Antimicrobial Fabric: Testing Methods and Industry Standards

Antimicrobial fabric finishing is a practical treatment for protective textiles. It uses several methods to test how well they perform. This article covers methods for testing textiles. It studies both qualitative and quantitative methods to assess how fabrics fight germs. It also looks at the main factors that affect antimicrobial fabric textile test results. Using the right procedures and trusted testing tools is crucial for accurate results.

Selection of Test Microorganisms

Microbial strains for testing antimicrobial performance in textiles include bacteria and fungi. When it comes to bacteria, we focus on Gram-positive and Gram-negative strains. Fungi, molds, and dermatophytes are the typical choices. To check the antimicrobial effectiveness of textile samples, choose relevant and representative microorganisms. The strains in Table 1 are common in nature. You can find them on human skin and mucous membranes. This makes them great for testing antimicrobial fabrics. To get accurate results, pick the right test organisms. It’s also important to use standardized textile testing tools.

Representative Microorganisms Used in Textile Testing

Staphylococcus aureus is a highly resilient bacterium that doesn’t form spores. It is a common Gram-positive strain used in textile testing. Researchers recognize Bacillus subtilis for its strong resistance due to spore formation. It often serves as an example of spore-forming bacteria. Bacillus megaterium is a spore-forming pathogen. It is commonly used in textile testing by researchers.
Escherichia coli is a Gram-negative bacterium that exists in various natural environments. It is a common standard in many textile testing protocols. The Chinese standard (GB2423.16-81) lists fungi like Aspergillus flavus and Chaetomium globosum for mold resistance tests. These fungi are often used to replicate real mold exposure on textiles and polymers.
Candida albicans is a yeast that can cause infections. It usually resides on human skin and mucous membranes. It is often used in antimicrobial textile testing. This is because it responds well to drugs and has a colony shape like bacteria. This makes it easy to count and observe during tests. Its special fungal traits and colony behavior make it a good choice for standard tests with modern textile tools.

Broad-Spectrum Antimicrobial Fabric Testing in Textiles

To see if an antimicrobial fabric works against many germs, use a mix of microbes. This culture should have specific strains in certain amounts. Right now, researchers are testing many antimicrobial products with three standard organisms: Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative), and Candida albicans (fungi). This method gives a glimpse, but it doesn’t fully reveal how textiles battle microbes in real life.
In textile tests with fungi, counting colony-forming units can be hard. This is because of how fungi grow. We often check antifungal performance by measuring fungal growth on the textile sample. This happens after someone keeps it in a controlled temperature and humidity. The evaluation uses a grading system from British Standard BS6085-81. This system helps assess fungal resistance. Consistent procedures are key to getting reliable results. You also need advanced textile testing tools. These tools mimic environmental conditions and track microbial activity on fabric surfaces accurately.

Antimicrobial Fabric Performance Testing Methods

We check how fabrics fight germs using three tests: qualitative, semi-quantitative, and quantitative. Each method is important for checking how well antimicrobial treatments work on textiles. They differ in accuracy and use.

Qualitative Testing Methods

• Qualitative textile testing methods include:
• AATCC Test Method 90 (Halo Test)
• JIS Z2911-1981 (Antimicrobial Test)
• AATCC 30 (Assessment of Antifungal and Rot Resistance)
• GB/T 20944.1-2007 (Textiles—Agar Diffusion Method for Antibacterial Activity)

Textile test methods usually involve putting test organisms on the fabric. Next, you perform a visual inspection for microbial growth. The evaluation checks how well antimicrobial agents move from the fabric to the agar. This method is great for leachable finishes. Still, it isn’t suitable for durable, wash-resistant treatments.

Advantages include low cost and fast results. Qualitative methods do not provide an accurate measurement of antimicrobial activity. As a result, the findings lack accurate numbers. To get precise and even results in product development or quality control, you often need advanced textile testing tools. Researchers use them for follow-up testing that relies on quantitative methods.

AATCC 90 Test Method

The AATCC 90 test is a quick textile test. It is primarily used to evaluate the effectiveness of antimicrobial agents. The process starts by adding test bacteria to an agar medium. Next, place the fabric sample right on the surface. After incubating at 37°C for 24 hours, check for microbial growth. Use a magnifying lens to see the size of the inhibition zone around the fabric. These results are then compared with a control sample.
This method is simple, quick, and can handle many samples at once. But it also has limitations. The incubation time is set, but the bacterial inoculum’s concentration isn’t clear. This could lead to inconsistent results. The size of the inhibition zone shows how well the agent spreads. It also indicates its effectiveness against microbes. This is useful for comparing it to a reference fabric. But it’s not a reliable way to measure antimicrobial activity quantitatively. Experts suggest using extra textile testing tools and measurable methods for better assessments.

AATCC 90 Spray Method

The AATCC 90 Spray Method is a changed version of the original AATCC 90 textile test. After incubation, spray a set amount of TNT reagent onto the fabric sample. We then check how well the antimicrobial works by looking at bacterial growth on the sample.
The color change happens due to a biochemical reaction. Succinate dehydrogenase from the test bacteria reduces the TNT reagent. This leads to an insoluble red pigment forming. This color change shows that the bacteria are active. It also helps us see how well the sample fights germs.
This textile test method has a major advantage. It can spot bacterial growth, even if there is no visible inhibition zone around the sample. Bacterial activity on the agar surface causes a red color. This change helps assess how well the antimicrobial works. This clear visual design makes it easy to use. So, it’s a helpful screening tool. You can use it with other textile testing devices for a better analysis.

AATCC 90 Colorimetric Method

The AATCC 90 Colorimetric Method builds on the original AATCC 90 textile test. It provides a semi-quantitative way to assess color. After incubation, wash the bacteria off the textile sample. Then, add a certain amount of TNT reagent to the bacterial suspension to start the color reaction. After 15 minutes, a spectrophotometer measures the absorbance at 525 nm. This absorbance value correlates with the number of viable bacteria present.
This method helps measure antimicrobial performance better than relying on visual observations. This method will not work for organisms that do not produce succinate dehydrogenase. The TNT reagent needs this enzyme for the color reaction.
For accurate and repeatable results, especially when measuring, this method needs precise control. Reliable textile testing tools, like calibrated spectrophotometers, are essential in testing labs.

JIS Z2911 Antifungal Test Method

The JIS Z2911 method tests how well textiles resist mold. It is one of the first standard ways to measure antifungal properties. The main idea is to spray a mixed spore solution evenly. Do this on both the fabric sample and the culture medium. We incubate the samples in controlled conditions. We check for mold growth at regular intervals during the set time.
The amount of mold that grows determines the textile’s antifungal effectiveness. This method offers a clear way to test textiles for fungal resistance. It focuses on materials that researchers will use in humid and microbe-prone areas.
To ensure accuracy and repeatability, use consistent methods and special textile testing tools. This is important, especially when checking resistant finishes or coatings.

AATCC 30 Test Method

Researchers use the AATCC 30 test to check how well textile materials resist mildew and rot. It determines the effectiveness of biocides in preventing fungal growth and material degradation.
The method has a few approaches:

• Soil burial method
• Agar plate method
• Humidity jar method

The soil burial method involves placing fabric samples of certain sizes in soil for a set time. After that, we measure their breaking strength. The loss in breaking strength indicates the material’s resistance to fungal attack.
The agar plate method evaluates the resistance of textiles to fungal growth. In this method, you take an agar plate with culture medium. Then, you inoculate it with a water suspension of Aspergillus spores. Fabric discs treated with a nonionic wetting agent go on the plate. They are also treated with the spore suspension. After incubation at a specific temperature, we check how much mold has grown on the fabric. We assess this by looking at the area covered by the fungus.
The humidity jar method uses treated fabric strips. These strips hang in a wide-mouth jar. The jar has a set amount of water mixed with a specific concentration of fungal spores. They keep the setup at a certain temperature. Then, we check the fabric strips for visible mold growth. We examine how much area the mold covers.

GB/T 20944.1-2007 – Textiles: Assessment of Antibacterial Activity, Part 1: Agar Diffusion Plate Method

This standard is the newest way to test how well textiles resist bacteria. Pour two layers of agar medium into a petri dish. First, add a sterile base layer. Then, pour an inoculated top layer. Someone places the fabric sample on top of both layers.
After a set incubation time, we check how well the textile fights bacteria. We do this by looking at the amount of bacterial growth where the sample meets the agar medium.

Semi-Quantitative Testing Method

The parallel streak method is a popular semi-quantitative test. It checks how well textiles resist bacteria. This method gives a quick and easy way to test how well-treated fabrics fight germs. It’s great for textiles with diffusible antimicrobial agents. It serves as a simpler alternative to the more complex AATCC 100 test.
AATCC 147 is a standard test method for evaluating antimicrobial fabric finishes on textiles. Researchers know it as a semi-quantitative method for textile testing. In this procedure, we take a specific volume of a bacterial suspension. This usually has spores of Staphylococcus aureus. Then, we inoculate it onto a nutrient agar plate by making five parallel streaks. Place the fabric sample across the streaks at a right angle. Press it gently to ensure it makes close contact with the agar surface. After incubation, we measure the antimicrobial activity of the textile. We do this by checking the width of the inhibition zones around the streaks that touch the fabric.
This textile test is efficient and reliable. It’s great for quality control and for developing antimicrobial fabrics.

Quantitative Testing Methods

Shake Flask Method: Fast but Limited to Non-Leaching Antimicrobials

Right now, the shake flask method and the absorption method are the top ways to test how well textiles resist bacteria.
The shake flask method places a textile sample in a bacterial suspension. Then, it agitates the mixture. This ensures good contact between the fabric’s antimicrobial agent and the microorganisms. The change in viable bacteria counts before and after shaking shows antibacterial effectiveness.

Absorption Method: Time-Consuming but More Accurate and Versatile

The absorption method needs a set volume of bacterial suspension. This applies to both the sample with antimicrobial agents and the untreated control. After incubation, rinse both samples with the elution solution. Count the viable bacteria in the eluent. Next, check the antimicrobial activity. Look at the change in bacterial counts before and after incubation.
The shake flask method is not simple. It mostly works with non-leaching antimicrobial fabrics. The absorption method takes more time. But, it works well for both leaching and non-leaching antimicrobial textiles. Also, its testing conditions closely mimic real-life wear. This makes it the most accurate method for textile testing available today.
Experts say the absorption method will be crucial for future textile tests.
Common standards for quantitative testing include:

• AATCC Test Method 100 (Quantitative assessment of bacterial reduction)
• JIS L1902-8:1998 (quantitative test method)
• FZ/T 02021-92
• Modified Quinn test
• ASTM E2149-2001 (Dynamic test method for the antimicrobial activity of immobilized agents)
• GB/T 20944.2-2007 (Evaluation of antibacterial fabric activity, Part 2: Absorption method)

The main benefit of quantitative textile testing tools is their accuracy. They are also objective and reproducible. However, they require more time and higher costs compared to qualitative approaches.
In this section, we will detail several common quantitative textile testing methods.

AATCC Test Method 100

AATCC 100: A Classic Standard for Antibacterial Textile Testing

The AATCC, or American Association of Textile Chemists and Colorists, created Test Method 100 in 1961. It was revised in 1965 and 1981. Now, it is a key standard for testing how well fabrics resist bacteria. It enables quantitative assessment of both bacteriostatic and bactericidal effectiveness in antimicrobial textiles.

Bacteria spread on both the treated (test) fabric and the untreated (control) fabric. After a set incubation time, we add a neutralizing solution. Then, we shake the samples hard to get out the leftover viable bacteria. We measure the bacterial count in the eluent with plate count methods. Next, we find the percentage reduction by comparing this count to the control sample.
Limitations of the original method:

• Limited number of samples per test
• Time-consuming procedure
• Unsuitable for evaluating non-leaching antimicrobial fabrics
• Lack of specificity in the neutralizing solution’s composition
• Nutrient-rich bacterial suspension may not reflect real-life wear conditions
• Large test containers make handling difficult

Improved AATCC 100: More Efficient and Suitable for Modern Antimicrobial Fabrics

Researchers have created a better version of AATCC 100. They used insights from testing done both at home and abroad to improve it. This new method is a strong textile testing framework. It can evaluate many types of antimicrobial fabric materials.
Key modifications include:

• Reducing the fabric sample size from a 4.8 cm diameter circle to a ~1.8 cm square
• Using 30 mL or 50 mL conical flasks with caps
• Replacing AATCC broth with 0.85% cold physiological saline (0–4°C) for bacterial suspension
• Diluting bacterial inoculum from ~10⁸–10⁹ CFU/mL to 1×10⁵–2×10⁵ CFU/mL
• Using 20 mL of cold 0.85% saline as the neutralizing rinse

Researchers calculate antibacterial efficacy as follows:

This improved method works for both leaching and non-leaching antimicrobial fabrics. Its nutrient conditions better match those found during real fabric use. It offers a solid, practical method for modern textile testing. More and more, labs around the world are using it in their testing protocols.

JIS L1902-8 (1998) Quantitative Test Method

Japanese scholars have improved the AATCC Test Method 100. They created new ways to count bacteria, test for bacterial growth, and use agar plates. They changed the bacterial inhibition tests and improved AATCC 100. The Japan Industrial Standards Committee updated the JIS L1902-1990 standard. They created the new JIS L1902-1998 standard for testing antimicrobial textiles. This method presents a clear and uniform way to assess how fabrics resist bacteria in Japan. It also supports the global effort to create dependable textile testing standards.

FZ/T 02021-92 Test Method

The FZ/T 02021-92 standard is a guideline from the Chinese textile industry. It explains how to test the antibacterial performance of fabrics. Put the test fabric and the control fabric in separate conical flasks. Use two flasks for the test samples and one for the control. Add the indicator bacterial solution first. Then, clean the control and zero-hour test samples using a buffer. Then, measure the bacterial count. The technician incubates the test samples at the right temperature for 20 hours. Then, the researchers wash them with the buffer solution. Finally, researchers measure the bacterial count. Calculate the bacterial reduction percentage by comparing counts taken before and after incubation.

This method is part of China’s commitment to high textile testing standards. It checks how well textile materials fight bacteria in various uses. This method shows manufacturers and researchers that antimicrobial treatments are effective in fabrics.

Modified Quinn Test Method

Principle: This method uses a set amount of bacterial suspension on the test fabric. This way, the bacteria fully contact the textile surface. After the set exposure time, add a nutrient medium to the sample. This lets any surviving bacteria grow. We check how well the fabric kills bacteria. We do this by measuring the drop in living bacteria. Bacterial colonies (CFUs) are counted using a magnifying lens. This method is a simple and effective way to test how well a fabric stops microbial growth after contact.

ASTM E2149–2001 Test Method

The ASTM E2149—2001 method is a shake flask test. It’s simple and easy to use, especially when compared to absorption-based methods. This technique uses a bacterial suspension. You immerse the fabric sample in it and shake gently to maintain contact. It’s perfect for both non-leaching and leaching antimicrobial textiles. Plus, it isn’t restricted by how much water the material can absorb. It can hold different types of textiles. This includes powders, down feathers, uneven fabrics, and oddly shaped samples.

This method works with fabrics. It also applies to powders, granules, and treated solid materials. Its broad scope and easy use make it a top choice for testing antimicrobial performance with standard textile tools.

GB/T 20944.2-2007 Textiles—Testing Antibacterial Activity: Part 2 Absorption Method

This national standard shows China’s new method for testing how textiles resist bacteria. The test principle is simple. First, you inoculate the test sample. Then, you also inoculate the control sample with a bacterial suspension. Someone carries out two procedures. The first elutes immediately after inoculation. The second elutes after incubation under specific conditions. We measure the number of viable bacteria in the eluted solutions. Then, we calculate the antibacterial value or inhibition rate. This helps us evaluate how effective the textile is against bacteria.

This method is very accurate. It works for both leaching and non-leaching antimicrobial textiles. So, it’s a reliable choice for testing smart textiles.

Key Factors Influencing Antibacterial Testing Methods for Textiles

This section discusses two main testing methods: the shake flask method and the absorption method.

Key Factors Affecting Shake Flask Test Results

The shake flask method’s accuracy and reliability depend on a few key factors.

Key factors are:

• Bacterial strains
• Washing agents
• Washing steps
• Test parameters

The criteria for measuring how effective the antibacterial agent is.

Test Bacterial Strains

Microorganisms from the same species can differ in origin. These differences can affect their sensitivity to the antimicrobial-treated textile. As a result, the antibacterial values may vary. Therefore, we recommend using standard bacterial strains for consistency.

Bacterial growth has a common pattern.

It has four phases:

• Lag phase
• Logarithmic (exponential) growth phase
• Stationary phase
• Decline phase

The duration of each phase varies between bacterial species. For example, Staphylococcus aureus is a strain often used in textile antibacterial tests. It multiplies at a rate much slower than Escherichia coli or Klebsiella pneumoniae. Results can differ based on the growth stage of the bacteria. Their metabolic activity changes at each phase.

Detergents and Washing Methods

Pre-washing the sample before testing is usually needed. This applies unless the textile is disposable or does not need washing for its use. This step has two roles: it clears dust and dirt from the fabric and gets rid of extra antimicrobial agents.

The type of detergent and its rinsing effectiveness can have a major impact on the test results. Therefore, we need to standardize the washing procedure and detergent formulation. Without such standardization, results from different testing institutions may not be comparable.

Test Parameters

Temperature is a critical factor influencing microbial growth. At low temperatures, bacteria stop growing. As the temperature rises, chemical and enzymatic reactions in the bacteria speed up. This leads to faster bacterial growth. Most bacteria in these tests are mesophilic. They grow best at about 37°C.

Bacterial reproduction is highly sensitive to temperature changes. Hence, temperature control is essential in antimicrobial fabric testing. The only standard with an incubation temperature of (24 ± 1)°C is “Antibacterial Knitted Fabrics.” All other standards use 37°C.

Researchers should standardize incubation temperatures to ensure comparability across testing methods.

Other test parameters can change results. Key factors include:

• Bacterial concentration
• Bacterial viability
• Nutrient availability
• Contact time with bacteria
• Incubation method
• Incubation duration

Establishing uniform test parameters enhances both intra-laboratory repeatability and inter-laboratory comparability.

Determination of Antibacterial Effectiveness

Currently, antibacterial effectiveness is typically expressed either as a percentage reduction or as a logarithmic reduction value. Percentage values are easy to grasp. However, they often miss key differences in antibacterial performance. For example: 90%, 99%, 99.9%, 99.99%, 99.999%. Each extra 9 after the decimal means a tenfold increase, even if the numbers look close. Also, test reports often round values to one decimal place. This makes it harder to see differences.

Since bacteria reproduce exponentially, percentage representation is not always scientifically appropriate. Log reduction values (like 1, 2, 3, 4, 5) show clear differences in antibacterial performance. However, non-experts might find them hard to understand.

We need to think about the environment and safety. So, we shouldn’t just chase very high antibacterial rates. Most antibacterial textiles touch human skin and get washed often. So, we must ensure they are safe and durable.

Taiwan Standard CNS 14945 looks at how well textiles fight bacteria. Applicants must submit safety and toxicity reports. Only then can they test antimicrobial fabric performance. These include:

• A skin irritation test report (pH < 2) and an allergenicity test with no allergic reactions in animals.
• An oral acute toxicity report for additives includes a lab-certified report. This shows no deaths or issues in mice at doses higher than 1,000 mg/kg.
• Or a third-party test report and declaration provided by the raw material supplier.

You can relax the safety rules for products that people won’t wash or touch with their bodies. So, we need to set clear antimicrobial fabric performance standards based on how the product is used.

Key Factors Affecting the Effectiveness of the Absorption Method

Test results from the absorption method depend on important factors. This covers how to make the bacterial suspension, the control sample, weigh the test sample, and how long to incubate. These elements play a decisive role in the success of the experiment.

Preparation of Bacterial Suspension

The first step in antimicrobial fabric testing is preparing the bacterial suspension. This step is crucial. It directly affects how well the bacteria grow during the experiment. Two main methods are used today. The first is the two-step method, which follows the U.S. AATCC 100 standard. The second is the three-step method, based on the Japanese JIS L 1902 standard.

The two-step method involves:

• Use an inoculation loop to streak bacteria from a stored culture onto a nutrient agar plate. Then, incubate the plate at 37 °C for a set time.
• Pick a typical colony from the agar plate. Then, add it to a nutrient broth. Incubate this broth at 37°C for a set time.
• The resulting bacterial suspension is then diluted to the required concentration for testing.

The three-step method adds one more step:

Take part of the bacterial suspension from the second step and put it into new nutrient broth. Incubate it at 37°C. Once done, dilute the culture to the desired concentration.

Importance of Bacterial Viability in Antimicrobial Fabric Testing

In antimicrobial fabric testing, it’s important to use bacteria with high viability. This helps show how well textile fabrics can fight off microbes. We check bacterial viability during testing. We do this by comparing growth on control samples before and after incubation. If bacteria grow well, they are more viable. But if growth is limited, they exhibit lower activity.

Two-Step vs. Three-Step Culturing Methods

Table 2 presents experimental data on the two-step and three-step methods. The findings show that the three-step method enhances bacterial viability in the suspension. This is especially true for Staphylococcus aureus. It tends to grow at a gradual pace. For Escherichia coli, which is generally highly active, the difference is minimal.

Strain-Specific Effects on Viability

The three-step method lowers the risk of using weakly preserved strains. It also ensures better results, giving a clearer picture of textile antimicrobial performance. It needs more time and effort than the two-step method. This is because it has three cultural stages. For active bacteria like E. coli, the extra step might not be necessary. The two-step method can yield similar results. This happens when the bacterial growth in the control sample meets the needed rate.

Choose the right method based on the bacterial strain for better efficiency.

Control Sample

Choosing the control sample is key in antimicrobial testing. This is because we compare the antimicrobial performance to the control. Bacterial growth in the control sample affects the final antimicrobial assessment. Some methods use the same fabric as the test sample. However, they do not include any antimicrobial agents. This serves as a control. This method is mainly for manufacturers. It works well for process optimization trials. But this is not practical for commercial testing. Companies often can’t provide matching control samples when they submit.

To make experiments easier and results easier to compare, we must choose a control sample that works for most textile test items. Antimicrobial textiles are usually things like socks and underwear. They are designed to fit closely against your skin. Because of this, the cotton backing fabric in GB 7565-287 was chosen as the control sample for color fastness testing.

Preliminary tests on the unwashed cotton backing fabric showed odd results. After 19 hours of incubation, the bacterial counts from the 10-fold dilution were surprising. This suggests there might be errors in the bacterial concentration readings.

Sample Amount

The amount of the test sample used has a significant effect on the test results. This happens because the washing step removes live bacteria from the fabric. But it can also wash out the antimicrobial agents on the fabric. More fabric means more antimicrobial agents go into the wash solution. These agents can stop bacteria from growing in the wash liquid. This helps lower the number of live bacteria in the end.

Larger test samples can show lower bacterial counts. This makes the antimicrobial effect seem stronger. However, this doesn’t truly represent the fabric’s real performance. So, it’s crucial to standardize the sample quantity. This will ensure reliable and consistent results in antimicrobial tests.

Incubation Time

Incubation time is the time it takes for bacteria to grow and reproduce. This happens under normal conditions until the number of live cells is at its highest. Experts regard eighteen hours as the optimal duration in theory. Current standard methods differ a bit here. Some say 18–24 hours, while others specify 18 ± 1 hour or 20 ± 2 hours. Still, they all fit within the general range of 18 to 24 hours.

 

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