This method can help answer key questions in the honey bee nutrition field, about the link between poor nutrition and hypopharyngeal gland growth and development. The main advantage of this technique is that one can use it to easily stain, visualize, and measure bee hypopharyngeal glands. Generally, individuals new to this method will struggle because the hypopharyngeal glands can be difficult to locate and dissect without visual guidance.
Before beginning the procedure, pour melted cool setting wax in a glass Petri dish. Add 20 microliters of freshly prepared Giemsa stain into one well, and 50 to 100 microliters of saline into an adjacent well of one microscope slide, per nurse worker bee to be processed. When the wax has set, use 10 millimeter micro spring scissors to remove the head from the first bee and use forceps, a dissecting microscope, and a wax carving pen to embed the head front-side-up in the wax dissection plate.
Insert pins in the eyes and mouth to secure the head to the plate, and use a sharp, breakable razor blade fixed into a pin vise to make a two to three millimeter incision between the eyes and mandibles on each side of the face plate. Gently run the micro scissors under the face plate and cut the antennal nerve that runs between the antenna and the brain. Using fine forceps, grab the face plate by the mouth, and pin the tissue face-side-down to the wax with fine point forceps and a pin.
Next, pipette 20 microliters of PBS onto the open section of the head. The hypopharyngeal glands look like a string of pearls and are located on top of the brain if intact. Using super fine point forceps, gently harvest one of the glands for immediate immersion in the Giemsa stain on the microscopy slide.
After a five minute incubation, transfer the gland to the pool of saline on the same slide, cutting the gland into smaller pieces under the microscope with the micro scissors as necessary. To measure the glands, open the measurement program for the light microscope, and select the 10 times magnification. Place the slide onto the microscope stage and manually locate the glands through the eye piece.
Increase the magnification to 60 to 80 times and manually adjust the focus of the glands at the new magnification. Further adjust the focus of the glands on the live image on the monitor and enter the image name and sample description into image name box. Click Acquire Image to obtain an image of the glands and under the Analysis tab, select the Area tool and un-select Value under Display tabs.
Unit will also be un-selected. Set all of the other settings to default. To measure the acini, continuously click or hold down the left mouse button while tracing one acinus perimeter at a time as carefully as possible.
Once all of the acini from a single image have been measured select Create Report, confirming that the appropriate options are selected, and click Export. Then save the report in a suitable format and clean up the work area and materials as appropriate. It is difficult to find a proper contrast and defined edges of the acini in unstained tissues.
In stained hypopharyngeal glands, the edges of the acini are sharp because of the improved contrast between the stained tissue and the white background. The hypopharyngeal glands grow over time in bees that are fed honey mixed with pollen, compared to bees that are fed honey alone. Hypopharyngeal gland size is also sensitive to the type of pollen provided.
For example, bees fed desert or almond pollen demonstrate glands of an equivalent size, while bees fed southeastern pollen diets exhibit glands that are larger than honey alone fed bees, but smaller than glands from bees fed either almond or desert pollen. While attempting this procedure, it's important to remember to carefully dissect the glands and to not over-or under-stain them. Don't forget that working with Giemsa stain can be hazardous and that precautions, such as keeping the stain away from flames, using proper disposal, and wearing gloves and eye protection should always be taken while performing this procedure.
