Dustin Edwards

Webbed Plate Calculator

Webbed plate calculator

1.11e10 or 1.11 x 1010.

What webs depends on plaque size; see the notes below.

Lab default: Phage Isolation and Purification Protocol: full plate titer.

Lab default: Phage Isolation and Purification Protocol: yields.

Webbed plates: 10 µl of tube 4 (11,100 pfu) on each of 6 plates

Lysate from flooding: About 30 to 42 ml

Webbed plates

  1. Lysate per plate: 11,100 / 1.11 x 1010 pfu/ml x 1,000 µl/ml = 1 x 10−3 µl.
  2. To plate that in 10 µl, the tube must hold 11,100 pfu / 10 µl x 1,000 = 1.11 x 106 pfu/ml.
  3. Total dilution: 1.11 x 1010 / 1.11 x 106 = 10,000-fold.
  4. That is 104: four 10-fold steps.
  5. Tube 1: 10 µl lysate + 90 µl phage buffer = 10−1, 1.11 x 109 pfu/ml.
  6. Tube 2: 10 µl tube 1 + 90 µl phage buffer = 10−2, 1.11 x 108 pfu/ml.
  7. Tube 3: 10 µl tube 2 + 90 µl phage buffer = 10−3, 1.11 x 107 pfu/ml.
  8. Tube 4: 10 µl tube 3 + 90 µl phage buffer = 10−4, 1.11 x 106 pfu/ml.
  9. Enough of the last tube: 6 plates x 10 µl = 60 µl.

Lysate from flooding

  1. Flooded with 8 ml of phage buffer each, a plate returns about 5 to 7 ml.
  2. 6 plates x 5 to 7 ml = 30 to 42 ml of lysate.

Dry or aged plates return less; one returned only 3.5 ml of an 8 ml flood.

Plates for a lysate volume

10 ml to archive plus 10 ml for DNA extraction is 20 ml.

Plates to flood: 3 to 4 webbed plates

Plates to flood

  1. At 7 ml per plate: 20 / 7 = 2.86, so 3 plates.
  2. At 5 ml per plate: 20 / 5 = 4, so 4 plates.

Pour a plate or two more than this: some plates do not web or yield less.

A webbed plate has so many plaques that they touch, leaving a lacy web of host. Flooding webbed plates with phage buffer gives the high-titer lysate. This calculator works out what to plate for a web from a lysate of known titer, and how much lysate the plates should give back.

Formula#

µl of lysate per plate = pfu wanted per plate / titer (pfu/ml) x 1,000 µl/ml
concentration to plate (pfu/ml) = pfu wanted per plate / µl plated x 1,000 µl/ml
lysate recovered (ml) = plates flooded x 5 to 7 ml
plates needed = ml of lysate wanted / 5 to 7 ml per plate

The volume from the first line is usually far too small to pipette, so it is plated by dilution: the lysate is diluted to the concentration in the second line (with the serial dilution planner's arithmetic) and a pipettable volume of the last tube goes on each plate.

Worked example: plating the web#

A lysate at 1.11 x 10^10 pfu/ml, aiming for 11,100 pfu on each plate, 10 µl per plate, 6 plates. The titer and target are from the lab's notebooks (Lab Calculations and Common Questions).

  1. Lysate per plate: 11,100 / 1.11 x 10^10 pfu/ml x 1,000 µl/ml = 1 x 10^-3 µl.
  2. To plate that in 10 µl, the tube must hold 11,100 pfu / 10 µl x 1,000 = 1.11 x 10^6 pfu/ml.
  3. Total dilution: 1.11 x 10^10 / 1.11 x 10^6 = 10,000-fold.
  4. That is 10^4: four 10-fold steps.
  5. Tube 1: 10 µl lysate + 90 µl phage buffer = 10^-1, 1.11 x 10^9 pfu/ml.
  6. Tube 2: 10 µl tube 1 + 90 µl phage buffer = 10^-2, 1.11 x 10^8 pfu/ml.
  7. Tube 3: 10 µl tube 2 + 90 µl phage buffer = 10^-3, 1.11 x 10^7 pfu/ml.
  8. Tube 4: 10 µl tube 3 + 90 µl phage buffer = 10^-4, 1.11 x 10^6 pfu/ml.
  9. Enough of the last tube: 6 plates x 10 µl = 60 µl.

Plate 10 µl of tube 4 on each of the 6 plates. In the notebook this lysate came from, six of seven plates made this way webbed in 24 hours, and the pooled lysate titered 2.2 x 10^11 pfu/ml.

Worked example: lysate from flooding#

The same 6 plates, each flooded with 8 ml of phage buffer:

  1. Flooded with 8 ml of phage buffer each, a plate returns about 5 to 7 ml.
  2. 6 plates x 5 to 7 ml = 30 to 42 ml of lysate.

The other way round, for 20 ml of lysate (10 ml to archive plus 10 ml for DNA extraction):

  1. At 7 ml per plate: 20 / 7 = 2.86, so 3 plates.
  2. At 5 ml per plate: 20 / 5 = 4, so 4 plates.

The lab plans 6 plates for this, because some plates do not web or yield less.

How many pfu make a web#

It depends on plaque size, so no calculator can supply it. In the lab's notebooks, on Mycobacterium smegmatis 1,000 to 4,000 pfu gave no web and 8,000 to 12,000 pfu webbed. For one Microbacterium foliorum phage, about 5,000 pfu gave few or no webs and about 20,000 pfu webbed, and targets of 280 to 1,500 pfu never webbed on M. foliorum. The dilution that webs also shifts between lysate batches, so the lab brackets: it plates a 10-fold series across whole decades, sees which one webs, and pours more plates of that one (the bracketing shortcut).

Where the numbers come from#

  • 10 µl plated per plate, with 250 µl of host: full plate titer.
  • No more than 25 µl of lysate on 250 µl of host (10% of the cell volume), or the cells lyse: the bracketing shortcut. The calculator warns above it.
  • 8 ml flood, 5 to 7 ml back per plate, and about 6 plates for 10 ml to archive plus 10 ml for DNA extraction: yields. Dry or aged plates return less; one returned only 3.5 ml of an 8 ml flood.