After I have reviewed the Haemocyte types in honey bee, therefor, I will provide you how apis mellifera haemocytes appear under the microscope, their basophilic, acidophilic colors and how Haemocytes Giemsa Staining appear.
In this article, we will start from the beginning, from collecting haemolyph, passing through preparation of giemsa, consequently, ending up with detection of basicity and acidity of each type of haemocyte.
Hemolymph Collection from Apidae Adults
This is based on the research “A New Method for Quick and Easy Hemolymph Collection from Apidae Adults” by Grzegorz Borsuk *, Aneta A. Ptaszyńska , Krzysztof Olszewski , Marcin Domaciuk Patcharin Krutmuang Jerzy Paleolog.
Biological material used in the AMHS Methods
- Immediately after emergence, Apis mellifera carnica workers and drones were individuallymarked with a paint marker and left inside their hive.
- Upon reaching 10 days of age, 40marked workers and 40 marked drones were captured, placed in two separate cages, and transported to the laboratory for hemolymph sampling.
- Hemolymph collection was performed immediately after the bees were delivered to the laboratory, no more than 30 minutes after their removal from their hive.
- We also removed 40 individually caged virgin 4-day-old honeybee queens, each accompanied by five workers, from a colony lacking a nurse queen.
- Hemolymph was collected from these queens within 30 minutes of their removal from the colony.
- Of the 10 hemolymph samples taken from the queens, extracted by AMHS .
Steps for Haemolymph Collection from adult workers
- Gas burner.
- Rectangular Styrofoam plate for holding an insect.
- An automatic pipette, pipette tips
- 70% ethanol.
- Cotton swabs.
- Ice-bath to cool 0.2-mL Eppendorf tubes.
- The equipment and laboratory room were sterilized with a UV lamp.
- The procedure was performed in the immediate vicinity of a working gas burner to maintain sterile air.
- MRS medium to confirm Hemolymph samples’ sterility.
- Sabouraud medium to prove Hemolymph samples’ sterility.
Some information you should know:
WHY DO WE USE 70% ETHANOL NOT 100% or 90% or 95%?
- Because you need also water to denature the proteins, enough water but not too much, not to dilute ethanol.
- 95% ethanol evaporates very quickly at room temp, thus 70% is more effective.
- 90% of alcohol has tendency to coagulate protein .Suppose the pure alcohol is poured over the bacterial cell .The alcohol will go through the cell wall of the bacteria in all directions ,coagulating the protein just inside the cell wall . So in this case the cell would get sealed & would become inactive but not dead.
- 70% of alcohol, if it is poured over a bacterial cell, it also coagulates protein but at slow rate .So that it can get penetrated all over the cell. Now the entire cell is coagulated & the cell finally dies.
- Bacterial growth medium is so-named by its inventors: de Man, Rogosa and Sharpe.
- MRS contains sodium acetate, which suppresses the growth of many competing bacteria.
- Samples’ sterility.
- Is named after,Raymond Sabouraud in 1892.
- A type ofagar growth media containing peptones.
- Used to cultivatedermatophytes and other types of fungi, and can also grow filamentous bacteria such as Nocardia.
- The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth.
- Samples’ sterility.
Method of the work
- 40 Honey bee workers, 10 days age, large in size.
- Hold the bee from its thorax on the Styrofoam plate.
- take cotton swaps, merge it in 70% ethanol; disinfect the area around the antenna.
- With the tweezers, detach the antenna, and then we’ll notice that a drop of haemolymph will appear.
- Press on the abdomen to get large drop of the haemolyphm.
- using the automatic pipette, collect the drops and put them in eppendorf tube.
- The eppendorf tube will be placed immediately in an ice-bath (2±4EC) to prevent melanization.
- Hemolymph sampled in this manner, could be immediately analyzed or frozen at −40EC for future studies (storage at −80EC is recommended if the storage period is to be longer than one month).
- These samples placed on MRS medium to confirm their sterility.
- Samples placed on Sabouraud medium to prove their sterility.
Why I preferred AMHS over other methods?
- AMHS provide short time for collecting haemolymph that decrease the risk of melanizattion.
- Low number of individuals used.
- Larger insect size was associated with easier and faster hemolymph sampling, and with greater volumes of collected hemolymph.
- In TCHS method, each of these procedures carries the risk that the ventriculus will be punctured, leading to sample contamination with intestine contents. Such contamination not only renders the sample unusable, but also requires that the capillary must be replaced with a new one or cleaned and disinfected before the next useذthus, increasing the overall hemolymph collection time.
- Hemolymph sterility can also be compromised by puncturing the membrane connecting abdomen segments.
- Moreover, hemolymph collection by decapitation may contaminate the hemolymph with nectar leaking from the bee’s stomach.
- Ethanol disinfection and limited contact between the hemolymph drop and the bee body would ensure sample sterility.
- The incubation of hemolymph acquired by AMHS on both MRS and Sabouraud agar mediums confirmed the hemolymph sterility.
Making a Peripheral Blood Smear
After we collected the blood sample, we will then prepare the blood smear so it will be ready for giemsa staining.
- A Romanovsky type of stain named after German chemist and bacteriologist Gustav Giemsa, and is often used in histopathological diagnosis and cytogenetics.
- Giemsa is a dye consisting of methylene blue-eosin and methylene blue. Named after Gustav Giemsa, a German chemist and bacteriologist.
- This dye solution is used in histopathologic diagnosis.
- It is used in staining blood films. Because of the basic and acidic dyes it contains it is favored in differentiating acid and basic granules in granulocyte.
- Essential in determining the presence of negri bodies, Tunga species, spirochetes and other parasitic protozoans in blood.
- Giemsa stain is a classic blood film stain for peripheral blood smears and bone marrow Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta.
- Used to visualize the classic “safety pin” shape in Yersinia pestis.
STAINING HAEMOLYMPH WITH GIEMSA STAINING
After we collect the haemolymph samples, we will stain the blood smear to know the acidity and basicity of each type of cells.
Watch the video
Basophilic cytoplasm (blue color) and acidophilic nucleus (dark color)
Acidophilic cytoplasm and basophilic nucleus
Whereas, Cytoplasm Acidophilic,we can find nucleus basophilic
Finally, Cytoplasm basophilic and nucleus acidophilic