Notes on microscopy for bee disease diagnosis
1. Tracheal mites (Acarapis woodi)
1.1. When to sample
When trying to detect tracheal mites, sampling time is crucial. Infestation by tracheal mites
(HBTM) varies through time. For detection of HBTM in colonies, bees should be collected in
winter or early spring when HBTM populations are highest because of the reduced bee brood
production. During this time, a high proportion of older, overwintering bees are present in the
colonies, and the mites have had a long time to reproduce. The large number of actively
feeding mites can cause the tracheae to turn black. Infestation of HBTM decreases in summer
due to the dilution of mite populations as they enter the large population of emerging host
bees.
1.2. Collecting bee samples
Because HBTM infestations are influenced by the age of bees, the location within the hive from
which bees are sampled should be considered. Since queens can be found on honey frames, it
is recommended to examine frames of the entire colony to find the queen before taking any
samples; this will ensure that the queen will not end up in the sample jar. Collect adult drones
for sampling as well, as they tend to have higher mite abundance than worker bees. However,
because drones are seasonal, adult worker bees are most often sampled for detection or
surveillance purposes.
Procedure:

Collect about 50 bees from frames in the honey super or from the inner covers where
older bees congregate. Highly-infested, older bees will have darkened trachea and many
stages of mites, although some of the younger female mites may have migrated out of
the tubes.

If young nurse bees (found in the broodnest) are present, these young bees may only
have foundress mites that just started reproducing. The presence of one foundress or a
foundress and an egg near the opening of the trachea may be difficult to detect. Thus,
to determine mite abundance (number of mites per bee) or mite intensity (number of
mites per infested bee), it is best to sample bees from the honey and pollen regions of
brood frames, where a good mixture of young and old bees is generally found.

Collect bees by scooping bees with a plastic cup or matchbox directly from the frames or
inner cover.

Place samples into vials or plastic bags or sample directly into a wide-mouth jar
containing 70% ethanol. Label each container or plastic bag with location, colony
number and the date the samples were collected.

Although bees can be preserved in 70% alcohol, fresh or frozen bees are easier to
dissect. Examination of tracheae is easier when no alcohol is inside them. Bees stored for
a long time in alcohol will have darker muscle and tracheal tissues, making the mites
harder to detect.
1.3. Number of bees to be examined to detect the presence of HBTM
About 30-50 bees should be examined per colony. Because the tracheal networks on the two
sides of the bees do not interconnect, this represents independent samples of 60-100 tracheae.
1.4. Microscopic detection of HBTM
The morphological technique most frequently used involves examining the prothoracic tracheae
under a microscope. Detection of low level infestation by A. woodi requires careful microscopic
examination of tracheae. When the infestation is heavy, the trachea will turn opaque and
discoloured and mites can be noticed without the aid of a microscope. One method is to pull off
the head and collar of a bee and examine the trachea. See Jamie Ellis video below.
Procedure:

The bee is placed under a dissecting microscope, held prone with forceps (across
abdomen) and the head and the first pair of legs is scraped off using a scalpel or razor
blade.

The ring of prothoracic sclerite (collar) is also removed using a fine forceps.

The exposed tracheae of both sides are removed after carefully detaching them from the
thoracic wall.

The tracheae are removed and placed on a glass slide and examined under a microscope
for mites; this technique is very time-consuming and also has the possibility to lose mites
while separating tracheae from the thoracic wall and transferring them to the slide.
A. woodi
2. Nosema spp.
2.1. Sampling
For the diagnosis and detection of Nosema spp. infected colonies, the oldest honey bees should
be the target population, since they are more frequently infected compared to younger bees.
Forager bees can be sampled outside the entrance. This method is useful in all areas during
flight and foraging conditions. Caution should be taken to avoid collection of young bees
performing their orientation flights. The time of the day of these flights could change in
different geographic areas. Bees that conduct their orientation flights are easily recognized by
the hovering behaviour in large numbers outside the entrance. When this behaviour is seen,
attempts to sample for foragers should be avoided.
During non-flight conditions, old bees may still be the target population for diagnosis. To
avoid sampling of newly emerged, uninfected bees, the samples can either be taken from
peripheral combs in the brood area, without hatching bees, or in a super above a queen
excluder. Samples should preferably be stored in the deep freezer until further processing.
2.2. Dissecting the ventriculus
With some training, it is easy to pull the ventriculus (D) out from bees using forceps.
Procedure:

Grip over the last abdominal dorsal and ventral segments with the forceps

Hold the abdomen in the other hand,

Slowly pull apart; the posterior portion of the alimentary canal comes out, sometimes
with, sometimes without, the honey sac attached to the anterior end of the ventriculus
A = sting apparatus; B = rectum; C = small intestine; and D = ventriculus (midgut). Line = 2
mm
2.3. Light microscopy
A compound microscope using 400X magnification is sufficient for observing Nosema spp.
spores in macerated bee preparations. Use of phase contrast light microscopy facilitates
distinguishing spores of microsporidia from yeast or other particles.
Although the differences in spore size between N. ceranae (A) and N. apis (B) are not
immediately apparent in light microscopy, there is a consistent difference. Spores of N. ceranae
are clearly smaller compared to spores of N. apis. Fresh, unfixed spores of N. apis measure
approximately 6 x 3 µm; whereas, fresh spores of N. ceranae measure approximately 4.7 x 2.7
µm. Although there is a slight overlap, with the smallest N. apis spores being smaller than the
largest N. ceranae spores, the average spore size of N. apis is approximately 1 µm larger in
length.
In contrast to spores of N. apis, the spores of N. ceranae are often slightly bent, and
appear less uniform in shape compared to N. apis spores. Although the difference in the size of
spores between these species is clear, it may still be difficult to detect the difference in routine
diagnosis of infected bees using light microscopy. This is particularly true because mixed
infections of both species can occur, even in individual bees.
Because of their light refractive properties, spores of Nosema spp. are easily seen
without contrast colouring in the light microscope in water squash preparations at 200-400 X
magnifications.
N. apis (B) and N. ceranae (A)
3. European and American foulbrood.
3.1. Microscopy
The laboratory diagnosis of European foulbrood is based on the identification of Melisococcus
plutonius in affected brood. The laboratory diagnosis of American foulbrood is based on the
identification of Pacenibacillus larvae in affected brood. One method for identification is the
microscopy of smears prepared from diseased brood.
3.2. Carbol fuchsin staining
Procedure:

Select larvae and/or pupae showing signs of foulbrood and place them on a microscope
slide.

Using a swab or stick, pulp the larvae together and spread over the slide pushing any
excess off one end, to leave a thin smear. Allow the smears to dry before processing.

Heat fix by flaming the slide over a burner a few times and flood with 0.2 % carbol
fuchsin for 30 seconds.

Wash off the stain and either air dry or gently blot dry before microscopic examination at
1000 times magnification.
A diagnosis of European foulbrood is made if examination revealed M. plutonius like
organisms. Organisms are considered to be M. plutonius if they are lanceolate cocci,
approximately 0.5 x 1.0 µm. E. faecalis is very like M. plutonius in appearance and has
frequently been confused as being the causative organism.
M. plutonius
A diagnosis of American foulbrood is made if examination revealed Paenibacillus larvae
like organisms.
P. larvae.
Further reading:Dade, H.A. (1994). Anatomy and dissection of the honey bee. International Bee Research
Association; UK. 196 pp. ISBN: 978-0860982142
Maurer, R. (2012). Practical microscopy for beekeepers. Bee Craft Ltd; UK. 96 pp.
ISBN: 978-0900147135
Meyer, O. (2010). Microscopy on a shoestring for beekeepers and naturalists. Northern Bee
Books; UK. 114 pp. ISBN: 978-0907908104
Stell, I.M. (2012). Understanding bee anatomy: a full colour guide. The Catford Press; UK. 208
pp: ISBN: 978-0957422803
YouTube videos from Dr Jamie Ellis, University of Florida:Tracheal mites:https://www.youtube.com/watch?v=6wy2PG_MB4Y
Nosema disease:https://www.youtube.com/watch?v=AMDN7r1SfbY
Foulbrood:https://www.youtube.com/watch?v=s74WIPpGRHs
Norman Carreck.
October 2016.
Science Director,
International Bee Research Association,
Laboratory of Apiculture and Social Insects,
School of Life Sciences,
University of Sussex,
Falmer,
Brighton,
East Sussex,
BN1 9QG,
United Kingdom.
Tel: +44 (0) 1273 872587
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Email: [email protected]
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