Disease Screening Under the Microscope

Beekeepers are advised during their beginners course and in many of the text books to check their colonies for acarine and nosema at the beginning and end of the season. Testing for these diseases involves observing samples of bees from each colony under a microscope and looking for the organisms causing the problem. The methods discussed below result in a simple yes or no, found it/didn’t find it. As many argue nosema in particular is endemic in British colonies it is possible to say “it is there if you look hard enough”. However, equipment such as haemocytometers used to count the density of spores to calculate the degree of infection in pooled samples is not available to many outside of professional laboratories.  It is therefore up to the individual beekeeper, on receiving a positive result, to decide whether to treat for the infection when it is found based on other factors- the strength of the colony, whether symptoms (dysentery) are present etc.

A detailed description of methods recommended for sampling for disease testing for use in professional laboratories can be found at  http://www.coloss.org/beebook

This is how we look for the infection at home:

First, catch your bees! The samples should be of mature bees, the thinking being that having been around the longest they are most likely to be infected. Foragers taken as they arrive at or leave the hive are the obvious candidates. You can block the entrance and scoop up the foragers as they congregate or hold something over the entrance such as a jar, until you have collected sufficient. To collect from a colony when the bees are not flying, take them from the outer combs of stores, trying to avoid the nurse bees that should be on the brood. Traditionally for nosema testing we take 30 per colony. Apparently this gives a 95% chance of detecting nosema in a colony in which 10% of the bees are infected!

Before they are dissected the bees must be killed and if the inspection is not to take place immediately they also need to be preserved. Samples of bees can be killed by placing them in the freezer or by asphyxiating them with Ethyl Acetate also know as Insect Killing Fluid (IKF). Ethyl Acetate is the active ingredient in non acetone nail varnish remover which is much cheaper to buy, although it takes slightly longer to do the job. To preserve the samples they can be left frozen or placed in alcohol until you are ready to test them.

To test for nosema, take 30 dead bees and cut off their abdomens. Add a little distilled water and grind the abdomens in a pestle and mortar to give a yellow/brown liquid. You can use a couple of spoons to do the grinding, but make sure you do a thorough job in order to release spores from the bee’s digestive tract. Take a drop of the liquid, using the pestle or a glass rod and put it on a slide and carefully drop a cover slip to spread the sample out.

The slide can then be viewed at x400 under the compound microscope. There will be quite a lot of debris from the ground bee. Nosema spores, if they are there, will look like grains of rice. The two types of nosema so far identified Nosema Apis and Nosema Ceranae are said to be of slightly different shape and size, but the differences are hard to distinguish. Putting Nigrosin stain on the slide before the sample can help make the spores stand out but it is not essential.

If nosema is found in your sample of bees and you decide to treat it there are not many options available to you. Nosema was until 2012 treated by an application of a syrup feed containing an antibiotic powder known as “fumidil B”. This is no longer available or approved for use in the UK. As Nosema has recently been classified as a fungus it may be susceptible to treatment with Thymol, which is fungicidal and is of course used by most of us already to combat the Varroa mite. You can also remove the bees from the source of the infection- spores in faecal matter from infected bees inside the hive and on brood frames. Remove the infected frames using a Shook Swarm or a Bailey Comb Change. Boxes floors and other hive parts should all be changed for cleaned and sterilised ones.  The infected frames can be either melted down and sterilised before reuse with fresh foundation or can be sterilised with Acetic Acid before reuse. Obviously changing frames can only be undertaken when conditions are suitable and the colony will need to be supported by feeding with syrup until fresh comb is drawn out.

Testing for Acarine is done under the dissecting microscope, one bee at a time. This is an infestation of the tracheae of the bee by the mite Acarapis woodi. The tiny mites enter the tracheae of the bee via the spiracles and breed there, eventually blocking the airways of the bee and reducing its lifespan and possibly introducing secondary infection from viruses.

We test for their presence by carrying out a thoracic dissection and looking for characteristic brown staining of the thoracic tracheae caused by the presence of many mites. This is done by removing its legs and mounting a dead bee on a cork at an angle allowing a view into the thorax from the neck end when the head is removed. Once the head has been removed the thoracic collar is carefully peeled away under the dissecting microscope to expose the tracheae. Normal tracheae will appear as milky white while a heavy infection will stain the tracheae with brown patches. Sometimes the individual mites can be seen as translucent shadows through the tracheae. Infected tracheae can also be dissected out and mounted in glycerine. Individual mites can then be seen under a compound microscope at x100 and above.

IBRA suggests that in order to get a statistically reliable indication of infection levels of Acarine at least 50 bees per colony should be inspected using the above method. See http://www.ibra.org.uk/articles/BEEBOOK-Vol-I-and-II for details of sequential sampling and the subsequent statistical analysis of results. Outside of the professional laboratory this would take a long time and would be extremely tedious, particularly if more than one colony was being tested! Fortunately for us the incidence of Acarine mites has apparently reduced significantly since the adoption by beekeepers of the use of chemical treatments for Varroa.