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Protocol for Growth Promotion Test

Trypcase Soy Agar Plates (Tryptone Soya Agar)


Document No. Effective date
Version No. Page No. Page 1 of 8
Prepared by
Name:

Sign & Date
Approved by:
Name:
Sign & Date

I. Objective:
Growth promotion test is performed on microbiological culture media to assess the ability of
the media to sustain growth. This protocol outlines the procedure for Growth promotion
testing at SPT. The micro-organisms used are recommended by the European
Pharmacopoeia & USP.

II. Scope:
The scope of this protocol is applicable to the Growth Promotion test, certification, usage
and reconciliation of Trypcase Soy Agar (TSA) plate received at MICRO lab.
III. Procedure:
Once a new lot of media from an approved vendor is received, it is necessary to certify the
lot. The lot may be certified by performing growth promotion tests.
Documentation of growth promotion tests will be done in this document including
corresponding certificate of analysis and usage and reconciliation. Each lot will have
separate protocol / report. The report will be completed and archived on exhaustion of
specific lot of the media.
Growth promotion test is performed by comparing one plate of new lot of media to two
plates of previously certified lot. A certified lot is a lot of agar of known fertility. In-case
of initial lot, this shall not be applicable. The colony forming unit (CFU) count in the new
lot of media is then compared to the mean count of the certified plates and the percentage
recovery is calculated.
A. Determination of microorganisms that are needed to test the medium:
Medium Organism Incubation
temperature
Incubation time
Trypcase soy Agar
(Tryptone Soya Agar)
Staphylococcus aureus ATCC 6538 -
represent as gram positive
Escherichia coli ATCC 8739 - represent
as gram negative
Candida Albicans ATCC 10231 -
represent as yeast
30
o
C to 35
o
C

30
o
C to 35
o
C

30
o
C to 35
o
C
3 days



Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 2 of 8

IV. Receipt, verification and storage of TSA Plates:
Name of the product (as mentioned on
Certificate, PO and product label)

Supplier Catalogue No.
Batch No.


Mfg. Date


Exp. Date
Quantity received
Purchase Order reference number & date
Supplier
Storage condition
CoA availability
Pack integrity check Satisfactory / Not satisfactory

Details of damage / remarks (if any):

Received and verified by: Name:

Signature and date:

Final remarks of consignment Accepted / Rejected
On satisfactory verification, apply quarantine label on the pack and store at respective
storage conditions / location.

V. Sampling Instruction:
Two plates to be tested for growth promotion tests. Two plate to be used a negative
control.



Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 3 of 8
Prepared by
Name:

Sign & Date
Approved by:
Name:
Sign & Date

Preparation of culture:

Organism Lot No.
Escherichia coli ATCC 8739
Candida Albicans ATCC 10231
Staphylococcus aureus ATCC 6538

Description
Escherichia coli ATCC 8739 Lot No.: Candida Albicans ATCC 10231 Staphylococcus aureus ATCC 6538
Date & time of
preparation
Performed by
(Sign & date)
Date & time of
preparation
Performed by
(Sign & date)
Date & time of
preparation
Performed by (Sign &
date)
Remove the hydrating fluid
vial and vial of lyophilized
strain preparation from
refrigerated storage. Allow
unopened vial of
lyophilized pellets to
equilibrate to room
temperature

While pellets are
equilibrating, pre-warm the
hydrating fluid to 35
o
C (at
least 30 minutes)

With a sterile forceps,
transfer two (2) pellets into
the 2 ml vial of hydrating


Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 4 of 8

Description
Escherichia coli ATCC 8739 Lot No.: Candida Albicans ATCC 10231 Staphylococcus aureus ATCC 6538
Date & time of
preparation
Performed by
(Sign & date)
Date & time of
preparation
Performed by
(Sign & date)
Date & time of
preparation
Performed by (Sign &
date)
fluid. (do not remove the
desiccant from vial). Two
pellets must be used to
obtain the challenge
concentration of <100 CFU
per 0.1 ml. Immediately
stopper and recap the pellet
vial and return the
remaining lyophilized
material to refrigerated
storage of 2
o
C to 8
o
C
Immediately re-cap the vial
with the hydrated material
and place into a 34
o
C to
38
o
C incubator for thirty
(30) minutes to assure
complete hydration

Immediately following
incubation vortex the
hydrated material to
achieve a homogeneous
suspension




VI. Testing Procedure:

Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 5 of 8

Description
Specification /
procedure
Observation
Done by (Sign
& date)

Escherichia coli ATCC 8739
Lot No.:
Candida Albicans ATCC
10231
Staphylococcus aureus
ATCC 6538

Date of testing (start date):
Date completed (after observation of
results):

Label plates with the organism name,
duplicate number, date of inoculation and
media type
To comply







Using sterile pipette, pipette-out 0.1 ml of
each microorganism solution to the media
plates
To comply Volume transferred:

Lot No. of sterile
Pipette:

Volume transferred:

Lot No. of sterile Pipette:

Volume transferred:

Lot No. of sterile Pipette:


Using sterile spreader spread the solution
evenly across the surface of the agar
Allow plates to dry before inverting and
incubation.
To comply Lot no. of sterile
spreader:

Lot no. of sterile
spreader:

Lot no. of sterile
spreader:


Two agar plate (of new lot) incubated along
with the test plates without any inoculation.
This will act as negative control

To comply
Record the time and date of incubation period
in the Growth Promotion Test Report of
individual medium.

To comply Incubator No.:

Date Incubated:

Time Incubated:

Incubator No.:

Date Incubated:

Time Incubated:

Incubator No.:

Date Incubated:

Time Incubated:


After incubation, remove all plates from the
incubator, record the time and date that the
To comply Date removed:

Date removed:

Date removed:



Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 6 of 8

Description
Specification /
procedure
Observation
Done by (Sign
& date)

Escherichia coli ATCC 8739
Lot No.:
Candida Albicans ATCC
10231
Staphylococcus aureus
ATCC 6538

plates removed. Time removed:

Time removed:

Time removed:

Inspect each plate for growth, counting all
CFU present on each plate, The percentage
recovery is calculated using the formula given
below:

For New Lot

Average Test count (new lot) (CFU)
% recovery = ----------------------------------------- X 100
Average count of approved plates

The average
count must
be between
10 to 100
CFU.

% recovery
must not
differ by a
factor greater
than 2 (i.e.,
50% to
200%)

No growth
must be
observed in
negative
control
New lot:
Plate 1:

Plate 2

Average:

Previously approved lot:
Plate 1:

Plate 2:

Average:

% recovery:

Negative control
observation:

New lot:
Plate 1:

Plate 2

Average:

Previously approved lot:
Plate 1:

Plate 2:

Average:

% recovery:

Negative control
observation:

New lot:
Plate 1:

Plate 2

Average:

Previously approved lot:
Plate 1:

Plate 2:

Average:

% recovery:

Negative control
observation:




Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 7 of 8
Prepared by
Name:

Sign & Date
Approved by:
Name:
Sign & Date

VII. Certificate of Analysis
Name of the Media Delivery Note No.
Batch No. / Lot No. Quantity received
Mfg Date Quantity approved
Expiry date Quantity rejected
Date of receipt Date of Certification
Organism
Specification / results
Remarks (Pass / Fail) Average test
count
% recovery
Escherichia coli ATCC 8739

Candida Albicans ATCC 10231

Staphylococcus aureus ATCC 6538

Review remarks of supplier CoA
To confirm the purity and
authenticity of supplied culture

Acceptance Criteria: Average test count: Not More Than 100 CFU; test plate count must not differ from
control plate count by factor greater than 2
Remarks: The received consignment Complies / Does not complies to the specifications. The material is
approved / rejected for further intended use
Prepared by (Signature and date) Approved by (Signature and date)




Protocol for Growth Promotion Test
Trypcase Soy Agar Plates (Tryptone Soya Agar)
Document No. Effective date
Version No. Page No. Page 8 of 8

VIII. Usage log: Stock control and reconciliation (use additional sheets
where needed)
Quantity Released
Product Name / Item B. No. Test Name Date used Quantity used


















Remarks:




Reconciliation performed by:

Name:

Signature & Date
Verified by:

Name:

Signature & Date