Antihacterial Finishes on Textile Materials: Assessment of

Antibacterial Finishes on Textile Materials: Assessment of

1. Purpose and Scope 1.1 This test method provides al quantitative procedure for the evaluation of the degree of antibacterial activity. Assessment of antibacterial nishes on textile materials is determined by the degree of antibacterial activity intended in the use of such materials. If only bacteriostatic activity (inhibition of multiplication) is intended, a qualitative procedure which clearly demonstrates antibacterial activity as contrasted With lack of such activity by an untreated specimen may be acceptable. However, if bactericidal activity is intended or implied. quantitative evaluation is necessary. Quantitative evaluation also provides a clearer picture for possible uses of such treated textile materials. 2. Principle 2.1 Swatches of test and control textile materials are tested qualitatively for antibacterial activity by AATCC Method 147, Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method. Those showing activity are evaluated quantitatively. Test and control swatches are inoculated with the test organisms After incubation, the bacteria are eluted from the swatches by shaking in known amounts of neutralizing solution. The number of bacteria present in this liquid is determined, and the percentage reduction by the treated specimen is calculated.

3. Terminology 3.1 activity, n.-of an antibacterial agent, a measure of effectiveness of the agent. 3.2 antibacterial agent, n-in textiles, any chemical which kills bacteria (bactericide) or interferes With the multiplieation, growth or activity of bacteria (bacteriostat). 4. Safety Precautions NOTE: These safety precautions are for information purposes only. The precautions are ancillary to the testing procedures and are not intended to be all inclusive. it is the users responsibility to use sale and proper techniques in handling materials in this test method. Manufacturers MUST be consulted for specic details such as material safety data sheets and other manufacturers recommendations. All OSHA Standards and rules must also he consulted and followed.

4.1 Both the qualitative and quantitative tests should be carried out by persons with training and experience in the use of bacteriological techniques. The U.S. Department of Health and Human Services publication, Biosafety in microbiological and biomedicaldical Laboratories, should be consulted (sec 13,1). 4.2 CAUTION: Some of the bacteria used in this test are capable of infecting humans and producing disease. There fore, every necessary and reasonahle preecaution must be taken to eliminate this risk to the laboratory personnel and to personnel in the associated environment. Wear protective clothing and respiratory protection that prevents penetration by the bacteria. 4.3 Good laboratory practices should be followed. Wear safety glasses in all laboratory areas. 4.4 All chemicals should he handled With care, 4.5 An eyewash/safety shower should be located nearby for emergency use. 4.6 Sterilize all contaminated sample and test materials prior to disposal. 4.7 Exposure to chemicals used in this procedure must be controlled at or below levels set by government authorities (e.g., Oecupational Safety and Health Administrations [OSHA] permissible exposure limits [PEL] as found in 29 CFR 1910.1000 of january 1, 1989). ln addition, the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Values (TLVs) comprised of time Weighted averages (TLV-TWA), short term exposure limits (TLVSTEL) and ceiling limits (TLV-C) are recommended as a general guide for air contaminant exposure which should be met (see 13.2). 5. Limitations 5.1 Por a qualitative, relatively quick and easily executed method to determine residual antibacterial activity of textile materials, refer to AATCC Method 147. 6. Test organisms 6.1 Test bacteria. 6.1.1 Staphylococcus aureus American Type Culture Collection No. 6538. Gram positive organism (seel3.3). 6.1,2 Klebsiella pneumoniae, American Type Culture Collection No. 4352. Gram negative organism (see 13.3). 6.1.3 Other suitable species can also be used. 7. Culture Medium 7.1 Suitable broth/agar media are Nutrient, Trypticase Soy and Brain-heart lnfusion.

Nutrient Broth: Peptone (Bacto-peptone) (see 13.4) 5 g Beef extract (see 13.5) 3 g Distilled water to 1000 ml 7.2 Heat to a boil to disperse ingredients. Adjust to pH1 6.8 0.1 With 1/V sodium hydroxide (NaOH) solution. (This is not necessary if prepared, dehydrated medium is used.) 7.3 Dispense in 10 ml. amounts in conventional bacteriological culture tubes (i.e., 125 X 17 mm). Plug and sterilize at 103 kPa (15 psi) for 15 min. 7.4 Nutrient agar, Add 1.5% bacteriological agar to nutrient (or appropriate) broth (see 7.1). Heat to boiling. Check pH and adjust to 7.1 0.1 using NaOH solution if necessary. Dispense in 15 1 mL amounts in conventional bacteriological culture tubos. Plug and sterilize at 103 kPa (15 psi) for 15 min. (May be sterilized in 1000 mL borosilicate glass asks and petri dishes poured from this.) 7.5 Slurry lnoculum Carrier (for hydrophobic fabrics) (see 7.2 and 7.3): Sodium Chloride 8.5 g Agar 3.0 g Distilled Water 1000 mL 8. Maintenance of Culture in Test Organisms 8.1 Using a 4 mm inoculating loop, transfer the culture daily in nutrient (or appropriate medium) broth for not more than two weeks. At the conclusion of two Weeks. make a fresh transplant from stock culture. incubate cultures at 37 2C (99 3F) or other optimal temperature. 8.2 Maintain stock cultures on nutrient or appropriate agar slants. Store at 5 1C (41 2F) and transfer once a month to fresh agar (see 13.6). 9. Qualitative Test (Screening or Presumptive Test) 9.1 For detection of bacteriostatic activity use AATCC Method 147 on a test specimen and control specimen using the organisms referred to above. For demon stration of bacterieidal activity, proceed to the quantitative test described below. 10. Quantitative Test (reference or Confirmatory Test) 10.1 Preparation. The following description will be in terms of fabric swatches. Textile materials not in fabric form can likewise be tested with the appropriate modication.

10.1.1 Size and shape of treated swatches Cut circular swatches 4.8 0.1cm (1.9 0.03 in.) in diameter, from the test Fabric (preferably With a steel die). Stack the swatches in a 250 mL widemouth glass jar with screw cap. The number of swatches to be used is dependent on the fiber type and fabric construction. Use that amount of fabric which Will absorb the 1.0 0.1 mL of inoculum, and leave no free liquid in the jar. For example, 4 swatches of cotton print cloth will absorb 1 ml.. The number of swatches used per jar should be reported. 10.1.2 Controls. Swatches of the same fiber type and fabric construction as test sample but containing no antibacterial finish (negative control). 10.1.3 Sterilization ot samples. This is Optional. The method to be used depends on the type of fiber and finish. Cotton, acetate and many manmade bers can be sterilized in the autoclave. Wool can be sterilized by ethylene oxide or by intermittent (fractional) sterilization in flowing steam. The latter is also least damaging to certain finishes. Report method of sterilization, if used. 101.4 Size of inoculum per sample. Apply 1.0 0.1 mL of an appropriate dilution of a 24 h broth culture of the test organism so that recovery from (1) untreated control fabric swatches or (2) treated test fabric swatches at 0" contact time (plated as soon as possible after inoculation) will show counts of -2 X 105 organisms. The dilution of the test organism should be made in nutrient (or appropriate) broth (see 7.1, 7.5 and 13.7). 10.2 Procedure. 10.2.1 Inoculaiion of tabrics. When sing Smphylococcus aureus, shake a 24 h culture and let stand for 15-20 min before preparing the inoculum* Place the swatches separately in sterile petri dishesid use 11 microliter pipette to inoeulate them making sure that there is even distribution of the inoculum (see 13.8). Transfer these swatches aseptically to the jar. Screw the jar tops on tightly to prevent evaporation. 10.2.2 As soon as possible after inoculation (0" contact time), add 100 1 mL of neutralizing solution to each of the jars containing the inoculated untrcated control swatches, the inoculated treated test swatches and the uninoculated treated test swatches. 10.23 The neutralizing solution should include ingredients to neutralize the specific antibacterial fabric treatment and to take care of any pH requirements of the fabrics (from finishes, antibacterial agents, etc.). The neutralizing solution employed should be reported (see 13,9). 10.1.4 Shake the jars vigorously for one minute. Make serial dilutions with water and plate (in duplicate) on nutrient (or appropriate) agar. Dilutions of 10 0 10 1, 10 2 are usually suitable. 10.2.5 lncubation over contact periods. incubate additional jars containing inoculated untreated control swatches and jars containing inoculated treated test swatches at 37 1 2C (99 3F) for 18-

24 h. Similar jars may be incubatecl over other periods (e.g., 1 or 6 h) to provide information about the bactericidal activity of the treatment over such periods. 10.2.6 Sampling of inoculated and incubated swatches. After incubation add 100 l ml. of neutralizing solution to jars containing untreated control swatches and to jars containing treated test swatches. Shake the jars vigorously for one minute. make serial dilutions and plato (in duplicate) on nutrient (or appropriate) agar, Dilutions of 10 0 101 - 102 are usually suitable for treated test fabrics. Several different dilutions may be required for untreated control fabrics depending on the incubation period . 10.2.7 lncubate all platos for 48 h at 37 2C (99 3F) or other optimal temperature. 11. Evaluation 11.1 Report bacterial counts as the numeber of bacteria per sample (swatches in jar) not as the number of bacteria per mL of neutralizing solution, Report 0 counts at 10 dilution as less than 100. 11.2 Calculate percent reduction of bacteria by the specimen treatments by one of the following formulas: 1) l00(B-A)/B=R where: R = % reduction A = the number of bacteria recovered from the inoculated treated test specimen swatches in the jar incubated over the desired contact period

B = the number ol bacteria recovered from the inoculated treated test specimen swatches in the jar immediately acr inoculation (at "0" contact time)

2) 100 (C-A)/C=R where: C = the number of bacteria recovered from the inoculated untreated control specimen swateltes in the jar immediately after inoculation (at 0 contact time)

lf B and C are not similar, the larger number should be used. lf B" and C are not significantly different, (B +C)/2 should be used as follows: 3) 100(D -A)/D = R

where: D= (B + C)/2

11.3 lf an untreated control is not available, use the following calculation which allows for any background organisms that might intcrfere with the test.

Bg = 100 [(B-E)-(A-F)/B-E] where: A, B: (see ll.2) E = the number of bacteria initially recovered from the uninoculated treated test sample (existing background organisms) F = The number of bacteria recovered from the uninoculated, pre-Wet treated test sample after incubation in the jar over the desired contact period (existing background organisms after contact period) Bg = background organisms

11.4 For a valid test there should be: (1) 0 colonies of test organism recovered from the uninoculated treated test specimen swatches and (2) a significant increase in the nurnbers of bacteria recovered from the inoculated untreated control specimen swatches incubated for the specied contact time over the numbers of bacteria recovered from the inoculated untreated speeimen swatches at O contact time (immediately after inoeulation). This applics only if dilution Was made in broth (see 10.1.4 and 13.7). 11.5 Report percent reduction of bacteria by the specimen treatment against each test organism. 11.6 The critcrion for passing the test must be determined by the interested parties. 11.7 Report the dilution medium used.

12. Precision and Blas 12.1 Studies (see 13.10) indicate the following within-laboratory precision of the Standard Plate Count (SPC) Test: (a) among-anulifsl variation of l8% and (b) within-analyst variation of 8%,

13. Notes and References 13.1 Publication available from U.S. De~ pal1memofHealtl1 and Human Services CDC/ NlH~HHS Publication No. (CDF) 84-S395; web site: wWw.l1hs.gov. 13.2 Booklet available 'om Publications Office, ACGIH, Kemper Woods Cemei: 1330 Kemper Meadow Dr., Cincinnati OH 45240; el: 5l3`742~2(2(J', web site: www,acgh.org. 13.3 Available from American Type Culture Collectinn (ATCC). P.O. Box 1549, Manassas VA 20108; tel: 703/365-2700; fax: 703/3652701; web site: wwW.zx1cc.0rg. l3.4 Bacm4Pcp1one may be obtained from Difco Laboratories 920 Henry St., Detroit Ml 4SZOl.

13.5 Beef extract may bs obtained (rom Ballimore Biological Laboramries, 250 Sclillmg Cr. Cockcysville MD 21030; Difco Labo-

ratorios (address above); or Oxoid (USA) Ltd., 9017 Red Branch Rd., Columbia MD 21045. 13.6 Conslstent and accurate testing 1'e~ quitas maintenance oa pure_ uncontamnatcd, nonmutam test culture. Avod comamination by use of good sterile leclinique in plating and transferring. Avoid mutation by Strict adhcr cnce to monthly stock t\'ansl`crs. Check cullure purity by making sireak plates periodically and obssrving for single species-chnractcribtc type ofcolonics. 13.7 The dilulion of the test organism may bc prepared in slerilc 0.85% salina solution or suitable buffer if a steadystale culture is de~

sircd during the comact perm; ' in the slurry inoculum carnft * _' bio fahrics are being Lesed. 13.8 A surfactant mn) ha ;;; tien medium to enhance \\ en' " bic fabrica. The surxctuni num ':; ~ cause es reduction in bacmrial n_:".?: testing al the intended uso concatf, the use and concemration uf >_';_

13.9 [f slexile distillcd uma? ;~ Nace of a ncutralzing soluuut. *f ways be the possibilm tha: :f' cide will be carried o\ er. 13.10 Pecer, J. T: Leslie. J. W. Replicate counting errors by and bacterial colony counters, _' E tion,Vol.45,1982, pp 238-240.

Acabados antibacterianos de las materias textiles: evaluacin de

1. Objeto y mbito de aplicacin 1,1 Este mtodo de ensayo proporciona al procedimiento cuantitativo para la evaluacin del grado de actividad antibacteriana. Evaluacin de acabados antibacterianos en los materiales textiles se determina por el grado de actividad antibacteriana destinado en el uso de tales materiales. Si slo actividad bacteriosttica (inhibicin de la multiplicacin) se destina, un procedimiento cualitativo que demuestra claramente la actividad antibacteriana en contraste con la falta de actividad por parte de una muestra no tratada puede ser aceptable. Sin embargo, si la actividad bactericida est previsto ni implcito. Evaluacin cuantitativa es necesario. La evaluacin cuantitativa tambin proporciona una imagen ms clara de los posibles usos de tales materiales textiles tratados. 2. principio 2.1 Muestras de materias textiles de prueba y control se analizan cualitativamente la actividad antibacteriana por el mtodo AATCC 147, Evaluacin de la actividad antibacteriana de los Materiales Textiles: Mtodo Streak paralelo. Aquellos que muestra la actividad se evalu cuantitativamente. Las muestras de prueba y control se inoculan con los organismos de ensayo Despus de la incubacin, las bacterias se eluyen de las muestras por agitacin en cantidades conocidas de solucin de neutralizacin. El nmero de bacterias presentes en este lquido se determina, y el porcentaje de reduccin de la muestra tratada se calcula. 3. terminologa 3,1 actividad, n.-de un agente antibacteriano, una medida de la eficacia del agente.

3,2 agente antibacteriano, n-en el sector textil, cualquier producto qumico que mata las bacterias (bactericidas) o interfiere con la multiplieation, el crecimiento o la actividad de las bacterias (bacteriosttico). 4. Precauciones de seguridad NOTA: Estas instrucciones de seguridad son para fines informativos nicamente. Las precauciones son accesorios a los procedimientos de prueba y no se pretende que sea definitiva. es responsabilidad del usuario utilizar la venta y las tcnicas adecuadas en el manejo de materiales en este mtodo de ensayo. Los fabricantes deben ser consultados para obtener detalles especficos, tales como hojas de datos de seguridad y recomendaciones de otros fabricantes. Todas las normas de OSHA y las normas que deben consultar y seguir. 4.1 Tanto las pruebas cualitativas y cuantitativas deben ser realizadas por personas con formacin y experiencia en el uso de tcnicas bacteriolgicas. Los EE.UU. Departamento de Salud y Servicios Humanos de publicacin, Bioseguridad en laboratorios microbiolgicos y biomedicaldical, debe ser consultada (s 13,1). 4,2 PRECAUCIN: Algunas de las bacterias utilizadas en este ensayo son capaces de infectar a los seres humanos y producir la enfermedad. Hay tanto, cada preecaution necesario y reasonahle deben tomar medidas para eliminar este riesgo para el personal de laboratorio y personal en el medio ambiente asociado. Usar ropa protectora y proteccin respiratoria que impide la penetracin de la bacteria. 4.3 Buenas prcticas de laboratorio deben ser seguidas. Use gafas de seguridad en todas las reas de laboratorio. 4.4 Todos los productos qumicos en caso de que manejarse con cuidado, 4.5 Una ducha lavaojos / seguridad debe estar situada cerca en caso de emergencia. 4,6 Esterilizar todas las muestras contaminadas y los materiales de ensayo antes de su eliminacin. 4.7 La exposicin a los productos qumicos utilizados en este procedimiento debe ser controlada en o por debajo de los niveles establecidos por las autoridades gubernamentales (por ejemplo, seguridad Ocupacional y Administracin de Salud [OSHA] los lmites permisibles de exposicin [PEL] que se encuentran en el 29 CFR 1910.1000 1 de enero de 1989) . En adicin, la Conferencia Americana de Higienistas Industriales Gubernamentales (ACGIH) Los valores umbral lmite (TLV) compuestos por tiempo promedios ponderados (TLV-TWA), los lmites de exposicin a corto plazo (TLV> STEL) y los lmites de techo (TLV-C) se recomiendan como una gua general para la exposicin a contaminantes del aire que debe ser cumplido (ver 13.2). 5. Limitaciones

Por un 5,1 cualitativo, mtodo relativamente rpido y ejecutado fcilmente para determinar la actividad antibacteriana residual de materiales textiles, se refieren a AATCC Mtodo 147. 6. Los organismos de ensayo 6.1 bacteria de prueba. 6.1.1 Staphylococcus aureus American Type Culture Collection N 6538. Gram positivo organismo (ver. 13.3). 6.1,2 Klebsiella pneumoniae, American Type Culture Collection N 4352. Organismo Gram negativo (ver 13.3). 6.1.3 Otras especies adecuadas tambin se pueden utilizar. 7. Medio de cultivo 7,1 adecuadas caldo / agar nutriente medios de comunicacin son la soja, tripticasa y lnfusion cerebro-corazn. Caldo Nutriente: Peptona (Bacto-peptona) (ver 13.4) 5 g Extracto de carne (vase 13.5) 3 g Agua destilada hasta 1000 ml 7,2 calor a ebullicin para dispersar los ingredientes. Ajuste a pH 1 6,8 0,1 con 1 / V hidrxido de sodio (NaOH). (Esto no es necesario si el medio preparado, deshidratado se utiliza.) 7,3 Dispensar en 10 ml. cantidades en tubos de cultivo bacteriolgicos convencionales (es decir, 125 X 17 mm). Enchufe y esterilizar a 103 kPa (15 psi) durante 15 min. 7,4 agar nutriente, Aadir 1,5% de agar bacteriolgico de nutrientes (o apropiado) caldo (ver 7.1). Calentar hasta que hierva. Verificar el pH y ajustar a 7,1 0,1 con solucin de NaOH si es necesario. Se distribuye en 15 1 ml en cantidades convencionales de tubos de cultivo bacteriolgicos. Enchufe y esterilizar a 103 kPa (15 psi) durante 15 min. (Puede ser esterilizado en 1000 ml frascos de vidrio de borosilicato y platos petri vertido de esto.) 7,5 Slurry inoculo Carrier (para tejidos hidrofbicos) (ver 7.2 y 7.3): Cloruro de Sodio 8,5 g Agar 3,0 g Agua destilada 1000 ml

8. Mantenimiento de la Cultura en organismos de prueba 8.1 Uso de 4 mm asa de inoculacin, transfiera la cultura cotidiana en nutrientes (o medio apropiado) de caldo por no ms de dos semanas. Al trmino de dos semanas. hacer un trasplante fresco de la cultura de valores. incubar los cultivos a 37 2 C (99 3 F) o una temperatura ptima otro. 8.2 Mantener en cultivos de agar inclinado de nutrientes o apropiado. Almacenar a 5 1 C (41 2 F) y transferir una vez al mes a agar fresco (ver 13,6). 9. Prueba cualitativa (examen o prueba presuntiva) 9,1 Para la deteccin de uso bacteriosttico Mtodo actividad AATCC 147 en una muestra de ensayo y la muestra de control utilizando los organismos mencionados anteriormente. Por de demostracin de la actividad bactericida, contine con la prueba cuantitativa se describe a continuacin. 10. Prueba Cuantitativa (referencia o prueba de confirmacin) 10,1 Preparacin. La siguiente descripcin se dar en trminos de recortes de tela. Los materiales textiles no en forma de tela de la misma manera se pueden probar con la modificacin apropiada. 10.1.1 Tamao y forma de muestras tratadas Cortar muestras circulares de 4,8 0,1 cm (1,9 0,03 cm) de dimetro, a partir de la tela de ensayo (preferiblemente con un troquel de acero). Pila de las muestras en un frasco de 250 ml de vidrio de boca ancha con tapn de rosca. El nmero de muestras para ser utilizados depende del tipo de fibra y estructura de tejido. Use la cantidad de tejido que absorbe el 1,0 0,1 ml de inculo, y no dejan libre del lquido en el frasco. Por ejemplo, 4 muestras de tela de algodn de impresin absorber 1 ml .. El nmero de muestras utilizadas por cada frasco debe ser reportado. 10.1.2 Controles. Muestras del tipo de fibra y de la construccin misma tela como la muestra de ensayo pero que no contiene acabado antibacteriano (control negativo). 10,23 La solucin neutralizante debe incluir ingredientes para neutralizar el tratamiento especfico de tejido antibacteriano y cuidar de los requisitos de pH de los tejidos (de acabados, agentes antibacterianos, etc.) La solucin neutralizante empleado debe ser informado (ver 13,9). 10.1.4 Agitar los frascos vigorosamente durante un minuto. Realizar diluciones seriadas con agua y la placa (por duplicado) en los nutrientes (o apropiado) agar. Las diluciones de 10 0 10 1 10 2, son generalmente adecuadas. 10.2.5 lncubation durante perodos de contacto. incubar frascos inoculados adicionales que contienen muestras de control sin tratar y frascos que contienen las muestras de prueba tratadas inoculadas a 37 1 2 C (99 3 F) durante 18 a 24 h. Frascos similar puede ser incubatecl sobre

otros perodos (por ejemplo, 1 o 6 h) para proporcionar informacin sobre la actividad bactericida del tratamiento durante esos perodos. 10.2.6 Muestreo de muestras inoculadas e incubadas. Despus de la incubacin se aaden 100 ml l. de solucin neutralizante a tarros que contenan muestras de control sin tratar y a tarros que contenan muestras de prueba tratadas. Agitar los frascos vigorosamente durante un minuto. hacer diluciones seriadas y Platn (por duplicado) en nutrientes (o apropiado) agar, diluciones de 10 0 101 a 102 son generalmente adecuadas para tejidos de ensayo tratados. Varias diluciones diferentes puede ser necesaria para los tejidos de control no tratados en funcin del perodo de incubacin. 10.2.7 lncubate todas Platos durante 48 horas a 37 2 C (99 3 F) o temperatura ptima otro. 11. evaluacin 11.1 Informe bacterianas cuenta como el Numeber de bacterias por muestra (muestras en frasco) no como el nmero de bacterias por ml de solucin neutralizante, Informe "0" cuenta en 10 "dilucin" menos de 100 ". Clculo de 11,2 por ciento de reduccin de las bacterias por los tratamientos de muestras por una de las frmulas siguientes: 1) 100 (B-A) / B = R donde: R =% de reduccin A = el nmero de bacterias recuperadas de las muestras de prueba tratadas inoculadas especimenes en el frasco se incubaron durante el perodo de contacto deseada

B = nmero de las bacterias recuperadas de las muestras de prueba tratadas inoculadas especimenes en el frasco inmediatamente inoculacin aficr (tiempo de contacto en "0")

2) 100 (C-A) / C = R donde: C = el nmero de bacterias recuperadas de la muestra de control no tratados inoculados swateltes en la frasco inmediatamente despus de la inoculacin (en el momento de contacto "0")

Si "B y C" no son similares, el mayor nmero se debe utilizar. Si "B" y "C" no son significativamente diferentes, (B + C) / 2 debe ser utilizado de la siguiente manera: