Question

Suppose you want to isolate viruses that infect marine cyanobacteria. Describe how you would do that compared to using the kinds of agar plates you were exposed to in this first laboratory session. Describe what parts of the procedure specifically must be changed. Would you need to consider incubation temperature differently? What kind of medium would you use to grow your cyanobacteria and phages?

Answer 1

Water samples containing the cyanophages are collected and must be filtered through a 0.2μm filter to remove bacteria from the samples. Then the water must be kept at 4 degree C in the dark. The basic principle of isolating viruses are that the viruses attach to a suitable host and then lyse the cell creating more number of progenis. This can be done by a plaque assay where host cells are lysed forming plaques and the budding viruses diffuse through the agar. The other method is to use a liquid medium in microtitre plate where the whole culture will get lysis as there is no agar to inhibit the progeny viruses. This well assay avoids the need to culture cyanobacteria on agar. Cyanobacterial cells are taken and added to the well followed by addition of the water samples A negative control with no water samples is taken. The samples are incubated at 25 degree C for 7-21 days. The lysate from a well where cells have become completely lysed are taken and centrifuged. The supernatant is serially diluted and used for further well assays. This is repeated 2 or 3 times to get conal isolates. Cellulose broth medium can be used to grow thew cyanobacteria. Temperature should be similar ie. optimum temperature for the growth of the cells.