International E co G en Incorporated



Fish Blood Sample Collection


Fish Greater than 20 gm in weight

Suggested Equipment

citrate buffer
3 cc 23 gauge 1" needle syringes
     (Fisher Cat. # 14-823-37);
tuberculin syringe - 1 cc 27 gauge 1/2" needle
     (Fisher Catalogue #14-826-87)
fish anaesthetic - MS222
     (Sigma Aldrich Cat.# A5040)
nalgene cryovials (Fisher Cat. # 03-337-7C)
nalgene cryovial closure colour coders
     (Fisher Cat. # 15-350-45 to 49)
cryovial storage box (Fisher Cat. # 15-350-1078)
cryovial holder (Fisher Catalogue # 03-337-7E)
several pairs of stainless steel tweezers
      (Fisher Cat #08-887-5)
stainless steel scissors (Fisher Cat # 13-806-2)

 

metal or glass container for sharp objects
paper towels
garbage bags
latex gloves (Fisher Cat #11-394-18)
ethanol for cleaning tweezers
scalpel handle (Fisher Cat #08-913-5)
scalpel blades (Fisher Cat #08-916-5A)
repetitive syringe dispenser
     (Fisher Cat. #13 686 38A)
3 ml syringe tips (Fisher Cat. #13 686 16)
variable pipette - 10 to 100 microlitres
     (Fisher Cat. #13 68641)
pipette tips (Fisher Cat. #21 1978E)

Preparation

Fish in this size range and larger can be anaesthetized and the blood drawn by syringe. In a standard plastic pail or fish tote 3/4 full of water add 0.50 gm MS222 (about a pinch of the powdered anaesthetic) and stir to dissolve. After the fish have been added, they should become sluggish and easily handled after 2 - 5 minutes. If this does not occur add another pinch of MS222. Repeat the procedure until the proper dosage has been reached. Do not add too much. Always wear rubber gloves when handling this chemical.

The size of the syringe will depend on the size of fish. On fish larger than 100 gm use a 3 cc 23 gauge 1" needle syringe. On fish ranging from 20 - 100 gm use a tuberculin syringe. The freezing media is placed directly into the syringe prior to use. This media prevents clotting and prepares the blood for rapid freezing. Shake the media frequently during transfer to the syringes to ensure that the DMSO is maintained in solution. Fill with exactly 100 microlitres of freezing media or an equal volume of the anticipated amount of whole blood to be collected. Where only small samples are to be collected (2 to 20 microlitres) fill the syringe with 25 microlitres of buffer. Replace needle cover and store in fridge or on regular ice until use. Pre-label the cryovials, but do not put any freezing media in them. All the freezing media required is placed in the syringes.

 

Collection

The anaesthetized fish may need to be held in order to prevent movement from muscle twitching. The syringe is inserted at an angle (needle of syringe pointed towards the head of the fish) in the side of the fish at a point in the tail region ventral to the anus and above the lateral line that is approximately half way between the lateral line and the dorsal surface of the fish. The objective is to tap into the caudal vein just below the backbone. The plunger of the syringe should be pulled partially out to create a small vacuum so that when the vein is encountered blood will flow into the syringe. Once the vein has been located then the plunger is pulled out further until sufficient blood has been collected. Finding the vein on the first few fish may take a while, but once you get experienced, the pace can quicken significantly. The fish should then be placed in a clean non-metal container of fresh river water to recover and then be released.

The sample should be fully drawn up into the needle so that there is an airspace in the barrel of the syringe at the needle end. The needle guard is then used to unscrew the needle. The needle and cover are placed in a glass or metal container for sharp object (including used scalpel blades) disposal. The pre-labeled chilled (on regular ice) cryovial is then opened and the contents of the syringe are expunged slowly into it. The cap is screwed back on and the cyrovial is then placed in a box within a cooler containing dry ice. If sub-sampling is to be done, it should occur just after the whole sample has been placed in the cryovial and before freezing. As above use a micropipette to transfer sub-samples to other pre-labeled cryovials kept in a cryovial holder.

Samples cannot be thawed out and then refrozen at any time. If this occurs then the samples will no longer be suitable for assay by flow cytometry.

Caution: DMSO can pass through the skin rapidly and may be genetically toxic - wear gloves when working with the citrate buffer.

 

Sample Management

The key to success of any project is in the organization of the samples once they are collected. The cryovial boxes should be clearly labeled as to:

  • species
  • location
  • range of sample #s inside
  • date of collection

Each individual cryovial should be labeled with:

  • sample #
  • species
  • date
  • location

The species and location can be coded to 1 or 2 letters. A master list of all samples collected by species location and date (with codes defined) should be maintained as sampling is done. The cryovials can be pre-labeled before proceeding to the field. The labeling should be double-checked by another observer. Correct labeling is of the utmost importance.

To aid in retrieving frozen samples, a master list of the sample contents of each cryovial box should be drafted, as each box is filled during the field sampling. When the samples are about to be placed in the freezer rack for long term storage in the -80oC freezer, the position of each box of samples within the rack and the rack’s location within the freezer should be noted on the appropriate freezer box master list.

 

Fish under 20 gm

Fish 20 gm and over

Citrate Buffer Freezing Media

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