Collection of eggs from tissues

 

Author Fred Lewis, Mei-Shei Su, Frances Barnes, and Yung-san Liang

Introduction

Schistosome eggs are collected for experimental use primarily from either livers or intestinal tissue of heavily infected animal hosts. This procedure describes the isolation of eggs from the livers of S. mansoni-infected mice. In order to obtain the cleanest preparation of eggs, the mice should first be perfused of adult worms, or the final egg preparation may contain many unwanted adult worm fragments.

Equipment

Spray apparatus (2-gal  pump-type, typically found in hardware stores)

Stainless steel sieves of decreasing pore sizes (Newark Wire Cloth; mesh openings of

420 µm, 180 µm, 105 µm, and 45 µm) (photos, sieves 1and 2)

Dissecting instruments

Waring blender, with variable speed control

Glass petri dishes (100 mm dia) with flat bottoms

Falcon #2340 cell strainer, 40 µm nylon (photo)

Pasteur pipettes

Light box

15 ml conical test tubes

 

Materials and reagents

1.2% NaCl (2 liters)

S. mansoni-infected mice

Procedure

·         Dissect the livers from mice that have previously been perfused of adult worms.

·         Place the livers in 1.2% NaCl.

·         Mince the livers with scissors, then homogenize in a Waring blender, first for 10 seconds at low speed, then at intermediate and, finally, at high speed for an equal amount of time.

·         Place the homogenate in the top tier of stainless steel sieves and allow it to pass through the tier of stacked sieves, from the largest pore size on top to the smallest pore size on the bottom tier while rinsing the tissue continuously on the top sieve with a spray apparatus containing 1.2% NaCl.  Agitate the sieves throughout the entire process to ensure that most of the eggs will pass through to the lowest sieve.

·         For best results, re-homogenize the homogenate trapped on the top sieve in the Waring blender and pass the homogenate through the sieves again, using the technique described.

·         Remove the upper three sieves; the fluid remaining in the lowest sieve (45 µm pore size) will contain the eggs.

·         Pour the suspension into a glass petri dish, and add 1.2% cold NaCl so that it is about ½ full. The egg suspension will contain eggs of several stages of maturation. Cold NaCl will keep most of the eggs from hatching into miricidia.

·         To enrich the population for mature eggs, gently swirl the dish over a light box (for better visibility).

·         Mature eggs will concentrate in the center of the vortex. These can be withdrawn with a pasteur pipette and placed in a 15 ml test tube on ice. Keeping the volume of the egg suspension in the petri dish constant by adding fresh cold 1.2% NaCl as needed, continue to swirl the dish and collect eggs from the center until no more can be seen concentrating in the center.

·         Using a Falcon #2340 40 µm nylon cell strainer (photo – egg screen) (or similar device of that pore size) to concentrate the mature eggs collected with the pasteur pipette significantly facilitates this procedure. Place the cell strainer in a second glass petri dish ½ full of cold 1.2% NaCl next to the plate you are swirling, and place each pipette-full of mature eggs into the strainer until no more eggs can be seen in the center of the petri dish.

·         Discard the waste contents (immature eggs, small pieces of liver, etc.) of the petri dish, add the collected suspension of enriched eggs from the strainer to its petri dish and repeat this procedure, using a fresh petri dish of 1.2% NaCl and the cleaned out strainer for the next cycle. Use the pipette to (gently) rinse remaining eggs from the strainer into its petri dish, and continue to use the same strainer for all cycles.

·         After 3-4 complete cycles, the resulting egg population will be highly enriched in mature eggs. Two photos show the “before” and “after” stages of egg purification by this method.

·         After the last cycle, gently pipette the clean, mature eggs from the strainer (while tipping it slightly onto its side to facilitate pipetting the eggs) into a 15ml conical centrifuge tube, on ice. Fill to the top with cold 1.2% NaCl and allow eggs to settle to the bottom of the tube. Alternatively, one may gently rinse the strainer of eggs with a small volume (>10 ml)  of cold 1.2% NaCl into a clean petri dish and then pipette into the 15 ml tube, rinsing the petri dish with another 5 ml of NaCl and adding this to the tube.

 

Follow-up comments/recommendations

The greatest yield of eggs from mouse livers can be obtained from mice that were exposed to approximately 200 cercariae, 7-8 weeks prior to harvesting. With practice, one can obtain about 20,000 mature eggs/liver at this level of infection,.

Harvesting eggs from the intestinal walls of the mice is possible, but only after the intestines are cleaned of feces and rinsed in copious amounts of 1.2% NaCl. At the level of infection above, one can expect to obtain roughly ½ the number of eggs from an individual mouse’s intestines as from its liver.

 

References

Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, in Current Protocols in Immunology 19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed).  Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.