SEA-PHAGES | Webbed plate hack for low-titer stocks and small plaques

Link to this post \| posted 26 Oct, 2024 14:52
nic.vega	If your students are still struggling with webbed plates, particularly for low-yield or small plaque phage, we've had good luck (1) increasing the incubation time of bacteria + phage before making plaque plates to allow a lysis cycle (I'm using 1h instead of 15 minutes with globi as host, your timing may vary) and (2) dropping the top agar concentration in PYCa to 0.2-0.3% by diluting with phage buffer - this also provides 2.5-5% glycerol, which can help increase plaque size (Santos, Sílvio B., Carla M. Carvalho, Sanna Sillankorva, Ana Nicolau, Eugénio C. Ferreira, and Joana Azeredo. 2009. “The Use of Antibiotics to Improve Phage Detection and Enumeration by the Double-Layer Agar Technique.” BMC Microbiology 9 (1): 148. https://doi.org/10.1186/1471-2180-9-148.) 73Kb

Link to this post | posted 26 Oct, 2024 14:52

If your students are still struggling with webbed plates, particularly for low-yield or small plaque phage, we've had good luck (1) increasing the incubation time of bacteria + phage before making plaque plates to allow a lysis cycle (I'm using 1h instead of 15 minutes with globi as host, your timing may vary) and (2) dropping the top agar concentration in PYCa to 0.2-0.3% *by diluting with phage buffer* - this also provides 2.5-5% glycerol, which can help increase plaque size (Santos, Sílvio B., Carla M. Carvalho, Sanna Sillankorva, Ana Nicolau, Eugénio C. Ferreira, and Joana Azeredo. 2009. “The Use of Antibiotics to Improve Phage Detection and Enumeration by the Double-Layer Agar Technique.” BMC Microbiology 9 (1): 148. https://doi.org/10.1186/1471-2180-9-148.)

Link to this post \| posted 19 Nov, 2024 00:44
mcclory	I just wanted to give a bump to this comment, as it was very helpful for our students working with G. rubripertincta phages this semester. The G. rub phages were consistently producing tiny, cloudy plaques that were barely perceptible to students when we were using standard PYCa top agar (we often only knew plaques were present from a spot test). We diluted our 25 mL PYCa top agar aliquots with 10 mL of PYCa media (for 0.29% agar) + 0.5 mL 30% glycerol (0.43%, I think this was a calculation error, but it worked so we continued with this after). I did a side-by-side comparison using a lysate one student was working with and got noticeably larger plaques. After that we had the whole class switch and all G. rub phages produced much more apparent plaques. Thanks for this tip.

Link to this post | posted 19 Nov, 2024 00:44

mcclory

I just wanted to give a bump to this comment, as it was very helpful for our students working with G. rubripertincta phages this semester. The G. rub phages were consistently producing tiny, cloudy plaques that were barely perceptible to students when we were using standard PYCa top agar (we often only knew plaques were present from a spot test).

We diluted our 25 mL PYCa top agar aliquots with 10 mL of PYCa media (for 0.29% agar) + 0.5 mL 30% glycerol (0.43%, I think this was a calculation error, but it worked so we continued with this after). I did a side-by-side comparison using a lysate one student was working with and got noticeably larger plaques. After that we had the whole class switch and all G. rub phages produced much more apparent plaques.

Thanks for this tip.

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Webbed plate hack for low-titer stocks and small plaques