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Staining microplastics with UV fluorescent Nile red

The search for microplastics in our environment continued

Almost a year ago I wrote about my plans to use Nile Red to detect microplastics following the paper I found before. It’s finally happening! There were several hurdles that I had to cross to get all the supplies necessary, and the project was put on hold by me for a while.

First, the most difficult to get was the Nile Red itself. I repeatedly ordered a vial of several milligrams or a gram on a suppliers website, but those orders got cancelled over and over again. Some suppliers first asked me to sign an agreement I wouldn’t use any of their chemicals to produce drugs before denying me based on the fact that I don’t have a registered laboratory, others outright refused orders or gave me back my money when they found out I was not registered as a company. When ordering the other lab equipment like, gloves, safety glasses and eppendorf tubes at a company that does supply to private individuals, I mentioned that I was looking for it. Their website did mention Nile Blue - which can be converted to Nile Red but is too complicated and unsafe for me to try - and they could also order from those same suppliers for me!

This may be a familiar story if you read my Minerals under uv light post, but when I was setting up the experiment back in september I nsoticed I was missing methanol. As that was readily available on a big Dutch web shop, I mindlessly ordered it, not looking up the safety information about it. But when it arrived by normal mail and I opened it, being greeted by big warning labels and triple packaging, I realised the danger and immediately regretted that I ordered something dangerous trough mail. As I don’t have a fume hood and I was not comfortable handling even slightly dangerous chemicals this project went on hold back then.

As I’m browsing for a house to buy and I just bought a new car, buying a fume hood has to wait a bit. Methanol can make you blind after ingesting, inhaling or absorbing through skin of only 10ml - with possible death after 30ml - so handling it requires a lot of care. When I got the bottle, I marked the fluid level in the bottle to make sure it was not leaking, and placed the bottle in a chemical cabinet, but now it was time to actually handle it.

The past few months, I’ve had a lot of time to think about how to do this experiment safely. With an evaporation rate of 2.9, compared to the evaporation rate of 2 from ethanol, ventilation was the most important factor. I decided to swiftly transfer the tiny amount of methanol I needed in open air. After buying some protective glasses and plastic gloves I was good to go.

The transfer of 1ml of methanol was anticlimactic. The Measuring pipet perfectly fit in the bottle attachment, so the methanol didn’t even have a change to evaporate. Worst case scenario after puting the bottle back into the cabinet, was me drinking the 1ml, which was far away from having the possibility to make me blind.

The paper mentioned an exact concentration of Nile Red to Methanol, at 1µg mL-1, with quenching - decreasing of fluorescence - occuring above twice that dose. With 1 gram at my disposal and no scientific scale I decided to guesstimate it and just put some Nile Red in the eppendorf tube.

As you can see, A lot of fluorescence was visible, and this can probably be optimized when you do have a scientific scale at your disposal. (you may notice I cropped the picture around my fingerprints). It looks like my UV light source is producing a lot of light outside the UV spectrum, but a visible light filter is attached and the blue hue you see on my hand is the result of the fluorescence of skin.

With my stereoscopic microscope and UV light set up I took a picture of a piece of plastic food wrapper:

After adding a drop of the Nile Red solution, the plastic was fluorescing!

As I live on a 5 minute walk to the beach, I had to get some sand again. When putting it under the same microscope and illuminating it with the UV flashlight, you are greeted by some already fluorescing bits and pieces. As most biological substances are already fluorescent in some way, there is already some kind of fluorescence going on. Most of these will be pieces of shell - when you walk around the beach at night with the UV light you are greeted by a world of shells mixed with some pieces of trash. More about this in a later post.

When the Nile Red solution was added the overall hue of the sample became purple, and one piece was lighting up bright orange/yellow. You can sadly see some surface movement, which was quite difficult to avoid using the night mode of my galaxy s9 and it’s long exposure.

There are also some smaller orange dots visible. These were suspended in the water, and not actually attached to any particles. I suspect that the proportion of the solution and the water was too high, causing the water to be over saturated with the Nile Red solution. More about this when I have some more updates for you!

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