Solutions With Formaldehyde-Water Solutions

APPLIED MICROBIOLOGY Vol., No., p. 9- May, 96 Copyright 96 American Society for Microbiology Printed in U.S.A. Comparison of Sterilizing Properties of Formaldehyde-Methanol Solutions With Formaldehyde-Water Solutions MYRA WILLARD AND ALICE ALEXANDER Materials Technology Department, Hughes Aircraft Company, Culver City, California Received for publication 6 January 96 ABSTRACT WILLARD, MYRA (Hughes Aircraft Co., Culver City, Calif.), AND ALICE ALEXANDER. Comparison of sterilizing properties of formaldehyde-methanol solutions with formaldehyde-water solutions. Appl. Microbiol. :9-. 96.-Sterilizing properties of formaldehyde in anhydrous methanol and formaldehyde in water were compared. Formaldehyde in methanol achieved sterilization in hr. Formaldehyde in water was found to sterilize in shorter periods of time and to have a longer shelf life than formaldehyde in methanol. Formaldehyde has been known for many decades to possess bactericidal properties. It has been used extensively to sterilize hospital equipment and to decontaminate sick rooms, clothing, and instruments (Finch, 959; Perkins, 956). The latest suggested application of the chemical is for the sterilization of space vehicles. Drawbacks to its even more frequent use have been its corrosive action on some materials and the fact that it sometimes leaves a white deposit on parts treated with its water solution. Reasoning that these undesirable properties of formaldehyde disinfectants were due to the reactions of formaldehyde with water, Opfell, MVIiller, and Louderback (96) developed a formaldehyde in anhydrous methanol sterilant. It was the purpose of this investigation to compare the sterilizing properties of this new sterilant with those of formaldehyde in water as functions of age of the sterilant, exposure time, and surface conditions of the parts to be decontaminated. Microorganism type and population were held constant. Short exposure times ranging between and hr were chosen so that differences in sterilizing abilities of the solutions would be readily discernible. MATERIALS AND METHODS The formaldehyde-water solutions consisted of 5 and % formaldehyde in distilled water, prepared from 7 % formalin solution (Baker analyzed reagent). Formaldehyde-methanol solutions evaluated were composed of.6 and 7.5 % formaldehyde in anhydrous methanol, prepared by the method described below. All solutions were assayed at the beginning of the test program according to the procedure below. To determine the sterilants' effectiveness on different surfaces, three types of test specimens were used: rough 9 and smooth aluminum coupons ( by by 6 in.) and stainless steel screws and a stainless steel block containing threaded holes. The smooth coupons were prepared by polishing with no. C fine emery paper. The rough coupons were roughened with no. 6C coarse emery paper. The test organism used was a stock water suspension of spores of Bacillus subtilis var. niger. Preparation of formaldehyde in methanol. The procedure of Opfell et al. (96) was followed in preparing formaldehyde in anhydrous methanol. The method consisted of pyrolyzing highly polymerized polyoxymethylenes which contain less than % water, and absorbing the vapor formed in anhydrous methanol. a-polyoxymethylene, which is the most suitable polymer because of its low bound-water and acid content, was prepared by the action of potassium hydroxide on an uninhibited aqueous solution of formaldehyde. A solution of,5 g ( moles) of paraformaldehyde and,5 ml of distilled water was heated to almost boiling and adjusted to ph 7 with dilute potassium hydroxide. Heating was continued for hr, and then the solution was filtered hot and allowed to cool. A solution of.5 g (. mole) of potassium hydroxide in 5 ml of water was added to the cold filtrate which was then stored for hr at approximately C. The white precipitated a-polyoxymethylene was separated by filtering, and washed until wash water was neutral. The precipitate was spread out in a thin layer, air-dried for hr, and then dried in a vacuum oven at C (5. mm of Hg) for 8 hr. The yield was 5 g ( %) of a-polyoxymethylene. Monomeric formaldehyde was prepared by pyrolysis and absorption in methanol. The apparatus was thoroughly dried. A mixture of 85 g of a-polyoxymethylene and 9 g of P6 (to absorb water given off during pyrolysis) in 55 ml of mineral oil was placed in a -liter threeneck flask. The flask was connected to a receiving flask by a heated tube (Fig. ). Dry nitrogen was passed through the apparatus for about min to flush out air (lower explosive limit for formaldehyde in air is 7 %). The oil mixture was heated to 6 C, and vapors were condensed in the collecting flask immersed in a Dry Iceacetone bath. By visual inspection of the oil mixture, pyrolysis was complete in min. The formaldehyde in the collecting flask was vaporized slowly by gradually removing the Dry Ice-acetone bath, Downloaded from http://aem.asm.org/ on May, 8 by guest

WILLARD AND ALEXANDER APPL. MICROBIOL. and the vapor was carried into a flask containing 5 ml of anhydrous methanol by the dry nitrogen gas at a flow rate of not more than.5 cc/min. The flask containing the methanol was cooled with a salt-ice slurry, since the absorption of the formaldehyde in the methanol is moderately exothermic. The yield was 5 ml of formaldehydemethanol solution. Assay of formaldehyde in methanol. The sodium sulfite method was employed for the quantitative analysis of formaldehyde (Kolthoff and Stenger, 97). It is based on the following reaction: HCHO (aqeuous) + NaSO + H -* NaOH + NaH(HCHO)SO (a slow reaction). Into an Erlenmeyer flask was placed an excess of a fresh M sodium sulfite solution. The solution was then acidified with M HCl until a ph of 7 was attained. A known volume of formaldehyde sample was added and completely mixed. The mixture was allowed to stand for 5 min, and then titrated with M HCl to a ph of 7. The end point was determined with a ph meter. Formaldehyde concentration in the sample was calculated as follows: per cent (w/v) HCHO -. X Va X Na X where. = meq weight of formaldehyde; Va = volume of acid used (ml); Na = normality of acid; and Vs = volume of sample (ml). Sterilization test. The test scheme for the evaluation of liquid sterilants is diagrammed in Fig.. Previously steamsterilized screws and coupons in covered petri dishes were inoculated with 6 spores of B. subtilis, by use of a sterile syringe. The petri dishes were opened slightly to allow the samples to dry. The test specimens were then thoroughly sprayed with the formaldehyde solution, and each coupon was covered with a sterile mating coupon. The screws were threaded into the steel block which had also FIG.. Apparatus for formaldehyde preparation. () Oil-pumped nitrogen. () Liquid nitrogen trap for moisture. () Pyrolysis flask with heating mantle. () Powerstat for heating mantle. (5) Powerstat for flexible heating strip. -(6) Formaldehyde-collecting flask in Dry Ice-acetone bath. (7) Methanol-absorption flask in ice-salt slurry. (8) Moisture absorbent in vent. (9) Flexible heating strip. () Thermometer. Vs been sprayed with the sterilant. The dishes were closed and set aside for,, or hr. After the exposure, the coupons were aseptically transferred to test tubes containing ml of sterile distilled water. The screws were removed from the block and also transferred to tubes of water. Each threaded hole was swabbed with a sterile cotton swab, and the swabs were placed in the tubes with the respective screws. The samples were scrubbed ultrasonically for 5 min, and -ml portions were aseptically removed from each to prepare pour plates with Tryptone Glucose Extract Agar. Plates were incubated at 7 C for days, after which colony counts were made with the aid of a Spencer counter. Spore recovery test. The spore recovery test is a control to determine that the method used for recovering the spores from the test specimens is adequate. Previously steam-sterilized coupons and screws in closed petri dishes were inoculated with 6 spores. After drying, each sample was aseptically transferred to tubes of sterile distilled water, and the samples were scrubbed ultrasonically. Portions were aseptically removed and diluted to spores per ml. Portions ( ml) of each diluted sample were then used to prepare pour plates with Tryptone Glucose Extract Agar. Plates were incubated at 7 C for 7 hr, after which colony counts were made. Inhibiting action of formaldehyde solutions. Instead of removing residual active formaldehyde at the end of the exposure, a special test was conducted to determine possible interference of the formaldehyde residue on bacterial I II I Sterilization Test Spore Recovery Test Formaldehyde Inhibition Test Coupons inoculated Coupons inoculated with 6spores. Coupons sprayed with liquid sterilant. Test coupon mated Coupon mated with anwith sterile coupon Coupons set aside with 6 spores. Sterile coupons sprayed with liquid sterilant. other sterile coupon. Coupons set aside hours.,, or hours. I, Jo Spores collected in Spores collected in Coupons placed in water water. water. and or spores Aliquots diluted to spores/ml. added. I ml aliquots incu- ml aliquots incu- ml aliquots incubated bated with growth bated with growth with growth medium. medium. medium. I Colonies counted. Colonies counted. Colonies counted. FIG.. Test scheme for evaluation of liquid sterilants. Downloaded from http://aem.asm.org/ on May, 8 by guest

VOL. ) 96 STERILIZING PROPERTIES OF FORAIALDEHYDE SOLUTIONS growth. The results give an effective spore population for this study. Previously steam-sterilized coupons and screws in petri dishes were sprayed with the liquid sterilant miixtures. The dishes were covered and set aside for hr. Each specimen was then aseptically placed in ml of sterile distilled water. The specimens were scrubbed ultrasonically, and or spores of B. subtilis were added to each tube. Portions ( ml) were aseptically removed from each tube to prepare pour plates with Tryptone Glucose Extract Agar. The plates were incubated at 7 C for 8 hr minimum after which colony counts were made. RESULTS AND DISCUSSION Spore recovery data are presented in Table. The expected number of spores, 6, were recovered from the test specimens. TABLE. Spore recovery for smooth, rough, and threaded surfaces* Type no. Plate Population Smooth Al coupon A. X 6 B. X 5 A 9. X ' B 9.6 X '. X 5 Rough Al coupon A 5. X 5 B 5.6 X 5 A. X 5 B. X 5. X 5 Screw A. X 6 B.8 X 5 A 6. X 5 B 6. X 5. X 5 * Inoculum: - spores per sample. No. TABLE. Inhibition controls of formaldehyde solutions Results of the inhibition tests show that inhibiting action of residual formaldehyde possibly transferred to the growth medium with the dilution samples was not sufficient to hinder interpretation of the sterilization test results with.6 % formaldehyde-methanol solution (Table ). However, inhibition was significant with the 7.5 % formaldehyde-methanol solution and the water solutions. From an original population of spores, the 7.5 % formaldehyde-methanol in the growth medium reduced the recoverable population to 8 with the smooth coupons, with rough coupons, and 65 with the screws. Formaldehydewater (5 %) caused a reduction from spores to 5 with smooth coupons, 7 with rough coupons, and 77 with screws. Similar inhibition was observed with the % formaldehyde-water solution, with a spore population of reduced to 89 with smooth coupons, with rough coupons, and,7 with screws. These results mean that in the sterilization tests with these solutions the following effective original spore populations existed: 7.5 % formaldehyde-methanol,. X to 6 X ; 5 % formaldehydewater, X to 9 X 6; % formaldehyde-water, X to X 5. Results of sterilization tests with the.6 % formaldehyde in methanol are presented in Table. The -hr test with the fresh solution showed very little lethal action on the rough coupons and screws. Exposure with this solution for hr initially produced sterility on the rough coupons, and a,-fold decrease in population to 5 on the smooth coupons and to 66 on the screws. After weeks of storage, lethal action in hr was negligible, but in hr was still fairly good. The number of spores recovered after hr of exposure increased a small but significant amount over that recovered in the initial test. After weeks of storage, spores recovered from the rough coupons and screws in the -hr exposure test increased still more, but.6% formalde- 7.5% formalde- 5% formaldehyde % formaldehyde Control (no formaldehyde) hyde in methanol hyde in methanol in water in water * Smooth Al,t 56, 89 coupon,5 6 66 _, 55 56 5 895 89 Rough Al 8 6 57 coupon 87 58 5 7 85 5 7 5 Screw 5 5 5,6 96 9 7, _, 5 79,7 86 8 95 _ 87 65 77 7,7 Spore suspension controls _,6, -,,6. X '. X * Number of spores added to sample. t Results expressed as number of spores recovered. Downloaded from http://aem.asm.org/ on May, 8 by guest

WILLARD AND ALEXANDER APPL. MICROBIOL. the number recovered from the smooth coupons was curiously less. Since the -hr exposure tests were generally showing decrease in activity with age of the sterilant, the exposure period was increased to hr after 6 weeks of storage of the.6 % solution. No viable spores were recovered after this exposure. In the initial - and -hr tests, 7.5 % formaldehyde in methanol was similar in activity to the.6 % solution (Table ). After weeks of storage, the solution was still very effective in hr, but decrease in activity was generally noted in the -hr test. After 9 weeks of storage, the -hr exposure test resulted in considerable decrease in lethal action on the screws. A total of,9 viable spores were recovered in this test, an increase of,8 over the number recovered in the test performed after weeks of storage. Results on the coupons remained good. No viable spores were recovered from the rough coupons, and spores were recovered from one of four smooth coupons. A -hr exposure test made after 6 weeks of storage resulted in apparent sterility of all test specimens. The initial, -, and -week storage tests on the 5 and % formaldehyde in water solutions employed - and -hr exposure periods. The -hr test had been deleted because of the poor results obtained for this exposure with the methanol solutions. However, apparent sterilization was achieved in all cases employing - and -hr exposures (Table 5). It was decided, therefore, to reduce the exposure in subsequent tests to hr until decreases in sterilizing activity were noted. After weeks of storage, the 5 and % water solutions TABLE. Sporicidal action and shelf life of.6% formaldehyde in methanol on smooth, rough, and threaded surfaces* Smooth Al coupon Rough Al coupon Screw and swab No. Initial test hrt 7 55 -, hr 9 5 * Inoculum: _6 spores per sample. Inhibition recovery: -6 spores per sample. I Too numerous to count. 5 5 66 weeks hr $ /-,6 hr 9 6 5 8 7 5 6 77 weeks 6 weeks ( hr) ( hr) 6 8 9 7,6 6 5 produced near sterility in all cases (Table 5). From an original recoverable population between and 5 spores, an average of.5 spores were recovered from the smooth coupons, none from the rough coupons, and from the screws after exposure to the 5 % solution. After exposure to the % solution, no spores were recovered from the smooth coupons, and fewer than ten were recovered from the rough coupons and screws. The % solution achieved apparent sterility in a -hr exposure after 5 weeks of storage, but not after 8 weeks. An average of spores were recovered from the smooth coupons, from the rough coupons, and 7 from the screws. In general, a small decrease in activity with sterilant age was found in the -hr tests on the 5 % formaldehyde in water solution after 6 and 8 weeks of storage. From 7 to 7 viable spores were recovered from the specimens treated with the 8-week-old sterilant mixture. The -hr exposure test on all specimens with 5 and % formaldehyde-water solutions stored for a maximum of weeks apparently resulted in sterilization in all cases. Formaldehyde in methanol was found to be an effective sterilant, although its action was not as rapid as that of formaldehyde in water. The formaldehyde in water solutions were found to be effective in achieving sterility under all conditions of test using -hr exposure periods, whereas the formaldehyde in methanol solutions were not. Near sterilization was accomplished in hr by the fresh.6 % formaldehyde in methanol, and by the 7.5 % solution stored up to weeks. Thereafter, sterilizing activity of both methanol solutions on the screws decreased significantly. Activity of the 7.5 % solution on the coupons appeared fairly TABLE. Sporicidal action and shelf life of 7.5% formaldehyde in methanol on smooth, rough, and threaded surfaces* No. Initial test weeks I hrt hr I hr hr 9 weeks 6 weeks ( hr) ( hr) Smooth Al 6 coupon 8.5 95 85.5 Rough Al coupon 6-5.5 Screws and 8 -,88,55 swabs, -, 67 7 -,,6,5 5,5, 57 -,7 7,9 * Inoculum: 6 spores per sample. Inhibition recovery between. X and 6 X spores per sample. Downloaded from http://aem.asm.org/ on May, 8 by guest

VOL. ) 96 STERILIZING PROPERTIES OF FORMALDEHYDE SOLUTIONS TABLE 5. Sporicidal action and shelf life of 6 and % formaldehyde in water on smooth, rough, and threaded surfaces* Formalde- hyde No. Initial test weeks weeks weeks 5 weeks 6 weeks 8 weeks weeks weeks concn hrt hrt ( hr) ( hr) ( hr) ( hr) ( hr) ( hr) ( hr) ( hr) ( hr) 5 Smooth Al coupons 6 8-7 it - - -.5 8 7 Rough Alcoupons 8 7 6 5-5 5 - - 9 7 7 Screws and swabs 7-85 Smooth Al coupons - - t - - - - - - - Rough Alcoupons - 6 7 - - - - - 5 Screws and swabs - 7 - - - - 7 * Inoculum: -6 spores per sample. Inhibition recovery: 5% solution, between X and 9 X 5 spores per sample; % solution, between X and X 5 spores per sample. t Contamination. constant for 9 weeks, although it is difficult to assess the ACKNOWLEDGMENT actual effectiveness on the rough coupons because of the The authors wish to thank John L. Brady for his excelhigh inhibiting action of the solution when these test speci- lent work in the preparation and analysis of the formalmens were used. Both methanol solutions sterilized all speci- dehyde solutions. mens in a -hr exposure after the solutions had been stored LITERATURE CITED for 6 weeks. When specimens were exposed to the water FINCH, W. E. 959. Disinfectants. The Macmillan Co., New York. solutions for hr, near sterility was obtained after weeks KOLTHOFF, I. M., AND V. A. STENGER. 97. Volumetric analvsis, of storage of the 5 % solution and after 5 weeks of storage Vol.. Interscience Publishers, Inc., New York. PERKINS, J. J. 956. Principles and methods of sterilization. Of the % solution. The 5 % solution exhibited a slight Charles C Thomas, Publisher, Springfield, Ill. reduction in activity after 6 weeks of storage, and the % OPFELL, J. B., C. E. MILLER, AND A. L. LOUDERBACK. 96. solution exhibited a slight reduction after 8 weeks of Semi-final report: evaluation of liquid sterilants, phase III, IV, V, VI and VII. Rept. No. R of P-b, Contract N- storage. 57 (JPL), Dynamic Science Corp. Downloaded from http://aem.asm.org/ on May, 8 by guest