TITLE: Enumeration and Propagation of Bacteriophage MS-2
AUTHORS: leili abkar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This Protocol guides through setp-by-step enumeration and propagation of MS-2 bacteriophage. It is  prepared and adjusted for </span><span style = "font-weight:bold;font-style:italic;">Escherichia coli</span><span style = "font-weight:bold;">(ATCC 15597) strain C-3000</span><span>and</span><span style = "font-weight:bold;">MS-2 phage (ATCC-15597-B1).</span></div></div>

[STEPS]
?. [Stock Preparation]
?. [1	Stock Preparation]
?. [Stock Preparation]
LB Broth Dilution TubesAdd 20 g of LB broth to 1 liter of milli-QHeat and stir until warm and fully dissolvedAutoclave at 121°C for 20 minutesAseptically transfer 900 µL of sterile broth to 1.5 mL microcentrifuge tubes
?. [Stock Preparation]
E.coli (ATCC 15597) Medium preparation – Medium 271Add 20 g of LB broth to / mL of DI waterHeat and stir until warm and fully dissolvedAutoclave at 121 C for 20 minutes.Let it cool down to 50C in water bath.Add 50 mL of SOL B aseptically.SOL A- LBSOL B – 1Glucose 10.0 g, Cacl2 2.94g, Thiamine 0.1 g (0.0005M), DI water 500 mL – filter sterilized using 0.22 µm filter in a sterilized vesselSOL B-2Thiamine HCL:3.73 gr in 10 mL of water (1M)Then add0.3 mL to 500 mL of Solution B-1SOL B: SOL B-1+SOL B-2
?. [Stock Preparation]
1.1Fresh Host Cell Stock: Escherichia coli strain B- using flasks 95 mL LB and 5 mL inoculationAt the end of the day (3pm) add 5 mL ofE.coliB stock to 95 ml of medium (LB broth+ SOLB)The following morning (at 9 am) dilute overnight stock 100 times in fresh medium (LB broth+ SOLB)Incubate for 3hrs and 15 min to get to the mid-exponential phaseat 37°C and200 rpmAdd 1.5 mL ofmid-exponential stockwith 500 µL of 50%glycerol(sterilized) in 2mL labeled cryotubeStore at -80°C until needed.
?. [Stock Preparation]
Bacteriophage MS-2 Stock preparationAdd 1 mL stockE. coliB into9mL of medium (LB broth+SOLB) and incubate for Mid exponential phase 3 hrs and 15 min (37°Cand200 rpm)Add500µL of bacteriophage MS-2 stock and 1 mL of sterilized 0.2 M CaCl2– [2.9402 g/ 100mL].Incubate overnight (20-24 hr) at37°Cand200 rpm.Cells and debris should be removed from the phage lysate by centrifugation at (3000 RPM) 3500 RPM for 15 min at room temperature.The phage-containing supernatant should be filter 0.45 µm- using sterilized syringe filters- into fresh sterilized falcon tubes.Stock solution should be near 1010-1011PFU/mL.Store at 4°C for 1-2 months.
?. [Stock Preparation]
Creating Stock from the vialAdd 1 mL stockE. coliB into9mL of LB broth and incubate for Mid exponential phase 3 hrs and 15 min (37°Cand200 rpm).Open the ampule and suspend freeze dried powder using (1.5 mL) sterile PBS; make sure is fully mixed. Transfer aliquots of 500 µL to sterile microcentrifuge tubes. They stay active for at least two months at 4 °C.Add100µL of bacteriophage MS-2 stock and 1 mL of sterilized 0.2 M CaCl2– [2.9402 g/ 100mL].Incubate overnight (20-24 hr) at37°Cand200 rpm.Cells and debris should be removed from the phage lysate by centrifugation at (3000 RPM) 3500 RPM for 10 min at room temperature.Recover stock by filtering at 0.45 µm- using sterilized syringe filters into fresh sterilized falcon tubes.Stock solution should be near 1010-1011PFU/mL.Store at 4°C for 1-2 months.
?. [Agar Plate preparation]
Prepare 450 mL – Part A bottom and upper layer agars as per Table 2.1. Autoclave at 121°C for 20 minutes.After autoclaving, let it cool down using the water bath at between 45-50CAdd the Part B solution aseptically. SOL B is heat sensitive and higher temperature may cause precipitation of CaCl2or disintegration of thiamin HCL.Add10 mLof bottom layer to Petri dish (100mm*15mm) and allow to harden. You should get ~50 plates. Make sure leaving the lid partially open not to get condensations,Condensation causes inaccuracy.Temper upper layer at range of 45-50°C, do not add SOL B if you are not ready to use it.If you are using in small portions, thenUse a 50 mL falcon tube, add 40 mL of top layer and 4 mL of SOL B. Vortex briefly.If you are using the whole 450 mL,Then add 50 mL of SOL BUse a 50 mL falcon tube, add 40 mL of top layer and 4 mL of SOL B. Vortex briefly.If you are using the whole 450 mL,Then add 50 mL of SOL B&nbsp;ComponentBottom Layer&nbsp;(1% agar)Upper Layer&nbsp;(0.9% agar)Part ALB Broth10 g10 gBacto Agar&nbsp;5 g&nbsp;&nbsp;&nbsp;&nbsp;4 gMilli-Q450 mL450 mLPart BSol B5050
&nbsp;ComponentBottom Layer&nbsp;(1% agar)Upper Layer&nbsp;(0.9% agar)Part ALB Broth10 g10 gBacto Agar&nbsp;5 g&nbsp;&nbsp;&nbsp;&nbsp;4 gMilli-Q450 mL450 mLPart BSol B5050
?. [MS-2 Enumeration]
PrepareE.coliB solution by incubating 1 mL ofE.coliB stock in 9 mL medium (LB broth+ SOL B).Incubate overnight for 16-18 hrs.Incubate for until in mid-exponential phase (3 hours and 15 min).After incubation is done add 1mL of sterilized CaCl2[brings the final Con to 20 mM]- quick vortex to mix it up.Serial dilution of MS-2 in LB using 1.5 mL microcentrifuge tubes (900 µL PBS + 100 µL of MS-2).Label plates based on the serial dilution, date and name (duplicates).Label fresh, sterile 15 mL tubes as serial dilution (10-1, 10-2, ….10-9)- These are for next step of addingE. coli.Add500 µLof preparedE.colito the labelled 15 mL tubes, then add 100 µL of serially diluted MS-2and 100 µL ofCaCl2Solutionto the correspondingE.colitubes – incubate for20 minutesatroom temperatureto allow for attachment of MS-2 to bacteria and before replication.Add 3 mL of upper layer to 15 mL falcon tubes (mixture of MS-2 andE.coli)mix it using your hand or vortex- avoid any bubbles.Pour it on the bottom layer plates and swirl it until fully spread.Allow for upper layer to harden.Keep control plate- justE.coliand no dilution of MS-2.Incubate at 37°C for 18-24 hr.Count Plaques.