Merck Webinar :

“Enhance Your Knowledge in

Microbiology Practices
acc to ISO 11133”

13 May 2020
Gintang Prayogi
Field Application
Specialist for

1
Biomonitoring - MERCK

Session :
“Quality Assurance of
Culture Media according to
ISO 11133:2014”
Topic

At this session, we’ll discussing about:

1. Media Classification and Terminology

2. Preparation and store of culture Media

3. Peformance testing of culture Media

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 Many tests and procedures depend upon culture media
being capable of providing consistent and reproducible
results. Culture media meeting established performance
criteria are therefore a pre-requisite for any reliable
microbiological work.

Sufficient testing should be carried out to demonstrate

a) the acceptability of each batch of medium,

b) that the medium is “fit for purpose”, and

c) that the medium can produce consistent results

This standard described – for the first time in story of

microbiology – the correct handling and the definition of
quality control for commercial culture media

3
 Developed by an ISO joint working group from food and
water microbiology

ISO 11133 Specify:

• The requirement for preparation of culture media

• Describes methods for performance testing of culture

media, intended for microbiological analysis of food
animal feed and water intended for consumption or used
in food production.

ISO 11133 Applies to producers:

1. commercial bodies producing and/or distributing ready-

to-use or semi-finished reconstituted or dehydrated
media;

2. non-commercial bodies supplying media to third parties;

3. microbiological laboratories preparing culture media for

their own use.

Its a mandatory standard for all ISO accredited laboratory

(ISO 17025)

4
 ISO 11133 requires:

Before use, the performance of each batch of culture

medium shall be tested, according to the media
categories

1. Ready to use: Might not need to carry out extensive

testing on media supplied with QC certificate, but shall
ensure that storage conditions are maintained.

2. Media prepared from commercialy available

dehydrated formulations: for enumeration media,
quantitative testing shall be performed; for other media,
qualitative testing may be sufficient.

3. Media prepared from basic individual components:

in addition to point 2, quantitative testing shall be
performed frequently in order to monitor trends in
quality of basic material

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• Amandment 1 containt correction and additional information
• Amandment 2 the test microorganism to be used, confirmation media, and reagent
a

02 Media Classification & Terminology

Culture Media Classified By Composition
Terminology Of Culture Media

1. Chemically Defined Medium

Culture medium consisting only of chemically defined constituents of known molecular structure and
degree of purity
2. Chemically Undefined or Partially Undefined Medium
Culture medium consisting entirely or partly of natural materials, processed or otherwise, the chemical
composition of which is not completely defined
3. Fluorogenic/Chromogenic Culture Medium
Culture medium containing one or more chromogenic/fluorogenic substrates
Culture Media Classified By Physical Consistency
Terminology Of Culture Media

1. Liquid Medium
Culture medium consisting of an aqueous solution of one or more constituents, such as peptone water
and nutrient broth. Liquid media in tubes, flasks or bottles are commonly called “broths”
2. Solid/Semi-Solid Medium
Liquid medium containing solidifying substances (e.g. agar-agar, gelatin) in different concentrations.
Solid media poured into Petri dishes are commonly called “plates”. Solid media poured into tubes or
small bottles that are kept in slanted positions while the media are solidifying are often called “slants”
or “slopes”. If the medium is dispensed to fill the bottom of the container, this forms a “butt”.

Butt

Slant/slope

Plate
Culture Media Classified According to their Use
Terminology Of Culture Media

1. Transport Medium Agar Stuart Transport Medium

2. Preservation MediumSolid/Semi-Solid Medium Agar Nutrient Agar Slope
3. Diluent/suspension medium Liquid Peptone Salt Solution
4. Resucitation Medium Liquid Buffered Peptone Water
5. Enrichment Medium Liquid Tryptone Soya Broth

6. Selective Enrichment Medium Liquid RVS Broth

7. Isolation Medium Agar mCCD agar

8. Selective Isolation Medium Agar Nutrient Agar

9. Chromogenic/Fluorogenic Selective Culture Medium Agar TBX Agar

10.Differential/Characterization Medium Agar Lactose Agar w/ Tergitol & TTC

Agar Baird Parker Agar
11.Enumeration Medium
Agar Kliger Iron Agar
12.Confirmation Medium
Agar TSA w/ LTHthio
13.Neutralizing Medium
Culture Media Classified According to their Use
Terminology Of Test microorganism
1. Test Organism
2. Reference strain
3. Reference stock
4. Stock culture
5. Working culture
6. Reference Material
7. Certified Reference Material

Batch/lot culture medium

Homogenous and fully traceable unit of a medium reffering to a defined amount of bulk,
semi finished product or end product, which is consistent in type and quality and which has been
produced within one defined production period, having been assigned the same batch
(or lot) number
02 Laboratory Preparation
of Culture Media
Quality Assurance Management: Documentation
Laboratory Preparation of Culture Media

Documentation from manufacturer or producer

The following information shall be available from manufacturer or producer
A. Name of the medium, individual components, batch code
B. Technical data sheet
C. Safety data sheet
D. Storage information and expiry date
E. Quality control certificate showing test organism used and result of
performance testing

Delivery acceptance of products

Upon receipt of media, laboratory shall pay attention on the following

A. Identity of product

B. Integrity of packaging

C. Expiry date of the product and other supplied documentation

D. Record the date of receipt

Quality Assurance Management: General Practice
Laboratory Preparation of Culture Media

The preparation of culture media is one of the fundamental steps

in microbiological examination and it shall given special care

I. Respect good laboratory practice (GLP)

II. Follow the manufacturers instructions regarding handling of

the media and other component, particularly those containing
hazardous materials i.e bile salts, sodium azide, antibiotics,
etc

III. Document all relevant data

IV. For media prepared from individual components follow the

recipe precisely and record all detail

When a new container is opened:

A. Check the seal,

B. Record date of first opening

C. Visually assess the content of opened containers.

D. Indicate a maximum storage time

Quality Assurance Management: Water Quality
Laboratory Preparation of Culture Media

1. Use purified water

i.e. Distilled, demineralised, deionised or water produced by reverse
osmosis or equivalent quality free from substances likely to inhibit or
influence the growth of microorganism
2. Conductivity
The used water shall be ≤ 25 µS, preferably below 5 µS, unless otherwise
required by design
3. Microbial Contamination
Should not exceed 10³ CFU/mL, preferably below 10² CFU/mL, and should
be monitored regularly
4. Container
The purified water shall be stored in tightly closed containers made from
an inert material (neutral glass, polyethylene, etc.) which shall be free
from all inhibitory substances.
It is however recommended that the water is used as soon as
produced.
Quality Assurance Management: Weighing, Rehydration and Dissolution
Laboratory Preparation of Culture Media

1. Weigh accurately the desired quantity

2. Use balance of sufficient discrimination, the maximum permissible errors
should be 1% or greater as given in ISO 7218 and ISO 8199
3. Take care not to inhale powder especially with media containing toxic
substances
4. Rehydrate with correct volumes of water and progressively mixed to prevent
formation of lumps*
5. Dehydrated media need rapid dispersion by instant and repeated or
continuous stirring followed by heating, if necessary, to dissolve*
6. Media containing agar should be allowed to soak for several minutes before
heating with mixing to dissolve and then dispensing if necessary before
autoclaving.
Quality Assurance Management: pH adjustment and Dispensing
Laboratory Preparation of Culture Media

1. Measure the pH using a pH meter and adjust before sterilization

if necessary, so that after sterilizing and cooling to 25 °C the medium
is at the required pH ± 0,2 pH units, unless otherwise stated.
2. The adjustment is normally carried out using a sodium hydroxide
solution of approximately 40 g/l [c (NaOH) = 1 mol/l] or dilute
hydrochloric acid of approximately 36,5 g/l [c (HCl) about 1 mol/l].
3. If adjustment is performed after sterilization, use a sterilized solution.
4. ISO11133 notes that pH adjustments before autoclaving are
usually not necessary, if provided good quality distilled or
deionized water is used
5. Dispense the medium into appropriate containers ensuring that Flat surface electrode
sufficient headspace is left to avoid boiling over during the cooling
process after heat treatment by autoclaving or remelting, or overflowing
after addition of supplements.
6. Additional information on pH measurement is given in ISO 7218 and
ISO 8199
Quality Assurance Management: Sterilization
Laboratory Preparation of Culture Media

Sterilisation
I. Dehydrated media are not free from contaminant
II. Be sure that no residual media covers the wall of container
III. Autoclave only 15 min at 121°C
IV. Avoid excessive autoclaving to prevent maillard reaction: degradation
and breakdown of nutrient constituents
V. Sterilize heat labile substrates by filtration (pore diameter 0,2 µM,
low protein-binding membrane)
VI. Add heat labile supplements with aseptic precaution to the cooled
medium (45°C)
VII. The performance of autoclave should be monitored (physical indicator
and biological indicator)

Sterikon® plus bioindicator for

checking steam sterilization
efficiency acc. to USP and Ph. EUR
Quality Assurance Management: Sterilization
Laboratory Preparation of Culture Media

Preparation of heat sensitive media

Media (e.g. For Enterobacteria) containing
- Briliant green BPLS (Salmonella in meat)
- Bile Salts VRBD (Enterobacteria in food)
- Iron Salts XLD (Salmonella in various food)

Those media should not be autoclaved, heat in boiling water until completely dissolve

i.e.
XLD agar is
most heat
critical
Quality Assurance Management: Storage of Prepared Media
Laboratory Preparation of Culture Media

1. Media should be protected from direct light and dessication

2. If not used immediately, keep media refrigated during recommended
time, unless otherwise specified in the specific standard or the results
of shelf life evaluation indicate a longer shelf-life.

Plates :2-4 weeks at 2-8°C

Sealed bottles and tubes :3-6 monts at 2-8°C

3. Perform Shelf life evaluation of stored media, Observe for colour

change, evaporation, or dehydration or microbial growth
Quality Assurance Management: Preparation for Use
Laboratory Preparation of Culture Media

1. Melt culture media by placing in a boiling water bath or by any other process
which gives identical results (e.g. a steam flow-through autoclave, as specified
in ISO 7218, ISO 8199).
2. Media that have previously been autoclaved should be reheated for a
minimum time to maintain media quality. Avoid over-heating and remove
when they have melted.
3. Molten medium should be used as soon as possible, but it is recommended that
it should not be retained (on 45-50°C) for more than 4 H. Unused medium
shall not be re-solidified and reused.
4. Heat-labile supplements should be added to the medium after it has been
cooled to below 50 °C. If the medium contains agar allow the sterile
supplement to equilibrate to at least room temperature before adding it
to the agar medium. Addition of cold liquid supplements may cause agar to
gel or form transparent flakes and prevent proper dispersion
 Quality Assurance Management: Preparation for Use
Laboratory Preparation of Culture Media

1. Pour the molten agar culture medium into Petri dishes so as to obtain a
thickness of at least 3 mm (e.g. for 90 mm diameter dishes, 18 ml to 20
ml of agar are normally required) or as specified in the appropriate
International Standard.
2. If plates are stored or if incubation is extended beyond 72 h, or incubation
temperature is above 40 °C, more culture medium may be required.
3. For surface inoculation of solid culture media, dry the plates shortly
before use until the droplets have disappeared from the surface of the
medium. Do not over-dry the plates.
4. In practice, the plates can be dried by placing them with the agar surfaces
downwards and with half open lids in a cabinet set at a temperature
of between 25 °C and 50 °C. Dry the plates until the droplets have
disappeared from the surface of the lids. Do not dry any further.
5. The agar plates can also be dried with the agar surface facing upwards in a
laminar-flow safety cabinet (at room temperature) for 30 min to 60
min, or overnight at room temperature with the lids in place.
Quality Assurance Management: Incubation
Laboratory Preparation of Culture Media

1. During incubation, agar media will lose moisture. This can affect the
growth of microorganisms in some circumstances
2. Factors influencing water loss are medium composition, amount of
medium in the plates, the type of incubator i.e. fan-assisted or
otherwise, humidity of the atmosphere in the incubator, the position
and number of the plates in the incubator and the incubation
temperature.
3. Water loss can be reduced by putting the plates, in piles of up to six,
in open-topped plastic bags (to avoid excessive condensation).
4. Alternatively, the humidity of air in incubators may be increased by
placing an open container of water in the bottom

Cultura mini Incubator

03 Performance Testing of
Culture Media
Test Organism For Performance Testing
Performance Testing of Culture Media

1. A set of test organisms should contain microorganisms with stable characteristics

representative of their species and which have been shown to be reliable for the
demonstration of optimal performance of a particular prepared medium.
2. The test microorganisms for each medium may include:
 robust positive strains with typical characteristics of the target organism;
 weakly positive strains;
 negative strains not showing expected characteristics of the target organism
(negative characteristics);
 partly or completely inhibited strains.
3. Refer to Annex E, Annex F, and Annex K of ISO 11133 for specified test
microorganism that can be used in certain application

26
 Test Organism For Performance Testing
Performance Testing of Culture Media

WDCM Numbers
Are given as universal strain identifier by the World data centre
for microogranisms (WDCM)
WDCM is part of WFCC (World Federation of Culture Collections)
they have initiated a system that will help users find local sources
of reference strains by citing all collections and providing contact
dtails and the collection’s unique reference
Information of the refernce strains can be accesed through
website of WDCM (http://refs.wdcm.org)

28
Preservation and Maintenance of Test Organism
Performance Testing of Culture Media

• Obtained from a Reference • A primary sub-

reference culture
collection, defined
Stock culture from an
aliquot of
to at least genus reference stock.
and species level, • A series of aliquots
of identical cultures Avoid to prepare
highly characterized reference stocks
from known origin obtained in the lab
by a single sub from subculture
Reference culture from strains
refernce strain
Strain Working
Culture

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 Preparation of Innocula and Innoculum level
Performance Testing of Culture Media
1. Prepare serial dilution in suitable diluent, and use most suitable dilution step for the desired number of
organism (CFU) in a specified volume
2. The suitable dilution to use should be determined from previous tests conducted under strictly
standardized condition for all steps
3. Use the innocula within a specified time (up to 2 hrs at room temp or within 24 hrs if stored 2-8°C)

Innoculum level

1. Productivity Testing
 Quantitative testing: 100 CFU Per plate (80-120 CFU, with minimum 50 cfu per plate)
 Qualitative testing: 10^3 – 10^4 CFU per plate
2. Selectivity Testing: 10^4 – 10^6 CFU per plate
3. Specificity Testing: 10^3 – 10^4 CFU per plate
 Quality Control
Performance Testing of Culture Media
1. Physical and Chemical Quality Control
 Fill volume and/or thickness
 Appearance, colour and homogeneity
 Gel consistency
 Moisture content

2. Microbiological Quality Control

 Microbial contamination
an appropriate quantity, depending on the size of the batch, shall be tested for absence
of microbial contamination by incubation under appropriate conditions

At least 1 plate or tube for small batches (<100 units)

 Methods for Performance Testing of Culture Media
1. Methods for solid culture media
 Quantitative test
 Media used for membrane filtration test
 Qualitative test
2. Methods for liquid culture media
 Quantitative test
 Qualitative test
3. Methods for diluents and transport media

32 Title of Presentation | DD.MM.YYYY

 Method for Quantitative Test
Performance Testing of Culture Media

A. Productivity B. Selectivity
Level of recovery of a target microorganism from the culture Degree of inhibition of a non-target microorganism on a
medium under defined condition selective culture medium under defined conditions.
Productivity ratio (Pr) shall reach a defined minimum limit; the Selectivity factor (Sf) is calculated as given by formula
productivity ratio is determined by
𝑆𝑓 =𝐷0 − 𝐷𝑠
𝑁𝑠
𝑃𝑟 = 𝐷0 : highest dilution showing growth on non selective
𝑁𝑜
reference medium
Ns: total count of colonies obtained on or in the culture
medium under test 𝐷𝑠 : highest dilution showing comparable growth on the
selective test medium
No: total count of colonies obtained on or in the defined
reference culture medium; obtained from one or more All expressed in 𝐿𝑜𝑔10 units
plates, and shall around 100 CFU
Productivity ratio shall reach a defined minimum growth of 0,7 The 𝑆𝑓 of non-target microorganism is at least 2
for microorganism on non-selective media, and minimum growth
of 0,5 for target microorganism on selective media

33
Method for Quantitative – Solid Media
Performance Testing of Culture Media

1. Interpretation of result > 70% recovery rate

2. e.g. BROLACIN (CLED) Agar – Non selective media

34
Method for Quantitative – Solid Media
Performance Testing of Culture Media

Interpretation of result > 50% recovery rate

e.g. CEREUS (MYP) Agar – Selective media

35
Method for Qualitative – Solid Media
Performance Testing of Culture Media

1. The growth on the plates after incubation were

assesed as
• “0” correspond to zero growth
• “1” correspond to weak growth
• “2” correspond to good growth

1. Target microorganism shall score 2 and have typical

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

36
Method for Qualitative – Solid Media
Performance Testing of Culture Media

Comparison of Qualitative test VIBRIO selective agar (TCBS) between

merck and competitor

37
Method for Quantitative – Liquid Media
Performance Testing of Culture Media

Quality Control of Selective media, RVS

Quantitative result on CLED agar (non-selective)

38
Method for Qualitative – Liquid Media
Performance Testing of Culture Media

Qualitative evaluation shall be carried out visually by allocating

growth scores e.g. from 0 to 2

“0” Refer to zero turbidity

“1” Refer to very light turbidity
“2” Refer to good turbidity

The score of a target microorganism shall be 2

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

39
Conclusions

1. ISO 11133 describe how to prepare, storage, perform quality control of culture media, used in food,
animal feed and water for consumption and production, as the first internationally accepted standard
2. ISO 11133 provide guideline to achieve highest quality and accuracy of culture media used in everyday
analysis, resulting the high quality and reliable analysis output
3. ISO 11133 is mandatory for ISO17025 certified Laboratories
Merck Expertise in Microbiology

A Pioneer in Microbiology
First commercial product of microbiology in 1892

Quality
Leading expertise in microbiology over 120 years with comprehensive
QC/QA since 1885
Pioneer of Dehydrated Culture Media (DCM) product acc to ISO 11133

Innovation
Pioneer of granulated media (1950)
Dry granulation since 1970
Pioneer of chromogenic media

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 Thank you For Your Attention!

If you need further assistance, kindly contact us at

Field Product Specialist for Biomonitoring:
[email protected]
Customer Service:
[email protected]
Visit our website:
Merckmillipore.com and Sigmaaldrich.com

42
 Merck Webinar :

“Enhance Your Knowledge in

Microbiology Practices

Q&A
Gintang Prayogi acc to ISO 11133”
Field Application
Specialist for
Biomonitoring - MERCK

Session : 1
“Quality Assurance of
Culture Media according to
ISO 11133:2014”

[email protected]