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Fish 424
Laboratory Exercises (Lab 4)
Please read through entire lab prior to beginning.
Collection and Preparation of Blood, Hematocrit, and Tissue Imprints
Introduction
In fish, components of the blood are formed and mature in the kidney, spleen, and thymus. Fish have no bone marrow or lymph nodes as in mammals. The components of fish blood are not easily categorized into those used for mammals. In addition, immature forms of various cell types are frequently present. Hematological techniques in fish health provide information on the general health of the animal and a method to assess some factors of non-specific resistance.
Collection of Blood for Hematocrit and Blood Smear
Materials for Blood Smearand Collection of Blood
Precleaned frosted-end type slides 25 mm x 75 mm x 1 mm (Fisher Scientific, Catalogue number 12-544-2)
Heparinized hematocrit tubes (3 per fish)
Pencil or Histology Shur Marker Pen (Fisher Scientific)
Marker pen
Scalpel and blades
Absolute methanol (Fisher Scientific, Catalogue number A452-1, Lot number 943778) in a Coplin jar
CritoSeal VWR#15407-103
Procedure for Blood Smear:
1. Label one slide (slide A) with fish number, tissue type, date and your initials with apencil and place the slide on a flat bench counter. Use clean frosted-end slides or the slides that had been cleaned with 95% ethanol.
2. Obtain a euthanized fish and sever the caudal peduncle with sharp scalpel.
3. Using 3 heparinized labeled hematocrit tubes, collect samples of blood directly from the severed artery of the fish. The blood will fill a hematocrit tube by capillary effect. Carefully lay samples flat on tray. Seal two tubes with critoseal and set aside for hematocrit.
4. With the third tube, place one small drop of the blood on the unfrosted end of the slide (slide A).
5. Hold another clean slide (slide B) with thumb and index finger at 45° angle to the slide A. Keeping the same angle, slowly move slide B toward the blood and contact these two slides with the blood. Spread the blood across to the edges of these two slides (45° angle wedge shape constructed with two slides should be filled with the blood).
6. Still holding slide B with 45° angle, move the slide toward unfrosted end of slide A to make a thin even blood smear on slide A. This should be done in one movement.
7. Air dry slide A and fix with Solution A for at least 10 min.
Determination of Hematocrit
Materials:
Heparinized hematocrit tubes
Critoseal
Centrifuge with hematocrit head
Hematocrit reader
Procedure for Centrifuging and Reading Hematocrit:
This step will be performed when the centrifuge is full (not individually).
1. Place 2 tubes for each fish in centrifuge (make sure that the centrifuge is equally balanced), and make sure that the critoseal end in to the outside edge.
2. Spin approximately 15 min.
3. Remove from centrifuge carefully and place the tube on the hematocrit chart with the top of the plasma fraction on the 100% line and the bottom of the red blood cells on line of the chart at 0%.
4. Read % red blood cells (red portion) to the bottom of the buffy coat (white blood cell layer) and record on data form. (For both tubes)
Preparation of Tissue Imprint
Materials:
Pre-cleaned frosted-end type slides 25 mm x 75 mm x 1 mm (Fisher Scientific, Catalogue number 12-544-2)
Pencil Scalpel
Forceps
Methanol
Procedure:
1. Label the slide with either pencil or Histology Shur Marker Pen with fish number, tissue type, date and your initials as in the preparation of blood smear.
2. Cut a small (about 2 mm3) piece of the head (or anterior) kidney with scalpel. Make additional imprints of trunk (or posterior) kidney, spleen, andliver.
3. Holding the tissue with forceps, gently press the cut edge onto a prepared slide repeatedly for several plots.
4. Air dry the slide and fix with Solution A for at least 10 min.
Staining Procedure (for blood smear and tissue imprints):
1. Dip the slide in Solution B 25 times.
2. Dip the slide in Solution C 25 times.
3. Rinse well with DI water.
4. Dry and examine the slides.
Safety
For your protection, read MSDS for chemicals used in this stain. Wear disposable gloves, lab apron or lab coat to protect clothing.
Hematocrit Results
Tube 1 ______
Tube 2 ______
Does this fish fall within the healthy range outlined by Klontz?
Evaluation of Blood Smears and Tissue Imprints
Using Microscopes, Locate Identify and Draw the Following Cell Types:
1. Erythrocytes
2. Lymphocytes
3. Neutrophils (Polymorphonuclear Leukocytes)
4. Thrombocytes
5. Monocytes and Macrophages