SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
1 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Date: 5/27/09
1. INTRODUCTION
Photochemical inactivation is a process used to crosslink nucleic acids of microorganisms in order to
prevent their ability to replicate. Photochemical inactivation is carried out using the synthetic
psoralen (S-59) and ultraviolet light. Killed but metabolically active (KBMA) vaccines are produced
by photochemical inactivation of nucleotide excision repair-deficient strains.
2. PURPOSE
The purpose of this SOP is to describe how to determine the concentration of S-59 psoralen that is
required to achieve inactivation of Francisella tularensis strains.
3. RESPONSIBILITY
It is the responsibility of those personnel who photochemically inactivate Franciscella tularensis
strains to understand and correctly follow the procedures outlined in this protocol. This SOP is
primarily directed to microbiology and process development technicians who may need to inactivate
Francisella tularensis strains.
4. SCOPE
This SOP describes the method for preparation of photochemically inactivated Francisella tularensis
ssp. novicida strains or Francisella tularensis ssp. holarctica LVS strains only. This protocol is not to
be used for fully virulent type-A Francisella tularensis strains.
5. PRECAUTIONS
This SOP may be performed in a BSL-2 Microbiology Laboratory. Gloves, safety glasses, and a lab
coat should be worn at all times. If the laboratory requires additional PPE, these requirements should
be observed as well.
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
2 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Date: 5/27/09
6. STUDY PERSONNEL
Name (Print)
Signature
Initial
7. EQUIPMENT AND MATERIALS: PLEASE DOCUMENT
Equipment
Bio-Safety Cabinet
Spectrophotometer
Shaker incubator
Incubator
P200, P1000
FX1019 UVA Device
CERUS Equipment #
Date
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
3 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Material
Date: 5/27/09
Manufacturer
Part #
F. tularensis cell bank
Cerus
Not Applicable
Sterile Chamberlain’s media
Cerus
Not Applicable
3mM S-59 solution
Cerus
Not Applicable
Sterile 0.5L Disposable
Erlenmeyer Flask
Corning
431145
6-well microplate
Corning
3471
Cerus
Not Applicable
Hyclone
SH30028.02
Cystine heart agar w/
hemoglobin
DPBS
Lot # used *
*Laboratory personnel enter the lot# used during the experiment
8. PROCEDURE
Day 1 Date = ___________________
Disinfect BSC surfaces with 70% ethanol
In BSC, using a 50mL sterile serological pipette, transfer 50mL of Chamberlain’s
media into a 500mL shaker flask.
Remove cell bank material from freezer, spray outside of vial with 70% ethanol, and
thaw contents by rubbing between gloved hands.
Initial
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
4 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Date: 5/27/09
In BSC, using sterile P1000 tip, transfer 1mL of cell bank material from vial into the
shake flask.
Incubate overnight at 37ºC shaking at 200 rpm
Start time = _______________
Start date = _______________
End time = _______________
End date = _______________
Flask turbid after incubation: __________ (Y/N)
Day 2
Measure OD of overnight. OD600 = __________.
Label two 6-well microplates “A” and “B”. Number each well “1” through “6”.
Transfer 3.15 mL of Chamberlain’s media into each well of 6-well A plate.
Transfer the following volumes of Chamberlain’s media into wells of 6-well B plate
so that the media and S-59 solution equal 3.15mL.
Well 1 (100nM S-59) = 3.14 mL
Well 2 (500nM S-59) = 3.1 mL
Well 3 (1M S-59) = 3.04 mL
Well 4 (13M S-59) = 3.12 mL
Well 5 (50M S-59) = 3.1 mL
Well 6 (10 M S-59) = 3.04 mL
Prepare a 0.3mM S-59 solution by mixing 100L of 3mM S-59 and 900L of sterile
Milli-Q water.
Prepare a 0.03mM S-59 solution by mixing 100L of the 0.3mM S-59 solution and
900L of Milli-Q water.
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
5 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Date: 5/27/09
Transfer the following volumes of S-59 solutions into wells of 6-well dish B.
Well 1 (100nM S-59) = 11.7 L of 0.03mM S-59
Well 2 (500nM S-59) = 58.3 L of 0.03mM S-59
Well 3 (1M S-59) = 117 L of 0.03mM S-59
Well 4 (3M S-59) = 35 L of 0.3mM S-59
Well 5 (5M S-59) = 583 L of 0.3mM S-59
Well 6 (10M S-59) = 117 L of 0.3mM S-59
Gently shake to mix.
Transfer 350L of the overnight into each well of Plates A and B.
Shake plates at 200 rpm and 37 ºC in the dark.
Collect a sample from a new well in Plate A periodically to check OD600.
Time Point
Start
Time Point 1
Time Point 2
Time Point 3
Time Point 4
Elapsed Time
(Hr:Min)
0
OD600nm

NA
Well
Initial
Effective date:
SOP No: Cerus_Ft_Protocol_005
version 1.0
Page
6 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Date: 5/27/09
Time Point 5
Time Point 6
Time Point 7
Time Point 8
= ln(OD/OD0) / (t-t0)
Where “OD” is the optical density at 600nm and “t” is the time in hours.
Once  drops to 0.5 of max, transfer 6-well plate B into FX1019 and illuminate at 6.5
J/cm2, lid off.
Dilute samples from each well of plate B 1:10 and 1:100 in DPBS.
Plate 100L of the neat illuminated broth and each dilution on agar plates(Cystine
heart agar w/ hemoglobin). 3-10 replicates should be plated for each dilution.
Incubate plates at 37ºC 2-3 days until visible colonies appear.
Start time = _______________
Start date = _______________
End time = _______________
End date = _______________
cfu
Well 1: 100 nM S59
Neat
1:10 dilution
1:100 dilution
CFU/mL
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
7 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
Date: 5/27/09
Well 2: 500 nM S59
Well 3: 1 M S-59
Well 4: 3 M S-59
Well 5: 5 M S-59
Well 6: 10 M S-59
CFU enumeration: If there are more than 300 colonies on a plate, then a plate with a
higher dilution should be counted. If the 1:100 dilution plate has more than 500 colonies,
then the results should be reported as too numerous to count TNTC. Cfu/mL shall be
reported as the average number of colonies on each plate x dilution factor x 10.
COMMENTS/DEVIATIONS
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
8 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
9.
Date: 5/27/09
QUALITY CONTROL
Media should be checked prior to use for visible growth. CDM should be clear and
CHAH plates should be free of colonies.
Determination of viable cfu prior to illumination: Prepare 8 serial dilution tubes by
placing 900ul sterile dPBS in each tube. At the time plate B is illuminated, serially dilute
100ul of bacteria from well 1 of plate A and plate 100ul of the 10-6, 10-7 and 10-8 on
CHAH plates in triplicate. After 2-3 days, enumerate the colonies on plates that contain
30-300 colonies and calculate the number of cfu/mL by multiplying the average number
of colonies x dilution factor x 10.
cfu
10-6
10-7
10-8
Plate A Well# 1:
10. REFERENCES
PRP01 ver.002: Preparation and Quality Control of Bacterial Culture Media
QCP-01-003: Determination of Viable Bacterial Count of a Bacterial Culture
11. CALCULATIONS AND FORMULAS
Bacterial growth rate calculation: µ= ln(OD/OD0) / (t-t0)
Where “OD” is the optical density at 600nm and “t” is the time in hours.
SOP No: Cerus_Ft_Protocol_005
version 1.0
Effective date:
Page
9 of 9
5/27/09
Title: Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
Approved: Justin Skoble
12. GLOSSARY
BSL-2 –biosafety level 2 laboratory
BSC – biosafety cabinet
dPBS – Dulbecco’s phosphate buffered saline
LVS – live vaccine strain
mL – milliliter
µL- microliter
µm- micron
µM-micromolar
mM-millimolar
nM- nanomolar
PPE – personal protective equipment
RPM – revolutions per minute
SOP – standard operating procedure
13. APPENDICES
None
Date: 5/27/09