DigsFish Services Recipes Page

Wet smears/blood smears/heart smears/digestive gland smears

Use the DiffQuick Stain Kit (Lab Aids, 3 Gondola Rd, Narrabeen Victoria 2101 phone (02) 9913 1396, fax (02) 9659 9559)

Fixatives

CAUTION: all fixatives contain toxic substances which can be harmful to humans. Please check the material safety data (MSDS) sheets which are available for each of the components of these fixatives and take appropriate precautions whenever they are being handled and used. MSDS data can be found online at MSDS.com or MSDSonline. For all fixatives, ensure that the volume of fixative used is at least 5 times that of the tissues being fixed.

Neutral buffered 10% formalin (for histology - freshwater fishes)
100 ml formalin, 900 ml tap water, 4g sodium dihydrogen phosphate, 6g disodium hydrogen phosphate pH 7), optimum tissue thickness 4-5 mm.

10% Seawater formalin (for histology, marine fishes and molluscs)
100 ml formalin, 900 ml of filtered seawater (filter to 10m or less), optimum tissue thickness 4-5 mm.

Davidsons fixative (for histology, crustaceans)
110 ml of glacial acetic acid, 220 ml of formalin, 330 ml of 95% ethanol, 340 ml distilled water. Optimum tissue thickness 4-5 mm. Inject fixative into the hepatopancreas of crustaceans to ensure rapid fixation.

70% ethanol (for DNA analysis)
737 ml of 95% (or 700 ml of 100%) ethanol, 300 ml of distilled water. Optimum tissue thickness approx. 1 mm.

Gluteraldehyde (2%) (for electron microscopy, store at 4C) 4 ml of 50% gluteraldehyde, 96 ml of filtered seawater (filtered to 0.22m) - optimum fixation time 24 hours at 4C. Optimum tissue thickness approx. 1 mm

Histopathology Cassettes

Use Simport M492 histology cassettes for large specimens, or Simport M493 cassettes for very small specimens.

Blood sampling

Needles and syringe combinations for fish of various sizes (please dispose of needles thoughtfully):
5-20 cm fish - 25g x 5/8 inch needle, 1 ml syringe
20-60 cm fish - 21g x 1 inch needle, 3 ml syringe
>60 cm fish - 18g x 1 inch needle, 3 ml syringe

Thioglycollate

1. Follow manufacturers instructions to make up thioglycollate (29.3 grams of powder to 1 litre of freshwater). Add 20 g of salt (NaCl) to each litre of thioglycollate when using freshwater.

2. Stir then sterilise thioglycollate and universal tubes in an autoclave or pressure cooker (121C for 15 minutes). Cool down to body temperature then add antibiotic/mycotic solution (50 ml for each 1 L of thioglycollate). Then dispense 10 ml aliquots into sterile universal tubes. Store the sterile tubes and media in the dark until use.

Lugols iodine (for identification of Perkinsus hypnospores)
1 gram of iodine granules, 2 grams of potassium iodide added to 100 ml of freshwater

Equipment Suppliers

DiffQuick Stain Kit Lab Aids, 3 Gondola Rd, Narrabeen Victoria 2101 phone (02) 9913 1396, fax (02) 9659 9559

Fixative reagents and equipment, histology cassettes, needles and syringes:

Australia - all equipment , try Biolab (phone 1300 735 292, fax 1800 067 639) email Biolab.com.au

New Zealand -