LOVELACE RESPIRATORY RESEARCH INSTITUTE
SOP No.: LBERI-1 v 1.0
Effective Date: 4/23/2009
Page 1 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks
Approved:
Robert Sherwood, PhD
LRRI Management
1
Date:
4/23/2009
Introduction
In order to ensure morphologically, phenotypically, and genetically consistent bacterial
propagation for animal challenges and bioaerosol studies, seed and working stocks must be
available. Seed and working stocks represent one and two passages from the primary stock,
respectively. Such stocks are created in order to maintain genetic stability (potentially lost
during multiple passages) throughout the course of studies related to this contract. This SOP
details the procedures for preparing and characterizing Francisella tularensis seed and
working stocks for live vaccine strain (LVS)- and SCHU S4-specific animal biological
safety level 3 (ABSL-3) studies. This SOP is a deliverable under Milestone 3 for LBERI’s
subcontract on the Tularemia Vaccine Development Contract under NIAID Prime Contract
#HHSN266200500040C and ADB Contract # NO1-A1-50040.
2
Purpose
The purpose of this SOP is to describe how to prepare F. tularensis seed and working stocks
to ensure consistent storage and growth of F. tularensis for later bioaerosol challenge studies
Seed and working stocks are prepared from primary stocks in order to maintain genetic
stability that could otherwise be compromised with multiple passages.
3
Responsibility

The Responsible Official (RO) and Alternate RO (ARO) for the Select Agents and
Toxins Program have the authority to act on behalf of LBERI with regard to select
agents and toxins. The RO/ARO are responsible for ensuring that facilities authorized
to possess select agents and toxins maintain control of them in accordance with
requirements set forth in U.S. Department of Agriculture (USDA) and Health and
Human Services (HHS) regulations on the possession, use and transfer of select agents
and toxins and this LRRI SOP.

The Animal Biosafety Level 3 (ABSL-3) Facility Director is responsible for ensuring
infectious agents are handled according to established SOPs and the LBERI Biological
Safety Manual.

Functional area directors/supervisors are responsible for oversight of their area of
responsibility to ensure that potentially infectious agents are handled properly and that
individuals working in these areas are trained in proper procedures.

Facility microbiologists are responsible for handling infectious or potentially
infectious agents according to established SOPs and the LBERI Biological Safety
Manual.
SOP No.: LBERI-1.1
Page 2 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks
4

Microbiology technicians are responsible for maintaining data generated using the
procedures described herein.

The Laboratory Supervisors are responsible for reviewing data generated under the
procedures described herein. The ABSL-3 Laboratory Supervisor, specifically, is
responsible for releasing F. tularensis seed and working stock lots for use in the
Facility.
Scope
This SOP details the procedures for preparing and characterizing Francisella tularensis seed
and working stocks for live vaccine strain (LVS)- and SCHU S4-specific animal biological
safety level 3 (ABSL-3) studies. This SOP applies to all individuals who prepare seed and
working stocks of Francisella tularensis in the ABSL-3 Microbiology Laboratory (Room
1031, Building 9223).
5
Precautions
Because of the inherent hazard to personnel resulting from handling highly infectious
microbial agents in the ABSL-3 Facility, individuals handling agents shall wear all required
personnel protective equipment (PPE), meet all occupational medicine requirements, and
handle agents in a manner that minimizes the potential for inadvertent infection or
contamination. PPE specific to the ABSL-3 Facility includes a powered air purifying
respiratory with associated hood, nitrile and latex gloves, Tyvek coveralls, safety shoes and
shoe covers.
6
Materials
6.1
Supplies

Francisella tularensis primary stock vial

LVS: Lot 703-0303-016 (13DEC05, Fisher Biosciences)

SCHU S4 Submaster Project 110419.2 (9MAR06, Fisher Biosciences)

SCHU S4 parent strain originated from the Salk Institute in Swiftwater, PA, Lot
623-42 (6SEP86)

The Salk Institute received the seed stock from Fort Detrick, MD.

In-house prepared blood cysteine glucose agar (BCGA), sterile

In-house prepared Chamberlain’s broth, sterile

20% (w/v) sucrose, sterile

1% peptone dilution blanks, sterile

Aerosol (filtered) pipette tips, sterile

250 mL plastic Erlenmeyer flasks, sterile
SOP No.: LBERI-1.1
Page 3 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks

Whatman BugStoppers

15 and 50 mL polypropylene centrifuge tubes, sterile

1, 5, 10, 25 and/or 50 mL pipettes, sterile

2.5 mL cryovials, sterile

Inoculation loops, sterile

Biohazard waste container

Disinfectant (5% acidified bleach solution, prepared in-house)
6.2
7
Equipment

Class II biological safety cabinet (BSC)

Variable speed vortex mixer

Variable speed refrigerated benchtop centrifuge with a sealable rotor

Variable volume pipettor, 100 L capacity

37°C incubator

37°C shaker/incubator

-80°C select agent freezer
Procedures
7.1
Preparation of Seed Stock
7.1.1
Confirmation of Primary Stock Purity

Allow a frozen primary stock vial to thaw at room temperature in the BSC.

Aseptically open the frozen primary stock vial from the external source, remove a
loopful of material with a sterile, disposable inoculating loop and streak onto a BCGA
plate the standard quadrant streak method as depicted below:
SOP No.: LBERI-1.1
Page 4 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks

Appropriately discard the loop and vial in a biohazard waste container. Record the vial
usage and passage on the appropriate tracking documentation

Incubate the BCGA plate (inverted) at 37°C ± 2°C for a minimum of 48 hours. Record
starting/ending incubation date, time, temperature, initials and other information as
outlined on a Francisella tularensis Stock Preparation Worksheet (example provided in
Appendix A).

Following incubation, confirm viability, purity and pigmentation of the culture against
the following criteria. F. tularensis (LVS and SCHU S4) colonies are 3-4 mm in
diameter, gray-white to slightly blue and opaque, demonstrate little or no hemolysis,
present a raised, convex appearance, and have a wet, shiny surface. Colony
morphology is shown in Figure 1 below:
Figure 1 – Francisella tularensis growth on BCGA. This image represents F.
tularensis LVS colonies after 48h of incubation at 37°C. F. tularensis SCHU S4
colonies present similar morphology.

7.1.2
If the culture is determined to be contaminated, repeat the above steps with another
primary frozen stock vial. If there are no remaining primary vials, request more vials
from the original source. If the source has no remaining primary vials, subculture the
contaminated culture via the quadrant streak method as described above (this should
ensure isolated, pure colonies).
Seed Stock Preparation

Using a sterile inoculation loop, pick an isolated colony from the primary stock purity
BCGA plate created according to section 7.1.1 above and aseptically inoculate 100 mL
of sterile Chamberlain’s broth in a sterile, plastic 250-mL Erlenmeyer flask.

Appropriately discard the loop and purity plate in a biohazard waste container.

Seal the flask using a Whatman BugStopper.

Disinfect the outside of the flask by wiping it down with an unexpired 5% acidified
bleach solution. Remove the flask from the BSC.
SOP No.: LBERI-1.1
Page 5 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks

Wipe the outside of the flask with a clean paper towel in order to remove the 5%
acidified bleach. Place the flask into a shaker/incubator set to 37°C. In order to ensure
proper rotation and prevention of spillage, tightly secure the flask within the shaker
using a spring clamp or equivalent.

Incubate the flask with shaking at 225 rpm for at target of 48h.

After incubation, remove the flask from the shaker and place in the BSC.

Using a sterile 50 mL pipette, aseptically transfer the F. tularensis suspension into two
sterile 50 mL conical tubes seal tightly.

While in the BSC, place the 50 mL tubes into an autoclavable, aerosol-tight centrifuge
rotor.

Centrifuge the tubes at 4100 rpm (2400 x g), 4-6°C for 10-20 min.

Remove the rotor from the centrifuge and place it in the BSC.
NOTE: In case of leakage of tubes within the sealed rotor, always open the rotor inside
the BSC.

Remove the 50 mL tubes and observe for pellet formation. If pellet has not formed,
perform additional centrifugation at the same conditions as stated above; an increase in
centrifugation time may be required to form the pellet.

Upon proper pellet formation, decant the supernatant into an appropriate liquid
biohazard container taking care that the pellet does not slide with the supernatant into
the waste container.

To each pellet, add a total of 25 mL of sterile Chamberlain’s broth; this will allow for a
total of 50 vials (1 mL per vial) to be created.

Mix thoroughly using a vortex mixer and combine the pellets into one tube.

Add an equal volume of 20% (w/v) sucrose and vortex for 10-15 sec on a medium to
high setting.

Aliquot the suspension equally (1.0 mL per vial) into 2.5-mL cryovials.

Label each seed stock cryovial with the organism name (Francisella tularensis), strain
(e.g., LVS or SCHU S4), “Seed Stock,” lot number (if applicable), date the stock was
created, aliquot volume and the cryomedium used (10% sucrose in Chamberlain’s
broth).

Place the seed stock vials in a -80°C select agent freezer.
7.2
Working Stock Preparation

Allow a frozen seed stock vial to thaw at room temperature in the BSC.
SOP No.: LBERI-1.1
Page 6 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks

Aseptically inoculate 100 mL of sterile Chamberlain’s broth with 100 L of the seed
stock in a plastic 250-mL Erlenmeyer flask. Discard the seed stock vial in a biohazard
waste container and record the vial usage on the appropriate tracking documentation.
NOTE: Inoculate a sufficient number of flasks to perform the planned work assuming
a final recovery of approximately 1 x 108 cfu/mL after 48h of proper incubation.

Seal each inoculated flask using a Whatman BugStopper.

Disinfect the outside of the flask(s) by wiping down with an unexpired 5% acidified
bleach solution. Remove the flask(s)from the BSC.

Wipe the outside of the flask(s) with a clean paper towel in order to remove the 5%
acidified bleach. Place the flask(s) into a shaker/incubator set to 37°C. In order to
ensure proper rotation and prevention of spillage, secure the flask(s) within the shaker
via spring clamps or equivalents. Incubate the flask(s) with shaking at 225 rpm for a
target of 48h.

After incubation, remove the flask(s) from the shaker and place in the BSC.

Using sterile 50 mL pipettes, aseptically transfer the F. tularensis solution into sterile
50 mL conical tubes (40 mL maximum per tube). Use one 50 mL tube per inoculated
flask.

While in the BSC, place the 50 mL tubes into an autoclavable, aerosol-tight centrifuge
rotor.

Centrifuge the tubes at 4100 rpm (2400 x g), 4-6°C for 10-20 min.

Remove the rotor from the centrifuge and place it in the BSC.
NOTE: In case of leakage, always open the rotor inside the BSC.

Remove the 50 mL tubes and observe for proper pellet formation. If pellets have not
formed, perform additional centrifugation at the same conditions as stated above.

Upon proper pellet formation, decant the supernatant from each tube into an appropriate
liquid biohazard container.

To each pellet, add 25 mL of sterile Chamberlain’s broth; this will allow for a total of
50 vials (1 mL per vial) to be created.

Mix thoroughly using a vortex mixer and combine the pellets into one tube. Use a
sterile plastic flask if a 50 mL tube is insufficient for the working volume.

Add an equal volume of 20% (w/v) sucrose and vortex for 10-15 sec on a medium to
high setting.

Aliquot the suspension equally (1.0 mL per vial) into 2.5-mL cryovials.

Label each seed stock cryovial with the organism name (Francisella tularensis), strain
(e.g., LVS or SCHU S4), “Seed Stock,” lot number (if applicable), date the stock was
created, aliquot volume and type of cryomedia used.
SOP No.: LBERI-1.1
Page 7 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks

8
Place the working stock vials in a -80°C select agent freezer according to Institute
policies.
Quality Control

Media
o Plates

From every 25 plates of prepared nutrient agar plates, remove one for
quality control (QC) purposes.

Incubate the selected plate(s) at 37°C for a minimum of 48h.

If microbial growth is observed, discard the batch of plates from which the
test plate was taken.

For any given study, DO NOT use plate media until QC has been
completed. Note that additional preparation time is required to perform
QC.
o Broth media


From every 100 mL of prepared nutrient broth, remove 5 mL for QC
purposes.

Aseptically dispense the removed volume into sterile 15-mL polystyrene
conical vials (5 mL per vial).

Incubate the broth media at 37°C for a minimum of 48h. If microbial
growth is observed (based on the qualitative presence of turbidity), discard
the batch of liquid from which the test aliquot was removed.

For any given study, DO NOT use broth media until QC has been
completed. Note that additional preparation time is required to perform
QC.
Pipette checks
o For each day of use, check pipettes according to LRRI SOP FCP-1036, Pipette Use
on GLP Studies

Seed stock
o After a minimum of 5 days at -80°C, remove 1 seed stock cryovial from the freezer,
allow to thaw at room temperature, and culture for isolation on BCGA as shown
in Figure 1. This is to ensure that the seed stock is viable, pure and presents
colony morphology consistent with F. tularensis. The plate culture is a quality
control measure on the new seed stock.
o Though optional, it is also recommended to perform the preceding step (reculturing from frozen seed stocks) at regular intervals (e.g., every 6 months) to
ensure the stability of the seed stock is maintained. If stability has been
compromised (as indicated by >1 log 10 decrease in titer), create a new seed stock
using the procedures outlined above.
SOP No.: LBERI-1.1
Page 8 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks

Working stock
o Remove a working stock vial from the select agent storage freezer and allow it to
thaw at room temperature.
o For purity confirmation, aseptically remove a loopful of material and culture for
isolation on BCGA as shown in Figure 1.
o To determine the working stock titer, perform serial 1:10 dilutions in sterile 1%
peptone (1:10 through 1:108) and plate in triplicate onto BCGA plates according
to LRRI SOP BSF-1632, Inoculation of Bacterial Cultures.
o Discard the working stock vial in a biohazard waste container and record the vial
usage on the appropriate tracking documentation.
o Incubate the BCGA plates (inverted) at 37°C ± 2°C for a minimum of 48 hours.
Record starting/ending incubation date, time, temperature, initials and other
information as outlined on the Francisella tularensis Stock Preparation
Worksheet (Appendix A).
o Following incubation, confirm colony morphology as described in Section 6.1.1.
Document plate counts on a plate count form (example provided in Appendix B)
and calculate the titer of the culture. If the culture is determined to be
contaminated, repeat the above steps with another aliquot of working stock. If
contamination continues to be observed, all of the working stock vials are
considered as contaminated and must be disposed of and prepared again
9
References
1. LRRI SOP BSF-1487, Use and Maintenance of Carbon Dioxide Gas Incubators for
the ABSL-3 Facility
2. LRRI SOP BSF-1599, General Microbial Dilution Technique
3. LRRI SOP BSF-1623, Operation, Maintenance, and Performance Verification of the
ABSL-3 Shaker Incubators
4. LRRI SOP BSF-1632, Inoculation of Bacterial Cultures
5. LRRI SOP BSF-1640, Class II Biosafety Cabinet Use in the Animal Biosafety Level
3 Facility
6. LRRI SOP BSF-1641, Disinfectant Preparation in the LRRI BSL-3 Facilities
7. LRRI SOP FCP-1036, Pipette Use on GLP Studies
8. LRRI SOP FCP-1124, Operation, Performance Verification, Calibration, and
Maintenance of Balances, Scales, and Weights
9. LRRI SOP TXE-1223, Operation and Maintenance of Benchtop Centrifuges
10 Calculations and Formulas
CFU/mL = Mean colony count on BCGA plates x dilution x (1/volume plated [mL])
SOP No.: LBERI-1.1
Page 9 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks
11 Glossary

ABSL-3 – animal biosafety level 3 laboratory

BCGA – blood cysteine glucose agar

BSC – Class II biological safety cabinet

mL – milliliter

mm- millimeter

PPE – personal protective equipment

rpm – revolutions per minute

SOP – standard operating procedure

w/v – weight to volume

h- hours

LVS- live vaccine strain of F. tularensis
12 Appendices
A. Example Francisella tularensis Stock Preparation Worksheet
B. Example Plate Count Form
SOP No.: LBERI-1.1
Page 10 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks
Appendix A: Example Francisella tularensis Stock Preparation Worksheet
A. Stock Name*
Type of Stock: Seed Stock
Working stock
(circle one)
Created on:
by:
Sample Label:
# of Cryovials Created:
Stock Titer:
* Complete Section A once the entire new stock has been created.
B. Primary Stock Purity
Cryovial Used:
Incubator ID:
Incubation Start Date:
Inoculation Date/Initials:
Type of Plate/XP:
Incubator Temp:
Incubation End Date:
Date Read/Initials:
Plate Description:
C. Stock Preparation
Type of stock to be created:
Seed Stock
Working Stock
Cryovial Used:
Chamberlain’s Broth XP:
Number of flasks:
Inoculation Date/Initials:
Incubator ID:
Incubation Start Date:
Incubator Temp/RPM:
Incubation End Date:
Centrifuge ID:
RPM/Temp/Time:
Cryovials created? N or Y
Cryovial Number:
Created by:
Cryomedia used:
List ingredients and expiration dates:
D. Stock Titer
Cryovial used:
1% peptone Lot Number/ XP:
Type of Plate/XP:
Inoculation Date/Initials:
Incubation Start Date:
Date Read/Initials:
Incubator ID:
Incubator Temp:
Incubation End Date:
Stock Titer:
E. Stock Purity
Cryovial Used:
Inoculation Date/Initials:
Type of Plate/XP:
Incubator ID:
Incubation Start Date:
Incubator Temp:
Incubation End Date:
Date Read/Initials:
Plate Description:
Reviewed by:
Date:
(circle one)
SOP No.: LBERI-1.1
Page 11 of 11
Title: Preparation of Francisella tularensis Seed and Working Stocks
Appendix B: Example Plate Count Form
Study/Purpose:
Stock ID:
Incubation Start Date/Time:
Incubation Temp
°C
Incubation Stop Date/Time:
Plate ID
Dilution
Reviewed by:
Vol. Plated (ml)
Plate Ct #1
Plate Ct #2
Date:
Plate Ct #3
CFU/mL
Comments