Validated GMO inactivation

(versionFR)

In order to more formally validate the usual GMO inactivation done with bleach for disposal of plates and cultures, we are adding this page. This validation was performed with two brands of commercial bleach and is applicable to both GMO bacteria and nematode worms transformed with antibioticR plasmids. It is directly specific for this SOP of the lab collection.

As always, precautions in using bleach and appropriate personal protective equipment (lab coat, gloves, and eye protection) are an essential part of this inactivation method.

The big white tray - labelled 'clean up' - was used, as usual for all manipulations.

The validation for bacterial plates was done upon bacteria transformed with a pJAMA-GFP plasmid, which make bright green colonies...

A fresh 2ml culture was used to make three plates of cultures on growth media carrying the selective antibiotic, ampicillin (LB-amp100 plates). A glass spreader was used - from a bent pasteur pipette, to distribute the following liquids.

1, with 100ul of culture (full lawn)

2, with 25ul of culture (less bugs)

3, with 5ul of culture (still too many to count)

These are shown below:

Plates used for bacterial colony validation

fluorescence of pJAMA-GFP colonies for inactivation test

Our 'DIY illuminator' with bright blue LEDs, and an amber filter (held in front of a phone camera) was used to get the fluorescence image.

Then, the plates were treated with bleach in the big white tray.

test of inactivation - bleach treatment

Three conditions of inactivation for this validation experiment were run: full strength bleach, for plate 1, 1/10 bleach in water for plate 2, and water only for plate 3, with the least colonies.

5ml of each solution was added to the appropriate plate, incubated for 5min and then swirled, as shown below, and incubated 5 more minutes.

swirling liquids on the plates after first incubations

Then, after waiting 5 more minutes, a couple of mls of the liquid from each plate was recuperated into small conical tubes (15ml) with sterile serological pipets and the old Drummond pipet-aid. On plate 1, all colonies appeared fully dissolved by the undiluted bleach, but plate 2 with 1/10 bleach, and plate 3, with only water, still had intact colonies, as shown.

After treatment, plate 1 colonies were dissolved away by the bleach.

After treatment with 1/10 bleach, colonies were still visible.

Colonies were still well visible after treatment with only water.

From the recuperated liquids, three more fresh plates were made by inoculating 50ul of each liquid taken after the treatment, as shown.

Fresh inoculations were made to new plates in triplicate from each of the liquids after the treatment.

After all that, a repeat of the inactivation of all three initial plates, as per the SOP, with 100% javel put also on plate 2 and 3 was performed (esp as it was clear even the 1/10 solution is not dissolving the colonies, like the full strength one. The liquids were inactivated with fresh bleach for disposal, and the 'clean up' tray was carefully washed in the big sink, as usual (around back, in the intermediate Polymeka space).

The 9 new plates were incubated overnight at 37^oC and checked for growth.

In the end, the experiment validated our ordinary inactivation method very well but provided some surprises.

No colonies were found on the new triplicate plates from either the 1st or 2nd treatment conditions.  That condition 2 worked so well, also giving no colonies on these secondary plates, as the original colonies did not dissolve with 1/10 javel, was quite a surprise. (In terms of reducing risk, however, it still seems better to use 100% bleach, as in the SOP.) Only the water treatment gave growth on the new plates.

Here is an image of all 9 plates, in ordinary light.

All the secondary plates, after incubation overnight.

Here is the fluorescence image (stacks of 3 plates).

fluorescent colonies were only found from the culture treated with water only.

No colonies were seen on these secondary plates even from liquid recuperated from the plate treated with 1/10 bleach.

We note in the experiment that we could also have tried to recuperate plasmids from the treated liquids, but that further test would only provide very useful information if very good competent cells were used. (sort of a waste)

No new colony growth even for the 1/10 treatment seemed quite satisfactory...

inactivation as usual, at the end

Finally, all secondary plates were inactivated as usual, according to the SOP, for proper disposal in the ordinary trash (one last picture here).

A similar validation was performed with transformed C. elegans 'worm' cultures, to be put on a new page.