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1, high-level biosafety laboratory transfer window characteristics and basic structure.

With the gradual development of the field of biosafety in China, the construction of biosafety laboratories is increasing, and the application fields of transfer windows are expanding, and specific requirements are put forward for the relevant national standards of transfer windows. GB19489-2008 "General Requirements for Laboratory Biosafety" puts forward specific requirements on the structural pressure-bearing capacity, airtightness, disinfection and sterilization of the transfer window in biosafety level 3 and level 4 laboratories: the structural pressure-bearing capacity and airtightness of the transfer window shall meet the requirements of the area where the transfer window is located, and have the conditions for disinfection and sterilization of the items in the transfer window. According to the provisions of JG/T 382-2012 transfer window industry standard, according to the use function is divided into basic type, purification type, disinfection type, negative pressure type, airtight type.

According to the protection level of high-level biosafety laboratory and whether to carry out animal living operation experiments, it is very important to choose the transmission window with reasonable function to prevent the leakage of pathogenic microorganisms. At present, the transfer windows of BSL-3 and ABSL-3 laboratories built in China are generally airtight (E1) and sterilized (C1, C2), while the transfer windows used in BSL-4 and ABSL-4 laboratories are generally airtight (E2) and sterilized (C1, C2). Taking BSL-4 and ABSL-4 as an example, the basic structure and function of the transmission window are as follows:

(1) Material: The sterilizable transfer window and inner cavity are made of 316L stainless steel with the required thickness, other parts can be made of 304L stainless steel with a thickness of 2mm, and the embedded frame can be made of 304L or 316L material.

(2) Airtight chamber: The inner cavity has the function of disinfection and is made of 316L stainless steel with the required thickness. When the pressure attenuation method inside the box is used for detection, when the pressure inside the box reaches -500Pa, the natural attenuation of negative pressure within 20min is less than 250Pa.

(3) Airtight door: there should be supporting mechanical and electronic interlocking. The window on the door is made of explosion-proof and fireproof glass with a material thickness of 5mm or more. When passing items, only when one side door is closed, the other side door can be opened. Door optional mechanical compression door or inflatable air-tight door.

(4) viewable window: the inner cavity is integrally formed, the weld seam is polished, the polishing degree is less than 0.4 μm, and the corner is smooth.

(5) UV lamp: the temperature can be -10 ℃ ~ +80 ℃; anti-corrosion, waterproof; no deformation within the working pressure range.

(6) Mechanical and electronic interlock: 3 sterilization lamps, which can sterilize and weld flush on three sides of the transfer window. The doors on both sides of the sterilizable transfer window cannot be opened within the set disinfection time, and the set time starts from the opening of the ultraviolet lamp.

(7) Pre-buried frame: Pre-buried frame can be made of 304L or 316L material. Due to the high requirements of biosafety laboratories, 316L stainless steel is recommended.

(8) fumigation disinfection unit: configure the standard interface with the international high quality hydrogen peroxide gas generator, dry gas disinfection. No need to reserve ventilation system, no corrosion and condensation. Configuration of 316L material sealing valve.

2. Influencing factors of sterilization effect

2.1 time

2002 disinfection technical specifications for the environmental conditions of ultraviolet disinfection are stipulated: indoor temperature should be controlled at 20 ℃ ~ 40 ℃, indoor humidity should be less than 60%. Indoor should be kept as dry as possible. And to ensure that the surface of the UV lamp without dust, no oil. The UV lamp should be wiped weekly with an alcohol cloth. The irradiation time of ultraviolet air disinfection is usually set to 30min ~ 60min. When the object with rough surface is irradiated with ultraviolet light, the irradiation time should be increased to ensure that both sides of the object can be irradiated.

2.2 irradiation intensity

According to the requirements stipulated in the Technical Specification for Disinfection, the irradiation intensity of the new high-intensity ultraviolet lamp should be greater than 180uW/cm3(253.7nm, 1m away), and the radiation intensity of the new ordinary ultraviolet lamp tube greater than 90uW/cm is qualified. The radiation intensity of the lamp tube in use should reach a minimum of 70uW/cm, which can be used temporarily, but the irradiation time must be extended. If the intensity of the ultraviolet light source is lower than 40uW/cm, the prolonged irradiation time cannot achieve satisfactory sterilization effect, that is, the use should be stopped. Because of the attenuation effect of the ultraviolet lamp irradiation intensity, the sterilization effect cannot be judged by whether the ultraviolet lamp is bright or not.

2.3 ambient temperature

the ambient temperature has a certain effect on the radiation output of the UV lamp, the ambient temperature of the biosafety laboratory is usually 22 ℃ ~ 26 ℃. Studies have shown that the temperature of the ultraviolet light source is about 40 ℃ when the ultraviolet output is the strongest. As the temperature decreases, the ultraviolet output is relatively reduced: and the temperature is too high, the ultraviolet radiation is absorbed and the output is also reduced. The output intensity of the lamp at 4 ℃ is only 20% ~ 30% of that at 27 ℃. When the temperature decreases, the bacterial resistance increases and the ultraviolet sterilization effect weakens. When the temperature is lower than 0 ℃, the sterilization effect is lost and the disinfection effect is affected. Therefore, for biosafety laboratories, according to the appropriate temperature for humans and animals, the ambient temperature should be controlled at about 24 ℃ ~ 26 ℃. At this time, the ultraviolet output of the ultraviolet lamp is also moderate.

2.4 ambient humidity

Similar to ambient temperature, ambient humidity is also one of the factors affecting the radiation output of UV lamps. Ultraviolet rays can be absorbed by water molecules in the air, so when the ambient humidity is high, the water molecules absorb a part of the ultraviolet rays, thereby reducing the disinfection effect. The research shows that when the ambient temperature is 24 ℃ and the ambient relative humidity is 35% ~ 85%, the radiation intensity of the ultraviolet lamp is inversely proportional to the moisture content of the air. Biosafety laboratory environmental humidity requirements of 40% to 70%. Therefore, according to the suitable humidity of human and animal, the ambient temperature should be controlled at 40%. At this time, the ultraviolet output effect of the ultraviolet lamp is also relatively good. And when the humidity is high, the irradiation time of the ultraviolet lamp should be increased.

3, the transmission window irradiation intensity verification method.

1) Determination of irradiation intensity: the voltage value is 220V, the ultraviolet lamp is turned on, and the measuring position should be 1.5m below the lamp tube. The irradiance value was measured using an ultraviolet intensity with a central wavelength of 253.7 nm.

2) For ordinary type or low ozone type straight tube ultraviolet lamp (30W), the measurement position should also be placed 1.5m below the lamp tube. It shall be replaced when its irradiance value is in accordance with Table 6.

3) Irradiation dose: For the object to be sterilized, the surface must be completely sterilized to meet the required irradiation dose. If the irradiation dose is insufficient, the expected sterilization effect may not be achieved.

4) Exposure dose calculation:

be calculated from the irradiation dose.

4. Determination of the efficacy of bacteria and their spores and fungi.

1) Preparation of bacterial solution (Table 2)

2) Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans culture, the bacteria liquid for viable count.

3) Place the sterilization carrier flat in the sterilization plate, drip each carrier with quantitative bacterial suspension, coat evenly, and put it in an incubator at 37 ℃ for drying.

4) Place the bacteria solution on 16 slides and place them in a sterilized plate. The UV lamp was turned on and the slides were irradiated after 5min. Placement position is 1.5m below the lamp. At 15min, 30min, 45min and 60min, 4 contaminated slides were taken out respectively, and placed in 5ml eluent test tube, shaking 80 times.

5) After appropriate dilution, take 1ml of eluent and pour it into a flat plate. Each stained slide is inoculated with two. Bacteria are cultured at 30 ℃ ~ 35 ℃ for 48h for viable count, and fungi are cultured at 23 ℃ ~ 28 ℃ for 72h for viable count.

6) Positive control, the bacteria solution was placed on 4 slides, and placed in a 5ml eluent tube, and shaken several times. Without UV lamp irradiation, they were cultured according to step 5.

7) Calculate the kill rate

8) judgment standard: the killing rate of indicator bacteria ≥ 99.9 is judged as qualified.

5. Inspection requirements for suppression

to the air tightness requirements of the transfer window, the air tightness test shall be carried out by pressure attenuation method under the condition that the initial pressure of the transfer window cavity is -500Pa, and the air pressure naturally attenuated within 20min shall not be less than -250PA.