BASED ON USFDA BAM 8th Edition

DETERMINATION OF INORGANIC PHOSPHATE

Reagents: 1. Trichloro – Acetic Acid 10% (in water),

2. 10 N Sulphuric Acid (Exactly),
3. Molybdate Sodium
4. Amino Nepthol Sulphonic Acid Reagent (ANSA)
5. 15% Sodium bisulphate
6. 20% Sodium Sulphite Solution (in water)
7. Standard Phosphate Solution.
Preparation of Molybdate Solution:
25 g Ammonium Molybdate reagent dissolve
in about 200ml water

In 1 lit. Volumetric Flask place

300 ml. 10N Sulphuric acid

Add the Molybdate solution and dilute with washings

to liter with water. Make stable indefinitely.
Preparation of ANSA Regent:
Place 195 ml. of 15% Sodium bisulphate
Solution in a glass- Stoppard cylinder

Add 0.5 g of 1, 2, 4-Amino Nepthol Sulphonic acid and

Shake until the powder is dissolved,
If solution is not complete add more sodium sulphite,
1ml.at a time with shaking but avoids an excess.

Transfer the solution to a brown glass bottle and store

in the cold condition. This solution is usable for
about 4 weeks, if kept as described above.
Preparation of Sodium Bisulphate:
To 30 g in a beaker add 200 ml. of water, stir to dissolve and if turbid allow to
stand well Stoppard for several days and filter. Kept well Stoppard.

Preparation of Standard Phosphate Solution:

Dissolve exactly 0.351 gm. of pure & dried mono-potassium

Phosphate in water and transfer quantitatively to 1 lit. Volumetric flask

Add 10 ml. of 10 N Sulphuric acid diluted to the mark with

Water and mix. This solution contains 0.4 mg.
of phosphorous in 5ml.it is stable indefinitely.
 BASED ON USFDA BAM 8th Edition
Procedure:
Start with protein free (10% TCA extract) Sample or digested filtrate as required.
Transfer 5ml. of sample to 10ml of graduated
Container and add 1ml of Molybdate and mix well.

Add 0.4ml of ANSA and again mix. Dilute to

the mark mix and allow to stand for 5 mints.

Transfer a portion of the colored solution to a suitable container

and read the photometer at 660 to 720nm, get the meter to
0 density with a blank prepared by treating 5ml of 10% TCA
with 1ml of Molybdate solution and 0.4ml of ANSA Reagent
followed by water to volume of 10ml establish the density of a
standard phosphate solution

Transfer 5ml of stock phosphate standard containing 0.4mg

of phosphate, to a 50ml of Volumetric flask make up to volume
with 10% TCA and mix.

Transfer 5ml of this dilute standard containing 0.04mg of phosphorous

to a suitable container (10ml)

Add 1ml Molybdate and 0.4ml of ANSA Reagent dilute to 10ml with
water and mix allow to stand for 5 mints. And determine the density in
photometer whose zero is set with blank as described above.
Phosphate Residue Testing:
1. 5 gms. Sample + 100ml. TCA – Make up 100ml. overnight.
2. 5 ml. from above TCA extract + 100ml. TCA make up.
3. 5ml. of above TCA extract + 0.4ml. of ANSA reagent + 1ml. Molybdate solution + make up to 10ml. with
distilled water.
4. Blank: - 5ml. of TCA + 1ml. of Molybdate solution + 0.4ml of ANSA reagent + make up to 10ml. with
distilled water.
5. Standard: - 5 ml. of stock standard phosphate solution + make up to 50ml. with TCA.
6. Working Standard: - from the above, 5ml. standard solution + 1ml of Molybdate solution + 0.4ml. of ANSA
reagent + make up to 10ml. with distilled water.
7. Switch on the spectrophotometer.
8. Set the % T, tune to set 0% T with set zero.
9. Keep the clean blank and set 100 at % T.
10. Then transfer % T to OD mode.
11. It will give you 0.000 OD.
12. Take out blank and insert standard and not the reading.
13. Likewise take the reading for sample also.
14. Then Calculate as per follow: Calculation:-
Sample OD value x (0.04 x 20 x 20 x 1/5 x 95/31) or 49.032
Standard OD value
Where: 0.04 = Factor value for P
20 = 1st dilution (5gm in 100ml TCA)
20 = 2nd dilution ( ” ” )
1/5 = to convert to per gm.
95/31= to convert P to PO4.
*****************************
 BASED ON USFDA BAM 8th Edition

ESTIMATION OF CHLORINE STRENGH

1. 1ml. of Chlorine.
2. 20ml. of Distilled water.
3. 1ml. of glacial acid ( CH3 COOH )
4. 1gm. Potassium iodide ( KI )

All the above four reagents are taken into a conical flash,
then titrated against 0.1 N Sodium thiosulphate (Na2 S2 O3)
Burette solution.

Appearance of pale yellow color is seen, stop the titration.

Add 1 or 2 drops of starch indicator solution then the

solution will change to dark brown color.

Proceed the titration with the same normality

Sodium thio-sulphate burette solution

Disappearance of dark brown color is the end point

(Colorless solution)

Note down the burette end point reading.

Calculation: - % of available Cl2 = Volume of Solution thio sulphate x 0.3546

Volume of Chlorine added

Preparation of Reagents:-

1. Preparation of 0.1 N Sodium thio Sulphate: 24.818 gm Sodium thio sulphate pellets in 1000ml Distilled
water.
2. Preparation of Starch Indicator: 10gms. Starch Powder in 100ml. boiling Distilled Water.

*********************
 BASED ON USFDA BAM 8th Edition
Aerobic Plate Count (APC) or TPC

25g Sample + 225 ml Buffer field’s Phosphate buffer

(10 -1)
10ml
1ml 1ml
90ml diluent 9ml.diluent 9 ml diluent
to make 10-2 10
-3
10
-4

1ml 1ml 1ml

Into Petri dishes + 12-15 ml. of PCA (45±1ºC) and gently shake well

Let solidify, invert and incubate at 35ºC for 48±2hrs.

Count no. of colonies on plates with 25-250 CFU present and calculate for APC/g of sample.

Plate with 25-250 CFU

Eg. Dil. 1:100 1:1000

Count 232,244 33, 28

Calculation:
εc
N = --------------------------------------------------------
[(1x n1) + (0.1x n2 )] x (d)

(232+244+33+28)
=
---------------------------------
[(1x 2) + (0.1 x 2)] 10-2

= 537
------- X 100 = 24409 = 24000 CFU/g or ml.
2.2

Where; N= no. of colonies/ml. or g of product

εc= sum off all colonies on all plates counted

n1 = no. of plates in first dilution counted

n2 = no. of plates in second dilution counted
d = dilution from which the first counts were obtained.
*****************
E. coli & Coliform Bacteria
 BASED ON USFDA BAM 8th Edition

25g sample + 225ml Buffer field’s Phosphate buffer (10-1)

Prepare 2 more consecutive dilutions (10-2 & 10-3)

Presumptive Test.
1 ml. of each dilution to 3 LST tubes, for 3 Consecutive dilutions

Incubate at 35ºC for 48±2 hrs.

Examine at 24 hrs. for gas production tubes.

According
For Coliform test to +ve test tube, will For E.coli test
Take test tube.

To BGLB broth To EC broth

Incubate at 35ºC for 48±2hrs. Incubate at 45.5±0.2ºC for 48±2hrs.

(Water Bath)

Examine tubes for gas production;

Record and calculate MPN of Coliform. Examine tubes for gas production,
Record and calculate MPN of Fecal Confirmative
Coliform. Test for
Identification
Streak loopful from each gassing tube to of Coliform.
L-EMB.

Incubate at 35ºC for 18-24 hrs.

Examine for dark centre & flat, with or without metallic sheen.
Transfer suspicious colony to PCA slant for Morphological &
Biochemical testing.

Incubate at 35ºC for 18-24hrs.

Morphological & Biochemical Testing for E.coli

 BASED ON USFDA BAM 8th Edition
1. Gram Strain: -

Gram –ve, short rod or cocci.

2. Indole Production:-
Inoculate tube of Typtone broth

Incubate at 35ºC for 24±2 hrs.

Add 0.2-0.3 ml of Kovac’s reagent.

Result: - appearance of distinct red color in upper layer is considered +ve.

3. Voges-Proskauer (VP) – reactive compounds:-

Inoculate tube of MR-VP broth Incubate at 35ºC for 1ml Add 0.6ml of α-napthol
48±2hrs. Solution and 0.2ml 40%
KOH.

Shake

Shake and Add few crystal of creatine

Let stand for 2hrs.
Result: - development of eosin pink color is considered +ve.

4. Methyl Red – reactive compounds:-

Incubate MR-VP tube additional 48±2hrs. Add 5 drops of methyl red solution.
At 35ºC after VP test.

Result: - Distinct red color is +ve, yellow is –ve reaction.

5. Citrate:-

Lightly inoculate tube of Koser’s citrate broth. Incubate at 35ºC for 96hrs.

Result: - Development of distinct turbidity is +ve reaction.

6. Gas from Lactose:-

Inoculate tube of LST broth and incubate at 35ºC for 48±2hrs.

Result: - Displacement of medium from inner vial is +ve reaction.

*****************************
Staphylococcus aureus
 BASED ON USFDA BAM 8th Edition
25g Sample + 225ml Buffer field’s Phosphate buffer (10-1)

Prepare 2 more consecutive dilutions (10-2 and 10-3)

1 ml. of each dilution to 3-TSB tube containing 10% Nacl and 1%

Sodium pyruvate for 3 – consecutive dilutions.

Incubate at 35ºC for 48±2hrs.

Streak 1 loopful of each tube showing growth (turbidity) to

Baird Parker Plate

Incubate at 35ºC for 48±2hrs.

Examine for circular, smooth, convex, moist, 2-3mm in diameter, gray to jet black,
Frequently with light –colored (off-white) margin, surrounded by opaque zone and
Frequently with an outer clear zone.

Transfer 1 suspected colony to BHI broth (0.2-0.3ml) for

Confirmation of Staphylococcus aureus

Incubate at 35ºC for 18-24hrs.

Add 0.5 ml reconstituted coagulase plasma with EDTA and mix thoroughly, incubate at
35ºC and examine for clot formation periodically over 6hrs. Period.

Report Staph. aureus / g as MPN /g

SALMONELLA IN RAW FLESH FOOD

25 g Sample + 225 ml LB and Blend for 2 mints.

 BASED ON USFDA BAM 8th Edition

Transfer to screw cap jar, let stand for 60 min. at room temp. With
Jar securely capped

Loosen jar caps ¼ turn and incubate at 35ºC for 24±2hrs.

Transfer 0.1 ml to 10 ml Transfer 1 ml to 10 ml

RV broth TT broth

Incubate at 42±0.2 ºC for Incubate at 35ºC for 24±2hrs or 43±0.2ºC

24 ±2hrs. (Water bath) (Water bath) for highly contaminated foods.

Streak loopful on BSA, XLD & HE agar

And incubate at 35ºC for 24±2hrs.

Examine for Typical Salmonella Colony Morphology:-

1. HE Agar: - Blue –green to blue colonies with or without black centre (may be appear as large glossy black
centre or almost completely black colonies.)
2. XLD Agar: - Pink colonies with or without black centers (may be appear as large glossy black centre or
almost completely black colonies).
3. BS Agar: - Brown, gray or black colonies (with metallic sheen sometimes), surrounding medium is usually
brown at first but may turn black for longer incubation (halo effect).

Typical colonies on BS Agar after 24±2 hrs. Pick 2 or more colonies.

Irrespective of whether or not BS agar are picked at 24±2hrs re-incubate BS agar plates an additional 24±2hrs after
48±2hrs., pick 2 or more typical colonies, if present.

Only if colonies picked from BS agar plates (24±2 hrs.) give a typical reaction in TSI & LIA that result in culture
being discarded as not being Salmonella.
Atypical Salmonella Colony Morphology:-
In the absence of typical or suspicious salmonella colonies, search for typical
salmonella colonies as follow:-
(A) HEA & XLD agars: - Yellow colonies with or without black centers, pick 2 or more a typical salmonella
Colonies.
(B) BS agar: - Green colonies with little or no darkening of surrounding medium.
 BASED ON USFDA BAM 8th Edition
Note: - If typical colonies are not present after 24±2 hrs. then do not pick any colonies but re-incubate
an additional 24±2hrs. If typical colonies will not present, then pick 2 or more atypical colonies.

Suggested Control Culture:-

3 additional Salmonella cultures are recommended to assist in selection of atypical salmonella
colony morphology on selective agars:-
Lactose – Positive; H2S – Positive S.arizonae (ATCC 12325)

Lactose – -ve ; H 2S – -ve S.abortus equi (ATCC 9842)

Lactose – Positive; H 2S – -ve S.diarizonea (ATCC 29934)

Presumptive testing for typical Salmonella and atypical Salmonella:-
1. Lightly touch the very centre of the colony by sterile needle and inoculate TSI slant by streaking slant &
stabbing butt.
2. Without flaming, inoculate LIA slant by stubbing deep butt (4cm) twice and then streak slant.
3. Incubate TSI & LIA slants at 35ºC for 24±2hrs. Cap tubes loosely to maintain aerobic condition.
4. For TSI: - Salmonella typically produces alkaline (red) slant and acid (yellow) butt with or without H2S
production.
For LIA: - Salmonella typically produces alkaline (purple) reaction in butt of tube. Consider only distinct
yellow in butt of tube as negative (acidic reaction).Do not iliminate cultures that produce discoloration in butt
of tube solely on this basis. Most salmonella culture produces H 2S in LIA.
Type of presumptive results which should be retained as potential salmonella and follow with the biochemical
and serological identification:
1. All cultures give an alkaline butt in LIA.
2. Cultures that give: acid butt in LIA and alkaline slant and acid butt in TSI.

Note: - Only cultures giving an acid butt in LIA and an acid slant and acid butt in TSI may be discarded. apply
biochemical and serological identification test to; 3 presumptive TSI cultures recovered from set to plates streaked
from SC or RV, if present, and 3 presumptive TSI agar cultures recovered from plates streaked from TT,if present.

If 3 presumptive +ve TSI cultures are not isolated from one set of agar plates, test other presumptive +ve TSI agar
cultures, if isolated, by biochemical & serological tests.
Examine a minimum of 6 TSI cultures for each 25g analytical unit.

*********************

LISTERIA MONOCYTOGENES

Enrichment:-

25 g Sample + 225 ml. EB and blend or stomach

for thorough mixing
 BASED ON USFDA BAM 8th Edition
Incubate at 30ºC for 4 hrs.

Add selective agents; acriflavin, nalidixic acid and cycloheximide

Continue incubating another 44 hrs. for a total of 2 days at 30ºC

Isolation:-
At 24 & 48 hrs. Streak EB culture on OXA, LPM or LPM plus
Esculin / Fe3+ or PALCAM agar.

Incubate LPM at 30ºC

Incubate OXA and for 24-48 hrs.
PALCAM at 35ºC for
24-48 hrs.

Examine for sparking blue (bluish

Examine for colonies have a black crushed glass) or white colonies by
Halo within 2 days. Using beamed white light to illuminate
Plates well.

Transfer 5 typical colonies from OXA, PALCAM or LPM to TSAYE,

Streaking for purity and typical isolated colonies. (Mandatory Step)

Incubate TSAYE plate at 30ºC for 24-48 hrs. Or at 35ºC if colonies

Will not be used for a wet mount motility observation.

Continue for identification Procedures.

Identification Procedures for L. monocytogenes:-

1. Examine TSAYE plate for typical colonies. With the oblique Henry illumination system, colonies appear
blue-gray to blue. The use of known controls on TSAYE is recommended.
2. Pick typical colonies from culture plate incubated at 30ºC or lower and examine in a wet mount, using 0.85%
saline for suspending medium, with the oil immersion objective of a phase- contrast microscope.
 BASED ON USFDA BAM 8th Edition
Choose a colony with enough growth to make fairly heavy suspension.

Listeria spp. are slim, short rod with slight rotating or tumbling motility.
3. Test typical colonies for catalase, Listeria sps. are catalase +ve.
4. Gram Stain 16 hrs. to 24 hrs. Cultures: All Listeria spp. are short; Gram +ve rods (however, with older cultures
the Gram Stain reaction can be variable and cells may appear Coccoidal).
5. Inoculate heavily 5% sheep blood agar or horse blood agar by stabbing plates that have been poured thick and
dried well. Draw grid of 20-25 sps. On plate bottom. Stab one culture per grid species. Always stab positive
controls and negative control.
L. ivanovii & L. monocytogenes - +ve.
L. innocua - -ve.

Incubate for 48hrs. at 35ºC.

Examine blood agar plate with bright light.

L.monocytogenes & L.seeligeri -------------------- Slightly clear zone around stab.
L.innocua--------------------- no zone of heamolysis.
L.ivanovii -------------------- well defined clear zone around stab.

Note nature of hemolytic reaction and resolve doubtful reactions by the CAMP Test.

6. Nitrate Reduction Test (Optional):-

Use TSBYE culture to inoculate nitrate broth.

Incubate at 35ºC for 5 days.

Add 0.2 ml reagent A, followed by 0.2 ml reagent B.

A red – violet color indicates presence of nitrate, i.e.; nitrate has been reduced. If no color develops, add
powdered zinc and let stand for 1 hr. A developing red-violet color indicates that is still present and has not been
reduced. Only L. grayi spp. murrayi reduces nitrates.

7. As an alternative procedure, add 0.2ml Reagent A followed by 0.2ml reagent C. An orange color indicates
reduction of nitrate. If no color develops, add powdered zinc as above. Development of an orange color
indicates unreduced nitrate.
8. Inoculate SIM or MTM from TSBYE; incubate for 7 days at room temp.

Observed daily: Listeria spp. are motile, giving a typical

Umbrella-like growth pattern. MTM gives better defined
 BASED ON USFDA BAM 8th Edition
Umbrellas.
Alternative, observe 30ºC TSBYE cultures by phase – contrast micro-scopy (1000 X)
for tumbling motility.
9. From TSBYE culture, inoculate the following carbohydrates as 0.5% (w/v) sol. in purple carbohydrate broth
(the use of Durham tubes is optional):- Dextrose, esculin, Maltose, rhamnose, mannitol and xylose.

Incubate 7 days at 35ºC.

Positive reaction Listeria spp. produces acid with no gas.

All species should be +ve for dextrose, esculin and maltose.
All Listeria spp. except L.grayi should be mannitol –ve.
Rapid Kits for Biological Testing & Identification:-
10. Purified isolates can be rapidly identified by conventional tests using commercial kits:-

• 20 S ™ or API-ZYM (Analytical Products, Plainview, NY) or

• Vitek Automicrobic Gram Positive and Gram –ve identification cards ( Bio
Merieux , Hazelwood, MO) or
• API Listeria (Bio Merieux sa Marcy- l’Etoile, France), which does not require an
additional CAMP test.
• The Micro - ID™ kit (Organon Teknika Corp; Durham, NC) permits speciation
of Listeria isolates if their CAMP reactions are known.
AOAC International, Gaithersburg, MD, has adopted the Micro-ID and the Vitek Automicrobic system as
official first action methods.
11. Isolates in pure broth culture may be identified to Genus level by using commercial
• ELISA kits (Organon; Bioenterprises pty Ltd, Reseville, NSW, Australia) or
• Non radio labeled DNA Probe kits (Gene Trak, Framingham, MA)
Note: - if such kits are used to screen enrichment cultures for Listeria spp.; cultures should be still
be streaked on selective agars regardless of screening results.
Non radioactive DNA prob specific for identification L. monocytogenes at the isolation and purification cultures
steps are available. These methods are highly recommended but with the indicated provisos.

• Accuprobe™ (Gen-Probe.Inc; Sen Diego, CA) or the L.monocytogenes assay (Gene Trak) is
recommended.
Purified isolates identified as L.monocytogenes should be retained for regulatory reference.

***************
VIBRIO CHOLERAE

Transfer 25g Sample to 225 ml APW

 BASED ON USFDA BAM 8th Edition

Incubate it for 18 hrs. at 37ºC Transfer four loopful into

10 ml. APW tube.

Streak one loopful of Surface

Growth on TCBS agar Incubate for 6 hrs. at 37ºC

Incubate for 18 hrs. at 37ºC

Observe for suspected V.C. colonies.

(Yellow flat smooth colonies with opaque centre and translucent peripheries having 2-3mm diameter)

Transfer 2-3 suspected colonies by streaking slant & stabbing butt into

Triple Sugar Iron Agar Kligler’s Iron Agar

(TSI) (KIA)

Observe the tubes for Acid Observe the tubes for Acid (yellow)
(Yellow) slant & Acid (yellow) butt and alkaline (red) slant. Neither
Butt neither gas nor H2S (blackening) gas nor H2S (blackening)
(Suspected reaction for V.C.) (Suspected reaction for V.C.)

Proceed for biochemical and

Serological tests.

S. No. Test Characteristic of V.C.

1. Motility Motile
2. Oxidase test +ve
3. Fermentation of Carbohydrates
 BASED ON USFDA BAM 8th Edition
a) Glucose Acid , no gas
b) Sucrose Acid , no gas
c) Mannitol Acid , no gas
d) Inositol Acid , no gas
e) Arebinose Acid , no gas
4. Amino Acid Decarboxylation
a) Lysine decarboxylation +ve
b) Ornithine decarboxylation +ve
c) Arginine dehydrolation -ve

• Serological test with Polyvalent O antiserum.

• If suspected, confirm by Ogawa, Inaba & Hikojima.

*************

VIBRIO PARAHAEMOLYTICUS

Transfer 25 g Sample to 225ml. of GSTB or APSW

Incubate at 37ºC for 18hrs.

 BASED ON USFDA BAM 8th Edition

Streak one loopful to TCBS agar

Observe for suspected V.P. colonies

(Round, 2-3 mm diameter with blue or green centre)

Transfer 2-3 suspected colonies to TSI agar by streaking slant and stabbing butt

Incubate for 24hrs. at 37ºC

Acid (Yellow) butt and Alkaline (red) with no production of gas &
H2S slant (blackening) will suspected reaction for V.P.

Proceed for biochemical tests

* Kanagawa Test:-

Take one pre dried Wagestuma Agar Plate

Spot several loopful of culture on the same plate in circular manner

Incubate for 24hrs. at 37ºC

Clear transparent zone around the colonies indicates +ve test

S. No. Test Characteristic of V.P.

1. Gram Staining Gram –ve , rods
2. Motility Motile
3. Oxidase +ve
 BASED ON USFDA BAM 8th Edition
4. Indole +ve
5. V.P. -ve
6. Fermentation of carbohydrates
a) Glucose (Acid) +ve
b) Sucrose (Acid) -ve
c) Mannitol (Acid) +ve
d) Arabinose (Acid) +ve
e) Inositol (Acid) -ve
7. Amino Acid decarboxylation
a) Lysine decarboxylation +ve
b) Ornithine decarboxylation +ve
c) Arginine dehydrolase -ve
8. Growth in Nacl
a) 0% -ve
b) 3% +ve
c) 6% +ve
d) 8% +ve
e) 10% -ve
 BASED ON USFDA BAM 8th Edition

****************

E.coli, Coliform test for food Samples by MPN - 3 tube Method

90 ml. NS + 10 gm. given Sample 9 ml. NS + 1 ml. of 10-1 Sol.

(10-1) (10-2)

1 ml

10 ml 1 ml
10 ml. Single Strength
MacConkey Broth (3-tubes)
10 ml. Double 10 ml. Single Strength
Strength MacConkey MacConkey Broth
(3-tubes) (3-tubes)

Incubate for 24 hrs. at 37ºC

Observe for Acid (Yellow color) & gas production

Note result as number of +ve tube in each set of tubes.

 BASED ON USFDA BAM 8th Edition
Compare with corresponding MPN Table

Inoculate one loopful from each Report as Presumptive Total Coliform/ gm.
+ve tube to BGLB 2% broth &
Mark the corresponding level.

Incubate for 24 hrs. at 37ºC

Observe for growth & gas production

Note result as number of +ve tube in each set of tubes.

Compare with corresponding MPN Table

Report as confirmed Total Coliform/gm.

Transfer one loopful to

EC Broth Tryptone Broth

Incubate at 44.5±0.5ºC Incubate at 44.5 ±0.5ºC for 24 hrs.

for 24 hrs.

Add 4-drops of Kovac’s

Observe for growth & gas production Indole reagent.

Note result as number of +ve tube Pink color at top shows +ve
in each set of tubes. Indole test

Compare with corresponding Note result as number of +ve tube

MPN-tube in each set of tubes.

Report as Faecal Coliform / gm. compare with corresponding MPN Table.

 BASED ON USFDA BAM 8th Edition

Report as E.coli/ gm.

*************
E.coli, Coliform test for Water Sample by MPN- 5 tube Method

50 ml. Water Sample 10 ml. Water Sample 1 ml. Water Sample

50 ml. Double 10 ml. Double Strength (DS) 10 ml. Single Strength (SS)
Strength (DS) MacConkey MacConkey Broth MacConkey Broth
Broth Flask (1) tube (5) tube (5)

Incubate for 24 hrs. at 37ºC

Observe for Acid (Yellow color) & gas production

Note result as number of +ve tube in each set of tubes.

Compare with corresponding MPN Table

 BASED ON USFDA BAM 8th Edition

Inoculate one loopful from each Report as Presumptive Total Coliform/ 100 ml.
+ve tube to BGLB 2% broth &
Mark the corresponding level.

Incubate for 24 hrs. at 37ºC

Observe for growth & gas production

Note result as number of +ve tube in each set of tubes.

Compare with corresponding MPN Table

Report as confirmed Total Coliform/ 100 ml.

Transfer one loopful to

EC Broth Tryptone Broth

Incubate at 44.5±0.5ºC Incubate at 44.5 ±0.5ºC for 24 hrs.

for 24 hrs.

Add 4-drops of Kovac’s

Observe for growth & gas production Indole reagent.

Note result as number of +ve tube Pink color at top shows +ve
in each set of tubes. Indole test

Compare with corresponding Note result as number of +ve tube

MPN-tube in each set of tubes.
 BASED ON USFDA BAM 8th Edition
Report as Faecal Coliform / 100 ml. compare with corresponding MPN Table.

Report as E.coli/ 100 ml.

*************

Evaluation of Bacterial Load of given surface by Swab Method

Take one template having inner area of 25 cm2

Sterilize it by blue flame and placed in on proposed surface

By the help of one sterilized Swab stick, swab the area under the template at right angles

Place the swab stick in a conical flask containing 100 ml of sterilized buffer solution

Pipette 1 ml solution (above) in duplicate Petri plates

Pour pre-molten, cooled TGBEA agar

Allow to set

Invert the plate & incubate at 37ºC for 48 hrs.

Count the number of colonies in Plates

 BASED ON USFDA BAM 8th Edition

Report as No. of colonies / cm2

No. of Colonies / cm2 = Av. No. of colonies in plates x 4 *

Where, * = 100/25, 100 = dilution factor

25 = area of swabbing

OR

No. of colonies / cm2 = Av. No. of colonies in plates X dilution factor (100)
Swab Area (25 cm2)

**********

Faecal Streptococci

Take 25 Gms. Of the sample

Macerate and Place it in 225 ml of buffer sol. 10-1 make two more consecutive dil. (10-2 & 10-3)

Place 1 ml of duplicate Petri dishes from last two dil. (10-2 & 10-3)

Take melted & cooled (45ºC) KF Agar (add 0.5% TTC) pour about 15-20ml. media to each plate

Allow it to solidify

Invert the plate; incubate it for 36-48 hrs. at 37ºC

Count all surface & sub – surface red to pink colonies

(Some will be with a thin white margin) as Faecal Streptococci
 BASED ON USFDA BAM 8th Edition

Report the result as cfu / gm.

Total Faecal Streptococci / gm = Av. Count X Dilution factor.

**********

Total Enterobacteriaceae Count

Take 25 Gms. Of the sample

Macerate and Place it in 225 ml of buffer sol. 10-1 make two more consecutive dil. (10-2 & 10-3)

Place 1 ml of duplicate Petri dishes from last two dil. (10-2 & 10-3)

Take melted & cooled (45ºC) VRBGA (Violet Red Bile Glucose Agar) pour about 15-20ml. media to each plate

Allow it to solidify

Invert the plate; incubate it for 24 hrs. at 37ºC

Count all surface & sub – surface pink-red to light pink colonies as Enterobacteriaceae
 BASED ON USFDA BAM 8th Edition
Report the result as cfu / gm.

Total Enterobacteriaceae Count / gm = Av. Count X Dilution factor.

*************
Sulphite Reducing Clostridia in Food & Salt Sample (Plate Method)

Aseptically wt. 10 gm. of given sample

Macerate & place it in 90 ml. of sterile NS (10-1)

(Shake well)

Make two more consecutive dilutions (10-2 & 10-3)

Pipette 1 ml from each dil. In duplicate Petri dishes

Pour molten & cooled DRCM agar

Allow to set the media

Incubate the plates in inverted position in an anaerobic jar at 37ºC for 48 hrs.
(Follow the procedure of anaerobiosis in the anaerobic jar)

Observe the plates for Black colonies.

 BASED ON USFDA BAM 8th Edition

If there is no black colony, incubate the plates for next 48 hrs.

Observe the plates for black colonies

Count the black colonies as Sulphite Reducing Clostridia

Report as Sulphite Reducing Clostridia / gm.

Sulphite Reducing Clostridia /gm = Av. Count X dil. Factor.

*****************

Sulphite Reducing Clostridia in Food & Salt Samples (MPN – 3 tubes Method)

90 ml NS + 10 gm given Sample 9 ml NS + 1 ml of 10-1 sol.

(10-1) (10-2)

10 ml 10 ml 1 ml

90 ml SS DRCM 10 ml SS DRCM 10 ml SS DRCM

Flask (3) Tubes (3) tube (3)

Pour sterile paraffin oil on the top of the media to prevent contact
with the air incubate at 37ºC in a Serological water bath

Observe for blackening of the media after 48 hrs.

If blackening is not found, incubate it for further 48 hrs.

 BASED ON USFDA BAM 8th Edition

Observe for blackening of the media

Note number of +ve tube in each set of tube

Determine the Sulphite Reducing Clostridia in sample by comparing

the reading with corresponding MPN table.

Report as Sulphite Reducing Clostridia / gm.

********

Sulphite Reducing Clostridia in Water Samples (MPN – 5 tubes Method)

50 ml Water Sample 10 ml Water Sample 1 ml Water Sample

50 ml DS DRCM 10 ml DS DRCM 10 ml SS DRCM

Flask (1) Tubes (5) tube (5)

Pour sterile paraffin oil on the top of the media to prevent contact
with the air, incubate at 37ºC in a Serological water bath

Observe for blackening of the media after 48 hrs.

If blackening is not found, incubate it for further 48 hrs.

Observe for blackening of the media

Note number of +ve tube in each set of tube

 BASED ON USFDA BAM 8th Edition

Determine the Sulphite Reducing Clostridia in sample by comparing

the reading with corresponding MPN table.

Report as Sulphite Reducing Clostridia / 100 ml.

********

GRAM STAINING

Take a oil – free, dust – free clean microscopic slide

Put a drop of Distilled water in the middle of the slide

Emulsify a spec of young culture with the water drop on slide

Dry in air

Fix the slide by passing 3-4 times through blue flame

Place the slide on staining bridge

Flood the smear with Gram’s Crystal Violet for 1 mint.

Wash with water

Flood with Gram’s Iodine for 1 mint.

 BASED ON USFDA BAM 8th Edition
Destin with drop wise addition of Alcohol until washings are free from Violet color

Counter stain with Safranine for 1 mint.

Dry in air

Observe the slide under microscope using oil immersion objective

Cells stained with Cells Stained with Red

Violet, Bluish violet
Or bluish purple

Gram -ve
Gram Positive

*****

MOTILITY TEST

Hanging Drop Method Plain Slide Method

Place a small drop of Place a small drop of Distilled

Distilled water on the water on a plain microscopic slide
Middle of a cover slip

Emulsify a spec of young Emulsify a spec of young culture

Culture with the water droplet with the water droplet

Take a cavity slide

Place a cover slip on the surface

Smear the margin of cavity

Slide with little paraffin jelly
 BASED ON USFDA BAM 8th Edition
Invert the slide on the
Cover slip

Now drop will hang in cavity Observe under Microscope

*******

CATALASE TEST

Take a clean Microscopic slide

Place a drop of distilled water on the slide

Emulsify it with the young culture

Take out 30% H2O2 from refrigerator (allow it to attain ambient temperature)

Flood the smear with 2 drop of 30% H2O2

Observe for result

Evolution of gas Gas not produced from

 BASED ON USFDA BAM 8th Edition
from the culture the culture

+ve Catalase test -ve Catalase test

******

CYTOCHROME OXIDASE TEST

Take one cytochrome oxidase test paper

Smear a little amount of the young culture on the paper

Observe for result

Development of blue color Blue color not developed

On the paper in few seconds on the paper

Cytochrome oxidase + ve. Cytochrome Oxidase –ve.

 BASED ON USFDA BAM 8th Edition

*******

HUGH & LEIFSON’S

Oxidative Verses Fermentative Reaction Test

Take a slant of H & L Glucose O/F Medium

Stab the culture using platinum niddle in such a way that

at least 2 cm long butt should be uninnoculated

Incubate for 18-24 hrs.

Observe for changes.

S.No. Observation Due to Result

1. Growth of bacteria along the line of Fermentative with Acid Fermentative
inoculation and yellow colour throughout but no gas (FANG)
the medium.
 BASED ON USFDA BAM 8th Edition
2. Yellow colours throughout the medium Fermentative with Acid Fermentative
and gas bubbles are also trapped in the & gas (FAG)
medium.
3. Yellow colour only at the top of the Acid production only Oxidative Non-
medium. at top area Fermentative

4. Deep pink colour only near the top area Increase in PH Alkaline Non-
Fermentative.

******

PREPARATION OF CYTOCHROME OXIDASE PAPER

Take 100 mg of N: N: N: N: Tetramethyl-P-Phenylene diamine

hydrochloride or, N: N: Dimethyl Paraphenylene diamine hydrochloride

Add 10 ml of Distilled Water to it.

Impregnate filter paper with the reagent and drain

Keep test paper in refrigerator away from light.

******

PENICILLIN SENSITIVITY TEST

Take one Pre-set, dried & cooled to RT Antibiotic Agar Plate

Smear a little of the culture over the agar plate on about 4 cm2
 BASED ON USFDA BAM 8th Edition

Place a 2.5 IU Penicillin disc on the surface of the Smear

Incubate the plate without inverting for 18-24hrs.

Observe for Result.

Clear zone of inhibition Clear zone of inhibition are not

are seen around the Smear seen around the smear

Sensitive to 2.5 IU Penicillin. Not Sensitive to 2.5 IU Penicillin.

*******

PREPARATION OF 2.5 IU PENICILLIN DISK

Take Whitman no. 1 filter paper

Cut into several pieces using standard paper pouch (Diameter 5 mm)

Keep small paper disks in one Petri dish & sterilize in hot air oven at 140ºC for 2 hrs.

Procure Penicillin G-sodium (Benzyl Penicillin) vial

Aseptically weigh 30 mg. into a sterile conical flask

Place 100 ml Sterilized Distilled water

Shake well to dissolve

Keep in refrigerator at 5-8ºC

 BASED ON USFDA BAM 8th Edition

When needed, pour few ml. to a sterile test tube

Dip each filter paper disc in the penicillin solution

Drain by pressing against the wall of the test tube

Each paper disk will contain 2.5 IU Penicillin

Use paper same day of dipping.

******

ESTIMATION OF SULPHUR DIOXIDE

Take 25 Gms of sample, made it into a fine paste using 200 ml of water. Connect into the apparatus
ensuring that all joints greased and tight when clamped. The outlet of the apparatus is kept immersed into 100 ml of
distilled water in a 250 ml conical flask. The conical should be placed into a beaker with chilled water and ice.
Then transfer the prepared sample into the round bottom flask and add 25 ml. of concentrated HCI. Immediate
connect –up the apparatus, switch – on the heating mantle to full heat, the liquid boils turns down the heat to
prevent frothing-up of the contents into the flask. Continue to boil till 5 minter until the first drop of the water
collects in the flask. Then switch off the heater and disconnect the conical flask and add 1 ml. of starch indicator
and titrate against 0.005N Iodine up to end point.

Calculation:-

Sulpher dioxide = Sample Titrate value X N X 32 X 100

Weight of the sample taken in Gms.
 BASED ON USFDA BAM 8th Edition
Where, N = Normality of titer Iodine solution.

32 =Atomic weight of Sulpher

*******

SALT RESIDUE TEST

Chemical Required:-

1. Burette Solution: - N/10 Silver Nitrate 16.987 gms. of AgNO3 in 1 lit. of water or 1.699 gms. of Ag NO3
In 100 ml of water.

2. Indicator Solution: - 5% Potassium chromate 50 gms of K2CrO4 in 1 lit. of water or 5 gms of K2CrO4 in
95 ml of water.

Procedure:-

Take 10 gms of sample in 90 ml of distilled water.

Take 50 ml of supernatant solution

Add 1 ml of Indicator Solution

Titrate it until the yellow colour changes to Orange (End Point)

 BASED ON USFDA BAM 8th Edition

Read T.V. & Calculate

Calculation:-

% of salt residue in sample = T.V. X N X 1.168

Where, T.V. = Titre Value

N = Normality of Burette Solution

1.168 = Conversion factor

************