Methods of chromosome preparation in fishes
Fish cytogenetics involves, arresting the chromosomes in dividing cells in metaphase stage using spindle inhibitor, like colchicine, followed by hypotonic treatment, fixation, dropping of cells onto microscope slides, chromosome staining, microscopy and analysis. Earlier chromosome studies were based on histological sectioned gonad material particularly testis, which provided only vague idea about number and morphology of chromosomes of a species. Subsequently, hypotonic treatment and squashing was practiced which provided much better information on chromosome morphology. Squashing of tissues resulted in many overlapping chromosomes and poor definition of chromosome morphology. The revolution in fish cytogenetics came only after the use of colchicine pre-treatment and hypotonic treatment followed by splashing technique, which facilitated chromosomal studies in both sexes. The improved method, based on preparation of cell suspensions by injecting colchicine to live specimens, dissecting out mitotically active tissue, hypotonic treatment for certain period, fixation of cells in Carnoy's fixative (acetic acid & methanol in 1:3 ratio) for an optimal period and preparation of slides by dropping single cell suspension onto the slides from certain height, followed by air drying/ flame drying the slides and staining the chromosomes with Giemsa, produces extremely satisfactory results.
A.1) In vivo chromosome preparation from Kidney / Gill tissues (Source: Barreto Netto et. al., 2007, Nagpure et. al., 2007)

1) Collect healthy fish specimens (preferably weighing 20-100 g).

2) Inject the fish with 0.05% colchicine intramuscularly @ 1 ml per 100 g of body weight.

3) Allow fish specimens to swim for 1- 2 h in a bucket after injection.

4) Anaesthetize the fish specimen with ethylene glycol (50 ppm) and dissect out the kidney/ gill tissues in a petri dish and cut into small pieces.

5) Homogenize the tissue in 6-8 ml hypotonic solution (0.56% KCl) in glass tissue grinder to make cell suspension.

6) Pour the cell suspension in 15 ml centrifuge tube and keep it at room temp for 20-25 min for cell swelling.

7) Stop the hypotonic action by adding 1-2 ml freshly prepared chilled Carnoy's fixative (methanol: acetic acid in 3:1 ratio) gradually.

8) Mix it gently with a pasture pipette.

9) Centrifuge cell suspension at 1,200-1,500 rpm for 10 min at room temperature (RT) to get cell pellet at the bottom.

10) Remove supernatant with a pipette leaving approximately 2-3 ml of supernatant. Add 6-8 ml freshly prepared chilled fixative slowly.

11) Keep the tube in refrigerator for 1-2 h for thorough fixation.

12) Mix the contents and centrifuge cell suspension at 1,200-1,500 rpm for 10 min at RT.

13) Remove the supernatant without disturbing cell pellet at the bottom and add fresh fixative.

14) Repeat steps 12 & 13, three times at 1 h interval till clear transparent cell suspension is obtained.

15) Take small quantity of cell suspension in a pasture pipette and drop it onto grease free, pre-cleaned glass slide from 1.5-2.0 feet height.

16) Allow the slide to air / flame dry.

17) Keep the slide for 1-3 days for ageing in dark and dust free environment.

18) Stain it with 4-6% Giemsa in phosphate buffer (pH 6.8) for 15-20 min.

19) Wash with DD water thoroughly.

20) Air dry and store the slides in a slide box.

21) Observe metaphase spreads in bright field microscope to ascertain the quality of staining.

22) Make the slides permanent by mounting with DPX mountant.

23) Screen the slides for good spreads and take photographs of metaphase spreads under oil immersion objective (100X).

24) For karyotype preparation, cut individual chromosomes from the photo prints.

25) Group the chromosomes into four categories - metacentric (m), submetacentric (sm), subtelocentric (st) and telocentric (t).

26) Paste the chromosomes on ivory sheet in decreasing order of size within the group (align centromeres of all chromosomes in each row).

27) Photograph the karyotype, which can be used as base line data for detection of chromosome aberrations.

A. 2) Chromosome preparation by In vitro colchicinization (Source: Nagpure and Barat, 1997)

1) Take kidney tissue from live/ freshly dead fish.

2) Homogenize to prepare cell suspension in 8 ml of RPMI 1640 culture medium.

3) Add 50 μl of 0.05% of colchicine.

4) Incubate the cell suspension in BOD incubator for 30-50 min at 27-32 ?C.

5) Centrifuge the cell suspension at 1200 rpm for 10 min.

6) Decant the supernatant.

7) Add 8 ml hypotonic solution (0.56% KCl) to the cell pellet.

8) Incubate the cells in this solution for 20-25 min at room temp.

9) Follow the steps 7-27 of 'in vivo chromosome preparation from kidney/ gill tissues' section.

A. 3) In vitro chromosome preparation from whole blood culture

1) Dispense 9.5 ml sterile RPMI 1640 media (fortified with 10% heat inactivated fetal calf serum and 10 μg/ml phytohaemagglutinin) to 15 ml sterile culture tube.

2) Collect 0.2-0.5 ml blood from caudal vein using heparinized syringe and mix the contents to the culture tube containing culture media and place the cap of tube tightly.

3) Incubate the culture at 28° C in CO2 / BOD incubator for 72 h.

4) For harvesting the cells for chromosome preparation, add 1.0 μg colchicine / ml of culture medium approximately 2 h of before the termination of culture and centrifuge at 1,000 rpm for 10 min.

5) Remove supernatant and gently add 5 ml of KCl (0.56%) solution, gently mix cells and incubate at RT for 20 min.

6) Follow steps 7- 27 of 'In vivo chromosome preparation from kidney/ gill tissues' section given above.

7) Add 8 ml hypotonic solution (0.56% KCl) to the cell pellet.

A. 4) Tips for improving quality of chromosomes
1) Time and temperature of hypotonic treatment needs to be standardized since optimum time varies in different climatic conditions and in different species. Over hypotonic treatment results in excessive swelling of cells and more scattering of chromosomes leading to difficulty in identification of complete metaphase complement. Insufficient hypotonic treatment results in poorly separated and overlapping chromosomes.

2) Fixative should always be fresh and chilled.

3) Concentration of cells in fixative should be optimum as higher concentration of cells result in overlapping of chromosomes.

B. Analysis of chromosomes (Source: Herbert and Varley, 1983)
A chromosome in mitotic metaphase has two distinguishing features: its length and a transverse constriction that marks the position of the centromere. From the chromosome length and centromere position, three parameters are calculated: the centromeric index, the arm ratio and the relative length. The first two factors, i.e. centromere index and arm ratio, describe about the chromosome itself and the relative length denotes about the size of the chromosome in relation to other chromosomes in the complement.
I. Centromere index: is defined as length of shorter arm of the chromosome divided by the length of whole chromosome and multiplied by 100.
II. Arm ratio: is defined as the length of longer arm of the chromosome divided by the length of shorter one. It is, therefore, always =1.
III. The Relative length: of a chromosome is defined as the length of the particular chromosome divided by the total length of all the chromosomes in the haploid set, including the one being measured and multiplied by 100.
The karyotype of a chromosome complement is prepared from the cells exhibiting the complete somatic chromosome number and characteristic chromosome morphology. The homologous chromosomes were paired based on their length, morphology and position of centromere. The chromosome pairs were arranged in decreasing order of morphology and size in the karyotype. In fishes, the method of chromosome classification based on arm ratio (Levan et. al., 1964) has been widely used, which is described below:

Chromosome classification by arm ratio

Centromeric position
Arm Ratio
Chromosome type/ Symbol
Median
1.00-1.70
Metacentric (m)
Sub-median
1.71-3.00
Submetacentric (sm)
Sub-terminal
3.01-7.00
Subtelocentric (st)
Terminal
> 7.00
Telocentric (t)
 
C. Chromosome banding (Sumner, 1990)
Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allows both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic blocks (C-banding) and nucleolar organizer regions (NORs) etc. These landmarks facilitate the assessment of chromosomal morphology, identification of sites of chromosome breaks and alterations, and location of specific genes on chromosomes.
C. 1) C-banding (Source: Sumner, 1972; Schweizer et al., 1978)

1) Prepare chromosome spreads and air dry.

2) Store slides containing metaphase spread in dust free box for aging for about 3-7days.

3) Place the slides in 0.2N HCl solution for 1 h in a coplin jar at room temperature followed by rinsing with DD water.

4) The rinsed slides are placed in coplin jar containing 5% aqueous saturated solution of barium hydroxide at 50°C for 3-4 min and slides are rinsed with DD water.

5) Then slides are again incubated for 1 h at 60°C in 2X SSC (0.3M sodium chloride; 0.03M tri-sodium citrate) in a water bath.

6) The slides are taken out and rinsed with DD water.

7) Then the slides are stained with 2% Giemsa in phosphate buffer (pH 6.8) for 20-30 min and rinsed with DD water.

8) The slides are then dried in air and mounted with DPX.

C. 2) Propidium iodide (PI) staining for C-banding

1) Treat the slide for C-banding as described above.

2) Stain the treated slides with PI (5μg /ml in PBS) for 20-30 min.

3) Wash the slides with 2X SSC.

4) Mount the slide with antifade and observe under fluorescent microscope using appropriate filter.

C. 3) Nucleolar organiser regions (NOR) staining (Source: Howell & Black, 1980; Toledo, 1996)

1) The reaction solution is made by mixing developing solution and silver nitrate solution in 1:2 ratio.

2) Place 6-8 drops (about 300 μl) of reaction solution on a chromosome slide and place a cover slip.

3) Incubated the slide at 50°C for 3-5 min till solution turns golden-yellow colour.

4) The slide is rinsed with DDW, air dried and permanently mounted with DPX using cover glass.

C. 4) NOR staining with chromomycin A3 (CMA3)

1) Prepare CMA3 working solution by mixing CMA3 solution and Buffer in 1: 9 ratio (49 μl CMA3 solution + 651 μl Buffer).

2) Place 100 μl of solution on a slide and cover with a cover slip and keep the slide for 1 h in dark place.

3) Rinse the slide with DD water.

4) DAPI counter staining: Place DAPI mixture (2 μg/ ml) on the slide and keep it in dark place for 30 min followed by washing with Mc Ilvaine's buffer.

5) Place antifade solution on the slide, cover with cover slip and observe under florescent microscope with suitable filter.

D. Preparation of reagents (Nagpure et. al., 2013)

1) 0.05 % Colchicine: Weigh 5 mg colchicine and dissolve in 10 ml DDW and store it in refrigerator for further use (use solution within a week).

2) 0.56% KCl: Weigh 0.56 g KCl and dissolve in 100 ml DDW (to be used fresh).

3) Fixative: methanol 3 parts: acetic acid 1 part (to be used fresh and chilled).

4) Giemsa stain (stock): Weigh 0.5 g Giemsa powder and dissolve in 33 ml glycerol, and incubate at 60° C for overnight in water bath, cool to room temperature and then add 33 ml methanol, properly mix and filter with Whatman filter No.1 and store at room temperature.

5) Giemsa stain working solution: 3% Giemsa stain is made by dissolving 1.5 ml stock solution in 48.5 ml working solution of phosphate buffer respectively.

6) Phosphate buffer: Stock solution of 0.5M disodium hydrogen orthophosphate (Na2HPO4) and 0.5M potassium dihydrogen orthophosphate (KH2PO4) can be prepared by dissolving 8.89 g Na2HPO4 and 6.8 g KH2PO4 in 1000 ml double distilled water separately and stored at room temperature. The working solution is prepared from 0.5M KH2PO4 31.3 ml and 0.5M Na2HPO4 22.8 ml by making total volume 500 ml with double distilled water.

7) 0.2N HCl: 1.78 ml conc. HCl is diluted with double distilled water to final volume of 100 ml to prepare 0.2N HCl solution.

8) 5% Barium hydroxide:5% Ba(OH)2 is prepared by dissolving 2.5 g Ba(OH)2 in 50 ml double distilled water (to be used fresh).

9) 2X SSC:The 2X SSC solution is made by dissolving 1.75 g sodium chloride and 0.89 g tri-sodium citrate together in 100 ml DD water.

10) 50% Silver Nitrate:It is prepared by dissolving 1.0 g AgNO3 in 2 ml DD water. Store the solution at RT in dark and use within a week.

11) Developing solution:The developing solution is prepared by dissolving 0.2 g gelatin in 9.9 ml warm DDW at 50°C, cool to solution to RT and add 0.1 ml formic acid and store at RT and use within a week.

12) Antifade solution:2.3 % 1,4 diazobicyclo-(2,2,2, Octane) in Glycerol or 10 μg/ml p-phenylenediamine in PBS: Glycerol (1:9).

13) Propidium iodide solution:Dissolve propidium iodide @ 5μg /ml in PBS.

14) Chromomycin A3 (CMA3):Stock Solution: Dissolve 0.5 mg in 1 ml DDW. CMA3 working solution and Buffer in 1: 9 ratio (49 μl CMA3 solution + 651 μl Buffer ).

15) DAPI solution:2 μg/ ml in Mc Ilvaine's buffer at 7.0 pH.

16) Mc Ilvaine's Buffer:

Citric Acid (0.1M) : 2.1014 g/100 ml Na2HPO4 (0.2M) : 3.5632 g /100 ml

Working Buffer:

0.91 ml citric acid (0.1M) solution + 4.09 ml Na2HPO4 (0.2M) solution + 5 ml DDW + 0.005075 g MgCl2, adjust pH 7.

References
  1. Barreto Netto MR, Pauls E, Affonso PRA (2007). A standard protocol for obtaining fish chromosomes under post-mortem conditions. Micron 38:214-217.
  2. Herbert CM, Varley JM (1983).Working with Animal Chromosomes. Chichester; New York : Wiley. xv, 250 p.
  3. Howell WM, Black DA (1980). Controlled Silver Staining of Nucleolar Organizer Regions with a Protective Colloidal Developer: a I-step method. Experientia, 36:1014-1015.
  4. Levan A, Fredga K, Sandberg AA (1964). Nomenclature for centromeric position on chromosomes. Hereditas, 52,201-20.
  5. Nagpure NS, Barat A (1997). A Simplified Method of Fish Chromosome Preparation by in vitro Colchicine Treatment. Indian J Expt Biol. 35: 915-916.
  6. Nagpure NS, Kushwaha B, Singh PJ, Kumar Ravindra, Srivastava SK, Lakra WS (2007). Genotoxicity assessment in Fishes- a Practical Approach. NBFGR Publication.
  7. Nagpure NS, Kushwaha B and Kumar Ravindra (2013). Fish Cytogenetics: Techniques and Current Status. Ed. UC Goswami, Narendra Publishing House, New Delhi (in press).
  8. Schweizer D, Ambros P, Andrle M (1978). Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A. Expl Cell Res 111:327-332.
  9. Sumner AT (1972). A Simple Technique for Demonstrating Centromeric Heterochromatin. Exp Cell Res 75 304-306.
  10. Sumner AT (1990). Chromosome banding, Unwin & Hyman, U.K. 434 pp.
  11. Toledo LFA, Bigoni AP, Bernardino G, Foresti F and Toledo-Filho SA (1996). Karyotype and NOR Conservatism with Heterochromatin Reorganization in Neotropical Bryconids. Caryologia, 49(1): 35-43.
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