Overlay plate isolation

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Acidophilic species that are cultivated by the overlay technique
Acidophilic species that are cultivated by the overlay technique


Movie showing Overlay plate isolation


‘Overlay’ Solid Media

Pyruvate etc. formed due to hydrolysis of agarose at low pH

Acidophiles very sensitive to organic acids, especially autotrophs e.g. Acidithiobacillus spp., Leptospirillum spp.

1990’s – BART – noticed that autotrophic colonies could be found growing in proximity to heterotrophic colonies on solid media

Devised ‘Overlay’ solid media to allow cultivation of many organisms previously uncultureable on solid media (Johnson, D. B. (1995). Selective solid media for isolating and enumerating acidophilic bacteria. Journal of Microbiological Methods 23: 205-218.)

Uses heterotrophic Bacteria to ‘detoxify’ the solid media

Selective solid media

Plate Target organisms pH
Inorganic iron overlay (iFeo)

(FeSO4 (25 mM))

Iron-oxidisers (autotrophs) ~2.6
row 2, cell 1 row 2, cell 2 row 2, cell 3
row 2, cell 1 row 2, cell 2 row 2, cell 3
row 2, cell 1 row 2, cell 2 row 2, cell 3
row 2, cell 1 row 2, cell 2 row 2, cell 3
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