Study On The Bio-aerosol Risk Assessment Of The Experimental Operation Of Pathogenic Microorganisms

Since 2003, with SARS, avian flu, H1N1 influenza outbreaks, testing and researching of pathogenic microorganisms in the laboratory are increased. Many laboratories were erected to face the need. The laboratories were engaged in human and animal contagious microorganisms such as bacteria, viruses and other microorganisms. If the preventive measures were ineffective, not only the laboratory staff but also the socity people may be leaded infectious diseases. The ordinary and accidental lab work of pathogenic microorganisms will produce a large number of infectious biological aerosols. According to statistics, the infectious diseases ratio of the staff of pathogenic microorganisms lab is 5 to 7 times higher than that of the general population. Therefore, the risk assessment of biological aerosols produced by experimental operation of pathogenic microorganisms is especially important.Objective: Using noninfectious bacteria, viruses to replace infectious bacteria, viruses. The biological aerosol risk of ordinary and accidental lab work of pathogenic microorganisms were studied to select the appropriate protective measures and methods.Content:1. Bacterium Serratia marcescens and phageΦX174 aerosol characters were studied to substitute bacterial and viral infectious diseases.2. The bioaerosol risk of ordinary and accidental laboratory research work were studied.3. Quantitative of phageΦX174 aerosol using SYBG GreenⅠreal-time QPCR was studied.Methods1. The damnification of Serratia marcescens and phageΦX174 in different medium using DV40 and BGI generator (10L/min flow) were studied. (Different liquid medium: nutrient broth, PBS, sterile saline) the generating time was 10min, to decided to use which liquid media. The indicator microorganisms survival rate according to the aerosol generating tine was studied (Aerosol generating 0min, 5min, 10min, 15min, 30min).2. The distribution of particle spectrum of BGI nebulizer were studied using Andersen sampler.3. Aerosol production quantity of the different generators were studied.4. The damage of AGI-30 sampler in different sampling media at the same time was studied (nutrient broth, PBS, sterile saline, which decided to use the media). The damage of AGI-30 sampler using the same sampling medium at different times was studied(0min, 5min , 10min, 15min which decided the sampling time).5. Bioaerosol risk of ordinary and accidental lab work of pathogenic microorganisms were studied in negative pressure laboratory with high concentrations of bacteria and virus (beat upon mix experiment, centrifugal experiments, accidental broken flask of high concentrations culture,Ⅱbiological safety cabinet leakage ) experiment, Anderson six sampler and settlement plate were used to take the bioaerosol. Aerosols risks were obtained under the various experimental operations.6. PhageΦ174 QPCR standard curve was established using SYBG GreenⅠ, the detection of specificity and sensitivity was studiJ ed and was applied to quantifying aerosol samples.Results1. Damage of different liquid nebulizing media of Serratia marcescens and phageΦX174 were stdied. The results was statisticed that the medium of PBS was the highest survival rate. There were no significant differences between the two generators, and there were significant differences of the survival rate at different times. PBS had the best impact resistance when AGI-30 sampler collects the aerosol.2. Bioaerosol risk of ordinary and accidental lab work of pathogenic microorganisms in simulation experimental conditiones were as below: In beat upon mix experiment the highest bacterial aerosol concerntration was 1823 cfu/m~3 , viral aerosol concerntration was 1852 pfu/m~3. Accidental broken flask of high concentrations culture can produce the highest bacterial aerosol concerntration was 9326 cfu/m~3 , viral aerosol concerntration was 9337 pfu/m~3. In centrifugal experiments the highest bacterial aerosol concerntration was 138 cfu/m~3 , viral aerosol concerntration was 134 pfu/m~3.The experimental results were very important to the risk management.3. The testing methods of evaluateing the biological safety cabinets protection against bacterial and viral aerosol were erected using Serratia marcescens and phageΦX174.4. PhageΦX174 traditional method for quantitative detection was double-layer agar culture method, counting from the cultivation of the host bacteria to the double-layer agar culture, the whole process needs 12-16 hours. Fast detection for phageΦX174 aerosol is of great significance. Fluorescent dye SYBR greenⅠmethod is simple, cheap. Ct value (Y) and number concentration lgN (X) the linear equation: Y =-3.38X +41.44, error is 0.014, the correlation coefficient was -0.999. The results of aerosol samples detected by QPCR was 32.62 times higher than double agar counting method, QPCR results was significantly higher than culture results. In fresh liquid culture sample of phageΦX174, QPCR method and culture method had good compliance. QPCR detection sensitivity was 101pfu / reaction. QPCR method can greatly improve the speed, from the collection of samples to obtain test results as long as 3-4 hours.ConclusionIn this study, Serratia marcescens 8039 and phageΦX174 aerosol characteristics were studied. Quantitative bioaerosol risk of ordinary and accidental lab work of pathogenic microorganisms were studied in negative pressure laboratory with high concentrations of bacteria and virus. The testing methods of evaluateing the biological safety cabinets protection against bacterial and viral aerosol were erected using Serratia marcescens and phageΦX174. QPCR Detection method of PhageΦX174 aerosol was established.