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.
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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I recall decades back trying to do “bottom-less” plating (as well as plate lysates, though the latter with the bottom agar in place). Just as they note in the article, it makes good sense. It didn’t work out well, though, and I didn’t have access to this article (unfortunately), so perhaps that’s part of the reason why. But that’s great that they are working out for you!
The article itself isn’t unknown, but according to Google scholar has only been cited 12 times over the past 50 or so years, and only three times according to PubMed, so perhaps deserves to be better known. 🙂
(http://scholar.google.co.jp/scholar?hl=en&q=Bacteriophage+plaque-count+assay+and+confluent+lysis+on+plates+without+bottom+agar+layer&btnG=&as_sdt=1%2C5&as_sdtp=)
(http://www.ncbi.nlm.nih.gov/pubmed/14098484)
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Oh my goodness! Awesome article dude! Thank you so much, However
I am going through issues with your RSS. I don’t know the
reason why I cannot subscribe to it. Is there anybody else having
similar RSS issues? Anybody who knows the answer can you kindly
respond? Thanks!!
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