t h e s l i d e s and then stained, which i s usually easier than staining t h e sections and then mounting them.
Also, t h e paraffin
There a r e d l s o some disadvantages t o the paraffin method. The sol- vents used t o dehydrate and i n f i l t r a t e t h e t i s s u e dissolve out the fat, so t h i s technic i s not suitable f o r studies of f a t d i s t r i b u t i o n. Also, the dehydration and t h e heat used i n i n f i l t r a t i o n tend t o shrink and d i s t o r t t h e muscle t i s s u e s , so i f one wishes t o measure s i z e o f muscle f i b e r s , f o r instance, another technic must be used.
The second technic f o r cutting t i s s u e s involves freezing the block with C02 and c u t t i n g on a freezing microtome. This microtome i s arranged so t h a t the knife t r a v e l s across t h e block, cutting t h e section. A s the b i f e is returned, t h e tissue c a r r i e r advances t o raise the block f o r the next section. A connection t o a C02 tank i s provided so t h a t CO2 under pressure i s released j u s t under t h e t i s s u e block t o^ freeze^ it. l y washed out of the t i s s u e before freezing s o t h a t it will freeze evenly. Tissues f o r frozen sections m a y o r may not be embedded. With cooked meat, em- bedding helps, as the t i s s u e i s so loose that t h e sections tend t o d i s i n t e g r a t e when put i n water. Ten percent g e l a t i n i s usually used f o r embedding. I n t h i s case, it i s not necessary t o i n f i l t r a t e t h e t i s s u e completely, j u s t t o form a secure frame around t h e block. This can usually be accomplished by holding t h e block i n the g e l a t i n solution f o r an hour o r so a t a temperature j u s t high enough t o keep t h e g e l a t i n melted, must be avoided, as t h i s a l s o hardens t h e t i s s u e and makes it d i f f i c u l t t o cut.
The f i x i n g solution must be thorough-
Prolonged exposure of the blocks t o heat
The frozen sections a r e removed from t h e knife w i t h a small brush and placed i n water. celloidin, then stained, o r they may be transferred through the s t a i n s i n a small basket, then mounted after staining,
Then they may be mounted d i r e c t l y on s l i d e s and covered w i t h
Slides prepared f’rom frozen sections a r e well adapted t o studies of fat distribution, s i z e of muscle fibers, and such items. be very laborious t o obtain serial sections. Also, much more handling of t h e sections i s required than with paraffin sections, and t h i s increases t h e d i f - f i c u l t y as t h e sections are very f r a g i l e.
However, it would
The t h i r d technic f o r preparing and cutting t h e blocks involves em-
bedding i n c e l l o i d i n and cutting t h e sections on a s l i d i n g microtome similar
i n action t o the freezing microtome, but without the freezing attachment. Celloidin embedding gives a firm block which i s e a s i l y cut,^ and causes much l e s s shrinkage and d i s t o r t i o n than paraffin embedding, d i f f i c u l t t o obtain serial sections. as it requires 1 0 t o 20 days t o properly i n f i l t r a t e and harden t h e blocks.
However, again it i s Also, t h i s method i s very time consuming
The methods of staining and mounting t o be employed depend on t h e things f o r which you are looking. hemotoxylin s t a i n s i s usually employed, use, as it can be prepared and used t h e same day. age up t o a c e r t a i n point.
To study j u s t t h e muscle t i s s u e , one of t h e Harris haemotoxylin i s t h e one we However, it improves with
Regular haemotoxylin must be aged 6 months t o a
f r u i t f u l is the use of the phase microscope f o r studying unstained material. It is necessary t o recognize the p o s s i b i l i t y t h a t each additional s t e p i n pre- paring materials f o r observation may cause chax@es i n the t i s s u e fromwhat it w a s originally. steps may yield r e s u l t s more indicative of t h e true state of the c e l l s under observation.
Therefore, a device which tends t o eliminate any of these
Histological work is^ very^ slow^ and^ time^ consumir?g.^ It^ i s^ not^ a^ tool s u i t a b l e f o r use with studies of short duration. It requires considerable ia- vestment of t i m e and e f f o r t , and a l s o a f a i r l y large investment of money f o r equipment. Zowever, i n work where there i s a continuity from year t o year, it may be profitably employed t o study charges which d e f i n i t e l y a f f e c t the pal- a t a b i l i t y of meat, and which cannot be observed macroscopically. It^ enables one t o g e t at some of the basic causes f o r the changes which occur i n m e a t due t o various treatments such as freezing, aging,^ and cooking,^ and due t o^ pro- duction aspects such a6 feed, age and sex.
References on Histological Technics as applied t o Meat Research. May 1 7 , 1949
A - General
- Conn, H.J. 1948 The history of staining. 2nd. Biotech Pub., Geneva, N.Y.
2. Mann, Gustav
1902 Physiological histolcgy - Methods and theory. C larendon Press
3. Maximow, A.A., W. Bloom 1948 Textbook of histology. 5th ed. W.B. Saunders Co.
- Sisson, S., J. D. Grossman 1948 Anatomy of the domestic animals. 3rd ed., rev. W.B. Saunders Co.
5. Dempsey, E.W., Wislocki, G. B. , Singer, M.
1946 Some^ observations of the chemical cytology^ of^ s t r i a t e d muscle. Anat. Rev. 96 #3 (Nov. )
6. Hmisch, C.M. 1947 Muscles, a review of anatomy, histology and physiology. Bull. Hosp. J o i n t D i s. 2 : 203-
- Murroughs, T.R. 1944 Muscle, fibrous and nerve tissue. Optic Journ. 81:^ 22,^ #18^ (Sept.^ 15)
- Rosenberg, L.E. 1947 Histological studies^ of^ muscle from crooked neck dwarf^ fowl. Anat. Rec. 97 #3, March
9. Schmitt, F. O., Bear, R.S.
1947 Electron microscope and x-ray d i f f r a c t i o n studies of muscle 8 tructure. Ann. New York Acad. Sc. g: 799-
B - Technic
1. Becker, E.R., R.L. Roudabush 1939 Histolagical Technique.
Collegiate Press Inc., Ames, Iowa
2. Benaley, R.R., S.H. Bensley 1941 Handbook of histolcgical and cytological technique. U. of Chicago Press 3. Conn, H.J., e 9 1940 Biolcgical s t a i n s. 4th ed. Biotech Pub., Geneva, N.Y.
4. Gatenby, J.B., J.S. Painter
1937 Microtomist's Vade-mecum. 10th ed. P. Blakiaton's Son & Co.
5. Guyer, M.F.
1945 Animal microlcgy, 4th ed., rev., U. of Chicago PXY 386
- L i l l i e , R.D. 1948 Histopathologic technic. Blakiston Co.
7. McClung, C.E.
1929 Handbook of microecopical technique.
Paul B. Hoeker, Inc.
C - E q u i p n t
1. Gage, S.H. 1943 The microecope. 17th ed. Comatock Pub. Co., Inc., Ithaca, N.Y.
- Richards, O.W. 1942 The e f f e c t i v e use and proper care of the microtome. Spencer Lens Co., Buffalo
D - Photomicrography
- Eastman Kodak Co. 1944 Photomicrography 4 14th ed.
2. Shillaber, C.P. 1944 Photomicrography. John Wiley & Sons, Inc,, N.Y.
12. Shrswebury, C.L., e $il&
1942 Chemical, histological and p a l a t a b i l i t y changes i n pork during freezirrg and storage i n the frozen state. Purdue U. Agrl. Expt. Sta. Bul. 472
13. Stewart, G.C., e t a 1945 Effects of aging, freeziw rate, and storage period on p a l a t a b i l i t y of b r o i l e r s.
Food R e s. - 10: 16-
14. Twoson, A.M.
1940 P a l a t a b i l i t y studies of beef rib roasts. I. As G f e c t e d
by high v e r ~ u slow oven temperatures. M.S. thesis. Iowa S-te College
C m BUTLER Thank you very much, Dr. Paul. ,
Professor Wanderstock w i l l please conduct the discussion on t h i s paper.
PROF. WANDERSTOCK: I an sure all of UB enjoyed Dr. Paul very
f i n e review of the histolc@cal techniques involved i n mat research. I think she did a n excellent j o b of Bummariziw t h e methods of imbedding t i s s u e , and the slides were c e r t a i n l y helpful t o most of U S.
There is^ a^ new^ method t h a t^ she did^ not^ mention,^ the so-called
"freezing - drying" method of t i s s u e preparation which involves very, very
l i t t l e chemical change. She mentioned t h a t a l l of these methods involve a d e p e e of chemical change. very l i t t l e chemical change.
The freezing-drying technique is one that involves
I thought you might be i n t e r e s t e d i n seeing some pictures of what t h e electron microscope shows, and s o I have a r e p r i n t here of some English
work t h a t I w i l l pass around, and you can see t h i s while we discuss Dr. Paul's
paper
Do any of you have any questions you'd l i k e t o d i r e c t t o Dr. Paul?
PROF. BIUMER: What method do you use i n l a b e l i x muscle %issue after it comes out of the picture? get a cross-section o r n o t i n the imbedding of the tissue.
It is r a t h e r d i f f i c u l t t o know whether you can
DR. PAuLr When we are collecting o u r samplea, we c u t 10% s t r i p s
about as big as my l i t t l e finger, and put them r i g h t i n formalin as w e are cooking, or whatever the treatslent is that we are doing. Then those a r e allowed t o sit over-night, or 24 hours. eize. We t i e them up i n cheesecloth and put a label on them, and then thoy c a n be put i n big b o t t l e s t o aave time and space. Otherwise, you are going t o have your whole laboratory o r a l l your space, covered with l i t t l e b o t t l e s , because they accumulate t e r r i f i c a l l y.
They are then c u t down t o a smaller
Then t h a t material is very easy t o work with. It i s good and firm, and from those s t r i p s w e c u t our blocks before w e start imbedding them. c u t very carefully e i t h e r along o r across the f i b e r s. t r a i n i n g t o l e a r n j u s t how t o do it, and then, of course,^ you^ look^ a t^ the^ first section, and if it did not t u r n out t o be what it is supposed t o be, you cro it out and start with another block.
We It takes considerable
We routinely c o l l e c t s u f f i c i e n t material s o t h a t we can go back and
If you do not have something t o go back to, then it is It is a good idea t o
start over again several times, i f we need t o , because you can g e t i n t o a l l kinds of d i f f i c u l t y. not too easy t o go back and start with another animal. g e t plenty while it is there, and then you have something t o work with.
PROF. BULL: Professor Wanderstock, several of us are j u s t poor, dumb meat men, and^ a^ few of u s do not h e s i t a t e t o show our ignorance^ at^ times, but what i s a Phase microscope.
DR. PAUL: Well, I have only seen one, and I w i l l t r y t o tell you about it. It is so arranged t h a t the l i g h t comes up underneath the stage through a very small, very carefully defined c i r c l e.^ It^ is^ s o r t of^ on the order of a dark f i e l d microscope, but the l i g h t comes through t h i s l i t t l e c i r c l e , it h i t s your material, and then everything is blocked out except j u s t t h i s one s p e c i a l ray. Up and above they have another c i r c l e. A n y w a y , some- t h i q about the optics of the system puts the l i g h t out of phase, s o t h a t p a r t of it comes through it a t one peak or point i n the wave, part of it come through at another point, s o t h a t you g e t your contrast, your dark and l i g h t , i n your material without h a v i n g t o s t a i n it.
You see, you s t a i n it s o you can w e it, and t h i s means of putting the l i g h t out of s t e p i n some way BO t h a t you can see it j u s t with the l i g h t. You do not have t o have any stains i n it. new. They have j u s t come out i n the last couple of years. I think both Bausch & Lomb and Spencer have them now, and they have a l o t of information on haw they work. I sm not a good enough physicist t o know.
The Phase microscopes a r e f a i r l y
PROF. BULL: Thank you. Nelson t e l l s me he has one,
PROF. WANDERSTOCK: J u s t one other thing on the Phase microscope:
It actually enables u s t o see s t r u c t u r e s t h a t we could not see with the ordi- nary l i g h t microscope. I n other words, it brings out some of the very minor constitutents; things t h a t we have been c a l l i n g a r t i f a c t s ; thinge t h a t w e can not describe and we guess at, and it has enabled the h i s t o l o g i s t t o actually see what those are.
PROF. NELSON: I have a question on that with reference t o the a b i l - i t y t o g e t your l i g h t through your t i s s u e sections. I have used the Phase microscope i n connection with protozoa and micro-organisms, (^) but there is a question i n my mind as t o the thickness of t h e t i s s u e s and the amount of l i g h t you can g e t through i n order t o g e t d i f f e r e n t i a t i o n.
DR. PAUL: I have not seen any work where it has been applied t o meat. micron.
I do know they have techniques f o r cutting sections for a t e n t h of a
PROF. NELSON: That c e r t a i n l y is t h i n enough.
