I have a Project i am trying to test out using DSMZ Bacteriophages and S. Aureus (Host), but it doesn't seem to work.
I've done multiple Tests but i can't seem to get any Plaques using Double layer Plates.
So far i have identified a couple factors that might be the problem:
1. I use a TSB Medium with 2mmol CaCl2 (maybe the Concentration is too low ?)
2. I let my Host grow till it gets to the exponential phase with an OD of 0.2-0.3 (might be too low of a bacterial count)
3. the temperature of the Top agar i use is around 45°C (might be too hot, but lower than that and it hardens too fast)
4. the Ratio i mix the Host Bacteria is 200µl of Host and 100µl of Phage solution.
I'm new to this method and i am Thankful for any Tips and troubleshooting Ideas you might have
Try the single layer variant of the method where you inoculate a plate with your bacteria to make a confluent lawn and then add your phages in 100-200 microlitres of buffer and distribute using a spreader while trying to avoid disturbing the lawn as much as possible. This can be a positive control to make sure your phages are able to infect your host. Also plaque assays only apply to lytic phages so make sure your phage strain does not need induction to become lytic or is a species that is shed without killing the host.
You might dot your phage sample directly to the bacterial lawn. That will also let you find out the susceptibility of many phage samples with a bacterial host. I think the temp and o.d. are all right. You can check if you see the growth of bacterial lawn.
Are you sure that you have phage in your stock?
Are you making appropriate dilutions so that you would see individual plaques if the phage titer was high?
Are you sure this phage will actually infect and replicate in this host strain?
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