Journal of Antimicrobial Chemotherapy (1978) 4,489-494

Early studies in the evaluation of disinfectants

W. B. Hugo

Department of Pharmacy, The University, Nottingham NG7 2RD, England

liquid disinfectants

Solutions of inorganic salts (especially common salt and nitre) have long been used as preservatives for meat products, and, OVCT 2 centuries ago, a test for the evaluation of the antiputrefactive power of salts was described. Because of its uncanny anticipation of a technique of disinfectant evaluation still in use today it is instructive to describe it in detail. It was due to John Pringle who was born in Roxburgh, Scotland on 10 April 1707. After a classical education at St. Andrews and Edinburgh Universities, Pringle studied medicine at Leiden and gained his M.D. degree in 1730. After a brief spell in academic life in Edinburgh he embarked upon a military career and became, in 1742, head of the British Military Hospital in Flanders. He was renowned for his treatise Diseases of the Army which earned for him the title of founder of modern military medicine. Pringle was elected a fellow of the Royal Society in 1745 and was its president from 1772-78. He was created a baronet in 1749. The test as described by Pringle (1750) consisted of adding lean meat to aqueous solutions of the salt, incubating and noting the presence or absence of smell (a practical end point), and indirectly measuring the presence of putrefactive micro-organisms. Pringle, however, did not leave the matter there. Using sea salt as standard he drew up a table of the relative antiputrefactive power of salts, giving the concept of a coefficient of relative activity, and was able to assign an antiputrefactive coefficient of 30 to alum It should be remembered that at the time that Pringle was writing, the role of bacteria in putrefaction had not been established. Pringle's results are shown in Table I. More than a century passed before the next report on the problem appeared, when Bucholtz (1875) published his determinations of the concentrations of, among other substances, phenol, creosote, salicylic and benzoic acids necessary to inhibit the growth of, and to kill mixed cultures of unnamed micro-organisms. Koch (1881) criticized the use of mixed cultures, a criticism perhaps not completely warranted as in practice disinfectants will, in most cases, be acting against mixed culture (Koch had evolved a technique of isolating pure cultures by the use of a solidified culture medium). Later, in his important epidemiological work on anthrax, he used silk threads as an inert support for anthrax spores which could be immersed in disinfectants and thereafter in a suitable liquid culture medium to determine if killing had occurred. This technique, however, led to an entirely false assessment of the sporicidal value of 489 O3O5-7453/78/O6O489+O6 $01.00/0

© (1978) Academic Press Inc. (London) Ltd.

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W. B. Hugo Table L Pringle's table of salt coefficients (1750) A table of the comparative power of salts in resisting putrifaction Sea salt 5a/ Gemmae Tartar vitriolated Spiritus mindereii Tartarus solubilis Sal diureticus Crude Sal ammoniac Saline mixture Nitre Salt of Hartshorn Salt of Wormwood Borax Salt of Amber Alum

1 1+ 2 2 2 2+ 3 3 4+ 4+ 4+ 12 + 20 + 30+

mercuric chloride for, due to carry over of disinfectant, the recovery fluid was rendered too toxic to permit the outgrowth and germination of anthrax spores, thereby giving the illusion that they had been killed. It was Geppert (1889) who drew attention to this error and advocated the use of ammonium sulphide as a neutralising agent for the excess mercuric chloride. As a result of this, the time of killing of anthrax spores by mercuric chloride had to be advanced from 'a few minutes' to 'several hours'. This early experiment of Geppert is the first recorded example of the use of a neutralizing agent, which is so important in the field of disinfectant evaluation and sterility testing. Koch's silk thread technique was used by otheT workers and Kronig & Paul (1897) used garnets as an inert support for micro-organisms. The paper of Kronig and Paul which may be read in translation in Brock (1961) layed down exemplary rules for disinfectant evaluation. After correspondence with K. Ikeda in Tokyo they adopted the proposal of the Japanese physical chemist to plot the logarithm of the bacterial numbers remaining viable after drug treatment against time and laid the foundation of the work on disinfectant dynamics which was to occupy research workers for the next half century and more. All workers in the field of disinfection should read Kronig and Paul's paper. A novel method which was designed to obviate the errors caused by carry over of residual disinfectant was described by Defries (1897). The test organism was dried on to the bottom of test tubes; the disinfectant to be tested was then added and allowed to act for the required time, when the disinfectant solution was poured from the tubes, which were then lightly rinsed with sterile distilled water before filling with a suitable culture medium and incubating. This test also has the merit of requiring no sampling stage but it is clear that some of the organisms which might include viable cells will be lost during the washing period and it is also possible that cells lying at the bottom of the dried film may escape contact with the disinfectant during the exposure period, especially if the latter is brief. Rideal and Walker introduced their technique, which is still in use today, in 1903. Semi-solid antibacterial preparations The use of the term semi-solid has been coined to embrace a group of pharmaceutical preparations known as pastes, ointments, creams and gels. The chief feature which

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distinguishes the first three is their viscosity, which decreases in the order given. They may consist of an intimate mixture of the active agent with either an oleaginous base or alternatively an emulsion with either water or an oleaginous substance as continuous phase. Gels are preparations in which the base is usually a carbohydrate polymer (starch, pectin, methylcellulose, tragacanth or sterculia gum) and water or more rarely a base of protein origin, such as gelatin, with a suitable quantity of water. More recently polyethylene glycols have been used; these occur in viscosity ranges similar to the paraffins and are water miscible. When formulating antibacterial preparations it is imperative to realize that the properties of the base may seriously modify the antibacterial activity of the medicament. It is quite useless to formulate a well-proven antiseptic into an otherwise elegant pharmaceutical preparation without determining if the final formulation is, itself, an effective antibacterial agent. This aspect of formulation is further discussed by Frank & Stark (1954) and Kolstad & Lee (1955). The early literature however shows that both testing techniques had been described and fundamental truths discovered before the beginning of this century. Gottstein (1889) for example studied the efficiency of an ointment containing 5% of phenol in wool fat (lanolin), using Pseudomonasfluorescensand Chromobacterium prodigiosum as test organisms, and concluded that an ointment of phenol dissolved in an oleaginous base was devoid of antibacterial properties. BreslaueT (1895) came to a similar conclusion. He used phenol in a number of different types of base. His method of testing was to dry the cultures of the organisms (Chr. prodigiosum and Staph. aureus) onto glass discs, immerse them in the preparation for a given period, then afteT washing off any adherent base with ether, incubate the washed discs in nutrient broth. He found that when soft paraffin was used as a base, the disc had to remain immersed for one day before they were sterilized, on the other hand, with an oil in water emulsion base, the discs were sterilized in 20 minutes. Cheyne (1915) and Keilty & Packer (1915) described further experiments on the formulation and evaluation of phenol ointments and reached the same general view. Despite the early recognition of the deleterious effect of oleaginous bases on the bactericidal properties of phenol an ointment consisting of phenol, 2 % in white soft paraffin, was described in the B.P. of 1948. Tests which measure bacteriostatic activity The first official test was published by the Food, Drug and Insecticide Administration of the United States Department of Agriculture using a method described by Reddish (1927) in which portions of the preparation were placed on the surface of nutrient agar inoculated with Staph. aureus. After incubation the zones of inhibition, if any, around the preparation were measured. This test was modified later by incorporating 10% of horse senim in the agar 'to simulate conditions in a wound' and a control consisting of unmedicated base was also used in each experiment. This test is known as the cup-plate test. Wood (1939), howeveT, when testing acriflavine emulsions obtained identical results whether serum was present or not. In addition to the placing of the test preparation on to sectors of seeded agar, it may be placed in a trough cut in uninoculated agar and test organisms, streaked in parallel lines up to the edge of the trough. Failure to grow up to the edge is indicative of inhibition. A feature of nearly all disinfectant testing is the adoption of a standard preparation. Bryan (1936) suggested that Ammoniated Mercury Ointment, U.S.P., might be used

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for this purpose and also that the minimum pTeformance of a preparation designated as an antiseptic was that it should produce a rone of inhibition of at least 2 mm on an agar plate seeded with Staph. aureus; this suggestion has not been adopted Tests for bactericidal activity A number of tests have been described which imitate, at least in part, the principle of the phenol coefficient test for liquid disinfectants. A culture of the test organism is mixed well with the semi-solid preparation and the mixture subcultured into a suitable broth designed to disperse the base and neutralize the antibacterial activity of the medicament. Fisher, Accousti & Thompson (1943) evaluated sulphanilamide ointment and Prusack & Mattocks (1949) cetrimide ointment by methods of this type. Foter & Nisonger (1950) mixed the culture and preparation and transferred it to a syringe surrounded by a constant-temperature jacket; at intervals the mixture is subcultured by ejecting small volumes from the syringe nozzle into subculture medium. Air disinfectants Apart from early empirical use of burning aromatics and sulphur in the counteraction of the odours associated with disease such as epidemic plague, the classical work of Joseph Lister represents the first attempt to destroy airborne bacteria. Pasteur had shown that fermentation and putrefaction were caused by 'living germs of which the air was full'. In 1865 Lister read Pasteur's paper and it occurred to him that the suppuration prevalent in surgical and maternity wards might be prevented by applying as a dressing some substance which would destroy floating 'germs'. In 1865 he introduced his carbolic spray in an attempt to destroy airborne bacteria. An account of Lister's contribution to microbiology may be read in the paper by Miles (1967). The action of ammonia, aromatic and aliphatic amines on airborne bacteria was studied by Trillat & FouassiCT (1912). These authors later examined the effect of radiations including a, P and y-rays on airborne bacteria (Trillat & Fouassier, 1914). The destruction of airborne bacteria is thought by some to be of importance in public health, and the hazards of cross infection in crowded environments such as cinemas, schools, factories or even the mess decks of warships might be reduced by treatment of the atmosphere with a suitable disinfectant. Many studies on this subject appeared in the 1940s and '50s but are now thought to be of dubious value. Methods of determining viable airborne bacterial populations Koch (1881) exposed plates of a solid medium to the air for varying periods and counted the colonies after incubation. This method is still used as a routine method of checking the bacterial flora of the air in such places asfillingrooms for injectable preparations, but it suffers from the defect of depending on the natural fall of bacteria on to the plate and can give no idea of the true viable population in a given volume of air. An attempt at a quantitative estimation of the airborne population was made by Petri (1888). Petri passed a known volume of air through a tube containing fine sand, the sand was then shaken with sterile saline and aliquots plated. Frankland (1887) substituted sucrose for sand, this having the advantage of being soluble in the saline. A modification of Frankland's apparatus was adopted by the American Public Health Association in 1909 as the standard for determining airborne bacteria.

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Rettger (1910) drew a known volume of air directly through saline. A similar device was described by Lemon (1943) the air being drawn through a nutrient broth. A device employing the principle of the centrifuge was described by Wells (1933). A cylinder of glass coated on the inside with nutrient agar revolved at high speed while held vertically in a suitable apparatus, air was drawn in at a constant rate and any bacteria present were thrown by means of centrifugal force on to the agar. The glass tube was then incubated and developing colonies counted. Luckiesh, Taylor & Halladay (1946) studied an electrostatic sampler and compared its performance with the exposed plate technique. Hollaender & Dal la Valle (1939) described a simple device which consisted of an inverted funnel secured in a box. The mouth of the funnel was arranged above a Petri dish containing nutrient agar. When a negative pressure was created in the box, air entered through the stem of the funnel and impinged on the agar. An instrument which bore a resemblance in principle to the last named device was the slit sampler described by Bourdillon, Lidwell & Thomas (1941). This instrument is available commercially. In summary, it can be seen that many of the methods in use today have a very early origin, a fact often discovered in other scientific spheres if the older literature is researched. References Bourdillon, R- B., Lidwell, O. M. & Thomas, J. C. A slit sampler for collecting and counting air-borne bacteria. Journal of Hygiene, Cambridge 41: 197-224 (1941). Breslauer, E. Ueber die antibacterielle wirkung der Salben mit besonderen berticksichtigung des Einflusses der Constitutionell auf den Desinfectienswerk. Zeitschrift fur Hygiene und Infektionskrankheiten 20: 165-97 (1895). Brock, T. D. (Ed.) Milestones in Microbiology. Prentice Hall, London (1961). Bryan, A. H. The comparative antiseptic action of ointments and related products. Journal of the American Pharmaceutical Association {Scientific Edition) 25: 606-20 (1936). Bucholtz, L. Antiseptika und bacterien. Archivfur experimented Pathologic und Pharmakologie 4: 1-81 (1875). Cheyne, Sir W. W. The Hunterian oration on the treatment of wounds in man. Lancet i: 419-30 (1915). Defries, W. Notes on the examination of disinfectants. Journal of the Royal Sanitary Institute 18:416-29(1897). Fisher, C. V., Accousti, N. J. & Thompson, M. R. Bacterial contamination in sulphonamide ointments. Journal of the American Medical Association 122: 855-8 (1943). Foter, M. J. & Nisonger, L. L. A new direct approach to the evaluation of the germicidal efficiency of scmisolid Pharmaceuticals. Annals of the New York Academy of Sciences 53: 1-219 (1950). Frank, R. & Stark, G. Ober die antibakterielle wirkung von acriflavin und cetyltrimethylammonium bromide in verschiedenen salbengrundlagen. Pharmaceutica Acta Helvetiae 29: 81-90 (1954). Frankland, P. F. A new method for the quantitative estimation of the micro-organisms in the atmosphere. Philosophical Transactions of the Royal Society 178: 113-52 (1887). Geppert, J. Zur lehre van den antisepticis. Berliner klinische Wochenschrift 26: 789; 819 (1889). Gottstein, A. Sublimat-land als antiseptic. Therpeutische Monatshefte 3: 102-6 (1889). Hollaender, A. & Dalla Valle, J. M. A simple device for sampling air-borne bacteria. Public Health Reports 54: 574-7 (1939). Keilty, R. A. & Packer, J. E. Experimental studies of various antiseptic substances for use in treatment of wounds based on the work of Sir W. Watson Cheyne. Journal of the American Medical Association 64: 2123-5 (1915).

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Koch, R. Zur Untersuchung von pathogenen Organismen. Mitteilungen aus dem Kaiserlichen Gesundheitsamte 1: 1-48 (1881). Kolstad, C. K. & Lee, C. O. The relative efficiences of various ointment bases as antiseptic vehicles. Journal of the American Pharmaceutical Association 44: 5-7 (1955). Kronig, B. & Paul, T. Die chemischen Gundlagen der Lehr von der Giftwirkung und Desinfection. Zeitschrift fur Hygiene und Infectionskrankheiten 25: 1-112 (1897). Lemon, H. M. A method for collecting of bacteria from air and textiles. Proceedings of the Society for Experimental Biology and Medicine 54: 298-301 (1943). Luckiesh, M., Taylor, A. H. & Halladay, L. L. Sampling devices for air-borne bacteria. Journal of Bacteriology 52: 55-65 (1946). Miles, Sir A. A. Lister's contribution to microbiology. British Journal of Surgery 54: 415-8 (1967). Petri, R. J. Eine neue Methode Bacterien und Pilzsporen in der Luft nachzuweisen und zu zahlen. Zeitschrift fur Hygiene, Leipzig 3: 1-145 (1887). Pringle, Sir J. Experiments on substances resisting putrifaction. Philosophical Transactions of the Royal Society 46: 480-7 (1750). Prusak, L. P. & Mattocks, A. M. The efficiency of cetyltrimethyl ammonium bromide in a series of ointment bases. Journal of the American Pharmaceutical Association, Scientific Edition 38: 67-69 (1949). Reddish, G. F. Examination of disinfectants. American Journal ofPublic Health 17: 320-9 (1927). Rideal, S. & Walker, J. T. A. Standardization of disinfectants. Journal of the Royal Sanitary Institute, London 24: 424-41 (1903). Rettger, L. F. A new and improved method of enumerating air-borne bacteria. Journal of Medical Research 22: 461-8 (1910). Trillat, A. & Fouassier, M. Action des doses infinitesimales de diverses substances, alcalines fixes ou volatiles, sur la vitalite des microbes. Comptes Rendus Hebdomadaire des Seances de VAcadimie des Sciences 155: 1184-6 (1912). Trillat, A. & Fouassier, M. Influence de la radio-activit£ de l'air sur les gouttlettes microbiennes de l'atmosphere. Comptes Rendus Hebdomadaire des Siances de VAcadimie des Sciences 159: 817-9 (1914). Wood, W. C. Acriflavine emulsions a study of their antiseptic value. Pharmaceutical Journal 142: 327-S (1939).