Feb 09, 2016
One-step growth experiments (cyanophages)
- Mathias Middelboe1,
- Amy M. Chan1,
- and Sif K. Bertelsen1
- 1Manual of Aquatic Viral Ecology
- VERVE Net
- Suttle Laboratory of Marine Molecular Microbiology and Virology
External link: http://www.aslo.org/books/mave/MAVE_118.pdf
Protocol Citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. One-step growth experiments (cyanophages). protocols.io https://dx.doi.org/10.17504/protocols.io.dra52d
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 04, 2015
Last Modified: November 10, 2017
Protocol Integer ID: 1538
Abstract
Described is the procedure used to perform a onestep growth curve for cyanophage BBC1-P1 via plaque assay on Synechococcus sp. BBC1 (Suttle and Chan 1993).
Guidelines
The procedure is similar to that for bacteriophages with one major difference. Where bacteriophage growth is measurable in the order of minutes, cyanophage growth curves are measured in terms of hours. The burst sizes are similar, being in the tens to hundreds.
As for bacteriophages, infection should be done at a MOI between 0.1 and 0.01. In this instance, the total infective center = the phage input because the proportion of multiplyinfected host cells is small. At higher MOI, the probability of cells infected by more than one virus would increase and the total infective centers (TIC = total number of infected cells + free viruses) become less than the phage input.
The dilution factor in going from the adsorption tube to FGT is chosen so that a reasonable number of plaques (ca. 50- 200) will form on the plates during the latent period. The dilution factor in going from the FGT (first growth tube) to SGT (second growth tube) is chosen on the basis of the expected increase in plaques at the end of the rise period so that later platings of SGT samples will also yield countable numbers. As mentioned in the bacteriophage section, it is recommended to perform a preliminary one-step experiment to estimate the possible length of the latent period and burst size. Then repeat the experiment with more frequent sampling with adjustments to the dilution factors to gain more precision. Sampling times may need to be adjusted for each phage-host system.
In summary:
1. Determine total phage input (Phageinput) and total cyanobacteria input (Cyanoinput).
2. At T = 0 determine the titer of unadsorbed phages (Free PhageT=0) and total infective centers (TICT=0 = initial total infected cells).
3. During the latent period and rise, determine the titer of total infective centers (TIC = total infected cells + any phages released).
4. At the end determine the titer of total progeny (TICfinal).
Calculations.
Adsorbed phages = Phageinput - Free PhageT=0
Percent adsorption = Adsorbed phages/ Phageinput x 100
Average MOI = Adsorbed phages/Cyanoinput
Burst size = (TICfinal/TICT=0)
Set up triplicate adsorption tubes (AT) in 1.5 mL microtubes.
Add phages of a known titer to the cyanobacteria host in exponential growth (cell concentration determined by microscopy; Cyanoinput) at an MOI of approximately 0.02.
Note
(e.g., 0.9 mL hosts [1 × 107 cells mL–1] + 0.1mL phages [2 × 106 PFUs mL–1] = MOI of ~0.02).
Allow the phages to adsorb to the hosts for 60 min. at room temperature and an illumination of 25µmol quanta m–2s–1.
01:00:00
Note
Flick tubes a couple of times at 30 min.
Set up control tubes (CT) as above, except:
a. positive controls: replace host cells with media (this gives input phage numbers).
b. negative controls: omit virus
After 60 min, remove unadsorbed phages (T = 0)
01:00:00
Centrifuge briefly to pellet host cells (e.g., 5 min, 16,000g, 4°C).
00:05:00
Remove supernatant, resuspend cells in fresh media.
Repeat washing step.
Assay washes to determine the number of unadsorbed phages (Free PhageT=0) for calculation of efficiency of adsorption.
Prepare nine 15-mL centrifuge tubes containing 10 mL algal growth media
Note
(6 labeled “FGT” [first growth tube] and 3 labeled “SGT” [secondary growth tube]).
Add 100 µL from AT to FGT (10–2 dilution).
Mix and remove 0.1 mL sample for T = 0.
Perform plaque assay immediately to determine total infective centers (TICT=0).
Add 100 µL from FGT to SGT (10–4 dilution).
Incubate FGT and SGT tubes at room temperature and ca. 25 µmol quanta m–2s–1
Remove 100 µL samples every 3 h from FGT (at 3, 6, 9, 12, 16 h) and SGT (from 12 h onwards) for ca. 30 h.
Determine the TIC via plaque assay.
Add 100 µL from positive control tube (CT) to FGT-control (10–2 dilution).
Determine the total phage input (Phageinput).