A PARAFFIN METHOD FOR REFRACTORY PLANT MATERIALS

CHARLOTTE PRAIT and R. H. WETMORE, Biological Laboratories, Haruard University, Cambridge, Mass.

Received for publication April 2, 1951

, ~ B S T R A ~ . - A modification of Zirkle’s n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and kilied and fixed in CRAF 111. It is dehydrated in Zirkle’s n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of tempera- tures. The butyl-alcohol-paraffin mixture is gradually replaced b y pure paraffin (melting point 56-58°C.). The material is embedded in Fisher Tissuemat (5658°C.) . Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.

The cutting of woody plant materials embedded in paraffin is always attended by difficulties. In this laboratory students of mor- phology and anatomy have found it necessary to prepare serial paraffin sections of plant parts as diversified and heterogeneous as the apical meristems and the mature rhizomes of ferns or as the stem tips and mature structures of both woody and herbaceous angio- sperms. The method here described is chiefly distinguished by long infiltration periods; it is important to note however that i t requires little of the technician’s time. The authors point out that it has been tested and modified over the past ten years for many types of plant materials and is unusually reliable. To the experienced technician there will be evident certain places in which the time schedule can be cut down; the schedule provided however is likely to give satis- factory results to the less experienced worker. Preparations obtained by this method have been found to be as good as those obtained in this laboratory by schedules requiring more attention, such as those of Ball (1941), Johansen (1940), Rawlins and Takahasi (1 947), and Sass (1940).

The authors wish to thank many workers of this laboratory, espe- cially Drs. J . E. Gunckel and Rhoda Garrison, for their snggestions.

Parts of plants, such as apices of fern rhizomes or buds of gymno- sperms and angiosperms, may be killed and fixed in CRAF 111 (Sass, 1940, p. 19). After trying a number of variations of the CRAF and F.A.A. types, CRAF 111 was found to be a very satisfactory killing and fixing solution. The material should be cut into as small pieces as

STAIN TECHNOLOGY, 1’01.. Zfi, So. 1. OCTOBFR 1951

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252 ST,ZIN TECHNOLOGY

convenient. It is recommended that pieces larger than 5 mm. in diameter be slashed or pricked. All material is benefited by aspira- tion immediately after being placed in the killing and fixing solu- tion. After fixation for 2+36 hours, the fixed pieces should be washed overnight in running water (Wetmore, 1932, pp. 47-8).

Hydrofluoric acid is frequently employed to soften woody mate- rials (Wetmore, 1932, pp. 49-50). When dealing with parts of plants containing both soft and woody regions, for example, the terminal portions of stems, the technician may oversoften the meristematic tis- sues while adequately softening the woody areas. The use of hydro- fluoric acid on such heterogeneous materials is therefore to be avoid- ed whenever possible..

In dehydration we have slightly modified Zirkle's n-butyl alcohol series (Zirkle, 1930): Distilledwater. . . . , , . . 857@ 70yQ SO?& 30% 15% 5T0 Oro 0% 07, 0% 95CT,cthylalcohol.. . . . 15 30 40 50 50 40 25 0 0 0 n-hutylalcohol.. . . . , , . 0 0 10 20 35 55 75 100 100 100

Small pieces of material need not be left more than 1-2 hours in any solution except the last two of 100% butyl alcohol. A longer time proves necessary if the pieces are large. However, regardless of size, all pieces should remain at least twelve hours in the second change of pure butyl alcohol.

The infiltration is carried on at gradually increasing temperatures. Butyl alcohol is a poor solvent of paraffin at room temperature, but dissolves or mixes with paraffin at the melting point of the latter. The assumption made in this schedule is that as the temperature in- creases more paraffin goes into solution and can enter the tissue. The use of a low-melting point paraffin at the beginning of infiltration facilitates this process. Partial evaporation of the butyl alcohol in steps 3 and 4 below permits a gradual concentration of paraffin in the mixture. Experience has shown that better ribbons result from long infiltration periods. The following schedule may be considered adequate for a piece of heterogeneous tissue, 1 cm. in thickness or larger, such as the apical region of a stout fern rhizome. For smaller pieces or softer material the periods may be shortened.

The detailed schedule of infiltration i s as follows: (1) Put shavings of paraffin melting at about 52°C. into the bottle

containing the material in the third change of 100% butyl alcohol. Stopper the bottle. Let it stand 1 day at room temperature (about

(2) Place the bottle in a warm place for 2 days.' If all the paraffin 20 C.) . goes into solution, add more shavings.

'In this laboratory for steps 2 and 3 we use a small uninsulated embedding oven set at 40°C. The bottles are placed on top of the oven for step 2 and inside it for step 9.

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REFRACTORY PLANT MATERIALS 253

(3) Unstopper the bottle for the rest of the schedule. Place it in an oven at about 40°C. for 2 days. If all of the paraffin goes into solu- tion, add more shavings.

(4) Place the bottle of material in a paraffin oven set at 59"C., or warm enough to keep 56-58' paraffin melted. Add more paraffin shavings. Lct it stand for I day.

(5) Pour off about one-quarter of the butyl alcohol and paraffin mixture a i d replace it with melted paraffin (56-58°C.).2 After 2 hours pour off about half of this and replace with melted paraffin. Follow this with 2 changes of melted paraffin at 2-hour intervals. Let it stand overnight. Next morning no odor nor taste of butyl alcohol should be present.

(6) Pour off this paraffin and replace with melted paraffin of 56- 58" melting point. Let it stand for one day in the 59°C. oven.

(7) Pour the paraffin off again and replace with 56-58°C. Fisher Tissuemat. Let this stand for 3 days.

(8) Embed. Before microtoming, the block of embedded tissue is trimmed so

that part of the specimen is exposed, and it is soaked in water until it cuts easily (Johansen, 1940, p. 145). Soft tissue is often damaged by soaking. I n heterogeneous material this difficulty may be overcome by exposing only the harder parts of the specimen to the action of water. For example, a groove may be cut through one side of the paraffin block and into the woody base of the terminal portion of a stem which is to be sectioned transversely. Water has been found to be as effective as and more convenient than glycerine-alcohol solu- tions (Baker, 1941) or hydrofluoric acid (Foster and Gifford, 1947).

REFERENCES

BAKER, J. R. 19.41. A fluid for softening tissues embedded in paraffin wax. J. Roy. Micr. Sac., 61, 75-8.

BALL, E. 1941. Microtechnique for the shoot apex: Amer. J. Bot., 28, 23343. FOSTER, A. S., and GIFFORD. E. hI., JR. 1947. Improvements in the paraffin meth-

JOHANSEN, D. A. 1940. Plant Microtechnique. McGraw-Hill Book Co., Inc.,

RAWLINS, T. E.. and TAKAHASI, W. N. Elimination of distortion and poor

SASS, J. E. 1940. Elements of Botanical Microtechnique. McGraw-Hill Rook

WmivoRE, R. H. 1932. T h e nse of celloidin in botanical technic. Stain Techn.,

ZIRKLE, C. 1930. T h e use of n-but71 alcohol in deh?drating \v\.otxl\- tissue for

od. Stain Techn., 22, 12S31.

New York.

staining in paraffin sections of plant tissues.

Co., Inc., New York.

7, 37-62.

19-17. Stain Techn.. 22, 99-102.

paraffin embedding. Science 71, 1034.

"Paraffin rather than Tissuemat is used for all steps ewept step 7 for the sake of economy.

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