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Antimicrobial & Biosafety

There are three important groups of microorganisms:

  • Fungi
  • Bacteria
  • Viruses

Vartest offers tests and studies to support antimicrobial efficacy:

Viruses

ISO 18184, the protocol from the International Organization for Standardization for measuring antiviral activity of porous materials, and ISO 21702, for non-porous materials. Vartest uses human coronavirus OC43, an especially close relative of and surrogate for human coronavirus SARS-CoV-2, which causes Covid-19 disease. The virus is exposed to the test material and then plated on MRC-5 cells in culture and scored either by cytopathic effect or immunostaining. Compared to inert controls, antiviral samples which have no or low cellular toxicity can score greater than 99.99% (Log 4) reduction in virus.

normal monolayer
infected monolayer
stained OC43 plaque

The left panel shows cells infected with a virus-containing extract at 10e6 dilution, the middle panel at 10e5 dilution (5 days incubation). This shows the abrupt loss of cytopathic effect upon dilution of virus extract and the certainty with which it can be identified. The right panel shows a virus plaque stained with antibody to the viral nucleoprotein (2 days incubation).

Client samples are exposed to virus in triplicate and each extract then titered in triplicate, generating a robust database to confidently describe antiviral activities. The test is readily adaptable by Vartest to other viruses such as influenza.

ISO 10993-5: 2009. Biological evaluation of medical devices – part 5: tests for in vitro cytotoxicity. A widely used test for cytotoxicity is the MTT assay, which is based on the reduction of MTT to insoluble formazan crystals by metabolically active cells. Crystals produced by metabolic activity are dissolved and measured by absorbance at 570 nM. Toxic agents will reduce metabolic activity and thus formazan production. The test is readily adapted to fabrics. Non-toxic polymer negative controls, toxic zinc-containing positive controls, and client samples are extracted in cell growth medium overnight at 37°C and 5% CO2. Vero monkey kidney cells are grown overnight in 96-well dishes to near confluence. The cell medium is then replaced with the extract-containing media and dilutions thereof made in growth medium and the cells incubated overnight. The MTT assay is then performed to assess the degree of growth (metabolic activity). A dilution series of a second toxic, positive control (sodium lauryl sulfate) is also used to confirm the test is within expected parameters.

Bacteria

AATCC 147. Antibacterial activity assessment of textiles using the streak method. This is a qualitative measure of textile antibacterial activity and most easily measures diffusible agents. Test bacteria are streaked on agar and overlain with strips of textiles. Antibacterial activity is shown by inhibition of bacterial growth underneath and in zones around the textile.

Psudomonas aeruginosa on cetrimide agar
Klebsiella pneumoniae on nutrient agar
Staphylococcus aureus on nutrient agar

Silver vapor deposition-treated multifiber strips laid on (left) Pseudomonas aeruginosa on cetrimide agar, (middle) Klebsiella pneumoniae on nutrient agar, and (right) Staphylococcus aureus on nutrient agar.

The assay is readily adaptable to other bacteria.

AATCC 100. Activity of antibacterial finishes on textiles. In this test, bacteria are incubated overnight with client samples, inert control, or known antibacterial control, recovered and titered. Comparison of the overnight growth with the inert control to the growth with the client sample gives a quantitative measure of the degree of antibacterial activity. Normally performed with Staphylococcus aureus and Klebsiella pneumoniae, the test is readily adaptable to other bacteria and can be done under a variety of growth conditions (e.g., 5% or 100% nutrient broth).

ISO 20743. Textiles — Determination of antibacterial activity of textile products, and JIS L 1902 (Japanese Industrial Standard), Textiles – Determination of Antibacterial Activity and Efficacy of Textile Products. These methods are similar to AATCC 100 but are routinely performed with triplicate samples. Limitations are placed on the acceptable degree of variability in bacterial number at zero time and after overnight incubation, resulting in a robust statistical outcome. Recommended bacteria are Staphylococcus aureus and Klebsiella pneumoniae.

ASTM 2180. Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials. In this test, bacteria are applied to triplicate client samples in an agar slurry to maximize contact with a hydrophobic surface. Antimicrobial and inert controls are also tested in triplicate. After incubation, surviving bacteria are recovered fully by disrupting the agar through three successive steps. Results are reported in triplicate and typically show very acceptable standard deviations. The test is usually performed with Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

ASTM E2149. Activity of Antimicrobial Agents Under Dynamic Contact Conditions. This method is intended to evaluate antimicrobial activity of non-leaching, antimicrobial-treated specimens under dynamic contact conditions, usually using Escherichia coli ATCC 25922. Bacteria are agitated in a minimum volume with the client sample or antimicrobial or inert controls, recovered and enumerated by dilution and plating. The method is readily adaptable to other organisms.

ASTM E1428-15a. Evaluating the Performance of Antimicrobials in or on Polymeric Solids Against Staining by Streptomyces species (A Pink Stain Organism). Pigments from bacteria can cause undesirable permanent stains. Streptomyces species, in particular, cause this problem. This test exposes fabric to Streptomyces growing on Malt Extract Agar. After 14 days growth, the fabrics are washed and assessed for the degree of pink stain.

Cotton fabric placed on a lawn of growing Streptomyces.
The cotton was removed, washed, and is shown exposed face up. The fabric is heavily stained.

Fungi

AATCC 30-3. Mildew and Rot Resistance of Textile Materials.

This tests for the growth of Aspergillus niger on the surface of a fabric. The fabric is placed on Salts Agar/3% glucose and the entire surface, including the fabric and the agar, is inoculated with Aspergillus spores. After 7 days incubation, the sample is scored for the presence or absence of macroscopic growth on its surface and for any zone of inhibition extending out into the agar. The sample is also examined stereomicroscopically and, if desired, by scanning electron microscopy. The figure (panel 1) shows A. niger growing on and around a cotton fabric square with no zone of inhibition. Panel 2 shows a zone of inhibition in a lawn of A. niger caused by a drop of concentrated antifungal cetrimonium bromide. Panel 3 shows the absence of growth on a square of polyethylene sheet with no zone of inhibition. Panel 4 shows a stereomicrograph of fruiting bodies. Panel 5 shows a scanning electron micrograph.

ASTM G21 (Resistance of Synthetic Polymeric Materials to Fungi) and MIL-STD-810G Method 508.7 (Fungal Resistance Test). While synthetic polymeric materials typically will not support fungal growth, additives such as plasticizers, dyes, etc., may support growth. In these tests, fabric is placed on Salts Agar which supplies minerals and salts but no carbon source. Spores are purified free of nutrient remnants from five widely different fungal species, mixed in equal concentration, and applied to the test sample and agar at high spore concentration. The plates are incubated at 28°C and 90% relative humidity for 28 days (ideal for fungal growth) and are then scored for the extent of the test sample surface covered by growth. The primary control is cotton fabric or paper placed on the agar, which should support growth following breakdown of cellulose. The primary negative control is the absence of growth on the agar itself. Method 508.7 has in addition control paper strips soaked in a growth promotion solution, inoculated with spore mix, and held in the incubation chamber to demonstrate that the incubation conditions will support growth. Vartest records both temperature and humidity.

Uninoculated cotton fabric.
G21 mix-inoculated cotton fabric.
Uninoculated control (soaked) cotton fabric.
Inoculated control (soaked) cotton fabric.

USP 61. Bioburden. A quantitative enumeration of bacteria and fungi on textiles or medical products. Client samples are extracted with a buffer to neutralize antimicrobials and this is plated on Tryptic Soy agar (for bacteria) and on Sabouraud Dextrose Agar (for Fungi). Efficiency of recovery of organisms from extracts with client samples is determined by inoculating with known numbers of C. albicans or S. aureus and measuring recovery compared to recovery from buffer alone. Efficiency of recovery of each of the five organisms listed in USP 61 (A. niger, C. albicans, B. subtilis, S. aureus, P. aeruginosa) can also be performed.

Vartest performs this test in accordance with ANSI/AAMI/ISO 11737-1, commonly referenced in the Radiation Sterilization standards (ANSI/AAMI/ISO 11137-1 & 2) and the EO Sterilization standard (ANSI/AAMI/ISO 11135).