CLF Microbiology Method Collection Web Version

METHOD GUIDE MICROBIOLOGY
Microbiological Reference Methods Collection 2014

Infant Nutrition Products and Related Raw Materials
A supporting document for Danone Nutricia Early Life Nutrition Global Safe Food Standards 014
Contents
 Introduction 1
 CLF Reference Methods
 M01_01ME Sample preparation 3

 M01_02ME TVC 30°C and 55°C 15
 M01_03ME Bacillus cereus enumeration 25
 M01_04ME Staphylococcus aureus detection 37
 M01_05ME Staphylococcus aureus enumeration 49
 M01_06ME Yeast and molds 59
 M01_07ME Enterobacteriaceae detection and MPN enumeration 69
 M01_10ME Clostridium perfringens detection 79
 M01_12ME Enterococci detection 95
 M01_13ME Enterococci enumeration 107
 M01_14ME Salmonella conventional method 119
 M01_15ME Salmonella Diasalm 133
 M01_16ME Salmonella Barrel 147
 M01_18ME Lactobacilli enumeration 157
 M01_21ME Sterility testing of acid and neutral sterilized products 165
 M01_22ME Listeria monocytogenes detection 173
 M01_25ME Enumeration of contaminating microorganisms in acidic 185
pasteurized products
 M01_26ME ISO based horizontal method of sulphite-reducing clostridia 193
 M01_30ME Cronobacter detection and MPN enumeration 203
 M01_32ME Pseudomonas detection 215
 M01_37ME Bifidobacteria enumeration 223
 M01_55ME Escherichia coli detection and MPN enumeration 233
 Annex: Comparison of CLF Reference Methods and ISO Standards 243

Introduction
Dear colleagues,
CLF Microbiology methods have already been in use for more than a decade now in several baby food
factories and have been used as the defined reference in discussions with raw material suppliers and
co-manufacturers.
The CLF Microbiology Method Collection is directly related to the Danone Nutricia Early Life Nutrition
Global Safe Food Standards. It provides explanations and background information to the method
references listed on page 24 of the 2014 version of the Danone document.
The CLF Microbiology Method Collection has been constantly updated according to changes in the
regulatory and scientific area. Most of the methods are based on relevant ISO norm standards.
However the ISO standards often leave options in the analyses regarding essential parts of the
analytical method like incubation temperature or the agars to be used. Moreover most of the ISO
methods are horizontal methods which indicate that they are applicable to all kinds of food in a full
range from human food to animal feed. This range has been indicated in the updated versions of the
CLF methods by extending the analytical scope to pet food. This is simply the reflection of the ISO
method and of no relevance for the analytical scope within Danone. In these cases the CLF
Microbiology Method Collection provides the necessary explanation and clarification. The option which
has been chosen in the CLF version is the one which has been found most suitable for infant formula
and related raw materials. In cases where no suitable ISO norm is available, the CLF Microbiology
Method Collection provides methods based on other international standards or scientific references.
At the end of each method, you will find “amendment and comments compared to ISO norm” in which
the most current updates are included, specific notes are highlighted, the comparison between CLF
method and ISO norm is made and the differences are illustrated in a flow diagram.
Validation and re-validation trials have been performed for each method in a broad spectrum of
bacteria as well as a wide range of products to ensure the reliability and robustness of the test
methods for infant nutrition products and related raw materials.
This booklet is the 2014 version of the CLF Microbiology Method Collection with the scope focused on
methods used for the quality control of baby food products and their raw materials. The document will
be updated on annual basis to keep the included methods up to date with changes in official methods
and with the scientific development in the field of analytical microbiology.
We are very interested in your feedback on the methods, because only with the experiences shared
with us from the operating laboratories is it possible to provide a method collection which is tailor made
to the requirements of infant nutrition release tests.
Microbiology and Biochemistry Team, Eurofins CLF
June 2014
1
2
SAMPLE PREPARATION – RESUSCITATION AND DILUTION
3
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No. M01_01ME_v04(Sample preparation).doc
Sample preparation
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Sample preparation –Resuscitation and Dilution
Reasons for amendment:

V3 (14.09.10)  Modifications with deviates from standard methods were added to 2
V4 (05.02.14)  Extension of the scope to pet food
 Liquid samples do not need to be resuscitated
 Added item 6.3 “Processing of butter/oil samples”
Name Signature Date

1st version written by: Marjan
2000
Bredius
revised by: Björn
2014
Hampel
verified (professional): Sha
2014
Zhu
verified (norm conformity): Nadine
2014
Wollnitz
approved: Matthias
2014
Fischer
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1 Scope and field of application

The method for the sample preparation can be applied to milk, milk powder, milk products,
cereals, thickeners and pet food. Additionally there is a description of how to dilute
samples of food products and water for microbiological analyses.
2 Principle of the method

Preferably relatively rich media are used for resuscitation such as trypton soy broth (TSB)
and buffered peptone water (BPW).
The samples which are to be analysed for the presence of vegetative cells, are
resuscitated.
Microorganisms can be sublethally damaged by heat, dryness or other processing or
storage conditions. These cells are viable and able to recover completely upon
reconstitution. Therefore they are as important as normal cells. However, as they are
damaged, they can often not survive or grow in the selective media. Therefore it is
necessary to suspend the sample into a non-selective medium and to give the sublethally
damaged cells time to repair. This process is called resuscitation. After resuscitation the
repaired cells will be able to grow on/in the selective media. In this way, an
underestimation of the number of microorganisms present in the sample is prevented.
Different dilution fluids can be used. For selective and elective media it is important that
the dilution fluid does not influence the selectivity or electivity.
Modifications
This method is based on EN ISO 6887-1, 1999.
The following modifications have been performed:
 Utilization of additional media (TSB and Ringer’s solution); resuscitation time for 2 h
NOTES:
Liquid samples do not need to be resuscitated.
Other media which can also be used for resuscitation are peptone salt solution (PSS) and Ringer’s solution
(RS).
During resuscitation, also growth of non damaged cells can occur. The resuscitation time is therefore always a
compromise between the resuscitation time necessary for sublethally damaged cells and the time in which
cells will start to grow.
In the past, BPS (buffered peptone salt solution) was used as well. Now PSS has replaced BPS. However, in
some methods the use of BPS is still mentioned.
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3 Definitions and abbreviations

Resuscitation Recovery of sublethally damaged cells in a non-selective medium.
Initial suspension Sample weighed in the resuscitation medium
Decimal dilution Dilution from the initial suspension or a further dilution, where 1 ml
start suspension or dilution is mixed with 9 ml dilution medium.
4 Reagents, materials, media

Medium/Reagent Abbreviation Advised maximum storage time1
Buffered peptone water2 BPW 1 month 4-10°C
Tryptone soy broth2 TSB 1 month 4-10°C
Peptone salt solution2,4 PSS 1 month 4-10°C
Ringer’s solution2 RS 1 month 4-10°C
NaOH 5% (w/v)3 NaOH 1 month 4-10°C
Amylase 1-5% (w/v) in water 1 month 4-10°C
Pectinase 1-5% (w/v) in water 1 month 4-10°C
Cellulase 1-5% (w/v) in water 1 month 4-10°C
Gelatinase 1-5% (w/v) in water 1 month 4-10°C

1
The shelf life of the prepared media/solutions is given when stored in the dark. Media with a shelf life longer
than 1 month have to be stored in well closed bottles (with screw cap).
2
One or more of these broths has to be chosen.
3
w/v means weight on volume e.g. g/L
4
PSS is also called “Maximal Recovery medium”
 Fill volume of resuscitation and dilution media

The resuscitation medium has to be filled in such a way that the volume after sterilization
is known. The maximum allowed uncertainty is ± 5%.
The dilution medium has to be filled in such a way that the volume after sterilization is
known: normally 9.0 ml. The maximum allowed uncertainty is ± 5%.
 Enzyme solutions
Filter and sterilize the enzyme solutions using a 0.2 m hollow fibre filter or use
commercially available sterile vials and dissolve in sterile water.
Use only media of good quality proven by supplier’s quality certificate.
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Follow the manufacturer’s description on:

1. Storage of the powdered media or reagent.
2. Preparation of the media.
3. Storage of the prepared media.
5 Equipment
Usual microbiological equipment
6 Procedure
A general procedure for resuscitation is described in 6.1 and for thickeners in 6.2.
6.1 General resuscitation procedure

1. Pre-warm the resuscitation broth to 18-25°C.
2. When necessary add 1 ml of the required sterile enzyme solution (described in 6.2).
3. Open the sample packaging in such a manner that the risk of contamination is
avoided: disinfect the packaging where it will be opened with 70% alcohol and use
disinfected scissors or other opening-equipment. Work in the vicinity of a flame.
4. Weigh an amount of sample (± 5%) into the resuscitation medium by shaking or using
a sterile spoon so that a 10% suspension (w/v) is obtained.
5. Shake the sample vigorously or mix using a stomacher. The sample is mixed till the
suspension is clump free.
For the analyses of spores, the sample is immediately analysed after mixing without
resuscitation as described in the respective method.
6. The resuscitation time between weighting the sample and start of the analyses is 2 h
at 18-25°C.
7. After resuscitation, continue as described in the method for the requested analysis.
NOTE:
It is allowed to warm the resuscitation medium shortly to 40°C (maximum 15 min, 40°C) to obtain a
homogenous suspension (for example for chocolate). After warming the sample has to be cooled to room
temperature quickly.
Masses shall be weighted with an uncertainty of max. 5%, volumes with an uncertainty of max. 5%.
Because no resuscitation is performed for the spore analyses, the sample can also be weight into a stomacher
bag, after which the necessary amount of medium is added to the sample.
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6.2 Resuscitation procedure for thickeners

1. Pre-warm the resuscitation broth to 18-25°C.
2. When necessary, add the given amount of the required sterile enzyme solution (see
table below).
3. Open the sample packaging in such a manner that the risk of contamination is
avoided: disinfect the packaging where it will be opened with 70% alcohol and use
disinfected scissors or other opening-equipment. Work in the vicinity of a flame.
4. Weigh an amount of sample (± 5%) into the resuscitation medium by shaking or using
a sterile spoon so that a suspension is obtained which can be handled by pipetting.
5. By the addition of a suitable enzyme, a 10% suspension can be obtained which is
liquid enough to be pipetted.
6. When enzymatic liquefaction is not sufficient or not possible, prepare a 5% or 1%
sample suspension.
7. For a spore analysis a 1% suspension has to be made when a 10% suspension is too
thick. Because no resuscitation is performed for the spore analyses, there is no
enough time for enzymatic liquefaction.
8. When no 10% suspension is made, this is noted on the working list or with the results.
9. Weigh an amount of sample into the TSB or PSS using a sterile spoon so that a 1%
suspension (w/v or v/v) is obtained. Sometimes even a further diluted suspension is
needed. For the analyses, no clumps should be present in the resuscitated sample.
10. Continue from 6.1.5.
Table 1: Directions for the use of enzyme solutions
Enzyme Amount Use for
amylase 1-5% w/v, 1 ml to 100 ml suspension starch (e.g. for cereals)
cellulase 1-5% w/v, 1 ml to 100 ml suspension cellulose (e.g. for guar gum)
gelatinase 1-5% w/v, 1 ml to 100 ml suspension gelatin
pectinase 1-5% w/v, 1 ml to 100 ml suspension pectin (e.g. for locus bean gum)
NOTES:
The weighed amount of sample may be maximum 5% above or below the set amount.
The growth of bacteria may be inhibited when the sample is thick, even when clumps are present. In those
cases even further dilution and/or a higher concentrated enzyme solution is necessary.
After resuscitation and/or before pasteurization the sample has to be clump free and liquid. This is achieved
by the combination of enzymes, the suspension concentration (10%, 1%, etc) and the time during which the
enzymes can break down the substrate. The more concentrated the sample, and the lower the enzyme
concentration, the longer it will take to liquefy the sample. Additionally enzymes are inactivated through
pasteurization and can therefore be less efficient or do not work at all in spore analyses.
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6.3 Processing of butter/oil samples

When the sample contains more than 20% fat, 1-10 g/l Tween 80 has to be added to the
resuscitation medium to emulsify the sample. For oil and butter samples, an addition of
Tween 80 of 5 ml in 90 ml BPW (50 g/l) in resuscitation medium is recommended. Tween
80 should be autoclaved before use.
6.4 Decimal dilution

1. Take 1 ml sample or initial suspension and add it to 9 ml dilution fluid (e.g. PSS or
RS).
2. Mix thoroughly using a vortex or a comparable shaking device.
3. From the dilution, further decimal dilution can be made in the same way.
NOTE:
For reference capsules and dry samples, the sample suspension (10% suspension) itself is regarded as the
first decimal dilution.
7 Expression of results, calculation

Not applicable.
8 Validation
See validation report M01_01VB.
9 References
The above mentioned method is based on the following references, but does not
necessarily reflect the exact methodologies.
1. Dijk, R, Grootenhuis A “Microbiologie van voedingsmiddelen - methoden, principes en
criteria” Keesing Noordervliet, Houten, 1999, p.33-34.
2. Everis L. “Significance of injured microorganisms in food” 1999, Review No 19,
Project No 42224, Campden and Chorleywood Food Research Association, UK.
3. Foegeding P.M., Bibek R. Chapter 7, “Repair and detection of injured micro
organisms” in: “Compendium of methods for the microbiological examination of foods”
C. Vanderzandt, D.F. Splittstoesser (eds), 1992, American Public Health Association,
Washington, p.239-249.
4. Mossel, D.A.A., Corry J.E.L., Struijk C.B., Baird, R.M. Chapter 9 “Recommended
monitoring procedures” in “Essentials of the microbiology of foods – A textbook for
advanced studies” John Wiley & Sons, Chichester etc, 1995.
5. International Organisation for Standardisation, “EN ISO 6877-1, Microbiology of food
and animal feeding stuffs – Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination”. 1999.
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6. International Organisation for Standardisation, “ISO/CD 6877-5, Microbiology of food

and animal feeding stuffs – Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination- Part 5: Specific rules for the
preparation of the initial suspension and decimal dilutions of products other than milk
and milk products, meat and meat products, fish products” 2000. ISO TC 34/SC 9,
2000-07-10. Nederlands Normalisatie Instituut Document Nr 2000-48.
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10 Attachments
Attachment 1: Composition of resuscitation and dilution media

Resuscitation/dilution media Ingredients Weight
PSS Enzymatic digest casein 1.0 g
NaCl 8.5 g
Water 1000 ml
RS NaCl 2.25 g
KCl 0.105 g
CaCl • 6H2O 0.12
Na2CO3 0.05
Water 1000 ml
BPW Enzymatic digest animal tissue 10.0 g
NaCl 5.0 g
Na2HPO4 • 12H2O 9.0 g
KH2PO4 1.5 g
Water 1000 ml
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Attachment 2: Resuscitation procedure for acid products and trace elements

The analysis of acid products and trace elements is out of the application of M01_01ME.
To analyze these products, M01_01ME with the following modifications are used.
The preparation of acid products such as lactat-casein, lab-casein and caseinat is
described in DIN EN ISO 6887-5:2011-01.
 Acid products
1. Weigh an amount of sample in resuscitation broth as described in M01_01ME 6.1/1-

4.
2. Mix the sample and measure the pH value.
3. Add sterile NaOH (5% w/v) to the sample until the pH value reaches 7. Note how
much NaOH has been added.
4. Add to a new resuscitation broth the noted amount of sterile NaOH (5% w/v).
5. Weigh an amount of sample in the resuscitation broth with NaOH as described in

M01_01ME.
NOTE:
For the analysis of acidophilic bacteria in acid products (like yoghurt or fruit-concentrate on Lactobacilli, see
M01_18ME or M01_02ME) a neutralisation of the resuscitation broth is not necessary.
 Trace elements
1. Proceed as described in method M01_01ME 6.1.1 till 6.1.3.
2. Weigh an amount of sample to get a 5% suspension (w/v or v/v) or a 1% suspension

(w/v or v/v).
3. Continue with point 6.1.5.
NOTE:
High trace-element-concentration is toxic for microorganisms. This effect works directly, a later suspension
from 10% to 5% or 1% is not satisfactory because the cells are already marred.
13
AMENDMENTS AND COMMENTS COMPARED TO ISO NORM
No resuscitation of liquid samples

Samples in liquid form do not need to be resuscitated. They can be directly poured or plated for
analysis after dissolved in non-selective broth.
Handling of butter/oil samples

The preparation of butter/oil samples is specified in DIN EN ISO 6887-5:2011-01 “Milk and milk
products. General guidance for the preparation of test samples, initial suspensions and decimal
dilutions for microbiological examination.”
Tween 80 must be added to BPW to enable a homogeny emulsion. The process of handling and
preparation of butter/oil samples is described in details in M01_02.
14
TOTAL VIABLE COUNT 30°C and 55°C
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No. M01_02ME_v05(TVC 30°C+55°C).doc
Total viable count (30°C and 55°C)
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V3 (14.09.10)  Description of the modifications is added
 New edition of norm method EN ISO 4833: 2003-02 has been added
V4 (07.05.12)  Type in the reference method corrected

 TTC as an option for TVC 30°C enumeration added
 Method extended for anaerobic total viable count: Schaedler agar
for the anaerobic TVC enumeration added with the reference strains
mentioned
V5 (28.01.14)  Preparation and enumeration process of butter as well as references
related have been added to 6.1/1.2, 6.1/2.2, 6.1/3.2 and 9
 Tween 80 has been added to 4 and 6.1/1.2 (reagents, material and
media)
 OSA agar has been deleted
 Application conditions of different pour plating agars have been
clarified (6.1/3.1)
 Influence of PCA agar brand on analytical results in non-butter
products (OXOID, CM 325) and recommendation for agar medium
for butter products (MERCK, 1.10878.0500) are specified in
6/6.1/3.3 and 3.4
Name Signature Date

2000
Bredius
revised by: Sha
2014
Zhu
verified (professional): Björn
2014
Hampel
2014
Wollnitz
approved: Matthias
2014
Fischer
17
of: 6 written: 6
05.02.2014
printed: 23.06.2014

The method for the total aerobic count can be applied to products intended for human
consumption and for animal foodstuff and environmental samples in the area of food and
feed production and handling.

This method is based on ISO 4833-1:2013.
A non-selective nutrient medium is used to culture a wide range of aerobic or anaerobic
microorganisms, resulting in visible colonies which can be enumerated.
For the enumeration of the aerobic total viable count at 30°C there is the option of adding
TTC to the Plate Count Agar (PCA). Through the bacterial dehydrogenase activity the
TTC will be reduced from a water soluble colorless substance to an insoluble red dye. The
red colorization facilitated the visibility of colonies in difficult matrices.
TTC may also have a toxic effect. Therefore the final concentration of 40 µg/ml PCA must
not be exceeded nor is the addition allowed for the enumeration of the aerobic TVC at
55°C.
Tetrazolium: colorless, soluble 2 reduction steps Formazan: red, insoluble in water
Figure 1: Chemical structure of TTC and Formazan

CFU colony forming units
TTC 2,3,5-triphenyl-tetrazolium chloride
TVC total viable count
ATCC American Type Culture Collection
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Plate count agar3 PCA 3 months 4-10°C
Milk plate count agar MPCA 3 months 4-10°C
Tween 80 (8.22187.2500, Merck)2 See manufacturer’s description

1% sterile filtered solution in water TTC 5 days 4°C
1
The shelf life of the prepared media/solutions is given when stored in the dark. Media with a shelf life longer
than 1 month have to be stored in well closed bottles (with screw cap).
2
Tween 80 is autoclaved at 121°C for 15 min before use.
3
Recommendations of PCA suppliers for TVC in butter and non-butter products are specified in 6/6.1/3.3 and
3.4.
Use only media of good quality, proven by supplier’s quality certificate.

1. Storage of the (powdered) media and reagents
2. Preparation of the media
3. Storage of the prepared media
5 Equipment
6 Procedure
6.1 Enumeration
1. Sample reconstitution
1.1 Prepare a resuscitated sample, a suitable dilution series dependent on the presumed
contamination level of the sample (see method M01_01ME). The number of colonies
on a plate should not exceed 300 colonies.
1.2 Preparation of butter sample:
Weigh 10 g butter into a container and place the container in a water-bath at 45°C.
Keep it in the water-bath until butter has just melted.
90 ml BPW is pre-warmed at 45°C and supplemented with 5 ml Tween 80. Add the
pre-warmed diluent to the butter test portion. Shake well until a stable emulsion is
obtained.
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2. Sample pipetting
2.1 Pipet 1 ml of the appropriate dilution into a Petri dish.
2.2 Pipetting of butter sample:
Butter emulsion is distributed evenly on the surface of dried, finished PCA agar
plates.
3. Pouring plate/Spreading method

3.1 Pouring plate method:
Pour approximately 15 ml of the suitable agar (tempered at 45 ± 2°C) into the Petri
dish, mix and allow solidification.
 PCA for all products
 MPCA for dairy products in case the dilution step plated is ≥ 1:1000
 Add TTC only if a differentiation of colonies from particular material of the sample
is difficult
 Schaedler agar for all products for the analyses of anaerobic TVC.
3.2 Spreading method for butter sample:

Carefully spread the inoculum as quickly as possible over the surface of the plate
using a sterile spatula.
3.3 The composition of the PCA agar medium is described in ISO 4833-1:2013 as
followed:
Enzymatic digestion of casein 5.0 g
Yeast extract 2.0 g
Glucose 1.0 g
1
Agar-agar 9 to 18 g
Water 1000 ml
1
Depending on the gel strength of the agar.
 Influence of the PCA agar brand on analytical results:

During the validation of this method (see M01_02VB2_v02), it has been observed that
the medium brand influenced the results of the total plate count tests. PCA brands
with agar concentration towards the lower limit of the required range (e.g. OXOID, CM
325; 9 g agar-agar/l medium) provide constantly slightly higher counts and less
deviation of analytical results. On the other hand, PCA of most suppliers contains 14
g agar-agar/l medium.
4. Incubate the plates upside down for 72 ± 3 h at the appropriate temperature:

30 ± 1°C aerobic mesophilic count
55 ± 1°C aerobic thermophilic count
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NOTE:
To avoid drying out of the plates they should be packed in a well-closed plastic bag or box. Add some tissue
paper to the plastic bag to absorb condensate water which helps to prevent spreading of the colonies.
5. Count all colonies. Spreading colonies are counted as single colonies. If less than
one quarter is overgrown by spreading colonies, count the colonies on the unaffected
part of the plate and calculate the corresponding number for the entire plate. If more
than one-quarter of the surface is overgrown by spreading colonies, re-analyze the
sample by using a surface layer.
NOTE:
When spreading colonies are expected, a thin surface layer of about 4 ml of the same agar can be poured to
prevent spreading after complete solidification. Allow to set.
6.2 Confirmation
Not necessary
6.3 Internal control

Positive controls: Reference materials containing aerobic microorganisms or
reference strains, e.g.
Enterococcus faecium ATCC 349 for TVC 30°C
Geobacillus stearothermophilus ATCC 12980 for TVC 55°C
Negative control: not necessary
Blank: -irradiated infant milk formula or a comparable product

Calculate the number of cfu as described in M01_01AA. Express the results as CFU per
volume or gram of sample.
8 Validation
See validation reports M01_02VB and M01_02VB2_v02.
9 References
1. Hatcher W.S. Jr., Weihe, J.L., Splittstoesser D.F., Hill, E.C. and Parish M.E. “Fruit
beverages” In: “Compendium of methods for the microbiological examination of
21
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foods”. 3rd ed., Vanderzant, C. and Splittstoesser, D.F. (Ed), American Public Health
Association, Washington, 1992, page 953-960.
2. International Organization for Standardization, 1990. ISO 7698, Cereals, pulses and
derived products – Enumeration of bacteria, yeasts and moulds.
3. International Organization for Standardization, 2013. ISO 4833, Microbiology of food
and animal feeding stuff – Horizontal method for the enumeration of microorganisms
– Colony-count technique at 30°C.
4. International Organization for Standardization, 1992. ISO 6610, Milk and milk
products – Enumeration of colony forming units of microorganisms - colony count
technique at 30C (IDF Standard 100B).
5. International Organization for Standardization, 1989. ISO 8261, Milk and milk
products – Preparation of samples and dilutions for microbiological examination.
6. International Organization for Standardization, 2010. DIN EN ISO 6887-5,
Microbiology of food and animal feeding stuffs – Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination – Part 5: Specific
rules for the preparation of milk and milk products.
7. International Organization for Standardization, 2002. ISO 13559, Butter, fermented
milks and fresh cheese – Enumeration of contaminating microorganisms – Colony-
count technique at 30°C.
8. Nederlands normalisatie instituut. 1998. Milk and milkproducts. Enumeration of
thermoduric micro organismen. 2nd draft, February 1998, NNI 98-05.
9. M01_01ME “Sample preparation and dilution”. CLF method, Microbiology Division.
10. M01_01AA “Calculations and expression of results” CLF method, Microbiology
Division.
11. M01_02VB Validation report.
12. M01_02VB 02 Validation report for the addition of TTC.
10 Attachments
Not applicable
22
TVC 30°C for butter sample

The preparation of butter sample is carried out according to DIN EN ISO 6887-5:2011-01 “Milk
and milk products. General guidance for the preparation of test samples, initial suspensions and
decimal dilutions for microbiological examination.”
TVC of butter samples consists mainly of three steps:

1. Dissolve butter sample in an empty container in a water bath of 45°C.
2. Add 90 ml BPW which is pre-warmed to 45°C to the butter material in the container.
3. Use the spreading method for TVC of butter sample
It has been found that the butter does not form a good emulsion in BPW. A fat layer is clearly
visible which might prevent the suspension of bacteria in the BPW. According to the ISO norm,
the emulsifier sorbitanmonooleat (Tween 80) is added to BPW in 1 to 10 g/l. However in the
internal validation test the concentration of 1 to 10 g/L was found to be insufficient to emulsify the
butter/oil-containing BPW solution. To obtain a well-soluble and homogenous emulsion, 5 ml
autoclaved Tween 80 (description of the product see below) has to be added to 90 ml BPW in a
correspondingly higher concentration of 50 g/l.
NOTES:
1. The most common commercially available sorbitanmonooleat is Tween 80. The Tween 80 used in CLF is in
liquid form (1 liter =1.07 kg; Merck, 8.22187.2500).
2. Tween 80 must be autoclaved before use.
3. In the butter sample treated in this method, Tween 80 is supplemented in 5 ml/90 ml BPW.
4. The dried pre-poured PCA medium should be warmed to 45°C before spreading.
Use of different media for different categories of non-butter product
 ISO based:
Generally PCA is used as the standard medium for TVC for all products.
 ISO based:
According to ISO 4833-1:2013, skimmed milk powder is added to PCA at 1.0 g/L of the
culture medium when dairy products are examined. The skimmed milk powder shall be
free from inhibitory substances. MPCA (Milk plate count agar) containing antibiotic free
skim milk in 1.0 g/L is commercially available and provided by producers like OXOID.
Because most milk and milk products contain skim milk in a relative high concentration
themselves, the MPCA agar is thus recommended only when the sample dilution is ≥
1:1000.
23
 Deviation from ISO:
The TTC supplementation in PCA represents an option for TVC determination in case the
colonies are difficult to read. The red colorization caused by reduction of TTC facilitates
the visibility of colonies in difficult matrices.

For anaerobe TVC determination in all kinds of products Schaedler agar is used.

OSA agar is deleted from the current version. It is exclusively mentioned in the method
M01_21 “Sterility testing of acid pasteurized and neutral sterilized liquid products”.
Influence of the PCA agar brand on analytical results

It has been observed in the ring trials of CLF that compared with PCA containing 14 g agar-
agar/L medium, PCA brands with agar concentration towards the lower limit of the required range
(e.g. OXOID, CM 325; 9 g agar-agar/L medium) provide constantly slightly higher counts and less
deviation of analytical results.
No specific norm for TVC 55°C in the ISO norm

TVC 55°C is not mentioned in the ISO norm. For TVC 55°C the ISO 4833-1:2013 is applied with
modification in incubation temperature.
24
BACILLUS CEREUS ENUMERATION
25
26
No. M01_03ME_v05(B.cereus enumeration).doc
B. cereus - Enumeration
of: 6 written: 25.01.2012
6
printed: 23.06.2014
Bacillus cereus – Enumeration

V2  Update to German version 2 which is accredited
 New edition of norm method ISO 7932: 2004-12 has been added.
 Update of reference material.
V4 (25.01.12)  Storage time of polymyxin
 API20E removed for confirmation
 Change of internal control strains
 Confirmation step optional
V5 (25.04.14)  Modern identification methods 16S rRNA gene sequencing and
MALDI TOF MS added to 6.2/B
Name Signature Date

1999
Bredius
revised by: Björn
2014
Hampel
2014
Zhu
2014
Wollnitz
approved: Matthias
2014
Fischer
27
of: 6 written: 25.01.2012
6
printed: 23.06.2014

The method for the enumeration of B. cereus can be applied to food products and water.

In contrast to most other Bacillus species, B. cereus is not able to produce acid from
mannitol. On mannitol egg yolk polymyxin agar with phenol red indicator (MYP) B. cereus
will grow as white-pink colonies due to the presence of the indicator phenol red. Mannitol-
fermenting microorganisms growing on MYP will change the color of the medium from red
to yellow. In most cases B. cereus produces a lecithinase which attacks the lecithin in the
egg yolk, resulting in a broad white precipitation zone. Polymyxin is inhibitory to gram
negative microorganisms and a number of gram positive cocci on MYP.
Modifications
This method is based on ISO 7932, 2004-12.
 MYP agar plates are stored for 4 days at room temperature
 confirmation through identification with biochemical test kit

B. cereus Gram positive, motile, aerobic spore forming rod, not able to ferment mannitol
and usually produces lecithinase activity.
CFU colony forming units
NOTE:
In accordance with ISO and AOAC, this method does not discriminate between B. cereus and the other
members of the B. cereus group, which are B. mycoides, B. anthracis and B. thuringiensis.
28
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Egg yolk EY See manufacturer’s description
Mannitol egg yolk polymyxin agar MYP 1 week 4-10°C
MYP- base MYP-b 1 month 4-10°C

Polymyxin solution See manufacturer’s description
Blood agar (sheep or horse) BA See manufacturer’s description
Biochemical identification test kit2 See manufacturer’s description
1
The shelf life of the prepared media/solutions is given when stored in the dark. Media kept longer than 1
month have to be stored in well closed (screw capped) bottles.
2
e.g. API or another comparable reliable test kit. When using the API test kit, the following is needed:
API 50 CH test strips, API 50 CHB medium

4.1 MYP
90 ml MYP-b + 10 ml EY+ 1 ml polymyxin. The polymyxin concentration in the MYP has
to be 100 IU/ml. Pour plates (Ø 14 cm) with about 40-50 ml MYP. Dry the plates before
use in an incubator at 37-55°C until the surface is dry (30-45 min. for a full incubator).
5 Equipment
Sterile spreaders
6 Procedure
6.1 Detection
1. Prepare a resuscitated sample, a suitable dilution series dependent on the presumed
contamination level of the sample (see method M01_01ME). The number of B.cereus
colonies on a plate should not exceed 50.
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2. Pipette the appropriate volume of (diluted) sample on one or more dried MYP plates
so that the sample can be absorbed by the MYP agar (for example pipette 2 times
0.5 ml onto 2 large plates).
3. Distribute the liquid evenly over the surface with a sterile spreader. During incubation
the surface of the plate should not be moist to prevent spreading of colonies.
4. Incubate the inverted plates for 42-48 h at 30 ± 1°C.
After 42-48 h the colonies and precipitation zone can be rather large. This is why
separate colonies are hard to distinguish. Therefore, count the typical colonies after
24 h of incubation after which the plates are further incubated.
5. Count the typical colonies:
Typical colonies are: large, white to pink, irregular, dull and ruffled or rhizoid (mould
like) of appearance and generally surrounded by a white to pink precipitation zone of
varying density.
Distinct yellow colonies are not B. cereus.
When the typical characteristics are not clear, suspected colonies can be confirmed
using one of the confirmation procedures described below.
NOTES:
Because spores can survive 70% alcohol, it is preferred to use disposable spreaders or dry air sterilised
spreaders.
As the colony morphology (form, surface, and color) is an important feature to distinguish between B. cereus
and competitive flora, spread plates have to be used instead of pour plates.
If the plates contain numerous mannitol fermenting microorganisms leading to the production of acid (and so
the color changes to yellow), the pink color of B. cereus colonies may be reduced or disappear entirely.
Some B. cereus strains produce only little or no lecithinase. Colonies of these strains will not be surrounded
by a precipitation zone. These colonies can be subjected to confirmation tests.
6.2 Confirmation
For colonies with typical growth, a confirmation is not necessary. In case the examination
is difficult due to the presence of many acid forming colonies, suspected colonies can be
transferred to fresh MYP agar to verify the typical characteristics.
Use well isolated colonies for the confirmation. When it is not possible to select well
isolated colonies, make a pure streak on MYP, tryptone soy agar (TSA) or blood agar
(BA) and incubate the plates at 30 ± 1°C.
Choose one of the two confirmation procedures described below:
 Haemolysis
1. The suspected colony is streaked onto BA.
2. The plates are incubated at 30 ± 1°C for 24 ± 3 h.
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3. The presence of ß-haemolysis is checked. ß-haemolysis is visible as a clear zone

around the colony with or without a greenish discolouration of the colony or the
medium.
B. cereus is ß-haemolysis positive.
 Biochemical or molecular biological identification

1. Perform an identification using a biochemical identification test kit e.g. API 50 CHB.
2. For further identification, 16S rRNA gene sequencing and MALDI TOF MS are
recommended.
NOTE:
Haemolysis test is the confirmation procedure proposed by the ISO Technical Committee at the moment.

Positive control: B. cereus ATCC 11778
Negative control: Escherichia coli ATCC 11775

Calculate the number of B. cereus as described in M01_01AA. Express the results as
CFU per volume or gram of sample.
8 Validation
9 References
1. AOAC Official methods of analysis, 1995, Microbiological methods, Chapter 17,
p. 52-55
2. Beumer R. 1998, Confirmation of B. cereus” Nederlands normalisatie instituut, Ref:
nnidoc. 370009/98-62, 5p.
3. Harmon, S. M., Goepfert, J.M. and Bennett, R.W. “Bacillus cereus” In: “Compendium
of methods for the microbiological examination of foods”. third ed., Vanderzant, C.
and Splittstoesser, D.F. (Ed), American Public Health Association, Washington, 1992,
page 593-604.
31
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4. Holbrook, R. „Consideration of the confirmation tests for the identification of colonies

of Bacillus cereus in ISO standard 7932“ ISO/TC 34/SC 9 N 3641998-12-14.
5. International Organisation for Standardisation, „Horizontal method for the
enumeration of low numbers of B. cereus – Most probable number technique “ISO
TC34/SC 9 N 424, 2000-04-17.
6. International Organisation for Standardisation, „ISO 7932, Horizontal method for the
enumeration of presumptive Bacillus cereus – Colony-count technique at 30°C“ 2004-
12.
7. M01_01ME “Calculations and expression of results“ CLF method, Microbiology
Division.
8. M01_01ME “Sample preparation and dilution“. CLF method, Microbiology Division.
9. Nederlands Normalisatie Institut. Doc. No 99-65 “ISO/TC 34/SC 9 N397, ISO/CD
7932/DAM 1 Microbiology of food and animal feeding stuffs – General guidance for
the enumeration of presumptive Bacillus cereus – Colony count technique at 30
degrees C“ 1999-09-17.
10. Nutricia Research, Notulen MRL9-11-1995, Ref: no0004. mlz.
10 Attachments
Not applicable
32
Presumptive Bacillus cereus group as target-microorganisms in M01_03 and

ISO 7932
The present Bacillus cereus enumeration method M01_03ME is based on DIN EN ISO
7932:2004 “Horizontal method for the enumeration of presumptive Bacillus cereus Colony-count
technique at 30°C”.
Typical presumptive colonies of Bacillus cereus colonies are generally large, a) pink (indicating
that mannitol fermentation has not occurred) and b) surrounded by a zone of precipitation
(indicating the production of lecithinase).
As indicated in the ISO 7932:2004, to make the test method practicable, the confirmatory stage
has been restricted to the typical aspect on MYP agar and the haemolysis test. The term
“presumptive Bacillus cereus” has been introduced to acknowledge the fact that the confirmatory
stage, if restricted to the MYP colony morphology level and haemolysis, does not enable the
distinction of B. cereus from other closely related but less commonly encountered Bacillus
species, such as B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides, B.
megaterium. All these Bacillus species are referred to as members of “presumptive Bacillus
cereus group” and are co-counted using the method.
The table below gives evidence that there are no significant differences between members of
presumptive Bacillus cereus group on the biochemical/morphological parameters lecithinase,
haemolysis and mannitol fermentation. An additional motility test may help to differentiate B.
cereus from other members in cases where the presence of the latter is suspected.
Using commercially biochemical test kits e.g. API 50 CHB a differentiation is possible between B.
cereus and other Bacillus group members, e.g. the extreme dangerous but rarely occurring B.
anthracis. Modern biological methods like 16S rRNA gene sequencing and MALDI TOF MS offer
better opportunities for further differentiation and identification.
Table 1: Biochemical and morphological differentiation characteristics of members of presumptive Bacillus cereus
group
B. cereus B. anthracis B. mycoides B. thuringiensis B. weihenstephanensis
Lecithinase + weak + + + +
Haemolysis + - + + +
Mannitol - - - - -
Motility + - - + Not determined
33
Atypical Bacillus cereus
Although formation of pink colonies surrounded by precipitate zone and positive reaction in
haemolysis test are regarded as typical for presumptive Bacillus cereus group, some strains of B.
cereus produce only little or no lecithinase. Colonies of these strains will not be surrounded by a
precipitation zone or surrounded by a narrow precipitation zone (see picture 1). However it has
been observed that the zone became bigger when the MYP plate is incubated longer. These
strains give an atypical morphological image on MYP medium and thus have to be subjected to
confirmation tests.
It is notable that the width of a haemolysis zone can vary: a narrow and unclear zone is shown in
a haemolysis test of these atypical Bacillus cereus strains (see picture 2). The zone area could
be enlarged in case of prolonged incubation of blood agar. For atypical Bacillus cereus strains a
confirmation using biochemical test kit or molecular biological tools is preferred.
Picture 1: growth of typical B. cereus (left) and atypical B. cereus (right) on MYP
Picture 2: haemolysis zone of typical B. cereus (left) and atypical B. cereus (right, in circle) on BA
34
Protocols for B. cereus enumeration: ISO method and CLF method
35
36
STAPHYLOCOCCUS AUREUS DETECTION
37
38
No. M01_04ME_v05(S.aureus detection).doc
S. aureus - Detection
of: 8 written: 28.02.2014
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printed: 23.06.2014
Staphylococcus aureus (coagulase positive Staphylococci)

– Detection

V3 (14.09.10)  Description of the modifications was added
 New edition of norm method EN ISO 6888-3: 2003
 Manufacturer information for agglutination test removed
V4 (15.05.12)  Title of test method amended („coagulase positive Staphylococci“)
 Table with other coagulase positive Staphylococcus species
included
V5 (28.02.14)  Coagulase reaction with test kit for the confirmation step added
 Molecular biological tools for the confirmation step added
Name Signature Date

Bredius 2000
revised by: Sha

Zhu 2014
Hampel 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
39
of: 8 written: 28.02.2014
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printed: 23.06.2014

The method for the detection of S. aureus and other coagulase positive Staphylococci can
be applied to food products and water.

Gioletti & Cantoni broth (GC) contains mannitol, glycine and sodium pyruvate to promote
the growth of S. aureus. As selective agents GC contains LiCl and K2TeO3 which inhibit
the growth of gram positive and gram negative bacteria respectively. S. aureus and other
Staphylococci can reduce tellurite to metallic tellurium which colours the medium black.
The selectivity of the GC is further improved by anaerobic incubation which inhibits the
growth of competitive flora.
Baird Parker Agar (BPA) also contains glycine, sodium pyruvate, LiCl and K2TeO3. The
BPA contains egg yolk which makes lypolysis- and proteolysis activity visible as a clear
zone around the colony. On BPA S. aureus is visible as grey-black colonies which are
mostly but not always surrounded by a double zone: a white precipitation zone (lecitinase
activity) surrounded by a clear zone caused by lipase and protease activity.
As an alternative method for isolation and confirmation, the Rabbit Plasma Fibriogen
(RPF) medium can be applied (without egg yolk). In this way the presence of coagulase
positive Staphylococci is immediately visible.
In addition to S. aureus, other coagulase positive Staphylococci are assessed with this
methodology as well (see table below):
oxidase catalase coagulase haemolyse
S. aureus - + + +
S. intermedius - + + +
S. hyicus - + +/- -
S. epidermidis - + - +/-
Modifications
This method is based on norm method EN-ISO 6888-3, 2003-09 + AC: 2005-03.
Following modifications have been performed:
 Utilization of GC broth without ingredient Tween 80 is also possible
 The heating process of the GC-tubes before usage to expel air as described in
ISO 6888-3 is not necessary
 Utilization of commercially available rapid latex test kit for the identification of
suspicious colonies
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Staphyloccocci Gram positive, catalase positive cocci
S. aureus A coagulase positive Staphylococcus. S. aureus can form typical as
well as atypical colonies on BPA
Baird parker agar base BPA-b 1 month 4-10°C
BPA-b + EYT6 BPA 1 week 4-10°C
Egg yolk + Tellurite6 EYT See manufacturer’s description
Giolitti & Cantoni-base GC-b 2 weeks 4-10°C
GC-b + K2TeO3 GC 1 week 4-10°C
1% (w/v) K2TeO3 in water5 K-tellurite 1 month 4-10°C
1.2% (w/v) K2TeO33H2O in water5 K-tellurite 1 month 4-10°C
(Paraffin or water agar)4 See manufacturer’s description
Coagulase plasma See manufacturer’s description
Staphaurex See manufacturer’s description
Trypton soya agar TSA 1 month 4-10°C
Trypton soya bouillon TSB 1 month 4-10°C
Rabbit plasma fibrinogen agar RPF agar 1 week 4-10°C

Rabbit plasma fibrinogen
RPF sup. See manufacturer’s description
supplement
1
2
e.g. API-Staph
3
Either the coagulase plasma test, the biochemical test kit or RPF agar (alternative method) is used for the
confirmation of S. aureus.
4
To obtain anaerobic conditions anaerobic jars are used or the tubes are covered with a layer of paraffin or
water agar. See note at 6.1
5
Do not use the solution when a white precipitation is observed within 1 month.
6
Used within the normal method but not when RPF is used (see 6.1.3, Method A)
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4.1 GC-base solution

When 1 ml (diluted) sample is inoculated, GC-b is prepared according to the manu-
facturer’s description (normal concentration) and 10 ml of the broth is filled in normal
20 ml tubes. GC-b prepared in this way is called standard GC-b.
When 10 ml (diluted) sample is inoculated, a double concentrated GC-b is prepared and
10 ml double concentrated GC-b is filled in larger 30 ml tubes.
4.2 GC
Add to 10 ml standard GC-b 0.1 ml filter sterilised potassium-tellurite solution (1.0 % filter
sterilised K2TeO3 solution or 1.2 % filter sterilised K2TeO33H2O solution, using a 0.22 m
filter).
Add to 10 ml double concentrated GC-b 0.2 ml filter sterilised potassium-tellurite solution.
The end concentration potasium tellurite in GC+ sample has to be 0.01%.
5 Equipment
Anaerobic jars in which tubes are incubated
6 Procedure
6.1 Detection
1. Prepare a suitable dilution series of a resuscitated sample (see method M01_01ME).
2. Pipette 10 ml of the initial suspension to double concentrated GC. Prevent the
introduction of air while performing.
3. The tubes are anaerobically incubated for 42-48 h at 37 ± 1°C.
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NOTES:
The introduction of air in the GC tubes before incubation should be prevented as much as possible: the
tubes are therefore not mixed.
To prevent the introduction of air when adding the sample, use one of the following techniques:
 Put the pipette on the bottom of the tube and pull the pipette carefully out again. Do not blow out the
pipette.
 Put the pipette against the wall of the tube, just above the surface of the GC. Carefully let the sample
flow out via the wall of the tube.
For the anaerobic incubation of the tubes either of the three methods has to be chosen:
 Incubation in anaerobic jars with anaerobic generation kit.
 The tubes are covered with an over layer (2 cm) of sterile, liquid paraffin.
 The tubes are covered with an over layer of sterile water agar (1.5-2% agar in water, tempered at
45 ± 2°C).
4. When water agar has been used, the agar-clump is cut with a sterile loop or spoon.
When paraffin has been used, the tubes are not mixed on a vortex but with a Pasteur
pipette. After that about 1 ml of the suspension is taken out with the pipette and
transferred to a sterile tube. By doing so, take care that as little as possible paraffin is
transferred to the sterile tube.
5. The tubes are mixed (e.g.) on a vortex to obtain a homogenous suspension.
6. For the isolation of S. aureus one of the methods described below is used.
 Standard isolation medium: BPA

1. Streak by means of a sterile loop (1 l) from the GC enrichment on a pre-dried BPA.
By using paraffin it is important to streak no paraffin on the plate.
2. The inoculated plates are incubated for 42-48 h at 37 ± 1°C.
3. Confirm the suspected colonies.
 Alternative isolation medium: RPF

1. Streak by means of a sterile loop (1 l) from the GC enrichment on a pre-dried RPF
agar.
2. The inoculated plates are incubated for 42-48 h at 37 ± 1°C.
3. Read the plate: coagulase positive Staphylococci (S. aureus and some other
Staphylococci spp.) show grey-black but sometimes white colonies with a turbid zone
(coagulase activity). Further confirmation tests are not necessary.
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S. aureus Blackening of GC tubes.

BPA: round, black or grey, shiny, convex colonies, surrounded by a double halo:
inside a white precipitation zone, surrounded by a clear zone.
RPF: coagulase positive Staphylococci (S. aureus and some other Staphylococci
spp) show grey-black but sometimes white colonies with a turbid zone.
Catalase positive, gram positive cocci, coagulase positive cocci.
Picture 1: Morphology of S. aureus on BPA medium
NOTE:
The double halo is sometimes invisible in atypical colonies. Atypical colonies are often seen in milk products,
shrimps and poultry which are contaminated with coagulase positive Staphyloccoci.
6.2 Confirmation
Suspected colonies are further confirmed by means of a coagulase test or a biochemical
identification test kit or molecular biological confirmation tools.
1. A purification streak is made of the suspected colony on TSA or another, general
medium. In general, coagulation tests and biochemical identification test kits are only
reliable when colonies grown on a non-selective medium are used (see also
manufacturer’s description).
2. Confirmation tools:
Suspected grey-black colonies (with or without zone) are tested by a rapid
agglutination test. Suspected colonies which give a doubtful or positive result in the
agglutination test are further confirmed. Such colonies are tested on catalase activity
(see M01_08AA for catalase test) or gram strain (M01_05AA).
Colonies which are negative in the agglutination test or which are no cocci or show
negative results for catalase reaction are no S. aureus.
 Coagulase reaction
The rabbit plasma coagulase reaction applies officially as the confirmation test in ISO
6888-1, 2 and 3. However the method is time-consuming and work-intensive.
44
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Commercially available, sufficient test kits (e.g. Staphaurex Plus*) are recommended
to use.
 Biochemical identification test kits e.g. API-Staph
 Molecular biological tools
For a further identification, MALDI TOF MS and 16S rRNA gene sequencing can be
applied.

Positive controls: Culti loop Staphylococcus aureus Oxoid
Staphylococcus aureus ATCC 6538
Negative controls: Escherichia coli ATCC 11775
Proteus mirabilis ATCC 29906
Blank: -irradiated infant milk formula or a comparable product

Express the result as the absence or presence of S. aureus per weight or per volume of
sample as described in M01_01AA.
8 Validation
9 References
1. Anon. 1993. Giolitti and Cantoni broth. Int. J. Food Microbiol. 17 (3), 247-248.
2. Gregson, D.B., Low, D.E., Skulnick, M., Simor, A.E. 1988. Problems with Rapid
agglutination methods for Identification of Staphylococcus aureus when
Staphylococcus saprophyticus is being present. J. Clin. Microbiol., 26 (7)., 1398-1399
3. International Dairy Federation. 1978. International Standard 60A, Detection of
coagulase positive staphylococci in dried milk.
4. International Dairy Federation. 1986. International Standard 138, Dried milk
enumeration of Staphylococcus aureus colony count technique at 37°C.
5. International Organization for Standardization. 1983. ISO 6888, Microbiology-General
guidance for enumeration of Staphylococcus aureus colony count technique.
6. Necker, D „Versuch zur St aureus Analyse“, 28-3-2000, CLF Central Laboratories
Friedrichsdorf GmbH, Microbiology Division, intern report ref. Nr: RD000903.
45
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printed: 23.06.2014
7. Nederlands Normalisatie Instituut. 2000. pr EN ISO 6888-3 Horizontal method for the
enumeration of coagulase-positive Staphylococci (Staphylococcus aureus and other
species) – Part 3: MPN technique for low numbers“ april 2000, Doc. ISO/TC 34/SC9
N415. NEN Doc. nr. 00-12.
8. Weers-Pothoff G., Moolhuijzen C.E.M., Bongaserts G.P.A. „Comparison of seven
coagulase tests for identification of Staphylococcus aureus“ 1987, Eur. J. Clin.
Microbiol. Vol 6, no 5, p. 589-591.
9. M01_01ME “Sample preparation and dilution”. CLF method, Microbiology Division
10. Catalase-Test, CLF-procedure M01_08AA.
Division.
12. International Organization for Standardization, 2003. EN ISO 6888-3: 2003-09 + AC:
2005-03 „Microbiology of food and animal feeding stuffs - Horizontal method for the
enumeration of coagulase-positive staphylococci - Detection and MPN technique for
low numbers”.
10 Attachments
Not applicable
46
Confirmation of suspicious colonies isolated from BPA agar

According to ISO 6888-3:2003, enriched cultures from GC broth can be streaked onto either BPA
or RFP medium. The inoculated plates are incubated at 37°C for 48 h.
A confirmation step is required for suspicious colonies isolated from BPA agar. In ISO 6888-1 the
rabbit plasma coagulase reaction applies officially as the confirmation method, which is very time-
consuming and work-intensive. Therefore a commercially available test kit for rapid latex
agglutination test (e.g. Staphaurex Plus*, Remel Inc.) is recommended.
Typical colonies on RPF agar are black, grey or sometimes even white surrounded by an opaque
halo. The RPF medium is based on coagulase reaction itself. Therefore a further confirmation of
colonies on RPF is not necessary anymore.
Protocols for S. aureus detection: ISO method and CLF method
47
48
STAPHYLOCOCCUS AUREUS ENUMERATION
49
50
No. M01_05ME_v03(S.aureus enumeration).doc
S. aureus - Enumeration
of: 6 written: 6
28.02.2014
printed: 23.06.2014
Staphylococcus aureus (coagulase positive Staphylococci)

 Enumeration

V2 (15.05.12)  Title amended (coagulase positive Staphylococci)
 Table with other coagulase positive Staphylococci added
 CFU added under abbreviations
 Ecometrie as a test method for media quality deleted
 Murex as a supplier for coagulase test kits deleted
 Validation report added
V3 (28.02.14)  Coagulase reaction with test kit for the confirmation step added
 Molecular biological tools for the confirmation step added
Name Signature Date

1st version written by: Björn
Hampel 2002
revised by: Sha

Zhu 2014
Hampel 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
51
of: 6 written: 6
28.02.2014
printed: 23.06.2014

This method for the enumeration of S. aureus and other coagulase positive Staphylococci
can be applied to food products and water.

Baird Parker Agar (BPA) contains glycine and sodium pyruvate to promote the growth of
S. aureus. As selective agents this medium countains LiCl and K2TeO3 which inhibit the
growth of Gram positive and Gram negative bacteria respectively. As elective agents egg
yolk is added to BPA. On BPA S. aureus is visible as grey-black colonies which are
mostly but not always surrounded by a double zone: a white precipitation zone (lecitinase
activity) surrounded by a clear zone caused by lipase and protease activity.
As an alternative option the Rabbit Plasma Fibriogen (RPF) medium can be applied
(without egg yolk). In this way the presence of coagulase positive Staphylococci is
immediately visible.
In addition to S. aureus other coagulase positive Staphylococci are assessed with this
methodology (see table below):
oxidase catalase coagulase haemolyse
S. aureus - + + +
S. intermedius - + + +
S. hyicus - + +/- -
S. epidermidis - + - +/-
This method is based on DIN EN ISO 6888-1 and 6888-2, 1999.

Staphyloccocci Gram positive, catalase positive cocci
S. aureus A coagulase positive Staphylococcus. S. auereus can build typical as
well as atypical colonies on BPA.
CFU Colony forming units
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Baird parker agar base BPA-b 1 month 4-10°C
BPA-b + EYT4 BPA 1 week 4-10°C
Egg yolk + Tellurite4 EYT See manufacturer’s description
Paraffin or water agar4 See manufacturer’s description
Coagulase plasma See manufacturer’s description
Catalase (H2O2) See manufacturer’s description
Staphaurex See manufacturer’s description
Rabbit plasma fibrinogen agar3 RPF agar 1 week 4-10°C

Rabbit plasma fibrinogen
supplement RPF sup. See manufacturer’s description
1% (w/v) K2TeO3 in water5 K-tellurite 1 month 4-10°C
1.2% (w/v) K2TeO33H2O in water5 K-tellurite 1 month 4-10°C

1
2
e.g. API-Staph.
3
Either the coagulase plasma test or the biochemical test kit or RPF agar (alternative methode) is used for the
confirmation of S. aureus.
4
Used within the normal method but not when RPF is used (see 6.1.3, Methode A)
5
Do not use the solution when a white precipitation is observed within 1 month.

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5 Equipment
Sterile spreaders
6 Procedure
6.1 Enumeration
 Standard isolation medium: BPA

1. Prepare a suitable dilution series of a resuscitated sample.
2. Pipette 0.1 ml of the initial suspension or of the appropriate dilution on a pre-dried
BPA agar.
3. Spread the sample over the plate by use of the pipette or by means of a sterile
spreader.
4. Incubate the plates for 42-48 h at 37 ± 1°C.
5. Count the number of suspected colonies.
6. Confirm a representative number of suspected colonies, but at least 5 colonies when
present.
 Alternative isolation medium: RPF

1. Prepare a suitable dilution series of a resuscitated sample.
2. Pipet 0.1 ml of the initial suspension or of the appropriate dilution on a pre-dried RPF
agar.
3. Spread the sample over the plate by use of the pipette or by means of a sterile
spreader.
4. Incubate the plates for 42-48 h at 37 ± 1°C.
5. Count the number of typical colonies.
S. aureus
BPA: round, black or grey, shiny, convex colonies, surrounded by a double halo: inside a
white precipitation zone, surrounded by a clear zone.
RPF: coagulase positive Staphylococci (S. aureus and some other Staphylococci spp)
show grey-black but sometimes white colonies with a turbid zone.
Catalase positive, gram positive cocci, coagulase positive cocci.
NOTE:
Validation test have shown that when spread plates were used, higher counts were obtained than when pour
plates were used. In addition it is easier to recognise and isolate suspected colonies from a spread plate.
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To decrease the detection limit to 1 cfu/ml for liquid products or to 10 CFU/g for dry products, 1 ml initial
suspension can be spread on 2 large agar plates.
6.2 Confirmation
Suspected colonies are further confirmed by means of a coagulase test or a biochemical
identification test kit or molecular biological confirmation tools.
1. A purification streak is made of the suspected colony on TSA or another, general
medium. In general, coagulation tests and biochemical identification test kits are only
reliable when colonies grown on a non-selective medium are used (see also
manufacturer’s description).
2. Confirmation tools:
Suspected grey-black colonies (with or without zone) are tested by a rapid
agglutination test. Suspected colonies which give a doubtful or positive result in the
agglutination test are further confirmed. Such colonies are tested on catalase activity
(see M01_08AA for catalase test) or gram strain (M01_05AA).
Colonies which are negative in the agglutination test or which are no cocci or show
negative results for catalase reaction are no S. aureus.
 Coagulase reaction
The rabbit plasma coagulase reaction applies officially as the confirmation test in ISO
6888-1, 2 and 3. However the method is time-consuming and work-intensive.
Commercially available, sufficient test kits (e.g. Staphaurex Plus*) are recommended
to use.
 Biochemical identification test kits e.g. API-Staph
 Molecular biological tools
For a further identification, MALDI TOF MS and 16S rRNA gene sequencing can be
applied.

Positive control: Culti loop Staphylococcus aureus Oxoid
Staphylococcus aureus ATCC 6538
Negative controls: Escherichia coli ATCC 11775
Proteus mirabilis ATCC 29906

Calculate the number of S. aureus as described in M01_01AA. Express the results as
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8 Validation
Necker, D. „ Test for Baird Parker (BPA) Agar count method“ 18-9-2000, CLF Central
Laboratories Friedrichsdorf GmbH, Microbiology Division, intern report ref. Nr: RD003603.
See Validation report M01_05VB.
9 References
1. Gregson, D.B., Low, D.E., Skulnick, M., Simor, A.E. 1988. Problems with Rapid
agglutination methods for Identification of Staphylococcus aureus when
Staphylococcus saprophyticus is being present. J. Clin. Microbiol., 26 (7)., 1398-
1399.
2. Europäisches Komitee für Normung. 1999. EN ISO 6888-1 deutsche Fassung,
Horizontales verfahren für die Zählung von koagulase-positiven Staphylkokken
(Staphylococcus aureus und andere Spezies).
3. Necker, D. „ Test for Baird Parker (BPA) Agar count method“ 18-9-2000, CLF Central
Laboratories Friedrichsdorf GmbH, Microbiology Division, intern report ref. No:
RD003603.
4. Weers-Pothoff G., Moolhuijzen C.E.M., Bongaserts G.P.A. „Comparison of seven
coagulase tests for identification of Staphylococcus aureus“ 1987, Eur. J. Clin.
Microbiol. Vol 6, no 5, p. 589-591.
6. Catalase-Test, CLF-procedure M01_08AA.
Division.
10 Attachments
Not applicable
56
Comparable enumeration results on BPA and RPF

BPA and RPF are recommended for the enumeration of coagulase positive Staphyloccoci (e.g. S.
aureus) in ISO 6888-1 and ISO 6888-2 respectively.
The validation tests (see M01_05VB) compared the bacterial counts on BPA and RPF for the
same panel of milk samples contaminated with S. aureus as well as the pure culture of S. aureus
and came to the conclusion that both media provided comparable results.
Higher bacterial count obtained using spread method than pour method
Spread method is defined officially in ISO 6888-1 using BPA medium, while pour method is
described in ISO 6888-2 where RPF is used.
However in the validation test significant higher numbers of S. aureus were detected on both
media BPA and RPF when using the spreading method (see M01_05VB). As a result the spread
method for S. aureus enumeration is adopted in the current method M01_05ME regardless of the
medium applied.
Confirmation of suspicious colonies isolated from BPA or RPF agar

A confirmation step is required for suspicious colonies isolated from BPA agar. In ISO 6888-1 the
rabbit plasma coagulase reaction applies officially as the confirmation method, which is very time-
consuming and work-intensive. Therefore a commercially available test kit for rapid latex
agglutination test (e.g. Staphaurex Plus*, Remel Inc.) is recommended.
Typical colonies on RPF agar are black, grey or sometimes even white surrounded by an opaque
halo. The RPF medium is based on coagulase reaction itself. Therefore a further confirmation of
colonies on RPF is not necessary anymore.
57
Protocols for S. aureus enumeration: ISO method and CLF method
58
YAEST AND MOLDS ENUMERATION
59
60
No. M01_06ME_v04(Yeast and Molds).doc
Yeast and Molds
of: 6 written: 03.05.2012
6
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Yeast and Molds  Enumeration (based on ISO 6611/IDF 94)

V2  Update to German version 2 which is accredited
V3 (03.05.12)  Ridacount as another option added in the method
 Definitions and abbreviations extended
 Norm method updated
 Ecometrie deleted
 Confirmation by microscopic preparation only optional
 Validation report for the Ridacount method included
 Molds should not be used as a positive control in order to prevent
contamination
V4 (05.02.14)  Remark that the current method is based on ISO 6611/IDF 94
 Modern identification tools added in 6.2
Name Signature Date

1st version written by: Thomas
2000
Koch
revised by: Björn
2014
Hampel
2014
Zhu
2014
Wollnitz
approved: Matthias
2014
Fischer
61
Yeast and Molds
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The method for the enumeration of yeasts and molds can be applied to food products
and water.

 Selective Agar
Chloramphenicol in Yeast extract-Glucose-Chloramphenicol agar (YGC) and oxy-
tetracycline in Yeast extract-Glucose-Oxytetracycline agar (YGO) inhibit the growth of
most bacteria. The growth of yeast and mold is not inhibited. The colonies of both
yeasts and moulds are counted after 3, 4 and 5 days. Yeast colonies are distin-
guished from mold thalli by their colony morphology. When necessary, the suspected
colonies are confirmed by microscopic examination. The final result is obtained after
4-5 days.
 RIDA®COUNT
RIDA®COUNT card consists of a dehydrogenated YGC media which is additionally
supplemented with TTC. The reduced form of TTC is visible by a red colorization.
These red spots appear before the actual colonies are visible. Consequently the
incubation time can be reduced to 72 h.
Chemical structure of TTC and Formazan
Tetrazolium: colorless, soluble in water 2 reduction steps Formazan: red, insoluble in water
Figure 1: Chemical structure of TTC and Formazan
Modification
This method is based on DIN EN ISO 6611:2004-10 with the following modifications:
 The enumeration is performed after 5 days but there is also an option to count the
colonies already after 4 days.
 If the plates are not countable after 5 days because of too much growth, the next
dilution will be taken or the result after 4 days will be used.
 Optional use of RIDA®COUNT Yeast and Mold card
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Yeast Mesophylic aerobic micro-organisms which form matt or shiny round
lenticular colonies, with a more or less convex surface and usually
having a regular outline. Yeasts are mostly large round to oval
budding cells.
Mold Mesophylic aerobic, filamentous, micro-organisms which grow on the
surface of the plates and produce fluffy and often pigmented, more or
less spread out thalli.
TTC 2,3,5-Triphenyl-tetrazolium chloride
NCIMB National Collection of Industrial and Marine Bacteria Aberdeen

Yeast extract-Glucose-
YGC 1 month 4-10C
Chloramphenicol agar2
Yeast extract-Glucose-
YGO 1 month 4-10C
Oxytetracycline agar2
RIDA®COUNT Yeast and Mold
See manufacturer’s description
(R-Biopharm)
1
2
Either YGC or YGO is used.

1. Storage of the powdered media or reagents.
5 Equipment
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6 Procedure
6.1 Enumeration
Enumeration by pour plate

1. Prepare of a resuscitated sample a suitable dilution series dependent on the
presumed contamination level of the sample (see method M01_01ME).
2. Pipette 1 ml of the liquid into a sterile Petri dish.
3. Add about 15 ml YGC or YGO (tempered at 45 ± 2°C), mix well and allow to solidify.
4. Incubate the plates for 4-5 days at 25 ± 1°C.
5. Count the yeast and mold colonies after preferably 5 days. Because the plates are
sometimes overgrown after 5 days, the plates can also be counted after 4 days.
When after 5 days the dishes are overgrown, the next dilution is counted or the
counts of day 4 are used.
Enumeration by RIDA®COUNT Yeast and Mold card

1. Prepare of a resuscitated sample a suitable dilution series dependent on the
2. Open the plastic foil of the RIDA®COUNT Yeast and Mold card, pipette 1 ml of the
liquid directly on a card and cover it with the foil.
3. Incubate the cards for 72 hours at 25 ± 1°C.
4. Count all the red dots. Yeast and molds cannot be clearly distinguished. Yeasts
usually grow with sharp edges and molds grow more with diffuse edges. But the
result can only be given as one total result for yeast and molds.
Mould
Mould
Yeast
NOTE:
®
Strongly-colored matrices like chocolate can not be analyzed with the RIDA COUNT method!
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6.2 Confirmation
The identity of doubtful colonies can be investigated by microscopic examination.
For a reliable identification on species level it is advised to perform the identification with
molecular biological tools e.g. 16S rRNA gene sequencing.
NOTE:
Prevent the opening of dishes on which molds or yeasts are grown because some
are infectious or provoke allergic responses. Furthermore, spreading of the mold
spores into the laboratory should be prevented. When the dish has to be opened
for confirmation, take precaution measures to prevent spreading.
Secondary mold colonies may develop when the plates are moved or turned before the
end of the incubation period. Secondary colonies develop by outgrowth of spores of the
molds. These secondary molds should not be counted.

Positive control: Yeast: e.g. Culti loop Candida albicans (Oxoid)
if necessary:
Molds: e.g. Culti loop Apergillus niger (Oxoid)
Negative control: Enterococcus faecium NCIMB QC 50032
Blank -irradiated infant milk formula or a comparable product
NOTE:
To prevent the contamination of incubators, it is forbidden to use molds as a positive control.

The number of yeast and the number of molds are counted separately. Calculate the
number of yeasts and molds as described in M01_01AA. Express the results as CFU per
8 Validation
See validation report M01_06VB and M01_06VB02.
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9 References
The above mentioned methods are based on the following references but do not
1. International Dairy Federation, “IDF standard 94B - Milk and milk products
Enumeration of yeasts and moulds (colony count at 25°C)”, 1990.
2. International Organization for Standardization, “ISO 7698 - Cereals, pulses and
derived products - Enumeration of bacteria, yeasts and moulds”, 1990.
3. International Organization for Standardization, “ISO 7954 - Microbiology - General
guidance for enumeration of yeasts and moulds - Colony count technique at 25°C”,
1987.
4. International Organization for Standardization, “ISO 6611/IDF 94 – Milk and milk
products – Enumeration of colony-forming units of yeasts and/or moulds – Colony-
count technique at 25°C, 2004.
5. Marquart, P.J.; Weenk, G.H “Comparison Mould count 22°C and 30°C”, 1991,
Nutricia report PJM/SN/4410.
6. Mislivec P.B., Beuchat L.R., Cousin M.A., Chapter 16, “Yeast and moulds” in:
“Compendium of methods for the microbiological examination of foods” C.
Vanderzandt, D.F. Splittstoesser (eds), 1992, American Public Health Association,
Washington, p.239-249.
Division.
9. DIN 10186 “Microbiological analysis of milk – Enumeration of yeasts and moulds –
Reference method”.
10 Attachments
Not applicable
66
ISO 6611/IDF 94 as the reference standard for the current method

The current method of enumeration and detection of yeast and molds M01_06ME is based on
ISO 6611:2004/IDF 94 “Milk and milk products — Enumeration of colony-forming units of yeasts
and/or molds — Colony-count technique at 25°C” which recommends the use of YGC/YGO as
culture media.
There is another ISO norm ISO 21527-2:2008 “Microbiology of food and animal feeding stuffs --
Horizontal method for the enumeration of yeasts and moulds -- Part 2: Colony count technique in
products with water activity less than or equal to 0.95”. In this ISO norm DG 18 medium,
Dichloran 18% glycerol agar (18%: mass concentration), is recommended to use for samples
with aw < 0.90. DG 18 is regarded as well suited for the enumeration of xerotolerant and
moderately exrophilic species. The aw of powdered infant formula is in the range from 0.2 to 0.5.
A new validation will be carried out on powdered milk samples using the DG 18 medium.
RIDA®COUNT card as an alternative method with shortened detection time

In ISO 6611:2004 the incubation time of inoculated YGC plate is 5 days (120 h).
RIDA®COUNT card provided by R-Biopharm is a dehydrated ready-to-use card containing YGC
medium. Additionally TTC solution is included in RIDA®COUNT card as well which makes the
colonies visible in red color.
Based on data in validation report (M01_06_VB02), the incubation time can be reduced to 72 h if
the RIDA®COUNT card is used for enumeration of yeast and molds, without hurting the recovery
rate. By contrast, the minimal incubation time of conventional method is 4 days (96 h).
Not all matrices are suitable for RIDA®COUNT card. Strongly-colored matrice like chocolate
makes the read and detection of red colonies difficult and can therefore lead to a decreased
recovery rate. For such matrices colonies are well visible only on pour-plated YGC medium.
It is not possible to distinguish between yeast and molds on the RIDA®COUNT card, while the
conventional method enables the visual separation between two different types of microbes.
Furthermore the conventional method represents the only option in case a further identification of
colonies is necessary.
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Protocols for yeast and molds: ISO method and CLF method
68
ENTEROBACTERIACEAE DETECTION AND MPN
69
70
No. M01_07ME_v05(EB detection MPN).doc
Enterobacteriaceae – Detection/MPN
page: 1 version: 5
of: 6 issued: 23.06.2014
printed: 19.05.2014

V3  Revision of ISO 8523:1991
 Changes in detection procedure: VRBG tube is replaced by
VRBG plate
V4 (16.04.10)  Culture media and reagent are not tested with ecometrie
V5 (17.02.14)  Modern biological Identification tools have been added to 6.2
 Most probable number (MPN) table added
Name Signature Date

2000
Bredius
revised by: Sha
2014
Zhu
verified (professional): Jana
2014
Melzer
2014
Wollnitz
approved: Matthias
2014
Fischer
71
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of: 6 issued: 23.06.2014
printed: 19.05.2014

The method for the detection of Enterobacteriaceae can be applied to products intended
for human consumption and animal foodstuff, and environmental samples in the area of
food production and food handling.

This method is based on ISO 21528-1: 2004.
For detection of Enterobacteriaceae an enrichment step is performed in
Enterobacteriaceae Enrichment broth (EEB). This selective medium contains brilliant
green and ox bile which are inhibitory to competitive flora.
The presence of Enterobacteriaceae in the EEB is detected on violet red bile glucose agar
(VRBG). In VRBG the selective agents are bile salts and crystal violet which inhibit the
growth of gram positive bacteria. Enterobacteriaceae are able to ferment the glucose
present in VRBG, which is visible by a deep-red/purple color change due to acid
production. In addition, a reddish zone is visible due to the precipitation of bile salts.
VRBG is however not completely specific for Enterobacteriaceae, other microorganisms
like Aeromonas and Pseudomonas species can also grow on the medium.
If the confirmation procedure is not applied, this method is not able to discriminate
between Enterobacteriaceae and other bile resistant microorganisms like Aeromonas and
Pseudomonas species.

Enterobacteriaceae Glucose fermenting, gram negative, oxidase negative and
facultative anaerobic rods. Glucose fermentation is often (but not
always) accompanied by gas formation.
Enterobacteriaceae Enrichment Broth EEB 1 month 4-10°C
Violet red bile glucose agar3 VRBG 2 days 4-10°C
Tryptone soy agar TSA 1 month 4-10°C

1
2
Confirmation is not always necessary. E.g. API 32E or API 20E can be used
3
VRBG is also called VRBD (violet red bile dextrose agar)
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5 Equipment
6 Procedure
6.1 Detection/MPN enumeration
An enrichment step is necessary to be able to detect low numbers of Enterobacteriaceae.
 Detection of Enterobacteriaceae in 100; 50; 25 or 10 gram

1. Take x ml of the resuscitated sample (1:10 dilution in BPW) (see M01_01ME) and
add x ml double concentrated EE.
2. Incubate the samples for 24  3 hours at 30 ± 1°C.
 Detection of Enterobacteriaceae in 4 x 1 gram/ 4 x 10 ml

1. Pipet 10 ml of resuscitated sample (see method M01_01ME) into a tube with 10 ml
double concentrated EE. For each sample 4 tubes are inoculated in such a way.
 Detection of Enterobacteriaceae in 4 x 0.1 gram / 4 x 1 ml or less

1. Pipet 1 ml of the resuscitated appropriated dilution (see method M01_01ME) into a
tube with 10 ml single concentrated EE. For each sample 4 tubes are inoculated in
such a way.
NOTE:
For MPN enumeration, inoculate an appropriate number of flasks/tubes with the same dilution depending on
the desired accuracy of the results. As a general rule, the technique specified requires three flasks or tubes
per dilution.
3. Each pre-enrichment has to be streaked on one VRBG plate. In case of the 4 x 1g

and 4 x 0.1g analyses one VRBG plate is used for the 4 straight streaks of the 4
enrichments.
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100; 50; 25; 10 g 4x1g 4 x 0.1 g

1 pre-enrichment 4 pre-enrichments 4 pre-enrichments
4. Incubate the inoculated VRBG -plates for 24  3 hours at 30 ± 1°C.

5. Read the (number of) positive streaks.
NOTE:
The selectivity of VRBG diminishes after 24 hours incubation and organisms previously suppressed may
exhibit growth.
6.2 Confirmation
The confirmation of Enterobacteriaceae is optional:
1. Make a pure streak of a well isolated colony onto a TSA plate and incubate at 30 ±
1°C for 18-24h.
2. Test the colony by oxidase- and catalase- reaction and gram staining.
3. Identify well isolated colonies using a biochemical identification test kit (API 32E or
API 20E).
4. For further identification, 16S rRNA gene sequencing and MALDI TOF MS are
recommended.

Positive controls: Escherichia coli ATCC 11775
Enterobacter cloacea ATCC 13047
Negative controls: Staphylococcus aureus ATCC 6538
 For detection in 100; 50; 25; 10 gram:

Express the results as the absence or presence of Enterobacteriaceae per weight or per
volume of sample (see M01_01AA).
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 For detection in 4 x 1 gram or smaller quantities:

Express the result as the number of positive VRBG plates per weight or per volume of
sample (see M01_01AA).
 For MPN enumeration:

MPN evaluation is performed according to MPN table (see 11).
8 Validation
9 References
1. International Organisation for Standardisation, “ISO 21528, Horizontal methods for
the detection and enumeration of Enterobacteriaceae – Detection and enumeration
by MPN technique with pre-enrichment” 2004.
2. Mossel, D.A.A, Eelderdink I. Koopmans, M, Rossem van F. “Influence of carbon
source, bile salts and incubation temperature on recovery of Enterobacteriaceae from
food using MacConkey-type agars” Journal of Food Protection, 1979, vol. 42, no. 6, p.
470-475.
3. Post D.E. “Food borne pathogens monograph number 5 – Escherichia coli, Shigella
species” Oxoid, March 1998, p. 23-24.
10 Attachments
Not applicable
75
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11 Most probable number (MPN) table for dilution in triplicate for 1 g, 0.1g and
0.01 g. (EN ISO 7218:2007)
Number of positive results MPN in CFU/ g Confidence interval ≥ 95%
0 0 0 < 0.30 0.00 0.94

0 0 1 0.30 0.01 0.95
0 1 0 0.30 0.01 1
0 1 1 0.61 0.12 1.7
0 2 0 0.62 0.12 1.7
0 3 0 0.94 0.35 3.5
1 0 0 0.36 0.02 1.7
1 0 1 0.72 0.12 1.7
1 0 2 1.1 0.4 3.5
1 1 0 0.74 0.13 2
1 1 1 1.1 0.4 3.5
1 2 0 1.1 0.4 3.5
1 2 1 1.50 0.5 3.8
1 3 0 1.60 0.5 3.8
2 0 0 0.92 0.15 3.5
2 0 1 1.4 0.4 3.5
2 1 0 1.5 0.4 3.8
2 1 1 2.0 0.5 3.8
2 2 0 2.1 0.5 4
2 2 1 2.8 0.9 9.4
2 2 2 3.5 0.9 9.4
2 3 0 2.9 0.9 9.4
2 3 1 3.6 0.9 9.4
3 0 0 2.3 0.5 9.4
3 0 1 3.8 0.9 10.4
3 0 2 6.4 1.6 18.1
3 1 0 4.3 0.9 18.1
3 1 1 7.5 1.7 19.9
3 1 2 12 3 36
3 1 3 16 3 38
3 2 0 9.3 1.8 36.00
3 2 1 15 3 38
3 2 2 21 3 40
3 2 3 29 9 99
3 3 0 24 4 99
3 3 1 46 9 198
3 3 2 110 20 400
3 3 3 > 110.00
76
30°C as incubation temperature for EE broth and VRBG agar in M01_07ME

In the current method an incubation temperature of 30°C for EE enrichment, VRBG and TSA is
recommended due to the fact that some psychrotrophic Enterobacteriaceae do not grow at a
higher temperature of 37°C.
In the ISO 21528-1:2004 the incubation temperature of 37°C is the priority recommendation
when the detection of Enterobacteriaceae is for a hygienic indicator. 30°C is left as option when
the enumeration is for a technical purpose or when the psychrotrophic Enterobacteriaceae are
involved.
Combination of pre-enrichment and selective enrichment

Compared to the ISO method, the current method combines the non-selective enrichment
including 2 h resuscitation and the selective enrichment in EE broth on the first day, which can
significantly shorten the detection procedure.
Bile resistant gram negative bacteria detectable using EB method

From the taxonomic point of view, some gram negative rods such as Aeromonas, Acinetobacter
and Pseudomonas do not belong to the family Enterobacteriaceae. As most common
opportunistic human pathogens, these bacteria have an optimal growth temperature between 30-
37°C similar to the temperature of human body and pose therefore risks of nosocomial
infections.
However from the analytical point of view Aeromonas, Acinetobacter and Pseudomonas are
grouped to the “bile resistant gram negative bacteria” which can be detected by using
Enterobacteriaceae method until the isolation step: they are able to grow in EE broth and form
typical rose pink colonies on VRBG like members of the Enterobacteriaceae family.
The term “bile resistant gram negative bacteria” has been introduced by the United States
Pharmacopeia and European Pharmacopoeia. It is no topic of ISO or food legislation. The
methods are not validated for the full group but side detections are as unacceptable as
Enterobacteriaceae.
These bile resistant gram bacteria can be differentiated from Enterobacteriaceae through
identification tests: While Enterobacteriaceae species are oxidase negative, Aeromonas and
Pseudomonas are positive for oxidase. Acinetobacter is oxidase negative like
Enterobacteriaceae, however by using further biochemical or molecular biological techniques
(API, 16S rRNA gene sequencing and MALDI TOF) a clear identification and differentiation of
Acinetobacter from the Enterobacteriaceae family is possible.
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Protocols for EB detection: ISO method and CLF method
78
CLOSTRIDIUM PERFRINGENS DETECTION
79
80
No. M01_10ME_v04(Cl.perfringens detection).doc
Clostridium perfringens - Detection
of: 11 written: 02.08.2010
11
printed: 27.06.2014

 DIN EN ISO 7937: 2004-08 replaces DIN EN 13401:1999-06
 Confirmation:
McClung test was replaced by the reverse CAMP test
Identification using a biochemical test kit (Rapid ID 32A)
V4 (02.04.14)  Addition of confirmation tools e.g. biochemical test kit 50 CHL and
16S rRNA gene sequencing and MALDI TOF MS
Name Signature Date

1st version written by: Marian
2000
Bredius
revised by: Sha
2014
Zhu
2014
Melzer
2014
Wollnitz
approved: Matthias
2014
Fischer
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of: 11 written: 18.06.2010
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This method describes a procedure for detecting Clostridium perfringens in products
intended for human consumption and animal foodstuff, environmental samples and water.
In compliance with ISO and the European standard, this method does not distinguish
between Clostridium perfringens and a few other related types that are not frequently
found, such as Cl. paraperfringens and Cl. absonum.

This method is based on the standard method EN ISO 7937: 2004-08.
The sample is pre-enriched in Rapid Perfringens Medium (RPM).
The RPM was developed to detect low numbers of Cl. perfringens in foodstuffs.
Neomycin and polymixin are the selective components: Growth of gram positive and gram
negative microorganisms is inhibited by neomycin. Polymixin inhibits the growth of gram
negative microorganisms.
The elective feature of RPM is the stormy fermentation of the Crossley milk. The addition
of thioglycollate, ferrous sulfate, and Crossley milk to the medium creates reducing
ambient conditions, necessary for good growth of Cl. perfringens.
Cl. perfringens forms stormy fermentation and causes coagulation of the milk. For
incubation, the selective temperature of 46°C is used.
RPM test tubes that indicate stormy fermentation with strong gas formation are streaked
further onto tryptose sulfite cycloserine agar (TSC). The selective reagent in TSC is
cycloserine (a broad-spectrum antibiotic) and the elective reagent is ferrous sulfite. After
streaking on TSC agar, a layer of TSC agar is applied. Suspicious colonies grow black on
TSC agar.
To confirm the suspicious colonies, at least one of three tests has to be applied:
A. Reverse CAMP test

The alpha toxin production of Cl. perfringens is verified in the reverse CAMP test1. As
indicator strain, Streptococcus agalactiae which belongs to the serogroup B of
streptococci is used. Streptococcus agalactiae secretes the CAMP factor, a cocytolysin.
Through the synergetic haemolysis of the alpha toxin by Cl. perfringens and the CAMP
factor of Streptococcus agalactiae, a characteristic, arrowhead-shaped haemolytic pattern
is formed.
B. Biochemical test kits or molecular biological tools
1
CAMP: abbreviation for the first persons to describe the co-hemolytic effect of the group B streptococci: Christie,
Atkins, and Munch-Petersen
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C. Biochemical tests
Lactose fermentation, gelatin liquefaction, nitrate reduction, and immobility are
characteristic of Cl. perfringens.
Modifications
This method is based on the standard method EN ISO 7937:2004.
Following modifications have been applied:
 Additional selective pre-enrichment in RPM for 18-24 h at 46°C (see Erickson JE,
Deibel RH, 1978, Appl. Environ. Microbiol. pp 567-571).
 Confirmation methods using reverse CAMP test which is based on the standard
method DIN 10103:1993 and/or biochemical test kits (Rapid ID 32A) and/or molecular
biological tools
Cl. perfringens Gram-positive, lactose fermenting, gelatinase-positive, nitrate

reducing, immobile anaerobic growing rods. Lactose fermentation
is associated with strong gas formation.
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1
Medium/Reagent Abbreviation Advised maximum storage time
Rapid Perfringens Medium base RPM-b 1 month 4-10°C
Crossley milk CM 1 month 4-10°C

Polymixin solution 0.1% (w/v) Pol 1 month 4-10°C
Neomycin solution 1% (w/v) Neo 1 month 4-10°C
RPM-b + CM + Pol + Neo RPM 1 month 4-10°C
2
Tryptose sulfite cycloserine TSC 1 month 4-10°C
Streptococcus agalactiae type strain
1 month 4-10°C
ATCC 13813
Clostridium perfringens type strain
1 month 4-10°C
ATCC 13124; 19408
Columbia agar with 5% sheep blood COS 1 month 4-10°C
Lactose gelatin medium LG 3 weeks 4-10°C
Motility nitrate medium MN 1 month 4-10°C
0.4% (w/v) sulfanilic acid in 15% Nit 13 See manufacturer’s description

acetic acid
0.1% (w/v) amino-2-naphtahalene Nit 23 See manufacturer’s description

sulfonic acid in 15% acetic acid
Zinc powder Zn 1 month 4-10°C

1
The shelf life of the media prepared refers to media stored in the dark. Media kept longer than 1 month must
be stored in closable bottles (with twist caps).
2
Before using the agar, depending on the manufacturer, cycloserine supplement must be pipetted into the
freshly poured agar (temp. 45 ± 2ºC). After adding cycloserine, the slides may not be kept more than 24 h.
3
Nit 1 and Nit 2 (or comparable reagents) are available commercially. They are used for nitrate reduction in
identification tests, e.g. API from Biomérieux.
4
e.g. Rapid ID 32A
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4.1 RPM
Prepare test tube of at least 40 ml with 10 ml RPM base according to manufacturer’s
instructions.
To 500 ml Crossley milk, prepared according to manufacturer’s instructions, 7.5 ml of a
1% filter sterilized neomycin-sulfate solution and 12.5 ml of a 0.1% filter sterilized
polymixin – B sulfate solution are added.
Pipette 10 ml of Crossley milk into each test tube with 10 ml RPM base. Before adding
the sample, the test tubes are warmed in a water bath (approx. 45 ºC) and the medium is
then mixed well.
5 Equipment
Anaerobic jars in which tubes are incubated
6 Procedure
6.1 Detection
1. Prepare a suitable dilution series from a resuscitated sample (method M01_01ME)
2. Pipette 10 ml of the (diluted) sample into the RPM test tube. Draw the pipette up
slowly to empty it into the test tube. The pipette is not purged.
3. Incubate the test tubes for 18-24 h at 46 ± 1°C.
4. Make a streak from suspicious test tubes (e.g. stormy fermentation, gas formation,
coagulation of the milk) using a sterile inoculation loop onto a freshly prepared and
dried TSC agar.
5. Apply an over-layer of TSC.
6. Incubate the agar at 37 ± 1°C for 18-24 h anaerobically.
7. Black colonies must be confirmed.
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Cl. perfringens: Forms gas (stormy fermentation) in the RPM test tubes.
Black colonies are obtained in TSC agar.
On the surface of TSC over-layer, white-grey colonies with or
without a black center are expected.
NOTES:
The TSC plate should be exposed to air as shortly as possible. Slides with suspicious colonies are returned
to anaerobic jars. The slides are held under anaerobic conditions until the analysis is completed.
Cl. perfringens can be inactivated if they are stored at low temperatures for a long period.
When the coating is placed on the TSC plate, it is possible that some of the colonies develop on the surface.
These colonies are white-grey, with or without a black center.
6.2 Confirmation
 Reverse CAMP test

1. The indicator strain Streptococcus agalactiae is applied with an inoculation loop to a
COS plate over the middle of the Petri dish as a continuous inoculation line.
2. Colonies suspicious for Cl. perfringens on TSC are streaked onto the COS plate at a
distance of 1 mm and at a right angle vertically to this indicator line.
3. Incubation of COS at 37°C for 18-24 h anaerobically.
4. Cl. perfringens is considered confirmed if an arrowhead-shaped light area (ß-
heamolysis) forms in the area where the inoculation lines come together at a right
angle (see picture 1).
Positive control: On another medium, the Cl. perfringens type strain is streaked at a right
angle to the S. agalactiae.
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S. agalactiae S. agalactiae
(indicator strain) (indicator strain)
“Arrowhead-
shaped” double
hemolysis
suspicious colonies suspicious colonies
Picutre 1: (a) Placement of the inoculation lines for the reverse CAMP test, (b) Diagram of the arrowhead-
shaped double hemolysis that confirms Clostridium perfringens
NOTES:
Cultivation conditions of the indicator strain Streptococcus agalactiae:

Cultivation medium: COS agar
Temperature: 37 ºC
Anaerobic incubation
A streak of Streptococcus agalactiae can be stored in a refrigerator at 4-8°C for 1 week and be used for
conducting the reverse CAMP test.
 Identification with a biochemical test kit (Rapid ID 32A)

Colonies suspicious for Cl. perfringens on TSC are streaked onto a COS medium and
incubated for 24 h at 37°C anaerobically.
Identification using the biochemical test kit (e.g. Rapid ID 32A).
 Identification with molecular biological tools

For a further identification the molecular biological tools e.g. MALDI TOF MS and 16S
rRNA gene sequencing can be applied.
 Biochemical characteristics
For every colony to be confirmed, a MN and a LG test tube is placed in a boiling water
bath for 10 min (to remove air bubbles) and cooled to room temperature. This is not
necessary if freshly prepared test tubes (same day) are used.
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1. Inoculate a suspected colony into a MN and a LG test tube by a deep straight stab.
2. The test tubes are incubated at 37 ± 1°C for 18-24 h in an anaerobic jar.
3. The test tubes are checked for growth along the puncture: mobile bacteria show
scattered growth in the MN test tube. Immobile bacteria show growth only at the
puncture in the MN test tube.
4. The MN test tube is checked for the presence of nitrite:
Two drops each of Nit 1 and Nit 2 reagent are added to the test tube. If a red color
appears in the medium within 15 min, the reduction from nitrate to nitrite is confirmed.
If no red colouring occurs after 15 min, a small amount of zinc powder is added to the test
tube.
If no red colouring occurs within 10 min after adding zinc powder, no nitrate was reduced.
5. The LG test tube is kept at 0-5°C for 1 h before reading.
6. The LG test tube is checked for lactose fermentation. Fermentation causes the color
of the medium to change to yellow. Fermentation is also frequently associated with
gas formation.
7. The LG test tube is tested for liquefaction of the gelatin:
If the test tube is fluid after the cooling phase, hydrolysis of gelatin has taken place.
If the test tube has solidified again after the cooling phase, it is incubated again for
18-24 h at 37 ± 1°C. The test tube is again kept at 0-5°C for 1 h and then analyzed. The
total incubation time of the LG test tube should not be longer than 44 h.
Cl. perfringens: nitrate positive, immobile, lactose and gelatinase positive.
NOTES:
As the MN medium is semi-solid, a straight stab is often difficult. Therefore, there is often growth in one
direction. This is assessed as immobile.
Some Cl. perfringens strains are not able to hydrolyze gelatin within 48 h. But if all other reactions are
positive (RPM, TSC, lactose, nitrate, and mobility), the sample is considered positive because of the
confirmation of the other tests.
Cl. absonum and Cl. sardiniensis can hydrolyze gelatin when incubated longer than 44 h. Therefore the LG
test tube should not be incubated any longer than 44 h.
Cl. bifermentans is lactose negative, gelatin positive, mobile, and nitrate negative .
If the result is not clear using one of the confirmation methods A, B, C or D, at least one more confirmation
method should be additionally applied.
The reliability of the confirmation reaction is 96% using the reverse CAMP test and 93.96% using
the Rapid ID 32A test kit.
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Positive control: Cl. perfringens ATCC 13124, ATCC 19408
Negative control: B. cereus ATCC 9139
7 Evaluation, calculation, interpretation

The result is expressed as the presence or absence of Cl. perfringens per weight or
volume of the sample (see method M01_01AA).
8 Validation
See validation report M01_10VB and M01_10VB_addendum 1.
9 References
The method described here refers to the references listed. But it does not necessarily
reflect the exact methods.
1. Boer E. de, Boot E.M. “Comparison of methods for isolation and confirmation of
Clostridium perfringens from spices and herbs”, Journal of food protection, 1983, vol.
46, No. 6 (June), p. 533-536.
2. International Organisation for Standardization, 2004. EN ISO 7937 “Microbiology of
food and animal feeding stuffs - Horizontal method for the enumeration of Clostridium
perfringens - Colony-count technique”, 2004-08.
3. Erickson J.E., Deibel R.H. “New medium for rapid screening and enumeration of
Clostridium perfringens in foods”, October 1978, Applied and environmental
microbiology, p. 567-571.
4. FAO “Chapter 17 - Clostridium perfringens” in: “Manual of food quality control, vol. 4:
microbiological analysis”, W. Andrews (ed), 1992, Rome, FAO, p. 207-212.
5. Harmon S.M., and Kautter D.A. “Media for confirming Clostridium perfringens from
food and feces” 1978, Journal of Food Portection, vol. 41, p. 626-630.
6. Jeffrey E., Harmon S. M.“Chapter 16 Clostridium perfringens” In: “FDA bacteriological
analytical manual” FDA, 1995, 8th. edition, p. 16.01-16.06.
7. Post D.E. “Food-borne pathogens monograph number 4 - Clostridium perfringens,
Bacillus cereus” Oxoid, Unipath Limited, Basingstoke, England.
8. Schalch B., Eisgrubber H., Geppert P., Stolle A.“Vergleich von vier Routineverfahren
zur Bestätigung von Clostridium perfringens aus Lebensmitteln” Archiv für Lebens-
mittelhygiene, Januar/Februar 1996 vol. 47, No 1 p. 27-30.
9. Weenk G., Brink J.v.d .“New confirmation test for Clostridium perfringens” Ref no
170/GW/mms, 19-4-1989, Internal memo Nutricia.
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10. Deutsches Institut für Normung, DIN 10103 (1993-08). “Mikrobiologische

Bestimmungen in Fleisch und Fleischerzeugnissen – Bestimmung von mesophilen
sulfitreduzierenden Clostridien“.
11. Munch-Petersen, E., and R. Christie. 1947. The effect of the interaction of
Staphylococcus b toxin and group B streptococcus substance on red blood
corpuscles and its use as a test for the identification of Streptococcus agalactiae. J.
Pathol. Bacteriol. 59:367–371.
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10 Attachments
Attachment 1: Motility nitrate medium and lactose gelatin medium in g/l

Media Ingredients Weight (g)
motility nitrate Enzymatic digest of casein 5.0
Meat extract 5.0
Galactose 5.0
Glycero 5.0
Potassium nitrate 1.0
Disodium hydrogen orthophosphate 2.5
Agar 1.0 to 5.0

lactose gelatin Enzymatic digest of casein 15.0
Yeast extract 10.0
Lactose 10.0
Gelatin 120.0
Phenol red 0.05
91
Selective enrichment in RPM for Cl. perfringens detection

The current method M01_10ME is based on ISO 7937:2004. However the ISO 7937 is an
enumeration method and no detection method for Cl. perfringens as international norm standard
is available.
The application of RPM is based on the publication of Erickson J.E. and Deibel R.H.: “New
medium for rapid screening and enumeration of Clostridium perfringens in foods”, Applied and
environmental microbiology, p. 567-571, 1978. This medium was developed to detect low
numbers of Cl. perfringens in foods. The elective feature of this medium is the stormy
fermentation of the milk. This reaction is also used by the FDA and FAO in the presumptive
confirmation of Cl. perfringens in Iron Milk Medium (see Chapter 17 - Clostridium perfringens in:
Manual of food quality control, vol. 4: microbiological analysis, W. Andrews (ed), 1992, Rome,
FAO, p. 207-212; Jeffrey E., Harmon S. M. Chapter 16 Clostridium perfringens In: FDA
bacteriological analytical manual” FDA, p. 16.01-16.06., 8th edition, 1995)
The selective enrichment in RPM followed by streaking on TSC agar has shown a high sensitivity
and a high specificity for Cl. perfringens detection in the validation tests (see M01_10VB).
CAMP test, API Rapid ID 32A and 16S rRNA gene sequencing as confirmation
options
The reverse CAMP test represents a reliable and easy test system to confirm a Cl. perfringens
strain with a sensitivity of 96%. Only one isolate from milk powder with cereals was identified as
Cl. perfringens with both API and 16S rRNA gene sequencing. In the validation tests the API
Rapid ID 32A showed a sensitivity of approximately of 94%.
In case of doubtful identification results, it is recommended to apply more than one identification
system to ensure a reliable outcome.
92
Protocols for EB detection: ISO method and CLF method
93
94
ENTEROCOCCI DETECTION
95
96
No. M01_12ME_v04(Enterococci detection).doc
Enterococci – Detection
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V3 (03.02.11)  Addition of the modifications compared to the reference method
 Deletion of the ecometrie method to test quality of media
 Deletion of reference capsules from SVM as internal control
V4 (06.02.14)  Application for water has been deleted
 Table of biochemical characteristics of Enterococci has been
added to 6.3
 Modern biological confirmation tools e.g. 16S rRNA gene
sequencing and MALDI TOF MS have been added to 6.3.
Name Signature Date

First version written by: Marjan
Bredius 1999
revised by: Angela
Lindenstrauß 2014
Zhu 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
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This method for the detection of Enterococci can be applied to food products.

For the detection of Enterococci Kanamycin Aesculin Azide Broth (KAAB) is used. KAAB
contains sodium azide and kanamycin as the selective agents. Sodium azide is inhibitory
to gram negative bacteria. Kanamycin is a broad spectrum antibiotic, but not inhibitory to
Enterococci. KAAB contains aesculin as elective agent. Enterococci are able to hydrolyse
aesculin into aesculetin. Aesculetin forms a black aesculetin-Fe-complex with the Fe
present in the medium. Suspected tubes are streaked onto Kanamycin Aesculin Azide
Agar (KAAA) which has the same selective and elective properties as KAAB.
Modifications
This method is based on: Dijk, R, Grootenhuis A “Microbiologie van voedingsmiddelen -
methoden, principes en criteria” Keesing Noordervliet, Houten, 1999, p.157-158.
Following modification has been performed:
 Selective pre-enrichment in KAAB

Enterococci Gram positive, catalase negative, facultative anaerobic cocci,
which was previously named as Lancefield D Streptococci


Kanamycin aesculin azide agar2 KAAA 1 month 4-10°C
Kanamycin aesculin azide broth2 KAAB 1 month 4-10°C
Trypton soy broth TSB 1 month 4-10°C
Bile broth (40%) BB See manufacturer’s description
Biochemical identification kits3 See manufacturer’s description

1
The shelf life of the prepared media/solutions is given when stored in the dark.
2
Dependent on the manufacturer, a Kanamycin supplement has to be added to the KAAA and KAAB.
3
API 20 Strep or rapid ID 32 Strep or other comparable kits. A confirmation is not always necessary.
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Sodium azide
KAAA and KAAB contain sodium azide. This substance is highly toxic. Precautions should
be taken to avoid direct contact. Especially the inhalation of fine dust during preparation
of dehydrated media should be avoided. Therefore gloves and a mouth cap should be
worn.
 Gloves
Gloves must be inspected prior to use. Use proper glove removal technique (without
touching glove's outer surface) to avoid skin contact with this product.
 Respiratory protection
Wear respiratory protection. Avoid the creation of dust. Avoid inhaling dust. Ensure
adequate ventilation.

4.1 Bile broth

Dissolve 5.0 g tryptone; 2.5 g yeast extract; 5.0 g glucose and 40.0 g dried ox-bile in 1
liter demineralised water.
Sterilise the BB for 15 min at 121°C.
The pH value after sterilisation should be 7.0 ± 0.1 at 25°C.
5 Equipment
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6 Procedure
6.1 Detection
1. Prepare of a 1:10 resuscitated sample, a suitable dilution series dependent on the
2. Pipette 1 ml or 0.1 ml of the appropriate dilution into a tube which contains 10 ml
KAAB.
3. Incubate the tube at 37 ± 1°C for 48  3 h.
4. Read the tubes: tubes which show any blackening and/or contain coagulated sample
together with a white color are suspected and need to be further analysed. All other
tubes are regarded as negative for Enterococci and do not need to be further
analyzed.
5. Streak a loopful of culture of the suspected tubes onto a pre-dried KAAA plate.
6. Incubate the KAAA plates up side down at 37 ± 1°C for 18-24 h.
7. Enterococci are small, round, smooth, grey- white colonies, surrounded by a black
zone.
NOTES:
Bacilli and Lactobacilli can give false positive results on KAAA.

Confirmation of the colonies is always necessary for fermented products.
6.2 Confirmation
A representative number of suspected colonies have to be confirmed if necessary. One or
more of the following tests have to be performed.
 Microscopic examination
 Catalase test
 Growth test at 45°C

1. Check for growth of the isolates in TSB at 45 ± 1°C and 37 ± 1°C after 24 ± 3 h.
2. When no growth occurs at both temperatures, it indicates that no viable colony has
been inoculated and the test is not valid.
 Growth test in bile broth

1. Check for growth in 40% bile broth and TSB at 37 ± 1°C for 24  3 h.
2. Streak a loopful of incubated bile broth and TSB on a KAAA plate.
3. Incubate the KAAA plates at 37°C for 24 ± 3 h.
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4. Read the KAAA plates as described above (6.1.4).

Identify the isolate by using a biochemical identification test kit (e.g. API 20 Strep or rapid
ID 32 Strep).
For further identification, 16S rRNA gene sequencing or MALDI TOF MS can be applied.
Table 1: Biochemical characteristics of Enterococcus
Biochemical reactions Results
Gram reaction Gram positive cocci (mostly in chains)
Oxidase reaction Oxidase negative
Catalase reaction Catalase negative (sometimes weakly positive)
Growth test Growth at 37°C and 45°C
Bile resistance Bile resistant (growth in 40% bile growth)
NOTE:
Lactobacilli are rods and do not grow at 45°C. Bacilli are rods and are catalase positive.

Positive controls: Enterococcus faecium NCIMB QC 50032
Enterococcus faecalis NCIMB QC 50030
Negative controls: Lactococcus lactis ATCC19435
Escherichia coli ATCC 11775

Absence or presence of Enterococci is expressed per weight or ml of sample (see also
M01_01AA).
8 Validation
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9 References
1. Anon. “Pharmacopeia of Culturemedia for Food Microbiology”. Int. J. Food Microbiol.
1987, vol. 5, p. 221.
2. Barrow, G.I. and Feltham, R.K.A. In: “Cowan and Steel's Manual for the Identification
of Medical Bacteria” Cambridge University Press, 1993
criteria” Keesing Noordervliet, Houten, 1999, p.157-158
4. Hardie, J.J..Genus Streptococci. In: Sneath, P.H.A. (Ed) “Bergey's Manual of
Systematic Bacteriology 2”, Williams & Wilkins, Baltimore, 1986, p. 1043-1071.
5. International Organisation for Standardisation, “ISO 7899/1, Waterquality - Detection
and enumeration of faecal Streptococci” 1984.
6. Mossel, D.A.A., Bijker P.G.H. und Eelderink I..”Streptokokken der Lancefield-Gruppe
D in Lebensmitteln und Trinkwasser - Ihre Bedeutung, Erfassung und Bekämpfung”
Archiv. für Lebensmittelhygiene, 1978, vol. 29, p. 121-127.
7. M01_08AA “Catalase test” CLF method, Microbiology Division.
10 Attachments
Not applicable
102
For Enterococci two ISO methods are available: ISO 7899-1:1998 for intestinal Enterococci in
surface and waste water (MPN in liquid medium) and ISO 7899-2:2000 for intestinal Enterococci
in surface and waste water (membrane filtration).
This means no suitable ISO reference is available for detection and enumeration of Enterococci
in infant formula. The relevant CLF methods for Enterococci are M01_12ME (Enterococci
detection) and M01_13ME (Enterococci enumeration).
The ISO references mentioned above are the only available methods; any lab will have to adapt
the methods somehow to the matrix infant formula.
Among the two ISO norms, the ISO 7899-2:2000 “Water quality - Detection and enumeration of
intestinal enterococci – Part 2: Membrane filtration method” is closer to M01_13ME but the
requested agars are different (Slanetz-Bartley agar combined with Bile Aesculine Acide agar, see
table 2 and 3) from the one used in CLF method (KAAA, see Table 1). The Slanetz-Bartley agar
is less specific compared to KAAA and the Enterococci count can be overestimated in case of
the presence of background flora or probiotic strains.
Table 1. Composition of KAAA in M01_12/13ME
Media Ingredients Weight (g/l) Reference
KAAA Peptone from casein 20.0 g M01_12ME
Merck, 1.05222 M01_13ME

Yeast extract 5.0 g
Sodium chloride 5.0 g
Sodium citrate 1.0 g
Sodium azide 0.15 g
Kanamycinsulfat 0.02 g
Aesculin 1.0 g
Ammonium iron (III) citrate 0.5 g
Agar 15 g
103
1
Table 2. Composition of Slanetz-Bartley medium in ISO 7899-2:2000
Slanetz-Bartley medium Tryptose 20.0 g ISO 7899-2:2000
Yeast extract 5.0 g
Glucose 2.0 g
Dipotassium hydrogenphosphate 4.0 g
Sodium azide 0.4 g
Agar 8 to 18 g
1
TTC solution: dissolve 2,3,5-triphenyltetrazolium chloride in water in 1 g/100 ml. Sterilize by filtration. Add
10 ml TTC solution to the Slanetz-Bartley medium.
Table 3. Composition of bile aesculin azide agar in ISO 7899-2:2000
Bile aesculin azide agar Tryptone 17.0 ISO 7899-2:2000
Merck, 1.05222 Peptone 3.0
Yeast extract 5.0
Ox-bile, dehydrated 10.0
Sodium chloride 5.0
Aesculin 1.0
Ammonium iron (III) citrate 0.5
Sodium azide 0.4
Agar 8 to 18
104
Protocols for Enterococci detection: M01_12ME and ISO norm
105
106
ENTEROCOCCI ENUMERATION
107
108
No. M01_13ME_v04(Enterococci enumeration).doc
Enterococci – Enumeration
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V4 (06.02.14)  Application for water has been deleted
 Spreading method as an option for enumeration to 6.1
 Table of biochemical characteristics of Enterococci has been
added to 6.3
 Molecular biological confirmation tools e.g. 16S rRNA gene
sequencing and MALDI TOF MS have been added to 6.3.
Name Signature Date

First version written by: Marjan
Bredius 1999
revised by: Sha
Zhu 2014
verified (professional): Thomas
Koch 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
109
of: 6 written: 03.02.2011
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printed: 23.06.2014

This method for the enumeration of Enterococci can be applied to food products.

This method is based on: Dijk, R, Grootenhuis A “Microbiologie van voedingsmiddelen -
methoden, principes en criteria” Keesing Noordervliet, Houten, 1999, p.157-158.
For the enumeration of Enterococci Kanamycin Aesculin Azide agar (KAAA) is used.
KAAA contains sodium azide and kanamycin as the selective agents. Sodium azide is
inhibitory to gram negative bacteria. Kanamycin is a broad spectrum antibiotic, but not
inhibitory to Enterococci. KAAA contains aesculin as elective agent. Enterococci are able
to hydrolyse aesculin into aesculetin. Aesculetin forms a black aesculetin-Fe-complex with
the Fe present in the medium.
Suspected tubes are streaked onto Kanamycin Aesculin Azide Agar (KAAA) which has
the same selective and elective properties as KAAB.

Enterococci Gram positive, catalase negative, facultative anaerobic cocci,
which was previously named as Lancefield D Streptococci


1
Kanamycin aesculin azide agar2 KAAA 1 month 4-10°C
Bile broth (40%) BB See manufacturer’s description

3
Biochemical identification kits See manufacturer’s description
1
2
Dependent on the manufacturer, a Kanamycin supplement has to be added to the KAAA and KAAB.
3
API 20 Strep or rapid ID 32 Strep or other comparable kits. A confirmation is not always necessary.
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Sodium azide
KAAA and KAAB contain sodium azide. This substance is highly toxic. Precautions should
be taken to avoid direct contact. Especially the inhalation of fine dust during preparation
of dehydrated media should be avoided. Therefore gloves and a mouth cap should be
worn.
 Gloves
Gloves must be inspected prior to use. Use proper glove removal technique (without
touching glove's outer surface) to avoid skin contact with this product.
 Respiratory protection
Wear respiratory protection. Avoid the creation of dust. Avoid inhaling dust. Ensure
adequate ventilation.

4.1 Bile broth

Dissolve 5.0 g tryptone; 2.5 g yeast extract; 5.0 g glucose and 40.0 g dried ox-bile in 1
liter demineralised water.
Sterilize the BB for 15 min at 121°C.
The pH value after sterilisation should be 7.0 ± 0.1 at 25°C.
5 Equipment
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6 Procedure
6.1 Detection
1. Prepare of a 1:10 diluted sample and resuscitate it for 2 h, a suitable dilution series
dependent on the presumed contamination level of the sample (see method
M01_01ME).
2.1 Pipette 1 ml of sample dilution into a Petri dish.
Add about 15 ml KAAA (tempered at 45 ± 2°C), mix well and allow to solidify.
2.2 Optional spreading method:
Spread 1 ml of sample dilution on a pre-dried KAAA plate and distribute the sample
homogenously with a sterile spatula.
3. Incubate the KAAA plates upside down at 37 ± 1°C for 48 ± 3 h.
4. Count the typical colonies:
Enterococci are small, round, smooth, grey- white colonies, surrounded by a black
zone.
6.2 Confirmation
A representative number of suspected colonies have to be confirmed if necessary. One or
more of the following tests have to be performed.
 Microscopic examination
 Catalase test
 Growth test at 45°C

1. Check for growth of the isolates in TSB at 45 ± 1°C and 37 ± 1°C after 24 ± 3 h.
 Growth test in bile broth

1. Check for growth in 40% bile broth and TSB at 37 ± 1°C for 24  3 h.
2. Streak a loopful of incubated bile broth and TSB on a KAAA plate.
3. Incubate the KAAA plates at 37°C for 24 ± 3 h.
4. Read the KAAA plates as described above (6.1.4)
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Identify the isolate by using a biochemical identification test kit (e.g. API 20 Strep or rapid
ID 32 Strep).
For further identification, 16S rRNA gene sequencing or MALDI TOF MS can be applied.
Table 1: Biochemical characteristics of Enterococcus
Biochemical reactions Results

Gram reaction Gram positive cocci (mostly in chains)
Oxidase reaction Oxidase negative
Catalase reaction Catalase negative (sometimes weakly positive)
Growth test Growth at 37°C and 45°C
Bile resistance Bile resistant (growth in 40% bile growth)
NOTE:
Lactobacilli are rods and do not grow at 45°C. Bacilli are rods and are catalase positive.

Positive controls: Enterococcus faecium NCIMB QC 50032
Enterococcus faecalis NCIMB QC 50030
Negative controls: Lactococcus lactis ATCC19435

Absence or presence of Enterococci is expressed per weight or ml of sample (see also
M01_01AA).
8 Validation
9 References
1. Anon. “Pharmacopeia of Culturemedia for Food Microbiology”. Int. J. Food Microbiol.
1987, vol. 5, p. 221.
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2. Barrow, G.I. and Feltham, R.K.A. In: “Cowan and Steel's Manual for the Identification
of Medical Bacteria” Cambridge University Press, 1993.
criteria” Keesing Noordervliet, Houten, 1999, p.157-158.
4. Hardie, J.J..Genus Streptococci. In: Sneath, P.H.A. (Ed) “Bergey's Manual of
Systematic Bacteriology 2”, Williams & Wilkins, Baltimore, 1986, p. 1043-1071.
5. International Organisation for Standardisation, “ISO 7899/1, Water quality - Detection
and enumeration of faecal Streptococci” 1984.
6. Mossel, D.A.A., Bijker P.G.H. und Eelderink I..”Streptokokken der Lancefield-Gruppe
D in Lebensmitteln und Trinkwasser - Ihre Bedeutung, Erfassung und Bekämpfung”
Archiv. für Lebensmittelhygiene, 1978, vol. 29, p. 121-127.
7. M01_08AA “Catalase test” CLF method, Microbiology Division.
10 Attachments
Not applicable
114
For Enterococci two ISO methods are available: ISO 7899-1:1998 for intestinal Enterococci in
surface and waste water (MPN in liquid medium) and ISO 7899-2:2000 for intestinal Enterococci
in surface and waste water (membrane filtration).
This means no suitable ISO reference is available for detection and enumeration of Enterococci
in infant formula. The relevant CLF methods for Enterococci are M01_12ME (Enterococci
detection) and M01_13ME (Enterococci enumeration).
The ISO references mentioned above are the only available methods; any lab will have to adapt
the methods somehow to the matrix infant formula.
Among the two ISO norms, the ISO 7899-2:2000 “Water quality - Detection and enumeration of
intestinal enterococci – Part 2: Membrane filtration method” is closer to M01_13ME but the
requested agars are different (Slanetz-Bartley agar combined with Bile Aesculine Acide agar, see
table 2 and 3) from the one used in CLF method (KAAA, see Table 1). The Slanetz-Bartley agar
is less specific compared to KAAA and the Enterococci count can be overestimated in case of
the presence of background flora or probiotic strains.
Table 1. Composition of KAAA in M01_12/13ME
KAAA Peptone from casein 20.0 g M01_12ME
Merck, 1.05222 M01_13ME

Yeast extract 5.0 g
Sodium chloride 5.0 g
Sodium citrate 1.0 g
Sodium azide 0.15 g
Kanamycinsulfat 0.02 g
Aesculin 1.0 g
Ammonium iron (III) citrate 0.5 g
Agar 15 g
115
1
Table 2. Composition of Slanetz-Bartley medium in ISO 7899-2:2000
Slanetz-Bartley medium Tryptose 20.0 g ISO 7899-2:2000
Yeast extract 5.0 g
Glucose 2.0 g
Dipotassium hydrogenphosphate 4.0 g
Sodium azide 0.4 g
Agar 8 to 18 g
1
TTC solution: dissolve 2,3,5-triphenyltetrazolium chloride in water in 1 g/100 ml. Sterilize by filtration. Add
10 ml TTC solution to the Slanetz-Bartley medium.
Table 3. Composition of bile aesculin azide agar in ISO 7899-2:2000
Bile aesculin azide agar Tryptone 17.0 ISO 7899-2:2000
Merck, 1.05222 Peptone 3.0
Yeast extract 5.0
Ox-bile, dehydrated 10.0
Sodium chloride 5.0
Aesculin 1.0
Sodium azide 0.4
Agar 8 to 18
116
Protocols for Enterococci enumeration: M01_13ME and ISO norm
117
118
SALMONELLA CONVENTIONAL
119
120
No. M01_14ME_v05(Salmonella conv).doc
Salmonella – conventional method
of: 10 written: 05.07.2010
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V3 (04.12.06)  Updated version based on ISO 6579:2002
V4 (05.07.10)  Addition of description of performed modifications varying from
norm method
 Addition of Amendment 1: 2007-07 to norm method DIN EN ISO
6579:2002-07
 Usage of MKKTn is mandatory , no longer optional
 Addition of validation report number
V5 (05.03.14)  Attachment 1 and 2 integrated in the text
 Addition of Bismuth sulfite agar as option for selective medium
 Addition of molecular biological identification methods 16S rRNA
gene sequencing and MALDI TOF MS
Name Signature Date

First version written by: 2000
revised by: Sandra
Scheuch 2011
Zhu 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
121
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The method for the detection of Salmonella spp. can be applied to products intended for
human consumption and animal foodstuff, environmental samples and water.

This method is based on ISO 6579:2002.
 Lysine iron agar is used additionally for confirmation.
 Hektoen enteric agar (HEA) or Brilliant green phenol red lactose saccharose agar
modified (BPLS) are used as the second selective medium.
To be able to detect sublethally damaged cells, the sample undergoes a pre-enrichment
step in a non-selective medium, buffered peptone water (BPW).
After the pre-enrichment, a selective enrichment is performed in Rappaport-Vassiliadis
soya broth (RVS) and Muller-Kauffmann tetrathionate novobiocin broth (MKTTn).
RVS which shows better growth characteristics and is less selective displaces the former
formulation Rappaport-Vassiliadis broth (RV). RVS contains bile salts as a selective
agent. The incubation temperature of 41.5 ± 1°C increases the selectivity as well.
MKTTn displaces the former Selenite-cystin broth. MKTTn is more selective, less toxic for
the environment and shows higher detection rates for Salmonella typhi and Salmonella
paratyphi.
The enrichment is streaked out onto XLD agar and a second agar of own choice. When
suspected colonies are found, two other media are inoculated. The electivity of these
media is based on sugar fermentation, sulphite reduction and/or lysine decarboxylation.
The decarboxylation of lysine is important to detect Salmonella which ferment lactose or
which are H2S negative.
The presence of Salmonella is confirmed via biochemical and serological tests

Salmonella Gram negative, oxidase negative, facultative anaerobic rods which belong
to the family Enterobacteriaceae. They are generally motile, non-lactose
fermenting and H2S positive.
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Buffered peptone water BPW 1 month 4-10°C
Buffered peptone water +0.01% (w/v)

BPWM 1 month 4-10°C
malachite green6
pH indicator strips See manufacturer’s description
7
Rappaport-Vassiliadis soya broth RVS 1 month 4-10°C
Muller-Kauffmann broth MKTTn 1 month 4-10°C

Brilliant green phenol red lactose
BPLS 1 week 4-10°C
sucrose agar modified + SM5
Hektoen enteric agar HEA 1 week 4-10°C
Sulpamandelate supplement
SM See manufacturer’s description
(optional)2
Xylose lysine deoxycholate agar XLD 10 days 4-10°C
Bismuth sulfite agar BSA 1 week 4-10°C
Triple sugar iron agar TSI 1 month 4-10°C
Lysine iron agar LIA 1 month 4-10°C
Trypton soy agar TSA 1 month 4-10°C
O-antigens serum (omnivalent)4 See manufacturer’s description

1
2
Can be added to BPLS to increase the selectivity in case of high numbers of competitive flora.
3
API 20E, API ID 32E or other comparable test kits are used only when TSI and or LIA are doubtful or
positive.
4
When LIA and/or TSI are (doubtful) positive
5
Dependent on manufacturer also called BGA.
6
Can be used for the analysis of environmental samples to suppress the growth of competitive flora.
7
RV with soya pepton is preferred. Dependent on manufacturer it is called RV or RVS.
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5 Equipment
6 Procedure
6.1 Detection
1. Weigh the sample in a 1:10 dilution in BPW.
For some samples this suspension has to be prepared according to a special
procedure (see Attachment 1).
2. Incubate the pre-enrichment at 37 ± 1°C for minimally 16 h and maximally 18 h.
3. Measure the pH value of the incubated BPW. In case the pH value decreases below
5.0 during incubation due to the growth of competitive flora, repeat the whole process
from point 1, but shorten the incubation time of the BPW to 8-10 h at 37 ± 1°C.
If the pH value decreased to less than 5.0 in spite of the shortened incubation time,
repeat the enrichment step with BPW + malachite green.
4. Transfer 0.1 ml of the pre-enriched culture into 10 ml RVS.

Transfer 1.0 ml of the pre-enriched culture into 10 ml MKTTn.
5. Incubate the RVS at 41.5 ± 1°C for 24 ± 3 h.
Incubate the MKTTn at 37 ± 1°C for 24 ± 3 h.
6. Streak a loopful of incubated RVS and MKTTn onto XLD or another second medium
of choice.
7. Incubate the plates at 37 ± 1°C for 24 ± 3 h.
8. Read the XLD and the second plate on the presence of Salmonella (see Table 1).
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Table 1: Salmonella spp. appearance on isolation media
Agar Salmonella Growth Description

characteristic
Pink-red colonies with a black center
XLD Normal typical
against a red background
Pink-red colonies against a red
H2S - half-typical
background, but without a black spot
Lactose + atypical Yellow colonies
Non-motile typical See normal

BPLS Normal typical Pink to red colonies surrounded by a
bright red medium.
H2S - typical See normal
Lactose + atypical Green-yellow colonies
BSA Normal typical Brown to black color with metallic sheen

H2S - half-typical Green to deep green colonies
Lactose + atypical Brown to green colonies
HEA Normal typical Metallic green colonies with a black

center against a pale green background
H2S - half-typical Metallic green colonies against a pale
green background but without a black
spot
Lactose + atypical Pink to orange colonies surrounded by a
zone of bile precipitation against a red
background
NOTES:
The chance to find lactose-positive Salmonella is small. Therefore, the decision to continue the confirmation
of non-typical colonies has to be based on the overall results of the analyses and the sample information (e.g.
the results of the Diasalm method) and of the results of the selective media.
Sometimes Pseudomonas are able to grow on the selective media as well. As they do not ferment lactose,
the appearance could be the same as H2S-negative Salmonella. This can be tested by an oxidase test.
Salmonella are oxidase negative, while Pseudomonas are oxidase positive.
For lactose-positive, H2S-positive Salmonella, the blackening may not be visible due to the lactose
fermentation (acidification of the medium).
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6.2 Confirmation
1. Perform the confirmation only with pure cultures or well isolated colonies. When no
pure culture is obtained and no well isolated suspected colonies are available, some
suspected material has to be streaked onto a new plate of one of the isolation agars.
2. Incubate the plate at 37 ± 1°C for 24 ± 3 h. Repeat step 1 and 2 until well isolated
colonies are obtained which can be used for further confirmation.
3. Stab suspected colonies twice into the butt of one LIA tube and streak the slant.
Stab the colony once in the butt of a TSI tube and streak the slant.
4. Streak the same suspected colony onto a TSA plate or another non-selective agar.
5. Incubate the TSI and LIA tubes and the TSA plate at 37 ± 1°C for 24 ± 3 h.
6. Read the TSI and LIA tubes as described in Table 2.
Table 2: Growth characteristics of Salmonella on TSI and LIA

characteristic
Yellow butt, with blackening and a
TSI Normal typical
red slant
H2S - half- typical Yellow butt, without blackening and

a red slant
Lactose + atypical Yellow butt, with or without
blackening and a yellow slant
Non- motile typical See normal
Purple butt with blackening and a
LIA Normal typical
purple slant
H2S - half-typical Purple butt without blackening and a

purple slant
Lactose + typical See normal
7. When the LIA tube is typical or doubtful or when the TSI tube is typical, perform an
identification using a biochemical identification test kit like API 20E, API ID 32E or a
comparable reliable test kit. Perform the test with a well isolated colony from an
overnight incubated non-selective plate e.g.TSA.
8. Serological tests can be carried out to obtain a quick result which can be used as a
direction whether the isolate will indeed be Salmonella or not. However, the results of
the serological test alone can not be used to exclude the presence of Salmonella.
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Carry out serological tests (omnivalent or polyvalent Salmonella O-group antiserum)

as prescribed by the manufacturer.
NOTES:
It is also possible to perform step 7 and 8 at the same time with or instead of the LIA and TSI tests.
Lactose-positive Salmonella will give none typical growth on TSI (yellow butt and yellow slant) but typical
growth on LIA. These isolates are also suspected for Salmonella.
Generally material of a non selective agar has to be used for serological tests. Perform the test exactly
according to the manufacturer’s description.
For lactose positive, H2S positive Salmonella, the blackening may not be visible due to the lactose

Positive controls: S. panama reference material ATCC 7378
S. enteritis NCIMB 11450
Negative control: Enterobacter cloacae ATCC 13047

Express the result as the absence or presence of Salmonella spp. per weight or per
volume of sample as described in M01_01AA.
8 Validation
9 References
necessarily reflect the exact methodology.
1. International Organization for Standardization “ISO 6579:2002 Microbiology –
Horizontal method for the detection of Salmonella spp.”
2. International Organization for Standardization” ISO 6785: Milk and milk products -
Detection of Salmonella” 1985.
3. Van Schothorst, M. and Renaud, A.M. (1985). Malachite green pre-enrichment
medium for improved Salmonella isolation from heavily contaminated samples, J.
Appl. Bacteriol. 59, 223-230.
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4. Watson, D.C. and Walker, A.P. (1978). A modification of Brilliant Green Agar for
improved isolation of Salmonella, J. Appl. Bacteriol. 45, 195-204.
5. M01_01ME “Sample preparation and dilution” CLF method, Microbiology Division.
Division.
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10 Attachments
Attachment 1: Special procedures for preparation of specific samples
Product Procedure
Cocoa and cocoa-containing BPW + 50g/L casein or + 100 g/L skim milk powder (or
products (> 20%) after ca. 2 h incubation add 0.018 g/Ll malachite green, if
high number of accompanying flora is expected)
Spices and herbs Prepare 1% suspension instead of 10%
Thickening agent Prepare 1% or even lower concentrated suspension

instead of 10% and/or add specific enzymes (see
M01_01ME)
Products with high fat BPW + 1-10 g/L Tween 80

content > 20%
Acidic products and The pH value of BPW is not allowed to fall below 4.5
acidifying products after incubation.
a) 1.0-2.0% (g/v) CaCO3 can be added during
preparation of BPW to prevent a too strong decrease
of pH.
b) The pH is more stable if double-strength BPW is
used.
Salmonella do not grow well in viscous BPW, even when the samples can be well suspended.
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Attachment 2: Interpretation of biochemical tests: General reactions of Salmonella

Reliable commercially available biochemical identification kits can be used following the
manufacturer’s instructions. Generally Salmonella show the reactions given in the table
below.
Test Positive or negative Salmonella inoculations, which

reaction show these reaction1 [%]
TSI glucose (acid production) + 100
TSI glucose (gas production) + 91.92

3
TSI lactose - 99.2
TSI sucrose - 99.5
TSI H2S + 91.6

4
Lysine decarboxylation + 94.6
1
These indications in percent serve only as indication and can differ in various countries and various
foodstuffs.
2
Salmonella typhi is anaerogenic.
3
Salmonella Arizone (subgenus III) can show a positive as well as a negative reaction. Subgenus II shows a
negative reaction.
4
Salmonella paratyphi A is negative.
130
Method M01_14ME identical to ISO 6579:2002

The method M01_14ME clarifies some points which are left optional in ISO 6579:2002:
 The ISO method requires as selective medium XLD and a second agar of choice, while
M01_14ME clarifies that the medium of choice has to be BSA, HEA or BPLS.
 Confirmation of positive results: The method M01_14ME specifies the biochemical

confirmation requirements including also diagnostic identification kits and molecular
biological techniques.
Salmonella colony morphology on BSA medium

Salmonella strains show brown to black-coloured colonies on BSA with a metallic sheen
(see picture 1). A typical Salmonella colony on BSA has a characteristic “fish eye” (see
picture 3) appearance: a black center, light edges surrounded by a black precipitate with
a metallic sheen (see picture 2).
Black center
Light edges
Black precipitate
Metallic sheen
Picture 1 (left top): Salmonella colonies on BSA

Picture 2 (right top): Fish-eyed Salmonella colony on BSA
Picture 3 (left bottom): Fish eye
131
Protocols for Salmonella detection: ISO method and M01_14ME
132
SALMONELLA DIASALM
133
134
No. M01_15ME_v05(Salmonella Diasalm).doc
Salmonella – Diasalm method
of: 9 written: 03.02.2011
9
printed: 23.06.2014

V3 (14.09.10)  Updated version based on ISO 6579:2002
 Enrichment in buffered peptone water with malachite green
(BPWM) only in the case of high amount of competitive flora
V4 (03.02.11)  Addition of Amd 1: 2007-07 to the method DIN EN ISO
6579:2002
 Explanation of modifications compared to the method DIN EN
ISO 6579:2002
 Deletion of ecometrial test results for quality testing of media
 Deletion of SVM capsules as reference material and addition of
ATCC numbers instead
 Addition of Citrobacter amalonaticus as a substitute for
pathogenic Salmonella positive control strains
 Deletion of API as a mandatory confirmation step. The strains
can directly be sent to an external lab for serotyping
V5 (05.03.14)  Addition of confirmation step
 Addition of molecular biological identification methods 16S rRNA
gene sequencing and MALDI TOF MS
Name Signature Date

First version written by: 2000
revised by: Thomas
Koch 2011
Zhu 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
135
of: 9 written: 03.02.2011
9
printed: 23.06.2014

The method for the detection of Salmonella spp. using the Diasalm method can be
applied to products intended for human consumption and animal foodstuff, environmental
samples and water.

To be able to detect sublethally damaged cells, the sample is soaked in buffered peptone
water (BPW). This non-selective medium allows the recovery and growth of gram
negative bacteria.
From the pre-enrichment broth a Diasalm plate is inoculated. Diasalm is a semi solid
medium, which allows Salmonella to migrate through the medium, forming a specific
migration zone. Competitive flora which is able to grow on Diasalm, does generally not
migrate. Diasalm contains malachite green, novobiocin and magnesium chloride as
selective agents. In addition the incubation temperature at 42°C increases the selectivity
as well. The diagnostic system of Diasalm consists of lactose, sucrose, bromocresol
purple, iron and thiosulfate. In contrast to many other Enterobacteriaceae species,
Salmonella cannot use sucrose as a substrate. Thiosulfate is reduced to H2S which leads
to a black precipitate in combination with iron.
On Diasalm plates Salmonella is visible as purple or purple-black colonies with purple or
purple-black migration zones.
From the Diasalm a further isolation onto two selective agars is performed. The electivity
of these media is based on sugar fermentation, sulphite reduction and some other
biochemical characteristics which are typical for Salmonella.
The presence of Salmonella is confirmed via biochemical and serological tests. The lysine
decarboxylation is important to detect Salmonella which can ferment lactose or which are
H2S negative (which are a-typical characteristics for salmonella).
Modifications
This method is based on DIN EN ISO 6579 (2002-07+ Amd. 1: 2007-07) “Microbiology of
food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp.”.
 The selective enrichment in Rappaport-Vassiliades soya broth (RVS) and Muller-
Kauffmann broth with Tetrathionate and Novobiocin (MKTTn) is replaced by a
selective enrichment on Diasalm.
 Lysin iron agar is used in addition for confirmation of suspicious colonies.
 Hektoen enteric agar (HEA) is used as a second selective medium.
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Buffered peptone water +0.01% (w/v)
6 BPWM 1 month 4-10°C
malachite green
Diagnostic semi solid Salmonella
Diasalm 5 days 4-10°C
medium
Brilliant green phenol red lactose BPLS 1 week 4-10°C
sucrose agar modified + SM5
Hektoen enteric agar HEA 1 week 4-10°C
2
Sulpamandelate supplement (optional) SM See manufacturer’s description
Xylose lysine deoxycholate agar XLD 10 days 4-10°C
Bismuth sulfite agar BSA 1 week 4-10°C
Triple sugar iron agar TSI 1 month 4-10°C
Lysin iron agar LIA 1 month 4-10°C
Trypton soy agar TSA 1 month 4-10°C
O-antigens serum (group A-S)4 See manufacturer’s description

4
H-antigens serum (Phase 1 and 2) See manufacturer’s description
1
2
Can be added to BPLS to increase the selectivity when much competitive flora is expected.
3
API 20E, API ID 32E or other comparable test kits are used only when TSI and or LIA are doubtful or
positive.
4
When LIA and/or TSI are (doubtful) positive
5
Dependent on manufacturer also called BGA.
6
Can be used for the analysis of environmental samples to suppress the growth of competitive flora.
137
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5 Equipment
6 Procedure
6.1 Detection
1. Weigh the sample in a 1:10 dilution in BPW.
For some specific samples the suspension is prepared according to a special
procedure (see Attachment 1).
2. Incubate the pre-enrichment at 37 ± 1°C for minimally 16 and maximally 18 h.
3. Measure the pH value of the incubated BPW. In case the pH value decreases to less
than 5.0 during incubation due to the growth of competitive flora, repeat the whole
process from point 1, but shorten the incubation time of the BPW to 8-10 h at 37 ±
1°C.
If the pH decreases to lower than 5.0 in spite of the shortened incubation, repeat the
enrichment step with BPW + malachite green.
4. Spot 3 drops of pre-enriched culture around the center of the Diasalm plate.
 When using normal pipettes or Pasteur's pipettes, spot a small drop of fluid.
 When using a plunger pipette, spot 15 l of fluid.
5. Incubate the Diasalm plates in a plastic bag at 42 ± 0.5°C for 24 ± 3 h. (Do not invert
the plates; incubate with cover up).
6. Read the Diasalm plates for the presence of Salmonella (see Table 1). If the plates
are negative after 24 h, re-incubate for another 24 ± 3 h at 42 ± 0.5°C.
7. When the Diasalm plates are negative after 48 h incubation, the sample is regarded
as negative for Salmonella spp.
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Table 1: Salmonella spp. appearance on Diasalm
Salmonella Growth Description

characteristic
Purple (and black spotted) colony and dull migration
Normal typical
zone
H2S - typical See above, but without black spots
Lactose + typical Purple (and black spotted) colony and migration zone
Purple (and black spotted) colony without migration
Non-motile half-typical
zone
NOTE:
The pH value of the BPW after incubation is expected to be between 4.5 and 7.5. Higher or lower pH values
may inhibit the growth of Salmonella in the BPW and therefore the results are not reliable.
6.2 Confirmation
1. Isolate migrated colony material by stabbing a sterile needle or loop at the edge of
the migration zone of positive or doubtful Diasalm plates. Whenever colonies are not
migrated, cells are picked up from the center of the spot.
2. Streak the loop with culture on XLD or another selective medium of choice. Make a
fraction streak to obtain well isolated colonies. Pick up some new culture material for
each plate.
3. Incubate the plates at 37 ± 1°C for 24 ± 3 h.
4. Read the plates as described in Table 2.
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Table 2: Salmonella spp. appearance on isolation media

characteristic
Pink-red colonies with a black center
XLD Normal typical
against a red background
Pink-red colonies against a red
H2S - half-typical
background, but without a black spot
Lactose + atypical Yellow colonies

BPLS Normal typical Pink to red colonies surrounded by a
bright red medium.
H2S - typical See normal
Lactose + atypical Green-yellow colonies.
BSA Normal typical Brown to black color with metallic sheen

H2S - half-typical Green to deep green colonies
Lactose + atypical Brown to green colonies
HEA Normal typical Metallic green colonies with a black

center against a pale green background
H2S - half-typical Metallic green colonies against a pale
green background but without a black
spot
Lactose + atypical Pink to orange colonies surrounded by a
zone of bile precipitation against a red
background
5. In case of suspicious positive colonies on selective media, stab suspected colonies

twice into the butt of one LIA tube and streak the slant.
Stab the colony once in the butt of a TSI tube and streak the slant.
6. Streak the same suspected colony onto a TSA plate or another non-selective agar.
7. Incubate the TSI and LIA tubes and the TSA plate at 37 ± 1°C for 24 ± 3 h.
8. Read the TSI and LIA tubes as described in Table 3.
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Table 3: Growth characteristics for Salmonella on TSI and LIA

characteristic
Yellow butt, with blackening and a
TSI Normal typical
red slant
H2S - half- typical Yellow butt, without blackening and

a red slant
Lactose + atypical Yellow butt, with or without
blackening and a yellow slant
Purple butt with blackening and a
LIA Normal typical
purple slant
H2S - half-typical Purple butt without blackening and a

purple slant
Lactose + typical See normal
9. When the LIA tube is typical or doubtful or when the TSI tube is typical, perform an
identification using a biochemical identification test kit like API 20E, API ID 32E or a
comparable reliable test kit. Perform the test with a well isolated colony from an
overnight incubated non-selective plate e.g.TSA.
10. Serological tests can be carried out to obtain a quick result which can be used as a
direction whether the isolate will indeed be Salmonella or not. However, the results of
the serological test alone cannot be used to exclude the presence of Salmonella.
Carry out serological tests (omnivalent or polyvalent Salmonella O-group antiserum)
as prescribed by the manufacturer.
NOTES:
The chance to find lactose-positive Salmonella is small. Therefore, the decision to continue the confirmation
of non-typical colonies has to be based on the overall results of the analyses and the sample information (e.g.
the results of the Diasalm method) and of the results of the selective media.
Sometimes Pseudomonas spp. are able to grow on the selective media as well. As they do not ferment
lactose, the appearance could be the same as H 2S-negative Salmonella. This can be tested by an oxidase
test. Salmonella are oxidase negative, while Pseudomonas are oxidase positive.
For lactose-positive, H2S-positive Salmonella, the blackening may not be visible due to the lactose
It is also possible to perform step 9-10 at the same time with or instead of the LIA and TSI tests.
Lactose-positive Salmonella will give none typical growth on TSI (yellow butt and yellow slant) but typical
growth on LIA. These isolates are also suspected for Salmonella.
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Positive controls: S. panama ATCC 7378.
Citrobacter amalonaticus ATCC 25405
Negative control: Enterobacter cloaceae ATCC 13047



Express the result as the absence or presence of Salmonella spp. per weight or per
8 Validation
For Citrobacter amalonaticus as positive control see M01_14VB2_v01.
9 References
1. Anon. (1993), Diagnostic Salmonella - Selective semisolid medium (DIASALM),
International Journal of Food Microbiology 17, 230-233.
2. Dijk, R, Grootenhuis A “Microbiologie van voedingsmiddelen - methoden, principes
en criteria” Keesing Noordervliet, Houten, 1999, p.189-196.
3. International Organization for Standardization “ISO 6579:2002 Microbiology –
Horizontal method for the detection of Salmonella spp.”
4. European Committee for Standardization “EN 12824 Microbiology of food and animal
feeding stuffs – Horizontal method for the detection of Salmonella (ISO 6579:1993
modified) “ 1997.
5. Landman W.J.M., Hartman E.G., Doornenbal P. “Salmonella-isolatie uit
pluimveemonsters: vergelijking diagnistic semi-solid salmonella agar (diasalm) en
rappaport vassiliades bouillon (rv)” De Ware(n) Chemicus, 1996, no 26, p. 234-237.
6. Oudenallen,v. A. (1992). Validation of non conventional methods for the detection of
salmonellae in foods. Nutricia Research report: RA004.mrd. 16-17.
7. Watson, D.C. and Walker, A.P. (1978). A modification of Brilliant Green Agar for
improved isolation of Salmonella, Journal of Applied Bacteriology 45, 195-204.
8. Zee v.d.H, de Boer E., and van Netten P (1990)., “Salmonella-isolatie met behulp van
modified semisolid rappaport-vassiliades (MSRV) medium” 1990, De Ware(n)
chemicus, vol. 20, p. 189-199.
142
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9. Zee, v.d.H. and Netten, v. P. (1991), Improved isolation of Salmonella enteridis from
poultry by the use of a semi-solid diagnostic salmonella medium (DIASALM), De
Ware(n) Chemicus 21, 180-189.
10. Zee v.d. H., Postma P., Vonk M. “Vergelijking van Porbelia PCR Salmonella testkit
met Diasalm en een gemodificeerde ISO methode” De Ware(n) Chemicus, 1996, no
26, p. 238-243.
10 Attachments
See Attachments 1 and 2 in M01_14ME.
143
Method M01_15ME as a rapid method validated against ISO 6579:2002

 The method is based on a semi-solid RVS agar (Diasalm) which is used as a screening
system to exclude the majority of samples as negative already after the selective
enrichment step in semi-solid RVS (Diasalm). The following steps are identical to
M01_14ME.
 M01_15ME does not include the second selective enrichment medium MKTTn. This is a
severe deviation from ISO 6579. However the number of strains which are not detected in
RVS is very limited but still includes some strains of S. typhi and S. paratyphi. Due to the
epidemiological situation a contamination with S. typhi and S. paratyphi is rather unlikely.
144
145
146
SALMONELLA BARREL
147
148
No. M01_16ME_v06(Salmonella Barrel).doc
Salmonella - Barrel
page: 1 version: 6
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printed: 2014-06-23
Salmonella − Barrel method

V4 (04.12.06)  Correct composition of BTW
 Addition of improvements of the method
V5 (16.04.10)  Analysis of 750 g sample has been added
 Addition of ISO 6579: 2002 + Amd. 1: 2007 as reference method
V6 (23.01.14)  This method is no longer considered as part of the accreditation
according to ISO/IEC 17025:2005
This method is not part of

the accreditation according to ISO/IEC 17025:2005
Name Signature Date

First version written by: Thomas
Koch 2000
revised by: Angela
Lindenstrauß 2014
Zhu 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
149
Salmonella - Barrel
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printed: 2014-06-23

This method can be applied to detection Salmonella spp. in dry food product samples of
750 g or 1500 g with a low Enterobacteriaceae contamination (<1000 CFU/g).

Following steps are necessary to detect Salmonella spp. in a sample:
1. Non-selective pre-enrichment to enable the detection of sublethally damaged
Salmonella cells.
2. Selective enrichment to support the growth of Salmonella spp. and to inhibit the
growth of competitive flora.
3. Isolation of the Salmonella spp. on selective media.
4. Confirmation of suspicious Salmonella isolates with several tests
In the current method, the first stage is described for samples of 750 g or 1500 g. The
first stage is split into two steps: The first step is the pre-enrichment in a 7.5 or 15 liter
barrel (depending on sample mass) at room temperature and the second step in 1 liter
bottles at 37°C. These two non-selective pre-enrichment steps allow the recovery and
growth of bacteria.
After the specific pre-enrichment stage described in this method, M01_14ME or
M01_15ME has to be followed from the selective enrichment step with RVS/MKTTn or
Diasalm to the end.

CFU Colony forming unit
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4 Reagents, materials, media of specific barrel pre-enrichment

Buffered tap water BTW

Potassium di hydrogen KH2PO4 See manufacturer’s description
phosphate
Disodium hydrogen phosphate Na2HPO4*2H2O See manufacturer’s description
Sodium chloride NaCl See manufacturer’s description

Malachite green oxalate, 10%
MG
solution in water
1
Table 1: Composition of BTW
Barrel with 15 L Barrel with 7.5 L BTW Composition

(tap water + sample) (tap water + sample) per L
NaCl 75 g 37.5 g 5.0 g
KH2PO4 22.5 g 11.25 g 1.5 g
Na2HPO4*2H2O 135 g 67.5 g 9.0 g
When thickeners, pre mixes or other products with a low nutrient value are analyzed,
about 75 g or 150 g (depending on sample mass) sterile milk powder is added to the
barrel to supply nutrients for microbiological growth.
Appropriate enzymes are recommended to use for thickeners (see M01_01ME) and/or
prepare a less than 10% sample suspension.
The BTW with sample has to be thin liquid.
5 Equipment
10 or 20 liter container (available at e.g. Nagle and Dispolab Asten)
Mixing device for 10 or 20 liter container (available at e.g. Heidolf or Boom Meppel).
1 or 2 L bottles
6 Procedure
6.1 Specific pre-enrichment

1. Let the tap run for 1 minute before filling the barrel.
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2. Weigh the appropriate amount of the salts into the barrel as described in Table 1
above.
3. Fill the barrel with about 2.5 L/5 L (lukewarm) water.
4. Add the sample. The barrel is first filled with about 2.5 L/5 L to prevent sedimentation
of the sample.
5. Fill the barrel with tap water to the appropriate volume.
6. For a 750 g/1500 g sample the barrel has to be filled up to 7.5 L/15 L (10% sample
solution w/v). It is also possible to use a 15% sample solution (w/v) which means 750
g/1500 g sample into 5 L/10 L of BTW. Compensate more or less sample with the
appropriate amount of water and salts.
7. After filling the barrel the temperature has to be minimally 20°C and may be up to
30°C.
8. The pH value of the suspended sample has to be 7.0 ± 0.5.
9. Mix the sample during 8 ± 0.5 h in the barrel at the local room temperature.
10. Transfer minimally 1.5 L/3 L from a 7.5 L/15 L filled barrel into a sterile container. The
sterile container may be filled for maximally 2 L. For other volumes in the barrel at
least 1 L for each 5 L in the barrel (20%) has to be transferred to a sterile container.
11. In case high amount of competitive flora is expected in the sample, add 1 ml 10%
MG solution per liter sample in the container. The final MG concentration has to be
0.1 g/L sample. The addition of MG suppresses the growth of gram positive flora.
12. Incubate the containers at 37 ± 1°C for 16-18 h.
NOTES:
During filling, the temperature of the barrel contents should never exceed 40°C when the sample is added.
Steps 3-6 describe the way the barrel will be filled in such a way that the sample is well suspended in the
BTW and the temperature is between minimally 20°C and maximally 30°C. Other ways of filling the barrel that
can achieve the same results are also possible.
When the incubation time in the barrel is shorter than 8 h, at least 3 l/ 6 l sample has to be taken from a 7.5 L
/ 15 L barrel for overnight incubation. In addition the temperature of the barrel during the complete incubation
has to be guaranteed to be at least 20°C. In any case it is not acceptable to incubate shorter than 7.5 h.
6.2 Selective enrichment, isolation and confirmation

13. Choose between the conventional method using RVS/MKTTn (M01_14ME) or the
Diasalm method (M01_15ME).
14. Start at the following point:
 Salmonella conventional method (M01_14ME):
Transfer 0.1 ml BPW into 10 ml RVS; 1.0 ml into 10 ml MKTTn.
 Salmonella Diasalm method (M01_15ME):
Transfer 3 drops BPW onto a Diasalm plate.
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15. Each container has to be handled as a separate sample.

16. Measure the pH value of the incubated container with BTW after inoculation of the
RVS/MKTTn or Diasalm. Use a pH measurement apparatus or pH strip. PH value <
5.0 has to be noted.
When the pH value has decreased below 5.0 during incubation due to growth of
competitive flora, then repeat from point 1, but shorten the incubation time of the
BPW to 8-10 h at 37 ± 1°C. If the pH-value decreased to lower than 5.0 in spite of the
shortened incubation, repeat the enrichment step with BPW + malachite green.
17. Isolation and confirmation
Details for isolation and confirmation step see M01_14ME and M01_15ME.
NOTE:
The pH value of the BPW after incubation has to be 4.5-7.5. Higher or lower pH values may inhibit the growth
of Salmonella spp. in the BPW and the results are therefore not reliable.

Due to the risk of contaminating the barrels, the use of a positive and negative control is
not advised for the barrel itself. A positive and negative control can be used according to
M01_14ME and M01_15ME from the moment that the sample is incubated in the 1 L/2 L
sterile containers at 37°C.
Positive controls: S. panama reference material ATCC 7378

Express the result as the absence or presence of Salmonella per weight or per volume of
sample as described in M01_01AA.
8 Validation
9 References
Division.
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2. M01_01ME “Sample preparation and dilution” CLF method, Microbiology Division.

3. M01_14ME “Salmonella - Conventional method” CLF method, Microbiology Division.
4. M01_15ME” Salmonella – Diasalm method” CLF method, Microbiology Division.
5. Kowalski W “Milupa Validation Rührstationen” Milupa Friedrichsdorf, 1997, Intern
report No: 3090 version 03. 08.01.97.
6. Schothorst, v. M. and Renaud, A.M. “Malachite green pre-enrichment medium for
improved Salmonella isolation from heavily contaminated samples”, 1985, Journal of
Applied Bacteriology 59, 223-230.
7. Woestemeyer I. “Salmonella validation for 1500g” CLF Central Laboratories
Friedrichsdorf GmbH, Microbiology Division , 13-08-1999, Intern report: ri993201.
8. International Organization for Standardization “ISO 6579: Microbiology - Horizontal
method for the detection of Salmonella spp.” DIN EN ISO 6579:2002-07 + Amd. 1:
2007-07.
9. Regulation of European Community (EC) No. 1441/2007 (2007-12) to the alteration
of EC regulation No. 2073/2005 about microbiological criteria for foods.
10 Attachments
Not applicable
154
Method M01_16ME to handle high sample volumes validated against the ISO
The method is based on a pre-enrichment step at room temperature over 8 h, followed by the
non-selective enrichment of only an aliquot of the pre-enriched sample.
The method is a severe deviation from the ISO requirements especially in the following points:
 Pre-enrichment at room temperature is not selective or elective for Salmonella.
Background flora can overgrow Salmonella and cause false negative results. Usually the
background flora in the samples is very low so that this is in practice not really an issue. It
is nevertheless important to limit the pre-incubation time strictly up to 8-8.5 h but at the
same time to ensure a minimum incubation of 7.5 h. Contamination of the water used for
the pre-enrichment can influence the method.
 The analysis of only an aliquot of the pre-enriched sample is not in line with the ISO and
although validated for the routine approach it cannot be guaranteed that the detection of
Salmonella is successful even in unlikely cases (e.g. exotic Salmonella strains).
 M01_16ME might not be accepted by some authorities.
155
156
LACTOBACILLI ENUMERATION
157
158
No. M01_18ME_v02(Lactobacilli enumeration).doc
Lactobacilli – Enumeration
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V2 (07.03.14)  Addition of confirmation tools e.g. biochemical test kit 50 CHL
and 16S rRNA gene sequencing and MALDI TOF MS
 Addition of Staphylococcus aureus as negative control

Name Signature Date

First version written by: Corinna
2009
Strolz
revised by: Björn
2014
Hampel
2014
Zhu
2014
Wollnitz
approved: Matthias
2014
Fischer
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printed: 27.06.2014
1 Area of application
The method for the enumeration of Lactobacilli can be applied to products for human
consumption and animal foodstuff and water.

This method is based on International Standard ISO 20128:2012 and ISO 5214:1998.
The sample is plated in MRS agar (deMan Rogosa Sharpe agar) which has a pH value of
6.2 ± 0.2. The samples are incubated at 37°C in a micro-aerophilic environment. The low
pH value and composition of MRS together with the micro-aerophilic incubation supply a
semi-selective environment for the growth of Lactobacilli.

Lactobacilli Gram positive, non spore-forming rods which can ferment lactose

Tryptone soya agar TSA 1 month 4-10°C

deMan Rogosa sharpe agar MRS 1 month 4-10°C
1
The shelf life of the prepared media/solutions is given when stored in the dark. Media which can be kept
longer than 1 month have to be stored in well closed (screw capped) bottles.

5 Equipment
Anaerobic jars and microphilic equipment
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6 Procedure
6.1 Enumeration
contamination level of the sample (see method M01_01ME). The number of colonies
on a plate should not exceed 300 colonies.
2. Pipette 1 ml of the appropriate dilution into a Petri dish.
3. Pour approximately 15 ml of the MRS agar (tempered at 45 ± 2°C) into the Petri dish,
mix and allow to solidify.
4. Incubate the plates upside down for 72 ± 3 h at 37 ± 1°C in a micro-aerophilic
environment. This is achieved by incubating the plates in an anaerobic jar with a
micro-aerophilic kit.
5. Count all colonies. Spreading colonies are counted as single colonies. If less than
one quarter is overgrown by spreading colonies, count the colonies on the unaffected
part of the plate and calculate the corresponding number for the entire plate. If more
than one-quarter of the surface is overgrown by spreading colonies, re-analyze the
sample (using a surface layer).
6.2 Confirmation
1. Pick up a representative number of well isolated colonies and make pure streaks on
MRS agar and TSA and incubate the agars at 37°C.
2. Examine the isolates under the microscope, perform test for gram stain and catalase
reaction.
Lactobacilli are gram positive, catalase negative, long, slim, non spore-forming rods.
3. For a further confirmation use a biochemical identification test kit (e.g. API 50 CHL).
Molecular biological confirmation tools such as 16S rRNA gene sequencing and
MALDI TOF MS can be applied as well.
NOTE:
To determine the number of lactobacilli in a fermented or probiotic product where the number of lactobacilli will
outnumber the amount of other microbiological flora to a great extent, confirmation may not be necessary or
can be performed up to step 6.2.2.

Positive control: Lactobacillus rhamnosus CC 800
Negative control: Staphylococcus aureus ATCC 6538
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Calculate the number of Lactobacilli as described in M01_01AA. Express the results as
8 Validation
9 References
Division.
2. International Standard ISO 15214, 1998: Microbiology of food and animal feeding
stuffs – Horizontal method for the enumeration of mesophilic lactic acid bacteria –
Colony-count technique at 30°C, Genève.
10 Attachments
Not applicable
162
Principle of Lactobacilli method

There are two ISO norms which describe the enumeration of Lactobacilli in food products.
The ISO 20128:2012 is applicable for the enumeration of presumptive Lactobacillus acidophilus
specifically in fermented and non-fermented milks, milk powders and infant formula. In ISO
20128, Lactobacillus acidophilus is the only target microorganism of interest. To suppress the
growth of other lactic acid bacteria and bifidobacteria in milk products such as Lactobacillus
delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Streptococcus
thermophilus, bifidobacteria, Lactococci, Lactobacillus casei, Lactobacillus paracasei subsp.
paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri and Leuconostoc species, the
antibiotics clindamycin and ciprofloxacin are added to MRS medium which is then incubated at
37°C for 72 h anaerobically.
Besides the ISO 20128, there is another ISO norm ISO 15214:1998 which specifies a horizontal
method for the enumeration of the viable mesophilic lactic acid bacteria group in products
intended for human consumption or animal foodstuff. No antibiotics are supplemented to MRS in
ISO 15214 because the aim of the method is to cover the whole group of mesophilic lactic acid
bacteria species. MRS is incubated at 30°C for 72 h aerobically in this ISO norm.
CLF validation data (see M01_18VB) has shown that a higher number of Lactobacillus spp. was
detected in the sample which was first resuscitated for 2 h in MRS broth under micro-aerophilic
condition. However a resuscitation step is required in neither of the ISO standards and hence not
adopted in the current method M01_18ME. In the same validation report, a micro-aerophilic
incubation of MRS agar at 37°C proved to support the growth of Lactobacillus spp. well. This
finding has been taken into the current method.
Protocols for Lactobacilli enumeration: ISO method and CLF method
163
164
STERILITY TESTING
165
166
No. M01_21ME_v02(Sterility test)
Sterility test
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Sterility testing of acid and neutral sterilized products

V2 (05.03.14)  Renaming of the method
 Strains recommended by European Pharmacopoeia added as
positive controls to 6.3
 Meat liver agar (MLA) as medium for anaerobic incubation
 Biochemical confirmation kits and molecular biological
confirmation tools are remarked in 6.2
Name Signature Date

1st version written by: Anabelle
2010
Reidel
revised by: Björn
2014
Hampel
2014
Zhu
2014
Wollnitz
approved: Matthias
2014
Fischer
167
Sterility test
page: 2 version: 2
of: 5 written: 05.03.2014
printed: 19.05.2014

This method describes the procedure for the sterility testing of liquid and semi solid
products. The method can be applied to acid pasteurized and neutral sterilized liquid or
semi solid products, irrespective of packaging material.

The method is based on the enrichment of potential, undesired microorganisms in the
product, followed by plating on a non-selective medium. The method can be used to
examine whether undesired contamination of the product has occurred. This method is a
modification of international recognised methodology: European Pharmacopoeia 5.0. Some
countries have their own national regulation for sterility testing (e.g. incubation time and
temperature). These regulations should be considered.

Semi solid products Products which are relatively solid in consistence but in which
microorganisms can still grow (e.g. jar food).

Orange serum agar OSA 1 month 4-10°C
Plate count agar PCA 1 month 4-10°C
Schaedler agar Schaedler 1 month 4-10°C
Meat liver agar MLA 1 month 4-10°C

1

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5 Equipment
6 Procedure
6.1 Detection
1. Incubate a representative number of samples from the batch under examination for 7
days at 30°C for examination of mesophilic and acidophilic microorganisms and at
55°C for thermophilic microorganisms. For milk-based products, incubate additionally 2
samples per batch for 15 days at 30C, to fulfill the EU legislation.
2. After incubation judge the appearance of the packed products before opening (gas
formation).
3. Open the packaging aseptically as follows:
 Crown cork bottle
Disinfect the crown cork and the opener with 70% alcohol.
 Jar
Upon opening observe the “click” of the vacuum.
 Tin
Disinfect the tin opening with 70% alcohol.
 Brick
Disinfect the brick opening with 70% alcohol and use disinfected scissors to open
the brick.
4. Depending on the type of product continue as followed:
 Liquid products
 Acidic product:
Streak the acidic product onto OSA plates and incubate the plates at 30°C
aerobically and anaerobically.
 Neutral product:
Streak the neutral product on two PCA [aerobic] and two MLA (or Schaedler)
[anaerobic] plates. Incubate the plates at 30°C or 55°C for mesophilic and
thermophilic microorganisms.
 Semi solid products
Transfer the product aseptically to TSB aerobically and BHI anaerobically in 1:10
w/v dilution. Incubate the TSB and BHI for 2 days at 30°C/55°C. After incubation
streak the products on PCA [aerobic] and on MLA (or Schaedler) [anaerobic] and
incubate the plates at 30°C or 55°C.
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5. Incubate the plates upside down as follows:

Aerobic incubation at 30  1C for 72 ± 3 h (OSA and PCA)
Aerobic incubation at 55  1C for 72 ± 3 h (PCA)
Anaerobic incubation at 30  1C for 72 ± 3 h (OSA and MLA or Schaedler)
Anaerobic incubation at 55  1C for 72 ± 3 h (MLA or Schaedler)
6. Judge the (neutral) products on smell and appearance and measure the pH.
7. Judge the plates for growth after 3 days (a first judgement after 1 and 2 days is also
possible).
6.2 Confirmation
For some of the spoiled products an identification using biochemical identification kit of the
microorganisms on the plates is necessary.
For further confirmation and identification, modern identification tools such as 16S rRNA
gene sequencing and MALDI TOF MS are recommended.
Positive controls1: S. aureus ATCC 6358
B. subtilis ATCC 6633
Cl. sporogenes ATCC 19404
C. albicans ATCC 2091

Negative control: not applicable
Blank: Incubated medium plates without inoculation streak
1
These strains are recommended as positive controls for sterility testing according to
European Pharmacopoeia norm for medical products.

The investigated sample is judged as non-sterile if:
 visual spoilage (smell or appearance), and/or
 a pH decrease, and/or
 bacterial growth on the plate
is observed.
Growth on the plate should follow the streak line.
170
Sterility test
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8 Validation
9 References
The above mentioned method is based on the following references but does not
1. L.P.M. Langeveld et al, Duration of the pre-incubation period in the sterility control of
UHT-sterilised milk in Netherlands Milk and Dairy Journal, 33, 1979, 172-180.
2. NEN. 1999 Analysis of the unwanted contamination of long shelf life liquid milk
products. 2nd draft.
3. EC Directive 92/46/EEC; laying down the health rules for the production and placing on
the market of raw milk, heat-treated milk and milk-based products.
10 Attachments
Not applicable
171
AMENDMENTS AND COMMENTS COMPARED TO EUROPEAN
PHARMACOPOEIA 5.0
Principle of sterility test

The sterility test is primarily applied to substances, preparations or articles which, according to
the European Pharmacopoeia 01/2005:20601, are required to be sterile. The European
Pharmacopoeia sterility test has been adapted to the current CLF method M01_21ME for testing
of sterilized products which should be free of any contaminating microorganisms.
Undesired microorganisms in such products are aerobes and anaerobes which can grow
optimally at temperatures of 30°C or 55°C. Therefore all possible growth conditions have to be
taken into consideration in this method. Additionally OSA is recommended for acidic liquid
product. The OSA is used for cultivation and enumeration of aciduric microorganisms associated
with the spoilage of products like fruit juices with low pH value.
The current method indicates the presence/absence of contaminating microorganisms in the
samples examined in the conditions of the test. The product is pre-incubated for 7 days at
appropriate temperatures to simulate the common turn-over time and conditions during the
transport from factory release to consumption by consumers. The pre-incubation temperature of
30°C applies for the examination of mesophilic and acidophilic microorganisms, while the pre-
incubation temperature of 55°C applies for thermophilic microorganisms.
Protocols for sterility test: EP method and CLF method
172
LISTERIA MONOCYTOGENES DETECTION
173
174
No. M01_22ME_v03(L. monocytogenes detection).doc
L. monocytogenes − Detection
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Listeria monocytogenes − Detection

V2 (11.12.09)  Description of the modifications is added
 Revision of DIN EN ISO11290-1: 1996-12: Addition of
Amendment 1: 2007-07
V3 (07.02.14)  Biochemical parameter for differentiation between L.
monocytogenes and L. innocua added to 6.2/5
 Modern identification methods 16S rRNA gene sequencing and

MALDI TOF MS added to 6.2/6
 Deletion of 6.3 SVM Reference strain due to its unavailability
 Deletion of OXOID Listeria rapid test kit
Name Signature Date

1st version written by: Sandra
Scheuch 2006
revised by: Corinna

Strolz 2014
Zhu 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
175
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The method for the detection of Listeria monocytogenes can be applied to products
intended for human consumption and for animal foodstuff.

This norm method is based on EN ISO 11290-1: 1996-12 + AMD 1: 2004-10.
The following modification has been performed:
 RAPID L’ Mono agar is used as selective medium
 streaking from first enrichment step is omitted
 API Listeria for confirmation
A semi-selective pre-enrichment of Listeria spp is made in ½ selective Fraser broth, to
give sublethally cells the possibility to recover. Next, a selective enrichment is made in
Fraser broth. Fraser broth contains as selective agents lithium chloride to suppress the
growth of Gram negative micro organisms and Enterococci, the antibiotics acriflavine
(inhibits many Gram positives) and nalidixic acid (inhibits Gram negatives). In ½ Fraser
broth the concentration of the last two agents is only half of that as in normal Fraser. As
elective agents Fraser broth contains aesculin and ferric ammonium citrate. All Listeria
spp. hydrolyse aesculin to aseculetin which reacts with ferric ions and results in
blackening. However, the absence of blackening is no guarantee for the absence of
Listeria spp.
The presence of Listeria spp. after enrichment can be detected by streaking on 2 selective
agar plates: Palcam and Rapid L’mono. Palcam contains as selecive agents lithium
chloride and acriflavine like in Fraser broth and polymixin and ceftazidime (inhibits Gram
negatives). Electivity is obtained by 2 systems: aesculin and ferric ammonium citrate (see
above), and D-mannitol and phenol red. Listeria spp do not ferment mannitol, which
distinguishes them from Enterococci and Staphylococci. On Palcam Listeria spp are
visible as grey-green/ olive green colonies with black haloes against a pink-purple
background. The center of the colony is somewhat sunken.
Rapid L’mono is based on the chromogenic detection phospholipase C activity (resulting
in blue colonies instead of white) and xylose fermentation (resulting in a yellow zone
around the colony). L. monocytogenes is phospholipase C positive and does not ferment
xylose, resulting in blue colonies without a zone.
The presence of L. monocytogenes is confirmed by biochemical characterisation.
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L. monocytogenes Gram positive, motile in a tumbling way at 25°C, but not when
incubated at higher temperatures, aerobic and anaerobically
growing non spore forming rod, has haemolytic activity, does not
ferment xylose.

Fraser broth base Frase-b 1 month 4-10°C
Fraser supplement, containing Fraser suppl. See manufacturer’s description
acriflavine and naldic acid
Fraser–broth with half the ½ Fraser 2 weeks 4-10°C
concentration of supplement
Fraser-broth with normal concentration Fraser 2 weeks 4-10°C
of supplement
Palcam base agar Palcam-b 1 month 4-10°C
Palcam supplement Palcam suppl. See manufacturer’s description
Palcam–with Palcam supplement Palcam 2 weeks 4-10°C
Rapid L’ mono2 L-mono See manufacturer’s description
Blood agar (sheep) BA See manufacturer’s description
Trypton soy broth4 TSB 1 month 4-10°C

1
The shelf life of the prepared media is based on storage in the dark. Media kept longer than 1 month have to
be filled in well closed bottles with a screw cap.
2
Rapid L’mono is a patented media of Bio-Rad. Other chromogenic media of comparable quality may also be
used.
3
e.g. API Listeria
4
Optional to check motility
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5 Equipment
The detection of L. monocytogenes must be performed in a level II laboratory (pathogen
lab).
6 Procedure
6.1 Detection
1. Weigh a suitable amount of sample into ½ Fraser in such a way that a 10% dilution is
obtained (e.g. 25 g sample in 225 ml ½ Fraser).
2. Mix the ½Fraser + sample and incubate at 30 ± 1°C for 24 ± 2 h.
3. Transfer 0.1 ml of the incubated ½ Fraser (regardless of its color) to a tube with 10 ml
full Fraser.
4. Incubate the full Fraser tube at 37°C for 48 ± 2 h.
NOTE:
Several 25 g samples can be pooled. Take care that the heating time to reach 30°C for bottles with larger
volumes is longer than for bottles with a smaller volume. Do not use volumes over 1.5 L.
5. Inoculate a Palcam plate and a second plate of choice (e.g. Rapid L’ mono) with a
loop from the incubated full Fraser. Inoculate in such a way that well isolated colonies
are obtained.
6. Incubate the Palcam and the second plate at 37°C for 48 ± 2 h.
7. Examine the plates for typical growth:
 Palcam:
After 24 h, Listeria spp. grow as small or very small greyish green or olive green colonies,
1.5-2 mm in diameter, sometimes with black center, but always with a black halo. After
48 h Listeria spp. appear in the form of green colonies about 1.5-2 mm in diameter, with a
central depression and surrounded by a black halo.
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 Rapid L’ mono:
L. monocytogenes: blue colonies without a yellow zone on a red background
L. ivanovii: blue colonies with a yellow zone on a red background
Other Listeria spp: white colonies
6.2 Confirmation
1. Check whether the colonies on the Palcam plate are indeed Listeria. For further
confirmation, take from the Palcam plate 5 colonies which are presumed to be Listeria
spp. Choose 5 colonies from L’ mono plate which are presumed to be L. mono-
cytogenes. When less than 5 suspected colonies are present, confirm all suspected
colonies.
2. Streak the colonies on a TSA or blood agar plate.
Optional: streak from the Palcam plate also on Rapid L’ mono agar.
3. Incubate the plates at 37°C for 18-24 h, or until the growth is satisfactory.
4. Confirm the isolates plates with a biochemical identification test kit e.g. API Listeria.
Follow the manufacturer’s description.
5. In API Listeria the only reaction which differentiates L. monocytogenes from the
harmless species L. innocua is the patented “DIM” in which the expression of
naphthylamidase is determined: While a strong orange-colored reaction for DIM is
obtained for the naphthylamidase positive L. innocua, L. monocytogenes shows a
very weak color change due to the absence of the enzyme (see picture 1).
Picture 1: L. innocua (upper) and L. monocytogenes (bottom) are differed only by DIM reaction using API
Listeria.
NOTE:
By using API Listeria for identification of isolates, the reference strain of L. monocytogenes must always be
tested in parallel for comparison.
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6. For further identification 16S rRNA gene sequencing and MALDI TOF MS are
recommended.
NOTES:
Typical Listeria colonies on TSA are 1-2 mm in diameter, convex, colorless and opaque with an entire edge.
To check whether the isolates are indeed Listeria spp, following tests can be performed:
1. Catalase test (M01_08AA). Listeria are catalase positive
2. KOH test (M01_06AA): Listeria are Gram positive
3. Motility: Inoculate a TSB tube with the isolate and incubate at 25 °C for 8-24 h, until a cloudy
medium is observed. When no 25 °C incubator is available, incubate at room temperature.
Look by microscope for slim, short rods with tumbling motility. Cultures grown above 25°C
may fail to exhibit this motion. Always compare them to a positive control. Cocci, large rods or
rods with rapid swimming motility are not Listeria spp.
4. L. monocytogenes is ß-haemolysis positive. On a sheep blood agar plate, incubated at 37°C
for 24 h this is visible as a narrow clear light zone. Compare to control strains. E.g. L. ivanovii
is also ß-haemolysis positive, showing a wide clear zone after 24 h incubation on sheep blood
agar.

Positive control: L. monocytogenes ATCC 35152
Negative control: Enterococcus faecium ATCC 8043

Express the result as the absence or presence of L. monocytogenes per weight or per
8 Validation
See validation file: M01_22VB.
9 References
1. International Organisation for Standardisation, “ISO 11290-1, Microbiology of food
and animal feeding stuffs – Horizontal method for the detection and enumeration of
Listeria monocytogenes. Part 1: Detection method”. EN ISO 11290-1: 1996-12 + AMD
1: 2004-10.
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2. International Organisation for Standardisation, “Report of the 20th meeting of ISO/TC

34/SC 9 << Agricultural and food products-Microbiology >> Vienna 14/16 June 2000”
doc ISO/TC 34/SC 9N436, Report of the Vienna meeting, July 2000, p. 11.
Division.
4. M01_06AA “KOH test” CLF procedure, Microbiology Division.
5. M01_08AA “Catalase test” CLF procedure, Microbiology Division.
6. Nederlands Normalisatie Institut Doc. No 2001-15, committee 370009 “Proposal for
the improvement of the isolation of L. monocytogenes“ 12. Feb, 2001.
7. Polivka, L. “Identification of Listeria with a new chromogenic medium RAPID
L’MONO® (Short communication)” Archiv für Lebensmittelhygiene, 2001, vol. 52, p.
22-23.
10 Attachments
Not applicable
181
In the DIN EN ISO 11290-1:2005 the enriched culture from not only the second enrichment broth
(full Fraser) but the first enrichment broth (½ Fraser) has to be streaked onto the agar. In the
current method only the enriched culture from the second enrichment broth (full Fraser) will be
streaked onto agar for isolation and identification.
In the DIN EN ISO 11290-1:2005 ALOA agar is recommended as the first selective medium,
combined with the second selective medium of choice.
In the current method Palcam and Rapid L’ mono are recommended as the selective mediums.
However either two of the three media (ALOA, Palcam and Rapid L’ mono) can be applied to the
L. monocytogenes detection.
Biochemical characteristic for differentiation between L. monocytogenes and

L. innocua
The bacterial genus Listeria spp. is currently taxonomically subdivided into six species: L.
monocytogenes, L. seeligeri, L. ivanovii, L. innocua, L. grayi, and L. welshimeri. Two of the
species are pathogenic: while L. ivanovii is found to be only pathogenic for animals, L.
monocytogenes refers to as a human pathogen which can cause meningitis in newborns born
through the vaginal delivery. L. innocua is a harmless Listeria species and it is widely found in the
environment and food sources.
All Listeria species are able to grow on selective media such as Listeria agar developed by
Ottaviani and Agosti (ALOA), Palcam and Rapid L’ mono. A further confirmation is necessary. It
is crucial to differentiate the pathogenic L. monocytogenes and other Listeria species, particularly
the harmless L. innocua.
It is known that all species of Listeria, except L. monocytogenes can hydrolyse substrate DL-
alanine-beta-naphthylamide due to the presence of naphthylamidase. Expression of
naphthylamidase activity can be determined with the DIM (differentiation of L. innocua and L.
monocytogenes) reaction of the API Listeria identification kit (bioMérieux). The naphthylamidase
positive L. innocua gives an orange-colored reaction in the first DIM reaction. By contrast, L.
monocytogenes shows a very weak color change due to the absence of the enzyme.
It is must be noted that in the API Listeria kit, the DIM reaction is the only test to differentiate
between L. innocua and L. monocytogenes. The reference strain of L. monocytogenes must
always be tested in parallel for comparison.
Deletion of Listeria monocytogenes reference capsule of SVM and OXOID

Listeria Rapid test kit
The Listeria monocytogenes reference capsule of SVM is deleted from the positive control list,
because it is not available anymore. Instead, the L. monocytogenes DSMZ 12464/ATCC 35152
is added to the list.
The OXOID Listeria Rapid test kit is not applied any more in CLF.
182
Protocols for L. monocytogenes detection: ISO method and CLF
method
183
184
ACIDIC PASTEURIZED PRODUCT
185
186
No. M01_25ME_v02(pasteurized product).doc
Acidic pasteurized products Enumeration
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Enumeration of contaminating microorganisms in acidic pasteurized

products
Reasons of amendment:
V2 (15.03.14)  Renaming of the method and additional comment in scope and field
of applications
 Meat liver agar represents as an alternative for Schaedler agar for
anaerobic total viable count
 Modern identification methods 16S rRNA gene sequencing and
MALDI TOF MS added to 6.2

Name Signature Date

First version written by: Anabelle
2011
Reidel
revised by: Sha
2014
Zhu
Koch 2014
2014
Wolllnitz
approved: Matthias
2014
Fischer
187
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5
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This method describes the procedure for the enumeration of aerobic and anaerobic total
viable count at 30°C and 55°C and the enumeration of acidophilic microorganisms in
acidic pasteurized jar food. The method can also be applied to juice, fruit preparation or
chilled products.

The method is based on the pre-enrichment of potential, undesired microorganisms in the
product, followed by the determination of the viable count at 30°C and 55°C under aerobic
and anaerobic conditions.


Trypton soya broth TSB 1 month 4-10°C
Orange serum agar OSA 1 month 4-10°C
Plate count agar PCA 1 month 4-10°C
Dextrose tryptone agar DTA 1 month 4-10°C
Schaedler agar Schaedler 1 month 4-10°C
Meat liver agar MLA 1 month 4-10°C

1

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5 Equipment
6 Procedure
6.1 Enumeration
6.1.1 Determination of aerobic and anaerobic total viable count at 30°C
1. Incubate the closed jar for 7 days at 30°C.

2. Prepare a 1:10 dilution of the sample in PSS (1 ml sample in 9 ml PSS).
3. Pipette 1 ml of the dilution into a Petri dish.
 Aerobic total viable count at 30°C

Pour approximately 15 ml of PCA into the Petri dish, mix and allow it to solidify. Incubate
the plates at 30°C for 72 h aerobically.
 Anaerobic total viable count at 30°C

Pour approximately 15 ml of MLA or Schaedler agar into the Petri dish, mix and allow it to
solidify. After solidification of the plates, pour an over layer with approximately 10 ml MLA
or Schaedler to obtain anaerobic conditions. Incubate the plates at 30°C for 72 h
anaerobically.
6.1.2 Determination of aerobic and anaerobic thermophilic microorganism

2. Prepare a 1:10 dilution of the sample in PSS (1 ml sample in 9 ml PSS).
 Aerobic thermophile microorganism

Pour approximately 15 ml of DTA into a Petri dish, mix and allow it to solidify. Incubate the
plates at 55°C for 72 h aerobically.
 Anaerobic thermophilic microorganism

Pour approximately 15 ml of MLA into the Petri dish, mix and allow it to solidification. After
solidification of the plates, pour an over layer with approximately 10 ml MLA to obtain
anaerobic conditions. Incubate the plates at 55°C for 72 h anaerobically.
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6.1.3 Determination of aerobic and anaerobic acidophilic microorganism

2. Prepare a 1:10 dilution of the sample in PSS (1ml sample in 9 ml PSS).
 Aerobic acidophilic microorganism

Pour approximately 15 ml of OSA into a Petri dish, mix and allow it to solidify. Incubate the
plates at 30°C for 72 h aerobically.
 Anaerobic acidophilic microorganism

Pour approximately 15 ml of OSA into the Petri dish, mix and allow it to solidify. After
solidification of the plates, pour an over layer with approximately 10 ml OSA to obtain
anaerobic conditions. Incubate the plates at 30°C for 72 h anaerobically.
6.2 Confirmation
The identification of suspicious colonies is carried out using appropriate biochemical
identification kits.
For further confirmation and identification, modern identification tools such as 16S rRNA
gene sequencing or MALDI TOF MS are recommended.

Positive controls: Enterococcus faecium ATCC 349 for 30°C
Geobacillus stearothermophilus ATCC 12980 for 55°C
Negative controls: not necessary
Blank: γ-irradiated infant milk formula or a comparable product

Calculate the number of colonies as described in M01_01AA. Express the results as CFU
per volume or gram of sample.
8 Validation
This method does not fulfill the requirements for a validated method according to
VA504_06 (CLF procedure) and is therefore not regarded as an accredited method by
CLF.
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9 References
The above mentioned method is based on the following references but does not
1. Baumgart, J.: Mikrobiologische Untersuchung von Lebensmitteln, Behr’s Verlag,
Hamburg, VII.11-3, 1994.
2. Bean, PG.: Microbiological techniques in the examination of canned foods,
Laboratory Practice 25, 303-305, 1976.
3. Sinell, H.-J.: Zur Methodik der mikrobiologischen Untersuchung von Voll- und
Halbkonserven, Fleischw. 54, 1642-1646, 1974.
10 Attachments
Not applicable
191
AMENDMENTS AND COMMENTS
Principle of acidic pasteurized product enumeration

The current method is a method for enumeration and identification of microorganisms which
survived the pasteurization process of acidic products. The low pH of these foods is selective for
yeast, molds and a few groups of aciduric bacteria such as lactic acid bacteria, primarily species
of Lactobacillus, which can cause spoilage of the product. OSA medium is suitable for cultivation
and enumeration of aciduric microorganisms.
On the other hand there are other microorganisms which can only survive but cannot grow in
acidic products. These bacteria will be cultivated and enumerated under all possible incubation
conditions (an-/aerobic; 30°C/55°C).
The product is pre-incubated for 7 or 5 days at appropriate temperatures to simulate the common
turn-over time and conditions during the transport from factory release to consumption by
consumers. The pre-incubation temperature of 30°C applies for the examination of mesophilic
and acidophilic microorganisms, while the pre-incubation temperature of 55°C applies for
thermophilic microorganisms.
Protocols for acidic pasteurized products enumeration
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SRC (ISO) DETECTION AND ENUMERATION
193
194
No. M01_26ME_v03(SRC ISO).doc
SRC (ISO)
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Horizontal method for the enumeration of sulphite-reducing clostridia

growing under anaerobic conditions

 Revision of evaluation: Confirmation of white colonies as SRB
 Revision of evaluation: Repetition in case of gas formation with a
decimal diluted sample
 Revision of incubation time referring to norm method
 Replaces SRC method as corporate method
V3 (28.01.14)  Method name changed from SRB to SRC
 Use recommendation of M01_43ME for milk products with high
background flora has been added to 1
 Confirmation procedure in 6.2/Note 2 in the last version has
been deleted and added to 6.2
 Optional heat treatment in 6.1/Note 3 in the last version has
been deleted and added to 6.1/2
 Modern confirmation and identification tools e.g. 16S rRNA gene
sequencing and MALDI TOF MS have been added to 6.2
Name Signature Date

First version written by: Miryam
Wolf 2007
verified: Sha
Zhu 2014
revised by: Thomas
Koch 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
195
SRC (ISO)
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6
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This method is a horizontal method for the enumeration of strictly anaerobic sulfite-
reducing clostridia (SRC), applicable to products intended for human consumption and
environmental samples in the area of food production (referring to ISO 15213: 2003).
For liquid milk and milk derived products with a high number of background flora, the CLF
method M01_43ME is recommended to use.

This method is based on: ISO 15213:2003 “Horizontal method for the enumeration of
sulfite-reducing bacteria growing under anaerobic conditions.”
The following modification has been performed:
 Optional pasteurization at 80°C for 10 min (applied only if necessary)
Sulfite-reducing clostridia have the ability to reduce sulfite to sulfide. This forms a black
precipitate with Fe3+ present in the medium due to formation of iron (II) sulphide as a
result of reaction between sulfide ions and trivalent iron [Fe(III)]. The colonies are visible
as grey or black colonies.
 Optional pasteurization:
The samples can be pasteurized at 80°C for 10 min to eliminate vegetative cells, not only
competitive flora but vegetative SRC cells as well. In addition, the heat shock caused by
pasteurization can activate the germination of some SRC spores. The method refers
hereby to a spore enumeration.
 Optional incubation at 50°C:

The plates can be anaerobically incubated at 50 ± 1°C for 24-48 h (final reading after
48 h) if thermophilic clostridia are present. Typical black-colored colonies are counted.

SRC Sulfite reducing clostridia, which form black colonies through the
reduction of sulfite
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Iron sulphite agar (OXOID) ISA 1 month 4-10°C

Anaerobic jars with equipment for
generating an anaerobic see manufacturer’s description
atmosphere
Columbia agar with 5% sheep
blood (for identification) see manufacturer’s description
Biochemical identification test kit2 see manufacturer’s description

1
The shelf life of the prepared media is based on storage in the dark.
2
e.g. API 20A or API rapid ID 32A

4.1 Iron sulphite agar (OXOID, CM0079) in g/l:
Tryptone 10.0
Sodium sulphite 0.5
Iron (III) citrate 0.5
Agar 12.0
5 Equipment
Anaerobic jars with equipment for generating an anaerobic atmosphere
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6 Procedure
6.1 Enumeration
1. Prepare a 1:10 suspension of the sample in BPW as described in M01_01ME.

Samples should be shaken no more than necessary, since germinated spores (after
resuscitation) are oxygen-sensitive.
NOTES:
Optional: Samples for SRC enumeration can also be analyzed without resuscitation.
One must be aware of that not too many samples are treated in one flow, since exposure time to oxygen could
be too long for the last samples, which could decrease quantity of SRC.
2. Pipette 10 ml of a suitable sample dilution into 2 empty Petri dishes. Pour 15 ml of

ISA (tempered at 46 ± 2°C) into the Petri dishes, mix and allow to solidifiy.
 Optional pasteurization:
Transfer 10 ml of sample suspension (not resuscitated) into an empty sterile tube. Heat
the samples in a water bath at 80 ± 1°C. As soon as a temperature of 80°C is reached in
samples, pasteurize the samples for exactly 10 min at 80 ± 1°C. After pasteurization the
tubes must be cooled down immediately under the tap water to 20°C. Dry the tubes from
outside and divide the sample over 2 Petri dishes. Pour plates as described above.
NOTES:
Check the temperature by keeping a thermometer in the middle of a tube with 10 ml blank sample or water.
Otherwise the heating time of sample has to be checked with a thermometer and validation data should be
available. After the heating time has been clearly defined, blank sample for temperature control can be
omitted.
Pasteurization can activate germination of spore and germinated spores are oxygen sensitive. Therefore the
cooling time after pasteurization should be as short as possible. Shake the tubes no more than necessary. Do
not treat too many samples in one flow.
3. After the medium has solidified, pour 10 ml of the same medium into the dish as an
over layer to ensure the anaerobic condition.
4. After solidification, incubate the plates in an anaerobic jar at 37 ± 1°C for 24-48 h.
5. Read the results after 24 h as a first control (depending on the degree of black color,
the growth rate and gas production, a first enumeration should be performed). The
final evaluation is carried out after 48 h. Black colonies, possibly surrounded by black
zone, are counted as SRC. White colonies have to be confirmed.
The presence of heavy gas formation (even if no colonies are visible) and/or the
presence of H2S smell are indications for the presence of SRC. Repeat the sample in
a higher dilution in such cases.
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6.2 Confirmation
1. Black colonies:
A confirmation of typical black colonies is not necessary.
2. White colonies:
White colonies have to be checked on anaerobic growth:
Streak typical white colonies on two blood agar plates in parallel and incubate one
plate aerobically and the other anaerobically at 37°C for 24 h. In case no aerobic
growth, but anaerobic growth is shown, the colonies are concluded as SRC and
counted.
For isolates growing anaerobically only, continue as described below:
Suspicious colonies are confirmed with biochemical test kits, e.g. API 20A or API
rapid 32A (see manufacturer’s description).
Molecular biological tools e.g. 16S rRNA gene sequencing and MALDI TOF MS can
be applied for further confirmation.

Positive controls: C. butyricum ATCC 16398
a sulphite concentration of at least 0.1% is needed to give pitch
black colonies
C. difficile ATCC 9689
sensitive to sulphite concentrations of 0.1% or higher
C. putrificum ATCC 25784
sensitive to sulphite concentrations of 0.1% or higher
C. perfringens ATCC 13124
Negative control: Bacillus licheniformis CLF 897
NOTE:
Fresh cultures of clostridia strains do not contain spores and will be inactivated through pasteurization
process. Therefore they cannot be used as positive control for the method, if an optional pasteurization is
applied.

Calculate the number of SRC as described in M01_01AA. Express the results as CFU per
199
SRC (ISO)
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8 Validation
9 References
1. Microbiology of food and animal feeding stuffs – Horizontal method for the
enumeration of sulfite-reducing bacteria growing under anaerobic conditions, ISO
15213: 2003-05-01.
2. Bredius. M. W. J.; de Ree. E. M.: Media for the detection and enumeration of
clostridia in foods. Handbook of Culture Media for Food Microbiology, S. E. L. Corry
et al. (Eds.) Elsevier Science B. V. 2003.
3. Clifford, W. J.; ABE ANELLIS; Ross. E. W.: Evaluation of media, time and
temperature of incubation and method of enumeration of several strains of Clostrium
perfringens spores. Appl. Micro. 27, 784-792 (1974).
4. Eisgruber, H.: Der kulturelle Nachweis Sulphitereduzierender Clostridien in
Lebensmitteln–kritische Anmerkung zur neuen ISO-Norm 15213. Archiv für
Lebensmittelhygiene 52, 72-112 (2001).
5. Eisgruber.H.; Reuter. G.: SCA – ein Selectivnährmedium zum Nachweis mesophiler
Sulphitereduzierender Clostridien in Lebensmitteln, speziell für Fleisch und
Fleischerzeugnisse. Archiv für Lebensmittelhygiene 42, 101-132 (1991).
10 Attachments
Not applicable
200
Iron Sulphite Agar (ISA) from OXOID replaces the ISO medium as standard
plating medium in the current method
In ISO 15213:2003, a so-called “Iron sulphite agar” is officially recommended. However the agar
named as “ISA” is not the same one described in the current method. From the aspect of
ingredients it corresponds to the commercially available Triptose-sulfite-cycloserine (TSC) base
provided by Merck KGaA (see Table 1).
In the validation test different commercially available agars were tested on the recovery of a
panel of SRC strains in various species. Among these media, the Iron Sulphite Agar (OXOID, CM
0079) has showed the highest efficiency in recovering SRC, while the growth of several SRC
species e.g. Cl. pasteuranium, Cl. sporogenes, Cl. pseudotetanicum, Cl. putrificum, Cl. novyi, Cl.
difficile, Cl. baratii and Cl. acetobutyricum was fully inhibited on the ISO-recommended TSC agar
base (see M01_26VB). The reduced sensitivity of TSC agar base is probably explained by the
higher (di)sulfite concentration (0.1%) (see Table 1). Sulfite-sensitive SRC have been reported
and a higher sulfite concentration can suppress the growth of those SRC strains. As a result, a
maximal sulfite concentration for medium of SRC detection of 0.05% is recommended (see Table
2).
Table 1: Composition of TSC agar base (Merck) (complying with “Iron sulfite agar” in ISO 15213) in g/l
Ingredients Weight
Tryptose 15.0
Pancreatic digest from soya 5.0
Yeast extrac 5.0
Sodium disulfite 1.0
Agar 12.0
Table 2: Composition of ISA (OXOID) in g/l
Ingredients Weight
Tryptone 10.0
Sodium sulphite 0.5
Iron (III) citrate 0.5
Agar 12.0
201
Confirmation of white colonies isolated from ISA
It has been shown that some SRC species like Cl. acetobutylicum and Cl. butyricum appeared
white on ISA agar (see M01_26VB). Hence a further confirmation of white colonies isolated from
ISA is obligatory: The isolate will be streaked on two blood plates and incubated at 37°C for 24 h
aerobically and anaerobically in parallel. Only the isolates which grow under anaerobic, but not
aerobic conditions are suspicious for SRC and have to be identified further using biochemical kits
or molecular biological tools.
Protocols for SRC enumeration: ISO method and CLF method
202
CRONOBACTER DETECTION AND MPN ENUMERATION
203
204
No. M01_30ME_v04(Cronobacter detection MPN).doc
Cronobacter spp. – Detection/MPN
page: 1 version: 4
of: 7 written: 2009-12-11
printed: 2014-06-27

V3 (11.12.09)  Description of performed modifications varying from norm
method has been added.
 Renaming of Enterobacter sakazakii to Cronobacter spp.
 Insertion of the composition of ESIATM
V4 (11.03.14)  Confirmation of α-glucosidase activity with paranitrophenyl-α-D-
D-glycopyranoside and confirmation of Tween 80 esterase
activity with Tween 80 agar are deleted
 Modern identification tools such as 16S rRNA gene sequencing
and MALDI TOF MS have been added
Name Signature Date

1st version written by: Suhad
Sanjaq 2004
revised by: Björn

Hampel 2014
Zhu 2014
Wollnitz 2014
approved: Matthias
Fischer 2014
205
page: 2 version: 4
of: 7 written: 2009-12-11
printed: 2014-06-27

This method for the detection of Cronobacter spp. can be applied to milk powder and
powdered infant formula as well as other food products, raw materials and environmental
samples.

This method is based on ISO/TS 22964:2006.
 DFI medium (Druggan-Forsythe-Iversen formulation) as chromogenic agar
 Confirmation: α-glucosidase activity and Tween 80 esterase activity
 Identification with API 32E and molecular biological tools

Enterobacteriaceae: Gram negative, oxidase negative, facultative anaerobic, glucose
fermenting rods
Cronobacter spp.: Microorganisms which produce yellow, sometime milky white
colonies on TSA, positive for α-glucosidase activity and tween 80
esterase activity and also give a corresponding biochemical
profile when tested with API 32E.
NOTE:
The α-glucosidase test is an essential differentiate criterion for identification and differentiation of Cronobacter
spp. from other closely related bacteria.

Buffered peptone water BPW 1 month room temperature
Lauryl sulfate tryptose broth LST 1 month room temperature
Tryptone soy agar TSA 1 month 4˚C-10°C

Druggan-Forsythe-Iversen agar DFI 1 month 4˚C-10°C
Enterobacter sakazakii isolation agar ESIA 1 month 4˚C-10°C
Biochemical identification test kit See manufacturer’s description

1
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4.1 modified LST (mLST) in g/l:

Tryptose 20 g
Lactose 5g
Potassium Di-Hydrogenphosphate 2.75 g
Di-Potassiumhydrogenphosphate 2.75 g
Sodium chloride 34 g
Sodium lauryl sulphate 0.1 g
Dissolve the components in the water and heat them if necessary. Adjust the pH if
necessary. Distribute the mLST with extra salt in flasks or tubes according to the
analytical needs and sterilize for 15 min at 121˚C.
Dissolve 10 mg of vancomycin in 10 ml distilled water and sterilize by filtration (0.2 µm).
The solution can be kept at 4 ± 1˚C for 14 days. Add 1 ml of sterile vancomycin to 100 ml
mLST to obtain a final vancomycin concentration of 10 μg/ml.
NOTE:
Vancomycin will be added to the mLST each time before use.

The most convenient way to prepare LST with 0.5 M NaCl is to use commercially available dehydrated LST
broth (weigh 35.6 g) and add 29 g of NaCl per liter.
4.2 Enterobacter sakazakii isolation agar (ESIATM)

ESIA medium is commercially available from suppliers e.g. AES Cheminux, France or
OXOID, UK (CM 1134).
Pancreatic peptone of casein 7g
Yeast extract 3g
Sodium chloride 5g
Sodium desoxycholate 0.6 g
5-bromo-4-chloro-3-indolyl α-D-glucopyranoside 0.15 g
Crystal violet 2 mg
Agar 12 to 18 g1
Water 1000 ml
1
Depending on the gel strength of the agar
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4.3 BrillianceTM Enterobacter sakazakii agar (DFI formulation)

DFI medium is commercially available from supplier e.g. OXOID, UK (CM 1055).
Tryptone 15 g
Soy peptone 5g
Sodium chloride 5g
Ferric ammonium citrate 1g
Sodium thiosulphate 1g
Sodium desoxycholate 1g
5-bromo-4-chloro-3-indolyl α-D-glucopyranoside 0.1 g
Agar 15 g
Water 1000 ml
5 Equipment
6 Procedure
6.1 Detection/MPN enumeration

1. 100 g of the product are resuscitated in 900 ml BPW and incubated for 16-20 h at
37°C.
2. Transfer 0.1 ml of pre-enriched sample into 10 ml mLST.
3. Incubate the mLST broth for 22-24 h at 43 ± 1°C.
4. Streak a loopful of the mLST culture onto the surface of a chromogenic agar plate (e.
g. ESIA or DFI medium).
5. Incubate the ESIA or DFI at 43 ± 1°C or 37°C respectively.
6. The chromogenic plate (e.g. ESIA or DFI) is examined for the presence of typical
colonies of presumptive Cronobacter spp..
 ESIA:
Typical colonies are small to medium sized (1-3 mm) green to blue colonies. Non-typical
colonies are slightly transparent and are colored slightly violet.
 DFI:
Cronobacter spp. forms easily distinguishable blue-green colonies.
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NOTE 1:
For MPN enumeration, inoculate an appropriate number of flasks/tubes with the same dilution depending on
the desired accuracy of the results. As a general rule, the technique specified requires three flasks or tubes
per dilution.
NOTE 2:
Strains of non-Cronobacter species such as Escherichia vulneris, Pantoea spp. and Citrobacter koseri etc.
appear blue-green on DFI as well.
6.2 Confirmation
Characteristic colonies on chromogenic medium are streaked onto TSA agar and
incubated for 24 h at 37°C. Most Cronobacter spp. strains show yellow pigment on TSA.
However some of them can appear as milky-white.
Isolated colonies are first tested with oxidase, catalase reactions and then confirmed
using biochemical identification test kits e.g. API ID 32E and VITEK 2 compact GN.
For further confirmation molecular biological tools such as 16S rRNA gene sequencing
and MALDI TOF MS can be applied.

Positive controls: Cronobacter sakazakii ATCC 29544
Enterobacter cloacea ATCC 13047
Negative control: Staphylococcus aureus ATCC 6538

 For detection in 100 gram:
Express the results as the absence or presence of Cronobacter spp. per weight or per
volume of sample.

8 Validation
See M01_30VB.
209
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9 References
1. ISO/TS 22964:2006. Milk and milk products--Detection of Enterobacter sakazakii.
2. Mossel, D.A.A, Struijk, C. “Standing operating procedure: boundary test for and
typification of Enterobacteriaceae in dried microbial dietary formula” EF 1998-250.
3. Nazarowec-White, M., Farber, J.M. (1997). Incidence, survival and growth of
Enterobacter sakazakii in infant formula” Journal of Food Protection, Vol. 60, No. 3.
4. Kandhai M.C. et al. (2004).: Occurrence of Enterobacter sakazakii in food production
environments and households, The Lancet, Vol. 363, p. 39 – 40.
5. Muytjens H.L. et al. (1988): J. Clin. Microbiol. 26,743-746
6. Nazarowec-White M. and Farber J.M. (1997a): Int. J. Food Microbiol. 34,103-113
7. Nazarowec-White M. and Farber J.M. (1997b): Thermal resistance of Enterobacter
sakazakii in reconstituted dried-infant formula. Lett. Appl. Microbiol. 24,9-13.
8. Breeuwer et al. (2003): Desiccation and heat tolerance of Enterobacter sakazakii, J.
Appl. Microbiol. 95,967-973.
9. Iversen et al. (2008): Development of a novel screening method for the isolation of
Cronobacter spp. Appl. Environ. Microbiol. 2550-2553.
10 Attachments
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11 Most probable number (MPN) table for dilution in triplicate for 100 g, 10 g
and 1 g. (EN ISO 7218:2007)
Number of positive results MPN in

Confidence interval ≥ 95%
CFU/100 g
3 x 100 g 3 x 10 g 3x1g
0 0 0 < 0.30 0.00 0.94

0 0 1 0.30 0.01 0.95
1 0 0.30 0.01 1
0 1 1 0.61 0.12 1.7
0 2 0 0.62 0.12 1.7
0 3 0 0.94 0.35 3.5
1 0 0 0.36 0.02 1.7
1 0 1 0.72 0.12 1.7
1 0 2 1.1 0.4 3.5
1 1 0 0.74 0.13 2
1 1 1 1.1 0.4 3.5
1 2 0 1.1 0.4 3.5
1 2 1 1.50 0.5 3.8
1 3 0 1.60 0.5 3.8
2 0 0 0.92 0.15 3.5
2 0 1 1.4 0.4 3.5
2 1 0 1.5 0.4 3.8
2 1 1 2.0 0.5 3.8
2 2 0 2.1 0.5 4
2 2 1 2.8 0.9 9.4
2 2 2 3.5 0.9 9.4
2 3 0 2.9 0.9 9.4
2 3 1 3.6 0.9 9.4
3 0 0 2.3 0.5 9.4
3 0 1 3.8 0.9 10.4
3 0 2 6.4 1.6 18.1
3 1 0 4.3 0.9 18.1
3 1 1 7.5 1.7 19.9
3 1 2 12 3 36
3 1 3 16 3 38
3 2 0 9.3 1.8 36.00
3 2 1 15 3 38
3 2 2 21 3 40
3 2 3 29 9 99
3 3 0 24 4 99
3 3 1 46 9 198
3 3 2 110 20 400
3 3 3 > 110.00
211
Chromogenic agars used in the current method

TM
ESIA is recommended officially in ISO/TS 22964 as standard medium for isolation of
presumptive Cronobacter spp. Inoculated EISATM medium is incubated at 44 ± 0.5°C for 24 h.
According to the validation data both ESIATM (44°C) and DFI (37°C) showed a high sensitivity of
100% for Cronobacter spp. The specificity of ESIATM is slightly higher (56%) than that of DFI
(44%) probably due to the higher incubation temperature of ESIATM. Both media are well suitable
for chromogenic detection of presumptive Cronobacter spp. and are therefore recommended to
use in the current method.
However it has been established that there are Cronobacter isolates which do not grow at an
elevated temperature of 44°C (Iversen et al., 2008). For such temperature-sensitive Cronobacter
isolates DFI medium might represent a better option. Also the incubation temperature of mLST is
adapted to 43 ± 1°C in the current method instead of 44 ± 0.5°C in the ISO norm.
API ID 32E for biochemical identification

API ID 32E proved to be reliable in Cronobacter spp. identification, showing obviously higher
sensitivity and specificity prior to API 20E. The latter system identified presumptive Cronobacter
isolates with both false positive and false negative outcomes.
212
Protocols for Cronobacter detection: ISO method and CLF method
213
214
PSEUDOMONAS DETECTION
215
216
No. M01_32ME_v03(Pseudomonas detection).doc
Pseudomonas spp.  Detection
of: 5 written: 2011-02-03
5
printed: 2014-06-27

 Analyses of Pseudomonas in 10 g and 1 g were aligned
 Burkholderia cepacia as a positive control was deleted
 Ecometrie as a test method for the quality of media was deleted
V3 (17.03.14)  negative control added to 6.3
Name Signature Date

First version written by: Miriam
2006
Wolf
revised by: Sha
2011
Zhu
Koch 2014
2014
Wollnitz
approved: Ron
2014
Wacker
217
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The method for the detection of Pseudomonas spp. can be applied to milk powder,
cereals and raw material and for special medical purposes.
Aeromonas spp.
Aeromonads are facultative anaerobe, oxidase-positive, gram-negative rods. They are
morphologically similar to Pseudomonas but their metabolism is fermentative. Most of the
Aeromonads are mesophil-psychrotroph, with a range of growth temperature of 4-45°C
and a growth optimal at 28°C. Aeromonas hydrophila is described as a new foodborne
pathogen (FDA 1984) which causes enteritis and vomiting.
Pseudomonas spp.
Pseudomonas are gram-negative rods, catalase and oxidase positive and motile by polar
flagella. Their metabolism is strictly aerob without fermentation. Most of the Pseudo-
monads are psychrotroph with a range of growth between 4–41°C.
Pseudomonas aeruginosa
Pseudomonas aeruginosa is known as a pathogen of nosocomial infection which often
proceeds to a sepsis. In cases of hospital food infections P. aeruginosa was found mainly
in milk, human milk, drinking water and sometimes in fruits and vegetables. Further
member of the Pseudomonas group which are attached food-hygienic importance are P.
fluorescens, P. putida and P. stutzeri. These three species are regarded as potential
enterotoxin producer.

The bacteria are pre-enriched in a liquid, high nutrient medium plus supplement which
contains all substances Pseudomonads required. After incubation a pure streak out of the
broth is made on a light-selective agar. Suspicious colonies on the agar plate were
identified biochemically.
Nutrient Broth No. 2 is a nutritious medium suitable for the cultivation of fastidious
pathogens and other microorganisms. This medium is comparable to a meat infusion and
is very rich in nutrients. It gives good growth from small inocula.
Pseudomonas agar base is designed so that by the addition of the appropriate

supplement CFC, the medium becomes selective for Pseudomonas spp. as well as other
gram-negative and oxidase-positive rods. The base medium is a modification of King’s A
Medium in which magnesium chloride and potassium sulphate are present to enhance
pigment production. The CFC supplement was described by Mead and Adams who
reduced the cetrimide concentration to allow the growth of all pigment forming and non-
pigment forming psychrophilic microorganisms. To enforce the selectivity cephaloridine
and fucidine was added.
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This method is based on ISO 13720:1995-12 (Meat and meat products – Enumeration of
Pseudomonas spp.).
 pre-enrichmeht in Nutrient No. 2+ CFC-Supplement at 30°C for 24 h
 incubation CFC agar at 30°C for 24 h

gram-negative/ Straight gram-negative rods, chemoorganothroph and facultative
oxidase-positive anaerobe. Growth occurs from 4°C to 45°C and they are catalase-
bacteria positive and oxidase-positive (e.g. Aeromonas)
psychrophilic Bacterial species that work in low temperature range (below 18°C)
and are mostly active around 12°C.

Cetrimide-Fucidin-
Cephalosporin supplement CFC See manufacturer’s description
(Oxoid SR0103E)
Nutrient broth No. 2 + CFC
NB+ Use directly after preparation
supplement (0.36 ml/90 ml)
Cetrimide agar base CA 1 month 4-10°C
Cetrimide-CFC agar (1 vial
Use directly after preparation
supplement/500 ml agar base)
Tryptone soy agar3 TSA 1 month 4-10°C
Plate count agar3 PCA 1 month 4-10°C
Oxidase Reagent1 Ox See manufacturer’s description
Catalase Reagent1 Cat See manufacturer’s description
Biochemical identification kit2 See manufacturer’s description
1
2
e.g. API 20 NE or API ID 32 E or a comparable reliable test kit.
3
Either TSA or PCA is used.
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5 Equipment
UV-lamp (wavelength 360 nm) for detection of fluorescent colonies
6 Procedure
6.1 Pre-enrichment and isolation

1. Prepare a 10% sample suspension in Nutrient Broth (NB) No. 2 + CFC supplement
(NB+)
 Detection of Pseudomonas in 10 g:
Weigh 10 g sample into 90 ml NB and incubate at 30 ± 1°C for 24 ± 3 h.
 Detection of Pseudomonas in 1 g:
Weigh 1 g sample into 9 ml NB and incubate at 30 ± 1°C for 24 ± 3 h.
2. Streak a loop full of each incubated broth on a sterile pre-dried Cetrimid-CFC-agar

plate and incubate at 30 ± 1°C for 24 ± 3 h.
3. Make a pure streak of all suspicious colonies on TSA or PCA for identification
The appearance of Pseudomonas spp. colonies on Cetrimid-CFC-Agar are round, smooth

or slimy. Most of them produce a blue-green and a fluorescent pigment but Aeromonads,
Burkholderia cepacia and some Pseudomonads do not have a blue-green pigment
formation and do not show any fluorescent signal under UV-light. But all species have one
common attribute, the positive oxidase reaction.
6.2 Confirmation
Suspicious colonies have to be characterized by gram straining, oxidase- and catalase-

test and have to be identified by a biochemical identification test kit. The biochemical test
kit is performed according to the manufacturer’s description.
For a further confirmation, 16S rRNA gene sequencing or MALDI TOF MS are
recommended.
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Positive controls: Pseudomonas aeruginosa ATCC 27853
Pseudomonas fluorescens
Pseudomonas putida ATCC 12633
Negative controls: Staphylococcus aureus ATCC 25923

Express the results as the absence or presence of Pseudomonas spp. per weight or
volume of sample (see M01_01AA).
8 Validation
9 References
1. Bergey’s Manual of Systematic Bacteriology, Volume 1, 1984
2. Oxoid, The Manual, 8th Edition 1998.
3. ISO 13720, First Edition 1995-12-15, Meat and meat products – Enumeration of
Pseudomonas spp.
4. Nutricia Corporate methods , 1988-12-28, Isolation of Pseudomonadaceae.
5. Nutricia Corporate methods , 1989-03-29, Detection of Pseudomonas aeruginosa.
6. Heeschen, W. 2003/12, Handbuch der Lebensmittelhygiene, Kap.2.3.2.11, Behr’s
Verlag Hamburg.
7. Europäisches Arzneibuch, 1997.
10 Attachments
Not applicable
221
Principle of Pseudomonas spp. detection method
The standard norm ISO 13720 “Meat and meat products – Enumeration of Pseudomonas spp.” is
an enumeration method. The method M01_32ME is a detection method.
Both methods include an enrichment step: in ISO 13720 the sample is resuscitated in BPW at 37
°C directly followed by spreading. In the present method the enrichment is carried out in nutrient
broth No.2 supplemented with CFC at 30°C for 24 h. The validation data (see M01_32VB1)
showed that the nutrient broth No.2 with selective supplement CFC followed by streaking on CFC
agar enabled a successful detection of Pseudomonas spp. and other gram negative, oxidase
positive rods with a high sensitivity of 1 CFU/g milk powder against a high number of competitive
flora.
In the ISO 13270 the incubation condition of inoculated CFC agar is 25°C 48 h. However it has
been found in the validation (see M01_32VB1) that the target microorganisms showed a better
growth, pigmentation and fluorescence at 30°C than at a higher (37°C) or a lower (25°C)
incubation temperature. Therefore the incubation temperature of CFC agar is adapted to 30°C.
Protocols for Pseudomonas detection: ISO method and CLF method
222
BIFIDOBACTERIA ENUMERATION
223
224
No. M01_37ME_v02(Bifidobacteria enumeration).doc
Bifidobacteria Enumeration
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Enumeration of presumptive bifidobacteria –

Colony count technique at 37°C

V2 (05.02.14)  The ¼ strength Ringer’s solution replaces the Tryptone salt
cysteine (TSC) as the diluent solution with information about the
reagent shelf life and preparation added
Name Signature Date

First version written by: Barbara
2011
Reiter
revised by: Sha
2014
Zhu
2014
Hampel
2014
Wollnitz
approved: Matthias
2014
Fischer
225
of: 7 written: 01.02.2011
7
printed: 27.06.2014

The method for the selective enumeration of presumptive bifidobacteria can be applied to
dairy products (fermented and non-fermented milks, milk powders and infant formula), and
starter cultures where these microorganisms are present, and in combination with other
lactic acid bacteria.
Bifidobacteria used in milk products usually belong to the species: B. adolescentis, B.
animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. infantis and B.
longum.

The antibiotic Li-Mupirocin (MUP) inhibits the growth of most lactic acid bacteria
commonly used in fermented and non-fermented dairy products.
Owing to the proven selectivity of the antibiotic supplement Li-Mupirocin added to the
medium, usually there is no growth of typical yogurt bacteria (Streptococcus thermophilus,
Lactobacillus delbrueckii subsp. bulgaricus), mesophilic cultures (e.g. Lactococcus lactis),
Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus rhamnosus on the
medium specified.
Additionally, TOS agar enhances the growth of bifidobacteria used in dairy products.
This method is based on norm method ISO 29981: 2010/IDF 220.

Bifidobacteria: anaerobic microorganisms that form lenticular or round colonies,
partially star-shaped or trilobate of diameter 1 mm to 4 mm on TOS-
MUP medium under the conditions specified in this International
Standard.
Bifidobacteria are non-acid-fast, non-spore forming, gram-positive,
non-motile and catalase-negative chemo-organotrophs, with the
production of acetic and lactic acid. Glucose is degraded exclusively
and characteristically by the fructose-6-phosphate shunt in which
fructose-6-phosphate phosphoketolase cleaves fructose-6-phospate
into acetyl phosphate and erythrose-4-phosphate.
The optimum growth temperature is between 37°C and 41°C.
 Microscopic appearance:
Rods of very varied shapes, usually curved and clubbed, often
branched, arranged singly, in pairs, in V arrangements, in chains, in
palisades of parallel cells, or in rosettes, occasionally exhibit swollen
coccoid forms.
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Ringer’s solution tablets3 5 years room temperature
Transgalactosylated
oligosaccharides propionate TOS 3 months 4-10°C2
agar base
Lithium-Mupirocin MUP See manufacturer’s description

1
2
TOS used in current method is supplied from Merck. Culture media from other suppliers may have other
maximal storage times
3
Ringer’s tablets from Merck Code: 1155250001. Tablets from other suppliers may have other maximal shelf
life

4.1 Diluent: ¼ strength Ringer’s solution in g/l:

NaCl 2.25
KCl 0.105
CaCl2 0.12
NaHCO3 0.05
The ¼ strength ringer solution is commercially available in form of tablets from suppliers
such as Merck, Thermor Scientific, Sigma Aldrich etc.
To prepare ¼ strength Ringer Solution, dissolve 1 tablet in 500 ml of distilled water.

Sterilize by autoclaving at 121°C for 15 minutes.
NOTE:
Adjust the diluent to room temperature before starting the analyses.
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4.2 Culture medium: TOS agar + mupirocin lithium (TOS-MUP)
4.2.1 TOS propionate agar base in g/l:

Peptone from casein 10.0
Yeast extract 1.0
KH2PO4 3.0
K2HPO4 4.8
(NH4)2SO4 3.0
MgSO4 • 7 H2O 0.2
R-Cysteine HCl• H2O 0.5
Sodium propionate 15.0
TOS 10.0
Agar-agar 15.0
NOTE:
TOS medium is heat sensitive and should be autoclaved in small portions (95 ml or 190 ml of dissolved
medium).
4.2.2 Li-Mupirocin supplement

Immediately before use dissolve the lyophilised MUP in deionized water and prepare a
solution with a concentration of 1 mg/ml (e.g. 50 mg MUP in 50 ml water or other amounts
in the same proportion). Sterilize the solution obtained by filtration through a membrane
(pore size 0.22 µm).
 Merck MUP selective supplement:

Each vial contains 25 mg Lithium-Mupirocin (Merck Cat. No. 1.00045.0010). Suspend the
lyophilised reagent in the original vial by adding 25 ml of sterile purified water resulting in
a supplement solution concentration of 1 mg/ml. Shake gently until the solution is clear.
4.2.3 TOS-MUP-agar
Aseptically add 5 ml MUP solution (1 mg/ml) to 95 ml of liquefied medium at 48°C +/1°C
(190 ml base medium is supplemented with 10 ml supplement solution).
Avoid air bubbles during addition of supplement, which can cause oxidation of the
medium.
The complete TOS-MUP medium contains a final Lithium-Mupirocin concentration of 50
mg/L.
After Lithium-Mupirocin supplementation the medium is used immediately.
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5 Equipment
Anaerobic incubation equipment, e.g. an incubation jar
6 Procedure
6.1 Enumeration
6.1.1 Preparation of test portion and primary dilution
 Dried milk products (e.g. infant milk formula)

1. Mix the closed sample by shaking and inverting.
2. Distribute 90 ml ± 0.1 ml of the Ringer’s diluent (room temperature) in a sterile bottle.
Pre-warm the diluent to 45°C in a water bath.
3. Weigh 10 g ± 0.1 g of the test sample directly into the bottle with the diluent at 45°C.
4. To dissolve the test portion, swirl slowly to wet the powder. Then shake the bottles
10 times, with a movement of about 300 mm, for approximately 7 s.
5. Place the bottles in a water bath at 45°C for 5 min and shake occasionally.
6. Immediately adjust to room temperature while shaking under running tap water for
2 min.
7. Prepare a serial 10-fold dilution.
 Probiotic yoghurt or yoghurt-like products

1. Distribute 90 ml ± 0.1 ml of the Ringer’s diluent (room temperature) in a sterile bottle.
2. Mix the closed sample by shaking and inverting (preferably 10 times, with a
movement of about 300 mm, for approximately 7 s) or with a sterile spatula.
3. Weigh 10 g ± 0.1 g of the test sample directly into the bottle with the diluent.
4. Shake the bottle 10 times, with a movement of about 300 nm, for approximately 7 s.
5. Prepare immediately a serial 10-fold dilution.
NOTE:
DO NOT prepare initial suspension by pipetting (1 ml).
6.1.2 Preparation of decimal dilutions
1. Shake (preferably 10 times, with a movement of about 300 mm, for approximately 7 s
the primary dilution (10-1 dilution) to obtain homogeneity.
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2. Pipette 1 ml of the primary dilution (initial suspension) into a test tube containing 9 ml
of Ringer’s solution.
3. Thoroughly mix the dilution by using a vortex mixer. For further dilution steps proceed
in the same manner.
NOTE:
Avoid filling the pipette with air bubbles.
6.1.3 Inoculation and incubation
1. Transfer by dripping, 1 ml of each appropriate dilution step (4 decimal dilution steps

within the countable area are recommended; in general 10-5 to 10-8 for dairy products)
into each empty Petri dish with two replicate plates per dilution
2. Pour approximately 15 ml TOS-MUP medium into the Petri dishes. Mix the medium
gently by rotating without incorporating air. Allow to solidify.
3. Immediately after solidification invert all Petri dishes and incubate under anaerobic
conditions for 72 ± 3 h at 37°C.
4. Count the colonies (identity bifidobacterial colonies by their whitish appearance) after
incubation (consider only plates with an amount of ≤ 300 colonies).
6.2 Confirmation
Identify bifidobacterial colonies by their whitish color. Select typical colonies from the
plates used for counting and examine microscopically.
Optionally, a fructose-6-phosphate phosphoketolase (F6PPK)-assay can be performed to
confirm the results.

Positive controls: Bifidobacterium longum subsp. longum ATCC 15707
Bifidobacterium animalis susp. animalis ATCC 25527
Bifidobacterium breve ATCC 15700
Negative controls: Streptococcus salivarius subsp. thermophilus ATCC 19258
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842
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Calculate the number of bifidobacteria as described in M01_01AA. Express the results as
8 Validation
9 References
1. International Organization for Standardization “ISO 2998: Milk products –
Enumeration of presumptive bifidobacteria – Colony count technique at 37°C” 2010-
02.
Division.
3. International Organization for Standardization “ISO 8261: Milk and milk products
General guidance for the preparation of test samples, initial suspensions and decimal
dilutions for microbiological examination” 2001
4. International Organization for Standardization “ISO 7218: Microbiology of food and
animal feeding stuffs - General requirements and guidance for microbiological
examinations” 2007.
5. Danone Research Center “Document MTH-06-202, Version 4: Enumeration of
presumptive bifidobacterium in dairy products – Colony count technique on TOS-MUP
media (ISO 29981 – IDF 220)” 2008.
6. Danone Research Center „Analytical Report & Expertise Report, AS 426-08:
Comparison of different diluents for bifidobacteria count” 2008.
10 Attachments
Not applicable
231
Ringer’s solution as diluent solution

¼ Ringer’s solution is recommended officially as a diluent in ISO 29981:2010. The older version
of M01_37 used tryptone salt cysteine (TSC) solution as diluent and is replaced by ¼ Ringer’s
solution in the updated current version. According to the validation data, no significant difference
between ¼ Ringer’s solution and TSC was shown for enumeration of target microorganisms in
powdered infant formula (see M01_37VB).
Protocols for Bifidobacterium enumeration: ISO method and CLF

method
232
E. COLI DETECTION AND MPN ENUMERATION
233
234
No. M01_55ME_v01(E.coli detection MPN).doc
E. coli - Detection/MPN
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Horizontal method for the detection and enumeration of presumptive

Escherichia coli (E. coli)

Name Signature Date

1st version written by: Sha
Zhu 2014
Bredius
Thomas
revised by: 2014
Koch
2014
Melzer
2014
Wollnitz
approved: Matthias
2014
Fischer
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The method for the detection and enumeration of E. coli can be applied to products
intended for human consumption and the feeding of animals and environmental samples.

This method is based on ISO 7251:2005 “Microbiology of food and animal feeding stuffs
– Horizontal method for the detection and enumeration of presumptive Escherichia coli –
Most probable number technique. The suspended sample is first diluted in 1:10 in lauryl
sulfate broth (LSB) and incubated for 48 h at 37°C. After the incubation in LSB inoculate
a tube of selective EC broth with a sampling loop and incubate the tube at 44°C for 48 h.
Modifications
Following modification has been performed:
 Isolation and confirmation of culture in EC broth using commercially available
biochemical identification kits e.g. API 32E and/or modern identification tools like
16S rRNA gene sequencing and MALDI TOF MS.

presumptive E. coli Bacteria which ferment lactose at 44°C with the gas production
and produce indole from tryptophan.
Some pathogenic strains of E. coli such as Escherichia coli O157
do not grow at 44°C. They are not detectable with the current
method.

1
Lauryl sulfate broth LSB 1 month room temperature
EC broth EC 1 month 4-10°C

Tryptone soy agar TSA 1 month 4-10°C
Biochemical identification test kit See manufacturer’s description

1
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4.1 LSB in g/l:

Double-strength medium Single-strength medium
Tryptose 40 20
Lactose 10 5
Di-potassium monohydrogen phosphate 5.5 2.75
Potassium dihydrogen phosphate 5.5 2.75
Sodium chloride 10 5
Sodium lauryl sulfate 0.2 0.1
4.2 EC broth in g/l (also commercially available):

Enzymatic digest of casein 20
Lactose 5.0
Bile salts No. 3 1.5
Potassium monohydrogen phosphate 4.0
Potassium dihydrogen phosphate 1.5
Sodium chloride 5.0
5 Equipment
6 Procedure
6.1 Detection
contamination level of the sample (see method M01_01ME). Pipette the appropriate
volume of (diluted) sample of e.g. 1 ml to 9 ml of single-strength LSB or 10 ml to 10
ml double-strength LSB.
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2. Incubate the single- or double-strength LSB for 42-48 h at 37°C. The Durham tube
must stick to the bottom of the tube.
3. After 48 h incubation period, if opacity, cloudiness or any visible gas is observed,
inoculate a tube of EC broth with a sampling loop and incubate at 44°C for 48 h.
4. After incubation, if opacity, cloudiness or any visible gas is observed, streak the EC
broth onto a TSA plate and incubate at 37°C for 24 h.
NOTES:
For MPN enumeration, inoculate an appropriate number of tubes with the same dilution depending on the
desired accuracy of the results e.g.:
Take three tubes of double-strength LSB and transfer to each tube 10 ml of initial suspension. These test
portions correspond to 1 g sample.
Take three tubes of single-strength LSB and transfer to each tube 1 ml of initial suspension. These test
portions correspond to 0.1 g sample.
Take three tubes of single-strength LSB and transfer to each tube a further decimal dilution of 1 initial
suspension. These test portions correspond to 0.01 g sample.
Incubate the tubes as described in 6.1 and interpret the results according to the MPN table.
6.2 Confirmation
Colonies on TSA are confirmed with biochemical kits e.g. API 32E. Particularly examine
the reaction of indole production. A red colour indicates the presence of indole which is
typical for presumptive E. coli.
For a further identification, 16S rRNA gene sequencing and/or MALDI TOF MS are
recommended.

Positive control: Escherichia coli ATCC 11775

 For detection:
Express the results as the absence or presence of presumptive E. coli per weight or per
volume of samples.
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8 Validation
9 References
1. International Organisation for Standardisation, „Horizontal method for the detection
and enumeration of presumptive Escherichia coli – Most probable number technique
“. ISO 7251:2005.
2. International Organisation for Standardisation, „Microbiology of food and animal
feeding stuffs – General requirements and guidance for microbiological examinations
“. ISO 7218:2007.
3. International Organisation for Standardisation, „Horizontal method for the detection of
Escherichia coli O157”. ISO 16654:2001.
4. M01_09ME “E. coli - Detection“. CLF method, Microbiology Division.
5. M01_01ME “Sample preparation and dilution“. CLF method, Microbiology Division.
10 Attachments
Not applicable
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11 Most probable number (MPN) table for dilution in triplicate for 1 g, 0.1 g and
0.01 g. (EN ISO 7218:2007)
Number of positive results MPN in CFU/g Confidence interval ≥ 95%

0 0 0 < 0.30 0.00 0.94
0 0 1 0.30 0.01 0.95
0 1 0 0.30 0.01 1
0 1 1 0.61 0.12 1.7
0 2 0 0.62 0.12 1.7
0 3 0 0.94 0.35 3.5
1 0 0 0.36 0.02 1.7
1 0 1 0.72 0.12 1.7
1 0 2 1.1 0.4 3.5
1 1 0 0.74 0.13 2
1 1 1 1.1 0.4 3.5
1 2 0 1.1 0.4 3.5
1 2 1 1.50 0.5 3.8
1 3 0 1.60 0.5 3.8
2 0 0 0.92 0.15 3.5
2 0 1 1.4 0.4 3.5
2 1 0 1.5 0.4 3.8
2 1 1 2.0 0.5 3.8
2 2 0 2.1 0.5 4
2 2 1 2.8 0.9 9.4
2 2 2 3.5 0.9 9.4
2 3 0 2.9 0.9 9.4
2 3 1 3.6 0.9 9.4
3 0 0 2.3 0.5 9.4
3 0 1 3.8 0.9 10.4
3 0 2 6.4 1.6 18.1
3 1 0 4.3 0.9 18.1
3 1 1 7.5 1.7 19.9
3 1 2 12 3 36
3 1 3 16 3 38
3 2 0 9.3 1.8 36.00
3 2 1 15 3 38
3 2 2 21 3 40
3 2 3 29 9 99
3 3 0 24 4 99
3 3 1 46 9 198
3 3 2 110 20 400
3 3 3 > 110.00
240
M01_55ME is based on ISO 7251 with deviation on confirmation step

The current method is principally based on ISO 7251:2005 which enables a detection and MPN
enumeration of presumptive E. coli after an incubation first at 37°C and then at 44°C. However
some E. coli strains including E. coli O157 do not grow at 44°C and are therefore not detectable
with this method.
In the ISO norm the confirmation is based on the examination of indole production through
transferring the culture from EC broth into peptone water followed by further incubation and
biochemical test. To obtain a more reliable and clearer identification result, in the current method,
the application of the biochemical identification kits which included the indole reaction is
recommended directly after incubation in EC broth.
Alternative E. coli method (M01_09ME) in CLF available

CLF provides another E. coli detection method M01_09ME with broader range which
combines the detection of E. coli and Enterobacteriaceae. From the pre-enrichment in
EE broth, a selective enrichment in brilliant green bile lactose broth and tryptone water
(44°C, 48 h) is performed. In parallel from the second pre-enrichment in BPW the
sample is streaked onto a chromogenic medium Tryptone Bile X-glucuronide (TBX). In
particular β-glucuronidase positive E. coli is detectable using this method. It must be
taken into consideration that some pathogenic E. coli strains which do not grow at 44°C
or which are negative for β-glucuronidase (E. coli O157) will not be detected.
M01_09ME can be provided by request.
241
Protocols for E. coli detection: ISO method and CLF method
242
243
244

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METHOD GUIDE MICROBIOLOGY

Microbiological Reference Methods Collection 2014

 CLF Reference Methods

 M01_01ME Sample preparation 3

 Annex: Comparison of CLF Reference Methods and ISO Standards 243

Microbiology and Biochemistry Team, Eurofins CLF

Sample preparation –Resuscitation and Dilution

Reasons for amendment:

Name Signature Date

1 Scope and field of application

2 Principle of the method

3 Definitions and abbreviations

4 Reagents, materials, media

Buffered peptone water2 BPW 1 month 4-10°C

Tryptone soy broth2 TSB 1 month 4-10°C

Peptone salt solution2,4 PSS 1 month 4-10°C

Ringer’s solution2 RS 1 month 4-10°C

NaOH 5% (w/v)3 NaOH 1 month 4-10°C

Amylase 1-5% (w/v) in water 1 month 4-10°C

Pectinase 1-5% (w/v) in water 1 month 4-10°C

Cellulase 1-5% (w/v) in water 1 month 4-10°C

Gelatinase 1-5% (w/v) in water 1 month 4-10°C

 Fill volume of resuscitation and dilution media

Use only media of good quality proven by supplier’s quality certificate.

Follow the manufacturer’s description on:

6.1 General resuscitation procedure

6.2 Resuscitation procedure for thickeners

Table 1: Directions for the use of enzyme solutions

Enzyme Amount Use for

amylase 1-5% w/v, 1 ml to 100 ml suspension starch (e.g. for cereals)

gelatinase 1-5% w/v, 1 ml to 100 ml suspension gelatin

6.3 Processing of butter/oil samples

6.4 Decimal dilution

7 Expression of results, calculation

6. International Organisation for Standardisation, “ISO/CD 6877-5, Microbiology of food

Attachment 1: Composition of resuscitation and dilution media

PSS Enzymatic digest casein 1.0 g

CaCl • 6H2O 0.12

BPW Enzymatic digest animal tissue 10.0 g

Na2HPO4 • 12H2O 9.0 g

Attachment 2: Resuscitation procedure for acid products and trace elements

1. Weigh an amount of sample in resuscitation broth as described in M01_01ME 6.1/1-

2. Mix the sample and measure the pH value.

5. Weigh an amount of sample in the resuscitation broth with NaOH as described in

1. Proceed as described in method M01_01ME 6.1.1 till 6.1.3.

2. Weigh an amount of sample to get a 5% suspension (w/v or v/v) or a 1% suspension

No resuscitation of liquid samples

Handling of butter/oil samples

Total viable count (30°C and 55°C)

Total viable count (30°C and 55°C)

Reasons for amendment:

V4 (07.05.12)  Type in the reference method corrected

Name Signature Date

Total viable count (30°C and 55°C)

1 Scope and field of application

2 Principle of the method

Tetrazolium: colorless, soluble 2 reduction steps Formazan: red, insoluble in water

Figure 1: Chemical structure of TTC and Formazan

3 Definitions and abbreviations

Total viable count (30°C and 55°C)

4 Reagents, materials, media

Plate count agar3 PCA 3 months 4-10°C

Milk plate count agar MPCA 3 months 4-10°C