Quality Assurance

and Performance
Testing
of Culture Media

Implementation of
ISO Standard 11133
Part 1 and Part 2

CONFIDENTIAL
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

CONFIDENTIAL
 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

its 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,
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
CONFIDENTIAL
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 assurance for
the preparation of culture media in the
laboratory

CONFIDENTIAL
Practices for quality control of culture media

CONFIDENTIAL
 Documentation

Any performance evaluation and test organism used

Technical data sheet

Quality-control certificate

Safety and/or hazard data where needed

CONFIDENTIAL
 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 121C

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 (45C)
CONFIDENTIAL
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

CONFIDENTIAL
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
CONFIDENTIAL
Melting of agar culture media

Wather bath versus Hot plate

Hot plate has the high risk that the

Culture Medium can be overheated
CONFIDENTIAL
 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.

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 5C 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

CONFIDENTIAL
 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. 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 35C
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%
CONFIDENTIAL
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

CONFIDENTIAL
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 quantitative methods

CONFIDENTIAL
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

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

BAIRD-PARKER AGAR
 Productivity
S. aureus ATCC 6538
 Selectivity
 Biochemical Characteristics
 Performance Evaluation and Interpretation of Result

CONFIDENTIAL
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

CONFIDENTIAL
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.

CONFIDENTIAL
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 characteristics.
CONFIDENTIAL
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

CONFIDENTIAL
Quantitative method Procedure
 Pre-culture the test strain in CASO Broth or Sab.-2%-Broth (Candida)
at 35C 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

CONFIDENTIAL
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

CONFIDENTIAL
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.

CONFIDENTIAL
Performance criteria (Annex B)
Enumeration Media Selective Media

Enumeration Media Non Selective Media

Enrichment Media Non Selective Media

CONFIDENTIAL
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 VRBD Agar Enterobacteriaceae

 ISO 4832 VRB Agar Coliforms

 ISO 16654 CT SMAC Agar E.coli 0157

CONFIDENTIAL
Certificate of Analysis Tryptic Soy Agar

all test strains

must have > 70 %
otherwise batch
is rejected

CONFIDENTIAL
Certificate of Analysis VRB Agar

CONFIDENTIAL
Certificate of Analysis Baird Parker Agar

CONFIDENTIAL
 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

CONFIDENTIAL
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.
CONFIDENTIAL
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).
CONFIDENTIAL
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.

CONFIDENTIAL
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.
CONFIDENTIAL
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.

CONFIDENTIAL
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

CONFIDENTIAL
Certificate of Analysis FRASER - Broth

CONFIDENTIAL
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 Approx. 1%

ATCC 14028
> 90 %
CONFIDENTIAL
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
CONFIDENTIAL
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.
CONFIDENTIAL
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).

CONFIDENTIAL
Control Card (Annex A)

CONFIDENTIAL
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 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

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 collections unique reference

ISO 11133 for quality control of culture media B. Gerten Merck KGaA 09-2013 48
ISO 11133 for quality control of culture media B. Gerten Merck KGaA 09-2013 49
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 Merck KGaA 09-2013 50
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

CONFIDENTIAL
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 Merck KGaA 09-2013 52
CONFIDENTIAL