TITLE: Photorepair Fluence Response Protocol
AUTHORS: Daniel Ma, David McDonald, NATALIE HULL

[DESCRIPTION]
Photorepair light exposures are performed on microorganism samples following UV light exposure (i.e. UV disinfection) to determine microorganism regrowth and repair kinetics. Photorepair is a light-dependent, enzyme mediated DNA repair process in bacteria that may reverse UV-induced DNA damage and decrease UV disinfection efficacy. Previously, photorepair kinetics were determined on a time-basis (i.e. exposure times). However, time-based photorepair determinations are not standardized. A photorepair fluence response protocol was developed here according to a standardized protocol proposed by Bohrerova and Linden (2007). If you cite your use of this protocol, you should also cite the relevant original literature.

[BEFORE_START]
For microbial samples, sanitize bench top surfaces with 70% ethanol.

[STEPS]
SECTION: Introduction and Pre-Experiment
1. The photorepair fluence response protocol is based on Bohrerova and Linden (2007).
 
To perform the photorepair fluence response experiment, you will first need to perform a UV fluence response experiment after the references below. Photorepair experiments and sample handling must be performed in a dark environment. Minimal red light may be used for visibility. This protocol assumes that the user has already performed the UV fluence response experiment. The Polychromatic Fluence (UV Dose) Response Protocol, linked below, can be used for performing the UV exposures.
 

 


SECTION: Before the Photorepair Experiment
1.1. UV Irradiation: Perform the UV fluence response experiment to prepare the UV irradiated samples for the photorepair fluence response experiment. Experiments must be performed in a dark environment with minimal red light for visibility.

 

After UV irradiation: Immediately remove sample from UV exposure. Keep samples in the dark and covered. Perform sample handling and plating in the dark with only red light for visibility. Take sample for microorganism enumeration and molecular analyses. Dilute samples and plate. Set aside samples for molecular analysis and store at -80°C.

Transfer the desired of the irradiated sample for photorepair and the remaining sample for dark repair into two identical, sterile Petri dishes (60 mm dishes).
SECTION: Performing the Photorepair Fluence Response Experiment
5. Sampling: After each time interval, remove the pre-determined sampling volume from the photorepair and dark repair samples. 

Handling: Transfer samples to empty, sterile microcentrifuge tubes. 

Dilute and plate organisms immediately: Perform serial dilutions with 1X PBS of samples and plate. Incubate plates under the desired growing conditions for the microorganism. Incubation times will vary per microorganism.

 

SECTION: Preparing the Photorepair Apparatus
2. Before performing the photorepair fluence response experiment, set up the photorepair fluence response experiment apparatus. A light source emitting the desired photorepair light (minimum 300-500 nm emission spectrum) should be suspended above a magnetic stirrer and dish containing the sample.

Warm up the photorepair light source (e.g. 30 minutes before performing exposures).
Start up spectroradiometer and acquisition software. 
If your spectroradiometer has not been set up to obtain spectral irradiance measurements, set it up, load calibration data (if necessary), and obtain background reading as part of the set up.
Obtain spectra irradiance measurements at the surface of the water sample (Figure 1).

 

SECTION: Post-Experiment
5.1. Optional if users account for the height adjustment and spectra measurements at different heights: After the experiment, calculate the photorepair fluence for each time interval. Use the irradiance measurements taken at each height for the corresponding time interval to account for the differences in light intensity at different distances from the lamp:   


SECTION: Post-Experiment
5.2. Perform enumeration of microorganism and calculate concentrations. The reference list below provides some sample equations used in the literature for determining repair kinetics.

Percent (Photo)reactivation
 
Survival Ratio
 





SECTION: Post-Experiment
5.3. Perform molecular analyses or molecular processing such as DNA extraction or protein extraction and perform further desired downstream molecular quantification methods, such as examples referenced below.

ATP Quantification
 
Cyclobutane Pyrimidine Dimer ELISA for DNA Damage
 
Endonuclease Sensitive Site (ESS) Assay for DNA Damage
 

 
Quantitative Polymerase Chain Reaction for DNA Damage
 
Quantification of Protein Damage 
 



SECTION: Performing the Photorepair Fluence Response Experiment
3. After obtaining measurements (optional: for each height required), export spectral irradiance data.

 

 

SECTION: Performing the Photorepair Fluence Response Experiment
4. Photorepair: Set the distance from the sample surface to the light source. Place the sample for photorepair exposure under the photorepair light at the pre-determined distance. Stir the sample continuously with the magnetic stirrer. 

Dark Repair: Place the dark repair sample on magnetic stirrer in a dark environment.

 

 

SECTION: Preparing the Photorepair Apparatus
2.2. Optional: To compensate for the change in sample distance from the light source with each subsequent volume withdrawal for sample analysis by calculating the height differential between sampling events, and measuring the light intensity at the center of the dish at the expected level at the start of the experiment to account for the changes in fluence calculation. The change in distance can be calculated using the volume withdrawn and the geometry of the dish (e.g. inner diameter and surface area). The number of measurements will be determined by the number of sampling events that should be pre-determined at the start of the experiment.
  

SECTION: Preparing the Photorepair Apparatus
2.1. Use the Photorepair Fluence spreadsheet to determine the photorepair fluence:  . The contents of each sheet are summarized in Table 1.

 


 

Data for the photolyase action spectra was digitized from Payne and Sancar (1990):
 

SECTION: Preparing the Photorepair Apparatus
2.3. Photorepair Irradiance Example Data: Measurements obtained using the method described in Figure 1 are shown in this example:   . A summary of the contents of this spreadsheet is provided in Table 2 below.