METHOD FOR TESTING BACTERIAL FILTRATION EFFICACY OF FABRICS

Abstract

The present invention relates to method of testing the bacterial filtering efficiency of a fabric, the method including the steps of preparing a solution including bacteria, peptone water and NaCl, feeding said solution including bacteria to a nebulizer, generating an aerosol of said bacterial solution and flowing said solution through a cascade impactor to provide a plurality of bacteria colonies in a plurality of plates present in a plurality of stages of said cascade impactor, characterized in that the concentration of NaCl in the peptone water used to prepare said bacterial solution is in the range of 30 g/L to 150 g/L and in that the temperature of said cascade impactor is in the range of −15° C. to 15° C.

Claims

1. A method of testing the bacterial filtering efficiency of a fabric, the method including the steps of preparing a solution including bacteria, peptone water and NaCl, feeding said solution including bacteria to a nebulizer, generating an aerosol of said bacterial solution and flowing said solution through a cascade impactor to provide a plurality of bacteria colonies in a plurality of plates present in a plurality of stages of said cascade impactor, characterized in that the concentration of NaCl in the peptone water used to prepare said bacterial solution is in the range of 30 g/L to 150 g/L and in that the temperature of said cascade impactor is in the range of −15° C. to 15° C.

2. A method according to claim 1, wherein said method is carried out according to EN 14683:2019+AC:2019 E modified to provide an NaCl concentration of the peptone water and a refrigeration of the cascade impactor as recited in claim 1.

3. A method according to claim 1, wherein said NaCl concentration is in the range of 60 g/L to 150 g/L.

4. A method according to claim 1, wherein the temperature of the cascade impactor is in the range of −4° C. to 15° C.

5. A method according to claim 1, wherein in the testing conditions said nebulizer is capable to generate an aerosol of a saline solution, free from bacteria, having Mean Particle Size in the range of 2.7 to 3.3 μm.

6. A method according to claim 5, wherein said nebulizer is selected from a jet nebulizer, an ultrasonic nebulizer, and a mesh nebulizer.

7. A method for bringing into a required size range the Mean Particle Size of particles generated by a nebulizer in a device for testing Bacterial Filtration Efficiency of a fabric, the device comprising a nebulizer for generating an aerosol of a bacterial solution and a cascade impactor including a plurality of stages, said method comprising the steps of: generating an aerosol of a solution including bacteria, peptone water an NaCl; flowing said aerosol through the cascade impactor to provide a plurality of bacteria colonies in a plurality of plates present in the plurality of stages of said cascade impactor, characterized in comprising the steps of: performing a positive control run by feeding said aerosol to said cascade impactor; determining the Mean Particle Size of the aerosol particles based on the resulting number of bacterial colonies; comparing the obtained Mean Particle Size with a required range of Mean Particle Size; modifying the control run conditions by increasing or decreasing the concentration of NaCl in said bacterial solution and/or the temperature of said cascade impactor; performing at least another positive control run under said modified conditions and repeating said steps until the resulting Mean Particle Size is within the required range.

8. A method according to claim 7, wherein said required range of said Mean Particle Size is from 2.7 μm to 3.3 μm.

9. A method according to claim 7, wherein said method is carried out according to EN 14683:2019+AC:2019 E.

10. A method according to claim 1, wherein said bacteria is selected from the group consisting of Mycobacterium tuberculosis, Streptococcus pneumoniae, Legionella pneumophilla, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli.

11. A method according to claim 1, wherein said fabric sample is placed between the first stage and the inlet cone of said cascade impactor.

12. A device for testing Bacterial Filtration Efficiency of a fabric, the device comprising a nebulizer for generating an aerosol of a bacterial solution and a cascade impactor including a plurality of stages, characterized in comprising refrigerating means to refrigerate said cascade impactor.

13. A device according to claim 12, wherein said device is a device according to EN 14683:2019+AC:2019 E.

14. A device according to claim 12, wherein said refrigerating means are provided at the exit portion of said cascade impactor.

15. A method according to claim 9, wherein the run conditions are modified by increasing or decreasing the concentration of NaCl in said bacterial solution and/or the temperature of said cascade impactor.

16. A method according to claim 7, wherein said bacteria is selected from the group consisting of Mycobacterium tuberculosis, Streptococcus pneumoniae, Legionella pneumophilla, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli.

17. A method according to claim 7, wherein said fabric sample is placed between the first stage and the inlet cone of said cascade impactor.

18. A device according to claim 13, wherein said device is provided with refrigerating means to refrigerate said cascade impactor.

19. A device according to claim 18, wherein said refrigerating means are provided at the exit portion of said cascade impactor.

Description

EXAMPLES

[0057] The following examples relate to exemplary embodiments of the present invention and are to be considered as illustrative and non-limiting with respect of the scope of the invention.

Example 1

Reagents and Materials

Tryptic Soy Agar (TSA)

[0058] Tryptic soy agar is a solid culture medium. The composition is reported in Table 1, with reference to the liquid solution used to produce the solid medium.

TABLE-US-00001 TABLE 1 (formula/litre) Enzymatic Digest of Casein 15 g Enzymatic Digest of Soybean Meal  5 g Sodium Chloride  5 g Agar 15 g Distilled water 1000 ml Final pH 7.3 ± 0.2 at 25° C.

Tryptic Soy Broth (TSB)

[0059] Tryptic soy broth is a liquid culture medium. The composition is reported in Table 2.

TABLE-US-00002 TABLE 2 (formula/litre) Enzymatic Digest of Casein  17 g Enzymatic Digest of Soybean Meal   3 g Sodium Chloride   5 g Dipotassium phosphate 2.5 g Dextrose 2.5 g Distilled water 1000 ml Final pH 7.3 ± 0.2 at 25° C.

Peptone Water—According to Standard Method EN 14683

[0060] Peptone water is a liquid medium used for diluting the bacterial culture before the test according to standard method EN 14683. The composition is reported in Table 3.

TABLE-US-00003 TABLE 3 (formula/litre) Peptone 10 g Sodium Chloride (NaCl)  5 g Distilled water 1000 ml Final pH 7.2 ± 0.2 at 25° C.
Peptone water—According to the Invention
Peptone water according to the invention is a liquid medium used for diluting the bacterial culture before the test according to the invention. The composition of an exemplary embodiment is reported in Table 4.

TABLE-US-00004 TABLE 4 (formula/litre) Peptone  10 g Sodium Chloride (NaCl) 100 g Distilled water 1000 ml Final pH 7.2 ± 0.2 at 25° C.

Example 2

Exemplary Procedure

[0061] According to the invention, the cascade impactor is cooled to a temperature in the range of —15° C. to 15° C., for example, by keeping the impactor for at least 5 minutes at —80° C. in a refrigerator.

[0062] Additionally, according to the invention, peptone water includes NaCl at a concentration of in the range of 30 g/L to 150 g/L.

1) Preparation of Media

[0063] a. Liquid media [0064] i. Tryptic Soy Broth (TSB) [0065] ii. Peptone Water

[0066] b. Solid medium [0067] i. Tryptic Soy Agar (TSA)

2) Bacterial Growth

[0068] a. Resuscitation of Staphylococcus aureus bacteria [0069] i. Growth of S. aureus in TSB medium at 37° C. overnight.

[0070] b. Inoculation of Staphylococcus aureus bacteria using streak plate method [0071] i. Growth of S. aureus in TSA medium at 37° C. overnight.

3) Test Phase

[0072] a. Conditioning of test fabric [0073] i. Hanging the test fabric in the climate chamber which provides constant temperature (21 ±5° C.) and relative humidity (85±5%) for at least 4 hours.

[0074] b. Inoculation of Staphylococcus aureus bacteria in TSB medium [0075] i. Selecting a single colony of S. aureus bacteria using inoculation loop, adding that colony in TSB medium, and growing bacteria at 37° C. for 4 hours.

[0076] c. Dilution of Staphylococcus aureus bacteria [0077] i. Confirmation of adequate growth of bacteria by measuring turbidity of bacterial solution using McFarland densitometer and/or OD600 spectrophotometer. [0078] ii. Diluting bacterial solution with peptone water to obtain 5×10.sup.5 colony forming unit (cfu).

[0079] d. Test operation [0080] i. Calibrating Bacterial Filtration Efficiency (BFE) device by setting vacuum flow rate to 28.3 L/min (1 cubic foot per minute) and running for 2 minutes (1 minute for bacterial challenge and the other one minute for maintaining the airflow without running the nebulizer). [0081] ii. Order of bacterial filtration efficiency tests is as follows (Table 5):

TABLE-US-00005 TABLE 5 ORDER OF BACTERIAL FILTRATION EFFICIENCY TEST 1 2 3 4 5 6 7 8 Positive Mask Mask Mask Mask Mask Positive Negative control 1 2 3 4 5 control control 1 2 [0082] iii. Numbering TSA petri plates from “Control 1-1” to “Control 1-6” with permanent pen, then locating them into Andersen Cascade Impactor (ACI) accordingly. [0083] iv. Pouring bacterial solution into nebulizer chamber. [0084] v. Running the test without any test fabric for 2 minutes to obtain positive control. [0085] vi. Removing TSA petri plates from ACI. [0086] vii. Taking the test fabric (i.e., the test mask) from climate chamber. [0087] viii. Removing ear loop and cutting two sides to open the pleated part of test fabric (i.e., the test mask). The test area shall be minimum 49 cm.sup.2. [0088] ix. Numbering TSA petri plates from “Mask 1-1” to “Mask 1-6” with permanent pen, then locating them into Andersen Cascade Impactor (ACI) accordingly. [0089] x. Locating test fabric to the top of ACI, and clamping tightly to prevent any leakage. [0090] xi. Running the test with test fabric for 2 minutes (1 minute for bacterial challenge and the other one minute for maintaining the airflow without running the nebulizer) to obtain mask 1 result. [0091] xii. Repeat the same protocol until 5.sup.th mask. [0092] xiii. Running the second positive control without any fabric (mask). [0093] xiv. Cleaning the nebulizer from bacterial solution, then running for negative control without mask and bacteria.

[0094] e. Growth of Staphylococcus aureus bacteria in incubator [0095] i. Locating all petri plates (8 set×6 plates=48 plates) at incubator at 37° C.±2° C. for between 20 hours and 52 hours.

4) Preparation of Report

[0096] a. Counting of Staphylococcus aureus bacteria [0097] i. Place TSA petri plate on a black surface, then starting to count by permanent marker or Promega Colony Count app depending on the urgency. [0098] ii. Write down all bacteria number on a paper report. [0099] iii. Transfer all data to Excel, then convert all data except petri 1 and petri 2 according to the positive hole conversion table from the cascade impactor manual. In the present example, an Andersen cascade impactor and the related “Table 1: Positive Hole Conversion Table”, reported in the paper of Ariel A. Andersen, “NEW SAMPLER FOR THE COLLECTION, SIZING, AND ENUMERATION OF VIABLE AIRBORNE PARTICLES”, J Bacteriol. 1958 Nov; 76(5): 471-484, at page 474 were used. In the mentioned table, the Actual bacteria number is represented as “r”, converted bacteria number is represented as “P”. For example, if actual bacteria number is 121, then converted bacteria number is 144. [0100] iv. Numbers in italics represents no conversion, underlines represent summation of converted positive control.

TABLE-US-00006 TABLE 6 CONVERSION OF ACTUAL POSITIVE CONTROL USING “POSITIVE HOLE CONVERSION TABLE” Petri Petri Petri Petri Petri Petri 1 2 3 4 5 6 (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ Total plate) plate) plate) plate) plate) plate) CFU Actual 124 210 251 312 283 9 1189 Positive Control 1 Actual 134 293 332 366 280 15 1420 Positive Control 2 Con- 124 210 395 606 492 9 1836 verted Positive Control 1 Con- 134 293 709 986 482 15 2619 verted Positive Control 2 Average 129 251.5 552 796 487 12 2227.5 Con- verted Positive Control

[0101] v. Calculation of mean total bacteria number [0102] 1. Summation of converted positive control 1. [0103] 2. Summation of converted positive control 2. [0104] 3. Calculate the average value of two converted positive controls. [0105] 4. According to EN14683 Standard, mean value of two converted positive control must be between 1700 and 3000 cfu.

TABLE-US-00007 TABLE 7 CONVERSION OF ACTUAL POSITIVE CONTROL USING “POSITIVE HOLE CONVERSION TABLE” Petri Petri Petri Petri Petri Petri 1 2 3 4 5 6 (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ Total plate) plate) plate) plate) plate) plate) CFU Actual 124 210 251 312 283 9 1189 Positive Control 1 Con- 124 210 395 606 492 9 1836 verted Positive Control 7 4.7 3.3 2.1 1.1 0.65 1 Hole Size coef- ficient Multi- 868 987 1303.5 1272.6 541.2 5.9 4978.2 plied con- verted positive control 1

[00002]$\mathrm{Mean}\mathrm{particle}\mathrm{size}1\left(\mathrm{MPS}1\right)=\frac{4978.2}{1836}=2.71$

TABLE-US-00008 TABLE 8 CONVERSION OF ACTUAL POSITIVE CONTROL USING “POSITIVE HOLE CONVERSION TABLE” Petri Petri Petri Petri Petri Petri 1 2 3 4 5 6 (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ Total plate) plate) plate) plate) plate) plate) CFU Actual 134 293 332 366 280 15   1420 Positive Control 2 Con- 134 293 709 986 482 15   2619 verted Positive Control 7 4.7 3.3 2.1 1.1 0.65 2 Hole Size coef- ficient Multi- 938 1377.1 2339.7 2070.6 530.2 9.8 7265.4 plied con- verted positive control 2

[00003]$\mathrm{Mean}\mathrm{particle}\mathrm{size}2\left(\mathrm{MPS}2\right)=\frac{7265,4}{2619}=2.77$$\mathrm{Average}\mathrm{Mean}\mathrm{particle}\mathrm{size}\left(\mathrm{MPS}\right)=\frac{2.71+2.77}{2}=2.74$ [0106] vi. Calculation of mean particle size [0107] 1. Convert actual bacteria number of two positive controls using the Positive Hole Conversion Table (in the present example, the “Table 1: Positive Hole Conversion Table”, reported in the paper of Ariel A. Andersen, mentioned above, was used). [0108] 2. Multiply converted bacteria numbers from positive control plate 1 to 6 with the respective hole size coefficient, i.e., in this case, 7, 4.7, 3.3, 2.1, 1.1, and 0.65 respectively. [0109] 3. Summation of multiplied numbers for positive control 1 [0110] 4. Summation of multiplied numbers for positive control 2 [0111] 5. Divide total value of the two multiplied numbers of the two control runs with the total value of the two converted positive controls, to obtain MPS for each positive control. [0112] 6. calculate the Mean value of MPS between the two MPS for positive controls. [0113] 7. According to 14683 Standard, mean particle size must be between 2.7 μm and 3.3 μm.

TABLE-US-00009 TABLE 9 CONVERSION OF ACTUAL MASK VALUE USING “POSITIVE HOLE CONVERSION TABLE” Petri 1 Petri 2 Petri 3 Petri 4 Petri 5 Petri 6 (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ (CFU/ Total plate) plate) plate) plate) plate) plate) CFU Actual 1 1 15 13 51 15  96 Mask Value Converted 1 1 15 13 55 15 100 Mask Value

[00004]$\mathrm{Bacterial}\mathrm{Filtration}\mathrm{Efficiency}\left(\mathrm{BFE}\right)=\frac{\mathrm{Average}\mathrm{converted}\mathrm{positive}\mathrm{control}-\mathrm{Converted}\mathrm{Mask}\mathrm{Value}}{\mathrm{Average}\mathrm{converted}\mathrm{positive}\mathrm{control}}\times 100$$\mathrm{Bacterial}\mathrm{Filtration}\mathrm{Efficiency}\left(\mathrm{BFE}\right)=\frac{2227.5-100}{2227.5}\times =95.51\%$ [0114] vii. Calculation of bacterial filtration efficiency [0115] 1. write down all bacteria number on a paper report [0116] 2. transfer all data to Excel, then convert all data except petri 1 and petri 2 according to “Table 1: positive Hole Conversion Table”, as above mentioned [0117] 3. Use converted mask data and average converted positive control for % BFE calculation for each fabric (mask) tested (an example of fabric data for one mask is reported in Table 9, wherein numbers in italics represents no conversion, underlines represent summation of converted fabric values). In order to obtain the final BFE result, 5 mask (i.e., 5 fabric samples) should be tested and averaged. [0118] 4. According to EN 14683 Standard, bacterial filtration efficacy must be ≥% 95 for type I masks, and ≥% 98 for type II/type IIR masks.

Example 3

Comparison Between EN 14683 and the Method of the Invention

[0119] In the present example, several samples of the same woven fabric were tested.

[0120] In the test carried out according to the invention, the same conditions of the test carried out according to EN 14683 were used, except for concentration of NaCl in peptone water and for the temperature of the impactor, that were according to the method of the invention.

BFE Test With EN 14683

[0121] All the solutions and conditions were according to EN 14683. It was found that average mean particle size is 1.81 μm and average BFE result is 78,15%.

TABLE-US-00010 TABLE 10 Total and converted cfu values Petri Petri Petri Petri Petri Petri Total Converted 1 2 3 4 5 6 cfu total (cfu) Positive 48 43 262 355 366 254 1328 2780 control 1 Sample 1 1 6 8 44 198 175 432 565 Sample 2 3 8 9 52 201 183 456 600 Sample 3 2 5 7 33 191 188 426 562 Sample 4 1 2 7 35 177 190 412 539 Sample 5 0 1 5 30 169 193 398 520 Positive 31 41 200 338 357 225 1192 2318 control 2

TABLE-US-00011 TABLE 11 Average Mean particle size and Converted total bacteria. Positive Positive control 1 control 2 Average Converted total bacteria (cfu) 2780 2318 2549 Mean particle size (μm) 1.84 1.76 1.81

TABLE-US-00012 TABLE 12 Bacteria Filtration Efficiency (%) according to EN 14683 method Sample 1 77.83 Sample 2 76.46 Sample 3 77.95 Sample 4 78.85 Sample 5 79.60 Average 78.14

BFE Trial With the Method of the Invention

[0122] As above mentioned, the method of the invention allows to maintain the mean particle size in the range between 2.7 μm and 3.3 μm. The method of the invention is different from EN14683 standard, in particular, in the percentage (%) of NaCl in the Peptone water, and in the Andersen cascade temperature.

In the present example:

[0123] Peptone Water Preparation: Peptone water according to the invention, having the composition disclosed in Table 4, here above reported was used. In particular, a commercial peptone water product (distributed by Condalab) was used. According to the formulation of such product,1.5 grams of powder contain 0.5 gram of NaCl and 1 gram of peptone. 9.5 grams of NaCl were added into the solution to make a 10 gram/100 ml NaCl solution which has a final concentration of 10% NaCl. Solution was autoclaved at 121° C. for 15 minutes.

[0124] Andersen Cascade Impactor Preparation: Six stages Andersen Cascade was cooled, in the present example, on the −80° C. refrigerator for 5 minutes.

TABLE-US-00013 TABLE 13 total and converted CFU values Petri Petri Petri Petri Petri Petri Total Converted 1 2 3 4 5 6 cfu total (cfu) Positive 120 235 314 272 292 11 1244 1961 control 1 Sample 1 0 3 10 93 72 0 178 198 Sample 2 10 9 75 149 3 0 246 291 Sample 3 2 1 18 83 41 0 145 157 Sample 4 13 1 26 49 83 0 172 186 Sample 5 1 2 37 71 82 2 195 214 Positive 201 211 375 302 156 4 1249 2286 control 2

TABLE-US-00014 TABLE 14 Average Mean particle size and Converted total bacteria. Positive Positive control 1 control 2 Average Converted total bacteria (cfu) 1961 2286 2123.5 Mean particle size (μm) 2.81 3.26 3.06

TABLE-US-00015 TABLE 15 Bacteria Filtration Efficiency (%) according to the method of the invention Sample 1 90.68 Sample 2 86.30 Sample 3 92.61 Sample 4 91.24 Sample 5 89.92 Average 90.15

[0125] It was found that average mean particle size is 3.06 μm and average BFE result is 90.15%.

CONCLUSION

[0126] A 12% BFE result differences between EN 14683 standard and the method of the invention was observed.

TABLE-US-00016 TABLE 16 Comparison of result of EN 14683 and the method of the invention. Method of the EN 14683 invention Sample 1 77.83 90.68 Sample 2 76.46 86.30 Sample 3 77.95 92.61 Sample 4 78.85 91.24 Sample 5 79.60 89.92 Average BFE result 78.14 90.15 Mean Particle Size (MPS)  1.81  3.06