**7. Methods for evaluating anti-microbiological activity of nonwovens**

The need to produce bioactive textiles containing biocides has led to the development of methods for evaluating antimicrobial activity. The final result of such a test is highly dependent on the testing method and the choice of test microorganism. Methods of evaluating antimicrobial properties can be divided into quantitative and qualitative methods (Dymel et al., 2008; Gutarowska et al., 2009).

Evaluation of the antimicrobial activity of textile products by qualitative methods is based on observation of the growth of microorganisms under and around a sample placed on an agar medium with a culture of the microorganisms. The effect of antimicrobial activity is indicated by the variously sized area in which the growth of the microorganisms is suppressed (Photographs 1–3).

Qualitative methods make it possible to evaluate the biocidal action of textiles both in the form of flat products, namely unwoven, woven and knitted fabrics, and in the form of fibres, threads, etc. The hydrophilic or hydrophobic nature of the textiles also has no effect on the final result. The only criterion for a textile product to be tested by qualitative methods is the diffusion of the active substance into the medium. Products must demonstrate at least minimal diffusion of the active component.

Photo 1. Growth inhibition zone around the *S. aureus;* polypropylene fibers containing 2% Ingaguard- method according to SN 195 920

Photo 2. Growth inhibition zone around the *C. albicans* polypropylene fibers containing 2% Ingaguard- method according to SN 195921

Microbial Degradation of Woven Fabrics and Protection Against Biodegradation 287

Quantitative methods are based on the general principle of inoculating the tested sample of material with a suspension of microorganisms of specified density, and then incubating them with the fabric. After some time, based on the number of microorganisms which survived contact with the fabric, the activity of the biocide in the sample is determined relative to a control sample not containing biocide. Quantitative methods are superior to qualitative ones, as the numerical results obtained for the biological activity of unwoven fabrics and textiles can be compared, to select the most effective solution for eliminating microorganisms. Table 11 lists the quantitative methods used for determining the

antimicrobial activity of bioactive unwoven fibres and textile products.

Testing hygienically-treated textile products for effectiveness against bacteria. Textile products hygienic finish council

Properties of textiles-Textiles and polymeric surfaces having antibacterial properties. Characterization and measurement of

Textile fabrics: Determination of the antibacterial activity: Germ

Antimicrobial products – Test for antimicrobial activity and

the selection of an appropriate testing method (Figure 3).

antibacterial activity

nonwovens and textiles

count method

efficacy

where:

fabric.

than 85%.

**Method / standards Standard number** 

Testing for antibacterial activity ISO/TC 38/-/WG23

The choice of method cannot be a random one; it is chiefly dependent on such criteria as the type of fabric, its properties and intended use, and the time of action on microorganisms. Based on these criteria and on analysis of the quantitative methods for evaluating antimicrobial properties of bioactive fabrics, a decision chart has been drawn up to enable

The test results Method AATCC 100 and Shake Flask Method are stated relative to the surface area or mass of the sample, in terms of reduction in the quantity of microorganisms:

N0 is the number of microorganisms per sample at time t0 with the bioactive fabric, and N is the number of microorganisms per sample after a time tn of exposure with the bioactive

A positive evaluation is given to fabrics on which the reduction in microorganisms is greater

% reduction = (N0-N)/N0100% (2)

Table 11. Methods for quantitative assessment of antimicrobial activity of bioactive

Shake Flask Method

XP G39-010

SN 195924

JIS Z 2801:2000

Assessement of antibacterial finishes on textile materials AATCC 100 Testing for antibacterial activity and efficacy on textile products JIS L 1902

Photo 3. Growth of bacteria respectively from the top: *S. aureus, E. coli; M.flavus, B. licheniformis* under polymer with nano-silver - method according to AATCC 147

Table 10 lists the most commonly used qualitative methods, including Swiss (SN), American (AATCC), Japanese (JI) and European (EN ISO) methods.


Table 10. Qualitative methods for assessing antimicrobial activity of bioactive nonwovens

Qualitative testing methods are similar to each other. They involve pouring out a layer of agar inoculated with a bacteria culture or fungal spores of specified density, or the application of microorganisms on an agar plate via linear inoculation. The tested material and a control sample of specified size are then placed on the inoculated medium. Following incubation, the action of the biocide is evaluated by measuring the area of suppression of growth, compared with a control sample not containing active antibacterial agent.

Quantitative methods are based on the general principle of inoculating the tested sample of material with a suspension of microorganisms of specified density, and then incubating them with the fabric. After some time, based on the number of microorganisms which survived contact with the fabric, the activity of the biocide in the sample is determined relative to a control sample not containing biocide. Quantitative methods are superior to qualitative ones, as the numerical results obtained for the biological activity of unwoven fabrics and textiles can be compared, to select the most effective solution for eliminating microorganisms. Table 11 lists the quantitative methods used for determining the antimicrobial activity of bioactive unwoven fibres and textile products.


Table 11. Methods for quantitative assessment of antimicrobial activity of bioactive nonwovens and textiles

The choice of method cannot be a random one; it is chiefly dependent on such criteria as the type of fabric, its properties and intended use, and the time of action on microorganisms. Based on these criteria and on analysis of the quantitative methods for evaluating antimicrobial properties of bioactive fabrics, a decision chart has been drawn up to enable the selection of an appropriate testing method (Figure 3).

The test results Method AATCC 100 and Shake Flask Method are stated relative to the surface area or mass of the sample, in terms of reduction in the quantity of microorganisms:

$$\text{\textquotedblleft reduction} = \text{\textquotedblright} \text{\textquotedblleft N}\_0\text{\textquotedblright} \text{\textquotedblleft} 100\%\tag{2}$$

where:

286 Woven Fabrics

Photo 3. Growth of bacteria respectively from the top: *S. aureus, E. coli; M.flavus, B. licheniformis* under polymer with nano-silver - method according to AATCC 147

(AATCC), Japanese (JI) and European (EN ISO) methods.

rot resistance of textile materials

streak method

diffusion plate test

Diffusion Plate Test

test

Antifungal activity, assessment of textile materials: Mildew and

Antimicrobial activity assessment of textile materials: Parallel

Standard Test Method for Using Seeded-Agar for the Screening

Textile fabrics: Determination of the antibacterial activity: Agar

Textile fabrics: Determination of the Antimycotic Activity: Agar

Standard Test Method for the Assessment of Antimicrobial

Resistance of Textiles to Microbiological Attack. Textiles – Determination of the antibacterial activity – Agar plate diffusion

Assessment of Antimicrobial Activity In Carpets

Activity In Carpets; Seeded-Agar Overlay Screen

Table 10 lists the most commonly used qualitative methods, including Swiss (SN), American

AATCC 30

AATCC 147

ASTM E2471-05

ASTM WK4757

SN 195920

SN 195921

CEN/TC 248/WG13

**Method / standards Standard number** 

Antibacterial activity of fabrics, detection of: Agar plate method AATCC 90

Antimicrobial activity assessment of carpets AATCC 174

Testing for antibacterial activity and efficacy on textile products JIS L 1902

Table 10. Qualitative methods for assessing antimicrobial activity of bioactive nonwovens

growth, compared with a control sample not containing active antibacterial agent.

Qualitative testing methods are similar to each other. They involve pouring out a layer of agar inoculated with a bacteria culture or fungal spores of specified density, or the application of microorganisms on an agar plate via linear inoculation. The tested material and a control sample of specified size are then placed on the inoculated medium. Following incubation, the action of the biocide is evaluated by measuring the area of suppression of

N0 is the number of microorganisms per sample at time t0 with the bioactive fabric, and N is the number of microorganisms per sample after a time tn of exposure with the bioactive fabric.

A positive evaluation is given to fabrics on which the reduction in microorganisms is greater than 85%.

Microbial Degradation of Woven Fabrics and Protection Against Biodegradation 289

reference strain, gram negative rods, a significant resistance to

reference strain, gram negative rods, a significant resistance to

reference strain, gram negative

reference strain, gram positive

gram positive coccus, exists on

gram positive bacilli produces spores, often found in the environment (air, soil),

widespread in the environment (mucous membranes, air, skin)

isolated from the air, high resistance to UV and

reference strain, yeast,

yeast, widespread in the environment (air, food)

mould, reference strain for testing of technical material resistance, present in the air

mould, often isolated from air

mould, often isolated from air

mould, reference strain

mould

mould

biocides

biocides

rods

coccus

the skin

disinfectants

**Microorganism Pathogenicity Characteristic** 

pathogen, various types of

and nosocomial infections

venous blood clots, ulcers, myocarditis, transmitted by air, common carriers in the nasal cavity and throat, nosocomial

infections

infections

*Bacillus subtilis* saprophyte, harmless to health,

disorders

*Candida albicans* a potential pathogen, systemic

hypoallergenic

skin infections

*Rhodotorula rubra* saprophyte, harmless to health,

*Aspergillus niger* saprophyte, harmless to health,

allergies

*Alternaria alternata* saprophyte, harmless to health, hypoallergenic

and nails

and nails

digestive disorders, urinary tract

infection, inflammation of the skin

pathogen, pneumonia, transmitted by air, nosocomial infections

pathogen, dermatitis, pneumonia,

saprophyte, harmless to health, sometimes skin infections

sometimes causes digestive

infections, skin, nail mucous membranes infections,

sometimes skin infections

sometimes respiratory, cornea and

saprophyte, harmless to health, sometimes upper respiratory tract infections, ear and nail infections,

pathogen, infections of hair, skin

pathogen, nail, skin and mucous

pathogen, infections of hair, skin

Table 12. Characteristics of test microorganisms for determination of the antimicrobial

membranes infections

activity of bioactive nonwovens (based on: Gutarowska et al., 2009)

*Micrococcus flavus* saprophyte, harmless to health gram positive coccus, often

*Escherichia coli*  ATCC 11229

*Pseudomonas aeruginosa* 

*Staphylococcus* 

*Staphylococcus epidermidis* 

*Penicillium chrysogenum* 

*Trichophyton mentagrophytes* 

*Scopulariopsis brevicaulis* 

*Epidermophyton floccosum* 

*Klebsiella pneumoniae*

*aureus*  ATCC 6538

Fig. 3. A decision tree for choose the method of quantitative evaluation of antimicrobial activity of bioactive nonwoven (based on Gutarowska et al. 2009)

The result may be given in the form of bactericidal activity and bacteriostatic activity (Method JIS L 1902, JIS Z 2801:2000).

Biostatic activity is calculated from the formula:

$$\text{biostatic activity (S)} = \log N\_k / N \tag{3}$$

where:

Nk is the number of microorganisms per sample after a time tn of exposure with the control fabric, and N is the number of microorganisms per sample after a time tn of exposure with the bioactive fabric.

Biocidal activity is calculated analogously:

$$\text{biocoidal activity (L)} = \log\_2 \text{N}\_0/\text{N} \tag{4}$$

where:

Fig. 3. A decision tree for choose the method of quantitative evaluation of antimicrobial

The result may be given in the form of bactericidal activity and bacteriostatic activity

Nk is the number of microorganisms per sample after a time tn of exposure with the control fabric, and N is the number of microorganisms per sample after a time tn of exposure with

biostatic activity (S) = log Nk/N (3)

biocidal activity (L) = log N0/N (4)

activity of bioactive nonwoven (based on Gutarowska et al. 2009)

(Method JIS L 1902, JIS Z 2801:2000).

where:

where:

the bioactive fabric.

Biostatic activity is calculated from the formula:

Biocidal activity is calculated analogously:


Table 12. Characteristics of test microorganisms for determination of the antimicrobial activity of bioactive nonwovens (based on: Gutarowska et al., 2009)

Microbial Degradation of Woven Fabrics and Protection Against Biodegradation 291

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Becker M.A., Williams P. & Tuross N.C. (1995) The USA first ladies gowns: a biochemical

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Brycki B. (2003) Chemiczne inhibiotory biodeterioracji (Chemical inhibitors of

Buchanan C.M., Gardner R.M. & Komarek R.T. (1993) Aerobic biodegradation of cellulose

Bucheńska J., Słomkowski S., Tazbir J. & Sobolewska E. (2003) Antibacterial poly(ethylene

Bujak S. & Targoński Z. (1990) Mikrobiologiczna degradacja hemiceluloz. *Postępy* 

Buschle-Diller G., Zeronian S.H., Pan N. & Yoon M.Y. (1994) Enzymatic hydrolysis of cotton, linen, ramie and viscose rayon fabrics. *Textile Research Journal* 64(5), pp. 270-279. Cain R.B. (1992) Microbial degradation of synthetic polymers. In: *Microbial control of* 

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N0 is the number of microorganisms per sample at time t0 with the bioactive fabric, and N is the number of microorganisms per sample after a time tn of exposure with the bioactive fabric.

A sample is taken to have bactericidal properties if the value of the coefficient of bactericidal activity (L) is greater than zero, and to have bacteriostatic properties if the value of the coefficient of bacteriostatic activity (S) is greater than 2 (Yu, 2003), which denotes a 100 fold reduction in the number of microorganisms.

The evaluation of activity is made with respect to selected potentially pathogenic (from Pure Culture Collections ATCC, NCTC) or saprophytic microorganisms occurring naturally in the human environment. Table 12 lists test microorganisms used for evaluation of the bioactivity of textiles and for their description.

The fundamental criterion for the selection of microorganisms for testing of antimicrobial activity is the intended use of the fabric. In the case of therapeutic fabrics, coming into contact with the human skin, or intended for use in hospitals and care centres, the microorganisms chosen for testing are those which are pathogenic and which are particularly resistant to chemical disinfection and antibiotic treatment, leading to hospital infections, for examples: *Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Bacillus licheniformis, Corynebacterium xersosis, Trichophyton mentagrophytes, Candida albicans*. Technical fabrics for uses such as air filtration, and for everyday uses (upholstery, blankets, carpets, net curtains, tarpaulin, etc.) usually come into contact with saprophytic microorganisms, not hazardous to human health, which are constantly present in the air in the form of bioaerosols. Such fabric is tested against the fungi: *Aspergillus niger, Penicillium chrysogenum, Alternaria alternata, Cladosporium cladosporioides* and bacteria*: Micrococcus flavus, Bacillus subtilis*.
