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 CONFIDENTIAL

ISO 11133
part 1 and 2
ISO 11133-1 (2000-06-01) ISO 11133-2 (2003-12-15)
Microbiology of food and Microbiology of food and
animal feeding stuffs — animal feeding stuffs –
Guidelines on preparation and Practical guidelines on
production of culture media performance testing of culture
media

This new standard describe for the first time in history of

Microbiology 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
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ISO 11133 – Part 1

Microbiology of food and animal feeding

stuffs – Guidelines on preparation and
production of culture media
Part 1:
General guidelines on
quality
the assurance of
preparation for culture media in
the laboratory

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Practices for quality control of culture
media

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 Documentation
✓Any performance evaluation and test
organism used

✓Technical data sheet

✓Quality-control certificate

✓Safety and/or hazard data where needed

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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 CONFIDENTIAL
Practices for Quality Control
➢ Sterilisation
✓ 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
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Practices for Quality Control
The performance of the autoclave should be monitored

Sterikon® plus
Bioindicator for
checking steam
sterilization efficiency
acc. to USP
and Ph. EUR

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Practices for Quality Control
Preparation of heat sensitive media
Media for e.g. Enterobacteriaceae
containing:
- Brilliant green
- Bile salts
- Iron salts
XLD Agar is
most critical
should not be autoclaved
These media have the highest risk of overheating
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Melting of agar culture media

Wather bath versus Hot plate

Hot plate has the high risk that the

Culture Medium can be
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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 CONFIDENTIAL
 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
media showing such changesorshould
microbial
not growth.
be CONFIDENTIAL
Shelf life of prepared VRB – Agar (200 ml in a bottle)

Internal validation study (Recovery %)

VRB – Agar
Test strains
TSA 8 months + 2 weeks
at RT at 35°C

Escherichia coli ATCC 8739 100 % 100 % 96 %

Escherichia coli ATCC 25922 100 % 97 % 97 %
Enterobacter cloacae ATCC 13047 100 % 99 % 83 %
Salmonella typhimurium ATCC 14028 100 % 100 % 100 %
Staphylococcus aureus ATCC 6538 100 % 0% 0%
Enterococcus faecalis ATCC 19433 100 % 0% 0%
Bacillus cereus ATCC 11778 100 % 0% 0%
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ISO 11133 – Part 2

Microbiology of food and animal feeding stuffs –

Guidelines on preparation and production of culture media

Part 2:

Practical guidelines on performance testing of culture

media

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General Quality Criteria

➢ The quality of culture media depends on the quality of

the basic ingredients, 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
to compare 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
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General Quality Criteria
Quality of Basic Media Components
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:
➢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

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General Quality Criteria
➢ Growth
- quantitative
- semi quantitative
- qualitative method

➢ Productivity BAIRD-PARKER AGAR

S. aureus ATCC 6538
➢ Selectivity
➢ Biochemical Characteristics
➢ Performance Evaluation and Interpretation of Result

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Productivity

➢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

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Selectivity

➢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.
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General
➢ 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
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Methods for Solid Culture Media
Quantitative method

➢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
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Quantitative method – Procedure
➢ 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

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Quantitative method - Interpretation

BROLACIN (CLED) Agar

GOLD Standard
Used only for internal control

Ref.Medium (TSA) NEW batch

Calculation : 85 / 100 = 85 %
recovery CONFIDENTIAL
Quantitative method – Interpretation

CEREUS Selective Agar (MYP)

GOLD Standard
Used only for internal control

Ref.Medium (TSA) NEW batch

Calculation : 84 / 123 = 68 % recovery rate

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Methods for Solid Culture Media

Productivity > 70% for non selective media

> 50% for selective media

Most important change !

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.

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Performance criteria (Annex B)
Enumeration Media – Selective Media

Enumeration Media – Non Selective Media

Enrichment Media – Non Selective Media

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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 VRBD Agar Enterobacteriaceae
8523
➢ ISO 4832 VRB Agar Coliforms

➢ ISO 16654 CT SMAC Agar E.coli 0157

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Certificate of Analysis Tryptic Soy – Agar

all test strains

must have > 70 %
otherwise batch
is rejected

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Certificate of Analysis VRB – Agar

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Certificate of Analysis Baird Parker – Agar

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 Recovery Rates – VRB Agar
120
110
100
90

% 80
70
60
50
40
30
643 806 541 906 476 306 370 506 291 306 Batch No
E.coli S.typhimurium
Enterob.cloaceae
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Qualitative streaking method

General

➢ The method is suitable for

supplementary performance
testing of solid culture media.

➢ The method is only

qualitative and scores are
therefore only indicative.
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Qualitative method – Interpretation

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

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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Methods for Liquid Media
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.
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Methods for Liquid Media
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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Methods for Liquid Media
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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Methods for Liquid Media
Quantitative method – Calculation / Interpretation

Target bacteria 106 to 108 cfu per ml

Unwanted below 104 cfu per ml

Extremely important to
avoid false-negative results

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Certificate of Analysis FRASER - Broth

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Methods for Liquid Media
Quantitative method – Mixed Culture
RVS / TTn / Selenite Cystine broth
Quality control Minimum level must be
105 cfu

Test strains Inoculum Growth after

24 hours

Escherichia coli Approx. 99 % < 10 %

ATCC 25922

Salmonella typhimurium
ATCC 14028
Approx. 1%
> 90 %
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Methods for Liquid Media
Quantitative method – Mixed Culture
Results on CLED agar after selective enrichment in
RVS broth
0 hr 24 hr

E. coli: Lactose (+) = yellow

Salmonella typhimurium: Lactose (-) = blue
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Methods for Liquid Media
Qualitative method - Interpretation
Qualitative evaluation shall be carried out visually
by allocating growth scores, e.g. from 0 to 2.

- 0 corresponds to zero turbidity

- 1 corresponds to very light
turbidity
- 2 corresponds to good
turbidity
The score of a target microorganism shall be 2.

Other characteristics such as gas formation, colour

change, etc. can also be assessed by this method.
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Documentation of Test Results

➢ 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).

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Control Card (Annex A)

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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 commonly

used culture media
contains tables with the media used for food microbiology
contains WDCM numbers instead of ATCC numbers
for all strains of microorganisms used for quality control
Reason:
ATCC did not allowed for future publications of ISO any
impression of equivalence of strains from different
culture 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 ?

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

Merck KGaA 2013
ISO 11133 – overview to actual parts

ISO TS 11133 Microbiology of food and animal feeding stuffs –

Guidelines on preparation and production of culture media

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 and
providing contact details and the collection’s unique reference

ISO 11133 for quality control of culture media B. Gerten 09- 48

Merck KGaA 2013
ISO 11133 for quality control of culture media B. Gerten 09- 49
Merck KGaA 2013
ISO 11133 – overview to actual parts

ISO TS 11133 Microbiology of food and animal feeding stuffs –

Guidelines on preparation and production of culture media

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)

ISO 11133 for quality control of culture media B. Gerten 09- 50

Merck KGaA 2013
Summary Part 1 and Part 2
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

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ISO 11133 – overview to actual parts

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

ISO 11133 for quality control of culture media B. Gerten 09- 5

Merck KGaA 2013 2
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