Wound dressings with antimicrobial properties ...

Wound dressings with antimicrobial properties ─ Requirements and test methods for determining bactericidal activity of antimicrobial wound care dressings A proposal for a European Standard test method for the determination of the antibacterial activity of wound care dressings containing antimicrobial substances

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Table of Contents 1 Introduction .................................................................................................................................. 4 2 Scope .......................................................................................................................................... 4 3 Normative references ................................................................................................................... 4 4 Terms & definitions ....................................................................................................................... 4 4.1.1 Control Dressing .................................................................................................. 4 4.1.2 Plate count method .............................................................................................. 4 4.1.3 Neutraliser ........................................................................................................... 4 4.1.4 Test Dressing ....................................................................................................... 4 4.1.5 Bactericidal .......................................................................................................... 4 4.1.6 Saturation Volume................................................................................................ 5 4.2 Safety precautions ............................................................................................................5 5 General requirements .................................................................................................................. 5 5.1 General ............................................................................................................................5 6 Apparatus..................................................................................................................................... 5 7 Reagents and culture media ........................................................................................................ 5 7.1.1 Water ................................................................................................................... 6 7.1.2 Tryptone Soya Broth ............................................................................................ 6 7.1.3 Tryptone Soya Agar ............................................................................................. 6 7.1.4 Maximum recovery diluent ................................................................................... 6 7.1.5 Test Medium - Simulated Wound Fluid ................................................................. 6 7.1.6 Neutraliser ........................................................................................................... 7 8 Reference strains ......................................................................................................................... 7 8.1 Storage of strains .............................................................................................................7 8.1.1 General ................................................................................................................ 7 8.1.2 Strains .................................................................................................................. 7 8.2 Preparation of Bacterial Suspensions ...............................................................................8 8.2.1 Stock Cultures ...................................................................................................... 8 8.2.2 Working Culture ................................................................................................... 8 9 Test Procedures ........................................................................................................................... 8 9.1.1 General ................................................................................................................ 8 9.1.2 Neutraliser ........................................................................................................... 8 9.2 Choice of experimental conditions ....................................................................................9 9.2.1 Contact Times ...................................................................................................... 9 9.2.2 Strains .................................................................................................................. 9 9.3 Saturation Volume ............................................................................................................9 9.4 Direct contact method .......................................................................................................9 9.4.1 Bacterial Suspension ........................................................................................... 9 9.4.2 Preparation of test samples.................................................................................. 9 9.4.3 Exposing the dressings to test organisms .......................................................... 10 9.5 Shaking method .............................................................................................................10 9.5.1 Bacterial Test Suspension .................................................................................. 10 9.5.2 Preparation of test samples................................................................................ 10 9.5.3 Exposing the test samples to the test organisms................................................ 11 9.6 Two compartment Method .............................................................................................. 11 This method allows the wound dressing to be in a moist environment although not totally soaked in the test medium. Absorbent dressings are allowed to absorb test medium from underneath, to mirror the clinical situation. ................................................................................................... 11 9.6.1 Bacterial Test Suspension .................................................................................. 11 9.6.2 Preparation of test samples................................................................................ 11 9.6.3 Exposing the test samples to the test organisms................................................ 12 9.7 Recovery of test organisms ............................................................................................12 10 Calculation and expression of results ....................................................................................... 12 2

10.1 Calculation of Viable counts (cfu/ml) .............................................................................12 10.2 Judgement of test validity .............................................................................................13 10.3 Calculation of antibacterial activity ................................................................................13 10.4 Effectiveness ................................................................................................................14 11 Test report ................................................................................................................................ 14 ANNEX A - (Normative) - Validation of dilution-neutralisation ............................. 15 1 Principle ................................................................................................ 15 2 Preparation of bacterial suspension ....................................................... 15 3 Test Validation ....................................................................................... 15 4 Neutraliser toxicity ................................................................................. 15 5 Dilution-neutralisation validation ............................................................ 15 6 Validity ................................................................................................... 15 ANNEX B - (informative) - Neutralisers .............................................................. 16 ANNEX C - (Informative) – Test Method Information .......................................... 17 1 Direct Contact method. .......................................................................... 17 2 Shaking Method..................................................................................... 17 3 Two compartment method. .................................................................... 17 4 Diffusion bed method. ............................................................................ 17 5 Quantitative adhesion method for bacteria on a binding dressing .......... 17 ANNEX D - Bibliography .................................................................................... 18

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1 Introduction

This standard describes requirements and a range of test methods for establishing whether a wound care dressing exerts antimicrobial activity. The laboratory tests simulate some conditions of application. Chosen conditions (test fluids, temperature, organisms, contact time) reflects parameters which are found in clinical situations including conditions which may influence the action of wound care dressings containing antimicrobial substances. The conditions are intended to cover general purposes and to allow comparison between laboratories and product types.

2 Scope

This standard specifies requirements and test methods recommended for the evaluation of antimicrobial activity of wound care dressings containing antimicrobial substances using a variety of methods. It is suitable for leaching and non-leaching antimicrobial dressings.

3 Normative references TO BE ADDED

4 Terms & definitions 4.1.1 Control Dressing A wound care dressing which is the same dressing as the dressing to be tested but without the

antibacterial treatment. If this is not available, then use a non-medicated dressing from the same product group preferably from the same manufacturer.

4.1.2 Plate count method A method in which the number of bacteria present after incubation is calculated by counting the number of colonies according to a ten-fold dilution method.

The results are expressed in CFU (colony forming units)

4.1.3 Neutraliser Chemical agents used to inactivate, neutralise, or quench the antibacterial properties of antibacterial agents.

4.1.4 Test Dressing Wound care dressing that contains antimicrobial substance(s)

4.1.5 Bactericidal Capability of the product to produce at least a 103 reduction in the number of viable bacterial cells [Gallant-Behm et al] from the challenge organisms required in Section 7.1.2 under the conditions defined by the appropriate method in Section 8.2.

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4.1.6 Saturation Volume Volume of fluid absorbed by the dressing as determined by the fluid absorption test. This volume of fluid is added to the dressing to prevent the dressing absorbing all of the challenge media

4.2 Safety precautions Test methods specified herein require the use of bacteria, therefore these tests should be carried out by persons with training and experience in the use of microbiological techniques. Appropriate safety precautions should be observed with due consideration given to country-specific regulations.

5 General requirements 5.1 General TO BE ADDED

6 Apparatus

Usual microbiological laboratory apparatus and in particular the following 1. Spectrophotometer, capable of measuring at a 600nm to 660nm wavelength, or McFarland's nephelometer 2. Incubator, capable of being controlled at 35°C ± 3°C 3. Shaking incubator or shaker in incubator, capable of being controlled at 35°C ± 3°C & 100 ± 5rpm 4. Water bath, capable of being controlled at 35°C ± 3°C 5. Mixer, (electromechanical agitator, e.g., Vortex® mixer) 6. Cutting templates a) Direct contact method - 2.5cm x 2.5cm, b) Shaking method - 5cm diameter, c) 2 compartment method - 1.8mm diameter. 7. Erlenmeyer flasks 250ml, sterile with caps or bungs 8. Containers, test tubes and flasks of suitable capacity 9. Petri dishes, of size 90mm to 100mm 10. Forceps, sterile 11. pH meter, having an accuracy of calibration of ± 0.1 pH units at 25°C 12. Autoclave, capable of being maintained at 121°C for a minimum holding time of 15 min 13. Pipettes, of nominal capacities 10ml, 1ml and 0.1ml. Calibrated automatic pipettes may be used. 14. 6-well plates 15. Cell culture inserts, suitable for 6-well plates with membrane pore size of 8μm.

7 Reagents and culture media

Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes. Dehydrated or ready-prepared products available on the commercial market are recommended for 5

use in preparing the culture media. The manufacturer's instructions for the preparation of these products should be strictly followed.

7.1.1 Water Shall be free from substances that are toxic or inhibiting to the bacteria. Demineralised water can be used for the preparation of dehydrated media that will be sterilised

7.1.2 Tryptone Soya Broth For the maintenance of bacterial strains, and growth of overnight cultures Tryptone, pancreatic digest of casein Soya peptone, papic digest of soya NaCl Dibasic potassium phosphate Glucose Water

17.0g 3.0g 5.0g 2.5g 2.5g to 1000ml

Sterilise in the autoclave. After sterilisation the pH of the medium shall be equivalent to 7.3 ± 0.2 when measured at 20°C ± 3°C.

7.1.3 Tryptone Soya Agar For the maintenance of bacterial strains, and performance of viable counts Tryptone, pancreatic digest of casein Soya peptone, papic digest of soya NaCl Agar Water

15.0g 5.0.g 5.0g 15.0g to 1000ml


7.1.4 Maximum recovery diluent Peptone NaCl Water

1.0g 8.5g to 1000ml


7.1.5 Test Medium - Simulated Wound Fluid Depending on the test method, a sufficient quantity of test medium should be prepared as follows: Maximum recovery diluent Foetal Calf Serum 6

50% 50%

For suspension of the test organisms when exposed to products containing antimicrobial substances. The test medium contains salts and proteins, which are known to interfere with some antimicrobial substances used for dressings, and also to simulate wound like conditions.

7.1.6 Neutraliser The neutraliser shall be validated for the active substance in the product under test in accordance with Annex ANNEX A - . The neutraliser shall be sterile. NOTE: Information on neutralisers that have been found to be suitable for some categories of products is given in Annex ANNEX B - .

8 Reference strains 8.1 Storage of strains 8.1.1 General The strains shall be stored in accordance with the supplier's recommendations or EN 12353. The identification and origin (culture collection) of the strains as well as the laboratory storage method shall be recorded.

8.1.2 Strains The following strains shall be used in all antibacterial activity tests:  Pseudomonas aeruginosa ATCC 15442, CIP 103467, NCIB 10421  Staphylococcus aureus ATCC 6538, CIP 4.38, DSM 799, NBRC 13276 or NCIMB 9518. NOTE:  ATCC is the American Type Culture Collection (USA);  CIP is the Pasteur Institute Collection (France);  DSM is the German Collection of Micro-organisms and Cell Cultures (Germany);  NBRC is the NITE Biological Resource Centre (Japan);  and NCIMB is the National Collection of Industrial Bacteria (UK). If additional strains selected do not correspond to the specified strains, their suitability for producing inocula of sufficient concentration shall be verified. If additional strains tested are not classified at a reference centre (eg clinical isolates), their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory for 5 years.

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8.2 Preparation of Bacterial Suspensions 8.2.1 Stock Cultures Stock cultures shall be kept in accordance with the requirements of EN 12353

8.2.2 Working Culture In order to prepare the working culture of the strain, subculture from the stock by streaking onto TSA plates and incubate. After 18h to 24h prepare a second subculture from the first subculture in the same way and incubate for 18h to 24h. From this second subculture, a third subculture may be produced in the same way. The second and/or third subcultures are the working cultures. If required a 48h subculture may be used for subsequent subculturing, provided that the subcultures have been kept in the incubator during the 48h period. For additional strains, any departure from this method of culturing or preparing the suspension shall be noted, giving reason in the test report.

9 Test Procedures 9.1.1 General Equilibrate all reagents (bacterial test suspension, diluents, neutralising solutions) to the test temperature of 20°C ± 3°C using a water bath controlled at 20°C ± 3°C. All reagents should be stabilized at 20°C ± 3°C before commencing the testing.

9.1.2 Neutraliser The method of choice is the dilution-neutralization method. To determine a suitable neutraliser the following procedure shall be adopted. 1. Carry out the validation of the dilution neutralization method (see Annex ANNEX A - ) using a suitable neutraliser, chosen according to laboratory experience and published data. 2. If this neutraliser is not valid (as per Annex ANNEX A - ), repeat the validation test using an alternative neutraliser as follows: ◦

polysorbate 80 30g/l,

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saponin 30 g/l,

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L-histidin 1g/l,

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lecithin 3g/l,

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sodium thiosulfate 5g/l

in either diluent (6.1.4) or in phosphate buffer 0.005 mol/l. 3. If both neutralisers are found to be invalid, neutralisation by dilution is used for each samples . 4. The inactivation of the bactericidal and/or bacteriostatic activity of the product shall be validated for each of the tested strains and for each of the chosen experimental conditions.

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9.2 Choice of experimental conditions 9.2.1 Contact Times  The standard contact time shall be 24h ± 1h.  For DIRECT CONTACT METHOD, additional contact times may be chosen from 30min ± 1min to 24h ± 1h  For SHAKING METHOD, additional contact times up to 72h ± 1h may be chosen. 9.2.2 Strains The strains shall include, as a minimum, the two standard strains as in 7.1.2 . Additional strains, including clinical isolates, may be used as long as details of the strains including the source is recorded in the test report.

9.3 Saturation Volume •

Samples of the wound care products are cut (see 5 - size dependant on the test method direct, shaking or 2 compartment) and weighed

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The samples are submerged in excess water (e.g. 40 times weight of sample) for 2 minutes,

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The sample is carefully removed (without squeezing), allowed to drip for 10 seconds and

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Then re-weighed.

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The weight of water absorbed is determined this is the saturation volume of the dressing.

9.4 Direct contact method 9.4.1 Bacterial Suspension 1. Take 9ml of diluent (6.1.4 ) and place in a test tube. 2. Take the working culture and transfer 1 loopful of the cells into the diluent (6.1.4 ). The cells should be suspended by mixing. 3. Transfer 5ml of the suspension from the test tube to a bottle containing 45ml test medium (6.1.5 ). The cells should be suspended in the test medium (6.1.5 ) by mixing. 4. Adjust the number of cells in the suspension to between 1.5 x 107 CFU/ml to 5.0 x 107 CFU/ml using the test medium (6.1.5 ), by spectrophotometer or McFarland's nephelometer.

9.4.2 Preparation of test samples 1. Details of the dressings to be tested as received shall be recorded including:

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batch number

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expiry date,

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manufacturer & size

2. Prepare 6 samples of each test dressing and control dressing (a total of 12 samples) for testing as follows: 2.1.

3 samples & 3 controls for testing at time = 0hrs, and

2.2.

3 samples & 3 controls for testing at time = 24hrs.

3. Prepare further samples of 3 test and 3 control dressings for every other time period to be investigated; 4. Cut samples using the 2.5x2.5cm template under aseptic conditions. 5. Absorbant dressings are pre-wetted with water under slight compression for 1 hour prior to the addition of inoculum . Dressings are placed in a petri dish with the saturation volume (see 8.3 and a weight of between100g to 200g (TO BE DETERMINED) placed on lid.

9.4.3 Exposing

the dressings to test organisms

1. Place each sample (test dressing and control dressing) in a separate petri dish. 2. Each product (including controls) should be tested in triplicate at T= 0hrs and T= 24hrs. 3. Pipette 0.2ml of the prepared inoculum ( 8.4.1) evenly at several points across the surface of the sample. 4. For T=0 samples, skip the next step and continue with step 6 5. For all samples place a lid on the the petri dish, place in a plastic grip bag and incubate at 35°C ± 3°C for 24 h. 6. At the end of the contact time (T=0 or T=24hrs), transfer each sample into a tube containing 10 ml neutraliser (6.1.6) and mix for 5s x 5 cycles. 7. Let the neutraliser react for an appropriate time (dependent on neutraliser – See Annex ANNEX A - ) at 20°C ± 3°C. This solution is considered as the 10-1 dilution. 8. See section 8,7 for recovery of test organisms 8.7

9.5 Shaking method 9.5.1 Bacterial Test Suspension 1. Pick up one colony of the test organism and inoculate it in 3 ml TSB (6.1.2 ). 2. Incubate overnight at 35ºC±2 ºC. 3. Mix the overnight culture for 10s, using a vortex mixer. 4. Dilute the pre-cultured bacteria from approximately 109 CFU/ml to 107 CFU/ml using MRD. 5. Dilute from 107 CFU/ml to approximately 1.5 - 5.0 × 106 CFU/ml using SWF (6.1.5 ). To count the bacterial start inoculum, perform a ten-fold serial dilution and plate as stated in (8.7 ). This is the initial inoculum level used in the calculations

9.5.2 Preparation of test samples 1. Details of the dressings to be tested as received shall be recorded including: ◦ 10

batch number

◦

expiry date,

◦

manufacturer & size

2. Cut three 5.0cm circular samples each of the test and control dressings.

9.5.3 Exposing the test samples to the test organisms •

Add 5ml plus the saturation volume (see of the dressing of inoculated SWF (see 6.1.5 ) to the Erlenmeyer flasks

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Add the test and control samples to separate flasks

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Incubate the flasks at 35°C ± 3°C & 100 ± 5rpm

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After the contact time remove a 0.5ml sample and add to 4.5ml of neutraliser (6.1.6 ) in a test tube.

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Let the neutraliser react for the appropriate time (dependent on neutraliser – see Annex ANNEX A - ) at 20°C ± 3°C. This solution is considered to be the 10-1 dilution.

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See section 8.7 for the recovery of test organisms

9.6 Two compartment Method This method allows the wound dressing to be in a moist environment although not totally soaked in the test medium. Absorbent dressings are allowed to absorb test medium from underneath, to mirror the clinical situation.

9.6.1 Bacterial Test Suspension 1. Pick up one colony of the test organism and inoculate it in 3 ml TSB (6.1.2 ). 2. Incubate overnight at 35ºC±2 ºC. 3. Mix the over night culture for 10s, using a vortex mixer. 4. Dilute the pre-cultured bacteria from approximately 109 CFU/ml to 107 CFU/ml using MRD (6.1.4 ). 5. Dilute from 107 CFU/ml to approximately 1.5 - 5.0 × 106 CFU/ml using SWF (6.1.5 ). To count the bacterial start inoculum, perform a ten-fold serial dilution and plate as stated in (8.7 ). This is the initial inoculum level used in the calculations

9.6.2 Preparation of test samples 1. Details of the dressings to be tested as received shall be recorded including: ◦

batch number

◦

expiry date,

◦

manufacturer & size

2. Cut three circular test pieces with a diameter of 18 mm of each of the test and control dressings. 3. Place each of the test samples into separate cell culture inserts, with the wound contact 11

surface facing down.

9.6.3 Exposing the test samples to the test organisms 1. Add 2.5 ml plus the saturation volume (8.3 ) of the piece of the test product and the control product of inoculated SWF (6.1.5 ) into wells of 6-well plates. 2. Place the cell culture inserts with test products and control products into the wells of 6-well plates. 3. Replace the lid of the 6-well plate.

4. Incubate at 35°C ± 3°C & 100 ± 5rpm for 24 hours 5. After incubation, lift up the cell culture inserts with a tweezers. Mix the SWF in the well using a pipette. 6. Remove a 0.5ml sample and add to 4.5ml of neutraliser (6.1.6 ) in a test tube. 7. Let the neutraliser react for appropriate time (dependent on neutraliser) at 20°C ± 3°C. This solution is considered as the 10-1 dilution. 8. See section 8.7 for the recovery of test organisms

9.7 Recovery of test organisms 1. For counting of the bacterial test suspension prepare 10-2, 10-3, 10-4 and 10-5 dilutions of the test suspension using diluent (see 6.1.4 ). 2. Mix by vortexing. 3. Take a sample of 1.0 ml of each dilution in duplicate and transfer each 1.0 ml sample into separate Petri dishes and add 20 to 25 ml melted TSA (see ), cooled to 45°C ± 3°C. 4. Spread plates using 0.1ml of each dilution across the pre-prepared TSA plates are acceptable, ensuring that the extra 10-fold dilution is taken into account in the calculations. 5. Incubate the plates at 35°C ± 3°C for 24-48h. 6. Remove plates from the incubator. 7. Discard any plates which are not countable for any reason. 8. Count the plates and determine the number of colony forming units. 9. Calculate the number of CFU/ml in the test suspensions (Vc ) using the method given in 9.1 10. Count only plates on which 15CFU to 300CFU have appeared, ignoring any others. 11. If no colonies are recovered on any of the plates from the dilution series, record the number as “= 0.05 x Bs, AND  Nd >= 0.5 x Nt

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ANNEX B - (informative) - Neutralisers Below is a table of examples of active agents and possible neutralisers Antimicrobial Agent

Neutralising Agent

Halogens

Sodium thiosulphate

Quaternary ammonium agents

Lecithin + polysorbate

Chlorhexidine

Polysorbate + egg lecithin

Polyhexamethylene biguanide

Polysorbate

Silver

Sodium Thioglycollate

Glutaraldehyde

Glycine

NOTE: the above list is not exhaustive and other neutralisers may be tried see BS EN 1040 for more examples.

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ANNEX C - (Informative) – Test Method Information This annex contains explanatory text about each of the test methods used in this standard, and explains the type of dressing they should be used for. In general, it is anticipated that manufacturers or test houses will use one of the first three methods described below to ensure that the product displays measurable antimicrobial activity. If measurable activity is seen, then they should consider using something like the diffusion bed method which simulates a wound bed more closely. 1

Direct Contact method.

The method is based upon a method referenced in Gallant-Behn et al. The method is suitable for both leaching and non-leaching antimicrobial agents. It is also suitable for most dressing types, but the recovery for super absorbant dressings may be limited 2

Shaking Method.

The method is based upon a method in Parson et al. The method is suitable for both leaching and non-leaching antimicrobial agents. It is also suitable for most dressing types, but the volume of fluid required may not represent the clinical situation for super absorbant dressings.

3 Two compartment method. This method is based on cell culture method in Ågren and Mirastschijski 2004. The method is suitable for leaching antimicrobial agents. The method allows the dressing to be in a moist environment although not totally soaked in the test medium. Absorbent dressings are allowed to absorb test medium from underneath, which mirrors the clinical situation

4 Diffusion bed method. This method (which is not presently part of this standard) is based on the paper published by Greenman et al, which describes an in vitro wound infection model that allows the comparison of the bacterial kill rate of antimicrobial wound dressings over the course of 3 days, with renewed microbial challenges each day, under realistic wound-like conditions.

5 Quantitative adhesion method for bacteria on a binding dressing Some modern dressings claim to exert an anti-microbial effect through the binding of bacteria to the dressing.

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ANNEX D - Bibliography

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EN 1040, Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics – Test method and requirements (phase 1)

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EN 12353 Chemical disinfectants and antiseptics – Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity

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Comparison of in vitro disc diffusion and time kill-kinetic assays for the evaluation of antimicrobial wound dressing efficacy. Wound Rep Reg 2005; 13:412-421 Gallant-Behn et al.

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Parsons_D, Bowler_P.G, Myles_V, Jones_S. Silver antimicrobial dressings in wound management: A comparsion of antibacterial, physical and chemical characteristics Wounds 2005 17 (8) 222-232.

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Greenman J, Thorn RMS, Saad S, Austin AJ. In vitro diffusion bed, 3-day repeat challenge ‘capacity’ test for antimicrobial wound dressings. Int Wound J 2006;3:322–329.