INDUSTRIAL A N D ENGINEERING CHEMISTRY
April 15, 1929
equilibrium would be reached between the amount of the volatile substance taken up by the agar medium and the amount of the air above it. The exact ratio would, of course, depend upon the solubility of the particular substance, its vapor pressure, temperature, and other factors. The opening of the flask for the purpose of making transfers also introduces an error which might be eliminated by preparing a number of such flasks from each of which only one transfer is made. On the whole, the method has little to recommend it either from a practical or scientific point of view. A modification of the Petri dish method to test the toxicity of relatively insoluble substances has been devised by Curtin.20 I n connection with certain tests of the toxicity of barium carbonate, a double-strength nutrient solution was prepared, and to 10 grams of this were added 5 grams of sodium carbonate solution containing the required quantity of carbonate. Then, after shaking, 5 grams of solution containing the proper amount of barium chloride were added, with shaking. The barium was immediately precipitated as carbonate in the hot solution and the cooling of the jelly held it in place with a minimum aggregation and settling out. This method is claimed to be very satisfactory in preparing cultures of certain relatively insoluble substances, but some compounds cannot be handled in this way, because other undesirable chemical reactions take place. Such materials must be treated in the powdered form. A very important advance in toxicity tests has been made by Bateman2l which greatly simplifies the determination of the toxic concentrations of any substance by the modified agar plate method. It has been shown that there is a definite relationship between the concentration of a preservative and the percentage retardation of the growth of the fungus. This relationship is such that a straight line is obtained when the logarithm of the concentration is plotted against the logarithm of the percentage retardation. FalckZ2had previously shown that under constant conditions the rate of radial growth of fungi is constant. Hence it is only necessary to prepare two concentrations below the toxic limit; of the substance and to plot the logarithm of the concentration used against the logarithm of the percentage retardation. This curve, being a straight line, may be extended to determine the concentration necessary to inhibit entirely the growth of the fungus. Modification of Agar Plate Method One of the outstanding difficulties in any of these methods of testing toxicities of wood preservatives is in connection with the sterilization of the preservative itself. This is particularly true when the preservative is volatile, as coal-tar creosote. When the preservative is a liquid, or a solid in a concentrated 20 21
I*
Curtin, IND. ENG.CHEM.,19, 1159 (1927). Bateman, Private communication on data to appear soon. Falck, Hausschwummforsckungen, 1, 53 (1907).
solution, it is not necessary to sterilize the preservative, because ordinarily these substances are toxic enough to be sterile. It is necessary, however, to sterilize the container in which the preservative is placed before addition of the nutrient agar. The common practice is to place known amounts of the preservative in tightly stoppered bottles or flasks and sterilize them. It is extremely difficult, however, to prevent the loss of more or less of the preservative by volatilization. During heating the air in the flasks expands, causing considerable internal pressure in the flasks. If a cork stopper is used, it softens during steaming, permitting the loss of some preservative. When glass-stoppered bottles are used and the stopper is tight, the internal pressures are often sufficient to burst the flask. I n order to overcome these difficulties and to prevent the loss of any preservative during sterilization, it is proposed that known amounts of preservative be placed in small glass bulbs or ampuls of known weight, which are then sealed and weighed. The sealed bulb is then placed in a glass-stoppered Erlenmeyer flask with a sufficient amount of nutrient culture medium to make the concentration desired. During sterilization the flask is plugged with cotton, the glass stopper being tied to the flask by means of a string. After sterilization, the cotton plug is removed and the bulb containing the preservative is broken by means of a sterile glass rod. The glass stopper is then inserted and, after very gentle shaking to permit some of the expanded air to escape, is tightly inserted and the flask violently shaken to emulsify the mixture. When about ready to gel, the upper portion of the flask is flamed in order to expand the air in it and thus permit the easy removal of the glass stopper, and the emulsion is poured into cold sterile Petri dishes. This method of testing the toxicity of wood preservative has been tried in this laboratory and found to be very satisfactory. Only one difficulty was experienced, which consists of the preservative getting into the neck of the bulb. It is quite difficult to break the small neck of the bulbs by means of a glass rod, as previously mentioned. It can, however, be easily broken by the use of sterile tongs. Care must be taken in filling the bulb not to get any preservative on the inner wall of the neck, since a portion of this would volatilize and be lost when the neck is sealed. The preservative can easily be placed in the bulbs by use of an ordinary capillary tube shown in Figure 1, C. After a little experience the amount of the preservative added to the bulb can be estimated fairly accurately by counting the number of drops from the capillary tube. This modification of the regular agar plate method of determining the toxicity of volatile wood preservatives is rapid, simple, and eliminates all possibility of loss of preservative during sterilization.
Modification of Hempel Gas Pipet' Geo. H. W. Lucas DEPARTMENT O F PHARMACOLOGY, UNIVERSITY
VERY analyst who has used a Hempel gas pipet knows how difficult i t is when employing mercury in the gas buret to prevent such substances as alkaline pyrogallol, fuming sulfuric acid, etc., from running up the capillary tubing in the pipet and coming into contact with the rubber a t the juncture between the pipet and the buret, when the level of the mercury in the buret and the leveling tube is being adjusted. Furthermore, as the gas is run back and forth through the pipet, droplets collect in the capillary tubing and these are frequently sucked 1
Received February 11, 1929.
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TORONTO, TORONTO, CANADA
back into the buret when the gas is being aspirated into it. The author has found that a small bulb about 0.5 cc. in volume blown in the capillary tubing, as shown in the accompanying sketch, causes these bubbles to be broken, and as it takes a few moments for the bulb to fill one has plenty of time to level the mercury in the buret.
