YY/T 1497-2016Evaluation test method for the viral filtration efficiency(VFE)of medical protective face mask materials—Test method using Phi-X174 bacteriophage (English PDF)

3.1 Virus

Virus refers to infectious micro-organisms that have no independent metabolic system and can only replicate in living host cells.

3.2 Bacteriophage

Bacteriophage refers to a type of virus that can infect bacteria.

NOTE: in this test method, bacteriophage refers to Phi-X174. Phi-X174 is not pathogenic virus to human beings, but it can be used to mimic pathogenic viruses to human beings.

3.3 Lysis

Lysis refers to lysis or destruction of whole bacterial cells.

NOTE: in this test method, lysis of Escherichia coli (as host cell) is caused by Phi-X174 invasion.

3.4 Plaque

Plaque refers to a clearly visible area theoretically (virology) formed by a single live virus's infection and lysis of host cells.

NOTE: in this test method, plaque refers to a clearly visible area in the colonies of E.coli C on the agar layer. Theoretically speaking, it is the result of a single live Phi- X174's infection and lysis of bacteria.

3.5 Plaque-forming Unit

Plaque-forming unit refers to plaque-producing virions through the infection and lysis of bacteria on the upper layer of agar.

3.6 Surrogate Microbe

Surrogate microbe refers to mode microbe used to mimic pathogenic microbes.

NOTE: in this test method, surrogate microbe refers to bacteriophage Phi-X174.

3.7 Viral Filtration Efficiency

Viral filtration efficiency refers to the percentage of virus suspended particles being filtered by test sample at a specified flow rate.

4.1 Test Principle

Make aerosol (with a certain virus concentration) pass through a sample at a certain flow rate. Through the determination of the number of viruses in the aerosol before and after penetrating the sample, calculate the viral filtration efficiency of the sample.

4.2 Instruments and Test Reagents

4.2.4.1 The formula of bacteriophage nutrient broth (Phi-X174) is as follows:

4.2.4.2 The formula of the lower layer of agar is as follows:

4.2.4.3 The formula of the upper layer of agar is as follows:

4.2.4.4 0.1% sterile peptone water.

4.2.4.5 Bacteriophage Phi-X174 (ATCC 13706-B1), whose concentration shall at least be 1.0 x 108 PFU/mL.

4.2.4.6 Escherichia coli (ATCC 13706).

4.2.1 Sampler

AGI-30 liquid impact sampler (2 samplers in each channel), which is able to endure a maximum flow impact of 12.5 L/min.

Tryptone 8 g Potassium chloride 5 g Calcium chloride 0.2 g Add water to 1,000 mL At 121 °C, after conducting pressure steam sterilization for 20 min, pH value is 7.3 ± 0.2.

Agar 15 g Nutrient broth 8 g Potassium chloride 5 g Calcium chloride 0.2 g Add water to 1,000 mL At 121 °C, after conducting pressure steam sterilization for 20 min, pH value is 7.3 ± 0.2.

Agar 7g Nutrient broth 8 g Potassium chloride 5 g Calcium chloride 0.2 g Add water to 1,000 mL At 121 °C, after conducting pressure steam sterilization for 20 min, pH value is 7.3 ± 0.2.

h) Use 0.1% sterile peptone water to dilute the bacteriophage culture solution to the concentration required for tests, so as to prepare bacteriophage challenging suspension liquid.

4.4.2 Test procedures

Add a proper amount of bacteriophage challenging suspension liquid for detection into the fluid container of the aerosol generator. The concentration of bacteriophage is controlled to be around 1.0 x 108 PFU/mL through the method described in 4.4.1.

Before each test, use purified air to fill the whole system for around 45 s, so as to balance the air channel. Then, activate the aerosol generator; set the time of delivering the bacteriophage suspension liquid to the atomizer to be 1 min; set the run time of air pressure and the sampler to be 2 min. Respectively add 20 mL of 0.1% sterile peptone water to the two AGI-30 liquid impact samplers to collect bacteriophage aerosol; form test solutions of the sample group and the positive control group. The positive control value of the sampler is calculated in accordance with the method in 5.1; it shall be not less than 106 PFU, otherwise, the concentration of bacteriophage shall be adjusted.

Respectively repeat the test of the sample group and the positive control group for 3 times.

4.4.3 Quantitative test of test solutions

Conduct gradient dilution of the test solution in the sample group to 10-3; conduct gradient dilution of the test solution in the positive control group to 10-7. Adopt the following procedure to quantitatively test the number of bacteriophages in the test solution (obtained in 4.4.2) after the dilution:

a) Transfer 2.5 mL of molten sterile upper layer of agar culture medium to a sterilized test tube; maintain the temperature of the upper layer of agar culture medium at (45 ± 2) °C;

b) Remove the test tube that contains the upper layer of agar culture medium away from the heat source; promptly add 0.5 mL of the diluted test solution, so as to prepare inoculation tubes;

c) Add 100 µL of Escherichia coli culture, which has been placed overnight, to each inoculation tube;

d) Thoroughly mix the test tube; pour it onto the surface of the plate of the lower layer of agar culture medium;

e) In terms of test solution collected from each test sample and control sample, prepare 2 plates (diameter of the plate: 90 mm);

f) Solidify agar; cultivate it at (36 ± 1) °C, till plaque that is clearly visible to the naked eye is generated (generally, around 3 h ~ 4 h);