Rev. sci. tech. Off. int. Epiz., 1995,14 (1), 75-79

Testing disinfectants for efficacy G. TAMÁSI *

Summary: Testing under laboratory conditions is undoubtedly useful in assessing the activity of disinfectants. However, such testing must be regarded as no more than a preliminary to field trials. This not only indicates the unreliability of laboratory tests, but also poses the wider problem of how any laboratory evaluation can be correlated with the field requirements. Many attempts have therefore been made to devise tests which are suitable to evaluate the in-use requirements of disinfection (e.g. onfarms). The aim of these techniques is to record the end-point of a disinfection procedure on the surfaces in animal houses. In view of the need to standardise testing conditions in the field, the author provides details of the optimum time and place for taking samples under field conditions. Samples for culture media should be taken from the floor when dry. The various techniques (i.e. swabbing, agar cylinder, agar carrier, 'ready-touse' set) should provide information on the end-point of the disinfection. The desired end-point is approximately one viable bacterium per cm of surface area. 2

The technique used should demonstrate whether the disinfection has been effective. K E Y W O R D S : Animal housing - Disinfection - Efficacy - Field trials Laboratory testing - Testing methods.

INTRODUCTION When animals are removed from animal housing, the surfaces within the premises should b.e regarded as contaminated areas which may serve as a source of infection for the next inhabitants of the premises. Therefore, animal housing should be thoroughly cleaned and disinfected during the interval between removal of one group of animals and introduction of the next group. Disinfectants are used on the interior surfaces of animal housing under a wide variety of conditions. Many factors influence the efficacy of the disinfection (e.g. temperature, pH, presence of organic materials, composition of the surface), making it difficult to predict efficacy; this should therefore be tested (6). The methods of performing the tests necessary for the registration of disinfectants are not described here. This paper deals with the means of testing the efficacy of disinfection using a registered disinfectant under field conditions. Another important focus here is

* Phylaxia Veterinary Biologicals Co., Szállás u. 5, P.O. Box 23, H-1486 Budapest 10, Hungary.

76 the testing of so-called 'preventive' disinfection - which is performed during the interval between removal from the premises of one group of animals and introduction of the next group - with special regard to day-old chicks and newborn animals.

WHERE AND WHEN SHOULD THE EFFICACY OF DISINFECTION BE TESTED? After the removal of animals and birds from a building and cleaning of the premises, the surfaces will still be c o n t a m i n a t e d with microbes. T h e extent of microbial contamination differs between the various surfaces. The floor can b e regarded as the most contaminated area of the animal house; the nature and irregularity of the floor usually make this more difficult to disinfect than other surfaces. Therefore, if a testing method shows that disinfection has been effective on the floor, the other surfaces are likely to have been satisfactorily disinfected. The timing of sample collection is another important aspect of testing. Samples for microbiological testing should not be taken from a wet surface. On the one hand, the disinfectant might still be acting on a wet surface, and the end-point of the disinfection has therefore not yet been reached. On the other hand, disinfectant residues in such samples might prevent the growth of microorganisms in culture media. Neutralising the disinfectant with an antagonist before taking samples from surfaces u n d e r field conditions would be nonsensical. The floor should therefore be allowed to dry before samples are taken for culture.

CULTURE SELECTION AND INTERPRETATION OF RESULTS Before discussing the principles involved in the selection of a test microorganism, it is useful to recall the function of disinfection: to kill contaminating microorganisms and thus prevent the incidence of a disease. Disinfectants are rarely required to act in an absolute capacity, and therefore the principle of all such testing is to d e t e r m i n e the degree of survival of microorganisms. A n ubiquitous microbe (i.e. found in stables, equipment, faeces, etc.) might be considered suitable for testing disinfection under field conditions. In practice, two such strains of test microorganisms are used: a strain of coliform bacteria and a strain of staphylococci, both cultured using a selective medium (3,4). The use of these organisms can u n d o u b t e d l y be of considerable value, but the results obtained must not be interpreted too broadly, as these strains do not exhibit a high degree of resistance to disinfectants (6). Determination of the total aerobic bacterial count provides a more reliable test m e t h o d . Samples are t a k e n from surfaces in animal housing, using a suitable method, to obtain a count of the number of viable microorganisms present. T h e extent of the survival of the total aerobic microorganisms can be used as a measure of the efficacy of disinfection. This is one of the most critical elements in the interpretation of results, and is of great significance. Many experiments in this field (7) have led to the following conclusion: the count of viable bacteria on the floor after a disinfection must not exceed the value of one viable bacterium per cm (or 100 bacteria per 100 cm ). 2

2

77 A n e n v i r o n m e n t a l microbiologist will be aware that this cannot serve as a strict definition. However, it should be noted that two separate, consecutive disinfections are usually necessary to reach such a low level of contamination on the floor of an animal house.

METHODS OF TESTING Direct swabbing techniques Several techniques have b e e n used for the assessment of disinfection efficacy, involving the collection of samples.from a given area (e.g. 100 cm ) by swabbing, and a subsequent quantitative (or semi-quantitative) assessment of surviving organisms. Even after a good disinfection, one or two of ten swabs may be positive in either coliform or staphylococci tests (e.g. in speed tests) ( 1 , 3 , 4 , 5 ) . 2

Agar cylinder techniques A stainless steel cylinder (e.g. 20 x 8 cm) is fitted with a piston, enabling a 3-4 m m contact the surface to be investigated. This Incubating the agar slice provides a microorganisms within a given area (2).

is filled with an agar medium. The cylinder layer of the m e d i u m to be p u s h e d out to layer is then cut and placed in a Petri dish. q u a n t i t a t i v e assessment of surviving

'Agar sausage' method One end of a teflon tube is sealed, and the tube is filled with agar medium to produce an 'agar sausage', which is then removed from the tube. One end of the 'agar sausage' is cut, and the cut surface is touched onto the surface to be investigated. A slice is then cut and placed in a Petri dish, providing a q u a n t i t a t i v e assessment of surviving microorganisms (9). Agar-carrying linen This technique has proved to be more practical than the other tests described above. A circle of linen is cut to the dimensions of a Petri dish, with a small flap which extends beyond the dish when the linen is positioned inside the base of the dish. The linen circle and the dish are sterilised. The Petri dish is then filled with agar medium. The cover of the Petri dish is removed, and the flap of linen can be used to lift the agar medium out of the dish and place this on the surface to be investigated. The flap should again be used to return the agar to the Petri dish (A. Forray, personal communication; 8). Ready-to-use test Although all of the above techniques are more or less feasible and can provide very useful information, they cannot be easily integrated into the disinfection practice of the premises, as they all require a laboratory background and skill in practical microbiology. Some 'ready-to-use' sets have therefore b e e n developed, which do not require such skills. The usé of one of these sets is described below. 2

A piece of linen (2 c m ) is impregnated with agar medium, mixed indicators and catalysers; this is placed in a small plastic bag. T h e set also contains wetting agent, tweezers, an incubation bag (all are sterilised) and a strip of glue. T h e bag containing the medium should be cut open, and the pieces of linen (with medium) should be placed on the floor using the tweezers. The pieces of linen should be wetted and then lightly

78 rubbed on the floor. The samples should be placed in the incubation bag, which should then be closed with the strip of glue and stored at room temperature in a dark place. The samples should be investigated after 4-16 h. If the violet colour of the medium has not changed, or has changed in only one of five pieces, this demonstrates the efficacy of the disinfection (representing a count of less than 100 viable bacteria per 100 cm ). If the colour of the media has changed from violet to yellow/brown in 4-6 h, or if more than one of five pieces has b e c o m e yellow/brown in 16 h, this d e m o n s t r a t e s that the disinfection has not been effective (8). 2

* * TESTER L'EFFICACITÉ DES DÉSINFECTANTS. - G. Tamási. Résumé : Les épreuves de laboratoire sont indubitablement utiles pour évaluer l'action des désinfectants. Cependant, elles ne doivent être considérées que comme préliminaires à des essais sur le terrain. Cela montre non seulement les limites de fiabilité des tests en laboratoire, mais pose également un problème plus vaste, à savoir comment étendre une évaluation faite en laboratoire aux conditions du terrain. C'est pourquoi de nombreuses tentatives ont été faites en vue de mettre au point des tests permettant d'évaluer les besoins concrets du terrain en matière de désinfection (par exemple dans les fermes). L'objectif de ces techniques est de fixer le seuil d'efficacité d'une méthode de désinfection des surfaces dans les bâtiments destinés aux animauxPrenant en considération la nécessité de normaliser les conditions d'études sur le terrain, l'auteur donne des détails sur le moment et le lieu optimums pour effectuer des prélèvements sur le terrain. Les prélèvements qui devront être mis en culture seront prélevés lorsque le sol est sec. Les diverses techniques (écouvillonnage, cylindre et conteneur de gélose, trousse ad hoc) devraient fournir des informations sur le seuil d'efficacité de la méthode de désinfection. Le seuil souhaité est approximativement d'une bactérie viable par centimètre carré. La technique utilisée devrait permettre de vérifier si la désinfection a été efficace. MOTS-CLÉS : Désinfection - Efficacité - Essais en laboratoire - Essais sur le terrain - Logement des animaux - Méthodes d'essais.

EVALUACIÓN DE LA EFICACIA DE LOS DESINFECTANTES. - G. Tamási. Resumen: Las pruebas de laboratorio son ciertamente útiles para evaluar la acción de los desinfectantes; pero no debe considerárselas sino como preliminares a las pruebas que se realizarán en el terreno. Lo cual no solamente muestra los límites de fiabilidad de las pruebas realizadas en laboratorio, sino que plantea a la vez el problema más vasto de cómo hacer la correlación entre una evaluación realizada en laboratorio y las condiciones del terreno.

79 Esto ha llevado a realizar varios intentos de desarrollar pruebas capaces de evaluar las necesidades concretas del terreno en materia de desinfección (por ejemplo en las granjas). Su objetivo es establecer el umbral de eficacia de un método de desinfección de las superficies en los locales destinados a los animales. Tomando en consideración la necesidad de estandarizar las condiciones de evaluación en el terreno, el autor ofrece detalles precisos acerca del momento y el lugar óptimos para obtener muestras de campo. Las muestras que se pondrán en cultivo serán tomadas del suelo cuando éste esté seco. Las distintas técnicas (escobillones, cilindro y contenedor de gelosa, kit ad hoc) deberían proporcionar informaciones sobre el umbral de eficacia del método de desinfección. El umbral deseable es aproximadamente de una bacteria viable por centímetro cuadrado. La técnica utilizada tendría que permitir verificar si la desinfección ha sido eficaz. PALABRAS CLAVE: Desinfección - Eficacia - Locales para animales Métodos de pruebas - Pruebas en el terreno - Pruebas en laboratorio.

* * * REFERENCES 1. ACKERMANN H.H., H Ä R T U N G J. & H I L L I G E R H.G. (1982). - Vergleich eines n e u e n

Verfahrens mit zweibekannten Verfahren zur Bestimmung der Keimzahl an Oberflächen im Stahl. Berl. Münch, tierärtzl Wschr., 95, 5-10. 2. KOVÁCS F. (1980). - Âllathigiénia, 2nd Ed. Agricultural Publishing Co., Budapest, 601 pp. 3. M E H L H O R N G., M E T H L I N G W., B E E R K., E R W E R T H W. & N E U P A R T H V. (1980). -

Mikrobiologische Schnellmethoden zur Desinfektionskontrolle. In Proc. 3rd International Congress for Animal Hygiene, Vienna (H. Willinger & G. Thiemann, eds), 188-189. 4. POLYAKOV A.A. (1979). - Veterinarnaya sanitariya. Kolos, Moscow, 618 pp. 5. STEIGER A . (1981). - Möglichkeiten und Gränzen der frischen Mh. VetMed., 36,725-728.

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6. SYKES G. (1967). - Disinfection and Sterilisation, 2nd Ed. Spon Ltd., London, 486 pp. 7. TAMÁSI G. (1983). - Uj készítmény 'DISINTEST' a fertôtlemtés ellenôrzésére. Magyar Allatorvosok Lapja, 38, 88-90. 8. TAMÁSI G. & NAGY A. (1971). - A preventiv fertôtlemtés szerepe az iparszerû sertéstartásban. Magyar Allatorvosok Lapja, 26, 605-614. 9. TEN CATE L. (1965). - A note on a simple and rapid method of bacteriological sampling by means of agar sausages. J. appl. Bacterio!, 28,221-223.