The Phage Cookbook by Dominik Refardt
The acronym LB has been incorrectly interpreted as Luria broth, Lennox broth, or Luria-Bertani medium, although according to its creator Giuseppe Bertani, the abbreviation LB was actually intended to stand for lysogeny broth (Bertani 2004).
5 g Yeast extract
10 g NaCl
10 g Tryptone (Casein peptone)
1 L double-distilled water
Often there are premade mixes of these ingredients available. Dissolve them in water by vigorous shaking or add a stirring bar and place them on a magnetic stirrer.
Some recipes recommend to adjust the pH. I have never done this and for the usual purposes this is not necessary.
If LB agar is required, add 15 g (for hard-agar, to pour plates) or 7 g (for top-agar) agar per 1 L medium. Autoclave. A thick plate (9 cm diameter) for counting plaques requires 25 mL, for other purposes you can go down to nearly half as much. Be careful to pour plates on an even surface when used for plaque counting! Uneven plates yield plaques of different size because the top-agar differs in thickness across the plate. The thinner the layer, the smaller the plaques. Let plates dry on the bench for three days before storing them in bags at room temperature. Drying the plates does not mean that you wait until all condensation droplets on the lid have disappeared. It concerns the agar. Fresh agar plates will "sweat" when incubated at 37 °C, which causes phages and/or bacteria on the plate to move around.
With 10 g NaCl (MW 58.4), the concentration of Na+ in the medium is 170 mM.
Make 1.5% agar (15 grams of agar in 785ml distilled H2O (you will add all ingredients to this bottle, so the volume of water is adjusted for this). Sterilize by autoclaving. Leave stir bar in while autoclaving.
Make 200 mL of 5x M9 salts: 11.28 g in 200 mL. Sterilize by autoclaving. You can make several of these bottles and store them at room temperature.
Make 100 mL of 1M MgSO4. Sterilize by autoclaving.
Make 100 mL of 1M CaCl2.
Make 200 mL of 20% glucose, i.e. 40 g in 200 mL. Sterilize by autoclaving.
Once everything is autoclaved, stir into the hot bottle of 1.5% agar: 200 mL of 5x M9 salts, 1mL of 1M MgSO4, 100 uL of 1M CaCl2 and 10 mL of 20% glucose. Add antibiotic if required.
Magnesiumsulfate is used to dilute bacteria and to store them in the fridge at 4 °C.
Prepare 1 M MgSO4 (molar mass 246.47 g/mol) and dilute it 100-fold to obtain 10 mM MgSO4. Autoclave the diluted buffer again before use.
Dissolve 123 g MgSO4⋅7H2O in 0.5 L double-distilled water
Autoclave
Store at room temperature
To prepare buffer, dilute the stock 100-fold in double-distilled water and autoclave
Phosphate buffered saline is an isotonic buffer that is very cell-friendly. Below is the recipe for 10x PBS. Dissolve all ingredients in 800 mL water, adjust the pH to 7.4 by adding HCl, then top up with water to 1 L. Sterilize by autoclaving. The buffer can be frozen to prevent unwanted growth, but salts may precipitate. The precipitate will dissolve again at room temperature.
80 g NaCl
2 g KCl
14.4 g Na2HPO4
2.4 g KH2PO4
Or even easier: Just use pre-made 10x PBS (Sigma 79383), or tablets (Sigma P4417).
Phage lysates are best stored or handled (e.g. in a dilution series) in SMG buffer. If it's only for a short time, 10 mM MgSO4 is also fine.
5.8 g NaCl
2 g MgSO4⋅7H2O
50 mL 1 M Tris HCl, pH 7.5
5 mL 2% gelatine solution (for SMG)
add H2O to 1 L, autoclave, store at room temperature.
The gelatine is supposed to stabilize the phage particles. I do not know how important this is. To be on the safe side, I never use SM, always SMG.