Titering Phage Stocks

CHP - last updated: January 19, 1999

(1) assemble components:

• many LB plates
• phage stocks
• dilution fluid (DF)
• 2.5 or 2.0 ml soft agar aliquots in sterile tubes, kept in heating block (~46degC)
• plating bacteria (7155 su+ and /or 7136su-)

(2) make serial dilutions of phage stocks:

• make estimate of phage titer (~10¹¹ might be an estimate)
• aliquot 5 ml of DF into many sterile glass culture tubes (at least 4 tubes/stock, 5 tubes if a high titer stocks)
• add 50 ul stock to first tube of DF - a 100 fold dilution (Cammie use fliter extra long pipet tips [USA 1011-0830])
• take 50 ul of this and add to next tube - now a 10⁴ dilution
• continue out until there is a 10⁸ or 10¹⁰ dilution

REMEMBER TO CHANGE TIPS

(3) decide plating levels:

plate on 7155 (su+) so ambers will grow.

A good assortment for an unknown phage stock concentration is:

6.1 (10⁶ dilution, 100 ul plated)
8.5 (10⁸ dilution, 500 ul plated)
8.1 (10⁸ dilution, 100 ml plated)
10.5 (10¹⁰ dilution, 500 ul plated)
10.1 (10¹⁰ dilution, 100 ul plated when plating high titer stocks )

To determine reversion frequency plate restrictive conditions
if examining ambers plate on 7136 if examining ts grow at higher temperature.

4.1 (10^-4 dilution 100 ml plated)
4.5 (10^-4 dilution 500 ml plated)

(4) spread plates:

• take one tube warmed with 2.0-2.5 ml soft agar
• add two drops plating bacteria (I do this first ,less likely to contaminate the plating bacteria)
• add specified amount of correct phage dilution
• vortex very briefly (mix not froth)
• pour mixture on LB plate, tip to spread evenly
  (NOTE: do this step as quickly as possible - before soft agar gels)

(5) perform controls:

make sure to plate 7155 alone (no phage) before and after all other plates, to check plating bacteria viability

(6) incubate plates overnight at 30degC

(7) count plaques on each plate

(8) calculate to obtain phage titer of stock