The absorption method for determining antibacterial activity is a quantitative microbiological test used to evaluate the antibacterial performance of textile products in accordance with standards such as GB/T 20944.2 and ISO 20743. By directly inoculating fabric specimens with a standardized bacterial suspension and counting surviving organisms after incubation, the method yields an antibacterial activity value and a bacteriostatic rate that objectively substantiate product claims. This article explains the technical principle, applicable samples, test organisms, step-by-step procedure, calculation logic, and typical applications of the absorption method.
Technical Overview
The absorption method (also called the absorption inoculation method) is a quantitative assay for measuring the antibacterial activity of antibacterial-treated textiles. In this method, a known volume of bacterial suspension is pipetted directly onto fabric specimens so that the inoculum is absorbed into the textile structure. After a defined incubation period under controlled temperature and humidity, the bacteria are eluted from the specimens in a neutralizing recovery medium and enumerated by agar plate counting. The difference between the logarithmic bacterial counts recovered from an untreated control fabric and the antibacterial-treated fabric is calculated as the antibacterial activity value, which reflects the ability of the finished textile to inhibit the growth of microorganisms.
Unlike qualitative techniques such as the agar diffusion plate method, which only shows zones of inhibition and is limited to diffusive agents, the absorption method provides numerical, reproducible results and is suitable for both leaching and non-leaching antibacterial treatments. It is one of the most widely used methods for compliance testing, product development, and quality control of antibacterial textiles, and it is routinely performed by accredited third-party laboratories.
Reference Standards
- GB/T 20944.2 — Textiles — Evaluation for antibacterial activity — Part 2: Absorption method
- ISO 20743 — Textiles — Determination of antibacterial activity of antibacterial finished products (absorption method among its quantitative options)
- JIS L 1902 — Textiles — Determination of antibacterial activity and efficacy (absorption method), the historical origin of this technique
Testing Samples
Applicable Materials
- Antibacterial-finished woven, knitted, and nonwoven fabrics
- Antibacterial fibers and yarns arranged into fabric-like test pieces
- Cut specimens from finished products such as towels, underwear, socks, bed linens, masks, wipes, and surgical garments
- Coated, laminated, or padded textiles carrying inorganic agents (silver, copper, zinc), organic agents (quaternary ammonium compounds, biguanides), or natural agents (chitosan, plant extracts)
- An untreated standard cotton reference fabric serving as the negative control
Specimen Preparation Requirements
- Specimens are typically cut to approximately 0.4 g per piece, with replicate pieces prepared for each test organism and each time point
- Specimens must be sterilized by an appropriate method (autoclaving, ethylene oxide, or irradiation) without damaging the antibacterial finish
- Samples should be homogeneous, representative of the production batch, and free of contamination before testing
- Hygroscopic specimens may be pre-moistened according to the standard to ensure uniform absorption of the inoculum
Testing Items
The absorption method evaluates antibacterial efficacy against standardized strains and reports the following quantitative parameters:
- Staphylococcus aureus (ATCC 6538) — Gram-positive bacterium, the primary indicator strain
- Escherichia coli (ATCC 11229 or ATCC 8739) — Gram-negative bacterium
- Candida albicans (ATCC 10231) — yeast strain used to assess antifungal activity
- Klebsiella pneumoniae (ATCC 4352) — additional Gram-negative strain under ISO 20743
- Growth value on the control fabric, used to validate the test
- Antibacterial activity value A, expressed as a logarithmic reduction
- Bacteriostatic rate (percentage inhibition of bacterial growth)
Testing Methods
The test procedure follows a controlled sequence of inoculation, incubation, recovery, and enumeration, with all operations performed under aseptic conditions in a microbiological safety cabinet.
Procedure Overview
- Culture preparation: test strains are cultured in nutrient broth for 18–24 hours and diluted to a standardized suspension, typically in the range of 1×10⁵ to 3×10⁵ CFU/mL
- Inoculation: a fixed volume of the bacterial suspension (commonly 0.2 mL) is pipetted onto each weighed specimen, ensuring the inoculum is completely and evenly absorbed into the fabric
- Zero-hour recovery: bacteria are immediately eluted from part of the control specimens to determine the initial recoverable count
- Incubation: the remaining inoculated specimens are held in closed sterile containers at 37 ± 2 °C in a humidified atmosphere for 18–24 hours
- Elution: after incubation, bacteria are recovered by adding a neutralizing broth such as SCDLP (soybean casein digest broth with lecithin and polysorbate 80) and agitating the specimens mechanically
- Enumeration: serial dilutions of the eluates are plated on tryptone soy agar (or appropriate agar for yeasts), incubated, and colony-forming units are counted
Calculation and Interpretation
Let U0 be the log of the viable count recovered from the control fabric immediately after inoculation, Ut the log count from the control fabric after incubation, and At the log count from the treated fabric after incubation. The growth value on the control is calculated as F = Ut − U0, and the test is valid only when F meets the minimum growth requirement specified by the standard (typically at least 1 log of multiplication) and the zero-hour recovery falls within the acceptance range.
The antibacterial activity value is calculated as A = Ut − At, and the bacteriostatic rate is calculated as (1 − 10^(At − Ut)) × 100%. In general interpretation:
- A ≥ 1 corresponds to a reduction of at least 90% and is regarded as evidence of antibacterial (bacteriostatic) effect
- A ≥ 2 corresponds to a reduction of at least 99% and indicates strong antibacterial performance
- A ≥ 3 corresponds to a reduction of at least 99.9% and approaches a bactericidal level of activity
Applications
The absorption method supports antimicrobial product development, regulatory compliance, and commercial claim substantiation across multiple industries:
- Validation of antibacterial finishing processes during research and development, including screening of silver-based, organic, and natural antibacterial agents
- Quality control of mass-produced antibacterial textiles to verify batch-to-batch consistency
- Medical and healthcare textiles such as surgical gowns, drapes, masks, wound-contact fabrics, and hospital bed linens
- Hygiene and intimate apparel including underwear, socks, stockings, and sanitary products where odor control and microbial suppression are claimed
- Home textiles such as towels, bedding, and upholstery fabrics marketed with antibacterial labeling
- Footwear linings, military clothing, sports apparel, and workwear intended for prolonged wear in demanding environments
- Third-party compliance testing and certification support against GB/T 20944.2, ISO 20743, and JIS L 1902 requirements
FAQ
What is the difference between the absorption method, the agar diffusion method, and the shake flask method?
The agar diffusion method is a qualitative screen suitable only for antibacterial agents that diffuse into agar, whereas the shake flask method (such as GB/T 20944.3) is designed for non-soluble antibacterial products tested in liquid contact. The absorption method is a quantitative technique in which bacteria are absorbed directly into the specimen, making it suitable for both leaching and non-leaching finishes and providing numerical activity values.
Why must the control fabric show sufficient bacterial growth?
The growth value on the untreated control fabric confirms that the test conditions allow normal bacterial proliferation. If the control does not multiply by the required log increment, the test is invalidated because an observed reduction on the treated specimen could not be reliably attributed to antibacterial action.
What sample size and form are required?
Each specimen is typically about 0.4 g, and multiple replicate pieces are needed for each strain, each condition, and the zero-hour control. Samples may be fabrics, nonwovens, or cut pieces from finished products, provided they can absorb the inoculum uniformly.
Can the absorption method evaluate antifungal performance?
Yes. Candida albicans is commonly included as a test strain to assess activity against yeasts, using appropriate culture media and slightly longer incubation for colony formation.
What does an antibacterial activity value of 2 mean in practical terms?
An activity value of 2 represents a 99% reduction in recoverable bacteria on the treated fabric compared with the untreated control after the same incubation period, which is generally regarded as strong antibacterial performance.
How is residual antibacterial agent interference controlled?
The recovery medium contains neutralizers such as lecithin and polysorbate 80 that inactivate residual antimicrobial agent carried over during elution, ensuring that counted colonies reflect bacteria that survived on the fabric rather than organisms killed after recovery.