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Tineid of Korea
Preparation of micromoth
venation, genitalia, larvae
All rights reserved. Copyright 2005 Tineid of Korea [Reuse]

Before starting the dissection
Be sure to take a specimen picture first before you start dissection. The dissection assumes that the specimen is damaged, so even if there are several same specimens, the specimen photograph must be taken. Sample photographs should be kept together with the serial number of the specimen. Photographs the front, back, and head (front, side) and takes a picture of what you think is necessary. Also should be done with sufficient data. It is usually limited only if the external form is difficult to identify or suspicious of another species.


Materials
vial(each reagent), reagent(50%, 70%, 90%, 100% EtOH, Xylene, surfactant+rinse mixture, 10% Acetic acid aqueous solution, D.W., Acetocarmine aqueous solution, Chlorazole Black E, Mercurochrome aqueous solution, Canada Balsam, 10% KOH aqueous solution, Spuit(each reagent), small brush (2-3 exs, no.1), pincette(very acute in terminal, 2-3 exs), Stirring hot plate, razor or mes or handy made knife, microscope for dissection, slideglass, coverclass, Schale(glass), watch glass, tissue(Kimspaper, general tissue) and additional tools for dissertion is hand-made.

Click! for original size Click! for original size Click! for original size
microscope, dissection⋅stain tool set

Preparation of wing venation
Wing venations were mounted on slide through following process:
  1. Remove wing from the thorax.
  2. Descaled in surfactant and decolorant (10%) for 5 minute, and treated in 100%, 70%, 50% alcohol, for 10 minute.
  3. Washed in D.W.
  4. Stained with acetocarmine (100%) for 3-4 hour.
  5. Dehydrated in 50%, 70%, 90%, and 100% alcohol.
  6. Fixed in xylene for 1 minute.
  7. Mounted with canada balsam on microscope slide.
  8. Dried at 50℃.

Click for original size

General method for genitalia preparation
Genitalia were mounted on slide after being treated by a modified method of the Robinson's (1976).

  1. Abdomen is carefully removed from body.
  2. Immersed the removed abdomen into the 10% KOH solution for 5-15 minutes.
  3. The softened abdomen is moved into watch glass containing 10% acetic acid and descaled by a sharpened dissection needle, tissue and micro brush pen.
  4. Abdomen is washed again in D.W. (Fullness volume) and transferred to 50% ethyl alcohol and descaled by a sharpen dissection needle and micro brush pen.
  5. Abdomen and genitalia are transferred to 70% ethyl alcohol and genitalia dissected by was pulling out gently and washed in water or D.W.(Fullness volume).
  6. Staining in mercurochrome (0.5∼1 minute), and washed in D.W. (Fullness volume) for 20-30 minute.
  7. Staining in chlorazol black E. (0.5∼1 minute), and washed in D.W. (Fullness volume).
  8. Genitalia and abdomen are dehydrated by 70%, 80%, 90% and absolute ethyl alcohol(100%).
  9. Genitalia and abdomen are transferred to xylene to exclude the impurities or fixations.
  10. Mounted with canada balsam.

Dissection manual for micro lepidoptera  modified by seok Kim
  1. Seperate an abdomen from the body (up and down, carefully)
  2. Softening and scaling in 10% KOH in hot water bath (100℃) for about 5 minutes.
    [notice] This part repeat until scales is removed copmpletely.
  3. Neutralization in weak 10% acetic acid at room temperature (3~4 minutes). Continue to scale.
  4. Washing in D.W.(more than 5 minutes)
  5. No move abdomen, reduce a little D.W.(by pipette) and add 50% ethyl alcohol. No move abdomen, reduce 50% ethyl alcohol partly, and add little pure ethyl alcohol.
  6. Seperate genitalia from the abdomen in about 70% ethyl alcohol (on microscope) Or separate genitalia after dying(mercurochrome + chlorazol black E.)
  7. Stain with mercurochrome after washing for about 5 minutes. (dying harden part)
  8. Stain with chlorazol black E. after washing for about 5 minutes. (dying soft part)
  9. Dehydration and fixation in alcohol in order(70%→90%→100%).
    [notice] No move abdomen & genitalia, reduce partly a little alcohol and add a little ethyl alcohol.
  10. Move in xylene after dehydration in 100% alcohol (fixation).
  11. Sealing off canada balsam and labeling.
  12. Attach a label to a preparat
  13. Record info of species in the preparat book(dried insect label data and dissection info etc. for cross-reference)
  14. Add dessection data label in dried specimen(with or without dissection, preparat number)
  15. Warming in temp. 40~45℃ for about 2 weeks. (bubble removal)

position of cover glass on slide glass

Position of cover glass on slide glass.
The way in which I use will help you in your slide arrangement.

Preserving of larvae and pupa
Both larvae and pupa can be killed by immersing in hot water for a few minutes.
If a small hole is then drilled in each, they may be gently dried without distortion. Alternatively they may be stored in 70% alcohol.

Preperation of larvae and pupa
  1. Heat larva or pupa in hot water bath (about 100℃) for short time.
  2. Keep in 70% ethyle alcohol vial.
  3. Labeling (by pencil)

Labeling
  • label for slide
  • UIB : University of Incheon, Dept. of Biology
    61424 : 6(Slide ID no.), 14(specimen ID no), 2(female genitalia), 4(4th slide specimen)
    ※ meaning of 4th number : 1(genitalia, male), 2(genitalia, female), 3(wing venation, male), 4(wing venation, female), 5(larve), 6(leg) etc.

  • preparat example
    [Permanent preparat]

Book record of preparat sample
Record the information of the permanent sample on the books. It is difficult for everyone to manage a sample using a database. Even if you have built a database, it is safe to create a book with original data. The format is unspecified. As you write, you can add items to organize by taxon. However, there are items that must be written on the books.
  1. Collecting label record
    - Record Collecting label data(date, collector, location info etc.) of specific specimen to be dissected.
  2. ID no
    - Unique number in original sample to be dissected
    - Unique number of slide specimen
  3. Dissection information
    - part to be dissected(genitalia, male/female, wing, etc.), Dissection date, Name of dissenter, Researcher id number or What distinguishes a researcher(Student ID, belong)
  4. Sealing material
    - Canada balsam etc.
    - There are many ways to make a permanent sample. Also a temporary seal for later. If you write down the material, it is possible to soften again later.
  5. Etc
    - There are cases in which dissection is not made as intended for various reasons.
    - If you lose a part, if the sample is unique, if there is no abdomen from the beginning, etc.
    - These records appear to be a very personal record, but they are also of great help to those who do research as well as themselves.


Arrangement and Storage
store
Dried specimen - adult, pupal case, host scar etc.
slide
Slide specimen(genitalia, wing etc) - by canada balsam
double vial
Alcohol sample(double glass vials) - alcohol(50%~70%)

Share know-how about insect specimen preparation
I have collected data about insect specimen preparation. Please share it with me if you have a data. I will post your data and its source on this website. Please send me your data by email.
tineidofkorea@gmail.com / tineid@naver.com

Other Links
Preparing Dissections and Slide Mounts of Moths, including Wing Venation, Genitalia, and Whole Bodies. by Sangmi Lee and Richard L. Brown
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