Practical Experiment on Culturing of Lambda (λ) Phage

The below mentioned article provides a Practical Experiment on Culturing of Lambda (λ) Phage.

Principle:

Bacteriophage λ is cultured in E.coli cell sus­pension. The phage is allowed to harbour and multi­ply till the complete lysis of bacterial cells. Then the bacterial cells are killed with chloroform and the phage particles are separated by centrifugation.

Materials:

A. E. coli LE 392 or other suitable strains

B. Stock λ phage (EMBL-3)

C. Chloroform

D. Pancreatic DNase and RNase

E. Sodium chloride

F. Polyethylene glycol (PEG 6000)

G. Cesium chloride

H. Luria Broth

i. Bactotryptone — 10 g

ii. Yeast extract — 5 g

iii. Sodium chloride — 5 g

iv. D-glucose — 1 g

v. Water — 1 l

vi. Sterilize and use.

I. SM Buffer

i. Sodium chloride — 5 g

ii. Magnesium sulphate — 1.2 g

iii. 1 M Tris (pH 8 0) — 50 ml

iv. Gelatin — 0.1 g

v. Water — 1 I

Sterilize and use.

Procedure:

1. Inoculate 1 ml of overnight grown culture of LE 392 E.coli strain to 100 ml LB containing 10 mM MgCl₂ and 0.2% maltose in triplicate. Grow for 2-3h till the OD 600 reaches 0.4.

2. With the assumption that 10D₆₀₀ = 8 × 10⁸ cells/ml calculate the cell concentration.

3. Centrifuge the cell suspension in autoclaved tubes at 4000 × g for 10 min at room temp.

4. Discard the supernatant and re-suspend the bacterial cells in 2 ml of SM.

5. Add bacteriophage from the stock to a concen­tration of 5 × 10⁸ phage/ml and mix rapidly.

6. Incubate at 37°C for 20 min. Shake intermit­tently.

7. To 125 ml of LB containing 10 mM MgSO₄ and 0.2% maltose, add one ml of infected cells (Step 4) in 500 ml flask. Incubate at 37°C with very vigorous shaking till it is lysed. Disappea­rance of silkiness is the indication of complete lysis. It takes 7-9 h for complete lysis.

8. To check complete lysis, take 1 ml of the culture from the flask and add few drops of chloroform. If it becomes clear proceed with the next step. If not, continue the incubation till complete lysis is achieved.

9. Add 2-5 ml chloroform to each flask and continue vigorous shaking for another 30 min at room temperature.

10. Collect the supernatant and pool for virus particles.

11. Bring the culture to room temperature and add pancreatic DNase and RNase, both to a final concentration of 1 µg/ml. Incubate for 30 min at room temperature.

12. Now, add NaCl to a final concentration of 1 M (29.2g/500 ml of culture). Dissolve by swir­ling. Let it stand for 1 h on ice.

13. Remove white silky mass of bacterial cells by centrifugation at 11,000 × g for 10 min at 4°C. Pool the supernatant and repeat this step.

14. To the supernatant in a flask, add solid polyethylene glycol (PEG 6000) in small quantities with slow shaking to a final concentration of 10% w/v (i.e. 50 g/500 ml).

15. Cool in ice water and keep it at 4°C overnight.

16. Centrifuge at 11,000 × g for 10 min at 4°C and recover the precipitated phage particles.

17. Re-suspend the bacteriophage pellet by gentle shaking in 7 ml SM buffer (i.e., 7 ml/500 ml of original supernatant).

18. To this suspension add equal volume (7 ml) of chloroform and mix thoroughly. Centrifuge at 1600 × g for 10 min at 4°C. Save the aqueous phase containing bacteriophage.

19. Measure the volume of this phage suspen­sion and add 0.5 g/ml of solid cesium chlo­ride. Mix gently. After completely dissolving cesium chloride, carefully layer this suspen­sion onto cesium chloride step gradients that are prepared in cellulose nitrate centrifuge tubes.

20. Centrifuge using SW 27 rotor at 25,000 rpm for 3 h at 4°C. A bluish band of bacteriophage particles will be visible at the interface between the 1.45 and 1.50 g/ml layers.

21. Collect the band of bacteriophage particles using siliconised Pasteur pipette. Store at 4°C in tightly capped tube.