Report ValidTestsGMOinact

Validation report

Standard Operating Procedure for Inactivation of Genetically Modified Organisms

Abstract

These validation tests were performed in the Biosafety level 1 laboratory of the Association Hackuarium. Both bacterial and nematode cultures were shown to be effectively inactivated by exposure to undiluted commercial bleach, as used in the standard inactivation method. In contrast, water had no effect on subsequent growth of cultures. Interestingly, while 10% bleach solutions completely prevented further growth of bacteria (even though, visually on plates, not much obvious change was apparent), some nematodes were able to survive exposure to such diluted bleach. Therefore, continuing the recommended use of undiluted bleach by the standard method for inactivation of genetically modified organisms in this community laboratory setting is required for safe subsequent disposal in the general waste for incineration.

Here is the French version of this report.

Introduction

All biological waste (genetically modified or not) from community lab, Hackuarium is inactivated by immersion in an appropriate disinfectant (e.g. bleach) for safe disposal in the general garbage, which is destined for incineration, in Switzerland. Biosafety level I (or P1) organisms, in addition to any possible contaminants of cultures (e.g. molds) must always be inactivated for safe disposal. In order to validate the usual method of inactivation of genetically modified organisms (GMOs) in the P1 laboratory, comparisons were made to experimentally determine whether the standard method was sufficient to inactivate bacterial or nematode genetically modified organisms. Only if the method succeeds by such empirical evidence can it be considered validated.

The standard operating procedure (SOP) for GMO inactivation is specifically in regards to plate cultures. The validation experiments were done with commercial bleach (Coop 'Prix Garantie') and are applicable to both bacteria and nematode worms transformed with antibiotic resistant plasmids. The chlorine of bleach can rapidly destroy bacteria, viruses and fungi, and recent work shows it kills cells by becoming the acid hypochlorite, which even in quite low concentrations can make proteins unfold and then stick together.  As a gas, chlorine can be very toxic and also attack the genetic material of cells, so lab users must always make sure to have sufficient ventilation when doing this inactivation protocol on their cultures for safe disposal.

These validation tests use bacterial ‘colonies’ and ‘lawns’ as very sensitive measures of the efficacy of the method of inactivation. A bacterial ‘colony’ consists of a discrete spot of bacterial growth, which started from a single cell on the solid media of the plate, while a bacterial lawn comes from having so many bacterial cells initially put on the plate, that no individual spots can be seen. The concentration of bacteria put onto the plate with sterile technique (‘inoculated’) is the main determinant of whether colonies or lawns are seen after overnight incubation at the appropriate temperature.  (There are exceptions, as highly motile bacteria can move around so you can’t know, unless you do time-lapse video microscopy, exactly how the colony arises. Some of these ‘swarmer’ bacteria have been used in the community lab context, and are quite interesting, even showing a ‘spiral galaxy’ morphology on occasion rather than a simple circle of growth. However, for the ‘workhorse’ of molecular biology, E. coli, used for these validations, one colony always arises from the clonal growth of a single cell.) In terms of limits of detection, and the objective to be able to observe a live bacterial cell, a single colony after overnight growth is readily observed, even if many millions of dead cells were inoculated on the plate.

A lawn of E. coli is generally used as the feeding substrate for C. elegans nematodes, and the presence of living animals is readily monitored by occurrence of ‘trails’ or ‘tracks’ left in the food source by the worms feeding on the bacteria.  If only one worm survives out of many thousands, its track from feeding will be observed.  To make the test even more informative for the nematode case, we began with starved cultures of C. elegans, in which a special ‘spore-like’ larval stage, termed the ‘dauer’ stage, and able to withstand many stresses to survive until conditions are better, were the initial basis for the test.

To note:

• Any liquid waste from experimental procedures, for instance supernatants from plasmid preps, are inactivated by addition of bleach to a final concentration of 10% and allowed to soak at least 10 mins before disposal down the drain in the big back sink of the cooperative space. As no culture larger than 500ml is allowed, and those quantities only rarely, this means not too much bleach is ever utilised. As the old Hackuarium autoclave was left behind in Renens, as it was huge, inefficient energetically, and quite ancient, this method for small volumes seems justified.
• The community lab reserves the use of its large pressure cooker for sterilization, not ever for waste treatments.
• The validation experiments were recorded in the on-line tool, Evernote, for the worm and bacterial experiments, and images from the process were also saved to a google drive folder.

Method

A comparison of three conditions, including two concentrations of bleach and water, was performed for both transformed bacterial cultures or nematodes, to evaluate the efficacy of the usual SOP with undiluted bleach for culture inactivation.  

Community lab members trained to use the P1 laboratory space must always remember that bleach solutions are classified as an irritant and a corrosive, and:

• Work in a well-ventilated area.
• Wear PPE consisting of a lab coat, eye protection, and gloves.
• Use cold water to prevent chemical decomposition.
• Label and date solution with preparation date and initials and discard after use.
• Remove PPE including gloves and wash hands thoroughly.
• Be aware that there is not only an eyewash station on the ‘safety shelf’ in the P1 lab, but also a shower around by the big sink in the space. Contact with bleach solutions can cause eye or mucous membrane irritation and burns. If bleach gets into the eyes, immediately rinse with water for at least 15 minutes. If skin comes in contact with bleach, rinse with lukewarm water for at least 5 minutes

For the validation tests, the most difficult conditions were chosen for the SOP procedure, a fully confluent law of bacteria or starved cultures of C. elegans, in which dauer larvae were expected to be found.

For bacteria:

Plates of a transformed and fluorescent E. coli strain were the starting point of the tests. Three different concentrations of these bacteria were plated, so a complete lawn, one less dense, and one with obvious individual colonies were obtained. These were treated on the plate, as in the usual SOP method, complete with 5 minute incubations and swirling.  However, the least dense plate was treated with only water, the more dense (or ‘confluent’) plate was treated with a 1 in 10 dilution of bleach, while the lawn of transformed and fluorescent bacteria was treated with the undiluted bleach.

For nematode strains:

Starved plates of two different C. elegans strains were the starting point of the tests. In this case, the organisms were first washed from the plates and then treated in small ‘microfuge’ tubes with 1) water, 2) a 1 in 10 dilution of bleach, or 3) undiluted bleach.

In both cases, secondary culture plates from each set of treatments were made and scored for growth.

Results

For the bacterial culture treated with undiluted bleach, the cell layer was basically dissolved away, while the dense colonies of the second plate treated with the 1 in 10 dilution remained obvious. Water treatment did not disturb the colonies on the third plate greatly, although many cells made the retrieved liquid cloudy.  From the secondary cultures, however, only the plates inoculated with bacteria from the water-treated plate were full of fluorescent and transformed colonies.  Both the diluted bleach and undiluted bleach conditions completely inactivated the cells, so no growth or fluorescence was observed.

For the nematode worms, only water treatment allowed ready observation of worms on the secondary plates.  However, a plate from one of the 1 in 10 dilution treatments did have a few worms that survived, including at least one Roller (sign of retention of the transgene in that strain).  No survivors were observed after treatment with undiluted bleach.  (All plates were re-checked over a few days, to confirm whether any further tracks were made in the thick bacterial lawn.)

Discussion

These results show that the inactivation protocol as given in the SOP with undiluted bleach is effective and can be considered validated.  While 10% treatments of these bacterial cultures might be considered safe from these results, it however is more prudent to stay with the current method, as other bacteria or culture contaminants, in particular those which might form spores, like Bacillus or molds, could be much less sensitive to diluted bleach.

For the work on the nematode worms, it is clear that only the full strength bleach should be used, as the dauer form can survive after exposure to a 1/10 dilution of bleach.

Further tests could be done to compare other commercial brands of bleach. However, as this validation was based on an inexpensive brand and it seems likely that most found in Switzerland are not going to be very much different, this does not seem to be an urgent need.  However, one must always make sure that bleach is stored in the dark and not used past the date of expiration for these inactivations to ensure safe disposal.

Another point discussed already in the Evernote experimental record has to do with the idea of seeing whether plasmids could be recuperated from the treated solutions, not just living organisms. Because such tests would require quite efficient competent cells, however, it also is only being considered…

References

Swiss legal basis: the Ordinance on Contained Use (OUC, RS 814.912) and the Ordinance on the Protection of Workers against Risks related to Microorganisms (OPTM, RS 832.231). These in turn are based upon the Laws to Protect the Environment (LPE, RS 814.012), and on Genetic Engineering (LGG RS 814.91) and against Epidemics (LEp RS 818.101), in addition to the convention to protect biodiversity (RS 0.451.43).