ncba nosema check protocol

7/31/2019 NCBA Nosema Check Protocol

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Checking forNosema Spores Using a

Compound Microscope

1. First check to see that all of the materials are present and that they weresuccessfully transferred to you (materials identification sheet attached).

2. Collect at least 10 forager bees, place them in a zip lock baggy, and freeze kill (putin freezer for at least 20 minutes) them until you ready for diagnoses.

3. Thaw bees 5 minutes prior to dissection.

4. Take the small scissors from the checked out dissecting kit and decapitate the bee.

5. Then take the fine forceps (tweezers) and pull the stinger and the last abdominal

segment out slowly. Typically the entire gut will come out as it is connected to thelast abdominal segment. You may have to stick the tweezers inside the abdominal

cavity and try to pull the gut out again if the gut breaks at all.

6. If you are having trouble removing the gut from the abdominal cavity (usually the

case for old or rotten bees) then just clip off the abdomen where it meets the thoraxof the bee and place the whole abdomen in the plastic grinder.

7. Add 2.5 milliliters (about teaspoon) of water per 10 bees to the grinder and grinduntil the solution looks uniform. If whole abdomen used the exoskeleton will

remain intact no matter how much grinding is done, which is fine.

8. Make a wet mount slide by taking one drop of solution and placing it on a glassslide, slowly lower a cover slip over the slide at an angle to prevent air bubble

formation.

9. Check to see that the microscope is on the lowest (4x) objective power (see

microscope diagram).

10. Take the wet mount slide and place it on the stage of the microscope, in order to do

this pull back the stage clip and then slowly release back into position so that the

wet mount slide is secured on the stage. Center the slide so that the cover slip is

below where the objective lens is.

11. Make sure the microscope is plugged in and switch on the light at the bottom of the

microscope.

12. Use the course adjustment knob and the fine focus to put what is in the water into

focus. MatureNosema spores at this magnification will be tiny specs at best.


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13. Then revolve the objective lens mount to switch to the next highest power (10x)

and bring the specs that you saw before into focus with the course adjustment and

the fine focus.

14. Lastly, revolve the objective lens mount to the next highest power (40x) and then

use the fine focus ONLY to bring the spores into focus because using the courseadjustment may drive the objective lens into the slide and destroy the lens.

15. When in focus the spores should look uniform, have bluish tinge, and be roundoval objects with thick spore coats. Under the highest power (40x)Nosema ceranae

spores should look like this if the bee is highly infected

Nosema apis spores are generally a little bit bigger and they are more roundish than oval.

Spores from each species look so similar molecular techniques are needed in the scientific

community to confirm the identity of the species. You can clean the slides and cover slipswith water and both can be re-used after drying.


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Microscope Diagrams

Stage

Power Switch

Course adjustment

Fine adjustment

Control for brightness

Objective lens (4x)

Objective lens (10x)

Controls to move thestage around

Stage clip


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Materials That Should be Present

1. Dissecting Kit with probes, forceps, scissors, scalpel, ruler, and scalpel blades

2. Compound Light Microscope

3. Glass Slides

4. Cover Slips

5. Tissue Grinder that includes a plastic tube and a pestle for grinding

Forceps

Ruler

Scalpel

Scalpel Blades

Scissors

Probes

Cover Slips

Pestle

GlassSlides

Tissue Grinder

ncba nosema check protocol

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