Department of Microbiology and Immunology


Shipping Fish to a Disease Diagnostic Laboratory

Before shipping any fish, telephone your disease diagnostic laboratory. Describe to the laboratory personnel the disease signs that you have observed and determine how they want you to ship your fish. Do not expect a diagnosis over the telephone. However, by informing the laboratory personnel of your problem and answering accurately their questions you can help to facilitate a rapid and accurate diagnosis. The more information you can provide to the diagnostic laboratory, the better will be the total evaluation of your disease case. Remember, the laboratory personnel recognize that you are in a difficult situation and they are anxious to help. Some general guidelines in specimen collection, preparation, and shipment follow.

I. The Specimen

The quality of specimen submitted to a fish disease diagnostic laboratory can greatly influence the ability of the fish health specialist to provide you with a diagnosis and recommendation for corrective action. Live fish showing the disease signs in question should be collected. Dead fish are of little value for disease diagnosis; because:

a) Fish decompose very rapidly once death occurs. If the disease in question was caused by a bacterium, other bacteria that take part in the normal decomposition process can quickly overgrow the pathogen and make its identification difficult or impossible.

b) Parasites require a live host for survival. Once the fish dies, the parasites will often quickly leave the fish in search of another live fish upon which to live.

c) Viruses also require a live host in which to live. Once the fish dies, the viruses will survive for only a limited period of time; sometimes only a few hours.

d) The time the fish has been dead is often impossible to know. Time is often wasted by providing a specimen that, although it may appear "fresh dead", is actually unsuitable for processing. The extra effort to collect live fish that show the clinical signs of the disease in question are well worth the effort.

It is best to collect 3-5 living fish that show the signs of the disease and submit them to the fish health specialist.

II. Packing and Shipping the Specimens

The best possible way to transport sick fish to a diagnostic laboratory is for the culturist to bring them to the laboratory alive. This will provide the fish health specialist with the best possible specimen and also the opportunity to obtain from the culturist additional information regarding the circumstances surrounding the mortalities. The culturist should also bring a water sample from the culture system if requested. This water sample should be collected in a clean container that can be tightly capped. If a chemical contaminant is suspected, the sample must be collected in a glass jar (not plastic) and handled according to instructions provided by the disease diagnostic laboratory.

If the culturist cannot transport the specimens and the fish must be shipped, the following methods (from most desirable to least desirable) can be used:

Live fish
Iced fish
Frozen fish
Formalin fixed fish

Most diagnostic laboratories prefer that they receive specimens that are not fixed in formalin because the formalin will also fix (kill) pathogenic microorganisms. Reaching a diagnosis may depend on the ability of the diagnostic laboratory to culture a bacterium or virus from the fish. This can only be done if the bacteria or viruses are not killed. In addition, a very important aspect of recommending a treatment for bacterial pathogens is to determine the antibiotic resistance of the bacterial isolate, which can only be done if the diagnostician can culture the microorganism in the laboratory. Formalin fixed materials can yeild important diagnostic information following histological examination, but because of its limitations, most diagnostic laboratories prefer to have access to materials from which they can culture pathogenic microorgansims. This issue should be discussed with the diagnostic laboratory with which you will interact.

Fish shipped by any of the above methods should be collected alive from those fish in the system that are showing the disease signs in question. It is best to collect the fish with a net or trap. Capturing fish by rod and reel will select individual fish that are still actively feeding. One of the first signs of disease common to many diseases is that fish stop feeding. Thus, collecting fish with a rod and reel will select the healthiest fish in the population. Accurate diagnosis of the disease may not be possible as these fish may not yet be infected.

A. Live Fish

Live Fish

1) Obtain a strong, waterproof, insulated shipping container. (e.g., a disposable styrofoam cooler in a sturdy cardboard box).

2) Fill a heavy plastic bag approximately 1/3 full of clean water from the culture facility. Place the bag in the shipping container and add the fish. Fill the bag with pure oxygen or air. Seal the bag by twisting the opening and securing it closed with several heavy-duty rubber bands or plastic tie-downs. An air tight seal is essential.

3) Place 3-5 pounds of crushed ice in a heavy plastic bag, seal the bag as described above, and place it in the shipping container next to the bagged fish.

4) In a separate, small plastic bag place a note that includes your name, address, telephone number and information describing the fish and the culture system from which they came (e.g., why you suspect a disease; number of mortalities, disease signs you observe on the fish, and their approximate size relative to other fish being cultured; when and how the shipped fish were collected; stocking density; any known water quality parameters). Place the bagged note inside the shipping container.

5) Seal the shipping container. Be sure to indicate which end is "up" and that live fish are enclosed.

CAUTIONS: Take extra care in making sure the container won't leak. "Double bagging" can sometimes help. Ship via a carrier that can provide overnight delivery. It is always best to contact the fish diagnostic laboratory to which you will submit materials prior to any shipment to coordinate the receipt of the fish.

B. Iced Fish

Iced Fish

1) Obtain a strong, waterproof, insulated shipping container. (e.g., a disposable styrofoam cooler in a sturdy cardboard box).

2) Wrap each fish individually with several sheets of newspaper and place each fish in a separate plastic bag and seal the bag. (note: wrapping in newspaper can prevent freeze burns that may obscure some signs of disease that could be important in reaching a diagnosis)

3) Place a larger, strong plastic bag in the shipping container fill the bag with 2-4 inches of crushed ice.

4) Place the individually bagged fish on the crushed ice in the larger bag and cover them with an additional 2-4 inches of crushed ice. Seal the larger bag by twisting the opening and securing it closed with several heavy-duty rubber bands or plastic tie-downs. An air tight seal is essential.

5) In a separate, small bag place a note that includes your name, address, telephone number and information describing the fish and the culture system from which they came (e.g., why you suspect a disease; number of mortalities, their appearance, and their approximate size relative to other fish being cultured; when and how the shipped fish were collected; stocking density; any known water quality parameters). Place the bagged note inside the shipping container.

6) Seal the shipping container. Be sure to indicate which end is "up" and that iced (perishable) fish are enclosed.

CAUTIONS: Adequate amounts of crushed ice, usually 10-15 pounds, will usually be satisfactory to keep the fish chilled during shipment.

C. Frozen Fish

NOTE: Recent regulations by the U.S. Department of Transportation (US DOT) have classified dry ice as a hazardous material when placed into the commercial shipping system. Only personnel with specialized training are authorized to prepare such packages for shipment and those shipments must be prepared in a very specific manner. Meeting these regulations may be difficult for some individuals and alternate methods of shipment should be chosen.

Frozen Fish

1) Obtain a strong, waterproof, insulated shipping container (e.g., a disposable styrofoam cooler in a sturdy cardboard box).

2) Place each fish in an individual plastic bag and seal the bag. Freeze the fish in the individual plastic bags.

3) Place a larger, strong plastic bag in the shipping container and fill the bag with 2-4 inches of crushed ice.

4) Place the individually bagged, frozen fish on the crushed ice. Cover the fish with additional crushed ice and tightly seal the bag by twisting the opening and securing it closed with strong rubber bands or a plastic tie down. If possible, use dry ice to insure that the specimens do not thaw during transit. Five pounds of dry ice will normally keep specimens frozen for 24 to 36 hours, if the shipping container is well insulated.

5) In a separate, small bag place a note that includes your name, address, telephone number and information describing the fish and the culture system from which they came (e.g., why you suspect a disease; number of mortalities, their appearance, and their approximate size relative to other fish being cultured; when and how the shipped fish were collected; stocking density; any known water quality parameters). Place the bagged note inside the shipping container.

6) Seal the shipping container. Be sure to indicate which end is "up" and that frozen (perishable) fish are enclosed.

CAUTIONS: Check with the commercial carrier for their policy regarding shipment of packages containing dry ice.

D.Formalin Fixed Fish

NOTE: Recent regulations by the U.S. Department of Transportation (US DOT) have classified formalin as a hazardous material when placed into the commercial shipping system. Only personnel with specialized training are authorized to prepare such packages for shipment and those shipments must be prepared in a very specific manner. Meeting these regulations may be difficult for some individuals and alternate methods of shipment should be chosen.

Formalin Fish

1) Make a 10% formalin solution. Neutral buffered formalin is best, but under practical field conditions water from the culture facility will usually provide adequate buffering capacity to the solution. (Mix 9 parts water with 1 part formalin).

2) Kill the fish before placing them in the formalin fixative. This can be done by with an "overdose" of MS-222 (tricaine methane sulfonate at 1 gm per 500 mL H2O). It is important that the fish be rapidly "fixed" so that the quality of tissue preservation will yield useful information. Formalin can normally provide for rapid fixation of tissues that are less than 1/2 inch thick. For this reason, the abdomen of larger fish must be opened for its entire length with one continuous cut. It is also important that adequate amounts of formalin be used to preserve the tissues. As a general rule, the ratio of formalin to tissue must be 10:1 (by weight or volume; ie. 1000 mL formalin : 100 gm fish).

3) The container with the formalin and the tissue must be tightly sealed. Care should be taken to prevent breakage of the container. Glass breaks and should be avoided -- Use plastic bottles such as empty, clean food containers (e.g., peanut butter, mustard, salad dressing -- use food service size for large fish) or containers obtained from scientific supply companies. The plastic bottles should also be sealed in leak-proof plastic bags as a further step to avoid leakage.

4) The sealed container should be placed in a shipping container filled with styrofoam pellets or other suitable packing material. Care should be taken to prevent breakage.

5) In a separate, small bag place a note that includes your name, address, telephone number and information describing the fish and the culture system from which they came (e.g., why you suspect a disease; number of mortalities, their appearance, and their approximate size relative to other fish being cultured; when and how the shipped fish were collected; stocking density; any known water quality parameters). Place the bagged note inside the shipping container.

6) Seal the shipping container. Be sure to indicate which end is "up" and that preserved fish are enclosed.

CAUTIONS: Formalin is irritating and toxic. Provide for good skin and eye protection as well as good room ventilation when using this chemical. A good practice to be employed when handling formalin or any other potentially irritating or toxic chemical would be to use rubber gloves to protect the hands, goggles to protect the eyes and a breathing mask to reduce inhilation of the chemical.

III. Summary:

In light of recent regulations by the U.S. Department of Transportation (US DOT) that have classified dry ice and formalin as hazardous materials when placed into the commercial shipping system, special considerations to such shipments apply (see above). In some cases, individuals wanting to ship fish should concentrate their efforts on methods for shipping live fish or iced fish.

The manner in which the fish specimens are prepared and shipped will influence what information can be obtained when the specimens are examined by the fish health specialist. Working with a live fish will provide the diagnostician with the best opportunity to gain useful information regarding the fish disease. The fish can be examined for live parasites. The identification of living microscopic parasites can be aided by observing the type of movement the parasites exhibit during examination of wet mounts of fresh materials. Microorganisms (bacteria and viruses) can be cultured from specimens that are delivered to the diagnostic laboratory alive. Should the diagnostician want to perform a histopathological examination, the diagnostician can take appropriate samples of organs and tissues, fix (preserve) them in formalin and have them processed for histopathological examination. In such an examination, the diagnostician looks for tissue changes that are indicative of a pathological process. It is critical that the tissues be preserved properly to insure that they are a true representation of the disease process and not of a condition of decomposition following death of the fish.

Specimens that are shipped on ice or frozen have some important diagnostic limitations. Living parasites may or may not be present on iced fish and histopathological examination may not yeild reliable information, depending upon the time during which the fish were maintained in an iced condition. The limitations of frozen fish are greater. When the frozen fish are thawed, the shearing action of melting ice crystals will destroy parasitic protozoa, making their identification very difficult if not impossible. The thawing of frozen tissues will also create a great deal of damage making those tissues of very limited or no use for histolopathological examination. Iced and frozen specimens are normally satisfactory for the culture of bacteria and viruses.

Specimens shipped in formalin are useful for histopathological examination as long as the specimens have been carefully preserved prior to shipment. As mentioned above, the diagnostician must be able to detect tissue changes that are indicative of the disease process and not of a decomposition process in an animal once death occurs. This can only be done with a carefully processed sample. The formalin fixed sample may not yield the identity of the disease organism and it will not yield information regarding antibiotic sensitivity, if the pathogen of interest is a bacterium.

The table below summarizes the impact of fish handling and preservation on disease diagnosis:

Shipment Method Parasitology Bacteriology Virology Histopathology
Live
+++
+++
+++
+++
Iced
+
++
+++
+/-
Frozen
-
++
++
-
Formalin Fixed
+/-
-
-
+++

Legend:
+++ no effect, excellent specimen for examination
++ negligible effect, good specimen for examination
+ moderate effect, specimen may be usable
+/- specimen may not be useful
- dramatic effect, specimen not useful

Therefore, as stated earlier, the methods of shipment for fish disease diagnosis, from most desirable to least desirable are:

Live fish
Iced fish
Frozen fish
Formalin fixed fish