How do scientists kill the bacteria they themselves made resistant?

Question

I was reading this article on researching bacteria resistance to silver by removing some of their genes.

Researchers then used "colony-scoring" software to measure the differences in growth and size of each plate's bacterial colony. E. coli strains with genes deleted involved in producing sensitivity, or toxicity, to silver grew larger colonies. Strains with genes deleted involved with resistance grew smaller colonies.

Once you end up with some resistant bacteria and you're done researching it, you can't just flush it down the toilet. How do you safely dispose those colony plates in a way that ensures those bacteria don't get out into the wild and reproduce?

Answer 1

You are absolutely right, flushing down the toilet (or the sink) or simply throwing them into the normal waste doesn't work for biosafety reasons. And it is also not allowed, depending on the country you would do this in, this can lead to hefty fines.

Biologically contaminated lab waste can be inactivated (=all potential dangerous organisms are destroyed) by two ways: Either by heat or chemically. Which ways is used, depends on the kind of waste.

The most commonly used way is autoclaving, meaning treating the waste with steam at high temperatures at higher pressure. The temperature used here is usually 121°C, the exposure time depends on the volume of the waste, since the temperature needs to be reached and kept for at least 20 minutes. See the references for more details.

Liquid wastes (like culture media) can also be inactivated chemically by adding chlorine bleach to decompose the cells. Bleach can also be used to decontaminate surfaces, although here more often alcoholic solutions (70% Ethanol or Isopropanol) are used. After chemical inactivation, the remaining solutions should not be autoclaved as the emerging fumes are either unhealthy (bleach) or explosive (alcoholic solutions) and this is unnecessary, too. Liquid wastes can also be autoclaved to inactivate them.

Autoclaving has the main advantage that it is rather simple (put the waste into the autoclave, close it and run a appropriate program), the waste can afterwards simply be discarded as normal waste, which may not be the case for chemically inactivated waste, which may need special precaution for disposal.

References:

1. Decontamination and Sterilization
2. Decontamination of laboratory microbiological waste by steam sterilization.
3. TECHNIQUES FOR STEAM STERILIZING LABORATORY WASTE
4. Decontamination of Laboratory Microbiological Wasteby Steam Sterilization

Answer 2

To address what seems to be the misconception underlying your question:

Killing pathogenic bacteria is not difficult; killing them without harming their (usually human) host is. This is why antibiotics are so precious: They are drugs that affect only bacteria¹ – by exploiting properties that are unique to them. It is usually resistance to antibiotics that we care about in bacteria, because it makes infections with such bacteria more difficult or even impossible to treat.

Once we investigate bacteria in vitro, e.g., in a Petri dish, there is no host that has to survive and thus we have far more options to kill them. The other answers have listed plenty of methods to do this – which are all lethal to humans as well.

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^{¹ This is of course somewhat simplified: Every drug has a side effect, and so do antibiotics. But their side effects on humans are more lenient than their primary effect on bacteria.}

Answer 3

Resistance Is...Reversible?

While people generally don't talk about bacteria losing antibiotic resistance, it does happen, and for a pretty obvious reason: the biochemical tools which confer resistance come at a metabolic/reliability/efficiency cost, or the bacteria would have had the resistance to begin with. In general, we can assume that bacteria are not adept at dealing with silver toxicity because silver does not occur often enough in their favorite environments. When you take the bacteria out of a silver-concentrated environment, there is no longer pressure to take extra measures to deal with it, and the resistance can be lost.

A Pound of Flesh

There are many ways to attack microbes, and even more ways for them to respond. The CDC provides a nice overview of some strategies.

Take fluoroquinolones, for instance. They kill bacteria by suppressing the enzymes which uncoil DNA. As you can imagine, this makes for a Very Bad Day^TM for the bacteria. If you're a bacterium, and you end up with some ciproflaxin inside your membrane, the most obvious thing to do is get rid of it. Some bacteria, like P. aeruginosa, which are resistant to ciproflaxin do just that: they build pumps in their membranes to pump it out. But as any engineer will tell you, adding new pumps to a system costs both space and energy which could have been used for other things (like finding food, reproducing, etc.), so you wouldn't do this unless you absolutely had to.

How do we know that they actually lose resistance? Well, we've observed it happening! We literally know which mutations and gene transfers are necessary to confer various resistances for P. aeruginosa, because wild strains are both resistant and susceptible, depending on their exposure to antibiotics (or laboratory conditions).

Gray Goo

Be glad that biology tends to find the most efficient solutions. This energy minimization tendency might be characterized as a kind of laziness, but it's really an optimal strategy for allocating your energy where it gives you the most value. For a microbe, that would be finding food and reproducing. That means that once the lab-grown freaks escape into the wild, whatever super-powers they gained from our mad scientist experiments are likely to be lost over time, once we stop bathing them in mutagenic toxins.

Unlike nanotechnology-gone-wild, we actually don't have so much to fear from lab-induced antibiotic resistance. It is the pernicious and casual use of antibiotics in our daily lives that will kill us in the end. These flow down our drains and into our streams and rivers and evolve those critters night and day, virtually guaranteeing that we will encounter resistant types in the wild.

Answer 4

Resistant to WHAT?

Scientists generally deal with bacteria resistant to some concentration of some particular substance (or combination thereof).

Those strains are still pretty much sensitive to great deal of other substances (e.g. etanol 70%), high temperature (boiling water at normal pressure or higher), etc generic ways to kill bacteria.

Answer 5

Autoclave at 120 degrees Celsius for 30 minutes. ^[ref]

Don't ever flush biological material, living animals, or anything other than sanitary-code approved waste down the toilet.

The proper way to dispose of used culture material is to sterelize it first, then dispose in sealed bags.

After counting, petri dishes should be secured in a plastic autoclave bag and autoclaved prior to disposal. During multi-day field trips, place used petri dishes in plastic autoclave bags, and close the bags for transport to the office. Then, following autoclaving, place the closed bags of petri dishes in dumpsters or other containers that will be emptied mechanically, to prevent accidental contact with people or animals. Never place petri dishes in motel or office wastebaskets.

Answer 6

On a more budget-restricted level, when I worked in a high school, the science lab tech would take all the old Petri dishes out of a dedicated disposals freezer, and take them to the campus coal fired boiler.

Since the boiler only ran in Winter, there was a lot of stuff to dispose in the first few cold days in Autumn. As such, the science department scheduled much of this work over winter. Work involving dissection also produced a lot of biowaste, but that was less-bad and could be disposed of in the general waste (it was the 80s, not a lot of recycling at the time.)

Once the furnace part of the boiler was at temperature, the thing was shuttered (somehow) and the access hatch opened. The frozen plastic dishes were thrown in, inside either paper or plastic shopping bags for ease of handling, then the hatch was closed and the dampers opened.

As for PPE, it wasn't much of a thing. Disposable gloves were worn for handling the dishes, and they went into the furnace once the dishes were all gone.

I remember once looking in the furnace when it was cold, before the ashes were raked out. There was zero trace of any material left behind, even metal items like staples or clips were vapourised.

In short - Burnination