Going Bottomless in the Plaque World

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

Bottom agar = Hard agar = Solid media

Top agar = Soft agar = Sloppy agar = Semi-Solid media.

I’m embarrassed to say that I’ve had this article in my reference database since August of 2007: Rizvi, S., Mora, P.T. (1963). Bacteriophage plaque-count assay and confluent lysis on plates without bottom agar layer. Nature 200:1324-1325. It is only today, however, and apparently as a form of avoidance behavior (there’s a talk I’m supposed to be working on), that I’ve obtained the reprint and set out to read it.

Their second sentence reads, “Having spent considerable time on preparation of ‘bottom’ agar plates for the agar layer assay by the plaque count method and for high-titre bacteriophage stock preparation on a large number of plates by the confluent lysis method…” And thus they are off striving to do something about this by investigating “…the possibility of saving time by using plates without ‘bottom’ agar for assay and stock preparation.”

The media they used for their ‘bottomless’ agar consisted of the following:

  • 10 g Difco bacto-casamino-acids (acid hydrolysed casein)
  • 15 g Difco bacto-nutrient broth
  • 10 g Sucrose
  • 1g Dextrose
  • 5g Crystalline magnesium sulphate
  • 5 g Sodium chloride
  • 8g Agar

Efficiencies of plating in testing phages T1 through T7 they found to be essentially 100% for T1, T3, and T7, and basically 50% for the rest. To the extent that my interpretation of the ‘smudges’ provided in Nature’s PDF can be trusted, the per plate yields for confluent lysis phage preps were more or less the same with versus without bottom agar. Consistently for the latter they note: “The yields obtained on plates without ‘bottom’ agar were slightly better than the yields obtained on plates with ‘bottom’ agar.”

They also note that, “Confluent lysis can be adapted for large-scale bacteriophage production by carrying it out on large stainless steel trays.”

Historical Referencing:

These authors also cite four, mostly Mark Adams-dominated publications for plaque count method (first three) and confluent lysis stock preparation (last). These are:

(1) Gratia, A. (1936). Des relations numeriques entre bactéries lysogenes et particules de bacteriophage. Ann. Inst. Pasteur, 57:652-676.

(2) Adams, M. H. (1950). Methods of Study of Bacterial Viruses. (Methods in Medical Research, 2:1) The Year Book Publishers, Inc., Chicago.

(3) Adams, M. H. (1959). Bacteriophages. Interscience Publishers, Inc., New York.

(4) Swanstrom, M., and Adams, M. H. (1951). Agar layer method for production of high titer stocks. Proc. Soc. Exp. Biol. and Med. 78:372-375.

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3 thoughts on “Going Bottomless in the Plaque World

  1. I have been doing both my plaque assays (in 6 well plates, 2 ml of phage-host-0.4 agar mixture) and my confluent lysis phage production (8 ml concentrated host, 2 ml phage lysate, 10 ml 0.27% agar) without bottom agar and it has been working perfectly. Great to have a reference though 🙂

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  2. Oh my goodness! Awesome article dude! Thank you so much, However
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    Like

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