Tilletia indica - The Food and Environment Research Agency - Defra
Tilletia indica - The Food and Environment Research Agency - Defra
Tilletia indica - The Food and Environment Research Agency - Defra
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Materials<br />
30% Bleach solution (3 parts household bleach: 7 parts water; c. 1.6% active NaOCl)<br />
Wash water = 0.01% aqueous Tween-20 (detergent)<br />
Large weigh boats (8 x 8 cm)<br />
Weighing balance<br />
250 ml Erlenmeyer glass flask<br />
100 ml measuring cylinder<br />
Parafilm‘M’ or clingfilm<br />
Laboratory flask shaker (alternatively, shaking flasks by h<strong>and</strong> is acceptable)<br />
500 ml Erlenmeyer glass flask x 2<br />
Funnel (approx. 13 cm diameter)<br />
53 µm mesh nylon sieve (11 cm external diameter): mesh from Spectrum Laboratories *<br />
20 µm mesh nylon sieve (4 cm external diameter): mesh from BDH or Spectrum Laboratories<br />
Aspirator bottle with distilled water<br />
Sterile disposable 3 ml Pasteur pipettes<br />
Pipettor (100 µl capacity) plus disposable pipettor tips (plugged)<br />
Pipettor (1000 µl capacity) plus disposable pipettor tips (plugged)<br />
15 ml sterile, disposable conical bottom centrifuge tubes<br />
Centrifuge (to take 15 ml centrifuge tubes above)<br />
Autoclavable, disposable waste bottle<br />
Autoclave bags<br />
Glass microscope slides (76 x 21 mm)<br />
Microscope cover slips (18 x 18 mm)<br />
Compound microscope (x 100–400 magnification)<br />
Dissecting microscope (x 10–70 magnification)<br />
Shear’s solution † (as an alternative mounting medium to water if slides are prone to drying;<br />
however, Shear’s starts to kill teliospores after a few minutes exposure <strong>and</strong> little<br />
germination can be expected after 1 hours exposure)<br />
* If 53 µm nylon mesh cannot be sourced, then an alternative mesh size could be<br />
used, e.g. 50 or 70 µm mesh<br />
† Shear’s solution: 300 ml Mc Ilvaine’s buffer ††<br />
6 ml Potassium acetate<br />
120 ml Glycerine<br />
180 ml Ethyl alcohol (95%)<br />
†† Prepare Mc Ilvaine’s buffer (Mathur & Cunfer, 1993) as follows:<br />
Dissolve 19.212 g citric acid (C6H8O7) in 1000 ml distilled water <strong>and</strong><br />
mix thoroughly.<br />
Dissolve 28.392 g of disodium phosphate (Na2HPO4) in 1000 ml of<br />
distilled water <strong>and</strong> mix thoroughly.<br />
Mix 8.25 ml of citric acid solution with 291.75 ml of disodium<br />
phosphate solution <strong>and</strong> mix thoroughly.<br />
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