ANALYTICAL CHEMISTRY
1038 Table I.
Recovery of Chlorine from Chlorides and Chlorine-Containing Anions
Compounds Present Sodium chloride Sodium chloride K S C S and K C S Sodium chloride K S C S and K C N Sodium chloride Potassium perchlorate Potassium perchlorate Potassium chlorate Potassium chlorate Sample MR-1
++
Chlorine Present,
Chlorine Found,
10.65 4.61 3.01 4.75 2.70 2.83 2.21 3.68 7.35%
10 58 4 59 3 01 1 76 2.71 2 84 2 28 3.70 7.317,
Mg.
Mg.
were analyzed by this method and all results were typical of those shown for sample MR-1. All samples contained cyanide, thiocyanate] chloride, and approximately ten different metal ions. Six determinations Tyere made on all samples similar to MR-1, using approximately 15 mg. for the fusion in each analysis. I n all cases it was necessary to add more sodium chloride before the titration. The average deviation of the mean was *0.05. The total chlorine present in sample MR-1 was also determined by the gravimetric method after a sodium peroxide fusion, a n d the result, 7.35%, represents an average of si.; determinations. LITERATURE CITED
5 ml. of a 0.01 A’ sodium chloride solution before titrating. A4n equivalent amount of silver nitrate is then deducted to obtain the blank. Results obtained by this method were in very good agreement with the theory as shown in Table I . DlSCUSSIOIV OF RESULTS
The method was first used with the chemically pure salts listed in Table I. I n addition, many samples of a smoke residue
( 1 ) Bullock. Burlingame. and Kirk, P. L., IND.ENG.CHEX.,.ISAL.
ED.,7, 178-80 (1935). (2) LaForce, J. R., Ketchum, D. F., and Ballard, A . E.. AXAL. CHEM.,21, 879 (1949). (3) Niederl, J. B., and Kiederl. Victor, “Organic Quantitative Microanalyds,” p. 160, Xew York. John Riley & Sons, 1947. (4) Preising, J. M., Slonek, 0. F., and Rudy, J. H., IKD.ENG.CHEM., ANAL.ED., 14, 875-7 (1942). (5) Scott, W. W.,“Standard Methods of Chemical Analysis,” Vol. I, pp. 271-9, N e w York, D. Van Nostrand Co., 1939. RECEIVED July 29, 1950.
Improved Toximetric Agar-Dish Test for Evaluation of Wood Preservatives ROBERT W. FINHOLT’ Union College, Schenectady, N . Y
Ii T H E past 30 years methods have been proposed for toximetric laboratory evaluation of wood preservatives. Two test methods have achieved prominence. The first, in point of view of age and use, is the agar-dish method of Humphrey and Fleming (6),Bateman and Henningson (Z), Schmitz (8, 9 ) , and Baechler (1). T h e newest is the soil-block test, which is an improved form of the standard agar-block European test. This soil-block test was developed by Flerov and Popov (4)and Leutritz (6),and has been studied by Richards and Addoms (7) and Duncan and Richards (3). These tests have been’developed to fill the need for an accurate and rapid method for the evaluation of possible wood preservatives. The agar-dish method gives results in 2 to 3 weeks; the soil-block method, in 2 to 4 months. Both methods give much more rapid results than the quickest outdoor test which, using wooden stakes, usually requires 1to 3 years. The chief criticism of the laboratory methods has come over their lack of similarity to actual conditions. The agar-dish test is rather far removed from natural conditions, but in spite of criticism it has held on because of its rapidity and low cost. The wood-soil test simulates natural conditions in giving the fungus its natural food and in providing a very favorable environment, but is both more expensive and timeconsuming than the agar-dish test. Actually, the two tests may seem to give the same approximate results in a qualitative sense, but they part company in most quantitative comparisons. I n spite of its limitations, the agar-dish method is a very useful tool in fundamental research on toxic materials, if results are used only after careful interpretation. I n this laboratory it has been found necessary to institute two changes in the prescribed method in order to get reliable results. The first modification was in the method of mixing the agar solution with the chemical being tested. Mixing had been done by swirling the toxic material with the agar solution (8) or by continued stirring with a motor-driven stirrer ( 1 ) when rather insoluble materials mere used; neither method gave adequate 1 Present address, Research Laboratory, General Electric Co.. Schenect a d y , N. Y.
and reproducible dispersions. -4 better method was found, ahich used a Waring Blendor for mixing the oil and agar solution. The Blendor, a high speed mechanical homogenizer, mixed creosote and agar so intimately that no droplets could be seen even under 300 magnifications. Use of this mixer under standard conditions made it possible to get good results even with oils and removed one of the sources of error that previous workers had recognized (1, 8) The second change v a s in the definition of a toxic point a t which the concentration of poison is high enough to halt the growth of a test fungus. Schmitz and others defined a “total inhibition point” and a “killing point.” The total inhibition point was taken as the minimum concentration that would allow “no signs of growth either on the nutrient agar or on the inoculum” (9). The killing point was taken as the minimum concentration of poison that would inactivate the fungus inoculum, so that when the plug of inoculum was removed and placed on an unpoisoned slant there would be no growth. These definitions had a fundamental error in considering the state of growth of t h e fungus on the inoculum plug as the criterion. It is only the growth or lack of growth on the surface of the poisoned gel itself that has any validity for many poisons. Because the plug contains no poison when placed in use, the only poison in it is that which diffuses into it from the main body of gel. If the test poison happens to be n-ater-insoluble, as is the case with most wood preservatives, the rate of diffusion is very slow and furthermore is dependent on contact between the plug and the main body of agar. Thus the previously defined “hilling point” is a function of both the toxicity of the test poison and its rate of diffusion. As a consequence, it was found that these values varied as much as 200% in testing oil-type poisons; sometimes a killing point was not found a t all, although the growth of the fungus was inhibited. The definition of total inhibition point did not make it clear whether growth meant an increase in the amount of fungus over the original planting or simply the presence of a visible mycelium, no matter how small. In either case, the judgment of the in-
V O L U M E 2 3 , NO. 7, J U L Y 1 9 5 1
1039
dividual experinienter was of key importance. I n actual laboratory tests, different experimenters varied by as much as 25% in their estimates on the same series of test flasks. Because of these difficulties a new definition was formulat,ed that seemed to avoid both the error and judgment factors of the previous definitions. The inhibition point was defined as the minimum concentration by weight percentage of poison necessary t o prevent any radial growth on the surface of the poisoned agar after a standard period of incubation. This point was called the apparent inhibition point (AIP) to differentiate it from the old total inhibition point (TIP). The incubation time was v:iried, depending on the fungus being used. Madison 517 (incorrectl>- called Fomes annosus) gave good results in the 211eek period that Schmitz had recommended, but Lentinus lepideus needed 3 weeks t o give conclusive results. There would undoubtedly be different times for other species. I n this laboratory use of the improved mixing method and the new apparent inhibition point gave results of high accuracy. The precision as a t least within *5% with any type of poison. Besides this improvement in experimental results, considerable work was saved in end point determinations, as there was no need for further transplanting to tell ivhether a fungus inoculum was dead or not. EXPERI3IENTAL
The procedure was based on previous test methods (1-4) hut was improved and simplified in several respects. The overall time for making the test has been reduced with no loss in accuracy or precision. The culture medium was prepared by adding 15 grams of Difco bacto-agar t o 1 liter of distilled water that had been heated to 95' C. The mixture was mechanically stirred for about 0.5 hour and the clear solution was filtered through cloth. T o t'he filtered solution were added 25 grams of Trommers malt extract (plain) and the hot solution was weighed into 500-ml. Erlenmeyer flasks, 100 grams per flask. A double-pan balance was used and the solution was added by means of a large buret. This gave an accuracy of about 1 0 . 1 gram. The flasks were stoppered with aluminum foil and sterilized at 15 p.s.i. gage pressure (121' C.) for 15 minutes, and the pressure was allowed to come back to normal in another 15 minutes. The average loss in weight on sterilization was 1 gram. Most of the poisons used in this laboratory were not sterilized, but the test chemicals that were t o be sterilized were sealed in pressure tubes and strrilized at the same time as the agar-malt solution.
Table I.
Sample Data for Determination of 4pparent Inhibition Point of a Creosote Fraction
Yol. of stock Solution 211.
0 0 0 0 0 0 0 0
10 20 30 37 40 43 53 90
Tf-1
Poison
Poison Rounded O f f 0 0 0 0 0 0 0 0
005
009 014 018 019 020 025 042
-1tJ-pical set of test results is shown in Table I . The apparent inhibition point is the first point where there is 1 cm. of growth, as that was the diameter of the inoculum plug. The diameter of the growth is the average of two flasks of similar composition, since all tests were run in duplicate and with unpoisoned control flasks. The stock solution contained 5 grams of creosote fractiori 3. The total volume of stock solution was 107 nil. and weighed 104 grams, a-hich made 0.1 ml. of stock solution containing 0.0047 gram of creosote. The weight of the final solutions \vas assumed to be 100 grams in each caw, although it varied from 99.1 t o 99.9 grams. This difference \vas negligible in this test. The apparent inhibition point was taken to be 0.019 * 5% of this valuc. SUMMARY
Fungitoxic materials can be evaluated as wood preservatives by mising the toxic substance with B malt extra(-t-agar solution and then testing the mix against standard fungi. The mixing can he done effectively using a Waring Blendor, and the effectiveness of the preservatives is judged by finding the least concentration needed to prevent growth on the' gel surface. This method is effective with oil-soluble preservatives which are ordinarily difficult to test, in agar solutions, and check results can usually be obtained to *5%.
Total Diameter of Growth
%
% 0.0047 0,0094 0,0140 0.0182 0.0188 0.0200 0.0247 0,0420
the bottom of t,he mixer, so as not to get, froth in the pipet. After addition of the poison emulsion, the flasks were reclosed with the original aluminum foil and given a slight swirlidg motion t o disperse the emulsion. It was found convenient to keep the flasks hot before addition of the poison b y using a large kettle canner. Eight flasks could be handled with ease this way, as the canner had a wire rack and lid. The mixed dispersions Lvere allowed to stand overnight in a 26.7" C. cabinet before inoculntion. The inoculum was a circular path of fungus taken from the outer portion of a culture grown on a plate by pouring 25 nil. of agar solution into a 90-mm. Petri dish. The culture was 14 days old when L e n t i n u s Lepidetcs (Madison 534) was used and 10 days old when ?*ladison 51i was used. The circular patch was cut using a sterile 1-cm. cork borer and the transplanting was done using a long thin spatula sharply bent at the ends. These procedures require a lengthy exposure t o air with consequent possible cont,amination, but in actual practice little trouble w a ~ encountered when suitable care was taken. Aft,er plating, the flasks were returned t o the constant temperature cabinet and incubated in the dark for 14 days with Madison 5 l i and 3 weeks with Madison 534. At the end of this time the flasks were lined u p in order and examined for signs of growth. The apparent inhibition point was taken as the lowest concentration that prevented any radial growth on the poisoned agar. I n most cases the apparent inhibition point was obvious if the concentrations differed by 5% or more.
ACKNOWLEDGMEYT
Cm 6.5 4.0 1.6 1.2
1 ,o, AIP 1.0 0.5, TIP None. killing Point
The author wishes to express appreciation for the financial support given by the American Creosoting Co. and for the technicnal help and assistance of C. D. Hoc-kei, formrily of Bell Telephone Laboratories, now of Union College. LITERATURE CITED
Fungus -Madison 517
To a single sterile flask, 5 grams of test chemical were added and this mixture was used as a stock solution from which dilutions were made up. The mixture was then poured into a sterile 250ml. Rlonel metal Waring Blendor cup and mixed for 2 minutes with the cover on. The flask was then twice rinsed with the niiu and hlended for an additional 30 seconds. The Blendor cup could be effectively sterilized by flaming or by ordinary sterilization. Calculated volumes of the hot (60" C.) solution were withdrawn from the mixer using a graduated 1-ml. pipet (previously kept warm in boiling water) and various amounts were added t o the other flasks. Care had to be taken to draw the liquid from
(1) Baechler. R., Proc. A m . Wood-Preserrew' d s s o c . , 1947, 94 -111. (2) Bateman and Henningson, Ibid., 19, 136 (1923). (3) Duncan and Richards, Ibid.. 1948, Preprint 1-5. (4) Flerovand Popov, A n g e w . Botan., 15, 386-406 (1933). (5) Humphrey and Fleming, U. S. Dept. Agr., Bull. 227 (1915). (6) Leutritr, BeZlSystem Tech. J . , 25, 102-35 (1946). (7) Richards and dddoms, Proc. d m . Wood-Preserurrr' --lsaoc., 1947, 41-58.
(8) Schmita, H., ISD.EXG.CHEM.,ASAL. ED.,1, 76 (1929). (9) Schmitr, H., et al., Ibid., 2, 361 (1930). RECEIVED August 17, 1950. Presented before the Subdivision on Economic Poisons, Division of Agricultural and Food Chemistry, a t the 118th Meeting of the . i \ r E ~ I c A sCHEXICAL SOCIETY, Chicago, 111.
