My phage belonged to P.aerugionsa PAO and was isolated from sewage. Pure phage picked up from middle of plaque and dropped into 200 microliter Cacl2 and 30 microliter chloroform. Phage transferred at cold chain for 12hrs to TEM Lab, but there is not any phage in micrograph?
Have the phage suspension shipped back from the TEM facility and titrate it, to check whether the phage is really 'dying'.
In such a case I would use, as controls, two or three well charcterized other bacteriophage samples (e.g. T4) prior to and after transportation, and apply the identical procedure. If these bacteriophages are detectable by standard negative staining investigatioin after transportation, it might be a matter of too low concentration of well preserved bacteriophages in your sample already prior to transportation. Even when bacteriophages are damaged by transportation I would still expect remnants of bacteriophages.
What the original titer was? Frequently microscopists fail to see the phage if there is not enough of it. Especially this happens with non-purified things due to poor adsorption to the grid. The direct visualization from the plaque seldom works well even if you do it with the fresh plate. Another point% CaCl2 alone may be quite alkalic solution. So try to grow higher titer stock, concentrate it on centrifuge at 70-100 Kg and resuspend in SM or similar buffer.
I used Cacl2 and Mg2so4 without chloroform for transport Siphoviridae and Povodiridae phage in respectively and both of them were alive and had proper micrograph.
Thanks a lot
My guess is you have too low concentration of and intact phage. Try to concentrate your phage using any of the availbale methods, let´s say precipitation with PEG, and after that you should have better luck with TEM. CaCl2 is used for a better adsorption of phage to the bacterial cell.
- Badges
- Science method