ISO Standard 11133 part 1 and 2

Guidelines on preparation and production of culture media in

Microbiological Laboratories

Dr. Sabine Müller

Head of Product Management Dehydrated Culture
Media & Rapid Testing Applications
Merck Millipore

July 2013
Merck - Tradition and Innovation

A Pioneer in Microbiology:
First commercial product in Microbiology 1892

Quality:
Leading expertise in microbiology for over 120 years
with comprehensive QC / QA since 1885.
Pioneer producing DCM acc. ISO 11133

Innovation:
Pioneer of granulated media (1950)
Dry granulation since 1970
Pioneer of chromogenic media
World’s largest collection of rapid tests in the
Lateral Flow format

1
 Why was ISO 11133 developed ?
1. No International Standard existed before in
which the Quality Control Specifications of
commercial Culture Media are described

2. Each Culture Media Manufacturer had established

it´s own QC Specification

3. User of Culture Media had no information which

growth limits are used by the Manufacturer

4. The new ISO 11133 described for the first time

quantitative growth limits for Productivity and Selectivity
Staph. aureus
which each Culture Medium MUST fulfill ATCC 6538 on
BAIRD-PARKER
AGAR
2
Other Supplier’s “Quality Assurance” certificates

Documentation

NO ISO 11133 compliance:

1. ONLY Qualitative testing
2. ONLY satisfactory performance of...

Page
 Example 1: High quality vs. cheaper DCM manufacturer
Merck Competitor 1

XLD Agar: Proteus vulgaris ATCC 13315

Merck: Yellow colonies as per specifications

Comp.: Black colonies lead to false-positive result
(indication of Salmonella)

XLD Agar: Salmonella typhimurium ATCC 14028

Merck: Luxuriant growth with black centers

Comp: Poor growth lead to false-negative result

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 Example 2: High quality vs. cheaper DCM manufacturer

Chromocult Listeria Selective Agar (ALOA)

Listeria monocytogenes ATCC 19111

Merck - Transparent agar (good visibility of Halo zone)

- Blue colonies with good colony size
- Efficient formation of Halo zone (as per specs and
ISO 11290)

Listeria Selective Agar (ALOA)

Listeria monocytogenes ATCC 19111
Comp. 2 - Intransparent agar (difficult read of Halo zone)
- Small pinpoint colonies -> poor growth
- No Halo zone
-> False-negative results (mix up with non-pathogenic
Listeria spp or Bacillus)

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 ISO 11133
ISO 11133-1 (TS: 2009-02-01) ISO 11133-2 (2003-12-15)
Microbiology of food and animal Microbiology of food and animal
feeding stuffs — feeding stuffs –
Guidelines on preparation and Guidelines on preparation and
production of culture media production of culture media
Part 1: Part 2:
General guidelines on quality assurance Practical guidelines on performance
for the preparation and production of testing of culture media
culture media in the laboratory

This standard described for the first time in history of Microbiology the correct
handling and 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
6
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 1:2009 published as Technical Specification (TS) 2nd edition

February 2009

Title: General guidelines on quality assurance for the preparation

of culture media in the laboratory

- Part 2: 2003 published as Technical Specification (TS) 1st edition

December 2003

Title: Practical guidelines on performance testing

of culture media
- Part 2 Amd 1: 2011 published as Amendment to TS 1st edition
February 2011

Title: Test microorganisms for commonly used culture media

7
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

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Practices for quality control of culture media
Part 1 of ISO11133
Scope of ISO 11133, Part 1

- Specifies requirements for the preparation of culture media to be

used for the microbiological analysis of products intended for human
consumption or animal feeding
- Also applicable to culture media to be used for microbiological analysis
of all kinds of water
- Requirements are applicable to four categories of culture media used
in labs that prepare and/or use culture media for performing
microbiological analyses:
• commercially manufactured ready-to-use media;
• media to be remelted, supplemented and distributed;
• media prepared from commercially available dehydrated formulations;
• media prepared from their individual components

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 Documentation

The following details should be available from the

manufacturer:

• Name of the medium, individual components + supplements,

product codes
• Batch codes
• pH value of the medium before use
• Storage information and expiry date

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Documentation

• Results of performance testing with criteria of acceptance

• Technical data sheet
• Quality-control certificate
• Safety and/or hazard data where needed

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Laboratory preparation of media

The preparation of culture media is one of the fundamental steps

in microbiological examination and it shall given special care

• Respect good laboratory practice and follow the manufacturers

instructions
• Document all relevant data
• For media prepared from individual components follow the
recipe precisely and record all detail

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 Laboratory preparation of media

Dating and storage of media

• Use principle first in first out

• Monitor entrance and first opening

• Respect the manufacturers expiry date
• After opening screw containers tightly
• Store below 25°C
• Avoid absorption of water, oxidation and contamination

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Laboratory preparation of media

Water for preparation of culture media

Microbial contamination should not exceed 103 CFU / ml and should

preferably be below 102 CFU/ ml.
A regular verification of microbial contamination should be established according to ISO 6222
(incubation at 22 °C for 68 h ± 4 h) or an equivalent validated method.
ISO 6222: Water quality – Enumeration of culturable micro-organisms -
Colony count by inoculation in a nutrient agar culture medium

• Preferred is fresh distilled water

if chlorinated water, then neutralise chlorine prior to distillation
• Store distilled water in inert material containers (glass, polyethylene)
• Resistive of 0.4 M cm
• Conductivity 25 µS/cm

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Laboratory preparation of media
Weighing and Rehydration of Media

• Weigh accurately the desired quantity

• Take care not to inhale powder, especially
with media containing toxic substances
• Rehydrate with correct volumes of water
• Use distilled or deionized water

Dispensing
• Use Containers with 1-3 x capacity of medium volume
• Let soak agar containing media in water to prevent excessive heating
at boiling point.

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Laboratory preparation of media
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
• Add heat labile supplements with aseptic precautions to
the cooled medium (45°C)

17
Sterilisation Time

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Preparation of heat sensitive media

Media (e.g. for Enterobacteria) containing:

- Brilliant green BPLS (Salmonella in meat)

- Bile salts VRBD (Enterobacteria in food)

- Iron salts XLD (Salmonella in various foods)

should NOT be autoclaved. XLD Agar is

Boil until complete dissolution only most critical

These media have the highest risk of overheating -> precipitations

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Practices for Quality Control

Monitoring performance of the autoclave

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

20
 Laboratory preparation of media

pH Adjustment

 Cool the autoclaved medium to 25°C and

check the pH with a glass-electrode
 Check pH with a temperature
compensating electrode
 pH level is +/- 0.2 at 25°C
 Adjust pH with 1 M NaOH or 1 M HCl

pH adjustment prior to autoclaving is not necessary

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Laboratory preparation of media

Storage of prepared liquid media

• Max. 3 Months in refrigerator w/o Supplements
• Max. 1 Months in refrigerator with Supplements
• Observe for colour change, evaporation, or dehydration or microbial growth

Pouring & storage of plates

• Pour after cooling to 45°C into Petri dishes
• Minimum thickness is 2 mm
• 90 mm Petri dishes 15 ml agar
• Storage in dark 4-12°C in sealed bags max 1 week
• Discard plates showing water loss, shrinkage, discolouration or
microbial growth

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Laboratory preparation of media

Drying of plates
• Oven at 25-55 °C (20 min.)
• Laminar air flow cabinet

Pre-incubation
Agar plates:
• Stacks of plates < 6
Liquid media:
• Depends on time to reach incubation temperature consider
volume (<500ml) loading (max. 6 in water bath), container
(glass), incubator (water bath)

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Guidance on preservation and maintenance of
control strains
Reference strain Obtained from a reference culture collection (WDCM)
lyophilised and kept under vacuum; or an
organism defined to at least genus and species
level, highly characterised from known origin.

Reference stock A series of aliquots of identical cultures

obtained in the lab by a single sub-culture
from the reference strain.

Working culture A primary sub-culture from an aliquot of reference stock.

Avoid to prepare reference stocks from subcultured
strains

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Reference strains from 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 and
providing contact details and the collection’s unique reference

26
27
Test organisms: WDCM numbers

on the new website of WDCM

(http://refs.wdcm.org)

it is also possible to search by

- species name
- WDCM strain number
- strain accession number
- number of standard
(ISO and other standards)

28
Test organisms, additional tips

• One person must be responsible for the strain collection.

• The storage condition and the place of storage must be defined.

• Separate area in the Lab should be reserved to handle only test

organisms

• Internal “shelf-life” of strains must be defined.

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Laboratory preparation of media

Melting of culture media

 Use volumes < 500ml
 Melt a culture medium by placing
it in a boiling water bath, but NOT
on a heat plate (risk of overheating)
 Media that have previously been autoclaved should be reheated for a
minimum time to maintain media quality.
 Avoid over-heating and remove when it has melted.
 Cool the molten medium to 47°C to 50°C in a thermostatically controlled
water bath.
 Molten medium should be used asap but it is recommended that it should not
be retained for more than 4 h.

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Laboratory preparation of media: Problems

Causes of Incomplete Solubility

• Inadequate soaking / heating
• Incomplete mixing
• Use of tap water
• Poor powder medium

Causes of Precipitates
• Repeated melting of solidified agar
• Long holding of molten agar at high temperatures

31
 Laboratory preparation of media: Problems

Causes of Improper Gel Strength / Soft Medium

• Insufficient solution of agar
• Incomplete mixing
• Incorrect amounts of dehydrated medium to volume of water
• pH drift due to acid hydrolysis
• Hydrolysis on prolonged heating or repeatedly remelting
• Effect of dilution of agar by too high inoculum

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Practices for quality control of culture media
Part 2 of ISO11133
Scope ISO 111333 part 2

This technical specification sets criteria and methods for the

performance testing of culture media

• Commercial manufacturer producing and / or distributing ready-to use

or semi finished or dehydrated culture media
• Microbiological laboratories preparing culture of their own

> 70% for non selective media

Productivity
> 50% for selective media

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Criteria for microbiological routine quality control

Growth
• quantitative -
• semi quantitative-
• qualitative method

Productivity
Selectivity
Performance evaluation and interpretation of result

35
Microbiological quality criteria

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 shall reach an defined minimum growth of 70% for

microorganisms on non-selective media

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Interpretation of Results > 70 % Recovery Rate

e.g. BROLACIN (CLED) Agar versus Non selective agar

GOLD
Standard
Used only for
internal
control

Ref.Medium NEW batch

(TSA)

Calculation : 85 / 100 = 85 % recovery rate

37
Microbiological quality criteria

Selectivity
To asses selectivity quantitatively, selective culture media and a
reference medium are inoculated with an appropriate inoculum of test
trains.

Selectivity shall reach a defined minimum growth of 50% for target

microorganisms on a selective medium

38
Interpretation of Results > 50 % Recovery Rate

e.g. CEREUS Selective Agar (MYP)

GOLD
Standard
Used only for
internal
control

Ref.Medium NEW
(TSA) batch

Calculation : 84 / 123 = 68 % recovery rate

39
ISO 11133 – Part 2
Methods for Solid Culture Media
Qualitative method – Procedure
 Agar plates are prepared in the usual manner
 Media normally used for the pour plate technique, for example Plate Count Agar
(PCA), may also be tested by surface plating on solidified media
 Use working cultures as described in the standard
 The test microorganisms are streaked in parallel straight lines
with a loop on the surface of the test medium
 Several test microorganisms can be streaked on the same plate without crossing
 The incubation times and temperatures stated in the standard methods are used

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ISO 11133 – Part 2
Methods for Solid Culture Media
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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ISO 11133 – Part 2 Methods for Solid Culture Media
VIBRIO Selective Agar (TCBS)

Merck

Vibrio parahaemolyticus Vibrio cholerae Enterobacter cloacae

42
ISO 11133 – Part 2
Methods for Solid Culture Media
Quantitative method – Procedure for Qualification

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

43
Specification of Tryptic Soy Agar Merck COA in
Compliance with ISO 11133

more than 70 % Recovery Rate for each test strain used

44
ISO 11133 – Part 2
Performance criteria (Annex B)
Enumeration Media – Selective Media

Enumeration Media – Non Selective Media

Enrichment Media – Non Selective Media

45
Culture Media in ISO 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 Enterobacteriaceae
VRBD Agar
ISO 8523
 ISO 4832 VRB Agar Coliforms
 ISO 16654 CT SMAC Agar E.coli 0157

45
ISO 11133 – Part 2
Methods for Solid Culture Media
Batch-to Batch Consistency: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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 ISO 11133 – Part 2
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.

47
Methods for use in performance testing of culture media

Quality Control of Selective Liquid Media

Target bacteria 106 to 108 c.f.u per ml

Unwanted below 104 c.f.u per ml

Extremely important to
avoid false-negative Results
48
ISO 11133 – Part 2
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 use in performance testing of culture media
Quality Control of Selective Liquid Media
RVS / TTn / Selenite Cystine broth

Quality control Minimum level must

be 105 c.f.u
Test strains Inoculum Growth after
24 hours
Escherichia coli Approx. 99 % < 10 %
ATCC 25922
Salmonella typhimurium Approx. 1 % > 90 %
ATCC 14028

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Methods for use in performance testing of culture media

Quantitative Results on CLED agar after selective enrichment step

0 hr 24 hr

Mixture of E. coli (unwanted) Lactose (+) = yellow

& Salmonella typhimurium (wanted) Lactose (-) = blue
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Summary
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 Millipore as manufacturer of culture media:

- MM 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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Quality of Culture Media:
Advantages of Granulated Media

• better flow properties

• easily weighed out without sticking to the side of
vessels or equipment
• dissolves rapidly and uniformly
• no lumps formed
• Homogenous distribution of the ingredients
guarantees reproducible composition even when
small amounts are produced.
• extended shelf life ( 5 years)
• storage at room temperature
• antibiotics included

53
TAT Broth - Powder vs. Granules

Other supplier Merck Millipore

Tween, Lecithin, Histidine are very hygroscopic

substances which cause rapid clumping of the
culture media.
54
TSB - Granules vs. Powder (1/2)

55
TSB - Granules vs. Powder (2/2)

56
Quality of Culture Media: Health Risks

Culture media are mixtures of substances of organic and inorganic origin with
different hazard levels.

Media may contain dyes and antibiotics.

Media are designed to boost or to inhibit bacteria growth.

At least culture media are nothing harmless even if they do not contain
hazardous substances .

57
Quality of Culture Media: Health Risks

Organic substances in media: Antibiotics in media:

• Peptones • Amphotericin
• Extracts • Gentamycin
• Agar • Acriflavin
• Bile salts • Cyloheximide
• Cholic Acid • Polymycin B Sulfate
• Deoxycholate • Nalidixin Acid
• Ox bile

58
Quality of Culture Media: Health Risks

Inorganic substances in media:

• Sodium Azide (very toxic)
• Tergitol (harmful, irritant)
• Triphenoletetrazoliumchloride (TTC) (highly flammable)
• Magnesium Chloride (irritant)
• Iron Chloride
• Lithium Chloride (harmful, irritant)
• Sodium Selenite (toxic)

59
Quality of Culture Media: Health Risks

Dyes in media:
• Malachite Green (harmful, irritant)
• Brilliant Green (harmful, irritant)
• Methyl Violet, Crystal Violet (harmful, irritant)
• Eosin (irritant)
• Methylen Blue (harmful)
• Fuchsin (carcinogenic)
• Pararosanillin (carcinogenic)
• Acriflavin (harmful)

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Cost Savings

In a number of granulated Merck DCM, antibiotics / inhibitors are already included

Make sure to compare OVERALL price of DCM & Supplement

Listeria Enrichment Broth

mECn+N and mTSB+N
MKTTn Broth
Selenite Cystine Broth
YGC Agar
DRBC Agar
RBC Agar
DG18 Agar
Pathogenic fungi Agar
Pseudomonas Agar (Cetrimide)
Yersinia Broth
SSDC Agar (Yersinia)
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DCM – Raw materials & Tailor made media
Not all DCM fit to every customer - sometimes a customized solution is required
Applications require the use of a modified DCM or a newly developed media
Customizations:: - Additions, deletions or substitutions of components
- pH or concentration changes in formulation of standard DCM
- Manufacture of a customer-designed medium

Merck Millipore is offering 2 solutions:

Raw materials Tailor made media

such as Agar-Agar, various designed to customers needs:
Peptones, Yeast Extract, Meat - incl. R&D work up
Extract, Gelatin, Tryptose, Ox - QC acc. customers test standards
bile, skim milk powder, etc; - Production from ~200 kg to 10 tons
mostly granulated - Confidentiality

Manufacturing of own media Optimised media by Darmstadt R&D &

using high quality raw materials manufactured by Darmstadt production site

62
If you have questions, please contact your local Merck Millipore subsidiary !!!
Thank you very much for your attention

63