D-113

Microbiological Media Validation

Section D — Laboratory Operations and Specifications Revision 7 11 pages

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3.0 Responsibility

yb
«
oy It is the responsibility of QC Laboratory Analysts to ensure all procedures and

3.2 {tis the responsibility of OC Laboratory Management to implementtis procedure and

to ensure that the procedure is being followed.

dee dapgi:T it is the responsibility of OC Laboratory Management to keep current this procedure

and to oversee validations and recov‘ery studies.

4.0 Definitions

44 OC ~ Quality Contral

[SOP

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4.16 420

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Dehydrated Media ~ Dry media consisting of best ingredients like, peptones,
vegetable extracts, animal tissue, sodium salts, inhibitory agents, agar content and
carbohydrate sources for the media preparation and growth of microorganisuis.
Ready-Made Media — Media which has been prepared and is ready to use upon
receipt.
TSB — Trvptic Sov Broth
RVS ~ Rappaport-Vassiliadis Soya Broth
ER — Enterobacteriaceae Enrichment Broth
MORS ~ De Man, Rogosa and Sharpe Agar
SDA — Sabourand Dextrose Agar
RYM — Rapid Yeast and Mold Petrifilm
TSA ~— Tryptic Soy Agar
LB Broth — Luria-Bertoni Broth
LB Agar ~ Luria-Bertoni Agar
LB Soft Agar — Luria-Bertont Soft Agar
EC — FE. coli/Coliform Count Petrifiin
bone

[SOP

Standard Operating Procedure | SOPNo | Rev
Mierobiclogical Media Validation | D-113 7
Page Sof 1

422 STAR ~ Staph Express Count Petrifilm

4.24 LD — Xylose Lysine Deoxycholate Agar

4.25 EB ~ Enterobacteraceac Count Petrifilm

4.26 BCSA ~ Burkholderia cepacia Agar

4.27 COA — Certificate of Analysis

428 DRC ~ Pichloran Rose-Bengal Chloramphenicol Agar

§.0 References

§a= 1 a- 718o, SaOoaP , 5M i>c robi«a l Li. miPtr T esati ng Usi” ng 32 May Mt >P ¥e tcnrbneia! fwieyl m'PTMM SWcy aseedtee e;m

3 D-716, SGP, Enumeration of Raw Materials for Probiotic Supplements

S4 -D-126, SOP, Non-Conforming Results in the GC Laboratory

?i
o
dem C-502, SOP, Record Storage, Retention, and Destruction
ia OO A-106, SOP, Documentation Guidelines for eGMP Records

C-S01, SGP, Document Contral Procedure tS

[SOP

Standard Operating Procedure } SOP No | Rev pave 4 of 11

Microbiological Media Validation | Bet ORL.

5.10 21 CFRPart 110 and 11]

Sf USP Chapter<20Z1>, <2022>, </> and <LPI6>

6.0 Required Supplies, Media, and Equipment

6.1 Ready-made and in-house made Media

G2 Control Organisnsinclude but are not limited ta:

62.1 Aerobie Bacteria

O21... Staphviococcus aureus, ATCC 6538

G25.2 Bacillus subsitix (spizenst), ATCC 6633

62.14 Burkholderia cepacia, ATCC 25416

G21.5 Salmonella enterica, ATCC 14028

6.2.1, Listeria monocytogenes, ATCC [3932

4.2.3 Probiotic

B23.2 Lactobacilins sop. fee. 1. rhamnosus, ©. acidophilus)

CON PODIEN TIAL: For ABT lon Labs use only

[SOP

Standard Operating Procedure _ SOP No : Rev
Page J of LI
Microbiologieal Media Validation

sa
’
)7 Aghustable Incubators

6.10 6.1]

enaLQ een
6.3.2 30°C - 34°C
6.3.3 35°C - 39°C
Adjustable Water Bath
6.4.1 42°C - 448C
Biological Safety Cabinet
Sterile Pre-saturated 70% Isopropyl Aleoho!l (IPA) Wipes
Sterile 70% Isopropyl Alcohol PA}
Cleanroom Indelible Marker
Sterile Gloves
74) Procedure
7 Growth Promotion Organism Preparation
]~
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1.1 Commercially prepared challenge organisms may be used. The ATCC control
strains disclosed in COA (Certificate of Analysis) from media vendor are
sufiable for use, in addition to the control organisms listed in this SOP.
7.1.2 Prepare the challenge organisms per manufacturer instruction, Use the

[SOP

Standard Operating Procedure — SOPNoe | Rey
ope ' nig oio7 | Pagebarll
Microbiological Media Validation

preparation within 8 hours.

7.1.3 Pull challenge organism into a | cc sterile syringe/needle or use a pipette to

aspirate the inoculurn inte a sterile pipette tip to inoculate plate or liquid media.
This is used to inoculate test media and control media,

Nete: The quantity of countable microbes must be between 10-100CFU per plate.

To Media Preparation

7.2.1 Por dehydrated media, refer to the instructions on the bottle label and sterilize
the media prior to use in accordance with SOP D-824: Operation and Cleaning

of the Tuttnaner E710 Auteclave.

V3.1 Each preparation of prepared media and each lot ofpurchased media is required

ta be challenged for growth promotion.

73.2 Obtain two test media for each organism to be tested. Label cach appropriately

with the organism name, date and initials.

dw daS )aw Obtain two control media(TSA or previous lot of the media type to test), which

have previously been tested and passed growth promotion. These are the
positive controls. Alternatively,AC petrifilm can be use as control media for

Y3.4 Inoculate the test media and cantrol media by placing an inoculum containing

S100 CFL, based on CFU/, [mL on organism's package label, in/on each test

item. For petrifilm, prepare lml of Butterfields Buffer or Peptone Salt diluent
containing [00a] of challenge organism, per film to inoculate.

734 Use a sterile inoculating needltye, loop, or plate spreader ta spread the organism
across any test plates or control plates to evenly spread the organism. Liquid

[SOP

SOPNe | Rev save Tak th
Standard Operating Procedure
ical Media Validation D-113 " Page 70
Mierobiolog

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Obtain at least two test media to be incubated as negative control of test lot.
Petrifilm negative controls are inoculated with sterile diluent only.
Incubate the tesi and control media at the appropriate temperature for the media
Record the organisms used on D-113-F1 Microbial Media Validation Testing

After Incubation

7.3.9.) Uf the test mecia is qualiiative media, examine ihe test media and
record growth or no growth on D-113-P1L Microbial Media Validation

Testuig Log. Qualitative media include broth or other liquid media, as
well as selective and differential media.

7.3.9.2 Uf the test media is quantitative media, examine the test media and

record the number of CFL per plate on D-113-F1 Microbial Media
Validation Testing Log.

7.3593 Examine the control media: record the number of CF Uplate on D-113-
Fi Microbial Media Validation Testing Log.

$39.4 Control media must have 10-100 CFU per plate per organism for the

VA95 Negative Control media must contain 0 CEU or no growth in order for

the tests to be valid.

7.3.10 Calculate the percent recovery for quantitative media. (.e., test count average

divided by control count average X 100).

/3.401 Percent recovery between the test plates and control plates must be 50-

[SOP

| SOP No Rev | _
DLS 7 Page 8 of Li
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200% for plates to be considered acceptable for use.

plates in quadruplicate. The control plates will be performed in duplicate.

T4414 The retest may be performed by the same or different lab personnel,

73412 Uf the media fails a seeond time, the media is rej«ected for use in the

73AZ Download and store manufacturer COA in digital folder labelled “COA Media
ve
a

7.4.1 Incubate the negative control for sterility check at specified temperature for time

specified as per Table 1.

TA There should not be any microbial growth recovered on the nevative control af

sterility media after incubation period.

Document results observed on Form D-113-F1 Microbial Media Validation Log. dm a

FSA Allow tesi media to be equilibrated at room temperature before pH

measurement. Petrifilm does not require pH readings.

The pH meter must be caliwebrated before use. Refer SOP D-706 Use and dam DL ah

calibration of pH Meters.

7h 3~. 93 AScEcPe epPotpeaarnircccee cSrpitt eeyr itea ffoory ep Hanh r ea e n a d l i y ngs a i e s d r ef Eie i wre n s ed e p e e e r e A a A c1h cult q ure - med e ia e , sce a T ie a bl t. e
q,

43.4 HY observed pH values do not meet these criteria, refer to COA vendor for
acceptable results. Document results observed on Form D-113-F 1.

[SOP

Standard Operating Procedure SOP No Rey
Page 9 of 11
BHiS 5 7
Microbiological Media VaHidation
Table 1- Media Sterility and Growth Promotion Testing, Incubation, and pl
Tomperatsre
Media Growth Pramoves Oreanicn Tooubation Tine pH
Reyuirement
u‘hwocalate A par doof BOR33°C 18.- 24 Hours é 2 ak 02
Sterifity incubation is NLT 18 Hours
18+ 24 Hours
BA Staphylococcus aureus ATCC 6338 Sterility Incubation is NLU 18 Hours FS OF
Escherichia coli ATCC 8736
Staphylococcus ancens ATCC 538
TSB Poendonwnas aeruginosa ATC 8027
Salmonella erdorioa ATCO 14028
Horkhalderia cepacin ATE 23é
do s om e pO.b tA ow
18 ~ 24 Tours
PsSetuapdhoymloancacss.e ausc ya ‘uarienonss.e A { fae
9027 ]8 «24 Hours
AC Petriftin Burkholderia cepacia ATCC 35416 3Q-3S°C Steriliv incubation is MILT 3 days
Bacillus subtilts ATCC 6633
18 ~ 24 Hours
Eschericheiaah ATCC £239
EC Petri Sterility aenbaticg is NLP TS hours
- 24 Hours
Escherichia coh ATO $739
MAC Broth Escherichia cah ATCC $739 mr
18-24 Hours
Sterility incubatian is NLT 18 Hours
SwUeaXM r SPtoAter siofa ri lmn Stapghyy locacens aurweuese A TCE 6338 Ritpem, sa Satenrieli ty incu[b§a-t i2 eo4n Hios ur e sMLT 1&8 Hours AMSAonly)
se a we “
Selronetia enterica ATCE 24028 38 SAT Sat erility ine abation |isORL° POUR Hours “
EB Peto Bim (ALD only}
18 ~ 24 Hours
EE Broth Messe! Saimionetin edieries ATCO }4028
Stenlity meubation 3s WET U8 Hours
18-24 Hours
RVS Breath Salmonella etiterica ATCC 14628
Sterility ine ” ation. isNLC 18 Hours
VREBG or suevility tnouet- i2o4n Htoeu rNsUT 18Hears
Salmonella entericaATCC 14928
ler CVRBG only}
aeo
23.48 hours
BCSA Agar Hurkholderia cepacia ATCC 28416
rea4e hou
LB Broth
Stscility Incubation is NLT 18-24 Hours
LB Agar, L.BSSant 18-24 hours
& Coli APCO 8739
Sterility ineuhation ig NLT 18-24 Hours
Prendomonas paracrugimoasa 18-24 ours
ATCC S027 Sterility incubation is NLT 18 ffours
HARDY CHROM 18 - 24 Hours
Sterility incubation is NET 18 Hours
—VARTIY CHRDM 24 Hours
Satmonedla. Sterility incubation is NUP 24 Hours
HARDY CHROM Listeria monocyiogenes
Listeria ATCC Tike
a = enaL a“ a snmor
24 Hours
Ste riliby ine ubation |is NLY 24 Faurs
BARDYCHROM 20- 28 how
Staphylncocens aurcus ATCC G438
y ineubstion is NET 20 Hours
A eet ea + en a )i WD
HARBYCHEROM 48 Hours
Candida, Sterility ineuation 1s NET 48 Houps
tes
SDA, DREC and Candnda albicans ATOR 1023)
RYM Petrifilm Aspergilus mger ATCC 16404 Sterility incubation is NLT 3 days {SDA and DRBC only)
MES Agar En
3 Days
TOS Agea r BifidebacteriurHU e sSpDgE.. > SRBC Sots eribty incubation tiss J. {2. days bs 25:2
OHYE Agar Bacillus coagnlans* SARE Stenbty metbation“ 1|S© Nt,YF 2 days Pts 0.2
* Treubate anaerobically
CONFIDENTPLIAL: For HB lon Labs use onby

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SOP No | Rey |
D3 | 7 | Page tO of 1
Microbiological Media Validation |

3.0 Use and Disposition

8} Media (prepared or purchased) once made or received will be labeled with a quarantine

sticker until testing is started. No media should be used in this state.

82. >2 Media (prepared or purchased) that does not pass pata, growth promoti*o n, or steriolyeity

will net be usedfor Microbial Tesating and immediately Hatscarded or returned to vendor.

8.3 Media (prepared or purchased)that meets all acceptance requirements will be t: geed

with a sticker indicating QC approval. Only use of approved media lots is allowed.

$0 Documentation Requirements

9] A POV check omst beeee for each completed pave of form D-113-F1

Microticiopical Media 9 Log as outlined in SOP A-106 Documentation

Oo AJ] documentation will be distribaied and maintained as eutlined in SOP C-401

Document Control and SOP C-502 Record Sterage, Retention, andDestruction.

10.0 Revision History

Revision | Hei Description ofChanges COR By

i O5/1] Added requiremnent to challengge all hatches ofprepared media for growth CC23-0242 | J. Sassraan
- M323 promotion. Other minor clarifications Updated loge Oe a Oe Bank eee on > WORE ears.
Adlusted incubation times and sterility incabation tines. Added numerous
5 O7/12/23 | arganisms that were missing. Adjustedforrn for more clear recordings and | CC-23-0337 CG. Shaw

6 06/11/24 | Updated SOP to follow current practices. CC 24-0269 A. Peres

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Standard Operating Procedure SOP Ne | Rev
Microbiological Media Validation D-LS | } i age Li of 1]