3.1.2. Preparation and measuring
- Dissect each bee individually on a flat glass surface or in a petri dish (Fig. 3).
- Mount the wings dry on microscope slides after rinsing in ethanol with decreasing concentrations (70% to 20%) and distilled water. Care has to be taken to mount the wings flat on the slide, without any folds or distortions. Taking care that all wings face the same direction will facilitate measurements. Cover with second microscope slide and fix together with masking tape. Label with sample ID.
- Carefully clean sternites and tergites from adhering tissue with forceps and/or a small spatula so as not to tear the chitin. If the chitin is very tender (e.g. because material is very old or has previously been subjected to freezing), tissue can be removed by immersion into concentrated KOH solution for a few hours (obey operator safety precautions: wear gloves and goggles) and subsequent rinsing in H2O.
- Embed flat body parts (legs, sternites, proboscis) in a solution of gum Arabic (not permanent), Euparal or Canada balsam (permanent) on microscope slides. Take care to avoid any air bubbles in the solution. Mounting body parts to face the same direction will facilitate measurements (Fig. 4).
- Cover with a second microscope slide and label with sample ID.
- When using Euparal or Canada balsam, keep the slides on a warming plate for several hours for the medium to harden.
- Because of their natural rounded shape, tergites are best glued to 10 mm glass rods covered with gum Arabic, Euparal or Canada balsam. Cover rod and tergites with clear tape after mounting. Use a spacer to suspend rods from surface to avoid sticking.
After mounting, the body parts can be measured. There is a wide range of measuring methods, including microscopes with ocular scales, online video measurements, and photographing the slides and analyzing them with commercial or freeware picture analysis programs. As long as devices are carefully calibrated, the specific methods do not matter too much. Precise calibration, however, is a critical prerequisite for a true representation of all measurements related to size and its importance cannot be emphasized enough!
- The calibration procedure greatly depends on the measuring method used (computer-aided techniques will mostly come with inbuilt calibration steps).
- Measure any distance using your own measuring device (microscope eyepiece, computer program etc.).
- Use a micrometer slide or similar exact measuring device to calculate the real distance from the units of the device used in step above.
- It is important to re-calibrate after each change of magnification.
Mostly, measurements are taken manually, but often with customized digital support as introduced by Daly (1982), which greatly improved precision. Any digitizing picture analysis program might be used, after verifying that it possesses sufficient precision. In geometric morphometry, a commonly used program is TPSdig (Rohlf, 2001), but efforts have been made to use automation for capturing wing morphometry. Quezada-Euán et al. (2003) and Steinhage et al. (1997) developed a semi-automated method to obtain wing measures that reduced the analysis time and improved precision for identifying bee species; this method was later improved to an automated system (Steinhage et al., 2007). The coordinates of the eighteen wing landmarks can also be determined automatically using the DrawWing software (Tofilski, 2004).
In addition, to achieve the best possible compatibility of data obtained
by different laboratories, great care has to be taken to always carry out
measurements or assessments in exactly the same way. This is of essential
importance, since particular personal habits or "measuring
traditions" within different laboratories can introduce biases that can
become troublesome and render data sets incompatible. It is thus essential for
inexperienced researchers, before starting a new morphometric project, to seek
advice from experienced laboratories and to use repeated measurements from the
same samples for cross-checking. Even between laboratories involved in
morphometry there is a need for crosschecking to standardize methods in detail.
Fig. 3. Dissection of bees for the full character suite of classical morphometry. Photo: Mohammed Al Sharhi.
Fig. 4. Mounted body parts for measuring. Photo: Mohammed Al Sharhi.