Preparing mites for study or photography
Several different techniques can be used for mounting and photographing mites. General recommendations can be found in Krantz & Walter, 2009. Below we briefly summarize the standard techniques we use to handle specimens for both molecular and morphological work at the University of Michigan Museum of Zoology.
Reagents and equipment needed:
- Nesbitt's clearing agent or lactophenol (Krantz and Walter, 2009, p. 89)
- Hoyer's mounting medium (Krantz and Walter, 2009, p. 91)
- 95-100% ethanol
- Electrical insulating varnish for ringing (e.g., Glyptal® or others)
- distilled water
- fine watchmaker's forceps
- minuten pin, finely sharpened, inserted in a wood matchstick base and fixed in place with glue
- 3-well glass plates
- capillary tubes and disposable plastic pipettes
- microscope slides and cover slips
- 1.5ml glass flat bottom vials
- heating and drying oven set to 50°C
- fume hood
- microscope equipped with differential interference contrast (DIC) and phase contrast optics, and a digital camera
- computer with image processing software installed
Mounting mites
- Prior to removing mites from bee specimens or nest material, we recommend you document the mite location(s) by photographing them and recording collection information (host species, location on bee, geographic locality, date, etc.)
- Collect mites with a pin (or by alcohol wash) and save them in 95-100% ethanol. Store in freezer at -80°C (in the field, regular household freezer/cooler with ice can be used). Avoid direct sunlight. Alcohol-preserved mites can be kept in the dark at room temperature for several days without noticeable effect on DNA quality.
- With a disposable pipette or capillary tube, move mites to a 3-well glass plate. For maceration/clearing, add 0.5-1mL of Nesbitt’s fluid, leave for 12-24 hrs or until mites are clear. Heating expedites clearing, but may damage specimens (setae can be broken off). Maceration is needed to soften specimen prior to mounting. Clearing removes highly refractive guanine crystals from mite specimens. Skip this step if mites are very delicate and soft (e.g., Tydeidae). Specimens can be put in Nesbitt’s fluid directly (no preservation in alcohol needed) but a subset of alcohol-preserved co-vouchers should be kept in alcohol for DNA work.
- Wash cleared specimens with distilled water (this step is optional). Washing is needed, especially for large specimens, to remove residuals of Nesbitt’s fluid which can crystalize over time in the mounting medium.
- With a pin, transfer mite specimen to a small drop of Hoyer's medium on microscope slide. Position mite so it is oriented upside down on the slide. Put a cover slip on the specimen, gently press it to relax the specimen (if needed), and evenly spread the mounting medium.
- Label the slide with a field number using a marker.
- Put a 1.5ml glass flat bottom vial filled with distilled water on the cover slip for 24 hrs. This will flatten the specimen and prevent the specimen from bending the appendages in the mounting medium.
- Remove the flat bottom vial and transfer the slide to oven. Dry for one week at 50°C.
- Using a fume hood, ring the cover slip with an insulating varnish and keep for 24 hrs at room temperature. Repeat this step one more time. Avoid using nail polish, house paint or other brittle materials for ringing because they crack over time.
- Put permanentpermanent:
associated exclusively with bees or their close relative, wasps; cannot live without these hosts
label on the right side of the slide.
Photographing mites
- Start with your microscope's brightfield settings. Adjust contrast using the iris diaphragm of the condenser to make sure it is sufficient to see all details of the specimen. Typically the lowest values are the best.
- If brightfield does not show transparent structures, consider using differential interference contrast (DIC) or phase contrast. See a discussion about the difference between these two. In my experience, DIC has a low depth of field (as compared to phase contrast and brightfield) but this may depend on the microscope. With DIC it is very difficult to make a picture of the entire specimen with all dorsal setae clearly visible – they usually can be clearly seen only when the picture is zoomed in. Phase contrast is typically the best choice for research pictures, but these look unnaturally dark and details may be obscured by phase contrast halos. As compared to brightfield, DIC, and especially phase contrast photos, are very difficult to digitally edit.
- In your camera settings, adjust the contrast, color temperature, and (rarely) gamma, so the background appears neutral gray to almost white (for brightfield and DIC).
- If possible, save the photo in raw format, so it can be further processed using image editing software.
- If the specimen is too large for your camera setup and you have Photoshop, you can take a series of overlapping pictures in the same focal plane and combine them (Menu File-Photomerge). Use the Reposition option with Blending as a starting point. Sometimes, the Auto option may work better but it can introduce artifacts (e.g., distorted shapes). If automatic photomerge fails, combine a subset of images at a time using the Photoshop menu command (Edit: Auto-Align Layers) or manual alignment, followed by blending (select layers and press Edit: Auto-Blend Layers).
