AB Mauri India Private Limited WI: CSL M12

MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 12/04/2016

Revision Date:04/01/2019

Method Title: MICROBIOLOGY- CLOSTRIDIUM PERFRINGENS

1. Purpose:
To enumerate Clostridium perfringens in spices and spice products

2. Apparatus:
2.1. Sterile Petri dishes (15 X 100 mm)
2.2. Pipettes (1 ml)
2.3. Test tubes (18 X 150 mm)
2.4. Conical flask (500 ml)
2.5. Incubators 35+/-2°C
2.6. Electronic Balance
2.7. Sterile spatulas or spoons
2.8. Marking pen
2.9. Spirit lamp/ Bunsen Burner
2.10. Inoculation loops
2.11. Microscope
2.12. Glass slides and cover slips
2.13. Culture tubes (13 X 100 mm)
2.14. Anaerobic gars
2.15. Anaerobic gas pack

3. Media and Reagents:

3.1. Peptone Water (218071 Difco)
3.2. Perfringens Agar Base (M837 Himedia)
3.3. Tryptose-Sulfite-Cycloserine (TSC) Supplement (FD014 Himedia)
3.4. Liver Veal Agar (M176 Himedia)
3.5. Egg Yolk Emulsion (FD045 Himedia)
3.6. Thioglycollate Fluid Medium (M009 Himedia)
3.7. Iron Milk Medium
3.8. Lactose-gelatin medium, modified (M987 Himedia)
3.9. Motility-nitrate medium, buffered (M630 Himedia)
3.10. Nitrate Test Reagents
3.9.1. Sulphanilic Acid (R015 Himedia)
3.9.2. Alpha-Naphthalamine (RM389 Himedia)
3.11. Gram Staining Reagents (K001 Himedia)
3.10.1. Crystal Violet
3.10.2. Gram’s Iodine
3.10.3. 95% Ethyl Alcohol
3.10.4. Safranin

ABM Analytical Methods Manual – Clostridium perfringens Page 1 of 4

AB Mauri India Private Limited WI: CSL M12
MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 12/04/2016
Revision Date:04/01/2019

3.12. Spore Staining Reagents (K006 Himedia)

3.11.1. Malachite Green
3.11.2. Safranin

4. Preparation of Media:
4.1. Preparation of Peptone Dilution Blank: Suspend 15.0g of peptone water to 1L distilled
water. Mix well and dispense 9ml & 225ml into required no. of tubes and bottles respectively.
Sterilize by autoclaving at 15lbs pressure for 15 minutes. pH 7.2+/-0.2
4.2. Preparation of Perfringens Agar: Dissolve 23.5g in 475ml distilled water. Sterilize by
autoclaving at 15 lbs pressure for 15 min. Cool to 50 oC, aseptically add one vial dehydrated TSC
supplement. Mix well and pour 7-10ml into sterile petriplates. pH 7.6 +/- 0.2
4.3. Preparation of Thioglycollate Fluid Medium: Suspend 29.75 in 1L distilled water. Dispense
10ml to test tubes. Sterilize by autoclaving at 15 lbs pressure for 15 min. Final pH 7.1 +/- 0.2.
4.4. Preparation of Iron Milk Medium : Heat iron nails in Bunsen burner to remove any oils. Add
one nail to tube containing 15ml milk. Or dissolve 1g ferrous sulphate, 7H 2O in 50ml distilled
water. Add slowly to 1L milk and mix with magnetic stirrer. Autoclave at 121 oC for 5-6 min.
Prepare fresh medium before use.
4.5. Preparation of Liver Veal Egg Yolk Agar : Suspend 48.5g in 500 ml distilled water. Sterilize
by autoclaving at 15 lbs pressure for 15 min. Cool to 50 oC, aseptically add 40 ml egg yolk
emulsion. Mix well and pour 15-20 ml into sterile petriplates. Keep the plates at 35 oC for 24 hrs
and check for sterility and store plates in refrigerators.
4.6. Preparation of Lactose-Gelatin Medium: Dissolve 16g in 100 ml distilled water. Dispense
10ml to test tubes. Sterilize by autoclaving at 15 lbs pressure for 15 min. Final pH 7.5 +/- 0.2. if
not use within 8 hrs, deaerate by heating at 50-70oC for 2-3 hrs before use.
4.7. Preparation of Motility-Nitrate Medium: Dissolve 23.5g in 1L distilled water. Dispense 5 ml
to test tubes. Sterilize by autoclaving at 15 lbs pressure for 15 min.

5. Procedure:
5.1. Sample Preparation
Aseptically weigh 25g of homogenate sample into a 250 ml conical flask containing 225ml
sterile Butterfield phosphate buffer. This dilution is 10-1. Shake at least 25 times in a gt.arc.
5.2. Preparation of Dilutions:
5.2.1. Using 1:10 dilution prepared above, make serial dilutions up to 10 -4 by transferring 1
ml diluent blank.
5.2.2. Mix each dilution thoroughly by gently shaking before each transfer.
5.2.3. Obtain uniform homogenate with as little aeration as possible.

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AB Mauri India Private Limited WI: CSL M12
MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 12/04/2016
Revision Date:04/01/2019

5.3. Plating
5.3.1. Pour 7-10 ml TSC agar without egg yolk into sterile petri dishes and allow solidifying.
5.3.2. Aseptically transfer 1 ml aliquots of each dilution to the center of agar plates.
5.3.3. Pour additional 15 ml TSC agar without egg yolk into dish and mix with inoculum by
gently rotating dish.
5.3.4. Control plates should be prepared for each lot of dilution blanks, medium, petri
dishes, pipettes and also a hood control to check the possibility of air
contamination.
5.3.5. When agar has solidified, place plates in upright position in anaerobic jar. Establish
anaerobic conditions and place jar in 35°C incubator for 20-24 h.
5.3.6. After incubation, remove plates from anaerobic jar and select those containing 20-
200 black colonies for counting. Count black colonies and calculate number of
clostridia cells/g food.
5.4. Confirmatory Tests
Select 10 typical C. perfringens colonies from TSC or TSC-egg yolk agar plates and
inoculates each into a tube of freshly deaerated and cooled fluid thioglycollate broth.
Incubate in standard incubator 18-24 h at 35°C.
5.4.1. Gram’s Stain - Prepare culture smear on glass slide. Heat fix. Cover the smear with
crystal violet solution, allow to stand for I min, rinse with water. Cover with Gram’s
iodine, stand for I min, wash off with 70% alcohol/acetone, wash off with water.
Cover with safranin; allow standing for 30 seconds, washing with water. Air-dried.
Observe microscopically. C. perfringens is a short, thick, Gram-positive
bacillus.
5.4.2. Spore Stain - Prepare culture smear on glass slide. Heat fix. Place the slide over the
boiling water bath. Cover the smear with Malachite green solution when water
condenses below the slide; allow standing for 5-7 min, rinsing with water. Cover
with safranin, let stand for 1 min. green spores with red vegetative cells.
Bacillus spores are ellipsoidal, central or sub terminal, and do not swell the
sporangium.
5.4.3. Iron-milk presumptive test - Inoculate modified iron-milk medium with 1 ml of
actively growing fluid thioglycollate culture and incubate medium at 46°C in a
water bath. After 2 h, check hourly for "stormy fermentation." This reaction is
characterized by rapid coagulation of milk followed by fracturing of curd into
spongy mass, which usually rises above medium surface. Remove positive tubes to
prevent spilling over into water bath. For this reason, do not use short tubes for the
test. Cultures that fail to exhibit "stormy fermentation" within 5 h are unlikely to be
C. perfringens. Only actively growing cultures are appropriate for this test.
5.4.4. Motility-Nitrate Medium - Stab-inoculate motility-nitrate (buffered) with 2 mm
loopfuls of pure fluid thioglycollate medium culture or portion of isolated colony
from TSC agar plate. Incubate inoculated media 24 h at 35°C.
C. perfringens is nonmotile. Examine tubes of motility-nitrate medium for type of
growth along stab line. Nonmotile organisms produce growth only in and along

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AB Mauri India Private Limited WI: CSL M12
MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 12/04/2016
Revision Date:04/01/2019

stab. Motile organisms usually produce diffuse growth out into the medium, away
from the stab.
C. perfringens reduces nitrates to nitrites. To test for nitrate reduction, add 0.5 ml
reagent A and 0.2 ml reagent B to culture in buffered motility-nitrate medium.
Violet color, which develops within 5 min, indicates presence of nitrites. If no color
develops, add a few grains of powdered zinc metal and let stand a few minutes. A
negative test (no violet color) after zinc dust is added indicates that nitrates were
completely reduced. A positive test after addition of zinc dust indicates that the
organism is incapable of reducing nitrates.
5.4.5. Lactose-gelatin media - Stab-inoculate with 2 mm loopfuls of pure fluid
thioglycollate medium culture or portion of isolated colony from TSC agar plate.
Stab lactose-gelatin repeatedly to ensure adequate inoculation, and then rinse loop
in beaker of warm water before flaming to avoid splattering. Incubate inoculated
media 24 h at 35°C. Examine lactose-gelatin medium cultures for gas production
and color change from red to yellow, which indicates acid production. Chill tubes 1
h at 5°C and examine for gelatin liquefaction. If medium gels, incubate an
additional 24 h at 35°C and examine for gelatin liquefaction. C. perfringens
produces acid and gas from lactose, and liquefies gelatin within 48 h.

6. Calculation:
Cl.perfringens = Average count obtained X Dilution X No. of +ve colonies
Total no. of colonies picked
The count shall be expressed in Organisms (Org) per g or ml, as applicable.

7. Reference:
6.1. FDA Bacteriological Analytical Manual
6.2. Compendium of Methods for Microbiological examinations of Foods.
8. Environmental aspects:
On completion of work, all used or unusable preparations relating to this analysis are to be disposed
of by autoclaving. Never mouth pipettes any liquid. Lab coats, gloves and mouth covers are must
when working with live micro-organisms or media containing hazardous ingredients such as dyes.

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