Staining Procedures

Gram stain

• Make smears of blood, fluid, tissue or bacterial colony from an agar plate, as described in previous sections and air dry. (Heat fix smears but do not heat excessively, i.e., slide should not be too hot to touch.)
• Place slide on a rack over a sink.
• Cover smear with crystal violet dye and let stand for 1 minute.
• Rinse briefly in tap water and drain. Flood smear with Gram's iodine, pour off and flood again. Let stand for 1 minute.
• Wash briefly in tap water and drain. Rinse with decolorizer (95% ethanol or ethanolacetone mixture from a pipet) until no more purple dye comes off the slide (usually 2-5 seconds). Excessive decolorization may give a false Gram-negative reaction.
• Wash briefly with tap water and drain. Flood smear with safranin stain and let stand 1 minute.
• Wash briefly with tap water, drain, and air dry.
• Examine the slide using the oil immersion lens (100x)
• Gram-negative bacteria will be pink to red; Gram-positive bacteria will be purple.

Diff-Quik

• Make smears of blood, fluids, or tissues.
• Dip 5 times in Diff-Quik solution 1, one second each time, and drain.
• Dip 5 times in Diff-Quik solution 2, one second each time, and drain.
• Dip 5 times in Diff-Quik solution 3, one second each time, and drain.
• Rinse in tap water and drain.
• Air dry and examine using 10x, 40x, or 100x.

NOTE: Diff-Quik solution 3 can become weakened with age or use. Check stain intensity on slides periodically. Slides may be re-stained with fresh solution 3 if necessary. Periodically pass solutions 2 and 3 through separate 0.45-μm filters to remove precipitates and contaminating bacteria.

Figure. Demonstration of how to make a thin blood smear.

1. On slide “A” express a drop of blood or hemolymph about one-half inch from the end.
2. The edge of a second slide “B” is placed on the surface of slide “A” at about a 45 angle and is moved to the right until contact with the drop of blood.
3. Contact with the blood will cause the drop to spread along the edge of slide “B” due to capillarity. Slide “B” is then pushed to the left, being careful to keep the edge pressed uniformly against the surface of slide “A”.
4. The size of the drop of blood and acuteness of the angle formed between the slides will determine the thickness of the film. A more acute angle results in a thicker film.
5. The smear is allowed to air dry for transport in a slide box and later staining.

General Necropsy Procedure

• Necropsy subjects should first be examined for external abnormalities or lesions that could include: pinheadedness or otherwise poor body condition; exophthalmia; cloudy cornea or lens opacity; hemorrhaging within the anterior chamber of the eyes, fins, body surface or body orifices (anus, nares, mouth, gill chamber), frayed or missing fins; gas bubbles within the fin rays or connective tissues of the eyes; ulcerations, abscesses, abrasions; body discolorations; excessive mucus; trailing fecal casts or rectal prolapse; external foreign bodies such as fungus, metazoan or protozoal parasites, cysts or tissue growths; potbelly or other protrusion or body malformations (spinal deformities, cranial swelling, shortened opercula, pugheadedness, microeye).
• External lesions such as ulcerations or abrasions should be struck onto TSA. Use of TSA with 1% NaCl may be necessary depending upon case information and whether fish are in saltwater and a halophilic bacterial pathogen is suspected.
• A peripheral blood smear should be made by excising the caudal peduncle (for small fish) and allowing a drop of blood to be deposited near the frosted end of a clean glass slide. The blood is smeared before clotting with a second glass slide by touching the drop with the slide at a 45o angle to the first slide and pushing the angled slide to the end of the first slide. Capillarity draws the smear across the first slide and the smaller the angle the thicker the smear (Figure, p. 3-6). Stain the smears in Diff-Quik (see staining procedures in this section) and observe on the microscope at 1000X for bacterial rods, erythrocytic inclusion bodies (EIB) and viral erythrocytic (VEN) cytoplasmic inclusions, necrobiotic bodies (IHNV) and erythroblastosis or other blood abnormalities in cell composition and morphology. Larger fish may be bled by caudal vein puncture into a heparinized syringe or Vacutainer and blood expressed onto a slide for subsequent smearing. For blood collection, the needle should be inserted at the location just below the lateral line that intersects with the rear margin of the anal fin. The needle should be inserted until just penetrating the vertebra (hemal canal) as indicated by slight resistance. Blood will automatically begin to flow when the Vacutainer is punctured by the needle base or when the plunger of the syringe is pulled back.

• Fish should be placed on their right sides for performance of the remaining necropsy procedures. Skin scrapes of normal and lesion areas mounted with a drop of PBS and coverslip on a glass slide should be made by using either the edge of the coverslip as the scraping instrument, or a scalpel. Bacteria or fungus from lesion areas or protozoal parasites such as Ichthyobodo and Trichodina are common subjects to look for beginning at 40x and then at 200X on a compound microscope using the aperture diaphragm.
• Wet mounts of gill filaments are made by using a small pair of surgical scissors to remove a portion of one gill arch. Gill filaments should be slightly teased apart for good viewing of filament and lamellar profiles and mounted in PBS with or without a coverslip. These should be examined immediately since branchial epithelium rapidly deteriorates causing postmortem artifact. Look for gas bubbles in the capillaries, telangiectasia, hyperplasia, external parasites (bacterial, protozoal, fungal, metazoan), or other foreign bodies. Should bacteria be observed or suspected the coverslip may be removed and used to mince the gill tissue. This is allowed to air dry for later Gram staining. After staining, the gill tissue is removed with forceps for viewing of the stained slide for bacteria by oil immersion.
• Disinfect the outer surface of the fish by flooding with 70% ethanol. Disinfect a pair of scissors, forceps and scalpel by immersion in 100% ethanol and passing the instruments through a Bunsen flame allowing the alcohol to ignite and burn off. Repeat one or two more times. Wipe instruments clean of any organic matter beforehand for effective disinfection.
• The abdominal cavity is entered by pulling the pectoral fin with sterile forceps while cutting into the abdominal wall at the base of the pectoral fin with a pair of small sterile scissors. The cut is continued dorsally to just below the lateral line where resistance is encountered. Start again at the base of the pectoral fin and continue the incision towards the posterior of the fish along the ventral abdominal wall to the vent. Stay slightly above the intestinal tract when making the incision so that it is not punctured, thereby contaminating the tissues. At the vent continue dorsally to just below the lateral line and continue cutting anteriorly to connect with the first incision. Remove the flap of abdominal tissue, thus exposing the internal viscera and cavity. When done correctly on a moribund specimen the air bladder should remain inflated and the GI tract completely intact. Instruments may need wiping of organic material and flaming repeatedly during this procedure.
• Visually examine viscera (heart, liver and gall bladder, kidney, pancreas, adipose tissue, spleen, air bladder, pyloric caeca and entire GI tract) for abnormalities such as: discoloration or mottled appearance; enlargement (hypertrophy); hemorrhage or erythema; abscesses or cysts; fluid in the abdominal cavity (ascites causing potbelly); foreign bodies such as fungus, metazoan parasites or tissue growths, etc.
• If bacteriologic samples are to be taken they should be struck onto TSA from the kidney and/or from visceral lesions before other samples are taken to avoid bacterial contamination (see bacteriology Chapter 4 for procedures). If Phoma is suspected, samples from the suspect lesion or air bladder should be struck onto PA. 
• Tissues to be taken for viral assay of larger fish (kidney/spleen pool) should also be placed into sterile tissue culture fluid for refrigeration and homogenization at a later time. Fry are generally processed whole for virology.
• Kidney smears for FAT detection of the BKD agent should be taken at this step. Generally, bacterial problems due to Gram-negative bacteria such as furunculosis and ERM agents can be detected more efficiently by isolation on prepared media. Kidney tissues for ELISA are generally not taken from a diagnostic case since clinical disease caused by the BKD agent can easily be detected using FAT or Gram stain when lesions are apparent.
• If the spleen has not been completely removed for virus assay, a spleen squash can be made by placing a cut section of the tissue with a drop of PBS on a glass slide and covering with a coverslip. Whole spleen squashes will be necessary when small fish are examined. Look for the presence of motile or non-motile bacterial rods and fungal hyphae. The coverslip may be removed and the squash Gram stained for confirmation of bacteria as described for gill tissues.
• A squash of a small section of the lower intestine (rectum) should also be made on a glass slide using PBS and a coverslip. Look for presence or absence of food and Hexamita or amoebae. Bacteria should obviously be abundant as part of the normal gut flora. Also look for fungal hyphae within the gut wall or lumen.
• A squash of lesion material from a visceral organ or organs may be warranted if present and if its cause is not readily discernible. Gram stains and/or Diff-Quik stains of this material may also be warranted (see staining procedures in this section). An example would be stained impression smears of kidney tissue to examine for possible BKD, PKD or Enterocytozoon salmonis.
• If the cause of mortality or morbidity is in question as to whether or not the above procedures will provide an answer, histology samples should be taken as a backup measure, but only if moribund fish are available. Fish that have been dead for several hours or longer are generally not suitable for histology due to postmortem tissue autolysis. If fry are involved, whole fish may be dropped into Bouin's fixative or 10%buffered formalin. Fingerlings should have the abdomens opened with scissors for better fixative penetration (see the histology section for more detail regarding fixation of tissues).
• If clinical signs suggest a central nervous system disorder the top of the cranial cavity should be opened and the brain included in bacteriologic sampling using TSA and cytophaga agar. Heads from additional affected fish should be severed behind the gill opercula and placed into fixative for later histological sectioning of the brain.
• During necropsy, occasional serial sectioning of skeletal muscle using a razor blade may be necessary should a lesion within that tissue be suspected. Examples would include abscesses, hematomas, neoplasms or encysted parasites causing a protrusion of the musculature. Depending upon the nature of the lesion, bacteriological sampling, Gram staining or fixation for histology may be necessary.

This necropsy procedure should include at least 5-10 moribund or otherwise affected fish. Control or healthy fish should be requested for comparison of whether abnormalities perceived are real or not. The number of control fish processed will depend upon the particular case and may range from 10 to none.
Necropsies are best performed as a 2-3 person team effort in which a microbiologist and/or technician can make gross external and internal observations and the bacteriologic and tissue preparations. The pathologist in charge can devote his or her time to interpreting the sample preparations on the microscope. In this approach a case can be processed in a minimum amount of time and provides further pathology experience to the support staff.

In summary, a standard necropsy should include:

• Accession number, case data information, laboratory worksheet
• External and internal gross observations which could include brain and serial sectioning of skeletal muscle.
• Wet mounts or squashes of:

1. gills
2. skin
3. spleen
4. lower gut
5. lesions (if any)

• Peripheral blood smear - Diff-Quik
• Bacteriology - TSA/PA from kidney and lesions (if any); Gram stains
• Virology sample (only necessary in some cases)
• Kidney smear for FAT
• Tissues for histology (only necessary in some cases)

NOTE: For Standard Laboratory Shellfish Necropsy Procedures see histology section for details by species.

Standard Necropsy Procedures for Finfish

After assigning an accession number to the sample, the case data information received with the sample should be used to fill out the laboratory worksheet that will accompany the case to completion. Live fish should be examined for behavioral abnormalities (spiral swimming, flashing, flared gill opercula, prostration, etc.) then anesthetized to avoid tissue artifacts caused by alternate methods of euthanasia such as pithing or a blow to the head. Some external abnormalities (whitened or eroded fin tips, cloudy cornea, body discoloration, excessive mucus) are best observed while the fish is submerged in water. In many cases postmortem change in fish received dead will prevent this latter opportunity.

Sample Shipment Instructions

• Pack samples in a small ice chest made of plastic or sturdy styrofoam which will not be damaged in transit. Ice chests (other than styrofoam) will be returned to the sender.
• Add pre-packaged ice substitutes. To prevent freezing, separate the samples from the ice with newspaper or other insulative material.
• Place completed Sample Submission Form(s) (page 1-8) (forms available from FPS) for each stock sampled within a waterproof plastic bag and enclose in ice chest.

NOTE: Clients should always include a Sample Submission Form (page 1-8) with a submitted sample which provides the species, brood year, clinical signs, time of onset, sample date, number of samples submitted and other very important information. Clients should not assume a previous telephone call will take the place of submitting this written information. In the future, samples submitted without this paperwork may be refused or at least delayed in processing. When in doubt about anything, clients should consult a pathology staff member and not forget the paperwork.

• Close, seal, and label the ice chest with appropriate instruction for the type of sample enclosed (i.e., “Live Fish – Do Not Freeze” for live samples
• Ship via express air or air freight (if you know it will not get bumped off the flight) as soon as possible. Instruct the airline to refrigerate the sample upon its arrival in Anchorage or Juneau. If sent early in the week, fewer air freight and delivery problems are encountered. Avoid shipping on Fridays.
• Contact the courier services currently used by the FPS in Anchorage or Juneau to have the sample(s) delivered. The delivery services must be told correctly on which airline the samples are being transported, flight number, air-bill number, the arrival time, and whether the package is traveling by Goldstreak or air freight. Unless special circumstances dictate otherwise, clients are expected to ship samples such that they may be received by the labs and therefore, processed during regular hours.

NOTE: The cost of shipping samples to the pathology labs is the direct responsibility of the user unless the samples are submitted as a courtesy request by lab personnel. This cost includes the delivery services used by both labs, which are inexpensive compared to the cost of pathology staff time and loss of laboratory processing time when pathology personnel have to pick up samples at the airport.

• Phone the Fish Pathology Lab to notify that the sample is enroute. Please provide the flight number, airbill number, and expected time of arrival. Subsequently check to see if it has arrived. It is the responsibility of the sender to ensure that the sample arrives at the laboratory in satisfactory condition.

Histology

Histological samples should be fixed in Bouin's solution, Helly's solution or 10% buffered formalin. Fix live fish after anesthetized. Use 10 moribund fish and 10 that are apparently normal from the same lot. Dead fish are unsuitable for histology. The volume of fixative must be 10 times the volume of tissue. For fish longer than 6 cm, slit the abdomen, detach the intestine at the anus, and pull the internal organ mass out slightly. For large fish, send only specified organs in fixative. Call the FPS for specific instructions prior to fixing. Further details regarding necropsy and histologic sampling for both finfish and shellfish are provided in the histology chapter (Chapter 6) of this manual.
Sample sizes for shellfish are: 30 live adult animals for a disease history; 60 live adults, 200 live spat and 1,000 live larvae for certification for import of seed from an out-of-state Pacific oyster stock; maximum of 10 live or fixed animals for diagnostic purposes.

Parasitology and General Necropsy

The same sampling procedures as in Bacteriology apply here. Live fish are preferred to frozen or preserved fish. This is especially true for detection of external protozoan parasites and general gross tissue lesions, which are usually lost during freezing. Fish may be fixed in 10% buffered formalin if live fish are not available. Fish longer than 6 cm should be opened along the abdomen to ensure adequate formalin fixation of all tissues. The FPS will not routinely process large numbers of fish ( 20) for purposes of establishing parasite (helminth) prevalences.

Fluorescent Antibody Test (FAT).

The following procedures are for Renibacterium salmoninarum (bacterial kidney disease, BKD), Yersinia ruckeri (enteric redmouth, ERM), and Aeromonas salmonicida (furunculosis). In disease outbreaks involving small fish, 10 moribund or freshly dead fish per affected lot(s) shipped in plastic bags on ice (not frozen) are sufficient for a disease diagnosis. A presumptive diagnosis of A. salmonicida may not be confirmed by bacteriologic culture if samples are frozen. Sampling is according to that described earlier under bacteriology. An additional sample of 60 randomly selected normal appearing fish from the same lot(s) may be required at a later date to determine the prevalence of sub-clinical disease within a given group of fish before release is approved.

In situations where a disease history and/or broodstock screening is desired, a minimum sample size of 60 fish will be required. Family tracking for BKD will require screening of all parent fish involved in the egg take. Whole fish should be sent when sampling alevins, fry and fingerlings. In situations where large fish are to be examined, only kidney tissues are required. Sampling procedures are identical to those described for virology sampling of male kidney tissues.

Although fresh-on-ice samples are necessary for successful isolation of certain disease agents, freezing is the least desirable, but necessary, alternative if there will be excessive delay in getting the samples to the FPS.

In situations where it is more practical for field personnel to prepare the slides for FAT rather than mail tissues, the appropriate materials will be provided by the FPS. Briefly, after collection of kidney tissues the procedure requires:

1. Homogenization of the kidney sample from each fish by kneading within the plastic sample bag.
2. A sterile wooden applicator stick is touched to an individual homogenized kidney sample and then mixed with a drop of phosphate buffered saline (PBS) deposited in a single numbered well on a multiple well slide.
3. The samples are allowed to air dry at room temperature and the slides may be mailed to the FPS in slide boxes.

Each kidney sample requires a separate applicator stick and well. Slides are prepared in multiples for parallel testing if fish are to be screened for BKD, A. salmonicida and Y. ruckeri (2 types require duplicate slides). Homogenization of the kidney is important to break open BKD pustules and distribute the causative organism or any other target bacteria for easier detection. It is also important to not make kidney smears too thick within the depressions, which makes interpretation difficult. Also, such smears may wash off the slide during

processing.