International E co G en Incorporated
Blood Sample Collection
Fish less than 20 gm in weight
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)
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)
Collecting blood from fish of this size requires that the fish be sacrificed. The fish after being killed by a blow to the head should be placed on a clean paper towel. With a clean scalpel (i.e. a new blade for each new specimen) cut off the tail. The same blade can then be used to cut out the head kidney, if required. Forceps that are rinsed in ethanol and air dried between specimens can be used to lift the head kidney and place it in a labeled (date, tissue, specimen #) cryovial for freezing. The nalgene cryovial closure colour coders can be used to differentiate the different stocks or treatment groups of the species sampled. There are 5 colours plus the no colour option to give 6 choices. These colour codings are especially useful when trying to locate specific samples in the freezer after they have been stored for a while.
Blood is collected from the caudal vein, located just beneath the backbone, in a heparinized blood collecting tube. The tube is held at an inclined angle to the vein and blood is drawn in by capillary action. This tube which can hold up to 250 microlitres. Although the flow cytometry assay can be successfully accomplished with as little as 2 microlitres of blood, we recommend that 50 microlitres be taken unless significant sub-sampling is required for archiving for future comparisons. The blood is then expelled (using an ear syringe or other type of squeeze bulb that has been adapted to fit the blood collecting tubes) into a pre-labeled (date, species, sample #) cryovial that has been pre-filled with 50 microlitres of freezing media (containing citrate buffer + DMSO). If large blood samples are required then ensure that the blood to freezing media ratio is 1:1. For smaller samples (from 2 to 20 microlitres), the receiving cryovials should be pre-filled with 25 microlitres of freezing media. A transfer syringe dispenser can be used to accurately transfer the required amount of freezing media. Freezing Media needs to be shaken frequently during transfer to the cryovials to ensure that the DMSO is maintained in solution. After the blood has been transferred to the cryovial containing freezing media and the cap screwed on, the cryovial should be gently rocked back and forth several times to mix the two solutions before freezing.
Multiple samples from the same fish may be required. These samples are derived by sub-sampling the citrate buffer and blood mixture from the initial sample. After the initial sample has been mixed by gently rocking the cryovial several times, use a variable pipette to transfer equal microlitre amounts (aliquots) to the required number of pre-labeled cryovials.
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.
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:
Each individual cryvial should be labeled with:
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 racks location within the freezer should be noted on the appropriate freezer box master list.
Fish under 20 gm