Basic endophyte isolation – testing the lower limits of aseptic technique

In the quest to isolate new endophytic fungi and build a strain library for screening, one of the main issues we face is still being unable to afford a laminar flow hood.  Sterility is a big concern in endophyte isolation, as it is very important to assure that any microorganisms found in the petri dish are coming from within the plant tissue, not on it, or outside of it.  A quick google search will return many home-built laminar flow hoods as well as more simple versions such as a “sterile hood” which consists of a fish tank or plastic container turned on its side and cleaned thoroughly with bleach/isopropyl alcohol.

In this experiment, we wanted to test the bare minimum (ie, no laminar flow hood, no make-shift sterile hood) and see if we could get a control with no growth and samples that looked like endophytic fungi were growing from them.

Samples were taken from:

English Ivy http://en.wikipedia.org/wiki/Hedera_helix

Rhododendron http://en.wikipedia.org/wiki/Rhododendron (only the fresh leaf was plated, the damaged batch of small leaves and stems were discarded)

Ginko http://en.wikipedia.org/wiki/Ginkgo and a few leaves with small branches/stems were selected from each.

Samples were washed for 30s under tap water, then cut into small sections and placed in 5% sodium hypochlorite for 30s, followed by 70% ethanol for 30s, followed by two sterile water washes for 30s each.  100uL of the final water wash was plated as the control.

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Afterwards, samples were plated on PDA (potato dextrose agar) supplemented with chloramphenicol to inhibit bacterial growth,and left in a dark cabinet at room temperature after being sealed with parafilm.

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Fast forward a week and here are how the plates look

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We’ll work on a follow up post on how the samples were re-plated via hyphal tipping to try to get pure cultures, but the good news is, the control doesn’t have any growth!  Also, on the Rhododendron leaf stem (the last two pictures) you can see the white mycelium growing out of the vascular tissue, pretty cool! In some of the cases, like leaving the outer bark on the ginko stem, contamination from epiphytes/ectophytes will occur, but it is still interesting observing the organisms grow.

There was at least one yeast colony on one of the ginko leaves, and a few samples were selected to re-plate to try to get pure cultures.  Ideally, we’d be observing the growth of the organisms daily and taking note of their growth rates, coloration, and other features, but we both have 9-5’s and sometimes cannot always get to the lab every day.  The Ivy sample was interesting to look at, but mostly discarded for further isolation because of the density of growth of the organisms.

Needless to say, it seems that with a little bit of practice and a very crude aseptic technique, you can isolate some endophytes of your own.

 

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