About Nematodes

Here you can discuss sample and specimen preparation issues.
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lperepol
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About Nematodes

#1 Post by lperepol » Mon Dec 17, 2018 5:18 pm

What would be a good procedure to preserve a nematode under a permanent slide? Stain to use, suspension liquid, slide sealer ....
Links to articles or comments appreciated.
And an aside question can nematodes categorized using a microscope?
Or better to record behaviour live with video?

Found a good resource here: https://nematode.unl.edu/index.html
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Re: About Nematodes

#2 Post by 75RR » Mon Dec 17, 2018 8:05 pm

Thanks for the link - great site!
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desertrat
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Re: About Nematodes

#3 Post by desertrat » Mon Dec 17, 2018 10:41 pm

I have made some permanent stained slides of tiny freshwater invertebrates, mounted in Canada balsam, but not nematodes.

I'm guessing you have some information on how to collect soil nematodes, if not, this link has has some information and another link.

https://www.ars.usda.gov/northeast-area ... l-samples/

I've done some reading in old editions of The Microtomist's Vade Mecum by Arthur Bolles Lee, editions from the late 19th and early 20th centuries. Nematodes are difficult, but not impossible to make permanent stained mounts of.

Nematodes are covered with a cuticle that is very difficult to penetrate with the chemicals used to dehydrate, stain, and mount specimens. There is mention of soaking the fixed nematodes in a weak solution of bleach, which makes the diffusion of fluids into and out of the nematode less difficult.

If you want to do more reading, you can download several editions from the Biodiversity Heritage Library:

https://www.biodiversitylibrary.org/sea ... DV#/titles

Other editions are available from other sources. The editions from 1893 to 1913 should have the most information for the methods I use.

After downloading several editions, I recommend scrolling to the end of each and looking at the index for Nematodes. Depending on the volume, you may be referred to a page number or paragraph number. The page numbers in the text will be different from the page numbers in the .pdf files, but not by more than about 20 pages.

The paragraphs on preparing the nematodes will refer to other paragraphs for bleaching and staining.

After you've studied these manuals, if you have more questions, I might be able help. You will see recommendations for some fixatives that contain some very poisonous chemicals. I've discovered that many tiny freshwater invertebrates can be effectively fixed with hot water that has just ceased boiling. Hot water works well on insect innards, too.

After fixation, the next step would be the dilute bleach solution for overnight, then through a series of alcohol of gradually increasing concentration, then the stain in one of the higher alcohol concentrations. The stain might take several days. The specimen will eventually be soaking in alcohol of at least 95%. After that it will be placed in a layer of 95% alcohol floating above a layer of cedar oil. After the specimen sinks to the bottom of the layer of cedar oil, it is placed on the slide and the excess cedar oil is carefully blotted away with small pieces of absorbent paper. Once the specimen is isolated in a very tiny drop of cedar oil on the slide, just barely bigger than the specimen, a drop or two of mounting medium is dropped on top of the specimen, and a cover slip is carefully lowered on top.

I use Canada balsam dissolved in xylene for mounting, and circular cover slips for zoological mounts. Other more modern resinous mounting media can be used and square cover slips are fine. Circular ones just look a bit better, but are more expensive to buy.

I'm not entirely sure this protocol will work, but it might. I may give it a try when winter is over.
Rick

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Re: About Nematodes

#4 Post by lperepol » Tue Dec 18, 2018 9:12 am

I love those older classics desertrat, thank you for the links!!!
Last edited by lperepol on Thu Dec 20, 2018 2:54 am, edited 1 time in total.
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Re: About Nematodes

#5 Post by desertrat » Tue Dec 18, 2018 4:24 pm

I did some more Googling after posting, and discovered that no one is processing nematodes using these older methods nowadays.

It seems the current consensus is that staining isn't very effective, and modern methods involve moving the fixed nematodes through gradually increasing concentrations of glycerin, which can be bought at any drugstore. The specimens are finally mounted, unstained, in pure glycerin.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411252/

Although not specifically stated, it looks like observation with oblique illumination would be fairly successful, and modern enhanced contrast systems like DIC even better.

When I tackle this subject, I will try the antiquated methods first, because that's what I'm set up for. If those don't work, I'll try the more modern methods.
Rick

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lperepol
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Re: About Nematodes

#6 Post by lperepol » Tue Dec 18, 2018 7:02 pm

Thank you for the links especially https://www.biodiversitylibrary.org/sea ... DV#/titles.
Will read the volumes!!!

I was also successful floating nematodes up with a 20% Blackstrap Molasses and 80% water solution and then add 50/50 soil/solution. I have so many nematodes in the soil they are easy to pick out of watered down soil. Using a funnel works well there is less debris. I use a grocery store plastic funnel set up like this -- https://www.plantpath.iastate.edu/tylka ... ann-funnel.
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I am wondering if there are stains that enhance different anatomical body parts of nematodes?

#7 Post by lperepol » Wed Dec 19, 2018 6:55 pm

1) I am wondering if there are stains that enhance a nematode's tooth?
2) I am wondering if there are stains that enhance a nematode's stylet?
3) I am wondering if there are stains that enhance different anatomical body parts of a nematode?

This would be to help in comparing nematode species/genus?

Found this: A Method for Staining Nematode Secretions and Structures --> http://journals.fcla.edu/jon/article/do ... 5912/63580
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desertrat
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Re: I am wondering if there are stains that enhance different anatomical body parts of nematodes?

#8 Post by desertrat » Wed Dec 19, 2018 8:28 pm

lperepol wrote:1) I am wondering if there are stains that enhance a nematode's tooth?
2) I am wondering if there are stains that enhance a nematode's stylet?
3) I am wondering if there are stains that enhance different anatomical body parts of a nematode?

This would be to help in comparing nematode species/genus?

Found this: A Method for Staining Nematode Secretions and Structures --> http://journals.fcla.edu/jon/article/do ... 5912/63580
Those are some exotic techniques! Way beyond anything that I will probably try doing.
Rick

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lperepol
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Re: About Nematodes

#9 Post by lperepol » Thu Dec 20, 2018 2:03 am

I have found a way of estimating nematode populations, stain with iodine, let them die. take picture(s) and count on picture them. Graph paper (squared paper) as background can help.

Now if I can get links to google photos to display as pictures.

https://photos.google.com/share/AF1QipP ... dUb2tMTjdB

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Practical plant nematology: A field and laboratory guide

#10 Post by lperepol » Thu Dec 27, 2018 10:41 am

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