I N D U S T R I A L A N D ENGINEERING CHEMISTRY
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tion is introduced into this tube from the buret, a length of 120 mm. of the solution being taken, followed by 150 mm. of ethyl ether. The open end of the tube is sealed and the contents are centrifuged back and forth several times. The tube is then cut open close to the surface of the ether. A capillary pipet with a mark a t the 150 cu. mm. point (this need not be exactly 150 cu. 1m, but the volume should be less than the total volume of the original acid solution) is prepared as shown in the figure. The capillar portion of the pipet is about 150 mm. long and 1.5 mm. in %ore. The wider portion is about 60 mm. long and 5 to 6 mm. in diameter. This is dipped down into the aqueous layer in the extraction tube. the upper end of the pipet being held closed with the finger. dmce even with this precaution some of the ether Iayer will enter the pipet, a bubble or two of air is carefully blown through the pipet to displace any ether solution. Then the required volume of the aqueous layer is drawn into the pipet. This sample is transferred to a microbeaker and there titrated, using 0.3 N sodium hydroxide and phenolphthalein as indicator. Using the same pipet, an equal volume of the original acid solution is titrated in the same way. The ratio Volume of alkali used for extracted sample Volume of alkali used for original sample
x 100
is:the partition constant.
Vol. 16, No. 7
The following values were obtained using the ratio of acid to ether 12 to 15 and a concentration of original acid aboyt 0.3 N . Formic acid Acetic acid Propionic acid Butyric acid n-Valeric acid
64.3 48.7 33.7 14.5 5.0
The values of the respective constants are so far apart that the acid can be identified without difficulty, even if small errors are made during the determination. LITERATURE CITED
(1) Behrens, W. U.,2.anal. Chem., 69,97 (1926). (2) Benedetti-Pichler, A. A., and Hybbinette, A. G., Mikrochemie, 30,15 (1942). (3) Craik, L.C.,IND.ENG.CHEM.,ANAL.ED.,8,219 (1936). (4) Mika, J., “Die exakten Methoden der Mikromassanalyse”,Stuttgart, Ferdinand Enke, 1939. ( 5 ) Osborn, 0. L., Wood, H. G., and Werkman, C. H., IND.ENG. CHEM., ANAL.ED., 5,247 (1933); 8,270 (1936). (6) Werkman, C. H., Ibid., 2,302 (1930); Iowa State Coll. J . Sei., 4, 459 (1930); 5,1, 121 (1930).
Staining Rubber in Ground or Milled Plant Tissues FERDINAND W. HAASIS Special Guayule Research Project, Bureau of Plant Industry, Soils and Agricultural Engineering, Salinas, Calif.
Following suitable pretreatment of samples, differential staining with Sudan IV and iodine greec has proved a useful and relatively rapid aid in the microscopic study of rubber in ground guayule tirsues, mounts in many cases being ready for examination within 90 minutes after starting the schedule.
A
CCESSORY to a comprehensive study of guayule produc-
tion now under way by the United States Department of Agriculture is an analysis of the rubber content of plants of varioua strains and ages grown under diverse environmental conditions. This analysis is currently made by chemical extraction of the rubber from finely ground tissues by a method based on that described by Spence and Caldwell (9). This method consists of a number of distinct steps and it is part of the program to test possible variants of these steps. As a check on the effectiveness of such tentative modifications, the ground samples are examined microsmpically, especially the spent charges remaining after extraction.
p. 212; 6 ) . For many workers, however, staining and counterstaining will prove an exceedingly useful aid in studying plant material,. It is for this group that these notes are intended. Suberin can be decomposed by treatment with potassium hydroxide in ethyl alcohol (8, p. 47),after which the cork cell walls no longer take up the rubber-staining dye. Any fats that may be present will likewise be saponified by the potassium hydroxide. For getting rid of resins and oils acetone or ethyl alcohol may be used (4, pp. 212, 214; 6). Pigments can be bleached out by means of oxidizing agents. Needless to say, the microscopic examination of ground samples is a far different matter from that of ordinary sections. I n the ground material the various structural elements are mostly much displaced from their original relative locations, many of the fragments are thicker than the usual run of sections, and the pieces are sometimes piled one on top of another. The face exposed under the cover glass of a microscopic mount is usually longitudinal, cross-sectional exposures being uncommon. Before extraction, stained rubber agglomerates are likely to be fairly plentiful, especially in the case of plants of high rubber content.
RUBBER-STAINING DYES
I n the microscopic inspection of tissues it is common practice t o use various dyes for staining diverse plant substances, sometimes singly, sometimes in combination (3, pp. 9-12, 167-174). For staining rubber in plant sectmiom,Lloyd (6) used alkanet, while Hall and Goodspeed (4, p. 212) and Artschwager ( 8 ) employed Sudan 111. Other dyes which stain guayule sections in patterns similar to those of Sudan I11 are Sudan IV, Sudan black B, and Calm oil blue NA. Unfortunately, these dyes are not specific for rubber but also stain various other materials, such as resins, oils, fats, suberin, and cutin (3,p. 210; 4, p. 213; 6, p. 63; 6; 8, pp. 47,48, 58; IO). Steps must accordingly be taken to eliminate these substances from the sample or to learn to recognize them by some distinctive character of form, color, or location. An experienced microscopist or technician might be expected to recognize the various tissues and substances in plants with which he is working, often without any staining a t all (4,
STAINING SCHEDULE
The choice of dye is partly a matter of personal preference. Sudan I11 colors rubber somewhat scarlet, while Sudan IV gives more of a crimson cast. Sudan black B gives an indigo or dark blue, approaching black, and Calco oil blue NA a much brighter blue. Addicott ( I ) has recently described a combination dye resulting in a blue staining of the rubber with red coloration of lignified and suberized tissues. I n working with ground material of guayule and other plants, the author found that Sudan IV combined with either iodine green or methyl green yields mounts showing striking contrast between the crimson of the stained rubber and the blue-green coloration of the woody tissues and cork cells. After considerable testing of various schedules, he tentatively settled upon the following as giving very satisfactory differential staining to the ground tissues studied, besides being comparatively fast in use:
A N A L Y T I C A L EDITION
July, 1944 Soak in potassium hydroxide eolution Add bleaohing eolution and soak Rinse with water Stain with a mixture of Sudan I V and iodine green (or methyl green) solutions Rinse with water Mount in glucose sirup Solutions u e d 1 . Potassium hydroxide 95% ethyl alcohol 2. A commercial bleaching agent (Cbrox) containing 5 2 5 % sodium hypochlorite and 94.75% inert ingredients (according to analysis puhlished on the label) 3. Sudan I V (dye content 86%) Acetone 70% ethyl alcohol 4. Iodine green Acetone 70% ethyl alcohol .5, Methyl green (dye content 6070) Acetone 70% ethyl alcohol G. A commercial table sirup [Karo (crystal white) 1 containing, according to the label, “corn sirup, sugar, salt, and vanilla”
15 minutes 10 minutes 0 . 5 hour
10 grams 100 ml.
0 . 0 9 5 gram 4 7 . 5 ml. 47.5ml. 0.095 gram 4 7 . 5 ml. 4 7 . 5 ml. 0 , 0 9 5 gram 4 7 . 5 ml. 4 7 . 5 ml.
The sample to be stained is placed in a small shell vial to cover the bottom to a depth of about 3 mm. To this is added about 1 ml. of the alkali solution. Sample and solution are thorou hly mixed, care being taken to wet all particles. At the end o f t h e sa onification treatment approximately 2 ml. of the bleaching sorution are added to the vial, wjthout removing the alkali, and the preparation is thoroughly stirred as before. The sample 1s then washed out into a cone of tough filter paper and rinsed with three funnelfuls of water. The sample mass is returned to its vial, dye solutions are added, about 0.5 ml. of each, and the preparation is once more thoroughly stirred. Good differential staining takes place in 30 minutes at room tem erature. After the sample is washed out into a Syracuse w a t c i glass, the water is drained off by capillary action of a small folded piece of bibulous paper, the dish is refilled, and the water again drained out. The wet sample, transferred to a microscope slide, is collected into a small pile and the excess moisture is drained away with a bit of bibulous paper. Mounting sirup is added, about 2 to 4 dro s, and thoroughly mixed with the sample. With application o f a cover glass the preparation is ready for examination. DISCUSSION
With the above-outlined schedule, rubber is stained crimson, lignified tissues and cork cells bluish green or blue-green. If suberin is not removed prior to staining, the cork cells stain purple or dark crimson or sometimes partly red and partly green. Although they can with practice be recognized by their distinctive structure, the purple or crimson color is not always clearly distinguishable from the stained rubber and inclusion of the alkali treatment is accordingly desirable. While Rawlins (8, p. 47) specifies boiling alcoholic solution of potassium hydroxide for removal of suberin and Miller ( 7 ) states that suberin is soluble in warm alkali, the present author has found the alcoholic solution fully effective a t room temperature in removing from the cork cell walls of guayule, mariola (Parthenium ineanum HBK), and coyote brush (Baccharis pilularis DC), the material stainable by Sudan 111, Sudan IV, Sudan black B, and Calco oil blue NA. Cold aqueous solution does not have this effect nor does 95% ethyl alcohol alone. With other plant species than those with which the author has worked the situation might be different. Each worker must, of course, study his own material. Although a short, treatment with the bleaching solution does not completely remove pigment from the larger tissue fragments, this is of relatively little significance when using Sudan IV as the rubber stain, since in guayule the residual color is a yellow green which is fully distinguishable from the stained rubber. Entire omission of the bleaching solution results in poorer staining with Sudan IV and iodine green. If both saponification and bleaching
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treatments are omitted, the Sudan IV-iodine green mixture a p parently fails to color rubber occurring in very small amounts, even with a $hour treatment. As far aa the final staining is concerned, it makes little, if any, difference whether the bleaching agent is used before or after the saponifying solution, but the sample is easier to handle when the alcoholic potassium hydroxide ’solution precedes the bleaching solution. I n this schedule cutin, when present, Stains a light pink or lavender color not readily confused with the crimson of the rubber. Leaf veins are dull light green or blue-green. The qhestion could be raised as to whether the stained agglomerates in unextracted ground samples might not be resin rather than rubber. The texture of these masses, however, is very definitely tough and elastic, indicating that they are at least mainly of rubber. Furthermore, in view of the fact mentioned above that resins and oils are soluble in acetone and in ethyl alcohol, it seems probable that these plant substances are at least in part removed by the solvents used for the alkali and dye solutions. Another application of this staining schedule is in studying factory bagasse as a check on t,he completeneM of rubber extraction in commercial milling. The sirup used in this technique, which was suggested by Johansen (5, p. 24), sets rather slowly, and slides must accordingly be kept flat for some days or weeks after specimens are mounted. I n preparations held for 2 months at room temperature the cover glass can be removed only with considerable d&cultv, while even those only a month old are fairly well set. Although this sirup mixes freely with water, an excess of water under the cover glass is undesirable because it tends to flow to the edges, subsequently drying out and leaving ragged spaces on the border of the preparation. No mold has developed on any of the author’s slides using this mounting medium, the oldest prepared 13months ago. It is the author’s practice to mix the two dye solutions together at the time of adding to the sample, but they may be combined as much as a week in advance without affecting the final stain. Premixed dyes, 4 weeks old gave differential staining, but much less brilliant than the fresher ones. There seems no change in color of the Sudan IV and iodine green staining in preparations even as much as 9 months old. While the time required for rinsing varies considerably with the material, for many samples mounted slides will be ready for microscopic examination within 90 minutes from the beginning of the schedule. ACKNOWLEDGMENT
The author is indebted to Hamilton P. Traub for the suggestion that Sudan I\‘ and Sudan black B might prove to be rubberstaining dyes. LITERATURE CITED
(1) Addicott, F. T.,“Differential Stain for Rubber in Guayule”, Stairb Tech., in press. (2) Artschwager, Ernst, U. S. Dept. Agr., Tech. Bull. 842,3 (1943). (3) Conn, H. J., et al., “Biological Stains”, 4th ed., Geneva, .N. Y., Biotech Publications, 1940. (4) Hall, H. M., and Goodspeed, T. H., Univ. Calif. Pub. Botanrl. 7 , 183-264 (1919). (5) Johansen, D. A.. “Plant Microtechnic”, New York and London, McGraw-Hill Book Co., 1940. Lloyd, F. E., Carnegie Imt. Wash. Pub. 139,176 (1911).
Miller, E. C.,“Plant Physiology:’, 2nd ed., New York and London. MoGraw-Hill Book Co.. .... 1938. Rawlins, T. E., “Phytopathological and Botanical Research Methods”. New York. John Wilev & Sons. 1933. Spence, D., and Caldwell, M. L., I ~ DENCL‘CIIEM., . ANAL.E D , 5,371-5 (1933). Whittenberger, R. T., “011Blue NA BS a Stain for Rubber i n Plants”, Stain Tech., in press. ~
