Maintaining Temperature for Infiltration

catalogue. The bath must be furnished with a ther- mometer for reading temperatures, and a thermostat for regulating the flow of gas and maintaining the A bath at a constant temperature. piece of paraffin about equal in bulk to the bergamot is placed in the vial containing the latter, and the vial put in the bath at the temperature of the room. The burner is lighted and the thermostat so regulated that the temperature of the oven rises from the room temperature to 45� C. in 3 to 4 hours. Infiltration is continued at this tem- perature for 1 to 3 hours longer in the case of root tips, and for 8 to 10 hours with harder tissues. As the paraffin dissolves, more should be added until the ber-. gamot will take up no more. IMBEDDING Imbedding is effected in the same bath. Halves of Petri dishes (50 mm., 3802, B. & L.) should be filled with paraffin melting at 45� 0. at the time the bath reaches 45� in order that paraffin may be melted by the time the specimens are ready to be transferred from the mixture of bergamot and paraffin. In order to remove all the bergamot, it is better to transfer the specimens from bergamot-paraffin to melted paraffin for two or three hours and then to place them finally in the dish in which they are to be imbedded. 26 MANUAL OF BOTANY Root-tips should be imbedded altogether for 5 to 6 hours, while stems, etc., will require 12 to 24 hours. The dish should be taken from the bath at the proper time and placed on top of the latter, where the speci- mens may be arranged with a warm needlepoint. It is then placed close at hand with as little jarring as possible and allowed to cool until a white film forms on the top, when it is floated carefully upon the surface of a dish of cold water. As soon as the par- affin forms a thick crust, the dish is sunk and the paraffin allowed to become hard. In order to remove the paraffin block from the Petri dish, the latter is placed upside down in a dish of ice water for 2 to 4 hours, when the block will either fall out or will be easily removed by means of a scalpel. The specimens should now be cut out of the paraffin. In cutting, it is best to leave about the specimen a wide margin, which may then be trimmed close, taking care to make the A faces of the block flat and parallel. margin should be left on one side to permit of easy attachment to the stick. Each block is then attached to a pine stick about 25 mm. long and 6 to 8 mm. square by dipping the end of the stick in the melted paraffin, placing the block squarely upon the end and building up about its base with melted paraffin taken up by a pair of tweezers. The stick with its block attached should be placed in cool water until it is ready to be sectioned. GENERAL DIRECTIONS 27 -- CUTTING THE SECTION THE MICROTOME The only microtome within the reach of most high schools is the Bausch & Lomb Student's Microtome, No. 2500 which, with the universal clamp, may be bought for about |20. The barrel microtome may be used for cutting paraffin blocks, but the long paraffin process is not worth while if a better microtome is not available. In sectioning on the microtome, the greatest care must be taken to see that the block is oriented properly in order that true sections may be cut and that the sections, as they are cut, may adhere in ribbons. If the sections will not hang together or ribbon, the block should be warmed slightly. More rarely, the block is too warm and must be cooled, though such a condition is usually indicated by a failure to cut a section each time. If the ribbon curls, the block is not placed squarely with reference to the edge of the knife, or it has not been trimmed so that the sides are parallel. The razor or knife should never be sharpened just before cutting as this will frequently cause the ribbons to become electrified, making it almost impossible to handle them. MOUNTING THE SECTION Sections are attached to the slide by means of al- bumen. This is prepared by shaking together 25 cc. of pure glycerin and 25 cc. of the albumen of eggs with A ^ gm. of sodium salicylate, and filtering. drop or two of the albumen is smeared over the portion of the slide to which the sections are to be attached and : 28 MANUAL OF BOTANY then rubbed off with a single stroke of the cloth. This albumen film is covered with water and the pieces of the ribbon of the proper length are floated upon the water. The slide is warmed very carefully in order to flatten the sections. Great care must be taken not to make the slide so warm as to melt the paraf&n. The water is then removed by means of a blotter and the slide is put away to dry for 2 to 4 hours or as long as need be. The removal of the paraffin, staining, v/ashing, etc., may be best carried on over a slop jar. The steps in the process are as follows Warm 1. the slide gently till the paraffin melts; wash with a pipette full of xylol. 2. Wash off the xylol with a half pipette full of 100 per cent alcohol. 3. Replace the 100 with 95 per cent alcohol. 4. Stain. If the stain used is safranin, methyl green, or haematoxylin, made up in 95 per cent or some other high grade of alcohol, simply cover the sections with the stain for 5 to 10 minutes. If the stain is made up in water or a low grade of alcohol, as in the case of iron-haematoxylin, the slide should be washed successively with 75, 55, 35, and 15 per cent. In staining with iron-haematoxylin, the slide should then be placed in water for a minute and then in a stain jar of iron alum for 10 minutes to several hours as desired. It is next rinsed in water for 2 to 3 minutes and then is placed in a stain jar of haematoxylin, in which it remains for the same length of GENERAL DIRECTIONS 29 time it did in the iron alum. The slide is now differ- entiated by placing again in iron-alum for a few minutes, glancing at it under the microscope every 30 seconds till the proper color is obtained. It is washed in water for 5 to 10 minutes and run up again to 95 per cent alcohol. 5. Wash with 100 per cent. 6. Cover with bergamot and clear for 5 minutes. 7. Kemove excess of bergamot and add one or two drops of balsam. Start the cover at one edge and lower it slowly to avoid bubbles. The slide should be placed, cover up, in a slide box to harden, which will require a short or long time according to the thinness of the balsam. REAGENTS The following simple formulae for reagents will be found useful to the teacher. Both safranin and methyl green should be made up in 2 per cent solu- tion, 2 gms. of the stain to 100 cc. of 95 per cent alcohol. Delafield's haematoxylin is made by dissolv- ing 1 gm. of haematoxylin crystals in 10 cc. of 95 per cent alcohol and adding it to 100 cc. of a satu- rated solution of ammonia alum. This mixture is allowed to ripen in a loosely stoppered bottle placed in the sunlight for three or four days, when it is filtered and 25 cc. of pure glycerin and 25 cc. of methyl alcohol are added. After standing for several days, the solution is filtered again and is then ready for use. The above solutions will keep indefinitely. For staining with iron-haematoxylin, a 2 per cent so- lution of ammonio-ferric-alum in distilled water is ; 30 MANUAL OF BOTANY used and a | per cent solution of haematoxylin in distilled water. Neither of these solutions keep very well and should be made up in small quantities. The formula for Flemming's solution (1) is 60 cc. 1 per cent solution of chromic acid in tap water, 5 cc. 1 per cent solution of osmic acid, and 1 cc. glacial acetic acid. Glycerin jelly may be prepared by soaking 5 gms. of pure white gelatin in 50 cc. of distilled water, adding 50 cc. of pure glycerin and 1 gm. of phenol. The usual solution of iodin consists of -| gm. of metallic iodin, 1 gm. of potassic iodid and 100 cc. of water. Glycerin is made up in 50 per cent or 12 per cent solution in water, according to whether it is used for plasmolysis or for general use. Potash is used in 10 per cent or 25 per cent solution in water. For ordinary use commercial balsam should be diluted A -J to 4 its volume with xylol or chloroform. con- venient and sufficiently accurate formula for diluting alcohol is the following : desired any per cent between 10 and 30, take as many cubic centimeters plus one of 95 per cent alcohol and add water to make up 100 cc. ; between 30 and 50, add two to the grade desired between 50 and 70 add four, and between 70 and 90 add five. REFERENCES IN THE TEXT Unless otherwise specified all references in the text will be to Bessey's "Essentials of Botany," seventh edition. References in Roman numerals are to plates in Part I and II of the Flora of Nebraska. The numher following the directions for drawing indicates the scale to ivhich the object is to be drawn. PLANT STRUCTURE OR HISTOLOGY THE CELL Cell wall (6)-- -- Primitive walls 1. Protococcus viridis, green slime: scrape off a little of the green growth on the outside of flower pots and carefully pick it apart ; note the extreme thinness of the cell-wall except in certain resting stages, where it is a thick . colorless band ; draw several cells or plants, showing wall and contents; 5.** The nucleus is not evident; the body which suggests it is usually a pyrenoid, a small bit of cytoplasm in which starch is stored, (f. 66, a. 135.) 2. Spirogyra sp., pond scum : mount a few filaments and note carefully the side and end walls; describe the wall, thinness, color, structure, etc., and draw one cell showing these points; 1. The nucleus usually shows very plainly in the center of the cell surrounded by radiating threads of protoplasm. The chloroplasts are green, ribbon-like bodies of protoplasm disposed spirally in the cell. Is the cell wall composed of distinct layers? -- ** Note remark in italics bottom of page 30. Spirogyra sp. The "sp." indicates any or no particular species. 32 MANUAL OF BOTANY Is it lined on the inner surface with cytoplasm or with cell sap? (141, f. 73, p. 142.) 3. Funaria hygrometrica^ moss : remove one or two leaves from the stem and mount them; the cells are here united into a tissue and the wall between two cell cavities is a double one formed by cementing the two walls together; draw a group of four or five cells, showing the wall but disregarding the green chloroplasts ; 1. Do you find any evidence of the double wall? (f.8,p.l3.) -- Modified walls 4. Hibiscus sp,^ mallow: mount a few pollen grains, taking care not to crush them ; draw one cell showing the external modifications of the wall; crush the cell and note the con- tents; 1. From the crushed cells it is pos- sible to tell whether the spines arise from the thickening or from a pushing-out of the wall. (f. 149, p. 252.) 5. Puccinia graminis, grain rust: remove a spore dot or sorus of the black rust or rest- ing stage and pick it apart ; note the varying thickness of the wall in different parts of the same cell and in corresponding parts of different cells; draw two or three spores showing various thicknesses of the wall : 2. How many cells in each spore? Has the stalk cell any protoplasm? Where is the HISTOLOGY 33 wall the thickest? Why? How does the growth of the spores in masses explain the varying thickness of wall in the same spore? (191, f. 113, II, III, p. 192; f. 114, p. 194.) 6. Pirus communis^ pear: cut longisections of the stems of mature pears, stain in safranin and mount in balsam ; note the square stone cells, describing minutely the structure of the greatly thickened wall; note the canals which radiate from the small cavity and the striations which run parallel to it ; 2. Explain the pits which appear in the back wall? Why do the canals of adjacent cells coincide? Why are the striations concen- tric? Do any canals branch? Can you find any indication of the original thin wall between the cells? (23, f. 14, p. 24.) Cell formation (9)-- -- Fission 7. Nostoc commune^ j^Hy chain : crush and separate the thallus before mounting; select a filament in which the cells are of difCerent length ; note that certain cells are just twice as long as others, while some intermediate in length show a slight constriction on either side ; draw 10 or 12 cells of a filament show- ing a variety of stages, preferably including the heterocyst; 4. The normal cells are globose or somewhat elliptical, those ready for fission about twice 2 34 MANUAL OF BOTANY as long. Fission starts by the pinching in of the side walls and is completed by the formation of an end wall between the points of constriction. In Anabaena, a related plant, the growth and pinching in of the wall occur at the same time. The heterocyst is a clear yellowish cell found at intervals in the filament, (f. 61, p. 126.) 8. Oscillatoria tenuis, thread slime: mount a few filaments and note the compact way in which the cells are united; draw one fila- ment, giving especial attention to the curved apical portion ; 1. Do all the cells undergo fission? What is the purpose of the apical cell? Is there a sheath about the filament? Explain why some cells are twice the length of others. How many movements has the filament? (f. 61, p. 126.) -- Budding 9. Saccharomyces cerevisiaey yeast: make a yeast culture by adding a small amount of "yeast foam" to a 10 per cent solution of cane sugar and keep at 30� to 35� C. for 24 hours; mount a drop of the solution and examine for budding cells; draw several cells showing different stages in the formation of a single bud, also one or two groups showing repeated budding; 3. The nucleus does not show ; the central bodv ---- . HISTOLOGY 35 is a vacuole. Are there any chloroplasts present ? Does one cell ever give rise to more than one bud? Distinguish between budding and fission. Is a new cross wall formed in budding? Stain lightly with iodin; what are the blue bodies? What part do they --play? (189, f. 112, p. 190.) Union 10. Splrogi/ra nit Ida ^ pond scum : mount a few fruiting threads and examine those parallel ones in which one thread is empty, the other filled with an elliptical resting spore or zygote; draw four cells, tAvo from each fila- ment, paying especial attention to the con- jugating tubes which connect them ; f How is the zygote formed? How does the protoplasm cross from one cell to the other? Explain the origin of the conjugating tubes. Why should the zygote have a thick wall? Is any protoplasm left in the clear cell? (143, f. 73, p. 142.) Cell contents Protoplasm (1) 11. Lycopersicum esculentuniy tomato : cut thin transections of a young flower stalk, taking care not to injure the hairs ; examine the basal cells of the latter for streaming protoplasm and nucleus ; draw a typical cell showing the threads of protoplasm, nucleus, nucleolus, vacuoles, and the pale green bodies, the plastids ; 1. 36 MANUAL OF BOTANY Is there a layer of protoplasm lining the wall of the cell? Why? Does the protoplasm move in a definite direction? Does it pass from one cell to another? Does the nucleus move? Note the effect of iodin. (f. 26, p. 43.) 12. yitella flexilis, stonewort: remove the whorl of cells from the apex of a growing leaf and mount with little pressure ; draw in optical section a cell in which the protoplasm is streaming actively, paying especial attention to the structure of the latter; 1. The cell contains a number of oblong nuclei which can not be seen without staining. What are the small green bodies along the inner surface of the wall? Do they move? Do the granules move more slowly at the sides or in the center of the cell? Does the protoplasm flow more than one way in the same cell? Contrast this movement with that of the protoplasm of the tomato hair. -- Chromatophoees (2,12) -- Chloroplasts 13. Funaria liijgroinetrica, moss: remove and mount two or three leaves ; the lower oblong cells of the base of the l^f show the individual plastids best ; draw a cell in which are shown the various stages of the fission of the plastids, giving to each its exact form and position ; stain with iodin ; 2. ; HISTOLOGY 37 Contrast the fission here with that of the cells of Nostoc. Do the plastids contain starch grains? Do they have a distinct wall or membrane? (f. 8, p. 13.) 14. Zehrina pendiila, wandering Jew : mount a thin longisection of the stem from just beneath the epidermis; stain very lightly with iodin; draw one cell showing the various stages of chloroplasts and starch grains; 1. How many starch grains in a plastid? In what part of the latter do they occur? Do you see any starch grain not in contact with a plastid? -- Chromoplasts 15. Tropaeolum majus, "nasturtium" : tear a piece of petal into small bits and mount ; the bright yellow color is due in most cells to numerous yellow balls of protoplasm, in a few cells the chromoplasts are needle- or crescent-shaped ; draw one cell of each kind 2. Stain with iodin. Do these chromoplasts contain starch? Do they show fission? Which are the normal ones, the spherical or needle-shaped? -- Leucoplasts 16. Zehrina pendida, wandering Jew : slip the point of the scalpel beneath the epidermis of the stem and strip the latter off free from green cells below; the nucleus usually occupies the center of the clear epidermal cells, -- ; 88 MANUAL OF BOTANY surrounded by a few threads of protoplasm wliicli run to the wall; the leucoplasts are round clear bodies lying upon the nucleus and occasionally found scattered in the cell 1. Stain with iodin. Do the leucoplasts divide? Do they contain starch? Is there protoplasmic movement in the threads? Has this anything to do with the leucoplasts? Starch (14) 17. Solaniim tuherosum, potato: cut a section from the fresh surface of a tuber ; draw two cells showing various grains and paying par- ticular attention to the striations of the latter; 2. Stain with iodin to demonstrate if possible the presence of protoplasm in the cell. Do you find any plastids in the cell? What is the hilum? Explain how the striations Why arise. is the gniin called excentric? Explain its form. 18. Fisiim sativum, garden pea : cut a few thin shavings from the flat surface of a split pea and mount them in weak alcohol; draw one cell showing the grains of starch with their striations and the small granules in which they are imbedded ; 1. Stain with iodin. Are the grains concentric or excentric? Is there a hilum? Note the thickened cell wall. Explain the triangular space at the corners of the cells, (f. 9, p. 15.) histology 39 -- Aleurone (16) 19. Phascolus vulgaris, bean : mount in weak alcohol a few shavings from the flat surface of a split bean; stain lightly with iodin; draw one cell showing wall, starch, and aleurone; 1. Replace the alcohol with water. What happens? Are the aleurone grains definite in size or shape? Do they have striations? Explain their position in the cell. Does aleurone give the same color with iodin that living protoplasm does? 20. Triticum sativum, wheat: divide a grain transversely and cut shaving sections across the edge; stain one section lightly with iodin to locate the aleurone and starch; draw a strip three or four cells wide extend- ing from the outer bran into the mass of starch; 1. Where is the aleurone found? Is it distinct from the starch? Do you find starch any- where in the aleurone cells or vice versa? Why is graham bread more nutritious than wheat bread? Contrast the position of the aleurone in the bean and the wheat. -- Crystals (17) 21. Zehrina pendula, wandering Jew: make a thin longisection of the stem; occasionally cells will be found filled with crystals, but usually the latter are scattered about; if 40 MANUAL OF BOTAXY possible draw both sorts of crystals in posi- tion in tlie cells ; with the long ones it may be necessary to draw several crystals sepa- ratelv; 1. "VMiat two shapes do the crystals assume? Do both forms occur in the same cell? Are the crystal cells found in definite positions in the tissue? Add a drop of hydrochloric acid at the edge of the cover. What hap- pens? (f. 12, p. IT.) 22. Begonia sp., begonia: cut a thin longisec- tion of the stem; draw a group of several cells, showing definitely the position of the crystal-bearing ones and paying especial at- tention to the structure of the crystal itself; a 3 Do the crystal cells have a definite position in the tissue? Does there appear to be any relation between this and the long bundles of fibers in the stem? How manv crvstals in each cell? Whv? Is the crystal a single one or a group of crystals? TSTiat is the usual shape of the individual crystals? (f.l2, p. 17.) THE TISSUES Primarj' tissue (21, 36)-- -- Meeistem 23. Hyacinthus orieniaUs, hyacinth: root tips are obtained by growing the bulbs in flower pots, or, better, in wide-mouthed bottles or -- ; HISTOLOGY 41 bulb glasses ; make very thin transections of the tips and, if possible, longisections also; note the characteristics of the individual cells, pay especial attention to their form and the way in which they are united to constitute a tissue ; draw a group of 8 or 10 cells 1. Stain with iodin. Explain the large nucleus and the abundance of protoplasm. Are the vacuoles few or many? Why should the cells show such regularity in size and arrangement? Do you find any intercellular spaces. Why? Do the cells contain starch? Plastids? Why? Do you find more than one nucleolus in any of the nuclei? Can you distinguish the two component walls in the double wall between two adjacent cell cavities? (f. 25, p. 37.) Secondary or modified tissues -- Parenchyma^ Soft tissue (21) 24. Begonia sp., be.2:onia: mount thin transand longisections of the stem; note the differences which the cells show in the two sections and draw a group of 4 or 5 cells from each; |. Stain with iodin. Contrast the cells of soft tissue with those of meristem with respect to size, shape, arrangement, amount of protoplasm, nucleus, wall, vacuoles, etc. Explain the intercellular spaces. Are they found in longisection? Why? Do you find starch or plastids? : 42 MANUAL OF BOTANY Explain why the transection of the cell is dif- ferent from the longisection. Is the cell wall thickened? Add a small drop of strong sul- phuric acid to sections stained with iodin; the cellulose walls turn a dark blue, the characteristic test for this substance, (f. 40, p. 62.) -- CoLLENCHYMA, Thick-augled tissue (22) 25. Begonia sp., begonia: mount a thin tran- section of the stem; the thick-angled cells are found directly beneath the single-rowed epidermis and are easily recognised by the bright thickened places in the corners of the cells ; draw a group of 8 or 10 cells showing how the cells with greatly thickened corners next the epidermis pass gradually into the soft tissue cells of the interior of the stem. Are there any cells in which the walls are thickened at the sides as well as in the corners? Can you find any trace of the original wall in the thickened angles? Do the thickenings show any layers? Why? Do you find any nuclei or plastids? What purpose does the thickening of the angles serve? Determine by the iodin-sulphuric acid test whether the angles are composed of cellulose. Beta vulgaris, beet, leaf-stalk Micrampelis lobata, wild cucumber, stem, (f. 13, p. 22.) * Cf. line 3 above. HISTOLOGY 43 -- ScLERENCHYMA, Stony tissue (23) 2(}. Pirns communis, pear : make small tliin trans- and longisections of the fruit-stalk of the pear; stain in safranin for 10 minutes and wash in 95 per cent alcohol ; the sections may be mounted in water, or they may be run up and mounted in balsam in the usual way; the stone fibers are found in dense white bundles arranged in a circle in the center of the stem, the stone cells are scat- tered here and there between the bundles and the outside of the stem; in the longisection they occupy the same places, but are best recognized by their difference in shape ; draw a group of 3 stone cells, preferably from the longisection and a group of stone fibers from each section; 1. Contrast stone cells and stone fibers in both sections, with respect to shape, size, posi- tion, arrangement, wall, canals, contents, etc. To which is the term isodiametric ap- plied? Do the radiating canals of two ad- jacent stone cells coincide? Why? Explain Why the striations in the stone cells. are they concentric? Explain the pits in the hack wall. Is there a nucleus? Why? Add anilin sulphate to the sections and, in 10 minutes, a drop of concentrated sulphuric acid; the yellow color is the characteristic test for stony tissue, (f. 14, p. 24.) -- 44 manual of botany Fibrous tissue (24) 27. Fraxinus lanceolata, green ash: split a small bit of twig and make thin trans- and radial longisections at the edge in such a way as to pass through the bark and the outer part of the wood ; the bark will show a ring of bright w^hite bundles of thick-walled fibers, bast, in the green parenchyma, and the woody portion will show a compact tissue made up of wood fibers; in the longisection the bast fibers are readily distinguished by their position and thick walls; draw from the transection a bundle of bast fibers, pay- ing especial attention to the striations of the wall, and a group of wood fibers ; draw from the longisection a s:roup of bast fibers, show- ing, if possible, the ends of some, and a number of wood fibers, showing the various ends and the way they join in the tissue; 1. Compare bast fibers and wood fibers in both sections. Show how each is best suited to its position. Do you find any indication of the original thin wall in the present thick wall of the fibers? Do the walls of the wood fibers have layers? Are there cells contents in either sort of fiber? Do the walls show canals or other markings? Explain why the ends are tapering. Make the lignin or wood test with anilin sulphate-sulphuric acid. (f. 15, p. 25.) histology 45 -- Sieve tissue (28) 28. Micrampelis lohata, wild cucumber: cut thin trans- and longisections of the stem, taking care that the latter pass through the fibrovascular bundles, the long threads which project into the central cavity and contain openings visible to the eye; in the transection, the center of each bundle is occupied by three or four large circles, crosssections of vessels ; next these on either side are several rows of very small, mostly rectangular cells, and imbedded in these, or just outside them, a row of larger polygonal or -- round openings the sieve tubes; in the longisections the tubes are recognized by their position on the outside of the bundle and by the broad masses of protoplasm, sometimes callose, on either side of the end parti- tion or sieve plate; if the transection is cut near a sieve plate, the perforations of the latter will appear as small black points in the cross-section of the tube ; draw from the transection the sieve portion of the bundle, showing the point just mentioned, and from the longisection a number of tubes showing the cross-section of the sieve plates and the funnel-like protoplasmic column on either side ; notice the small rectangular companion cell, touching the sieve tube, with its nucleus and densely granular protoplasm ; 1. -- 46 MANUAL OF BOTANY Are the two protoplasms on either side of the sieve plate in connection? Are the sieve tubes nucleate? Do you find either starch . or plastids present? Make the cellulose test. Cucurbita pepo, pumpkin, leaf-stalk, (f. 18, p. 29.) Milk tissue (26) 29. Euphorbia splendens, spurge: make transand longisections of the stem; in the tran- section, the milk tubes appear as small thick-walled circles just outside the ring of woody tissue in the center, in the longisection as branching tubes filled with a sticky granular liquid ; draw several tubes in longisection, showing position in the tissue, con- tents, etc. ; 1. Stain with iodin. What are the bone-shaped granules in the tubes? Do you find plastids or nuclei? Do you find striations in the wall of the tube? Asclepias syriaca, milkweed, stem. (f. 16, p. 27.) -- Tracheary tissue (30) -- Tracheae or vessels 30. Impatiens halsaniina, balsam, touch-menot : cut radial longisections of the stem ; the fibrovascular bundles will show a succession of cylindrical vessels with the walls thickened in various ways ; the simplest have circular or spiral thickenings on the inner surface, these