International Standard @ 7402

INTERNATIONAL ORGANIZATION FOR STANDARDIZATION*ME)KAYHAPOAHAR OPrAHM3AUMR no CTAHAAPTM3AUMM*ORGANISATlON INTERNATIONALE DE NORMALISATION

a Microbiology - General guidance for the enumeration

of Enterobacteriaceae without resuscitation -
M P N technique and colony count technique
Microbiologie - Directives générales pour le dénombrement sans revivification des Enterobacteriaceae - Technique NPP et
méthode par comptage des colonies
iTeh STANDARD PREVIEW
First edition - 1985-10-15
(standards.iteh.ai)
ISO 7402:1985
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bab7cfcd329d/iso-7402-1985

-w UDC 579.67.087.23: 579.842.1/.2 Ref. No. I S 0 7402-1985 (E)

%
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Descriptors : microbiological analysis, detection, micro-organisms, enterobacteriaceae, test results, test equipment, bacteria count methods.

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Foreword
IS0 (the International Organization for Standardization) is a worldwide federation of
national standards bodies ( I S 0 member bodies). The work of preparing International
Standards is normally carried out through IS0 technical committees. Each member
body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, govern-
mental and non-governmental, in liaison with ISO, also take part in the work.

Draft International Standards adopted by the technical committees are circulated to

the member bodies for approval before their acceptance as International Standards by
the I S 0 Council. They are approved in accordance with I S 0 procedures requiring at
least 75 % approval by the member bodies voting.
iTeh STANDARD PREVIEW
International Standard I S 0 7402 was prepared by Technical Committee ISO/TC 34,
Agricultural food products. (standards.iteh.ai)
Users should note that all International Standards undergo revision from time to time
ISO 7402:1985
and that any reference made herein to any other International Standard implies its
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latest edition, unless otherwise stated,
bab7cfcd329d/iso-7402-1985

O International Organization for Standardization, 1985 0

Printed in Switzerland
 INTERNATIONAL STANDARD I S 0 7402-1985 (E)

Microbiology - General guidance for the enumeration

of Enterobacteriaceae without resuscitation -

e
M P N technique and colony count technique

iTeh STANDARD PREVIEW

O Introduction
(standards.iteh.ai)
1 Scope and field of application
This International Standard is intended to provide general
ISO 7402:1985 This International Standard gives general guidance for the enu-
meration of Enterobacteriaceae present in products intended
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guidance for the examination of products not dealt with by
bab7cfcd329d/iso-7402-1985
existing International Standards and for the reference of bodies for human consumption or feeding of animals
preparing microbiological methods of test for application to - by calculation of the most probable number (MPN)
food products or to animal feeding stuffs. In view of the large
after incubation a t 35 OC or 37 O C in liquid medium
variety of products within this field of application, these
guidelines may not be appropriate for some products in every - by counting colonies in a solid medium after incubation
detail, and for some other products it may be necessary to use at 35 OC or 37 O C .

0
different methods. Nevertheless, it is hoped that, in all cases,
every attempt will be made to apply these guidelines as far as For low numbers, the MPN method is preferable, otherwise the
possible and that deviations from them will only be made if colony count method is preferred.
absolutely necessary for technical reasons.
This International Standard does not include resuscitation pro-
When this International Standard is next reviewed, account will cedures and the results should not, therefore, be related to
be taken of all information then available regarding the extent criteria or specifications based on the assumption that resusci-
to which the guidelines have been followed and the reasons tation has been carried out.
which necessitated deviation from them in the case of par-
A limitation on the applicability of this International Standard is
ticular products.
imposed by the susceptibility of the methods to a large degree
of variability. The methods should be used and the results inter-
The harmonization of test methods cannot be immediate, and preted in the light of the information given in 10.3.
for certain groups of products, International Standards and/or
national standards that do not comply with the guidelines may
already exist. In cases where International Standards already 2 Reference
exist for the product to be tested, they should be followed, but
it is hoped that, when they are reviewed, they will be aligned IS0 6887, Microbiology - General guidance for the prep-
with this International Standard so that, eventually, the only aration of dilutions for microbiological examination.
remaining departures from these guidelines will be those
necessary for well established technical reasons.
3 Definitions
For any particular product, the method to be used will be
specified in the International Standard dealing with that For the purpose of this International Standard, the following
product. definitions apply.

1
I S 0 7402-1985 (E)

3.1 Enterobacteriaceae : Micro-organisms which ferment From the number of confirmed typical colonies per plate, calcu-
glucose and show a negative oxidase reaction when the test is lation of the number of Enterobacteriaceaeper millilitre or per
carried out according to the method specified. gram of the test sample.

3.2 count o f Enterobacteriaceae : The number of Entero-

bacteriaceaefound per millilitre or per gram of the test sample 5 Diluent, culture media and reagent
when the test is carried out according to the method specified.
5.1 Basic materials

In order to improve the reproducibility of the results, it is

4 Principle recommended that, for the preparation of the diluent and
culture media, dehydrated basic components or complete
4.1 Preparation of dilutions dehydrated media be used. Similarly, commercially prepared
reagents may be used. The manufacturer's instructions shall be
Preparation of decimal dilutions from the test sample. rigorously followed.

Chemical products shall be of recognized analytical quality.

4.2 Enumeration of Enterobacteriaceae
The water used shall be distilled or deionized water, and shall
be free from substances that might inhibit the growth of
4.2.1 M o s t probable number (MPN) technique
Enterobacteriaceaeunder the test conditions.
NOTE - This technique is recommended when the number sought is
expected to be in the range 1 to 100 per millilitre or per gram of the test If the diluent and media are not used immediately, they shall,
sample. unless otherwise stated, be kept in the dark at a temperature
between O and 5 OC, for no longer than 1 month, in conditions
Inoculation of three tubes of double-strength medium with a that prevent any change in their composition.
specified quantity of test sample if the product is liquid, or with
iTeh STANDARD PREVIEW
a specified quantity of the initial suspension in the case of other
5.2 Diluent
products.
(standards.iteh.ai)
See IS0 6887 and any specific standard dealing with the
Inoculation of three tubes of single-strength medium with a product to be examined.
specified quantity of the test sample if the product is liquid, ISO or 7402:1985
with a specified quantity of the initial suspension in the case of
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other products. Then, under the same conditions, inoculation 5.3 Culture media
bab7cfcd329d/iso-7402-1985
of three tubes of single-strength medium with the first decimal
dilution of the test sample or of the initial suspension. 5.3.1 Buffered brilliant green bile glucose broth (E.E.
broth)
Incubation of the tubes at 35 OC or 37 O C ' ) for 24 h.
Composition a) b)
From the number of confirmed positive tubes, calculation of Double-strength Single-strength
medium medium
the most probable number of Enterobacteriaceaeper millilitre
or per gram of the test sample using the MPN table (see an- Peptone 20,o 9 10,o 9
nex A). Glucose 10,o g 50 g
Disodium hydrogenortho-
phosphate (Na2HPOS 129 g 6,45 g
4.2.2 Colony count technique Potassium dihydrogenortho-
phosphate (K H2P04) 4,O g 2,o 9
NOTE - This technique is recommended when the number sought is
expected to be greater than 100 per millilitre or per gram of the test
Ox bile, dehydrated 0 0 9 20,o 9
sample.
Brilliant green 0,030 g 0,015 g
Water 1 O00 ml 1000 ml
Inoculation of two plates of violet red bile glucose agar (poured
plate technique) with a specified quantity of the test sample if Preparation
the initial product is liquid, or with a specified quantity of the
initial suspension in the case of other products. Covering with Dissolve the components or the complete dehydrated
an overlayer of the same medium. medium in the water by boiling. Do not heat the medium for
longer than 30 min. Cool the medium rapidly.
Inoculation of other pairs of plates under the same conditions,
using decimal dilutions of the test sample or of the initial Adjust the pH so that after boiling it is 7,2 at 25 OC.

suspension.
Transfer 10 ml portions to sterile tubes or bottles (6.5).
Incubation of the plates at 35 OC or 37 OC for 24 h.

1) The temperature should be agreed between the parties concerned and recorded in the test report.

2
 I S 0 7402-1985 (E)

Do not sterilize the medium. Preparation

The medium may be stored for up to 1 week at O to 5 O C . Dissolve the components or the complete dehydrated
medium in the water by boiling.

5.3.2 Violet red bile glucose agar Adjust the pH so that after sterilization it is 7,O at 25 OC.

Transfer the culture medium in quantities of 15 ml to tubes

Composition
or bottles (6.5).
Peptone Sterilize the medium for 15 min at 121 f 1 O C .
Yeast extract
Bile salts Leave the tubes or bottles in a vertical position.
Glucose
Sodium chloride The medium may be stored for up to 1 week at O to 5 OC.
Neutral red
Crystal violet Just before use, heat in boiling water or flowing steam for
Agar 15 min, then cool rapidly to the incubation temperature.
Water
5.3.4 Nutrient agar
Preparation
Cornposition
Dissolve the components or the complete dehydrated
medium in the water by boiling. Beef extract 3,O g
Peptone 5,O g
Adjust the pH so that after boiling it is 7,4 at 25 OC.
Agar 8 t o 18 gl)
Water 1 O00 ml
Transfer the culture medium to sterile tubes, flasks or
iTeh STANDARD PREVIEW
bottles (6.5) of capacity not more than 500 ml.
Preparation
Do not sterilize the medium. (standards.iteh.ai)
Dissolve the components or the complete dehydrated
medium in the water by boiling.
Prepare this medium just before use (see 9.2.2 andISO
9.3.1).
7402:1985
Adjust the pH so that after sterilization it is 7,O at 25 O C .
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Preparation of agar plates (required only for MPN technique,
bab7cfcd329d/iso-7402-1985
see 9.2.2) Transfer the culture medium to tubes, bottles or flasks (6.5)
of capacity not more than 500 ml.
Transfer immediately approximately 15 ml of the culture
medium, cooled to approximately 45 O C , to Petri dishes Sterilize the medium for 15 min at 121 + 1 OC.
(6.71, and allow to solidify.
Preparation of agar plates (see 9.4.1)
Just before use, dry the plates, preferably with the lids off
and the agar surface downwards, in the oven (6.31, main- Transfer portions of about 15 rnl of the culture medium,
tained at 50 O C , for 30 min. melted and cooled to approximately 45 O C , to Petri dishes
(6.7) and allow to solidify.
If prepared in advance, the undried plates shall not be kept
for longer than 4 h at room temperature or 1 day a t O to Just before use, dry the plates, preferably with the lids off
5 oc. and the agar surface downwards, in the oven (6.31, main-
tained at 50 O C , for 30 min.

5.3.3 Glucose agar If prepared in advance, the undried plates shall not be kept
for longer than 4 h at room temperature or 1 day at O t o
5 OC.
Composition

Tryptone 10,o g 5.4 Oxidase reagent

Yeast extract 1,5 g
Glucose 10,o 9 Composition
Sodium chloride 5,O g
Bromocresol purple 0,015 g N, N, N’, N-tetrarnethyl-p-phenylene-
Agar 8 to 18 g’) diamine dihydrochloride 1,o g
Water 1 O00 ml Water 100 ml

1 i According to the manufacturer’s instructions.

3
I S 0 7402-1985 (E)

Preparation 7 Sampling

Dissolve the reagent in the cold water. Carry out sampling in accordance with the specific standard
appropriate to the product concerned. If no such specific stan-
Prepare the reagent just before use. dard exists, it is recommended that agreement be reached on
this subject by the parties concerned.

6 Apparatus and glassware

NOTE - Disposable apparatus is an acceptable alternative to reusable 8 Preparation of the test sample
glassware if it has suitable specifications.
See the specific standard appropriate to the product con-
Usual microbiological laboratory equipment and in particular : cerned. If no such specific standard exists, it is recommended
that agreement be reached on this subject by the parties
6.1 Apparatus for dry sterilization (oven) or wet ‘ concerned.
sterilization (autoclave)

Apparatus that will come into contact with the diluent, culture
media, or the sample, except for apparatus that is supplied 9 Procedure
sterile (in particular plastics apparatus), shall be sterilized by
one of the following methods :
9.1 Test portion, initial suspension and dilutions
a) by being kept at 170 to 175 OC for not less than 1 h in an
oven ; See IS0 6887 and any specific standard appropriate to the
product concerned.
b) by being kept at 121 f 1 O C for not less than 20 min in
an autoclave. Prepare a single decimal dilution series from the test sample if
iTeh STANDARD PREVIEW
the product is liquid, or from the initial suspension in the case
6.2 Incubator, capable of being maintained at 35 f 1 or of other products.
OC

at 37 f 1 OC. (standards.iteh.ai)
6.3 Drying cabinet, oven or incubator, capable of being ISO 7402:1985
9.2 MPN technique
maintained at 50 f 5OC, for drying the surfaces of agar
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plates. bab7cfcd329d/iso-7402-1985
9.2.1 Inoculation and incubation
6.4 Water-bath, capable of being maintained at 45 OC, for
Take three tubes of double-strength medium L5.3.1 ail.
cooling the melted culture media.
Transfer to each of these tubes, using a pipette (6.81, 10 ml of
the test sample if the product is liquid, or 10 ml of the initial
6.5 Test-tubes, of dimensions approximately suspension in the case of other products.
16mm x 160mm and 20mm x 200mm, and flasks or
bottles, for the sterilization and storage of the diluent and Take three tubes of single-strength medium 15.3.1 bil. Transfer
culture media. to each of these tubes, using another pipette (6.81, 1 ml of the
test sample if the product is liquid, or 1 ml of the initial suspen-
6.6 Petri dishes, made of glass or plastics, of diameter 90 to sion in the case of other products.
100 mm.
Take three more tubes of single-strength medium L5.3.1 b)l.
Transfer to each of these tubes, using another pipette (6.81,
6.7 Loop, of diameter approximately 3 mm, and wire, of
platinum-iridium or nickel-chromium, and/or a glass rod. 1 ml of the first decimal dilution of the test sample if the
product is liquid, or 1 ml of the first decimal dilution of the
NOTE - A nickel-chromium loop is not suitable for use in the oxidase
initial suspension in the case of other products.
test isee 9.4.2.1 i.
Incubate these nine tubes at 35 O C or 37 OC1) for 24 h.
6.8 Total delivery graduated pipettes, of nominal
capacities 1 and 10 ml, graduated respectively in 0.1 ml and 9.2.2 Isolation
0,5 ml divisions, and with an outflow opening of diameter 2 to
3 mm. Streak a ioopfui (6.7) from each of the nine incubated cultures
(see 9.2.1) on the violet red bile glucose agar plates isee 5.3.2)
6.9 pH-meter, accurate to 0,l pH unit at 25 O C . and incubate the plates at 35 OC or 37 O C 1 ’ for 24 h.

1) The temperature should be agreed between the parties concerned and recorded in the test report.

4
 9.2.3 Selection o f colonies for confirmation Consider the determination to be void if half or more than half
the surface area of a plate is overgrown. If less than half of the
From each of the plates incubated in 9.2.2on which typical surface area of a plate is overgrown, count the colonies on the
pink to red colonies (with or without precipitation haloes) or clear part and extrapolate so that the number corresponds to
colourless, mucoid colonies have developed, select at random the total surface area of the plate.
five such colonies for biochemical confirmation (see 9.4.2)
after
subculturing (see 9.4.1).
9.4 Confirmation

9.3 Colony count technique 9.4.1 Subculturing

Streak on nutrient agar plates (5.3.4)each of the colonies

9.3.1 Inoculation and incubation selected for confirmation (see 9.2.3and 9.3.2).

9.3.1.1 Take two sterile Petri dishes (6.6). Using a sterile Incubate these plates at 35 O C or 37 O C ') for 24 h. Select a well
pipette (6.81,transfer to each dish 1 ml of the test sample if the isolated colony from each of the incubated plates for bio-
product is liquid or 1 ml of the initial suspension in the case of chemical confirmation (see 9.4.2).
other products.
9.4.2 Biochemical confirmation
0 Take a further two sterile Petri dishes. Using a fresh sterile
pipette, transfer to each dish 1 ml of the first decimal dilution
(10-I)of the test sample if the product is liquid, or 1 ml of the 9.4.2.1 Oxidase reaction
first decimal dilution of the initial suspension (lop2)in the case
of other products. Using the platinum-iridium loop or wire or glass rod (6.71,
take a
portion of each well isolated colony (9.4.1)and streak on a filter
Repeat this procedure with successive decimal dilutions, using paper moistened with the oxidase reagent (5.4) or on a com-
a fresh sterile pipette for each decimal dilution. mercially available disc. A nickel-chromium loop or wire shall
iTeh STANDARD PREVIEW not be used.

9.3.1.2 Pour into each of the dishes approximately 15 ml of

(standards.iteh.ai)Consider the test to be negative if the colour has not turned
the violet red bile glucose agar medium (5.3.2)
which has been dark purple in 10 s.
melted and cooled to approximately 45 O C in the water-bath
(6.4).The time elapsing between completion of the preparationISO 7402:1985
of the initial suspension (or of the 10-1dilution if the product is 9.4.2.2 Fermentation test
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liquid) and the pouring of the medium (5.3.2) into the dishes
bab7cfcd329d/iso-7402-1985
shall not exceed 15 min. Stab, using a wire (6.71, the same colonies selected in 9.4.2.1
into tubes containing glucose agar (5.3.3).
Incubate at 35 O C or
Carefully mix the inoculum with the medium by horizontal 37 OC for 24 h.
movements of the plates and allow the mixture to solidify by
If a yeltow colour develops throughout the contents of the

a
placing the plates on a cool horizontal surface.
tube, the reaction is regarded as positive. Most strains produce
gas.
9.3.1.3 After solidification of the mixture, add a covering layer
of 10 to 15 ml of the violet red bile glucose agar medium (5.3.21,
melted and cooled as described in 9.3.1.2, to prevent spreading
growth and to obtain semi-anaerobic conditions. 10 Expression of results

9.3.1.4 Allow the second layer to solidify. Incubatethe plates, 10.1 Calculation of the most probable number
bottom uppermost, at 35 O C or 37 O C for 24 h. IMPNI

9.3.2 Counting and selection of colonies 10.1.1 Count the number of positive tubes for each dilution.

Select, if possible, two plates (9.3.1.4)containing 15 to 150

10.1.2 If one of the selected typical colonies (9.2.3)of a sub-
typical colonies (see 9.2.3)
of diameter 0,5 rnm or more; count
culture (see 9.4.1) is oxidase-negativeand glucose-positive, the
these suspect colonies. Select at random five such colonies
tube from which the subculture is derived shall be regarded as
from each plate for biochemical confirmation (see 9.4.2)after
being positive.
subculturing (see 9.4.1).

If two successive dilution levels gave plates containing 15 to 10.1.3 Using the MPN table (see annex A), determine from
150 typical colonies, select the two plates inoculated with the the number of positive tubes in the different dilutions, the most
greater amount of test sample. probable number (MPN) index.

1) The temperature should be agreed between the parties concerned and recorded in the test report.

5
I S 0 7402-1985 (E)

10.1.4 In the case of liquid products, the number of Entero- 10.2.5 Calculate the number of Enterobacteriaceaeper milli-
bacteriaceae per millilitre is calculated by dividing the MPN litre (in the case of liquid products) or per gram (in the case of
index by 10. In the case of other products for which initial other products) by multiplying the number obtained according
suspensions are prepared, the number per gram is equal to the to 10.2.4by the dilution factor.
MPN index.
10.2.6 Express the result as a number between 1,0 and 9,9
multiplied by the appropriate power of IO.
10.2 Calculation of colony count
In the event of finding less than 15 typical Enterobacteriaceae
10.2.1 If at least 80 % of the selected typical colonies (see colonies per plate, express the result as calculated (in 10.2.3
9.3.2)are oxidase-negative and glucose-positiveand thus con- and 10.2.41,noting that the 95 % confidence limits (annex B)
firmed as Enterobacteriaceae, the number of micro-organisms widen for smaller numbers of colonies, but that they are still
present will be the same as that given by the count made in better than those for corresponding MPN counts (see annex A).
9.3.2.
10.3 Precision of the method

10.2.2 In all other cases, the number shall be calculated from It is known that wide variations in results may occur with the
the percentage of oxidase-negative and glucose-positive col- MPN technique. Results obtained from this method should

e
onies in relation to the total number of selected colonies therefore be used with caution. Confidence limits are given in
(see 9.3.2). annex A.

For statistical reasons alone, in 95 % of cases the confidence

limits of the colony count technique vary from i l 6 % to
10.2.3 Calculate the arithmetic mean of the number of f52 % I ) ; for colony counts of less than 15 per plate, the con-
Enterobacteriaceae colonies counted on the duplicate plates in
fidence limits are given in annex E. In practice, even greater
9.3.2. variation may be found, especially among results obtained by
different workers.
iTeh STANDARD PREVIEW
10.2.4 Retain only two significant figures, rounding the result
as follows :
11 Test report
a)
(standards.iteh.ai)
if the number is less than 100, round to the nearest
it The test report shall show the method used, the temperature of
multiple of 5; incubation and the results obtained. It shall also mention any
ISO 7402:1985
operating details not specified in this International Standard, or
b) if the number is more thanhttps://standards.iteh.ai/catalog/standards/sist/37664ff0-5606-44e1-a8af-
100,and does not end with a regarded as optional, together with details of any incidents
5, round it to the nearest multiple of IO; bab7cfcd329d/iso-7402-1985
likely to have influenced the results.

c) if the number is more than 100 and ends with a 5,round The test report shall include all the information necessary for
it to the nearest multiple of 20. the complete identification of the sample.

1) J. Sci. i d . Agric. 20 : 573.

See COW ELL^^^ MORISETTI,

6
 I S 0 7402-1985 (E)

Annex A

Determination of most probable number

Table 1 - MPN indexes and confidence limits’)

Category2) when Confidence limits3)

Number of MPN number of tests is
positive results index I 2 3 5 1 0 >95 % >99 %
O 0 0 < 0.30 0,oo 0,94 1,40
O 0 1 0,30 3 2 2 2 1 0.01 0,95 1.40
O 1 0 0,30 2 1 1 1 1 0.01 1,O0 1.60
O 1 1 0.61 O 3 3 3 3 0,12 1,70 2.50
O 2 0 0,62 3 2 2 2 1 0,12 1,70 2,50
O 3 0 0,94 O 0 0 0 3 0,35 3.50 4,60
1 0 0 0,36 1 1 1 1 1 0,02 1,70 2,50
1 0 1 0.72 2 2 1 1 1 0.12 1,70 2,50
1 0 2 1.1 O 0 0 3 3 0,4 385 46
1 1 0 0,74 1 1 1 1 1 0.13 2,oo 2,70
1 1 1 1,l 3 3 2 2 2 0.4 3,5 4,6
1 2 0 1,l 2 2 1 1 1 0.4 3,5 4.6
1 2 1 1 3 3 3 3 3 2 0,5 3.8 52
1 3 0 1,6 3 3 3 3 2 05 3,s 52
2
2
0
0
0
1
iTeh STANDARD PREVIEW
0,92
1,4
1
2 1
1
1
1
1
1
1
1 0,15
0,4
3.50
385
4,60
4.6
2
2
0
1
2
0
(standards.iteh.ai)
2.0
1.5
O
1
3
1
3
1
3
1
3
1
03
0.4
3.8
3,8
5.2
52
2 1 1 2,o 2 2 1 1 1 03 3.8 52
2 1 2 2.7 O ISO 3 7402:1985
3 3 3 or9 9,4 14,2
2 2 0 2,l 1 1 1 1 1 0.5 4,o
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2 2 1 2.8 3 2 2 2
bab7cfcd329d/iso-7402-1985 1 0.9 9.4 14.2
2 2 2 3.5 O 0 0 0 3 0.9 9.4 14,2
2 3 0 23 3 2 2 2 1 03 9,4 14,2
2 3 1 3.6 O 3 3 3 3 089 9,4 14,2
3 0 0 2,3 1 1 1 1 1 0.5 9,4 14,2
3 0 1 33 1 1 1 1 1 0,g 10,4 15,7
3 0 2 64 3 3 2 2 2 1.6 18,l 25,0
3 1 0 4.3 1 1 1 1 1 03 18,l 25.0
3 1 1 7.5 1 1 1 1 1 1.7 19,9 27,0
3 1 2 12 3 2 2 2 1 3 36 44
3 1 3 16 O 0 0 3 3 3 38 52
3 2 0 9.3 1 1 1 1 1 1,s 36.0 43,0
3 2 1 15 1 1 1 1 1 3 38 52
3 2 2 21 2 1 1 1 1 3 40 56
3 2 3 29 3 3 3 2 2 9 99 152
3 3 0 24 1 1 1 1 1 4 99 152
3 3 1 46 1 1 1 1 1 9 198 283
3 3 2 110 1 1 1 1 1 20 400 570
3 3 3 > 110
I From DEMAN,J.C. Eur. J. Appt. Microbiol. Biotechnol. 17 1983 : 301-305.
2) See table 2.
3) The confidence limits given in table 1 are meant only to provide some idea about the influence of statistical variations on results. There wilt always
also be other sources of variation, which may sometimes be even more important.