Phage titers

Count the colonies and calculate the phage titer as colony-forming units (cfu). 

Mix the cells and take an aliquot for phage titering. The titer should be at least 10 times higher than the library diversity. 

Take an aliquot of 10 pL for measuring the input phage titer and proceed immediately to the capture with the remaining 990 pL. 

On the magnet, discard the supernatant containing unbound phages. Eventually take an aliquot for measuring the phage titer. 

Take 300 pL for measuring the output phage titer and transfer the rest of the culture to a 1-L flask containing 200 mL of LB medium with the appropriate antibiotic. Proceed as described in Section 6.3.1.3. 

Tissues can now be stored at -80°C for up to 2-3 days without significant lose of phage titer. 

Nontarget organs can be harvested and phage titer determined for the purpose of monitoring the selection. 

Set up amplification of output phage as well as titration of input and output phage. Titer input, acid wash (if desired), and output. Amplify output or acid wash (if desired). 

Between successive rounds of phage biopanning, the output phage sample (and/ or the acid wash sample, if desired) must be amplified. This procedure may be performed in parallel with the phage titering as detailed in Subheading 3.3 or may be performed independently. 

The phage titers should change between rounds of selection the phage recovered should increase with each selection cycle, as should the output-to-input ra-... 

On the following day, count the number of colonies on each titration plate to determine the phage titer. Phage titer (in pfii/mL) = average of (dilution factor x number of colonies) X 10 (to have per mL, because we plated 100 pL) x 2 (dilution with TGI cells). 

Count the blue plaques on each plate especially those having about 10 plaques. Phage titer is obtained by multiplying each number with dilution factor for the given plate and is referred to as plaque forming units (pfu) per 10 pL. 

It is necessary to estimate the phage titer for the purchased or prepared display library, and to proceed with the rounds of enrichment for interacting phage. This keeps the multiplicity of infection (MOI) less than 1. A low MOI is needed to assure that each plaque contains only one DNA sequence. 

Fig. 1. Growth curve of T7 bacteriophage after multiple infection of P Mabeled bacteria in nutrient broth. , light transmission of the whole culture at 660 m , phage titer o, radioactivity of the supernatant solution after centrifugation of aliquots of the infected cells. Note that the physical methods give the same stepwise curve as does biological assay (262).

Two sets of conditions were used to study phage adsorption (a) The cells were plasmolyzed in sucrose before addition of the phage, (b) The cells and phage were mixed before plasmolysis. Subsequent to adsorption (1-4 min at 37°C), the mixtures were diluted in the same medium, cells were sedimented by low-speed centrifugation, and fixed as described in Section 3.3. The supernatants were tested for phage titers in order to ascertain the extent of adsorption. 

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