Sampling for viruses in drinking water

Question 1

If water is chlorinated, what is needed to stop the process?

Answer 1

Sodium thiosulfate: neutralizes the chlorine

To stop chlorine from killing viruses so the concentration can be counted in the sample

Question 2

how is a sample of drinking water collected?

Answer 2

collection filter, connect hose, pass water through filter, pathogens stick to filter, bring filter into lab to count

Question 3

how do positively charged filters work?

Answer 3

viruses (capsid=protein=amino acid) in basic pH are negatively charged

COO-

NH2

Question 4

How are viruses removed from positively charged filters?

Answer 4

add beef extract of pH 9.5 to cause filter to become negatively charged

-negatively charged viiruses detach

Question 5

how do negatively charged filters work?

Answer 5

add HCl + water + virus

-under go protonation

= COOH, NH3+

-filter through neg filter

just in case add Mg2+, Ca2+ (divalent cation) to act as a bridge in case not all are protonated

want pH not to acidic or basic

dont want viruses to die

Question 6

removal (elution) from negative filter

Answer 6

add beef extract pH 9.5, viruses become negative

Question 7

process after 1L sample after filter

Answer 7

add acid to decrease pH to 3.5

organic material (with viruses attached) precipatates

centrifuge

viruses pellet out in centrifuge tube

resuspend pellet in buffer

Question 8

ways to analyz sample after concentrate

Answer 8

cell culture

PCR

Question 9

what are the two methods of cell/tissue culture

Answer 9

plaque assay (quantitative)

cytopathic effects (observes whether present or not)

Question 10

process of plaque assay

Answer 10

grow sample in single surface , nutrient agar on top of cells, when viruses release from cell, attach onto adjacent cell -> reproduce, spread

dead cells=clear area.

stain to see dead cells- white dot=plaque

1 pfu= 1 virus

Question 11

advantages of cell culture

Answer 11

detects only ineffective particles/capable of infecting

ability to quantify viruses pressent in sample

highly sensitive: sample+cells=replication

entire sample can be analyzed

Question 12

disadvantages of cell culture

Answer 12

detects only viruses that grow in cells

need to know what virus looking for, so know which cells to infect (virus coresponds to specific cell)

relatively nonspecific- can only detect general type of virus

up to 2-4 weeks for analysis

cost of anaylysis

• one virus for one cell