International Standard
~~ ~
0 7937
INTERNATIONAL ORGANIZATION FOR STANDARDIZATIONWEWYHAPOIIHAR OPrAHHBAUHR no [Link] INTERNATIONALE DE NORMALISATION
Microbiology - General guidance for enumeration of
Clostridium perfringens - Colony count technique
Microbiologie - Directives générales pour le dénombrement de Clostridium perfringens - Méthode par comptage des colonies
First edition - 1985-07-01
iTeh STANDARD PREVIEW
([Link])
ISO 7937:1985
[Link]
375cfcf0dcda/iso-7937-1985
UDC [Link] : 579.852.13 Ref. No. I S 0 7 9 3 7 - 1 s (E)
Descriptors : microbiological analysis, detection, micro-organisms, food products, bacteria count methods.
Price based on 7 pages
IS0 (the International Organization for Standardization) is a worldwide federation of
national standards bodies (IS0 member bodies). The work of preparing International
Standards is normally carried out through I S 0 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 IS0 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 7937 was prepared by Technical Committee ISO/TC 34,
, Agricultural food products.
([Link])
ISO 7937:1985
[Link]
375cfcf0dcda/iso-7937-1985
INTERNATIONAL STANDARD I S 0 7937-1985 (E)
Microbiology - General guidance for enumeration of
Clostridium perfringens - Colony count technique
O Introduction 1 Scope and field of application
This International Standard gives general guidance for the
O. 1 This International Standard is intended to provide general enumeration of viable Clostridium perfringens in products in-
guidance for the examination of products not dealt with by ex- tended for human consumption or feeding of animals.
isting International Standards and for the reference of bodies
preparing microbiological methods of test for application to
food products or to animal feeding stuffs. In view of the large 2 Reference
variety of products within this field of application, these
guidelines may not be appropriate for some prod- IS0 6887, Microbiology - General guidance for the prepara-
ucts in every detail, and for some other products it may be tion of dilutions for microbiological examination.
necessary to use different methods. Nevertheless, it is hoped
that, in all cases, every attempt will be made to apply these
iTeh STANDARD 3PREVIEWDefinitions
guidelines as far as possible and that deviations from them will
only be made if absolutely necessary for technical reasons.
([Link])
For the purpose of this International Standard, the following
definitions apply.
When this International Standard is next reviewed, account will
be taken of all information then available regarding the ISOextent
7937:19853.1 Clostridium perfringens: Bacteria that form black col-
to which the guidelines have been followed and the reasons
[Link]
which necessitated deviation from them in the case of par- onies in the specified selective medium and which give positive
ticular products. confirmatory reactions when the test is carried out by the
375cfcf0dcda/iso-7937-1985
method specified in this International Standard.
The harmonization of test methods cannot be immediate, and
for certain groups of products, International Standards and/or 3.2 enumeration of C. perfringens: Determination of the
national standards that do not comply with the guidelines may number of viable and confirmed C. perfringens bacteria per
already exist. In cases where International Standards already millilitre or per gram of sample, when the test is carried out by
exist for the product to be tested, they should be followed, but the method specified in this International Standard.
it is hoped that, when they are reviewed, they will be aligned
with this International Standard so that, eventually, the only re-
maining departures from these guidelines will be those 4 Principle
necessary for well established technical reasons.
4.1 Inoculation of Petri dishes with a specified quantity of the
test sample if the initial product is liquid, or a specified quantity
0.2 For practical reasons, the definition of Clostridium per-
of the initial suspension in the case of other products.
fringens given in clause 3 and used as the basis for the pro-
cedure does not exclusively describe strains of Clostridiumper- Inoculation, under the same conditions, using decimal dilutions
fringens. In particular, the confirmatory tests are inadequate to of the test sample or of the initial suspension.
distinguish between Clostridium perfringens and other closely
related but less commonly encountered Clostridium species Mixing with a selective medium (poured plate technique) and
such as C. paraperfringens and C. absonum. adding an overlayer of the same medium.
0.3 For statistical reasons, the lowest limit for the number of 4.2 Anaerobic incubation of the plates at 35 O C or 37 OC1)
colonies counted per dish has been set at 15, but, for practical for 20 h.
purposes, a count of lower numbers of Clostridiumperfringens
is often required. The confidence limits of such determinations 4.3 Calculation, from the number of black colonies appearing
(estimated counts) are given in the annex. on the plates, of the number of characteristic colonies.
1) The temperature should be agreed between the parties concerned and recorded in the test report.
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I S 0 7937-1985 (E)
4.4 Subjection of characteristic colonies to confirmatory pro- Store in a refrigerator at 4 f 2 O C .
cedures and calculation of the number of C. perfringens per
millilitre or per gram of sample. Discard unused medium 2 weeks after preparation.
Preparation of agar plates for confirmation
5 Diluent, culture media and reagents
Transfer portions of about 15 ml of the base, melted and
cooled to approximately 45 O C , to Petri dishes and allow to
5.1 Basic materials
solidify.
In order to improve the reproducibility of the results, it is
recommended that, for the preparation of the diluent and Immediately before use, dry the plates, preferably with the
lids off and the agar surface downwards, in a drying cabinet
culture media, dehydrated basic components, or complete
(6.2) at 50 O C for 30 min.
dehydrated media, be used. Similarly, commercially prepared
reagents may also be used. The manufacturer's instructions If prepared in advance, the undried plates shall not be kept
shall be rigorously followed. longer than 4 h at room temperature or 1 day at 4 OC.
Chemical products shall be of recognized analytical quality.
5.3.2 D-Cycloserine solution
The water used shall be distilled or deionized water, free from
substances that might inhibit the growth of Clostridium per- Composition
fringens under the test conditions. 0-Cycloserine (usewhite crystalline powder only) 4,O g
Water 100 ml
Measurements of pH shall be made using a pH meter (6.51,
measurements being referred to a temperature of 25 OC. If the Preparation
diluent and culture media are not used immediately, they shall,
unless otherwise stated, be stored in the dark at approximately Dissolvethe o-cycloserine in the water and sterilize the solu-
4 OC, in conditions which do not produce any change in their tion by filtration.
composition.
in a refrigerator at 4 f 2 OC.
iTeh STANDARDStorePREVIEW
5.2 Diluent Discard unused solution 4 weeks after preparation.
([Link])
See I S 0 6887 and the specific standard dealing with the
product to be examined. 5.3.3 Complete medium
ISO 7937:1985
Before plating (see 9.2). add 1 ml of the sterilized D-cycloserine
5.3 Egg-yolk-free tryptose-sulfite-cycloserine
[Link]
solution (5.3.2) to each 100 ml of sterile melted base (5.3.1 1 at
agar (SC11) 375cfcf0dcda/iso-7937-1985
50 OC.
5.3.1 Base 5.4 Fluid thioglycollate medium
Composition Composition
Tryptose 2) 15,O g Pancreatic casein digest
Soytone2) 5,O g L-Cystine
Yeast extract 5,O g Dextrose (D-glucose)
Disodium disulfite (Na2S205),anhydrous 1,og Yeast extract 5,O
Ammonium iron(ll1)citrate3) 1.09 Sodium chloride
Agar 12,O to 18,O 94) Sodium thioglycollate (mercaptoacetate)
Water 1000ml Agar
Resazurin
Preparation Water
Dissolve the components in the water by boiling. Preparation
Adjust the pH so that it will be 7,6 after sterilization. Dissolve the components in the water by boiling.
Transfer the base to tubes or flasks or bottles of capacity Adjust the pH so that it will be 7,l after sterilization.
not more than 500 ml.
Dispense 10 ml portions into test-tubes and sterilize at
Sterilize for 10 min at 121 O C . 121 O C for 15 min.
1) This was originally designated EY-free TSC (HAUSCHILD Appl. Microbiol. 27 1974: 78-82].
and HILÇHEIMER.
2) The names tryptose and soytone are used at present only by certain producers of media. Any other pancreatic casein or soybean digest giving
comparable results may be used.
31 This reagent should contain at least 15 % ( m / mof
) iron.
4) According to the manufacturer's instructions.
2
I S 0 7937-1985 (E)
5.5 Motility-nitrate medium 5.7 Nitrite-detection reagent
Composition 5.7.1 5-amino-2-naphthalenesulfonicacid (5-2 ANSA)
solution
Peptone 5,O g
Beef extract 3,O g Dissolve 0,l g of 5-2 ANSA in 100 ml of 15 % (VI V ) acetic acid
Galactose 5,O g solution. Filter through a filter paper.
Glycerol 5,O g
Potassium nitrate (KNOJ) 1,og Store in a well-stoppered brown bottfe (preferably with a bulb-
Disodium hydrogenorthophosphate( Na2HP04) 2 3g type dropper) at 4 O C .
Agar 1,O to 5,O g l )
Water 1 000ml 5.7.2 Sulfanilic acid solution
Preparation Dissolve 0,4 g of sulfanilic acid in 100 ml of 15 % ( V / V ) acetic
acid solution. Filter through a filter paper.
Dissolve the components in the water by boiling. Store in a well-stoppered brown bottle (preferablywith a bulb-
type dropper) at 4 O C .
Adjust the pH so that it will be 7.3 after sterilization.
0 '. Transfer the medium to culture tubes in 10 ml quantities and
sterilize at 121 O C for 15 min.
5.7.3 Preparation of complete reagent
Mix equal volumes of the two solutions (5.7.1 and 5.7.2) just
before use.
If not used the same day, store in a refrigerator at 4 f 2OC;
just prior to use, heat in boiling water or flowing steam for Discard unused reagent immediately.
15 min and then cool rapidly to the incubation temperature.
Zinc dust
iTeh STANDARD 5.8 PREVIEW
Discard unused medium 4 weeks after preparation.
5.6 Lactose-gelatine medium ([Link])
6 Apparatus and glassware
NOTE - Disposable apparatus is an acceptable alternative to
Composition ISO 7937:1985glassware if it has suitable specifications.
[Link]
Tryptose 2) 15,O g Usual microbiological laboratory equipment and in particular
375cfcf0dcda/iso-7937-1985
Yeast extract 10,o 9
Lactose 10,o 9
Gelatine 120,o g
6.1 Apparatus for dry sterilization (oven) or wet
sterilization (autoclave) (autoclave operating either separ-
Phenol red 0,05 9
ately or as part of an apparatus for preparing and distributing
Water 1000ml
media).
0 Preparation Apparatus that will come into contact with the diluent, culture
media, or the sample, except for apparatus that is supplied
Dissolve the components, except the lactose and phenol sterile (in particular plastics apparatus), shall be sterilized by
red, in the water. one of the following methods :
a) by being kept at 170 to 175 O C for not less than 1 h in an
Adjust the pH so that it will be 7,5 after sterilization.
oven;
Add the lactose and phenol red, dispense 10 ml portions b) by being kept at 121 f 1 OC for not less than 20 min in
into test-tubes and sterilize at 121 O C for 15 min. an autoclave.
If not used the same day, store in a refrigerator at 4 rf- 2 OC. 6.2 Drying cabinet or oven, ventilated by convection, (for
drying the surface of agar plates), capable of being maintained
Just prior to use, heat in boiling water or flowing steam for at 50 f 1 OC.
15 min, then cool rapidly to the incubation temperature.
6.3 Incubator, capable of being maintained at 35 f 1 OC or
Discard unused medium 3 weeks after preparation. 37 f 1 OC, depending on the temperature adopted 3) (for main-
1) According to be manufacturer's instructions.
2) The name tryptose is used at present only by certain producers of media. Any other pancreatic casein digest giving comparable results may be
used.
3) The temperature should be agreed between the parties concerned and recorded in the test report.
3
IS0 7937-1985
taining the inoculated media, plates and tubes within one of 9.2 Inoculation and incubation (poured plate
these temperature ranges). technique)
6.4 Anaerobic jars or any other apparatus appropriate for Transfer, by means of a sterile pipette, 1 ml of each dilution of
anaerobic culture. the initial suspension, or of the test sample if the initial product
is liquid, in duplicate, to the centres of empty Petri dishes, pour
15 to 20 ml of the SC agar (5.3.3) into the dish and mix well
6.5 pH-meter, accurate to i 0,l pH unit at 25 O C . with the inoculum by gently rotating each dish. When the
medium has solidified, add an overlayer of 10 ml of the same
6.6 Loops, of platinum-iridium or nickel-chromium, of SC agar. Allow to solidify and place the plates with the lid up-
diameter approximately 3 mm, and stab-inoculation needle of permost in anaerobic jars or other suitable containers (6.4) and
the same material. incubate at 35 O C or 37 OC2) for 20 h. Longer incubation may
result in excess blackening along the bottom rim of the plates.
6.7 Filtration apparatus, for sterilization of solutions.
9.3 Counting of colonies
6.8 Test-tubes, of diameter 18 mm and length 180 mm and
culture bottles or flasks,l) for sterilization and storage of
culture media and incubation of liquid media. 9.3.1 After the specified period of incubation (see 9.21,count
and record the number of characteristic colonies on the plates
6.9 Measuring cylinders, for preparation of complete in accordance with 9.3.2,9.3.3,9.3.4and [Link] of
media. C. perfringens are black.
6.10 Graduated pipettes, calibrated for bacteriological use 9.3.2 If both plates corresponding to a particular dilution con-
only, of nominal capacities 1 ml and 10 ml, graduated in 0,l ml tain between 15 and 150 characteristic colonies, count the
and 0,5ml divisions, respectively, and with an outflow opening characteristic colonies on each plate and record the arithmetic
of diameter 2 to 3 mm. iTeh STANDARD PREVIEW mean of the counts from the two plates; otherwise record the
count on one plate.
6.11 ([Link])
Rubber bulbs, for use with the graduated pipettes for
distributing the components of the nitrite-detection reagent.
9.3.3 If there are plates containing between 15 and
ISO 7937:1985
6.12 Petri dishes, made of glass or plastics material, of
150 characteristic colonies at two consecutive dilutions, count
[Link]
the characteristic colonies on each plate from each respective
diameter 90 to 100 mm. 375cfcf0dcda/iso-7937-1985
dilution and determine the arithmetic mean of the counts from
the two plates from each of the two dilutions as specified in
7 Sampling [Link] the arithmetic mean of the two values obtained
except when the ratio of the higher value to the lower value is
Carry out sampling in accordance with the specific standard ap- greater than 2;in this case, record the lower value of the two.
propriate to the product concerned. If no such specific stan-
dard exists, it is recommended that agreement be reached on
this subject by the parties concerned. 9.3.4 If parts of the plates (see 9.3.2)are completely black-
ened, or if it is difficult to count well isolated characteristic
colonies, count colonies on plates at the next higher dilution,
8 Preparation of the test sample even though their number may be less than 15,and proceed as
in 9.3.5.
See the specific standard appropriate to the product
concerned. If no such specific standard exists, it is recom-
mended that agreement be reached on this subject by the par- 9.3.5 If there are fewer than 15 characteristic colonies on
ties concerned. plates from the initial suspension or the test sample (if the initial
product is liquid), count the actual number of characteristic col-
onies on each plate and record the arithmetic mean of the
9 Procedure counts from the two plates.
9.1 Test portion, initial suspension and dilutions
9.3.6 If there are no characteristic colonies on plates from the
See IS0 6887 and the specific standard appropriate to the initial suspension or the test sample (if the initial product is
product concerned. liquid), record the statement "No colonies observed".
1) Bottles or flasks with metal screw-caps may be used.
2) The temperature should be agreed between the parties concerned and recorded in the test report.
4
I S 0 7937-1985 (E)
9.4 Confirmation Examine the tubes of lactose-gelatine medium for the presence
of gas and of a yellow colour (due to acid) indicating fermenta-
9.4.1 Selection and purification of colonies for tion of lactose. Chill the tubes for 1 h at 5 O C and check for
confirmation gelatine liquefaction. If the medium has solidified, reincubate
for an additional 24 h and again check for gelatine liquefaction.
Select a total of 10 characteristic colonies from the plates
counted in accordance with either 9.3.2, 9.3.3 or 9.3.4. If less 9.4.3 Interpretation
than 10 colonies are available on the plates counted, select all
the characteristic colonies present. Confirm these colonies as Bacteria which produce black colonies in SC medium, are non-
described in 9.4.2. motile, reduce nitrate to nitrite, produce acid and gas from
lactose, and liquefy gelatine in 48 h are considered to be C.
If the surface area of the plates is overgrown and it is not perfringens.
possible to select well-isolated characteristic colonies, in-
oculate 10 characteristic colonies into fluid thioglycollate Cultures that show a faint reaction for nitrite (i.e. a pink colour)
medium (5.4). Incubate under anaerobic conditions at 35 O C or should be eliminated, since [Link] consistently gives an
37 O C 1 ) for 18 to 24 h. Streak the colonies on SC base agar intense and immediate reaction.
plates (see 5.3.1 1, and add an overlayer of 10 ml of the SC base
agar.
10 Expression of results
0 Allow to solidify and incubate anaerobically at 35 O C or 37
for 18 to 24 h.
OC')
10.1 Calculation of colony count
Select from each plate at least one characteristic and well
separated colony. 10.1.1 If 80 % or more of the selected characteristic colonies
(see 9.4.1) are confirmed as C. perfringens, the number of
Confirm this colony as described in 9.4.2. these micro-organismspresent shall be taken to be the same as
the number of C. perfringens given by the count made in 9.3.2,
iTeh STANDARD PREVIEW
If necessary, repeat the streaking and inoculation on SC base 9.3.3 and 9.3.5.
agar plates until well isolated, characteristic black colonies are
obtained.
([Link])
10.1.2 In all other cases, the number shall be calculated on
the basis of the percentage of the count of characteristic col-
9.4.2 Biochemical confirmation
ISO 7937:1985onies (9.3.2, 9.3.3 or 9.3.5) that were confirmed (in 9.4.1 and
9.4.2) as C. perfringens.
[Link]
375cfcf0dcda/iso-7937-1985
[Link] Confirmation using motility-nitrate medium Example :
Stab-inoculate the selected colonies (see 9.4.1 1 into motility- If the mean colony count was recorded as 75 in accordance
nitrate medium (5.5). Incubate under anaerobic conditions at with 9.3 and six of the 10 colonies (60%) tested were confirm-
35 O C or 37 O C 1 ) for 24 h. ed as C. perfringens, the number of C. perfringens colonies
should be reported as 75 x 0,60 = 45.
0 Examine the tubes of motility-nitrate medium for the type of
growth along the stab line. Motility is evident from diffuse 10.1.3 Retain only two significant figures for the expression
growth out into the medium away from the stab line. of results, proceeding as follows :
Test for the presence of nitrite by adding 0,2 to 0,5 ml of the a) if the number is less than 100, round it to the nearest
nitrite-detection reagent (5.7) to each tube of motility-nitrate multiple of 5;
medium.2)The formation of a red colour confirms the reduction
of nitrate to nitrite. b) if the number is greater than 100 and ends in 5, round it
to the nearest multiple of 20;
If no red colour is formed within 15 min, add a small amount of c) if the number is greater than 100 and does not end in
zinc dust (5.8) and allow to stand for 10 min. If a red colour is 5, round it to the nearest multiple of 10.
formed, after the addition of zinc dust, no reduction of nitrate
has taken place. For estimating low numbers (see 9.3.4 and 9.3.51, take the
average of the counts of confirmed characteristic colonies and
[Link] Confirmation using lactose-gelatine medium round to the next highest whole number.
Inoculate the selected colonies (see 9.4.1 1 into lactose-gelatine Multiply this value by the reciprocal of the corresponding dilu-
medium (5.6). Incubate under anaerobic conditions at 35 O C or tion of the test sample to obtain the number of Clostridiumper-
37 O C 1 1 for 24 h. frhgens per millilitre or per gram of product, as appropriate.
1) The temperature should be agreed between the parties concerned and recorded in the test report.
2) For health reasons, it may be desirable to carry out this test under a fume hood.
5
I S 0 7937-1985 (E)
10.2 Reporting of results even greater variation may be found, especially between results
obtained by different workers.
Report the result as the number of C. perfringens per millilitre
or per gram of product, expressed as a number between 1,0
and 9,9 multiplied by the appropriate power of 10. 10.3.2 Precision of the count of low numbers
(less than 15)
If there were no characteristic colonies on plates from the initial
suspension (if the initial product is solid), the number of
Clostridiumpetfringensper gram of product should be reported Confidence intervals for estimated counts of low numbers
as fewer than IO. (10.3) are given in the annex.
If there were no characteristic colonies on plates from the
test sample (if the initial product is liquid), the number of C.
perfringens per millilitre of product should be reported as fewer 11 Test report
than 1.
The test report shall show the method used, the temperature of
incubation, and the results obtained. It shall also mention any
10.3 Precision of the count operating details not specified in this International Standard, or
regarded as optional, together with details of any incidents
10.3.1 Precision of the count of high numbers (between
15 and 150) likely to have influenced the results.
For statistical reasons, the confidence intervals of this method
vary, in 95 % of cases, from f 16 % to i 52 % . I ) In practice,
The test report shall include all the information necessary for
the complete identification of the sample.
O
iTeh STANDARD PREVIEW
([Link])
ISO 7937:1985
[Link]
375cfcf0dcda/iso-7937-1985
and MORISETTI.
1) See COWELL J. Sci. i d . Agric. 20, 1969 : 573.
6
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