A better method to get live insects to hold still

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MarkSturtevant
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A better method to get live insects to hold still

Post by MarkSturtevant »

I have found a solution for taking staged shots of live, active insects. The method is simple, and anyone can do it. Of course chilling insects is commonly done, but I personally have very mixed results with that approach and sometimes chilling is fatal to the subject. The method described below does no harm and the results have been good.

CO2 gas is a quick anesthetic of insects. It knocks out most insects immediately (although some species can close their breathing spiracles and resist it longer). They quickly recover once the gas is removed, but for a time they stand and move normally while also being rather dopey. This is the time window in which you can take pictures. The effect is quite safe, and can be done repeatedly on the same subject.
Some species take longer to recover than others. Some recover very quickly and fly off. But all species that I have tried so far do have at least a brief period where pictures can be taken.

How is this done? The principle is to use an Alka Seltzer tablet in water. This releases a lot of CO2. I invented a little gas chamber to knock out insects with Alka Seltzer, and the apparatus is shown here.

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The above picture shows the chamber that holds an Alka Seltzer tablet. It is a 2 oz. serving cup (I use these for a lot of other insect-related things). The center of the lid is cut out and replaced by a piece of screen. The piece of paper on top is explained next.

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This picture shows that the serving cup is placed inside a larger cup. The insect is placed inside this larger cup. The serving cup fits snugly inside so the insect cannot slip down to the bottom. There is a piece of paper on the lid of the serving cup to cut down on the spray of water once water is added. If the insects wings get wet, that can detract from the desired effect of making the pictures look natural. Finally, the lid of the larger cup has a hole near one edge. Once the insect has been placed in the larger cup I can aim water through the screen top of the inner cup, activating the Alka Seltzer. I fill the inner cup to about 1/3 full. By then, the insect is usually completely knocked out. I then wait several seconds, take the insect out (that is what the paint brush is for), and place it on my stage which has been prepared in advance. Then I wait for recovery. This is a good time to take get the camera aimed and to take some test shots to check on lighting and so on. Once the insect is standing and looks normal enough I can start taking pictures. I recommend that this be done near a window so that if the insect flies away it will likely turn up there to be retrieved.

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Two examples of insects that were photographed this way are shown here. The first is a tiny Chalcid wasp (I think it is Conura side). This one wanted to clean itself constantly after recovering, and I could take a lot of pictures. The second insect is a small Ichneumon wasp (Cratichneumon sp). This insect recovered very quickly and flew to the window after about a minute. Still, that was a minute I would not have any other way with this perky, constantly moving subject.
Mark Sturtevant
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Lou Jost
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Post by Lou Jost »

What an excellent idea! I hope there are Alka Seltzer tablets in Ecuador, I am eager to try this.

Pau
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Post by Pau »

CO2 anaesthesia is a routine method at Genetics labs to manage Drosophila. There are commercial CO2 emitting stages intended to work at the stereomicroscope named fly pads like
http://www.tritechresearch.com/dros.html
It's safer for both the fly and the people than the classic ether anaesthesia
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Post by johan »

That's excellent, thanks for posting!
My extreme-macro.co.uk site, a learning site. Your comments and input there would be gratefully appreciated.

MarkSturtevant
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Post by MarkSturtevant »

Pau wrote:CO2 anaesthesia is a routine method at Genetics labs to manage Drosophila. There are commercial CO2 emitting stages intended to work at the stereomicroscope named fly pads like
http://www.tritechresearch.com/dros.html
It's safer for both the fly and the people than the classic ether anaesthesia
This is the basis for this idea. I had spent years doing just that. It was remarkable how flies can be knocked out, kept knocked out for a very extended time, and they recover quickly and normally.
Mark Sturtevant
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rjlittlefield
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Post by rjlittlefield »

MarkSturtevant wrote:recover quickly and normally.
For some definition of "normally"...

Google search on "drosophila co2 behavioral studies" will return quite a bit of info. At this moment, the first hit is to https://www.nature.com/articles/srep15298, which reports:
Title: Impaired climbing and flight behaviour in Drosophila melanogaster following carbon dioxide anaesthesia

Abstract:

Laboratories that study Drosophila melanogaster or other insects commonly use carbon dioxide (CO2) anaesthesia for sorting or other work. Unfortunately, the use of CO2 has potential unwanted physiological effects, including altered respiratory and muscle physiology, which impact motor function behaviours. The effects of CO2 at different levels and exposure times were examined on the subsequent recovery of motor function as assessed by climbing and flight assays. With as little as a five minute exposure to 100% CO2, D. melanogaster exhibited climbing deficits up to 24?hours after exposure. Any exposure length over five minutes produced climbing deficits that lasted for days. Flight behaviour was also impaired following CO2 exposure. Overall, there was a positive correlation between CO2 exposure length and recovery time for both behaviours. Furthermore, exposure to as little as 65% CO2 affected the motor capability of D. melanogaster. These negative effects are due to both a CO2-specific mechanism and an anoxic effect. These results indicate a heretofore unconsidered impact of CO2 anaesthesia on subsequent behavioural tests revealing the importance of monitoring and accounting for CO2 exposure when performing physiological or behavioural studies in insects.
The message I take away is that CO2 anesthesia has some very attractive features, but it's not completely benign.

--Rik

MarkSturtevant
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Post by MarkSturtevant »

@ rjlittlefield:
Acknowledged! I should not say there is no effect. But to the eye they seem ok enough.
Lou Jost wrote:What an excellent idea! I hope there are Alka Seltzer tablets in Ecuador, I am eager to try this.
It occured to me just now that of course baking soda and vinegar would also release a lot of CO2. But I don't know if the there are acidic vapors that would be bad for the insect somehow.
Mark Sturtevant
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Lou Jost
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Post by Lou Jost »

I bet there is an Alka-Seltzer knock-off here.

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Post by zzffnn »

MarkSturtevant wrote:@ rjlittlefield:
Acknowledged! I should not say there is no effect. But to the eye they seem ok enough.
Lou Jost wrote:What an excellent idea! I hope there are Alka Seltzer tablets in Ecuador, I am eager to try this.
It occured to me just now that of course baking soda and vinegar would also release a lot of CO2. But I don't know if the there are acidic vapors that would be bad for the insect somehow.
You should be able to dilute vinegar to a concentration that produces minimal vapor and still releases CO2 from baking soda. I don't know exactly what that concentration/dilution is though. It should be easy to figure out with serial (2 fold) dilutions and experimentation.

For example, hydrochloric acid fumes a lot at 37%, but significantly less at 30% and such vapor reduction is not linearly proportional to concentration - when you dilute more, you get much much less vapor, disproportionally.
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gpmatthews
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Post by gpmatthews »

Why not use a solid acid such as tartaric acid or citric acid? That way all you have to do is add water as with the tablets.
Graham

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MarkSturtevant
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Post by MarkSturtevant »

gpmatthews wrote:Why not use a solid acid such as tartaric acid or citric acid? That way all you have to do is add water as with the tablets.
Dunno. In the end one would want to use reagents that are easy to get, and ones that evolve CO2 without harming the subject very much.
Mark Sturtevant
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gpmatthews
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Post by gpmatthews »

I think the advantage of a solid acid is that there are no volatiles as with acetic acid or hydrochloric. They are also easy to handle and can be used in solution or as a powder. Citric acid, for example, is non toxic and easily available.
Graham

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anvancy
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Post by anvancy »

This is a very good option.

Generally for studio stacking, at times I find the freezing option dries out the insects. If I forget the insects in the moisture relaxing chamber they get squishy and the specimen is of no use.

The tablet that is discussed here,is it those aspirin kinds one used for headache?

It seems if the tablet option is not available I will have to go with the vinegar and baking soda option or the citric acid option. What do we need to add in citric acid powder to trigger the Co2?
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gpmatthews
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Post by gpmatthews »

Use citric acid with sodium carbonate (washing soda) or bicarbonate. They react to produce carbon dioxide. They will only react usefully in solution, so at least one of the components has to be dissolved in water. They mix ok as dry powder, and will effervesce if wetted, producing carbon dioxide.
Graham

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Post by rjlittlefield »

gpmatthews wrote:Citric acid, for example, is non toxic and easily available.
And in fact Wikipedia writes:
Alka-Seltzer contains three active ingredients; aspirin (acetylsalicylic acid) (ASA), sodium bicarbonate, and anhydrous citric acid.
I always wondered what was in that stuff. Thanks for the tickle to check it out!

--Rik

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