Are you sure that the bacterial host you are using for your lawn is an appropriate host for any phages you are trying to identify? Remember that phages are very specific for their host.

When you say your get bacterial growth in the phage zones, do you just mean the underlying lawn? Or are there new colonies coming from the sample spot, which would signal that your sample is not sufficiently sterilized.

I passed the sample through a 0.22 filter multiple times. my sample is sterile but I don't know which bacterium the phage is specifically for. i am trying the efficacy of phage obtained from feces on s.aureus

Phage are very strain specific, so you could have hundreds of different phages in your sample but if none are specific for your plating strain, then you will not see them and they will have no effect. Staph aureus is not typically found in the GI tract of humans (although it can be found occasionally) therefore you might not expect to find any Staph aureus phages in stool samples, or perhaps only very rarely.

So if your goal is to find S. aureus phages, you might want to test different samples (skin or nasal swabs for example), but if your goal is find phages in stool, you might want to try different host strains for your lawn.

Hi Bahar Öztürk,

I fully agree with Dr. Michael J. Benedik . It is hard to isolate S. aureus-specific phages from feces. Since this pathogen is not GI borne, it may prove very unyielding. Instead, you may try samples from skin, nares (nose), and some environmental samples.

However, I may want to tell you that S. aureus phages are very delicate in nature and yield very small and tiny phages that may be very hard to identify on a plate. Although these phages are lytic in nature, one may find bacterial growth in the background of these plaques. To avoid this, you should set the OD600 of your culture to 0.15-0.2 (not more than this), take 100ul of this bacterial culture, and mix it with an equal amount of your test sample (eg: sewage sample supernatant, centrifuged, and filtered). Add this 200ul mix to your soft agar and then overlay it onto your plates. Also, for dealing with SA phages, use Nutrient broth and Nutrient agar and not nutrient-rich media like BHI broth/agar or MH broth/MH agar. These fastidious media will enhance bacterial growth and reduce the visibility of your plaques (if they're in the sample). Nutrient broth/agar allows regular bacterial growth. This comes from my own experience while handling SA phages. You should also incorporate Calcium chloride (~2-5mM) in your media (broth and agar) and may also incorporate antibiotics to increase the size of your plaques. See this: Methicillin-resistant Staphylococcus aureus phage plaque size enhancement using sublethal concentrations of antibiotics. Applied and environmental microbiology

I wish you Good Luck!

Bacterial growth occur on phage plate due to strain specifity and O.D culture.