Verication Protocol

Protocol Reference:
2
Contents
1. About BioBall

Multishot 550 3
2. About This Protocol 4
3. Safety Precautions 4
4. Protocol Pre-approval 4
5. Protocol Scope 5
5.1. Intended Location Of Use 5
5.2. Strains To Be Validated 5
5.3. Documentation 6
5.4. Comments 6
6. Verifcation Of Counts And Purity 7
6.1. Materials 7
6.2. Method 7
6.2.1. BioBall Preparation 7
6.2.2. Inoculation Of Agar Plates And Incubation (Initial Counts) 8
6.2.3. Verifcation Of 8hr Stability At 2-8c 9
6.2.4. Interpretation Of Results 10
6.2.5. Acceptance Criteria 10
6.3. Documentation 11
6.4. Comments 11
6.5. Section Review And Approval 11
7. Confrmation Of Organism Identity 12
7.1. Materials 12
7.2. Method 12
7.2.1. Macroscopic And Microscopic Identifcation 12
7.2.2. Biochemical / Molecular Identifcation (Optional) 13
7.2.3. Acceptance Criteria 14
7.3. Documentation 14
7.4. Comments 14
7.5. Section Review And Approval 14
8. Final Review And Approval 15
8.1. Final Comments 15
8.2. Protocol Review And Approval 15
9. Discrepancies 15
3
1. About BioBall MultiShot 550
The pharmaceutical industry is required to perform QC with inocula containing less than 100 cfu for several tests including growth
promotion, sterility and method validation.
Once validated, BioBall

may be used as a precise, reliable and cost effective alternative to traditional reference culture methods
such as seed lot maintenance systems.
BioBall contains a precise number of microorganisms in a water soluble ball delivering unprecedented accuracy for Quantitative
Microbiological Quality Control. Lot specifc certifcates of analysis confrming counts and organism identity are supplied with each
lot number.
BioBall MultiShot 550 has a batch mean of between 500 and 600 cfu with a standard deviation less than or equal to 10% of
the batch mean. When re-hydrated with 1.1mL of re-hydration fuid provides 10 x 100uL doses, each containing 50 cfu.
It has an 8 hour stability when re-hydrated with BioBall Re-Hydration Fluid and stored between 2 to 8C, making it ideal when
several controls are needed at the same time or within the same day.
BioBall is available in a wide range of microorganisms, including equivalent strains to those required by the British
Pharmacopoeia, (BP), European Pharmacopoeia, (Ph.Eur.), Japanese Pharmacopoeia, (JP) and United States Pharmacopoeia,
(USP). BioBall reference strains are recommended by BTF for use as an alternative to traditionally derived reference cultures in
the following pharmacopoeial chapters:
British Pharmacopoeia & European Pharmacopoeia
Appendix XVIA Tests for Sterility, (Ph.Eur 2.6.1)
Appendix XVIB Tests for Microbial Contamination, (Ph.Eur. 2.6.12, 2.6.13)
United States Pharmacopoeia
<61> Microbiological Examination of Non-sterile Products: Microbial enumeration tests
<62> Microbiological Examination of Non-sterile Products: Tests for specifed Microorganisms
<71> Sterility Tests
<1227> Validation of Microbial Recovery from Pharmacopoeial Articles
<2021> Microbial Enumeration Tests Nutritional and Dietary Supplements
<2022> Microbiological Procedures for Absence of Specifed Microorganisms Nutritional and Dietary Supplements
Japanese Pharmacopoeia
35. Microbial Limit Tests
36. Microbial Limit Test for Crude Drugs
54. Sterility Test
All reference strain identifcations are confrmed by genetic typing.
BTF as a Reference Materials producer is the frst company worldwide accredited under the ISO Guide 34 Standard for
quantitative microbiologial reference standards, including BioBall.
BioBall products have up to 24 months* shelf life when stored frozen.
*Depending on the organism.
4
2. About this protocol
This protocol outlines the process used to demonstrate the suitability of BioBall

as a replacement for traditionally derived culture

collections. The protocol within is designed to meet the requirements of regulatory bodies, with regards to documentation and
traceability of the verifcation exercise.
The verifcation process involves fve main steps;
1. Pre-approval of the verifcation protocol by quality representatives and personnel executing the protocol, (section 4).
2. Defnition of the scope of the verifcation, including intended location of use and reference strains to be tested,
(section 5).
3. Verifcation of BioBall organism counts, strain purity and 8hr stability at 2-8C, (section 6).
4. Identifcation of BioBall organisms, (section 7).
5. Final review and approval of verifcation protocol, (section 8).
Verifcation is based on adequate recovery of each reference strain grown on culture media petri dishes. Once growth is evident
the recovery is enumerated and purity visually confrmed. The organism recovered can then be further identifed by traditional
methods.
Once completed this protocol should be kept on fle for future reference. A reference identifer may be entered in the box on the
cover of this protocol for ease of reference.
3. Safety precautions
BioBall MultiShot 550 contains viable and potentially pathogenic bacteria and should only be handled by experienced laboratory
personnel trained in the safe handling of these micro-organisms. The number of micro-organisms contained within BioBall
MultiShot 550 is low. However, BioBall MultiShot 550 should be handled as if potentially infectious. Refer to your national safety
guidelines. After use, dispose of packaging in accordance with appropriate biohazard disposal practices. Do not use the product if
the packaging is damaged. Do not use the product after the expiry date. The product should be used according to the procedure
described in this verifcation protocol. Any modifcations may affect the results.
4. Protocol pre-approval
Before commencing this protocol, personnel responsible for performing the verifcation should be nominated and approval
should be sought from the companies quality department. This ensures that the content of this document meets any validation
policies that may be effective in the intended location of use.
Name (Print) Title Signature Date
Protocol to be performed by
Protocol reviewed by
Protocol approved for use by
5
5. Protocol scope
Record below the details of the site at which BioBall

is to be used.
5.1 Intended location of use
Company Name: __________________________________________________________
Address: __________________________________________________________
__________________________________________________________
__________________________________________________________
__________________________________________________________
__________________________________________________________
Department: __________________________________________________________
SIGN / DATE_____________________________
5.2 Strains to be validated
Indicate in the table below which BioBall strains are to be validated as part of this protocol. Comments may be made in section
5.4 regarding organisms not included in this protocol.

Table 1. Strains included in BioBall Verifcation Plan.
SIGN / DATE_____________________________
*Strains are sourced from NCTC, NCPF and ACM.
ATCC is a trade mark of the American Type Culture Collection.
Organism
BioBall MultiShot 550
Product Code
Designation* Included (Yes/No)
Aspergillus brasiliensis (niger) 56011 NCPF2275 / ATCC16404
Bacillus subtilis 56012 NCTC10400 / ATCC6633
Candida albicans 56013 NCPF3179 / ATCC10231
Clostridium sporogenes 56014 NCTC12935 / ATCC11437
Escherichia coli 56016 NCTC12923 / ATCC8739
Pseudomonas aeruginosa 56017 NCTC12924 / ATCC9027
Salmonella abony 56018 ACM 5080 / NCTC 6017
Staphylococcus aureus 56019 NCTC10788 / ATCC6538
6
5.3 Documentation
The documentation as listed in table 2 below should be attached to the end of this protocol. Documentation for BioBall

is
available from www.bioball.com. Additional documentation may be added if required.
Document Title Document led (YES / N/A)
BioBall Material Safety Data Sheet

Table 2. Documentation
SIGN / DATE_____________________________
5.4 Comments

SIGN / DATE_____________________________
7
6. Verication of Counts and Purity
6.1 Materials
Required BioBall

strains, (section 5.2)

BioBall Re-Hydration fuid, (Catalogue No. 56021)
Calibrated 100L pipette and sterile tips
Tryptone soy agar plates, (TSA), or equivalent
Sabouraud dextrose agar plates, (SDA), or equivalent
Columbia +5% blood agar plates or equivalent
Vortex
Sterile spreaders
Incubators (20 -25C & 30-35C)
6.2 Method
6.2.1 BioBall preparation
1. Using table 3 below, record the batch number of each strain to be used. A Certifcate of Analysis should also be
retained from the BTF website www.btfbio.com for each batch and should be attached to the end of this protocol.
Organism Catalogue No. Batch Number Certicate of Analysis
Aspergillus brasiliensis (niger) 56011
Bacillus subtilis 56012
Candida albicans 56013
Clostridium sporogenes 56014
Escherichia coli 56016
Pseudomonas aeruginosa 56017
Salmonella abony 56018
Staphylococcus aureus 56019

Table 3. Organism batch numbers and Certifcates of Analysis
SIGN / DATE_____________________________
2. Label the BioBall Re-Hydration Fluid with the spare label included in the box of BioBall MultiShot 550.
3. Remove cap from BioBall Re-Hydration Fluid.
4. Remove the stopper from the glass vial containing the BioBall.
5. Tip the BioBall into the BioBall Re-Hydration Fluid, replace the cap and wait 30 seconds.
Note: BioBall Re-Hydration Fluid needs to be at room temperature when the BioBall is added. It is important
NOT to pour the rehydration fuid into the glass vial. Each re-hydrated BioBall in 1.1 mL BioBall Re-Hydration Fluid
contains 10 doses of 100 L
6. Vortex for 5 seconds.
7. Use in aliquots of 100 L.
8. Discard the fnal 100 L
9. Can be used up to 8 hours after re-hydration, if the re-hydrated BioBall is stored in a refrigerator at 2 to 8C.
Re-vortex the re-hydrated BioBall for 5 seconds before each use.
SIGN / DATE_____________________________
8
6.2.2 Inoculation of agar plates and incubation (initial counts)
1. Select correct type of agar plate for each reference strain according to table 4 below. Label plates accordingly with the
organism name and date of inoculation.
2. Using a 100L pipette, inoculate the surface of 5 agar plates with 100L of rehydrated BioBall

solution onto
each plate.
3. Using a sterile spreader spread the solution evenly across the surface of the agar.
4. Repeat steps 1 to 3 for all desired organisms.
5. Once completed, transfer all inoculated plates and incubate at the temperature, time and environmental conditions
indicated in table 4 below.
6. Record the time and date incubated in table 4 below.
7. Replace the cap onto each strain used and place the remainder of the re-hydrated BioBall solution into a
refrigerator at 2 to 8C. Record the time and date at which the solution is refrigerated in table 4 below.
Organism Agar Incubation
Time/Date
Incubated
Performed
by (Initial)
Time/Date
removed
from
incubator
Performed
by (initial)
Time/Date
regerated
Performed
by (initial)
Aspergillus brasiliensis (niger) SDA
20-25C
5 d
Bacillus subtilis TSA
3035C
24 4 hrs
Candida albicans SDA
20-25C
5 d
Clostridium sporogenes
Columbia
+5% blood
3035C
48 4 hrs
(Anaerobic)
Escherichia coli TSA
3035C
24 4 hrs
Pseudomonas aeruginosa TSA
3035C
24 4 hrs
Salmonella abony TSA
3035C
24 4 hrs
Staphylococcus aureus TSA
3035C
24 4 hrs
Table 4. Organism strains, agar types and incubation conditions, (initial counts).
9
6.2.3 Verication of 8hr stability at 2 to 8C
1. Following 8 hours of refrigeration at 2 to 8C remove all suspensions from the refrigerator. Record the time and date at
which the solution is removed from the refrigerator in table 5.
2. Vortex each suspension for 5 seconds.
3. Select correct type of agar plate for each reference strain according to table 5 below. Label plates accordingly with the
organism name, date of inoculation and 8hr Stability.
4. Using a 100L pipette, inoculate the surface of 5 agar plates with 100L of rehydrated BioBall

solution onto
each plate.
5. Using a sterile spreader spread the solution evenly across the surface of the agar.
6. Repeat steps 3 to 5 for all desired organisms.
7. Once completed, transfer all inoculated plates and incubate at the temperature, time and environmental conditions
indicated in table 5 below.
8. Record the time and date incubated in table 5 below.
Organism Agar Incubation
Time/Date
removed
from
refrigerator
Performed
by (Initial)
Time/Date
Incubated
Performed
by (Initial)
Time/Date
removed
from
incubator
Performed
by (initial)
Aspergillus brasiliensis
(niger)
SDA
20-25C
5 d
Bacillus subtilis TSA
3035C
24 4 hrs
Candida albicans SDA
20-25C
5 d
Clostridium sporogenes
Columbia
+5%
blood
3035C
48 4 hrs
(Anaerobic)
Escherichia coli TSA
3035C
24 4 hrs
Pseudomonas aeruginosa TSA
3035C
24 4 hrs
Salmonella abony TSA
3035C
24 4 hrs
Staphylococcus aureus TSA
3035C
24 4 hrs
Table 5. Organism strains, agar types and incubation conditions, (8hr stability).
10
6.2.4 Interpretation of results
1. Remove all plates from the incubators, recording the time and date that the plates were removed in the applicable
table above.
2. Inspect each plate for growth, counting all colonies present on each of the fve replicate plates. Whilst doing so, taking
note of the purity of each plate, and record all results in the relevant table below.
Organism
Plate count (CFU)
Mean count
(CFU)
Pure
Growth
(YES/NO)
Recorded by
Sign/Date
1 2 3 4 5
Aspergillus brasiliensis (niger)
Bacillus subtilis
Candida albicans
Clostridium sporogenes
Escherichia coli
Pseudomonas aeruginosa
Salmonella abony
Staphylococcus aureus
Table 6. Results for organism counts and purity, (initial counts)
Organism
Plate count (CFU)
Mean
count (CFU)
Pure
Growth
(YES/NO)
Recorded by
Sign/Date
1 2 3 4 5
Aspergillus brasiliensis (niger)
Bacillus subtilis
Candida albicans
Clostridium sporogenes
Escherichia coli
Pseudomonas aeruginosa
Salmonella abony
Staphylococcus aureus
Table 7. Results for organism counts and purity following refrigeration of solution at 2-8C
6.2.5 Acceptance criteria
1. The counts obtained for all organisms must be within 10 -100 colony forming units on each agar plate, (CFU) for the
recovery to be valid.
2. Cultures must be visibly pure that is all colonies should be macroscopically identical with no evidence of
plate contamination.
11
6.3 Documentation
The documentation as listed in table 8 below should be attached to the end of this protocol. Documentation for BioBall

is
available from www.btfbio.com. Additional documentation may be added if required.
Document Title Document led (YES / N/A)
QC certicates for all BioBall strains used in the verication test
QC certicates for SDA media used in the verication test
QC certicates for TSA media used in the verication test
QC certicate for BioBall Re-Hydration Fluid (Catalogue No. 56021)
Table 8. Documentation
SIGN / DATE_____________________________
6.4 Comments
SIGN / DATE_____________________________
6.5 Section Review and Approval
Once the results for counts and purity are recorded a review of the results against the acceptance criteria should be made.
Counts acceptable Yes / No*
Purity acceptable Yes / No*
* circle as applicable
Name (Print) Title Signature Date
Results reviewed by
Results approved by
12
7. Conrmation of Organism Identity
7.1 Materials
Inoculated agar plates from section 6 (with growth)
Glass slides
Gram stain reagents
Microscope
Biochemical microorganism identifcation kit, e.g. API, Vitek II (optional)
DNA/RNA microorganism identifcation kit (optional)
7.2 Method
7.2.1 Macroscopic and Microscopic Identication
1. Using plates showing growth from section 6 of this protocol, examine growth present for each organism type, recording
both macroscopic, (colony) and microscopic characteristics in table 9 below. Microscopic identity should be performed
using staining techniques appropriate to the organism.
Organism Macroscopic description
Conforms
(YES/NO)
Microscopic
description
Conforms
(YES/NO)
Performed by
Aspergillus brasiliensis
(niger)
White / pale yellow mycelium, spherical,
lamentous colonies, become black
with conidia (spores)
Long, smooth
conidiophore, biseriate
phialides, round vesicle.
Bacillus subtilis
Irregular, dull, dry, erose, opaque cream
colonies
Gram positive rods
Candida albicans Circular, smooth, convex and bright white
Ovoid/circular budding
cells
Clostridium sporogenes
Grey/translucent irregular colonies, dry, dull
in appearance, with an undulate margin
Gram positive rods
Enterococcus faecalis
O-white, entire, circular, smooth, glistening,
convex
Gram positive cocci
Escherichia coli
Circular, low convex, smooth, entire,
translucent and cream in colour
Gram negative rods
Pseudomonas aeruginosa
Irregular, glistening, can have a greenish
colour on large colonies, with amucoid edge
Gram negative rods
Salmonella abony Entire, circular, cream, smooth, glistening Gram negative rods
Staphylococcus aureus
Entire, smooth, golden yellow, circular
colonies with sometimes also a paler cream/
white colony variant
Gram positive cocci
Table 9. Macroscopic and microscopic colony morphology.
13
7.2.2 Biochemical / Molecular Identication (optional)
1. As an option, the laboratory may wish to further identify each microorganism strain isolated. Details of the method
used and the results should be entered into the space below.
SIGN / DATE_____________________________
14
7.2.3 Acceptance criteria
1. Identifcations of all organisms should match the descriptions listed in table 9.
2. If biochemical or molecular methods are used to identify each strain, the results must correctly confrm the
organism identity.
7.3 Documentation
The documentation as listed in table 10 below should be attached to the end of this protocol. Documentation for BioBall

is
available from www.bioball.com. Additional documentation may be added if required.
Document Title Document led (YES / N/A)
Biochemical / molecular identication result reports (if applicable)
Table 10. Documentation
SIGN / DATE_____________________________
7.4 Comments

SIGN / DATE_____________________________
7.5 Section Review and Approval
Once the results for culture identity are recorded a review of the results against the acceptance criteria should be made.
Identifcations acceptable Yes / No*
* circle as applicable
Name (Print) Title Signature Date
Results reviewed by
Results approved by
15
8. Final review and approval
8.1 Final Comments

SIGN / DATE_____________________________
8.2 Protocol Review and Approval
Once sections 5 to 7 are completed a review of the protocol against the acceptance criteria should be made.
Sections 5 to 7 completed and signed Yes / No*
All supporting documentation attached Yes / No*
Protocol complete Yes / No*
* circle as applicable
Name (Print) Title Signature Date
Protocol performed by
Protocol Reviewed by
Protocol approved by
9. Discrepancies
Any discrepancies discovered during this protocol should be addressed here. Technical information and advice regarding protocol
discrepancies may be obtained from your bioMrieux sales representative.

SIGN / DATE_____________________________
bioMrieux S.A.
69280 Marcy lEtoile - France
Tel. 33 (0) 4 78 87 20 00
Fax. 33 (0) 4 78 87 20 90
www.biomerieux.com
www.biomerieux-industry.com
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