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है”ह”ह

IS 5887-4 (1999): Methods for Detection of BacteriaResponsible for Food Poisoning, Part 4: Isolation andIdentification of Clostridium perfringens (clostridiumWelchii) and clostrid/um botul inum and enumeration ofclostridium perfringens [FAD 15: Food Hygiene, SafetyManagement and Other Systems]

Indian Standard

METHODS FOR DETECTION OF BACTERIARESPONSIBLE FOR FOOD POISONING

PART 4 ISOLATION AND IDENTIFICATION OF CLOSTR/D/UMPERFR/NGENS(CLOSTR/D/UM WELCH//) AND CLOSTR/D/UMBOTUL/NUMAND ENUMERATION

OF CLOSTRIDIUM PERFRINGENS

(Second Revision )

ICS 07.100.30

0 BIS 1999

BUREAU OF INDIAN STANDARDSMANAK BHAVAN, 9 BAHAD~ SHAH ZmM M~G

~W DELHI 110002

June 1999 Price Group 2

Food Microbiology Sectional Committee, FAD 46

FO~WORD

This Indian Standard ( Part 4 ) ( Second Revision) was adopted by the Bureau of Indian Standards, afterthe drafi finalized by the Food Microbiology Sectional Committee had been approved by the Food andAgrictiture Division Council.

Several micro-organisms contaminating food give rise to clinical symptoms, such as abdominal pain, nausea,vomitting, diarrhoca and sometimes pyrexia. A well-known exception is that of botulism where the symptomsare those of diKc@ty in swallowing, diplopia, aphonia and difflcdty in respiration. Poisoning through foodis characterized by symptoms of explosive nature which occur in otherwise healthy indiviatis. Such explosivenature of food poisoning helpsindifferentiating conditions from those of out-breaks of food-borne infectiousdiseases which generally spread over a period of several days. This part of the standard covers the methodfor isolation and identification of some clostridium species responsible for food poisoning.

This standard-was published in 1970 and was first revised in 1976

This standard ( Part 4 ) is being revised in order to cover the method for Bacillus cereus in a separate ‘part(Part 6 ) and-to align with 1S0 7932:1993 ‘Microbiology — General guidance for the enumeration of Bacjllwscereus — Colony-count technique at 30°C.’ ,

In reporting the restit of a test or analysis made in accordance with this standard, if the final value, observedor calcdated, is to be rounded off, it shall be done in accordance with IS 2 : 1960 ‘Rules for rounding offnumerical values ( revised)’.

IS 5887 (Part 4 ) :1999

Indian Standard

METHODS FOR DETECTION OF BACTERIARESPONSIBLE FOR FOOD POISONING

PART 4 ISOLATION AND IDENTIFICATION OF CLOSTR/D/UM PERFR/NGENS( CLOSTRIDIUM WELCHII) AND CLOSTRIDIUMBOTULINUMAND ENUMERATION

OF CLOSTRIDIUM PERFRINGENS

(Second

1 SCOPE

This standard ( Part 4 ) prescribes methods forisolation and identification of Clostridium perfringens(Clostridium welchii) and ~ostridium botulinum andenumeration of Ciostridium perfringens in foods.

2 WFE~NCES

The following Indian Standards contain provisionswhich through reference in this text, constituteprovision of this standard. At the time of publication,the editions indicated were valid. All standards aresubject to revision and parties to agreements basedon this standard are encouraged to investigate thepossibility of applying the most recent editions of thestandards indicated below:

IS No.

1070:1992

5404:1984

6850:1973

6851:1973

6853:1973

7004:1973

7128:1973

7536:1975

10232:1982

Title

Wagent grade water ( third revision )

Code of practice for handling ofsamples for microbiological analysis(jrst revision)

Agar, microbiological grade

Meat extract, microbiological grade

Peptone, microbiological grade

Yeast extract, microbiological grade

Proteose peptone, microbiologicalgrade

Soluble starch, microbiological grade

Guidelines for preparation ofdilutions for microbiologicalexamination of food

For microbiological examination the samples shouldbe handled carefilly. For this purpose, IS 5404 shallbe followed.

Revision )

4 QU~ OF WAGEN~

Unless specified otherwise, pure chemicals shall be -employed in tests and reagent grade water ( seeIS 1070) shall be used where use of water as a reagentis intended.

NOTE — ‘Pure chemicals’ shall mean chemicals that

do not contain impurities which affect the test results.

-5 GE~W cmcTE~HCS

5.1 Clostridiumperfringens (Cl. welchii)

Anaerobic, Gram-positive rods, about 4 to 6 w x 1 y

with square ends. Spores are formed when grown inEllner’s medium ( 6.6 ), and are oval, subterminal andnot bulging. Typical food-poisoning strains ofClostridiurn perfringens are non-haemolytic onhorse blood agar ( 6.3 ), and produce lecithinaseas demonstrable on the egg-yolk plates ( 6.5 ) inshowing precipitates around the colonies. The sporesare heat resistant at 100°C for one hour; however, someworkers have reported reduced heat resistance of thespores when cultures are grown in Ellner’s medium( 6.6). The classification of Clostridiumperfringensinto various types have been made on the basis of‘toxins’. The three types of Clostridium perfringenscommonly associated in food poisoning are Types A,C and D.

5.1.1 In practice, the method of identification of food-poisoning strains of Clostridiumperfringens arebasedon colonial characters, morphology and the Naglerreaction. It shotid be noted that the frequency of spore-bearing Clostridium perfringens is low and reducesthe diagnostic values of this criterion.

5.2 Clostridium botulinum

Anaerobic, Gram-positive-rods, large and stout withstraight sides and rounded ends. Spores are oval,central or subtermiti and distend the bacillary body.On blood agar medium ( 6.3 ), growth is associatedwith haemolysis which may not be larger than the

1

IS 5887( Part 4 ) :1999

colony. On egg-yok medium ( 6.5 ), colonies produceopalescence and a pearly layer and are lactose negative.

5.2d Guinea pigs inoctiated intra-peritoneally withbotulinum toxin show difflcdty in breathing, flaccidparalysis of the abdominal muscles and salivation. Atautopsy, marked congestion of internal organs,extensive thrombosis and hemorrhages are noted. Thetoxic activity -may be neutralized by botulinumantitoxin, and the toxin is thermolabile.

5.2.2 Toxin of Cl. botulinum Type E is also thermo-labile, is activated by trypsin and may convenientlybe demonstrated using white mice.

6 ~DW

6.1 Nutrient Broth

Mix and dissolve by heating 10 g peptone ( seeIS 6853 ), 10 gmeat extract ( see IS 6851 ),5 g sodiumchloride in 1000 ml water. When cool, adjustpH from7.5 to 7.6. Remove precipitate by filtration throughfilter paper. Sterilize by autoclaving at 120°C for1j min.

6.2 Nutrient Agar

To the medium as in 6.1 add agar ( see IS 6850 ) insuch a concentration as to solidi~ and produce asufficiently firm surface when poured in sterile petridishes. The concentration of agar to be added variesfrom batch to batch and needs to be adjustedaccordingly. US~ concentrations required vary from1.5 to 3.0 percent. Dissolve the agar in the nutrientbroth and sterilize by autoclaving at 120°C for15 min. Plates and slopes are prepared from sterilenutrient agar.

6.3 Blood Agar with Neomycin

Melt sterile nutrient agar (6.2) and hold betweenjo to 55°C in a water-bath. Add sterile blood free frompreservatives to give a concentration of 10 percent.Mix well and pour plates. Horse blood is commonlyused but when not available, that of sheep, human orrabbit maybe used, Add aseptically neomycin sulphate,70 Vg per ml, before pouring in plates.

6.4 Cooked Meat Medium

Mince 500 g freshbeefheart and place in 500 rrdWineboiling water contining 1.5 ml 1N sodium hydroxidesolution. Simmer for 20 min. Drain off the liquidthrough muslin filter while still hot and partially drythe meat in the cloth or on filter papers. To 500 ml ofthe liquid filtered from the cooked meat add 2.5 gpeptone (see IS 6853) and 1.25 g sodium chloride.Steam at 1000c for 20 min and add 1 ml concen-trated hydroc Moric acid and filter. Bring the reactionoft he filtrate to pH 8.2 and steam again at 100°C for

30 rein; adjust to pH 7.8. Place meat in test-tubesor in about 30 ml screw-capped bottles to a depth ofabout 2.5 cm and cover with 10 ml of the brothobtained. Autoclave at 120°C for 20 min. A layer ofsterile paraffin may be added to cover the surface, ifnecessary.

6.5 Wilfis and Hobb’s Medium with Neomycin

Bre,ak fresh eggs which have been scrubbed and thensterilized by immersing in 0.1 percent aqueous solutionof mercuric chloride with precaution to keep thecontents sterile and separate the yolk from the white.Discard the egg white and suspend yolk in equalvolume of sterile 0.9 percent solution of sodiumchloride. Prepare separately sterile milk by auto-claving skimmed milk at 120°C for 15 min. Skimmedmilk is prepared by centrifuging ordinary milk toremove the cream. Dissolve by steaming, 4 to 5 g agar(see IS 6850) and 4.8 g lactose in 400 d of nutrientbroth ( 6.1 ) of pH 7.0 to which has been added1 percent solution of neutral red, 1.3 ml. Steritize at120”C for 15 min. Cool to 50to 55°C and add 15 mlegg yolk solution, 60 ml sterile skimmed mik andsterile solution of neomycin sulphate to a finalconcentration of 70 ~g per ti.

6.6 Spore Inducing Medium ( Ellner’s Medium )

Dissolve salts and ingredients separately in 1000 mlwater by brief steaming at 10O°C, 10 g proteompeptone ( see IS 7128 ),3 gyeast extract (see IS 7004 ),3 g soluble starch ( see IS 7536 ), 0.1 g magnesiumsulphate ( MgS04 ), 1.5 g potassium dihydrQgenphosphate (KH2P04), and 50 g disodium hydrogenphosphate (Na2HP04,7H20). Adjust to pH 7.8.After dispensing in test tubes of 150 mm x 20 mm totwo-thirds full, sterilize by autoclaving at 120°Cfor 15 min.

6.7 Medium for CL Botulinum me E

To the ingredients as in cooked meat medium ( 6.4 ),add 5 percent trypticase, and 0.5 percent peptone(see IS 6853). AdjustpH to 7.0. Stedize at 120°Cfor20 minutes and add a sterilized solution of 10 percentsodium thiuglycollate and 20 permnt glucose to havefinal concentrations of 0.2 and 0.4 percent -vely.

7.1 Clostridium.perfringens

Where necessary, the sample is blended in a sterileblender/jar for 2 minutes or macerated with sterilesand in a sterile mortar using approximate~ 200 d ofdiluting fluid per approximately 25 g of the sample.The diluting fluid shodd be 0.1 percent peptone(see IS 6853) in water sterilized at 120°C for 20 ~final pH 6.8 + 0.1 or 3.4 percent of potassium

2

{[w

IS 5887 (Pati 4) :1999

dihydrogen phosphate (KH2POQ) in water, pHadjusted to 7.2 and sterilized at 120”Cfor 20 min. Analiquot of the specimen is inoculated into cookedmeat medium (6. 4 ) and the inoculated tube heated ina steamer at 100°C for one hour and incubated over-night at 37°C. An aliquot of the specimen is alsoinoculated directly onto blood agar medium ( 6.3 )and the egg-yolk medium ( 6.5 ) and incubated in ananaerobic jar at37°C overnight. Subcultures are madefrom the growth in medium ( 6.4 ) on to the two solidmedia ( 6.3 and 6.5 ) and incubated in an anaerobicjar at 37°C overnight.

7.2 Cl. botulinum

Preheat the sample at 80°C for 30 min andinoculate into cooked meat medium (6.4), and the twosolid media, 6.3 and 6.5. The solid media are incubatedanaerobically, and all the three inoculated media areincubated at 37°C for 5 to 10 days.

The type Estrains exhibit low thermal resistance, andare missed in specimens which have been heated priorto inoculation. For the isolation of these strains, theprocedure as in 7.2.1 should be followed.

7.2.1 Isolation of Cl. Botulinum Type E

Inoculate the specimen in duplicate tubes of themedium ( 6.7 ), and incubate at 30°C for 3 days. Insterile test tubes take diquots of 2 d samples of grotihand mix with equal volume of absolute ethanol. Letstand at 25°C for one hour with occasional mixing.Streak onto egg-yolk medium ( 6.5) and inoculate intomedium ( 6.7 ). Incubate overnightat37°C, the solidmedium being incubated in an anaerobic jar. Examinethe solid medium for presence of colonies withopalescence zones indicating growth of CI. botulinumType E. If such colonies are present, carry out testfor toxin using the growth in medium ( 6.7 ) inoculatedwith ethanol treated culture.

8 ~STS FOR~EN~CA~ON

8.1 Clostridiumperfringens

is fixed by passing to and fro over a flame and cooled.Cover the smear with the stain ( a ) for 30 s, pour offthe stain and wash with ( b ) and then cover with(b)and allow to remain for 30 s. Wash off with ethanoluntil the dye ceases to stream out. Wash in runningtap water and apply ( c ) for about one minute. Washin tap water and dry for examination.

8.1.2 Colonial Characters

By growth on blood agar medium ( 6.3 ) and egg-yolkmedium ( 6.5), as described in ( 5.1 ).

8.1.3 Spore Production

Inoculate growth from any of the media as in 6.3 and6.5 into spore inducing medium (6.6 ). The growth incooked meat medium ( 6.4 ) after following theprocedure as in 7.1 may also be inoculated into mdium(6.6 ).

NOTE — Some workers have noted reduced heat

resistance of spores when cultures are grown in 6.6.

8.1.4 NaglerReaction — In fitm Test for Alpha Toxin

One half of a plate of egg-yolk medium ( 6.5 ) is spreadover with two or three drops of standard Clostridiumperfringens antitoxin and dried. The area isdemarcated. The two halves of the plate are inoculatedwith the suspect strain and incubated at 37°Canaerobically. Lecithinase activity is shown byprecipitates around colonies in the half tithoutantitoxin and this reaction is inhibited in the other halfwith specific antitoxin. The production of the enqmelecithinase C, as demonstrated in the Nagler reaction,by all types of Clostridium perfringens is used todistinguish Clostridium perfringens from other spiesof Clostridia. However, Cl. bt~ermentes also producelecithinase and maybe dfierentiated from Clostridium

perfringens by Cl. bifermentes showing proteolyticactivity, ready sporulation and non-fermentation oflactose. Lactose fermentation is carried out in 1percentpeptone water sugar medium incubated anaerobicallyat 37°C.

8.1.1 Gram k Stain 8.2 Cl. botulinum

The stain consists of

a ) 0.5 percent methyl violet or crystal violet inwater,

b) iodine solution ( 1percent iodine and 2 percentpotassium iodide in water), and

c) counterstain ( 0.1 g neutral red, 0.2 ml of1 percent acetic acid and 100 ml water ).

On a clean grease-free slide, ve~ light and thin smearcovering a sdl area is made di&ly from liquid cultureand in clean tap water if from solid media. The smear

8.2.1 Gram k Stain

See 8.1.1.

8.2.2 Colonial Characters

By growth on blood agar medium ( 6.3 ) and egg-yokmedium ( 6.5 ), as described in 5.2, and for Type Estrains as in 7.2.1.

8.2.3 In Evo Test for Toxin

Grow suspect strain in cooked meat medium ( 6.4 ) for5 to 10 days. Obtain filtrate and divide into two

3

IS 5887( Pati 4 ): 1999

portions, one of which is heated at 10O°Cfor 10 min.Use three guinea pigs for intraperitonal injection withfiltrate as follows:

a) One animal is protected with polyvalentbotulinum antitoxin and injected with 2 ml ofunheated filtrate;

b) One animal as injected with 2 ml of unheatedfiltrate and is unprotected; and

c) One animal is injected with 2 ml of heatedfiltrate.

Death with paralytic symptoms of the unprotectedanimal receiving unheated filtrate and survival of theother two animals diagnose the presence of botulinumtoxin.

8.2.4 Demonstration of Toxin of Cl. BotulinumType E

The procedure as in 8.2.3 may fail to demonstratetoxin of Cl. botulinum Type E. For such strains theprocedure shall be as follows.

8.2.4.1 To filtrate from growth in medium (6.7) asobtained after procedure described in 7.2.1 is addedtrypsin to a final concentration of 0.1 percent.Incubate at 37°C for 60 min. Dilute specificT~e E antitoxin 1 in 5 with 0.1 M phosphate bufferofpH 6.5 containing 0.2 percent gelatin. To 1.5 ml ofdiluted antitoxin, add equal volume of trypsinizedfiltrate, mix and keep at room temperature for 30 min.Inject 1 ml of the mixture intra-peritoneally into a pairof white mice. Mso inject a pair of mice with 0.5 ml ofthe filtrate heated at 100°C for 10 min and anotherpair of mice with 0.5 ml of unheated trypsinizedfiltrate. Observe the mice up to 96 h. Death of theunprotected mice and survival of the mice receivingneutralized toxin and the heated toxin diagnose toxinof Cl. botulinum Type E.

8.2.5 Since bacteriological diagnosis of food-poisoningdue to Cl. botulinum is based on the demonstration

ofthe toxin in the food or in~tind mnten~ the premceof the toxin in such materials need to be demonstrated.

8.2.5.1 Procedure

The material is soaked overnight in equal volume ofsterile normal saline. The suspension is centrifugedand the supernatant sterilized by filtration. This isthen directiy used to note the presence of botulinumtoxin by animal inoctiation as with titure filtratedescribed in 8.2.3 and 8.2.4.

9 .SERO~~G

Food poisoning strains of Clostridiumperfringens maybe serotyped by slide agglutination using coloniesfrom blood agar ( 6.3 ), and testing with specificagglutinating sera, if these are available.

10 E-RA~ON

10.1 Clostridiumperfringens

25 to 50 g of the sample is taken in-a sterile blender/jarand to this is added diluting fluid ( see 7.1 ) to havedilution of 10-1. Blendat8000 to 10000 rev/rein for2 min. Alternatively, macerate the sample withdiluting fluid in a sterile mortar with sterile sand.Make serial ten-fold dilutions with the diluting fluidin duplicate series up to 10-7. Streak 0.1 d from mchtube evenly onto blood agar medium ( 6.3 ) and dseonto egg-yolk medium ( 6.5). Incubate in an anaerobicjar at 37°C for 18 to 24 h. It is useful to incubateaerobically duplicate plates similarly inocdated forcomparison. The suspect colonies (5. 1 ) are countedand the number of viable colonies per gram of sampledetermined bymultiplying by the dilution factor(s )and dividing by the mass of the sample.

10.1.1 Since food-poisoning occurs due to thepresence of large number of Clostridium perfringens,usurdly millions per gram; therefore, counts of ,mloniesshall be of considerable help partictiarly in surveysfor clostridial invasion of food producing factories.

4

c

.*

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This”Indian Standard has been develoDed from Doc: No. FAD 46 ( 544).

Amendments Issued Since Publication

Amend No. Date of Issue Text Affected

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