quality assurance - mikrobiyoloji.org 20140327 c.pdf · confidential quality assurance and...

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Quality Assurance and Performance Testing of Culture Media

Implementation of ISO Standard 11133 Part 1 and Part 2

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Contents of Quality Control Lecture

1. Introduction of new ISO Standard 11133-1

2. Practices for quality control of culture media

3. Introduction of new ISO Standard 11133-2

4. Summary

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Why was ISO 11133 developed ?

1. No International Standard exist in

which the Quality Control Specifications of

commercial Culture Media are described

2. Each Culture Media Manufacturer has established

it´s own QC Specification

3. User of Culture Media have no information which

Growth limits are used by the Manufacturer

4. The new ISO 11133 describes now for the first time

which limits for Productivity and Selectivity each

Culture Medium MUST fulfil,

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ISO 11133-1 (2000-06-01)

Microbiology of food and animal feeding stuffs —

Guidelines on preparation and production of culture media

ISO 11133-2 (2003-12-15)

Microbiology of food and animal feeding stuffs –

Practical guidelines on performance testing of culture media

This new standard describe for the first time in history ofMicrobiology the definition of quality control for commercial

culture media.

Quantitative testing and reporting of results ( CoA )with recovery rates is the new preferred testing method

ISO 11133 part 1 and 2

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Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media

Part 1:

General guidelines on quality assurance for the preparation of culture media in the laboratory

Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media

Part 1:

General guidelines on quality assurance for the preparation of culture media in the laboratory

ISO 11133 – Part 1

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Practices for quality control of culture media

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�Any performance evaluation and test organism used

�Technical data sheet

�Quality-control certificate

�Safety and/or hazard data where needed

Documentation

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Documentation - ISO 11133 part 1

� Check list by the laboratory Laboratory

checks upon receipt of the medium

� name of the medium and batch code

� date of receipt

� expiry date

� condition and integrity of packaging

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�� SterilisationSterilisation

� Dehydrated media are not free of contaminants besides

heat-resistant microoganisms

� Be sure that no residual media covers the wall of container

� Autoclave only 15 min. at 121°C

� Sterilisation duration depends on the size of load and containers

� Avoid excessive autoclaving to prevent Maillard reactions degradation and breakdown of nutrient constituents

� Sterilise heat labile substrates by filtration

� Add heat labile supplements with aseptic precautions to the

cooled medium (45°C)

Practices for Quality Control

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Sterikon® plus

Bioindicator for

checking steam

sterilization efficiency

acc. to USP

and Ph. EUR

The performance of the autoclave should be monitored


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These media have the highest risk of overheating

- Brilliant green

- Bile salts

- Iron salts

Preparation of heat sensitive media

XLD Agar is most critical


should not be autoclaved

Media for e.g. Enterobacteriaceae

containing:

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Hot plate has the high risk that the Culture Medium can be overheated

Wather bath versus Hot plate

Melting of agar culture media

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Preparation of media in Petri dishes

�Pour the molten agar culture medium into Petri dishes so as

to obtain a thickness of at least 2 mm (e.g. for 90 mm

diameter dishes, 15 ml of agar are normally required) or as specified in the International Standard.

�Allow the agar to cool and solidify by placing the plates with lids in place on a cool, horizontal surface.

�During incubation, a loss of moisture of the agar media will

occur. A loss of water content can affect the growth of microorganisms in some circumstances.

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Storage of media in Petri dishes

�Use the solidified medium immediately or store under conditions which

prevent its composition from being modified, i.e. in the dark and / or in the refrigerator at 5°C ± 3 °C in sealed bags

� Label the plates on the base with date of preparation and/or expiry date and identity

�In general, for the surface inoculation of a solid culture medium, dry the plates, preferably with the lids removed and with the agar surface facing downwards

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Shelf life of prepared media

�The shelf-life of media varies. Specific International or National Standards may stipulate specific conditions and shelf life.

�It is generally recommended not to exceed 2 to 4 weeks of

storage for plates and 3 to 6 months for bottles and tubes,

unless otherwise specified in specific standards or results of the laboratory shelf life validation indicate a longer shelf life.

�A validated expiry date for stored media should be

established. Observe any colour change, sign of

evaporation/dehydration or microbial growth. Batches of media showing such changes should not be used.

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Shelf life of prepared VRB – Agar (200 ml in a bottle)

100 %

100 %

100 %

100 %

100 %

100 %

100 %

96 %

97 %

83 %

100 %

0 %

0 %

0 %

100 %

97 %

99 %

100 %

0 %

0 %

0 %

TSA

VRB – Agar

Test strains

Escherichia coli

Escherichia coli

Enterobacter cloacae

Salmonella typhimurium

Staphylococcus aureus

Enterococcus faecalis

Bacillus cereus

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

8739

25922

13047

14028

6538

19433

11778

Internal validation study (Recovery %)

8 months

at RT

+ 2 weeks

at 35°C

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Part 2:




Part 2:


ISO 11133 – Part 2

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� The quality of culture media depends on the quality of the basicingredients, correct formulation, quality of preparation

procedures, elimination of contaminant microbial agents and appropriate packaging and storage conditions.

� Quantitative, semi-quantitative or qualitative evaluations shall be performed by the methods described in this Standard or by another generally accepted technique.

� For interpretation of the results of testing, it is necessary tocompare the amount of growth on the test medium with that on a

reference medium. The use of a specific reference medium is therefore mandatory for quantitative methods

General Quality Criteria

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Quality of Basic Media Components

�peptones and meat or yeast extracts variable in their nutritive properties

�agar variable in its gelling properties

�buffering substances

�bile salts, bile extract and deoxycholate, antibacterial dyes, depending on their selective properties

�antibiotics depending on their activity


Culture media described in the International Standards were judged

satisfactory; however, due to the variability of their quality, it may be

acceptable for media manufacturers to modify the concentration of

some basic biological ingredients, as listed below:

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� Growth

- quantitative

- semi quantitative

- qualitative method

� Productivity

� Selectivity

� Biochemical Characteristics

� Performance Evaluation and Interpretation of Result


BAIRD-PARKER AGAR

S. aureus ATCC 6538

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�Solid, semi solid or liquid culture medias shall be

inoculated with an appropriate inoculum of the working

culture of each of the defined test microoganisms using

an appropriate device e.g. spiral plater

�Productivity has to reach a fixed limit

Productivity

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�To asses selectivity quantitatively, selective

culture media and a reference medium are

inoculated with an appropriate inoculum of

test trains.

�Selectivity has to reach defined values.

Selectivity

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� For isolation and enumeration media, at least semi-quantitative testing should be performed. For characterization test media, qualitative testing may be sufficient.

� Quantitative tests will give greater assurance of media quality.

� For those media which contain no indicators or selective agents the use of a single positive test strain is appropriate. For those media which do contain indicators or selective agents, strains which demonstrate the function of the indicator(s) and selectivity must be utilized.

� For complex media, i.e. with added supplements, each batch should be verified with strains with characteristics.

General

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�It is a general method suitable for most solid culture

media

- A spiral plater can be used

- The pour plate method for enumeration can also be used

in this way

�It may not suitable for testing types of moulds

Methods for Solid Culture Media

Quantitative method

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� Pre-culture the test strain in CASO Broth or Sab.-2%-Broth (Candida) at 35°C for 24 h

� Dilute the pre - culture with 1/4 Ringer - solution

� Inoculate the test medium, an older reference lot and a reference medium with the Spiral Plater

� Count the colonies in comparison to an older reference batch and to a

reference medium

Quantitative method – Procedure

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Ref.Medium (TSA)

GOLD StandardUsed only for internal control

NEW batch

BROLACIN (CLED) Agar

Calculation : 85 / 100 = 85 % recovery

Quantitative method - Interpretation

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Ref.Medium (TSA) NEW batch

CEREUS Selective Agar (MYP)

Calculation : 84 / 123 = 68 % recovery rate

Quantitative method – Interpretation

GOLD StandardUsed only for internal control

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> 70% for non selective media

> 50% for selective media

Most important change !

Productivity

These values are generally achievable, however less rigorous criteria can be accepted for certain combinations of media and test

microorganisms.

For media which are not specified in Annex B, e.g. those described in local standards, these criteria may not be applicable.

Methods for Solid Culture Media

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Enumeration Media – Selective Media

Enumeration Media – Non Selective Media

Enrichment Media – Non Selective Media

Performance criteria (Annex B)

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Culture Media in ISO Food Norms with a recovery rate > 50%

� ISO 6888-1 Baird Parker Agar Staphylococcus aureus

� ISO 7954 OGY Agar Yeast & Moulds

� ISO 15214 MRS Agar Lactic acid bacteria

� ISO 7932 MYP Agar Bacillus cereus

� ISO 11290 Oxford Agar L. monocytogenes

� ISO 11290 PALCAM Agar L. monocytogenes

� ISO 7938 TSC Agar Clostridium perfringens

� ISO 7402

ISO 8523

� ISO 4832 VRB Agar Coliforms

� ISO 16654 CT SMAC Agar E.coli 0157

VRBD Agar Enterobacteriaceae

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Certificate of Analysis Tryptic Soy – Agar

all test strains must have > 70 %otherwise batchis rejected

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Certificate of Analysis VRB – Agar

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Certificate of Analysis Baird Parker – Agar

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30

40

50

60

70

80

90

100

110

120

643 806 541 906 476 306 370 506 291 306

E.coli S.typhimurium Enterob.cloaceae

Recovery Rates – VRB Agar

%

Batch No

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Qualitative streaking method

� The method is suitable for

supplementary performance testing

of solid culture media.

� The method is only qualitative

and scores are therefore only

indicative.

General

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The growth on the plates after incubation is assessed as:

� 0 corresponds to zero growth

� 1 corresponds to weak growth

� 2 corresponds to good growth

Qualitative method – Interpretation

Target microorganisms shall score 2 and have typical appearance,

size and colony morphology. The growth of non-target microorganisms shall be partly or completely inhibited (0 or 1).

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Quantitative dilution method for target and non – target microorganisms

� To determine the productivity of a liquid medium an

appropriate inoculum shall be used.

� The method is also appropriate for evaluation of new culture media, broths or diluents.

Methods for Liquid Media

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Quantitative method – Procedure

� Select an appropriate number of tubes or 10 ml portions of each batch of liquid medium to be tested.

� Inoculate test broth and reference broth for each test organism with a small number (e.g. 10 cfu to 100 cfu).

� Inoculate test broth and reference broth for each test organism with a higher number (>1000 cfu).

� For testing mixed cultures in selective media inoculate test broth and reference broth with a small number of target

microorganisms and in the same tube with a higher number of non-target microorganisms.


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Quantitative method – Reading

� Remove an aliquot volume or if necessary a dilution from

each broth after the incubation step and spread to a

non-inhibitory agar plate.

� After incubation colonies of target and non-target

microorganisms are counted, in the case of mixed cultures

distinguishing the different types.

� Calculation and interpretation shall be done with respect to

the aim of examination.


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Quantitative method – Calculation / Interpretation

106 to 108 cfu per ml

below 104 cfu per ml

Target bacteria

Unwanted

Extremely important to avoid false-negative results


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Certificate of Analysis FRASER - Broth

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RVS / TTn / Selenite Cystine broth

Quality control Minimum level must be

105 cfu

Test strains Inoculum Growth after

24 hours

Escherichia coli

ATCC 25922

Approx. 99 % < 10 %

Salmonella typhimurium

ATCC 14028

Approx. 1 % > 90 %

Quantitative method – Mixed Culture


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24 hr

E. coli: Lactose (+) = yellowSalmonella typhimurium: Lactose (-) = blue

Quantitative method – Mixed CultureResults on CLED agar after selective enrichment in RVS broth

0 hr


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- 0 corresponds to zero turbidity

- 1 corresponds to very light turbidity

- 2 corresponds to good turbidity

Qualitative method - Interpretation


Qualitative evaluation shall be carried out visually by allocating growth scores, e.g. from 0 to 2.

Other characteristics such as gas formation, colour change, etc. can also be assessed by this method.

The score of a target microorganism shall be 2.

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� The manufacturer or supplier shall provide, on

request, the specific microbiological characteristics

and general information relating to the specific batch

of culture medium.

� The use of control sheets for documenting and

evaluating the results of the routine performance

testing is recommended (see Annex A).

Documentation of Test Results

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Control Card (Annex A)

ISO 11133 for quality control of culture media B. Gerten Merck KGaA 09-2013 47

ISO 11133 – overview to actual parts

ISO TS 11133 Microbiology of food and animal feeding stuffs –Guidelines on preparation and production of culture media

Part 2 Amd 1: 2011 Test microorganisms for commonlyused culture media

contains tables with the media used for food microbiology

contains WDCM numbers instead of ATCC numbersfor all strains of microorganisms used for quality control

Reason:ATCC did not allowed for future publications of ISOany impression of equivalence of strains from differentculture collections compared to ATCC(planned as a table for revised ISO 11133);

also only allowed to use ATCC numbers cited as ATCC™

using of ™ is only possible in ISO standards, if equivalent substance / material can also be used – but how can we show equivalence of the strains from different culture collections ?




WDCM numbers

are given as universal strain identifiers

by the World Data Centre for Microorganisms (WDCM)

WDCM is part of WFCC (World Federation of Culture Collections)

they have initiated a system that will help users find local sources

of the reference strains by citing all collections andproviding contact details and the collection’s unique reference




WDCM numbers

on the new website of WDCM

(www.wfcc.info)

it is also possible to search by

- species name

- WDCM strain number

- strain accession number

- number of standard

(ISO and other standards)

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Demands to a reliable manufacturer of culture media:� Media manufacturer must be certified according to at least ISO 9001 standards

� Media manufacturer should produce culture media in full compliance with

ISO 11133 Quality control methods

Merck as manufacturer of culture media:� Merck is certified as culture media manufacturer acc. various ISO norms.

� Merck has available a clear documentation about the performance of their culture

media with values that exceeds ISO 11133

Customers:� Customers labs which are certified acc. ISO standards need:

- To use validated culture media

- To follow validated enrichment procedures

Summary Part 1 and Part 2



Availability of ISO standards: ANSI web store (http://webstore.ansi.org/)

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