126.96.36.199. Individual bees or pooled samples
To determine the degree of infection in colonies from pooled samples of bees, the required precision of the diagnosis should first be determined (see section 188.8.131.52).
- Measure 1 ml of water per bee in the pooled sample.
- Grind the required number of bees, their abdomen or ventriculus (see section 184.108.40.206.) thoroughly in water (1/3 of the measured amount) in a mortar with a pestle.
- Add the remaining water and mix thoroughly.
- Add a small droplet to a haemocytometer and allow spores to settle
before counting (see the miscellaneous methods paper of the BEEBOOK (Human et al., 2013).
By examining infection in each bee in a sample, the proportion of infected bees can be determined. For N. apis, this is a better measurement than the average spore count per bee in a pooled sample (pool of individual bees), if the objective is to measure the influence of infection on honey yield (Fries et al., 1984). To determine the proportion of infected bees within a colony, individual bees must be examined.
- Use 1 ml of water per bee. If other amounts of water are used it is necessary that the dilution factor is stated. Depending on the content of the bees’ intestine with sometimes massive pollen amounts, dilution may be necessary to actually see the spores in light microscopy.
- Grind each bee, their abdomen or ventriculus separately (see section 220.127.116.11.) thoroughly in water in a mortar with a pestle.
- Look for the presence of spores in the macerate under a microscope (see section
The use of 96-well PCR plates with a single bee or abdomen or ventriculus per well for maceration can be useful for high throughput analysis. If using a 96-well plate, a system (manual or automatic) to guarantee that every bee or abdomen is completely macerated to release the spores to be detected needs to be developed. Nevertheless, there is a highly significant and positive correlation between the proportion of infected bees and average spore counts per bee. Thus, under most circumstances, the less labour intensive use of pooled samples can be used, rather than determining the infection status of at least 59 individual bees (see section 18.104.22.168. and the statistics paper of the BEEBOOK (Human et al., 2013)).
Use of dead bees from the floor board of live colonies should be avoided, since the correlation with the health status of the live bees is too low (Fries et al., 1984).
It remains to be determined whether the relations described here are the same for N. ceranae.