SEA-PHAGES | 2X smeg top agar

Link to this post \| posted 19 Sep, 2022 18:56
edoddmoh@uottawa.ca	Hi! Is there is a reason the smeg top agar is prepared at 2X, and then diluted to 1X? I'm wondering if I can prepare it as a 1L volume, autoclave, and then aliquot into smaller bottles (as with the PYCa top agar). We are phage-hunting with smeg for the first time this semester. Thanks! Liz

Link to this post \| posted 21 Sep, 2022 17:09
viknesh	edoddmoh@uottawa.ca Hi! Is there is a reason the smeg top agar is prepared at 2X, and then diluted to 1X? I'm wondering if I can prepare it as a 1L volume, autoclave, and then aliquot into smaller bottles (as with the PYCa top agar). We are phage-hunting with smeg for the first time this semester. Thanks! Liz Hi Liz, Part of the reason top agar is prepared at 2x is so that experiemnts that require the use or large samples of phage (in buffer) do not overly dilute the nutrient in the medium. For our general PHAGES protocols, you should be fine preparing top agar at 1x. 7H9 medium has lots of salts in it, and calcium chloride tends to precipitate out over time. For this reason, calcium chloride is only added before use. Even if you prepare 1x top agar, you might want to only add calcium chloride before use. If you do add it early, please let us know how long it takes for the precipitates to form so that we can share with others. Vic

Link to this post | posted 21 Sep, 2022 17:09

viknesh

edoddmoh@uottawa.ca
Hi! Is there is a reason the smeg top agar is prepared at 2X, and then diluted to 1X? I'm wondering if I can prepare it as a 1L volume, autoclave, and then aliquot into smaller bottles (as with the PYCa top agar). We are phage-hunting with smeg for the first time this semester. Thanks!
Liz

Hi Liz,

Part of the reason top agar is prepared at 2x is so that experiemnts that require the use or large samples of phage (in buffer) do not overly dilute the nutrient in the medium. For our general PHAGES protocols, you should be fine preparing top agar at 1x. 7H9 medium has lots of salts in it, and calcium chloride tends to precipitate out over time. For this reason, calcium chloride is only added before use. Even if you prepare 1x top agar, you might want to only add calcium chloride before use. If you do add it early, please let us know how long it takes for the precipitates to form so that we can share with others.

Vic

Link to this post \| posted 23 Sep, 2024 16:31
e.guevara	Hi, I too was wondering about the 2X 7H9 Top Agar as we have always prepared it as a 1X solution. We are currently working with smeg and have a reoccurring issue with our top agar appearing grainy while working with it in the lab. I thought perhaps the students were letting the top agar cool down (leaving it on the bench for too long), but I myself poured a couple of plates and found the same problem - grainy top agar. We prepare a 1X 7H9 top agar, autoclave and keep in the fridge until we need it. We then microwave the 1X 7H9 top agar and add the CaCl2 after warming and right before our lab. To clarify, these plates were for our first plaque assay where we added a direct (soil) isolation phage sample with our smeg. We are currently testing our smeg directly added to our L-agar plates (no top agar) to see if any grainy particles appear, in which case the grainy texture would be from our smeg. Has anyone had any similar problems with the 7H9 Top Agar? Thanks! Elena

Link to this post | posted 23 Sep, 2024 16:31

e.guevara

Hi,

I too was wondering about the 2X 7H9 Top Agar as we have always prepared it as a 1X solution. We are currently working with smeg and have a reoccurring issue with our top agar appearing grainy while working with it in the lab. I thought perhaps the students were letting the top agar cool down (leaving it on the bench for too long), but I myself poured a couple of plates and found the same problem - grainy top agar. We prepare a 1X 7H9 top agar, autoclave and keep in the fridge until we need it. We then microwave the 1X 7H9 top agar and add the CaCl2 after warming and right before our lab.

To clarify, these plates were for our first plaque assay where we added a direct (soil) isolation phage sample with our smeg. We are currently testing our smeg directly added to our L-agar plates (no top agar) to see if any grainy particles appear, in which case the grainy texture would be from our smeg.

Has anyone had any similar problems with the 7H9 Top Agar? Thanks!

Elena

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2X smeg top agar