Nosema sampling and preparation technique

From: James C. Bach
Date: 3/8/98
Time: 8:26:11 PM
Remote Name: 206.129.139.58

Comments

I wonder about your technique of preparing your sample. You used a sieve to strain the sample after crushing it. Did you filter your sample subsequent to the sieving? What container did you put the strained mix in? Did you properly measure the mix volume? Where in the mix did you take your sample to put on the haemacytometer?

The way I prepare the mix is as follows:

Randomly remove 25 bees from a bee sample of 200 or so bees.

Remove the abdomens of the bees.

Crush the abdomens in just enough water to make possible the separation of the gut contents from the organs and abdomens.

Strain this mix through a Kimwipe filter paper. This is a type of paper that does not produce fine reisdue but is porous enough to allow the Nosema spores to pass through. To do this I put a small glass funnel into the open mouth of a graduated 150 ml test tube. The mix is poured into the Kimwipe and the liquid allowed to flow into the graduated test tube. The amount of material from the 25 bees usually amounts to about 80ml.

I then add water to make the total quantity (110ml?) suggested in the paper I sent you.

Nosema spores tend to settle to the bottom of the test tube over a short period of time.

I shake up the test tube prior to taking a sample to put on the haemocytometer.

Using a capillary tube I remove my sample from an area at the 55ml point in the test tube.

I place the sample on the haemocytometer and look at all squares to do my count. If there are a lot of spores found then I do the selected square count as you did.

Since my haemocytometer has two measuring locations I put material from the capillary tube on both squares, make my counts, and average them for the final data of spores per bee..