suppl Berghammer et al

Electronic Supplementary Material

Andreas Berghammer · Gregor Bucher · Florian Maderspacher · Martin Klingler

A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum

Dev Genes Evol (1999) 209: 382–389

Manufacture of Tribolium stockkeeping tools

Note: Figs. 1 to 3 mentioned below refer to the printed article

Large and small blocks
To assemble large and small blocks, three polystyrene vials are tied together with rubber bands, upon which the contacts between the vials are moistened with some drops of acetone. After drying in a hood for a few minutes, the vials remain firmly fused together, and additional vials can be added until, in several steps, the block is complete. Care must be taken to ensure that the openings of all vials are level such that the contents of one block can be poured into another block with minimal loss of flour. By this technique, one person can fabricate about 50 blocks in one day.

Small and large beetle sieves
These sieves consist of a polyamid screen of 0.80 mm mesh width, which is glued onto a support made of polyacryl (polymethyl-methacrylate) plates, 6 mm strong, into which openings 22 or 30 mm wide are cut, respectively. Strips of polyacryl are affixed as rails above and below this support to prevent the sanded blocks from slipping. Figure 4 provides the measures for the small beetle sieve (rails are depicted as gray rectangles). The corresponding measures for the large beetle sieve are 0.0 - 20.0 -38.1 - 56.1 - 74.3 - 92.2 - 110.6 - 128.5 - 146.5 - 164.6 - 182.7 - 200.7 - 218.8 - 238.8 mm (top row) and 201.3 - 181.3 - 163.1 - 131.8 - 100.6 - 69.3 - 38.1 - 20.0 - 0.0 mm (right column).

Fig. 4 Design and measures of the small beetle sieve

Egg sieve
The egg sieve is mill-cut from a block of polyacryl sized 50 x 141.5 x 169.7 mm. 25 wells are cut through this block, each 25 mm wide, such that the walls separating the wells are at least 2 mm strong (Fig. 2D). The arrangement of these wells is the same as in the top plate of the egg funnel (see below). Therefore, the exact drilling positions are the same as those depicted in Fig. 5 for the top plate of the egg funnel (top). To lock small sanded blocks in place on top of the egg sieve, a rectangle of 150 x 122 mm is mill-cut into the top of the egg sieve (Fig. 2C). The same rail also fits to a corresponding elevation on the top plate of the egg funnel to ensure proper positioning of egg sieve and egg funnel during transfer of egglays to maturing blocks (Fig. 2E). At the bottom of the egg sieve, a screen of 0.30 mm mesh size (stainless steel) is affixed by a plastic glue (for example "Tangit" glue from Henkel in Düsseldorf, Germany).

Egg funnel
The egg funnel consists of two polyacryl plates which are mill-cut to either fit the egg sieve (top plate) or the maturing sieve (bottom plate). The two plates are attached by four support poles made of aluminum. 25 flexible tubes (polyvinyl chloride) connect corresponding wells of the two plates. The design of this device is shown in detail in Fig. 5. Cones cut into the top plate serve as funnels to reduce the large diameter of the egg sieve wells to the narrow inner width of the tubes. The diameter of cones, borings and tubes are such that no steps are formed where eggs falling through might get caught. Therefore, the borings at the center of the cones are 6 mm in diameter, smaller than the inner diameter of the tubes (8 mm). On the bottom side of the plat, the borings expand to 11 mm, fitting the outer diameter of the flexible tubes which are affixed to the plate by silicon glue. Into the bottom plate of the egg funnel, a rectangle of 85 x 71 mm is mill-cut to ensure proper positioning of the maturing block. 25 borings are arranged within this rectangle (for exact positions see Fig. 5). These borings again are 11 mm wide on top of the plate to hold the tubes. On the bottom of the plate, the borings decrease to 9 mm, i.e. a diameter smaller than that of the wells of the maturing sieve.

Fig. 5 Design and measures of the egg funnel device. In the center, a side view is shown; outlines of the top and bottom plates are depicted above and below, respectively

Maturing sieve
These devices are cut from 70 x 85 x 20 mm polyacryl blocks. The diameter of the 25 wells is 10 mm, and their arrangement is identical to that of the corresponding borings in the egg funnel (as described in Fig. 5 bottom). At bottom of the maturing sieves, a polyamid screen of mesh size 0.25 mm is affixed by glue (Tangit). At one end, two additional wells of 4.1 mm diameter serve to lock with the clearing blocks which carry corresponding knobs of 4.0 mm diameter (Fig. 2F).

Clearing block
The clearing blocks are similar in design as the maturing blocks, but of more resistant material (polyvinyl chloride) and only 8 mm high to allow easier access to cleared embryos. At bottom, a custom-cut microscope slide (77 x 65 x 1 mm) is glued on with silicon glue. Care must be taken to properly seal all wells when affixing the slide in order to prevent leakage of lactic acid during over night incubation. Note that this glue should be allowed to dry for about one week prior to first contact with lactic acid.

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