TITLE: Modified one-step growth (mOSG) assay
AUTHORS: Eva JP. Lievens

[DESCRIPTION]
This protocol describes a modified one-step growth assay, as presented in the manuscript "Efficient assays to quantify the life history traits of algal viruses" (Lievens et al. 2023, Applied & Environmental Microbiology, doi.org/10.1128/aem.01659-23). It is designed to quantify the adsorption constant, depolarization probability, lysis time, and burst size of chloroviruses (large, lytic dsDNA viruses). The protocol should also be applicable to other lytic viruses that are large enough to be counted by flow cytometry. 

Compared to a classic one-step growth assay, this protocol uses multiple MOPs (virion:host ratios), miniaturization, and flow cytometry to increase efficiency. Briefly, viruses and host cells (algae) are mixed at a high concentration and allowed to adsorb, infections are synchronized by 1:1000 dilution, diluted suspensions are aliquoted, and independent aliquots are sampled every 2h. The samples are then measured with flow cytometry, and non-linear statistical models used to estimate the adsorption constant, lysis time, burst size, and depolarization probability. See the associated manuscript for a full description and schematic figures, as well as the statistical model.

[BEFORE_START]
This version is for two adsorption plates, which corresponds to max. 36 different viruses when using 5 MOPs/virus sample. It is assumed that the virion concentration of the virus samples is known (see dx.doi.org/10.17504/protocols.io.6qpvr6q93vmk/v1).

Use sterile techniques throughout this protocol. Using materials that don't bind viruses is important; see Materials for more information.

Definitions:
MOP = Multiplicity of Particles, the ratio of virions to host cells
BBM = modified Bold's Basal Medium, see the associated publication
growth conditions = the conditions under which the one-step growth assay is being run (e.g. a specific temperature)
culture plates = 96-well flat-bottomed tissue culture plates (see Materials)
deep well plates = 96-well 2.2ml PP deep well plates (see Materials)
transparent seal = transparent PCR plate seal (see Materials)
if the type of seal is not specified, it can be any adhesive plate seal that does not breathe (e.g. aluminum, thermo, ...)

[STEPS]
SECTION: Preparation (can be done the day beforehand)
1. Prepare the 1:1000 dilution plates.
SECTION: Preparation (can be done the day beforehand)
2. Prepare the 1:10000 dilution plates.
SECTION: Preparation (can be done the day beforehand)
3. If following the alternative 1:1000 dilution option (see step 9.1):
Prepare the 1:10 intermediate dilution plates.
SECTION: Preparation (can be done the day beforehand)
4.1. In two culture plates, mix viral suspensions so they have a volume of 50μl and varing virion concentrations:
final MOP 0.5: concentration 1.5*107 virions/ml at this stage; will be 0.75*107 virions/ml when the volume is 100μl
final MOP 1: concentration 3*107 virions/ml at this stage; will be 1.5*107 virions/ml when the volume is 100μl
final MOP 2: concentration 6*107 virions/ml at this stage; will be 3*107 virions/ml when the volume is 100μl
final MOP 5: concentration 15*107 virions/ml at this stage; will be 7.5*107 virions/ml when the volume is 100μl
final MOP 10: concentration 30*107 virions/ml at this stage; will be 15*107 virions/ml when the volume is 100μl
Avoid reusing tips for virus pipetting; reusing tips leads to lower accuracy (possibly because viruses bind to the plastic) (EJPL, unpublished data).
                plate  column  row  strain  MOP  virus to add (ul)  BBM to add (ul)    1  1  A  NE-JV4  0.5  1.03  49.0    1  1  B  NE-JV4  1  2.07  47.9    1  1  C  NE-JV4  2  4.14  45.9    1  1  D  NE-JV4  5  10.3  39.7    1  1  E  NE-JV4  10  20.7  29.3    1  1  F  MA-1E  0.5  1.04  49.0    1  1  G  MA-1E  1  2.07  47.9    1  1  H  MA-1E  2  4.15  45.9    1  2  A  MA-1E  5  10.4  39.6    1  2  B  MA-1E  10  20.7  29.3    1  2  C  PBCV-1  0.5  0.72  49.3    1  2  D  PBCV-1  1  1.44  48.6    1  2  E  PBCV-1  2  2.89  47.1    1  2  F  PBCV-1  5  7.21  42.8    1  2  G  PBCV-1  10  14.4  35.6    1  2  H  ctrl        50  

SECTION: Preparation (can be done the day beforehand)
5. Prepare 8ml of "flow cytometry positive control" sample in a 15ml tube (use any sample with a known virion concentration). Store at 4°C.
SECTION: mOSG
6. Get the adsorption plates out of 4°C storage, vortex & spin down. They will warm to room temperature during the next step.
SECTION: mOSG
7. Prepare an intermediate algal suspension.
SECTION: mOSG
7.1. Centrifuge the algae for 5min at 4500rpm.
SECTION: mOSG
7.4. Using the algal pellet and BBM, prepare 10ml of an intermediate algal suspension at 3*107 cells/ml.
SECTION: mOSG
8. Adsorption.
SECTION: mOSG
8.1. Using a 200μl multichannel pipette, aliquot 50μl of the intermediate algal suspension into wells 1A-12D of the adsorption plates. Make sure the algal suspension is added into the virus suspension (don't leave drops on the side of the wells), and use new pipette tips for every column to avoid contamination.
SECTION: mOSG
8.3. Shake the adsorption plates at high speed: 15min at 1200rpm on a microplate shaker with orbital diameter 1mm, or 15min at 500rpm on a shaker with orbital diameter 10mm.
Background info: These conditions led to ≥ 80% adsorbed virions in preliminary tests with PBCV-1 (EJPL, unpublished data).
SECTION: mOSG
9. Do a 1:1000 dilution to synchronize infections.
Notes: Do this step as quickly as possible: the time of dilution should be as close as possible to time point 0.
Background info: 1:100 dilutions were insufficient to prevent secondary infection (EJPL, unpublished data).
SECTION: mOSG
9.1. Using a 10μl multichannel pipette, transfer 2μl from adsorption plate "1" into each of the 1:1000 dilution plates "1". Do the same for adsorption plate "2" and the 1:1000 dilution plates "2".

Alternative 1:1000 dilution option, using a 200μl multichannel pipette:
Transfer 20 µl from adsorption plate "1" into 1:10 intermediate dilution plate "1". Do the same for adsorption plate "2" and 1:10 intermediate dilution plate "2".
Mix the 1:10 intermediate dilution plates by pipetting each column up and down 3x.
Transfer 20 µl from 1:10 intermediate dilution plate "1" into each of the 1:1000 dilution plates "1". Do the same for intermediate dilution plate "2" and the 1:1000 dilution plates "2".
SECTION: mOSG
10. Do a 1:10000 dilution, which will be used to look for signals of secondary infection.
SECTION: mOSG
10.1. Mix the remaining 1:1000 dilution plate "1" by using a 1000μl multichannel pipette to pipette each column up and down 3x. Do the same for the remaining 1:1000 dilution plate "2".
Transfer 200μl from the remaining 1:1000 dilution plate "1" into the 1:10000 dilution plate "1*". Do the same for the remaining 1:1000 dilution plate "2" and the 1:10000 dilution plate "2*".

If following the alternative 1:1000 dilution option (see step 9.1):
Using a 10μl multichannel pipette, transfer 2 µl from the 1:10 intermediate dilution plate "1" into the 1:10000 dilution plate "1*". Do the same for the 1:10 intermediate dilution plate "2" and the 1:10000 dilution plate "2*".
SECTION: mOSG
11. Time point 0 (0h) sampling.
Notes: Due to the time taken by steps 9 and 10, the "time point 0" samples are typically collected ~30min after the actual time point 0 (which is the end of the adsorption period, i.e. the idealized dilution time). As long as the infected cells don't start lysing within ~30min, this doesn't affect the results.
SECTION: mOSG
11.1. Centrifuge the remaining 1:1000 dilution plates "1" and "2" for 15min at 2000g, to separate free virions from algae.
SECTION: mOSG
11.2. Transfer 100μl of the supernatants from the centrifuged 1:1000 dilution plates "1" and "2" into culture plates. Label these "supernatant plate 1 0h" and "supernatant plate 2 0h".
SECTION: mOSG
11.3. Add 100μl BBM to wells 12E and 12F of the supernatant plates, and 100μl of the "flow cytometry positive control" to wells 12G and 12H of the supernatant plates.
SECTION: mOSG
11.4. Seal the supernatant plates and store at 4°C.
SECTION: mOSG
12. Every hour after time point 0, invert the 1:1000 dilution plates to mix the contents.
Background info: Several mixing methods were tested; inverting was similar to mild shaking (EJPL, unpublished data).
SECTION: mOSG
13. Every two hours after time point 0, sample one "1" and one "2" 1:1000 dilution plate as in step 11. After 16h, sample the "1*" and "2*" plates as well.
SECTION: Flow cytometry
14. Between 1-7 days after the mOSG (ideally 1-4 days), measure the virion concentration in the supernatant plates using protocol dx.doi.org/10.17504/protocols.io.6qpvr6q93vmk/v1 (for low-concentration virion samples).
Tip: To avoid conflating flow cytometer errors with mOSG results, don't measure the supernatant plates in chronological order.
SECTION: mOSG
8.4. Note the time at the end of the 15min adsorption period. This is time point 0.
SECTION: mOSG
8.2. Close the adsorption plates with their lid.
SECTION: Preparation (can be done the day beforehand)
4. Prepare the adsorption plates.
SECTION: Preparation (can be done the day beforehand)
4.2. Seal the adsorption plates (with the lids on top of the seal), vortex, and store at 4°C.
SECTION: Preparation (can be done the day beforehand)
3.1. Prepare two culture plates with 180μl in wells 1A to 12 D (i.e. wells 12E, 12F, 12G, 12H free). Label one plate "1" and the other "2".
SECTION: Preparation (can be done the day beforehand)
3.2. Seal the 1:10 intermediate dilution plates and store at room temperature.
SECTION: Preparation (can be done the day beforehand)
2.1. Prepare two deep well plates with 1800μl BBM in wells 1A to 12D (i.e. wells 12E, 12F, 12G, 12H free). Label one plate "1*" and the other "2*".

If following the alternative 1:1000 dilution option (see step 9.1):
Prepare two deep well plates with 1998μl BBM in wells 1A to 12D (i.e. wells 12E, 12F, 12G, 12H free). Label one plate "1*" and the other "2*".
SECTION: Preparation (can be done the day beforehand)
2.2. Seal the 1:10000 dilution plates and store at room temperature.
SECTION: Preparation (can be done the day beforehand)
1.1. Prepare 34 deep well plates with 1998μl BBM in wells 1A to 12D (i.e. wells 12E, 12F, 12G, 12H free). Label half of these plates "1", and the other half "2".

If following the alternative 1:1000 dilution option (see step 9.1):
Prepare 34 deep well plates with 1980μl BBM in wells 1A to 12D (i.e. wells 12E, 12F, 12G, 12H free). Label half of these plates "1", and the other half "2". 
SECTION: Preparation (can be done the day beforehand)
1.2. Seal the 1:1000 dilution plates and store at room temperature.
SECTION: mOSG
7.2. Gently pour off the supernatant and resuspend the pellet.
SECTION: mOSG
7.3. Count the concentration of algal cells in the pellet using a haemocytometer.
SECTION: mOSG
9.2. Seal all but one of the 1:1000 dilution plates "1" and all but one of the 1:1000 dilution plates "2". Use a transparent seal, and make sure that the seal sticks very well. Invert the sealed plates to mix, and leave under the growth conditions.
SECTION: mOSG
10.3. Seal the 1:10000 dilution plates. Use a transparent seal, and make sure that the seal sticks very well. Invert the sealed plates to mix, and leave under the growth conditions.
SECTION: mOSG
10.2. Seal the remaining 1:1000 dilution plates.
SECTION: mOSG
11.5. Discard the 1:1000 dilution plates.