October 15, 1930
INDUSTRIAL AND EA'GINEERING CHEMISTRY
From the measurements a t the wave length where, for example, the spectral transmission curves of T 1and Tz intersect, one determines the proportion of TS in the mixture. With this proportion known, the proportions of T I and TZin the mixture are derived from the transmission factors found a t any other wave length.
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While it is theoretically possible to determine the proportions of the components in a mixture containing four or more, the practical difficulties in securing the greater required number of precise measurements a t different wave lengths is so great as to destroy the practical value of this method of quantitative analysis for such complicated mixtures.
Methods of Testing Mothproofing Compounds' William Moore AMERICAN CYANAMID SALESCOMPANY, 535 FIFTH AvE., NEW YORK,N. Y .
URING the past few years, with the development of various preparations to render woolen materials mothproof, the effectiveness of these materials has been tested frequently by chemists with little or no biological training. In working with a living organism one encounters various reactions which vary according to the conditions of the experiment. These reactions and habits have not been fully recognized in all cases. The purpose of this paper is t o bring out some of the peculiarities of the webbing clothes moth in relation t o the standard methods of testing mothproofing preparations, Although the final answer of the effectiveness of any mothproofing preparation is its ability t o stand up under home conditions, it takes a long time to carry out a test under such conditions. I n order to accelerate the methods of testing preparations, various artificial tests have been devised. The results of such tests are considered to give a final answer as to the effectiveness in the home.
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Half-Circle Test
One of the common tests to determine the mothproofing properties of a compound is that generally known as the halfcircle test. This consists of putting into a Petri dish two half-circles of woolen material, one treated and one untreated. A number of larvae of the clothes moth are then liberated in the dish and it is put away under suitable conditions for a period of time. Such a test shows whether the treated piece is repellent to the larvae. If no further care is taken than indicated above, erratic resultsmay be obtained. First, one must be careful that the treated piece of cloth is cut from the same piece of cloth as the untreated, and is exactly the same in all details. Clothes moths will show a preference for a loosely woven over a tightly woven piece of cloth. They prefer a piece of cloth with a long nap to one with little or no nap. A certain piece of dyed cloth may be mothproof when compared with a sample of undyed woolen cloth, but attractive to moths when compared with a piece of wool dyed with different dyes, Even with these precautions one can run an experiment, cutting the half-circles from the same piece of cloth, and, without treating either sample, show that they prefer one half to the other half. The writer has run such experiments with a piece of gray-green woolen dress goods. This material is a very fine grade of cloth and has a very fine soft nap on one side. By placing one half-circle with the nap side down and the other with the nap side up, it is possible to concentrate all the feeding on the nap on the under side of the cloth. (Figure 1) The same can be done with mohair. Such an experiment merely indicates that the larvae of the clothes moth like to work between two surfaces. On repeating the mohair 1 Received April 19, 1930 Presented as a part of the Insecticide Symposium before the Division of Agricultural and Food Chemistry at the 79th Meeting of the American Chemical Society, Atlanta, Ga., April 7 to 11, 1930.
experiment and placing a circle of ordinary muslin over the top of two pieces of mohair, greater injury was obtained on the piece of cloth with the nap side up, indicating that, although they like to work between two surfaces, they prefer working between two surfaces of cloth rather than between a surface of wool and one of glass. The cloth should be placed in the dish so that the moth larvae do not show a preference for one piece because of its arrangement in the dish. The next precaution deals with the selection of larvae for the test. Clothes-moth larvae cause two types of d a m a g e damage due to feeding, and damage due to the cutting of fibers to be incorporated in the cocoon which they form when ready to pupate. The larvae normally feed upon animal fibers, such as wool and feathers, but when ready to make their cocoons they will cause damage to almost any material available which has suitable fibers for incorporation in these cocoons, whether wool, cotton, silk, rayon, or even paper. The following experiment illustrates this point. A Petri dish is made up consisting of a half-circle of wool and a halfcircle with a cotton material having a nice nap. By using partly grown feeding larvae the damage will be confined entirely to the wool. Repeating the experiment in exactly the same way, but using larvae which are practically ready to pupate, damage will result to the cotton due to cocoon formation. (Figure 2) I n determining the feeding preferences of clothes-moth larvae, partly grown individuals should be used, since with fully grown larvae erratic results may be obtained. In addition to the size of the larvae, the number employed has a decided bearing. There is a general tendency to use an abnormally large number of larvae in running a test, thinking thereby to accelerate the results. Under natural conditions the heaviest infestation which the writer has ever encountered did not represent more than one larva per square inch. On this basis ten larvae per dish would represent natural conditions. A material which is repellent to clothes moths will show no damage, even from as high as fifty feeding larvae, over a period of 4 weeks. The same material gave protection against fully grown larvae for 4 days, but when the experiment was run for 3 weeks the nap from the untreated piece was totally destroyed, with the result that when the last larva was ready to form its cocoon the most suitable place was on the treated sample, with the result that one cocoon was formed on the treated portion. The presence of abundant nap, whether treated with a mothproofing preparation or whether present on a material which is not eaten by a clothes moth, is a powerful attrahent for cocoon formation. I t would be well t o see how the larvae of the webbing clothes moth react to several different types of materials used for mothproofing purposes. Phenylsalicylate is sometimes used as a moth-proofing agent. When a piece of cloth recently treated with a carbon-tetrachloride solution of phenylsalicylate is placed in a half-circle test with an untreated piece of
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cloth, it shows excellent protection. Whether this protection is due to the odor of the phenylsalicylate or t o its taste is not known but, if the piece of cloth is exposed to atmospheric conditions for several months, during which time most, if not all, of the odor has disappeared, a half-circle test will show that much of the protection has been lost. Cinchona alka-
Figure 1-Both Pieces Untreated. Right-Hand Half-Circle Placed w i t h Nap Side Up(and Left w i t h Nap Side Down All feeding upon half-circle with nap turned down.
loids combined with a fatty acid show excellent protection, all of the larvae feeding upon the untreated sample. Since this preparation is not volatile, the experiments can be repeated 6 months or several years later with the same result. (Figure 3) Some mothproofing preparations contain arsenic or silicofluorides. Experiments run with both of these materials show that they have little or no repellent effect upon the clothes-moth larvae. (Figure 4) I n some experiments one will find a larger number of larvae on the untreated piece than on the treated piece. I n other tests they will be equally divided and occasionally there are even more larvae on the treated piece then on the untreated.
Another interesting experiment would be a modification of a half-circle test in which both pieces of cloth are treated, but with different preparations. One can use a half-circle t'reated with the cinchona alkaloids in comparison with a half-circle treated with a silicofluoride preparation. (Figure 5) The
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larvae will all leave the piece treated with the cinchona alkaloids and will be found upon the half-circle treated with the silicofluoride. This test does not mean that one of the materials is more effective as a mothproofing agent than the other, but merely shows the relative repellent effect of different preparations.
Figure 2-Half-Circle of Wool on Left; Half-Circle of Cotton o n Right Note feeding damage on wool, and damage due to formation of cocoons on cotton.
In brief, the half-circle test, if carried out with suitable precaution and with a suitable number of feeding larvae, answers the question as to whether the material under consideration is a repellent to the clothes-moth larvae, and, by comparison with other materials, the degree to which it is repellent. Whole-Circle Test
The whole-circle test consists of putting a whole circle of treated material in a Petri dish and introducing larvae as in the half-circle test. With the whole-circle test there is no opportunity for the larvae to show a preference, and since the larvae are fully confined they are forced to feed upon the material. To determine the reactions of partly grown larvae when placed on a whole circle of unsuitable food material, such a test was conducted using a circle of cotton cloth. Although these larvae were only about half grown, after wandering around for a time they finally spun small resting cocoons and became quiescent. It is known that larvae may remain in such a resting condition for more than two years. When fully grown larvae are used, they quickly form their pupal cocoons, cutting fibers from both ends of the cocoon to incorporate in it, thus actually causing damage to the cotton. When partly grown larvae are used in a whole-circle test of a piece of wool treated with a silicofluoride or with a preparation containing arsenic, they quickly die. Although webbing will be found upon the treated piece of cloth, this webbing may be removed and the actual damage will be negligible. This shows clearly that both the silicofluoride and the arsenic preparations are poisonous and that larvae feeding upon cloth treated with these preparations will die, usually before they have sufficient time to cause appreciable damage. If fully grown larvae are used, the larvae will spin cocoons just as they did on the cotton, and in so doing will damage the cloth by cutting fibers to incorporate in the cocoons, but these fibers are not eaten, and consequently the larvae are not killed. It is difficult always to select larvae which are fully grown and ready to pupate. For this reason, in running such a test a certain number of larvae will be killed due to feeding.
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In the case of a test conducted with a piece of cloth treated with a silicofluoride, twenty apparently fully grown larvae were introduced. Of these, fourteen formed cocoons, cutting fibers to be incorporated in the cocoon. Moths emerged from twelve of these cocoons, showing clearly that the cutting of
causing noticeable injury to the cloth. Whether these larvae are poisoned due to having fed slightly upon the cloth or whether they die of starvation is not known. In the case of the untreated cloth, 10 per cent developed, causing considerable damage. It is possible that a higher percentage could
Figure 4-Right Half-Circle Thoroughly Sprayed on B o t h Sides w i t h Solution of Fluosilicate; Left Side Untreated
Figure 5-Right Half -Circle Treated with Cinchona Alkaloid; Left Half-Circle Soaked in Solution of Fluosilicate
fibers for the formation of cocoons has nothing to do with the eating habits of the larvae and that a poison will not prevent this damage nor will it kill the larvae and thus prevent the adult moths from emerging. In one whole-circle test a piece of cloth was treated with an arsenic preparation containing 1.5 per cent of arsenious o x i d e a n enormous dose of arsenic, since in spraying operations for foliage insects the concentration of lead arsenate does not as a rule exceed 0.25 per cent. In spite of this enormous dosage of arsenic, out of twenty larvae used in the test, five formed cocoons, cutting fibers to place over the cocoon, and from these five cocoons four adult moths emerged. (Figure 6) The whole-circle test answers the question as to whether or not the compound is poisonous. In this work again it should be pointed out that feeding larvae should be used, since with fully grown larvae damage may result from the habit of cutting fibers in the formation of cocoons. The cutting of these fibers is not poisonous to the larvae and does not kill the larvae, but permits them to pupate and finally emerge as adults. Two Whole-Circle Tests
be raised using fewer larvae per dish. A study is being made to determine the best method of carrying out this test so that the highest percentage of larvae may be raised to maturity.
A moth-proofing preparation containing naphtha as a solvent will kill the larvae reached by the treatment. Old larvae not killed by the spray are able to migrate, but the question arises as to what will happen t o the young larvae hatched from eggs laid upon the treated material a t some later dates. These larvae are so small that the range of their migration is quite limited and they must either feed or die of starvation. To answer this question, a whole-circle test may be conducted, using a number of larvae that have just hatched. This method insures the absence of predacious mites which may be introduced if adult moths are placed in the dish to lay eggs. Since under normal conditions the mortality of baby larvae is rather high, it is necessray to run a whole circle of untreated cloth to compare with the whole circle of treated cloth. Using one hundred newly hatched larvae in a test with cloth treated with cinchona alkaloids, it has been found that the larvae die without developing and without
Cupboard Test
The cupboard test requires a long period of time, but more nearly answers the question of the value of the preparation in the home. It requires a room maintained under suitable conditions for rearing moths so that it is heavily infested a t all times. Treated and untreated pieces of cloth are placed in the cupboard, subject to the infestation in the room. The experiment may be conducted by having one drawer contain-
Figure 6-Full-Circle Treated w i t h 1.5 Per C e n t Solution of Arsenious Oxide Fibers cut off in the formation of five cocoons; four adults emerged.
ing nothing but treated pieces of cloth, another drawer with nothing but untreated pieces of cloth but exactly similar to the treated pieces in the other drawer, and a third drawer containing treated and untreated samples of cloth. A moth-
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proofing material which will protect a piece of cloth from damage under such conditions for a year or more will also be effective in the home. Conclusions
I n running Petri dish experiments considerable care is necessary that the moths are answering the question which is asked, and not some other question. This is particularly true of a half-circle test. The half-circle test, if properly planned, will indicate whether the material is repellent to clothes-moth larvae. A modification of this method enables one to develop the degree of repellency exhibited by different materials. Such a test, however, does not necessarily give any information as to whether the material is poisonous to
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clothes-moth larvae. The whole-circle test answers the question as to whether the material is toxic t o clothes-moth larvae when they feed upon it. It also shows clearly that a material may be highly toxic but unable to prevent the cutting of fibers in the formation of cocoons. The whole-circle t e i t does not clearly show the repellent properties of a mothproofing preparation. The experiment with two dishes, one with a whole-circle of treated and one with a whole-circle of untreated cloth, may be used to show the effect of the preparation on newly hatched larvae. Although these quick tests will give considerable information concerning the action of a moth-proofing material, the cupboard test probably more clearly represents what will actually happen in the home, but has the disadvantage of requiring a long period of time.
Chemical Micrurgy’ A Method for Studying the Characteristics of Microscopic Quantities of Material Robert N. Titus and Harry LeB. Gray EASTMAN KODAK COMPANY, ROCIIESTER, N. 1‘.
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HE literature contains many references to methods for the analysis of small amounts of material. Especially in the examination of spots in paper, several regular tests hare been devised which require the use of the microscope (6). The composition of these specks of material is determined mainly by their appearance, their physical characteristics, and a few chemical tests on a semi-microscopic scale. I n the case of metallic elements such as iron and copper there are color tests in which the particle reacts n-ith the reagent to form a colored zone. Typical of these is the use of 2, 4-dinitrosoresorcinol to distinguish betyeen metallic or ferrous iron and ferric iron in paper by the formation of a colored lake ( 2 ) . Chemical mfcroscopy provides a method for the identification of comparatively small amounts of material, and the spectroscope has also found application in the analysis of specks. There are many occasions when even elementary information concerning the character of particles too minute to be satisfactorily studied by any of these methods may he of great value. For these problems, where for some reason the material cannot be concentrated and the scattered methods do not prove adequate, chemical micrurgical technic offers a means of investigation. The term “micrurgy” was originated by PBterfi (3’) from “micros” meaning small and “ergon,’ work, and applied by him to the science of microdissection and injection in the fields of physiology and biology. While these methods are not rapid in comparison with the usual methods of chemical microscopy and require considerable patience and manipulative ability, they are rapid and surprisingly accurate compared with the time required and effort expended in obtaining the same results, provided they could be obtained, by indirect means.
veloped a micromanipulator for their microdissections, which is a device for holding and manipulating under the microscope a variety of tools. Several designs of micromanipulators are available (1,3’,4). Figure 1 shows the instrument designed by P6terfi and manufactured by Zeiss. I n general they all incorporate a substantial base to which the microscope is firmly anchored. The tools are held by suitable clamps and their motion is controlled by accurate micrometer adjustments. In addition to the fine movements in each of the three planes, the P6terfi instrument has two
Micromanipulator
The fundamental prerequisite in dealing with particles of microscopic dimensions is t o be able to handle them directly, individually, and separated from any contaminating substances. This has usually been accomplished with dissecting needles held in the hands. The biologists, however, have de1 Received 4ugust 4, 1930. Presented before the Division of Industrial and Engineering Chemistry at the 80th Meeting of the American Chemical Society, Cincinnati, Ohio, September 8 to 12, 1930. Ccmmunication KO 451 from the Koddk Reasearch Laboratories
Figure 1-Micromanipulator
rapid adjustments in the up-and-down and lateral directions. This instrument also provides a rocking or tilting adjustment which enables a tool to be carried a t an angle which is especially important in making the tools. Chemical investigations by this means place some limitations on the microscope and its optics. Any stand provided with a mechanical stage may be used. A binocular
