Wednesday, November 17, 2010

Clearing insects: Lactic Acid vs. KOH

Using Google searches, I have found little information on clearing of insects to observe their structure. What information that is available does not relate the benefits and detriments of various clearing agents. Here I will summarize these issues, especially to show the benefit of using lactic acid contrasted with hydroxides.

Potassium hydroxide (KOH: also known as caustic potash or lye) is a strongly basic chemical that has historically been most often used for insect clearing. It is stored as a solid and dissolved in distilled water for use, usually as a 10% solution. When heated, soft tissue is quickly destroyed as well as lightly sclerotized cuticle. Continued exposure to KOH will cause the chitonous matrix of cuticle to become sticky and soft, and a specimen left in KOH for extended periods will become clear and dissolve into invisibility. The destructive process can be slowed down by clearing in room temperature solution rather than heated, but a specimen exposed to KOH cannot ever be neutralized completely. Many old slides of specimens, including holotypes, today seem to contain nothing due to the long term caustic action of this chemical. In addition, once a specimen is placed in glycerin for viewing, it cannot be returned to KOH for more clearing.

Sodium hydroxide (NaOH) has similarly been used in clearing. It has the same properties of KOH, except it is reported that it is less caustic on weakly sclerotized tissue and membrane (Gurney et al. 1964).

Lactic acid (2-hydroxypropanoic acid) is a weakly acidic chemical now coming into favor as a clearing agent. It is stored as a fluid, usually at 80% concentration, and can be used directly. Unlike the previous hydroxides, lactic acid will not indefinitely clear material, even when heated. Membraneous and weakly sclerotized structures are retained while muscle and fat are dissolved. Lactic acid also will cause membraneous structures to expand under heat, which can make the treatment useful for eversing an aedeagus stuck within a phallocrypt. In addition, the specimen can be passed through glycerin and returned to the clearing solution if needed.

I have begun using lactic acid for all my clearing. The benefits are obvious, with some precautions. First, since heating causes expansion of tissue, sometimes specimens will explode. Therefore, only low circulating heat rather than boiling should be used. Second, while weakly sclerotized structures will be retained, intersegmental membrane is more likely to be broken over time. Gentle care when using probes and forceps will insure the parts stay connected.

Reference:

Gurney, A. B.; Kramer, J. P.; Steyskal, G. C. 1964. Some techniques for the
preparation, study, and storage in microvials of insect genitalia. Annals of the
Entomological Society of America 57:240-242.

11 comments:

Caitlin said...

This post was INCREDIBLY helpful to me. Thank you very much.

-Purdue entomology BS graduate

Anonymous said...

Clear with lactic acid after fixing I'm assuming. Correct?

Kai said...

Anon,

I seldom fix specimens, as almost all of my specimens are to be pinned or collected in alcohol.

It may be time to update this post. I've been using a nifty new method for genitalia dissections.

~Kai

Anonymous said...

Nice job Kai. I was looking for something just like this. Clearing some caddis from Sleep Bear Dunes NLS.

Ed DeWalt
INHS

Kai said...

Ed,

I have been using a new method combining sodium hydroxide, acetic acid, and lactic acid. There is a new post, including photographs, forthcoming.

~Kai

Jerry Cates said...

Good information, Kai. Much appreciated. Looking forward to your information on the sodium hydroxide, acetic acid, and lactic acid formulas you are using. I presume that combination is superior to lactic acid alone. I'm about to use lactic acid to clear mites in the Macronyssidae. Any words of wisdom?

Kai said...

It depends on the size of the specimen and how sclerotized. I use the NaOH for softening tachinid fly abdomens for dissection. After dissection, I buffer the strong base by soaking in acetic acid, and use hot lactic acid for final clearing. The ides is to use the strong base for as short of a time as possible to avoid future problems of invisibility.

The problem with lactic acid is it often causes tissues to expand and can deform cuticle. I've found ths to be an issue with some genital structures. In particular, the surstyli tend to bend lateral in a dorsal view and can distort the shape in lateral view. It may be related to heat, I don't really know. Thats all the wisdom I've got. Despite doing a goodly number of genitalic dissections, I'm still a novice compared with people like Jim O'Hara of the CNC.

Jerry Cates said...

Thanks, Kai

You are brave to venture into the heady world of the Tachinidae...

The mites are in the genus Ornithonyssus and have been preserved in ethanol for less than 24 hrs. Even the mature specimens are not well sclerotized. I'll try working the strong base, acetic acid, and lactic acid, in sequence, at relatively low temp at first, and see how it goes.

Anonymous said...

Completely different kingdoms... but I use both KOH and 85% lactic acid as clearing agents (also for certain reactions with KOH) for fungal specimens. Lactic acid is viscous, which is great for slowing down spores to take pictures (Brownian motion or evaporating water can be a pain). It also works nice for making semi-permanent slides.

I often leave specimens mounted in lactic acid on a hot place set to very low overnight to remove air bubbles and clear the tissue properly. Good stuff.

Brian Brown said...

Kai: Don't forget:

Cumming, J. M. 1992. Lactic acid as an agent for macerating Diptera specimens. Fly Times 8: 7.

I regularly dissect into lactic acid, heat about 30 seconds in the microwave (in 10 s spurts, as suggested by Terry Wheeler). It works beautifully.

Brian

Kai said...

Brian,

Yes, I have used the "10 second" method before, but often times I find it overheats the tissues, and causes genitalia sclerites to expand and break. Usually I prefer a slower clearing process for this reason.

Thank you for recommending that paper. I did not know of it when I originally wrote this.

~Kai