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Biological and Biochemical Analysis of Bacteria and Viruses
Published in Christopher S. Cox, Christopher M. Wathes, Bioaerosols Handbook, 2020
Andreas Hensel, Klaus Petzoldt
The most common test system to determine infectivity of a given virus is the plaque assay. Monolayers of host cells are inoculated with appropriate dilutions of virus and to prevent the spreading of virus throughout the layer, after a short adsorption period, the culture is overlaid with a medium containing agar. After an appropriate incubation period, cytopathic effects start, usually focally, producing small plaques indicating infected cells. Theoretically, a single infective virus can induce one plaque-forming unit (PFU), but in most cases the ratio between the number of infective virus particles and the host cell is about 1,000:1. The main reasons for this phenomenon are (i) inhibition of viral replication caused by the agar overlay, (ii) aggregation of virus particles, and (iii) the presence of defective viral particles in the sample. When an air sample with unknown virus content has to be screened without focusing on a particular species, primary or secondary cell cultures should be given preference, because they allow a broad spectrum of virus species to multiply.79 Preferably, cell cultures derived from the host or related species should be utilized (see also Chapter 6). Rehumidification of the air sample is known to increase the number of PFU of some virus species.53 Infectivity assays to detect virus can be performed with collecting fluids, but in most cases large amounts of air have to be sampled before detectable numbers of infective virus particles can be provided for test performance.80 To prevent bacterial growth, 200 units of penicillin and 100 μg of streptomycin per mL may be added to the sample. Because collecting fluids are usually highly contaminated, the suspension has to be filtered through 0.45 μm and 0.22 μm pores. Aliquots are placed on cellular monolayers (limiting dilution of the sample may be done, if necessary) and incubated for 1 hour or more at 37°C to allow absorption of the virus particles. The collected specimens then may be removed or left on the cell culture, but fresh media should be added to the cultures. These should be observed for cytopathic effects following a 1 to 2 week incubation period. Most species of cytopathic viruses take 24 to 72 hours to induce the visible cytopathic effects (CPE), but this is a function also of the inoculated dose, while titration may be performed to evaluate the TCID50 (i.e., the dose required to infect 50% of the tissue cultures). Furthermore, for low initial virus concentrations in a specimen, up to 5 blind cell passages are recommended to achieve detectable morphological changes.
Short-range bioaerosol deposition and recovery of viable viruses and bacteria on surfaces from a cough and implications for respiratory disease transmission
Published in Aerosol Science and Technology, 2021
C. T. Wang, S. C. Fu, Christopher Y. H. Chao
In this work, real bacteria E. coli and virus bacteriophage (phage for short) were employed. We first prepared a 200 mL E. coli or phage solution with concentration of 107 colony forming unit (CFU)/mL or plaque forming unit (PFU)/mL. Then, we load the mixture of artificial saliva solution and the microorganisms into the cough generator for aerosolization. The air jet together with aerosolized droplets directly collided with and spread along the PVC plate surface. After droplet deposition, the PVC plate was cut into small pieces with size of 4 × 4 cm2 according to the pre-marked grid with sterilized scissors. Each small piece was further cut into two symmetrical pieces. One part was used to count the size-resolved bioaerosol by a microscope (Ni-E, Nikon, Japan). The other part was put into a tube with 5 mL sterilized water to extract the deposited E. coli or phage in water. Then, the pour plate method and top agar layer method were used to measure the viable E. coli and phage concentrations in water respectively and were described in detail in Section 2.4. The deposited viable E. coli and phage numbers on surface could then be calculated.