Document #:
Title:
Version:
PRP-01.002
Preparation and Quality Control of
Bacterial Culture Media
4
Effective Date:
1/30/09
1
ANZA Therapeutics, Inc.
Page:
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INTRODUCTION
Several types of growth media can be used to support the growth of strains of Francisella
tularensis (Ft). This SOP details the procedure for preparing liquid and agar culture media
for the robust growth of bacteria, including Francisella tularensis subspecies novicida,
including strains U112 and mutant strains of F.t. novicida received from UTSA. Careful
quality control assures the integrity and purity of the bacterial culture.
2
PURPOSE
This document describes how to prepare liquid and agar culture media for the growth and
maintenance of bacteria.
3
RESPONSIBILITY
3.1
Personnel involved with the preparation and quality control of bacterial culture media
are responsible for following this procedure.
3.2
Microbiology department management, or designee, is responsible for implementing
this procedure and revising it as necessary
3.3
4
Data generated from use of this SOP may be reviewed by Quality Assurance
department
SCOPE
This procedure applies to cysteine heart agar supplemented with hemoglobin (CHAH) plates
for growing F. novicida strains and Chamberlains Defined Medium (CDM). This media is
either made at Anza Therapeutics or Cerus’ microbiology department.
5
PRECAUTIONS
Gloves, safety glasses, and a lab coat should be worn at all times when preparing media.
A surgical mask may be worn as an option to prevent inhalation of media dust.
6
MATERIALS AND EQUIPMENT NEEDED
6.1
Materials
6.1.1
Deionized (DI) or MilliQ water
6.1.2
Appropriate powdered culture media components (listed in Attachment #1
under recipe for particular medium being made)
6.1.3
Appropriate sterile liquid supplements (listed in Attachment #1 under recipe
for particular medium being made)
Document #:
Title:
Version:
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Preparation and Quality Control of
Bacterial Culture Media
4
Effective Date:
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6.1.4
Spatulas
6.1.5
Weigh boats
6.1.6
0.2um filter flasks, sterile
6.1.7
Pipettes, sterile
6.1.8
1 N HCl
6.1.9
Disinfectant, 10% bleach
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6.1.10 Stir bar
6.2
7
Equipment
6.2.1
Magnetic Stir Plate
6.2.2
Pipette Aid
6.2.3
Incubator, 37oC
6.2.4
Vortexer
6.2.5
pH Meter, calibrated prior to usage
6.2.6
Shaker incubator
6.2.7
Calibrated scale (capable of a weighing accuracy of at least one significant
digit beyond weight to be weighed)
6.2.8
Spectrophotometer
6.2.9
Autoclave
PROCEDURE
7.1
Preparation of Broth or Agarose Media
7.1.1
See Attachment #1 for recipes for making various types of media. If recipe
does not exist in Attachment #1, follow the formulation recommendations of
the media manufacturer, an appropriate literature reference, or a recipe
prepared by the requestor of the media.
Document #:
Title:
Version:
PRP-01.002
Preparation and Quality Control of
Bacterial Culture Media
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7.1.2
Agar (plate) media can be made from any of the recipes in Attachment #1 by
adding the manufacturers suggested amount of agar to the broth media before
sterilizing.
7.1.3
Media Sterilization
7.1.4
Media sterilized with the AES Laboratoire S8000 autopreparator and/or
ASP300 Pourer Stacker
7.1.5
7.2
Page:
7.1.4.1
Media that will be sterilized and dispensed using the S8000
autopreparator and/or ASP300 Pourer Stacker can be made and
mixed directly in the internal vessel.
7.1.4.2
Refer to INS 00313 for instructions for using the S8000
autopreparator and ASP300 Pourer Stacker.
Media sterilized with an autoclave
7.1.5.1
Media that will be sterilized with an autoclave should be prepared
and thoroughly mixed on a stirring plate in an appropriately sized
container with magnetic stir bar for 10 minutes .
7.1.5.2
Transfer the liquid into clean autoclavable glass bottles. Fill each
bottle to no more than 80 of its total volume, leaving an adequate
air space.
7.1.5.3
Attach caps loosely with autoclave tape.
7.1.5.4
The autoclave should be run so that the media is sterilized at a
temperature of 121C for a minimum of 15 minutes, or as specified
in the recipe.
7.1.5.5
Swirl the media after taking it out of the autoclave. At this point
media containing agar can be placed in a waterbath at 37o C before
pouring plates that day, or allowed to harden and then remelted in
microwave until completely melted for pouring plates in the future.
Lot Numbers, Storage, Expiration, and Labeling
7.2.1
Lot numbers are assigned to uniquely identify each separate preparation
(batch) of media. Lot numbers should be MMDDYY followed by a letter to
represent the batch order for that day (starting with A and proceeding to Z for
each batch made that day). These lot numbers will be recorded on the data
capture sheets. If more than 26 batches are made in a day proceed to using
2 letters (i.e., AA, BB, CC).
Document #:
Title:
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Preparation and Quality Control of
Bacterial Culture Media
4
Effective Date:
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7.2.2
Expiration dates are 6 months for nonselective broth media, 3 months for
nonselective plate media, and is determined separately for selective media. If
not otherwise known, selective media should have an outdate of 3 months.
7.2.3
Broth media can be stored at RT or 4C, unless other storage requirements are
recommended by the manufacturer. Agar media will be stored inverted in
plastic sleeves at 4C.
7.2.4
Media will be labeled with a minimum of: Type of media, lot number, date of
preparation, expiration date, storage conditions, initials of preparer.
7.2.5
Plate media will be labeled on the outside of the plastic sleeve, however all
plates of a particular lot should be marked down the sides of the plates with a
combination of colored lines to help identify the type of media and/or
antibiotics present in the media. The lines used for a particular lot should be
recorded on the data capture sheets. The convention for lines is as follows:
Number of
Lines
Meaning
Red
1
Streptomycin
Blue
1
Chloramphenicol
Brown
1
Erythromycin
Black
1
Carbenicillin
Line Color
Black
2
VPP
NOTE: BHI (brain heart infusion) and LB (Luria-Burtani) agar are both
indicated by having no colored lines. LB agar is distinguished from BHI agar
by being significantly lighter in color.
8
QUALITY CONTROL
8.1
pH
8.1.1
8.2
For broth media, pH should be checked after prepared media has cooled to RT
by sterilely aliquoting a small amount of media into a separate container. The
pH should be as specified in the recipe being followed.
Growth Controls
8.2.1
Each new lot of media will be tested for it’s ability to support growth of the
intended organism as well as the ability to suppress growth if it is a selective
media. Appropriate Quality Control organisms to use can be found in
Attachment #1 for the media being prepared. If a recipe does not exist for the
Document #:
Title:
Version:
PRP-01.002
Preparation and Quality Control of
Bacterial Culture Media
4
Effective Date:
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5 of 19
particular media being prepared, the media preparer and requestor of the
media decide on appropriate Quality Control organisms.
8.2.2
For agar media, plate a bacterial suspension of known titer on the test plates
and a reference lot (previously shown to support adequate growth) according
to QCP-01.003. After overnight incubation, if the titer on the test plates
differs from the titer on the reference lot by 0.5 log, the growth promotion
quality control will be repeated. If a subsequent retest yields similar results,
the test lot will be rejected.
8.2.3
For broth media both the overnight OD and viability, and an abbreviated
growth curve will be evaluated.
8.2.3.1
To determine the overnight OD and viability, a small aliquot of a
fresh or frozen suspension of the appropriate test organism will be
inoculated into a 50 mL aliquot of the test lot and a reference lot
(this should be a lot of the same type of media which has been
shown previously to support adequate growth). After overnight
incubation with shaking, the OD600 will be determined for both the
test and control lots. In addition, both test and control lots will be
diluted and plated to determine the viability of the organisms after
overnight incubation. If the test lot does not show growth of at
least 0.5 OD units below the control lot, and the final titer is not
within 0.5 logs of the control lot, the quality control will be
repeated. If a subsequent retest yields similar results, the test lot
will be rejected.
8.2.3.1.1
8.2.3.2
To perform the abbreviated growth curve, 50 L of the
test and control overnight cultures from Section
7.4.2.3.1 above will be inoculated into 50 mL of fresh
test and control lots of media respectively. The OD600
will be taken at the time of inoculation and at two time
points at least 3 hours apart during the next 8 hours (i.e.
at 3hours and 6hours). Both the time taken and the OD
will be recorded. If any of the test OD readings is not
at least 80 of the control reading, the quality control
will be repeated. If a subsequent retest yields similar
results, the test lot of media will be rejected.
For selective media, an inhibition control will be performed. For
agar media, a colony or frozen stock of a bacteria whose growth is
expected to be suppressed will be streaked onto the test lot. This
same organism will also be streaked onto a non-selective media
that has been shown to support growth of the bacteria. For broth
media, a small aliquot of fresh or frozen suspension of a bacteria,
Document #:
Title:
Version:
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Preparation and Quality Control of
Bacterial Culture Media
4
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whose growth is expected to be suppressed, will be inoculated into
50 mL of both the test lot and a control lot of non-selective media
and incubated overnight with shaking. Any evidence of growth of
the bacteria on the selective media warrants a repeat of the quality
control. If the subsequent repeat yields similar results, the lot will
be rejected. If no growth is observed on the nonselective media,
the test should be repeated.
8.2.4
8.2.5
Sterility Control
8.2.4.1
For plate media, 2 to 10 of the lot will be set aside as sterililty
controls. The plates will be incubated at 37C overnight and at RT
for an additional 48 hours. Any evidence of growth on the media
warrants further investigation of contamination. In this case, a
larger sampling of the plates (at least 3X) should be incubated to
determine if the lot has a contamination problem and needs to be
rejected. Although the lot of media can be released for general use
after passing this initial sterility quality control, the sterility plates
will be held at room temperature for a total of 1 month before
being discarded. The plates will be checked for growth weekly,
and if growth is noticed on any of the plates, any remaining plates
in general circulation will be recalled and checked for growth. A
determination will be made at that point as to whether the plates
will remain in general circulation depending on the number of
sterility plates that showed growth and the results of the recheck of
plates that had been in circulation.
8.2.4.2
For broth media, all containers of broth from the test lot will be
placed in the incubator at 37C overnight and then at RT for an
additional 48 hours. Any container of media that shows growth
(evidenced by turbidity) will not be released for general use. Broth
media for which turbidity cannot be used as a sterility guide (i.e.
2 hemoglobin) will be tested for sterility using a blood culture
system following manufacturers instructions.
Quarantine
8.2.5.1
While results for the growth and sterility tests are pending, the
media will be put in quarantine. GLP or GMP use of the media at
this time is not allowed. If a user elects to use a lot of media, for
research purposes, that is still in quarantine, the user acknowledges
the risk that the media may not be suitable for use. For research,
usage of media still in quarantine is discouraged.
Document #:
Title:
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Preparation and Quality Control of
Bacterial Culture Media
4
Effective Date:
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8.2.5.2
9
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When all the QC tests have been passed, a label will be affixed to
the bottle of broth or sleeve of media indicating that it has passed
QC.
REFERENCES
9.1
Attachment #1, Media Recipes
9.2
Attachment #2, Media Preparation Data Capture Sheet
9.3
QCP-01.003, Determination of the Viable Bacterial Count of a Bacterial Culture
9.4
INS 00313, Media Preparation Using the S8000 Autopreparer and APS300 Pourer
Stacker
10
CALCULATIONS AND FORMULAS
11
N/A
GLOSSARY
mL- milliliter
PPE- personal protective equipment
DI- deionized
QCP- Quality Control Procedure
PRP- Preclinical/Research Procedure
RT- room temperature
C- centigrade
VPP-veggie peptone phosphate
BHI- brain heart infusion
LB-Luria-Burtani
OD- optical density
12
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APPENDICES
12.1
Attachment 1: Chamberlain’s Defined Medium
12.2
Attachment 2 Luria Burtani (Miller)
12.3
Attachment 3: Yeast Extract with Glucose or Yeast No Glucose
12.4
Attachment 4: BHI
12.5
Attachment 5: VPP
12.6
Attachment 6: 2% Hemoglobin Solution
12.7
Attachment 7: Cystine Heart Agar with Hemoglobin
Document #:
Title:
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PRP-01.002
Preparation and Quality Control of
Bacterial Culture Media
4
Effective Date:
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12.8
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Attachment 8: Example of Data Capture Sheet for Media Preparation
8 of 19
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Page 1 of 11
12.1 Attachment 1:
Media Name: Chamberlain’s Defined Medium (CDM)
Media Type: Broth
Ingredients:
Ingredient
Chamberlain Medium
Suggested
Manufacturer
Teknova
Catalog Number
C0712
g/L
24.2
Recipe:
For each ingredient multiply the g/L required by the number of liters to be made. Weigh out the
appropriate amount of the ingredients and stir to dissolve in MilliQ water. Using a magnetic stir bar
and stir plate, adjust pH to 6.26 by dropwise addition of 10N NaOH. Sterilize by filtration though
0.22uM filter apparatus.
Quality Control:
pH: 6.26 ±0.2
Positive growth control: Ft novicida
Negative growth control: None
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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12.2 Attachment 2
Media Name: Luria Burtani (Miller)
Media Type: Broth or Agar
Ingredients:
Ingredient
Bacto tryptone
Yeast extract
Sodium chloride
Suggested
Manufacturer
BD
Catalog Number
211701
g/L
10.0
BD
212750
5.0
Spectrum
S0160
10.0
Q-Biogene
3002-021
See
manufacturer’s
instructions
or
LB powder or capsules
Recipe:
For each ingredient multiply the g/L required by the number of liters to be made. Weigh out the
appropriate amount of the ingredients and stir to dissolve in MilliQ water. Sterilize at 121C for 15
minutes and aseptically dispense into appropriately sized sterile vessels or Petri dishes.
Quality Control:
pH: 7.0 ±0.2
Positive growth control: Escherichia coli
Negative growth control: None
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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12.3 Attachment 3:
Media Name: Yeast Extract with Glucose or Yeast No Glucose
Media Type: Broth
Ingredients:
Ingredient
Yeast Extract
Potassium phosphate
monobasic
Suggested
Manufacturer
BD
Catalog Number
212750
g/L
25.0
Fisher
P285-500
9.0
a.
Glucose
Sigma
G-5767
Glucose is left out of the formulation if Yeast No Glucose is being made
10.0
a.
Recipe:
For each ingredient multiply the g/L required by the number of liters to be made. Weigh out the
appropriate amount of the ingredients and stir to dissolve in MilliQ water. Use 10N NaOH to adjust
to a final pH of 7.2. Filter through a 0.2 μ filter to sterilize.
Quality Control:
Positive growth control: Escherichia coli
Negative growth control: None
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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12.4 Attachment 4
Media Name: BHI
Media Type: Broth or agar
Ingredients:
Ingredient
Powdered BHI broth
Suggested
Manufacturer
Catalog Number
EMD
1.10493.5007
g/L
See
manufacturers
instructions
Recipe:
For each ingredient multiply the g/L required by the number of liters to be made. Weigh out the
appropriate amount of the ingredients and stir to dissolve in MilliQ water. Sterilize at 121C for 15
minutes and aseptically dispense into appropriately sized sterile vessels.
Quality Control:
pH: 7.4 ±0.2
Positive growth control: Escherichia coli
Negative growth control: None
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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12.5 Attachment 5
Media Name: VPP
Media Type: Broth or agar
Ingredients:
Ingredient
Powdered VPP broth
Suggested
Manufacturer
Catalog #
Oxoid
VG0200
g/L
See
manufacturers
instructions
Recipe:
For each ingredient multiply the g/L required by the number of liters to be made. Weigh out the
appropriate amount of the ingredients and stir to dissolve in MilliQ water. Sterilize at 121C for 15
minutes and aseptically dispense into appropriately sized sterile vessels.
Quality Control:
pH: 7.3 ±0.2
Positive growth control: Escherichia coli
Negative growth control: None
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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12.6 Attachment 6
Media Name: 2% Hemoglobin Solution
Media Type: Additive solution
Ingredients:
Ingredient
Freeze dried bovine
hemoglobin
Suggested
Manufacturer
Catalog #
g/L
BD
212392
20.0
Recipe:
After determining the amount of media to be made, add the required amount of hemoglobin to a dry
beaker and add cold MilliQ water while stirring vigorously. Continue intermittent stirring for 10 to
15 minutes until all of the hemoglobin is dissolved. Bring to 121°C for 15 minutes to sterilize, cool
to approximately 50°C, and dispense into sterile bottles. Store prepared solution at 1C to 10°C for
up to 6 months.

Quality Control:
Sterility only
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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12.7 Attachment 7
Media Name: Cystine Heart Agar with Hemoglobin
Media Type: Agar
Ingredients:
Ingredient
Cystine Heart Agar
2% Hemoglobin solution
Suggested
Manufacturer
Difco
Catalog #
24711
Amount
102 g/L
See recipe above
NA
50%
Recipe:
Suspend the required amount of Cystine Heart agar in MilliQ water. Bring to 121C for 15 minutes.
Cool to 50C to 60C and aseptically add an equivalent volume of 2% hemoglobin solution to the
sterilized Cystine Heart agar. Cool to ~45C and dispense ~20 mL aliquots into sterile Petri dishes.
Quality Control:
Growth promotion: Francisella LVS
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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1. ATTACHMENT 8: EXAMPLE DATA CAPTURE SHEET FOR MEDIA PREP
2. SUMMARY
Data capture sheet for Preparation of Chamberlain’s medium and Chamberlain’s medium w/
20% w/v sucrose.
3. STUDY PARTICIPANTS
Name (Print)
Signature
Initial
Date
Tae Kim
4. EQUIPMENT AND MATERIALS
Equipment
Balance
Milli-Q water system
pH meter
Biosafety cabinet
Incubator
ANZA
Equipment #
Calibration
Expiration
ANZA Therapeutics, Inc.
Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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Material
Confidential
Page 9 of 11
Manufacturer
Part #
Falcon
352051
Chamberlain’s media
powder
Exp Date: _____________
Disposable bottles
1 N or 10 N NaOH
Sucrose
0.2 m sterile filter sets
Sterile tubes
Lot #
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5.
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Page 10 of 11
PROCEDURE
Date = ___________________
Initial
1
Clean BSC with 70% alcohol solution.
2
Calculate amount of Chamberlain’s media powder needed:
24.2 g x _____ L media = _____ g Chamberlain’s powder
3
Weigh out Chamberlain’s powder, __________ g
4
Transfer powder to a sterile bottle containing a stir bar and add Milli-Q water
equivalent to the volume of desired media stated in Step 2. __________ L
Milli-Q water. Stir until all powder is dissolved.
5
Adjust pH to 6.25 using NaOH. pH = __________
6
Transfer ½ of media into a 2nd sterile bottle.
7
Calculate amount of sucrose needed for cryopreservation solution.
200 g x _____ L media = _____ g sucrose
8
Weight out sucrose, _________ g
9
Add sucrose to 1st media bottle and stir until dissolved.
10
Filter sterilize each bottle through a 0.2 m filter. Label each bottle with
contents, NB #, “Store at 2-8 °C”, initials, date of manufacture, and an
expiration date 3 months from the date of manufacture.
11
From each bottle, remove two 5 mL aliquots into individual sterile tubes.
12
For each bottle, place one tube in a 37 °C incubator and the second tube at
ambient temperature. Incubate for 3-5 days then inspect for growth.
Start time/date: _______________
End time/date: _______________
Chamberlain’s media sterile (circle one): Y or N
Chamberlain’s media w/ 20% sucrose sterile (circle one): Y or N
6. COMMENTS
_________________________________________________________________
__________________________________________________________________
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Document Number: PRP-01.002, Ver: 4, Effective: 1/30/2009
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__________________________________________________________________