ZEIN
A New Industrial Protein L. C. SWALLEY Corn Products Refining Company, Argo, Ill.
Zein, an alcohol-soluble protein, is available only as a by-product of corn processing. It is extracted commercially from gluten meal with aqueous isopropyl alcohol. The process consists of extraction, clarification, oil extraction with hexane, precipitation by water, and flash drying. Zein solvents and plasticizers have free hydroxyl, carboxyl, ketone, amino, or amide groups. Usually, but not always, addition of water or secondary solvent is required.
EIN is the alcohol-soluble protein of corn. It was first isolated by Gorham (4) in 1821 and was given the name "zein" by him. Being a readily available protein, it has been the subject of considerable investigation. Further early work was done by Bizio (1) in 1822, Ritthausen (7) in 1869, and Osborne and co-workers (a, 5 , 6) in the period 1891 to 1908. However, the reports of these men and a considerable number of other more recent investigators have not been a satisfactory guide for further work on the development of zein as a commercial product. Apparently much of the zein described in the literature was denatured because of overenthusiastic attempts a t purification, and did not represent the original zein in physical properties. Even today we cannot state definitely that we are dealing with zein which has not been more or less damaged by incipient denaturation. During recent years the preparation and uses of zein have been investigated by the companies which process corn in large quantities for starch production. I n the starch process the zein content of the corn is concentrated in the gluten fraction which contains 50 per cent or more of protein, of which 70 per cent can be classified as zein. The potential yield of zein at present is about 1 pound per bushel of corn. The zein, however, is present in corn in varying amounts, depending on the variety of the corn and other conditions. ShoWalter and Carr (8) discuss the literature on this subject and show in their own work analyses of corn varying from 8.06 to 18.43 per cent in total protein content (N X 6.25) and 2.21 to 10.44 per cent in zein content (dry basis). The present investigation has checked the fact that various varieties of hybrid corn exhibit similar differences, but the number of samples analyzed has been insufficient to justify detailed discussion. A maximum of 6.4 per cent zein was found. These differences are apparently eliminated for practical purposes by the fact that the corn purchased represents an average sample of t h a t normally produced.
Z
Extraction Conditions As practical extraction solvents, only the lower aliphatic alcohols have the required properties of solvent power, availability, low cost, and ease of recovery. Methanol is unsuitable because of insufficient solvent power and tendency to promote denaturation of the zein. Ethyl and isopropyl alcohols fulfill all the requirements. The higher alcohols are unsuitable 394
Zein is dispersed i n aqueous solutions of a wide variety of soaplike compounds. Zein is cured by formaldehyde; the reaction is catalyzed by acids and promoted by ammonia or primary amines. Formamide cures zein. Zein is compatible with some highly acidic and alcohol-soluble resins. Zein is applicable to many uses including plastics, paper coatings, adhesives, laminated hoard, solid color printing, films, etc.
because of cost, poor zein solubility, difficulty in recovering the zein in pure form, or difficulty in solvent recovery. The fundamental characteristics of the extraction are shown in Figure 1. These show the yields obtainable by extraction with various concentrations of ethyl or isopropyl alcohol a t different temperatures. The gluten meal used contained 51 per cent protein on a dry basis. The meal passed a 14mesh screen but was retained on a 30-mesh screen. The calculated alcohol concentration of the solvent included consideration of the moisture in the meal. The extraction time was 5 hours. However, since the zein dissolves slowly under certain conditions, rate of extraction may still be a deciding factor in the determination of some of the points showing low yields. The following points are to be noted: 1. Isopropyl and ethyl alcohols give curves of similar shape. They can be compared directly, provided the isopropyl alcohol concentration is 5 per cent by volume less than for ethyl alcohol, and the curve for isopropyl alcohol is compared with the 10" C. higher curve for ethyl alcohol. 2 . There is a wide region of high yield with a rather flat maximum at 60 to 65 per cent alcohol concentration, on either side of which the yield drops rapidly with change in alcohol concentration. 3. Little is gained by raising the temperature above 65' C. This is due in part t o the increased denaturation at the higher temperatures. Zein, as it exists in the available raw material, consists of two or more chemical individuals characterized by differences in solubility. The main fraction dissolves readily in alcohols containing only a small amount of water. The other fraction, insoluble in strong alcohols, dissolves in weaker aqueous alcohols. The quantitative details of these relations have not yet been investigated completely. It is preferable to extract with alcohol of high concentration in order to obtain a zein which is more readily soluble in strong alcohols, leaving behind the fractions which are soluble only in the weaker alcohols. The extraction with 85 per cent isopropyl alcohol by volume or with 92 per cent ethyl alcohol at 60" C. yields zein of satisfactory solubility characteristics. It gives a reasonable yield amounting to about 50 per cent of the protein in the gluten meal, or about 70 per cent of the total zein content. It may eventually prove feasible to recover the remainder of the zein. This product will have characteristics different from those of the main fraction but is expected to be entirely suitable for many uses, suoh as in plastics.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
CQrnmercial Preparation The process of isolating zein which has now been advanced to the commercial state is as follows: Gluten meal, containing 50 to 55 per cent protein on the dry basis, is screened to remove particles finer than 40 mesh and is fed continuously into a seven-stage countercurrent extractor where it is extracted with 85 per cent isopropyl alcohol a t
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difficulties in drying due to the fact that wet zein becomes a doughy plastic mass when heated t o temperatures above 15"C. This requires that the zein be kept cold until it is actually dispersed for drying, and that the drying be carried out as rapidly as possible a t the lowest possible temperature. The entire process is made continuous to decrease operating costs and t o avoid storage of materials a t the various stages.
Properties
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A s c o m m e r c i a l l y produced, zein is a fine powder, slightly yellow in color, containing 7 or 8 per cent of volatile matter and not over 2 per cent of n o n p r o t e i n s o l i d s . Ash and oil contents are each less than 0.5 per cent. It is between 98 and 99 per cent soluble in 92 per cent ALCOHOL CONCENTRATION- 4~ s i VOLUME ALCOHOL CONCENTRATION- % 0Y VOLUME ethyl alcohol by volume a t 25" C. FIGURE 1. ZEIN EXTRACTION YIELDS WITH ETHYL ALCOHOL(left) AND ISOPROPYL ALCOHOL (right) Watson, Arrhenius, and Williams (IO) report that the greater part of the zein has a molecular weight of approximately 35,000, while 55" to 60' C. The contact time is 1.5 t o 2.5 hours. The that of another constituent is approximately half this separation in each unit of the extractor is by settling. Exvalue. tract is pumped from stage to stage while the meal, in the Zein is characterized in general by relative deficiency of form of a suspension in extract, passes by gravity in the opactive groups in comparison with most proteins. Based on a posite direction. The residual meal is steamed to recover the molecular weight of 38,000, there are apparently twelve free solvent, dried, and returned for use in feed. The extract is a acid groups per molecule (3). However, these are relatively somewhat viscous liquid of deep yellow color, containing weak since they cannot be neutralized sufficiently to effect the about 6 grams of protein per 100 cc., practically all of the solution of zein below a pH of about 11.8. Some investioil present in the meal (about 1per cent based on the extract), gators report tRat zein has no free basic groups, while others the xanthophyll pigments, and some water-soluble materials. report a maximum acid binding power of 9 moles per mole of The extract is cooled to 15" C. to precipitate certain undezein, baeed on the above molecular weight. sirable substances and is then filtered on an Oliver precoatFigure 2 shows a typical titration curve obtained in the type filter. The conditions of cooling and filtration are impresent investigation. The point of inflection is a t a pH of portant for the production of zein of satisfactory solubility and 7.5, and the curve indicates absorption of 5.3 moles of acid solution stability. The clear extract is mixed with 80 to 120 per mole of zein at pH 3 and 12.2 moles of alkali a t p H 11. per cent of its volume of hexane. The mixture separates into These are not maximum absorption figures. two layers, the upper containing almost all of the hexane, I n view of the relatively unstable nature of zein solutions, most of the isopropyl alcohol, at least 97 per cent of the oil, it is somewhat surprising that zein in the dry form is very and 90-95 per cent of the xanthophyll pigment. The lower stable toward heat. It can be heated for several hours a t layer consists of a 15 to 20 per cent solution of zein in ap100" C. without any noticeable change in properties. It proximately 60 per cent (by volume) isopropyl alcohol, and does not decompose a t temperatures as high as 200' C., alcontains only small amounts, as indicated above, of hexane, though the absence of chemical change a t this temperature oil, and xanthophyll pigrpents. The phase relations are quite sensitive to variations in ,q.ater content of the extract and, t o has not been determined. One troublesome characteristic of zein is that it tends to a lesser degree, to the proportion of hexane to extract. The become denatured when in solution. The denaturation, which layers are separated by continuous centrifuges. Any hexane is a change in the chemical nature of the zein rendering it inremaining in the zein layer is removed by vacuum distillation, soluble in the usual solvents, is autocatalytic; the denatured and the zein is precipitated from the viscous solution by insoluble material catalyzes the reaction to such an extent spraying into a rapidly moving body of refrigerated water, that in some cases, where considerable amounts are present, using nozzles of special design. The zein precipitates in rather an otherwise stable solution may gel in a few hours. This refibrous form which becomes brittle on complete extraction of action is also accelerated by agitation, even in comparatively the solvent and which rises to the surface of the precipitating dilute solutions of zein. The solvent composition has a tank, from which it is removed continuously by drag paddles. marked effect on the rate of the reaction. I n general, the more The zein is filtered and washed continuously on a string-type nearly the solvent becomes anhydrous while still remaining a filter. The filter cake, which has a spongy structure, is dried satisfactory solvent for zein, the more stable will be the soluin a flash-drying system. I n this system the filter cake is distion. I n the case of ethyl alcohol solutions, alcohol concenpersed and mixed with refrigerated dried product. The mixture trations of 90 per cent by volume or higher are relatively is ground and fed into a hot air stream. Practically instable; below this point the stability decreases rapidly. While stantaneous drying takes place without aubjecting the mano satisfactory method of completely stabilizing zein soluterial to actual temperatures high enough to fuse it in the tions has been developed, the stability is sufficiently improved moist condition. The chief difficulties in the process are the by the presence of highly acid resins such as rosin so that prevention of denaturation, which is acoomplished by careful in practical formulations alcohol concentrations as low as temperature control and by keeping the system clean, and the
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85 per cent by volume can be used without undue difficulties from instability. Instability due to denaturation is to be distinguished definitely from the separation of a solution into two phases due to the use of improper solvent mixtures. Solvents The general type of solubility behavior of zein is the same as for other high-molecular-weight or colloidal materials such as cellulose nitrate, acetate, or highly polymerized resins. That is, the same general types of relations hold true for all of these materials in viscosity, in the improvement of solvent power by the addition of certain secondary solvents which in themselves are not active solvents, and in the tolerance of the solutions for certain amounts of diluents which if added in excess cause precipitation. The aliphatic alcohols are the solvents having the greatest value in practical applications of zein. Zein is insoluble in the anhydrous alcohols except methanol, in which the solutions are relatively unstable, but it is soluble in the other alcohols provided a certain amount of water is present. Solvents of the alcohol type in which zein is soluble without added water are the glycols, such as ethylene glycol or diethylene glycol, the ethyl ether of ethylene glycol, furfuryl alcohol, and tetrahydrofurfuryl alcohol. Zein is soluble without added solvents in certain organic acids, such as acetic or lactic. Some phenols dissolve zein without added solvent, although with phenol itself a small amount of alcohol or water is needed to give complete solubility. Aliphatic amines dissolve zein but yield very unstable solutions. Aromatic amines require only a small amount of alcohol to give satisfactory solutions. Methyl and ethyl lactates alone are solvents for zein, while the other esters of hydroxy acids require the addition of small amounts of alcohol or water. Ketones alone are not zein solvents, but their solutions in water dissolve zein readily. Acetone-water mixtures between the limits of 60 t o 80 per t e n t acetone by volume dissolve zein.
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0.0 0.1 02 HCI tt--r NaOH MlLLlMOLS PER GRAM OF P R O T E I N
03
OF FIGURE 2. ALKALIA N D ACIDABSORPTION ZEIN
Anhydrous solvent mixtures are of interest because of the possibility of using resins and plasticizers which would not be compatible in solutions containing water. Solvent mixtures containing denatured ethyl alcohol with various secondary solvents have been studied in detail. The various classes of compounds when used in this manner show the following general types of behavior: The aliphatic hydrocarbons have no actual solvent power; they act purely as diluents and are therefore probably unsuited for practical use except in special cases. The aromatic hydrocarbons improve the solvent power of the anhydrous alcohol; the order of decreasing efficiency is
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benzene, toluene, and xylene, the latter being relatively ineffective. None of these solvents when used alone with anhydrous alcohol will yield a solution that is satisfactory for practical use, and the addition of a third solvent which may be a plasticizer or a resin, or water is required to improve the solvent power. Aliphatic fatty acid esters, such as ethyl or butyl acetate, act as diluents and add practically no solvent power for zein, but in some cases are useful for blending other constituents or for adjusting the drying rate of coating solutions. The esters of hydroxy acids such as the lactates or isobutyrates are powerful solvents, 10 to 20 per cent of these materials being sufficient to give with anhydrous alcohol tj satisfactory solvent mixture. The chlorinated hydrocarbons are excellent secondary solvents and give satisfactory solutions within the range of 10 to 20 per cent by volume on the high alcohol side; if used in excess they require between 20 and 50 per cent alcohol to produce satisfactory solutions. Many of these solvent mixtures are remarkable for the stability which is imparted to the zein solutions. Ethylene dichloride is probably the most promising of these solvents. Aliphatic nitro compounds are of interest, yielding satisfactory solutions between the limits of 15 and 55 per cent nitro compound. The addition of 10 per cent or more of ethylene glycol or diethylene glycol to anhydrous alcohol produces a satisfactory solvent. These materials may also be regarded as temporary plasticizers. These various anhydrous solutions, in general, have a higher viscosity than the aqueous alcoholic solutions of zein, and the addition of a small amount of water to lower the viscosity usually yields a more practical solution. Moderate amounts of water are adsorbed by the zein and are not effective in precipitating water-insoluble materials from the composition. A convenient measure of solvent power of a given solvent mixture is the temperature a t which the solution separates into two phases. The exact relations depend somewhat on the particular grade of zein used, but the general conclusions can be applied to any of the grades which have been tested. Figures 3 and 4 show typical curves of this nature, relating the composition of the solvent to the temperature a t which separation into two phases occurs. The mixtures used in determining these curves contained 1gram of zein to 5 cc. of solvent mixture. The tolerance as ordinarily determined for lacquer solvents can be determined directly from these curves by noting the solvent composition a t which the curve crosses the desired temperature line. Zein is soluble in aqueous solutions of alkaline materials. A p H of a t least 11.5 is required, which eliminates solvent solutions of weakly alkaline character. The total alkali requirement is about 1.2 per cent of sodium hydroxide based on zein. Solutions can be made as concentrated as 20 per cent of zein and may find application as coating or impregnating materials. The solutions in sodium hydroxide are subject to rapid deterioration due to reaction of the zein with the alkali, with the formation of insoluble material consisting presumably of sodium salts. Potassium hydroxide solutions are usable for a longer time. Zein disperses easily in water solutions of a wide variety of soaps or soaplike substances, including fatty acids and rosin, These dispersions appear to have many characteristics of solutions and may be diluted with considerable amounts of water without precipitation of the zein. Such solutions or emulsions are useful as coating materials and are economical to use because the volatile solvent has been eliminated.
Curing Reactions Zein reacts slowly with formaldehyde, yielding a product of improved toughness, mechanical strength, and water re-
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sistance. The reaction, however, if not catalyzed, is slow compared with the same reaction in the case of other proteins such as casein. It is also inhibited by alcohols. The slowness of the reaction makes i t possible to compound the curing agent directly into plastics and to use formaldehyde in the solutions of zein for coating or impregnating purposes. The reaction with formaldehyde is catalyzed by acids. Organic acids such as acetic or lactic are effective, but the strong mineral acids, such as hydrochloric, are superior where other considerations permit their use. Small amounts of ammonia or primary amines act as promoters for the reaction but are effective only in the presence of an excess of acid.
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is not important. The substituted sulfonamides are efficient plasticizers for some uses. Uses Work on zein was first undertaken in this laboratory to provide a raw material for plastics of the casein type. Zein has the advantage over casein in that the formaldehyde used for curing can be incorporated directly into the plastic before forming; thus the lengthy curing in a formaldehyde bath which is required by the more rapidly reacting proteins is eliminated. A plastic is most readily made from zein by mixing the raw material with about 20 per cent of its weight of water and 5 per cent of formaldehyde, together with any pigments, plasticizers, or other material which may be desired. The mixture is plasticized on rolls and can then be formed in any convenient manner such as pressing into sheets in a hydraulic press. I n this operation a partial cure is obtained by pressing for a short time a t about 100" C. Preliminary forming operations, such as cutting into button blanks, punching, or stamping, may be carried out on the partly cured material. The cure is completed by baking for a rather long period a t 60" to 100" C., the time depending on the temperature and the size of the pieces. The cured material is extremely well suited for finishing by automatic machining operations; it is easily machinable and produces but little wear on the cutting tools. An important property which improves its value for use in buttons and in similar novelties is that it can be dyed from an aqueous dye bath after all forming operations have been completed. The zein plastic is very tough and has a transverse strength of approximately 15,000 pounds per square inch. I t s water resistance is somewhat better than that of the casein plastic; i t absorbs about 10 per cent moisture when immersed in water long enough to obtain equilibrium. Where the type of article manufactured permits, a material saving in cost may be gained by cold pressing the raw zein powder, with or without modifying agents, into desired shapes in a pill machine provided with dies of the proper shape
SECONDARY SOLVENT-% BY VOLUME
FIGURE 3. ZEIN SOLUBILITY IN BINARY MIXTURES OF ANHYDROUS SHELLACOL AND SECONDARY SOLVENTS
Zein can be cured by formamide. At least 30 per cent by weight based on zein is required if the zein film is heated to effect the cure, or 40 per cent if the film is not heated. The formamide is simply added to the zein solution used for coating or other purposes. Formamide-cured zein bodies have, however, a tendency to become brittle on aging.
Resins and Plasticizers Zein is compatible with acid resins such as rosin, Manila, or shellac, and also with some relatively neutral resins such as alcohol-soluble ester gum. These materials in zein solutions serve the same purposes as in the case of the cellulose derivatives, giving gloss and body to the film and decreasing the cost on a dry solids basis. The perfect plasticizer for zein has not yet been found. Fatty acids of high molecular weight are preferred. Esters of hydroxy acids such as dibutyl tartrate are applicable. The higher boiling glycol derivatives, such as diethylene glycol or Carbitol, or high-molecular weight glycol esters, such as glycol phthalate, are suitable where water resistance
ALCOHOL-9- BY VOLUME
FIGURE 4. ZEINSOLUBILITY IN AQUEOUSALCOHOLS
and subsequently curing the piece by immersion in formaldehyde containing a curing catalyst. By using a vacuum pressure system, thin pieces can be cured throughout. The outstanding use for zein appears to be in the paper coating field. As a decorative coating for magazine covers, labels, and similar uses, it gives a pleasing surface without the high gloss characteristic of some other coating materials. It can also be formulated with a high gloss. It is particularly notable for resistance to scuffing. Compositions for this use may contain rosin or other resins and are most successfully
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plasticized with a fatty acid, preferably rather highly unsaturated, such as soybean fatty acid. It can be applied either as an alcoholic solution or as an aqueous dispersion made as previously described. Zein is outstanding in its resistance to penetration by greases or oils, and therefore has a large potential use in the making of food wrappings or containers where this property is of importance. This property is also useful in that a very thin layer on paper or other absorbent material will permit the application of a wax coating without penetration of the paper. It is also effective as a sealing coat against asphalt or phenolic resins which are very difficult to seal with other available materials. Fatty acids, or fatty acids and a resin, are the preferred nonzein constituents. Because of its cost zein can be used as an adhesive only in cases where its specific properties can justify its use. I n the application of high-grade wood veneers it is very useful in that the adhesive does not strike through the wood. A waterproof bond for this purpose, joining the veneer either to a wood or transite base, is made by using a solution of zein containing formaldehyde and an organic acid (9) and curing under pressure at an elevated temperature. A laminated board made from zein-impregnated paper is tough and fairly flexible, can be sheared and punched easily, or can be embossed by hot pressing. Uses for this material will be chiefly of a decorative nature where low cost, toughness, and ease of producing satisfactory colors are important, and where water resistance is not essential. Because of its high molecular weight, the impregnation of paper with zein is rather difficult, but a satisfactory product can be made by using an absorbent paper and applying the zein in a fairly concentrated solution a t a slightly elevated temperature. The use of rosin, ester gum, shellac, or resins of similar nature in amounts up to 50 per cent of the weight of the zein improves the flow in the laminating process, and in many cases reduces the cost without impairing the mechanical properties of the finished product. The impregnated paper is pressed dry at
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about 100" C. under a pressure of 500 to 2000 pounds per square inch, depending on the plasticizers used. Zein is useful in solid color printing where the chief object is to coat a paper or cardboard surface as evenly as possible with a dye. It aids in the spread of the dye and brings out the true color of the dye in the solution rather than the metallic appearance due to dye crystals which is characteristic of many vehicles used for this purpose. I n many cases the fastness of the dye to light is improved. Bleeding of the dye may be decreased or eliminated. The production of films and fibers from zein is expected to provide outstanding uses. However, these developments are being deferred until more satisfactory methods of curing zein have been developed.
Acknowledgment The writer wishes to acknowledge the assistance of numerous members of the Research, Engineering, and Sales Departments of the Corn Products Refining Company in the development work described in this paper, Literature Cited (1) Bizio, Giorn. di fisica, chimica, storia naturale, medicine ed arti Brugnatelli, [2] 5 , 127-35, 180 (1822). (2) Chittenden and Osborne. Am. Chem. J.. 13. 453-68. 629-62 (1891); 14, 20-44 (1892). (3) Cohn, C. J., Berggen, R., and Hendry, J., J. Cen. Physiol., 7,8198 (1924). (4) Gorharn, J., Quart. J. Sci., Literature & Arts, 11. 206-8 (1821). (6) Osborn, T. B..J. Am. Chem. SOC.,19, 524-32 (1897). (6) Osborn, T. B., and Clapp, S. H., A m . J . Physiol., 20, 477-93 (1908). (7) Ritthausen, J. prakt. Chem., 106, 471-89 (1869). (8) Showalter, M. F., and Carr, R. H., J. Am. Chem. SOC.,44. 2019-23 (1922). (9) Sturken, O., U. S. Patent 2,115,240 (April 26, 1938). (10) Watson, C. C., Arrhenius, S., and Williams, J. W., Nature, 137, 322 (1936).
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PRESENTED before the Division of Industrial and Engineering Chemistry s t the 98th Meeting of the American Chemical Society, Boston, Mass.
Antiseptic and Germicidal SAMUEL S. EPSTEIN AND FOSTER DEE SNELL Foster D. Snell, Inc., Brooklyn, N. Y.
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N RECENT years, medical and food-processing authorities have shown a growing interest in air-borne infections and in air-borne contaminations. The use of antiseptic and germicidal paints has been proposed as a helpful factor in reducing atmospheric microbial pollution. The purpose of this paper is t o review the literature on the subject of antiseptic and germicidal paints, particularly with respect to methods of testing, and to present some of our own test methods and results. Frequent washing of painted surfaces is recognized as a good sanitary practice. Robb ( 8 ) found that thorough scrubbing of painted walls effected a greater reduction in the number of air bacteria in the operating room than washing the floors with 0.1 per cent solution of mercuric bichloride or 5 per cent carbolic acid. In tracing the causes of occasional severe staphylococci infections originating in an operating room, Hart (6) always found Staphylococcus aureus on walls and ceilings despite frequent painting and daily washing.
Two approaches to the preparation of germicidal paints have been used. The first is simply the addition of antiseptic agents to otherwise standard paints. The second is modification of the vehicle by addition of halogen to the oil. Practically speaking, the latter means chlorination of linseed oil to a 4 per cent halogen content. Portier and Kling (7) found that the incorporation of chlorophenols rendered paints antiseptic, as based on tests with Escherichia coli and Staphylococcus aureus. One drop of a broth culture was deposited on a painted metal surface and allowed to dry in light and in darkness for 24 hours. A drop of sterile water was placed on the dried bacterial culture and allowed to remain for 15 minutes, after which the moistened area was swabbed with sterile cotton and the cotton inoculated into broth. Lack of growth indicated antiseptic power. Normal development of fresh inoculations of test cultures in such broth excluded the factor of inhibition. Using substantially the same technique, Troussaint (9) re-
