Experiment One
Nosema Sampling Test Results

Purpose: 

1. To establish a baseline measurement for nosema in our hives for further experiments and 

2. To determine our own ability to measure nosema consistently using an hemacytometer (specialized microscope slide) and a child's microscope.   

This experiment is intended to determine whether a 50 bee sample, taken from a ~100 bee sample collected from 12 hives in a yard of 24 hives will give consistent  results that can be used to determine the success or failure of a treatment.

Method and materials: 

50 whole or partial bees were mashed with a spoon in 50 ml of water measured with kitchen utensils. The resulting slurry was then shaken and strained through a fine kitchen sieve to remove debris and then the number of spores were counted using a Hemacytometer and a 400 power  microscope. 

Initially a child's cheap microscope was used, however when we were unable to find any spores, a high school type microscope was borrowed. Total nosema spores were counted on an hemacytometer in 5 blocks of 16 squares each and the result multiplied by 4 X 10^6 and divided by 80 to estimate average spore count per bee. (In simple terms, that means 50,000 spores per bee were estimated for each spore actually counted on the hemacytometer).

Samples of about 150 bees were taken from 12 hives each in four bee yards. Duplicate samples were taken at the same time and place from the same hives by the same person and were intended to allow evaluation of the meaningfulness of the results. Samples were blind coded by a second party before lab work and tabulation to avoid inadvertent operator bias. As it turned out, the samples were not well mixed up in initial tests, and therefore the results were unduly affected by a few hives in each yard.  

The samples were analyzed twice by the same operator.  After considerable discussion about why we were not getting consistent results, the samples were done a third and final time.  The third effort reflects the learning that took place, and are likely consistent with the actual average levels in the bees sampled.  

Note: We did not count anything on the re-test that was not a definitely a nosema spore. In earlier counts, we counted anything that we thought might be one.

Code

Yard Name

Hemacytometer
Spore Counts*

Average
Spores/bee

Comments

5

DRSouth

28

16

2,200,000

Abdomens Only

1

DRSouth

0

4

200,000

Whole Bees

2

Zieglers'

1

50,000

Whole Bees

3

Zieglers'

3

150,000

Abdomens Only

6

DRNorth

1

50,000

Abdomens Only

8

DRNorth

3

150,000

Abdomens Only

7

Brian's

0

1

50,000

Abdomens Only

4

Brian's

5

10

750,000

Abdomens Only

Observations: 

Initially, comparisons of any two samples taken at the same time etc. did not seem very consistent. Counts range from near zero to millions from samples that should have been identical. Later tests showed that nosema levels in all apiaries sampled were very low, so that only one to three (total) actual spores were found in any one measurement.  This low level made it hard to find something that was not there.  

With the exception of sample #5, the results were quite consistent.

Notes:: Examination of samples sent by Eric for calibration of our technique resulted in identical counts to his, and demonstrated that --  with the exception of our failure to mix our samples well (we took bees from the top of the jar the first time) -- our microscope methods were good.

* These were two different counts from the same sample of ground up bees. (+/-1) allows for the difficulty of deciding exactly whether a shape in the microscope is truly a spore or just trash.  '-' in the table means that no second measurement was made from the slurry.

Sampling with this small number of bees (50/sample) is subject to considerable variation inasmuch as it appears that when most bees sampled have no nosema, but occasional individual bees have may have high nosema counts, the random factor may skew the results between two samples taken at the same time and place. For increased accuracy larger samples are necessary. 

Experiments to measure success of various treatments for nosema would be meaningless based on these gross measurements. We do not here have good, accurate measurement to proceed with meaningful tests of nosema treatments. If we wish to proceed, we must use much larger samples. 

Revised Conclusions: We had such low levels of nosema in the yards we sampled that it was difficult to reach conclusions about the sampling technique.  However we are now reasonably satisfied that we do get consistent results, at least at this infestation level in terms of magnitude, if not exact spore counts. In the case of the vastly anomalous result in sample #5, we postulate that one highly infested bee in the sample could affect the results as was observed there.