The history of each group of oysters being examined should be closely followed, particularly information related to age, line number, stocking rate, growth performance, survival rates, grading history, rejection rates and parasite or pest burdens. Any abnormalities in any of these areas should be noted when they are observed so that these data are available for future reference if required.

Water quality

Basic water quality parameters such as temperature and dissolved oxygen should be regularly monitored. Other water variables such as salinity, pH, microbiology (enteric virus or faecal coliform counts), lagoon primary productivity and presence or absence of toxic phytoplankton should also be recorded Ideally these records should be available for the entire history of each group of oysters being examined.

Gross examination and histopathology

1. Try to examine any shells which appear diseased first, then make up the remaining oysters (if any needed) randomly by picking oysters from a variety of lines/racks/bags (i.e. every second line) and taking oysters from different sites along each line. For example, if you are sampling 30 oysters from a site with 6 lines of 1000 oysters, and there are 10 apparently diseased shell, sample these first, then the remaining 20 oysters can be collected by taking 3 or 4 random oysters (for example, every 50th or 100th oyster ) from each of the 6 lines.

2. Ensure the oysters are dissected promptly. Dead shell are of little use as the changes which occur after death often obscure the true cause of death. It is best to sample diseased shell while they are still alive, even if only just alive (moribund).

3. The site, oyster number and shell measurement of each oyster (measured to the nearest millimeter) should be recorded on the data sheet .

4. The presence of grossly evident infections with metazoan parasites and fouling organisms (mudworms, fungi, boring sponges, drill hole infections etc.) should also be noted Figures 1, 2, 3, 4.

5. The oyster is opened by severing the adductor muscle.

6. The nacre of inside of each shell valve should be examined for boring sponges, mudworm blisters or abnormal, brown coloured conchiolin deposits associated with mantle recession Figures 5, 6. The severity of conchiolin deposits can be described using the following semiquantitative scale:

Abnormal conchiolin lesion severity grading
    0 - apparently healthy , no lesions evident
    1 - 1 or 2 focal lesions
    2 - less than 25% of shell valve perimeter affected
    3 - 25 to 50% of shell valve perimeter affected
    4 - 50 to 75% of shell valve perimeter affected
    5 - more than 75% of shell valve perimeter affected

7. An oblique transverse section, approximately 5 mm thick, is cut from each oyster using a scalpel. The section should be oriented to include mantle, gonad, digestive gland, gills foot and kidney Figure 7. Place the section into a histology cassette (Simport M492) and place it into 10% seawater formalin (see Recipes page).

8. A number of oysters sampled from each site should also be examined for the presence of Perkinsus sp. by placing excised samples of mantle, digestive gland, gills, foot and kidney into Rays fluid thioglycollate medium (RFTM) (see Recipes page).

9. Any remains from diseased oysters examined can either be frozen at -20C for short term storage, then disposed of in landfill, and/or buried or incinerated - do not put them back into the water.

Thioglycollate (also see Recipes page)

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. 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.

3. Place tissues of interest (mantle, digestive gland, gills, foot and kidney) into tube and incubate in dark at room temperature for 7 to 14 days. If you are sending the samples by mail to DigsFish Services for analysis, this is the time to send them.

4. Remove tissues from thioglycollate, place in petri dish and flood the tissues with dilute lugols iodine. Macerate the specimen to allow iodine to penetrate.

5. Examine the stained tissues using a dissecting microscope at 40 x magnification for blue or black spheres of Perkinsus sp. Figure 8.

Bacteria testing

Bacteriology uses specialised techniques which should be conducted using methods recommended by the diagnostic laboratory which will undertake the sample screening. Contact DigsFish Services or the laboratory responsible for processing your samples for more information on their desired methods of sample collection.

Electron microscopy

Excise small sections (1-2 mm3) of digestive gland, gill, mantle, gonad or any other organ of interest and place them in a 1.5 ml eppendorf tube containing 2.5% gluteraldehyde (see Recipes page). Store them at 4C for 24-48 hours. Rinse once in seawater filtered to 0.22 microns and send to DigsFish Services for processing.

Molecular techniques

Excise small sections (1-2 mm3) of digestive gland, gill, mantle, gonad or any other organ of interest and place them in a 1.5 ml eppendorf tube containing 70% ethanol (see Recipes page). Store them at 4C for 24-48 hours, then send to DigsFish Services for processing.