TITLE: Polychromatic UV  Dose Determination
AUTHORS: Daniel  Ma, NATALIE  HULL

[DESCRIPTION]
For polychromatic UV light sources, UV irradiance throughout the sample depth can be calculated by changing variables the “Germicidal Fluence (UV Dose) Calculations for a Low Pressure UV Lamp” obtained from Bolton Photosciences Inc. (Edmonton, Alberta, Canada) modified to account for polychromatic UV radiation sources (Linden and Darby, 1997). Because UV LEDs are polychromatic, the sensitivity of the radiometer (calibration data supplied by International Light) must be adjusted to the relative lamp emission of the LED (Bolton et al., 2015).

[BEFORE_START]
Sanitize bench top surfaces with 70% ethanol.

[STEPS]
SECTION: Introduction
1. The purpose of this protocol is to document the steps used for determination of UV doses for polychromatic UV sources such as UV LEDs and medium pressure mercury lamps. The method is modified from Bolton and Linden (2003). Gather equipment and materials. Sterilize materials and prepare agar for enumerating microorganisms.


SECTION: Microorganism preparation
3. Prepare microorganism cultures for UV exposure experiment.
 

SECTION: Microorganism preparation
3.1. Bacterial culture: Add 200 µL of E. coli K-12 cryostock to 20 mL sterile Tryptic Soy Broth. Incubate   for 240 min .

SECTION: Microorganism preparation
3.2. Remove culture from incubator and transfer to 50 mL centrifuge tubes. Centrifuge 5000 rpm, 10 min.
 


SECTION: UV Dose Determination
5. Determine UV exposures by importing UV absorbance scan data into a UV Dose Determination spreadsheet.

 

 

3.3. Decant and discard supernatant. Transfer sterile 1X Phosphate Buffered Saline (PBS) solution to the bacterial cells to balance the tubes. Vortex to mix.

4. Additional Cell Preparation: If using bacterial cells, aliquot working stock into as many 15 or 50 mL centrifuge tubes as needed for the number of UV exposures that you will perform. Sonicate for 30 s.
 


3.4. Centrifuge 5000 rpm, 10 min. Decant and discard supernatant. Suspend cells in 20 mL fresh 1X PBS.

 

3.5. Dilute the E. coli re-suspension into the desired volume of sterile 1X PBS to create working stock for UV exposures.
 

3.6. Take absorbance scan from 200-800 nm of working stock using UV-VIS Spectrophotometer.


 

Turn on UV-VIS Spectrophotometer. Warm up lamps at least 10 minutes or longer (e.g. turn on before removing culture from incubator).
Blank using distilled water. 
Take absorbance scan of distilled water. Empty cuvette.
Rinse cuvette three times with working stock. 
Take absorbance scans for at least duplicate samples of working stock.
Export absorbance scan spectra to flash drive.

SECTION: UV Dose Determination
5.1. Select UV doses for constructing UV dose response curve.

 


SECTION: UV Dose Determination
5.3. Set up radiometer.


 


Obtain the Petri Factor by measuring the irradiance at the plane level that the sample surface will be at. Take measurements in a 0.5 cm grid in both the x- and y-direction starting from the center of the Petri dish. Take enough measurements in all directions so that the exterior of the Petri dish falls within the outer most measurements.

Enter measurements in the UV Dose Determination Spreadsheet in the Determination of the "Petri Factor" Section.


 

SECTION: UV Dose Determination
5.2. Set the distance between the UV source and the surface of the sample. Enter the height in the UV Dose Determination spreadsheet.


 

SECTION: UV Dose Determination
5.4. Follow steps in the Instructions and Notes Section of the UV Dose Determination spreadsheet.
SECTION: Performing UV Exposures
6. Perform UV exposures by transferring samples to a sterile Petri dish, adding a stir bar, and placing on the magnetic stir plate. Expose the sample to UV irradiation for the calculated exposure times.
 

 

 

SECTION: Performing UV Exposures
6.1. Collect samples after exposures. Dilute and plate samples. Record the volume used for plating each dilution. Plating may be done with spot plating or spread plating. After samples are dried, incubate plates.

E. coli: 37 °C 960 min  (time may vary based on growth media)


 


SECTION: Performing UV Exposures
6.2. Calculation concentrations in non-irradiated and irradiated samples to determine log inactivation. Construct UV dose response curve by plotting log inactivation vs. UV dose.
SECTION: Introduction
2. UV Dose Determination Spreadsheet. The example loaded is for UV LEDs but other polychromatic light sources can be added to and used in this spreadsheet. Append the date of the experiment and other details as appropriate.