Snehalaxmi Choudhury

SABRI FOOD PRODUCTS PRIVATE LIMITED

 HACCP PROCEDURES AND MICROBIOLOGICAL TESTING

HACCP: HAZARD ANALYSIS CRITICAL CONTROL POINT

A system that identify, evaluates and control hazards which are significant for food safety.

HAZARD

A biological, physical or chemical agent as condition of food with the potential to cause an adverse
effect is called hazard.

HAZARD ANALYSIS

A process of collecting and evaluating information on hazards and condition leading to their presence
in order to decide which are significant for food safety and therefore should be adjusted in HACCP
plan.

HACCP PLAN

A document of food processing unit which spells out significant hazards, there critical control points,
the controls, monitoring, inspection procedure and record keeping in the approved unit to produce
hazard free food product.

CCP: Critical Control Point

A step at which control can be applied which are essential to prevent or eliminate a food safety hazard
or reduce it to an acceptable limit.

HAZARDS ARE DIFFERENT TYPES

1. Biological Hazard
2. Chemical Hazard
3. Physical Hazard

Or it can be

1. Process specific any type of foreign particles

2. Product specific like Histamine found in Tuna fish

LIST OF MEDIAS
 1. PLATE COUNT AGAR
2. HECTOEN ENTERIC AGAR
3. TERGITOL 7 AGAR
4. LACTOSE BROTH
5. THIOSULPHATE CITRATE BILE SALT
6. ALKALINE PEPTONE WATER
7. MCKONKEY BROTH
8. XYLOSE LYSINE DEOXYCHOLATE AGAR
9. BAIRD PARKER AGAR
10. RAPPAPORT VASSILIADIS MEDIUM
11. BRILIAN GREEN BILE BROTH
12. BISMUTH SULPHITE AGAR

CULTURE MEDIUM

It is a mixture of chemicals that can support the growth of microorganism (contains a source of
carbon and energy for organisms to grow)

AGAR

A complex sulphated polysaccharide visually extracted from red algae that is used as solidifying agent
in preparation of culture media.

STERILIZATION

A process of removing or killing all microorganism (bacterial spore) or material or object.

DISINFECTION

A process of sufficiently reducing the no. of pathogenic organism (not bacterial spore) so that they no
longer represent a hazard.

MICROBIOLOGICAL TESTING

1. Salmonella
2. Vibrio cholerae
3. Vibrio Parahaemolyticus
4. Total Plate Count
5. Listeria

CALCULATIONS

Total RM BLACK TIGER/ SEA TIGER/SEA WHITE/BAMBOO/FLOWER/ CHILKA WHITE :

Total MCS *PACKING/65%
Total RM VANNAMEI = Total MCS *PACKING/70%
Total MCS VANNAMEI= Total RM /PACKING*70%
Sampling = √Total No Master Cartons + 1

2
SSOP SANITARY STANDARD OPERATING PROCEDURE

1. Safety of water and ice.

2. Condition and cleanliness of food contact surfaces.
3. Prevention of cross contamination.
4. Maintenance of hand washing, hand sanitizing and toilet facilities.
5. Protection of food, food packing material and food contact surfaces from adulteration.
6. Labelling, storage and use of toxic compounds.
7. Employee health condition.
8. Exclusion of pests.

HACCP HAZARD ANALYSIS CRITICAL CONTROL POINT

Seven Principles of HACCP

1. Conduct hazard analysis and identify preventive measures.

2. Identify critical control point in the process.
3. Establish critical limits.
4. Monitor each CCP.
5. Establish corrective action
6. Establish verification procedure.
7. Establish record keeping and documentation procedure.

CURRENT GOOD MANUFACTURING PRACTICES

1. Personnel
a. Disease control
b. Cleanliness
c. Education and training
2. Plant building and facilities
a. Plant and Grounds
b. Plant construction
3. Sanitary operations
4. Sanitary Facilities and control
5. Equipment and utensils
6. Production, process and control
7. Ware housing and distribution

DECISION TREE

A sequence of questions applied to each process step with an identified hazard to identify which
process are CCPs.
 PROCEDURE FOR MICROBIOLOGICAL TESTS

TOTAL PLATE COUNT

 2.3 gm of PCA was taken in 100ml distilled water

 10gms Shrimp homogenized with 90ml of sterile working buffer (1.34ml stock buffer added to
1000ml distilled water)
 Stock Buffer- 3.4gm Potassium dehydrogenase orthophosphate taken in 50ml of distilled water
and the pH is adjusted to 7.2
 Then 10¯1 dilution is done. Serial dilution is done up to 10¯4.
 Pipette out 10ml from 10¯1 dilution & add 90ml of sterile working buffer to make 10¯2 and
accordingly 10¯4.
 1ml (10gm sample+90 ml buffer) +9ml buffer, and accordingly up to 10¯4
 8 sterile petriplates taken.
 Add 15-18 ml of PCA/TGA to each plate.
 Leave for 30mins (for solidifying)
 Transfer 1ml each from 10¯1 - 10¯4 flask to two sterile petriplates
 Keep plates for incubation at 37°C for 48hrs
 Count Colonies

Calculation of TPC (cfu/gm) =

€colonies

(1 x n1) + (.1 x n2) dilution factor

SALMONELLA

Stage1- 25 gm of sample+225ml of lactose broth

 Incubate at 37°C for 24hrs.

Stage2 -Selective enrichment: 1ml from stage1+ 9ml of medium

(i) Selenite cysteine broth / R.V. (ii) Tetrathionate Broth

Stage 3- Presumptive identification on

 BGA- light pink transparent colonies surrounding media(red)

 HEA- green blue colonies, with or without black centre
 XLDA- red colonies, black centre
 BSA- Black colonies, silvery white metallic

Streak loopful from enrichment broth and stab on the BSA, XLDA, HEA, BSA plates.

Incubate at 37°C.
VIBRIOCHOLERAE

 25gm of Sample and homogenized with 225ml of alkaline peptone water (APW) {pH=8.8}
 Incubate at 37°C.
 After 1618 hours
 After 6 hours takeout the flask without shaking.
 Pick up the growth on the surface layer and streak on TCBS agar.
 Incubate at 37°C for 24 hours.
 Yellow colonies are seen in the plate.

VIBRIO PARAHAEMOLYTICUS

 25 gm of sample was homogenized with 225 ml APW having 3% NaCl.

 Incubate at 37°C for 24 hours.
 Streak on TCBS agar.
 Green colonies are visible.

E.Coli

 10 gm of Sample taken with 90ml of stock buffer

 100ml Tergitol7 agar is prepared.
 Sterilize at 115°C for 20min
 Before plating, add 0.25ml of 1% solution of sterile Triphenyl Tetrazolium Chloride
 Dried it for 30-45mins.
 0.5ml of diluent is pipetted onto the respective agar plates and plating is done by spread plate
method using sterile bent glass rod.
 Incubate at 37°C for 18-24hrs
 Observation: - lime yellow, sometime with rust brown center and yellow zone around.
STAPHYLOCOCCUS AUREUS TEST:

 Sterile 100ml of BPA medium cooled to 50°C.

 Before inoculation, add 1ml of sterile 1%potassium tellurite followed by 5ml of 50% egg yolk
emulsion.
 Mix well, poured in to sterile petriplates.
 Allowed to set and cool to room temperature.
 0.5ml of each dilution is pipetted into respective marked petriplates. Plating is done by spread
plate method.
 Incubate at 37°C for 36-48hrs.

SOP FOR ANTIBIOTCS

 SAMPLE EXTRACTION PROCEDURE FOR AOZ, AMOZ, SEM, AHD

CHEMICAL PREPARATION:

 Preparation of 1X sample extraction buffer: mix 1 volume of 10 x sample extraction buffer

with 9 volumes of distilled water.
 DIPOTASIUM HYDROGEN PHOSPHATE: 0.4gm of K2HPO4 and 100 ml of distilled water.
 Sodium hydroxide (NaOH):4 gm NaOH and 100 ml distilled water
 HCl: 8.3 ml of HCl and 91.7 ml distilled water
 Preparation of 1x wash solution: mix 1 volume of 20x wash buffer concentrate with 19
volume of distilled water.

SAMPLE EXTRACTION FOR NITROFURANS:

1 gm of composite sample homogenized with 0.5 ml of 1X sample extraction buffer
 Add 3.5 ml of distilled water, 0.5 ml of 1 M HCL & 20µl of 50Mm 2-Nitrobenzaldehyde to it and vortex for
30 sec.

Incubate samples for 3 hours at 600c. Vortex the samples for 30 seconds every hour during the incubation

After incubation to each tube add 5 ml of 100mM K2HPO4, 0.4 ml of 1M NaOH and exactly 6.0 ml of ethyl
acetate. Vortex the sample for at least 30 seconds

Centrifuge the sample for 10 minutes at 4500 rpm

Transfer exactly 3.0 ml of the upper layer supernatant to a new tube for drying

Dry the samples by slowly blowing nitrogen gas over it in a 600 o C water bath for about 25 minutes at 5-10
psi pressure

Dried residue is dissolved in 1 ml of n- hexane & vortex for 30 seconds

Add exactly 1.0 ml of 1X sample extraction buffer, vortex for 30 seconds

Centrifuge the samples for 10 minutes at 4500 rpm

Completely remove the lower aqueous layer & 100µl is used for assay.

SAMPLE ASSAY PROCDURE:

100µl standards of each (AOZ/ AMOZ/ SEM/ AHD) are added to the plate in the order from lower
concentration to higher concentration.
 100µl of each sample in duplicate into different sample wells

50 µl of HRP conjugate (AOZ/ AMOZ/ SEM/ AHD) and mix well by gently rocking the plate manually for 1
minute

Incubate the plate for 30 minutes

Wash the plate 3 times with 250 µl of 1X wash solution. After the last wash, invert the plate and gently tap
the plate dry on tissue.

Add 100µl of TMB substrate. Incubate the plate 20 minutes at temperature (20-25 0c) in the dark.

After incubation add 100µl of stop buffer to stop the enzyme reaction

Read the plate as soon as possible following the addition of stop buffer on a plate reader with 450nm
wavelength.

SAMPLE EXTRACTION FOR CHLORAMOPHENICOL

 Weight 3 gm of the homogenized sample into conical tubes

Incubate sample at 500 c for 30 minutes

Add 6.0ml of ethyl acetate to it

Vortex the sample for 5 minutes

Centrifuge it for 10 minutes at 4500 rpm

Transfer exactly 4.0 ml of supernatant in to a 15 ml test tube and dry it

Dissolve the dried residue in 2 ml of n-hexane.

Add 1 ml of 1x sample extraction buffer and mix by vortex for 1 minute

Centrifuge for 10 minutes at 4500rpm at room temperature

100µl of the lower aqueous layer is used for assay

SAMPLE ASSAY FOR PROCEDURE FOR CHLORAMPHENICOL:

100µl of each CAP standards in duplicate into different wells from lower to higher concentration
 100µl of each sample in duplicate into different sample wells

50 µl of CAP- HRP conjugate and mix well by gently rocking the plate manually

Incubate the plate for 30 minutes at room temperature

Wash the plate 3 times with 250 µl of 1X wash solution. After the last wash, invert the plate and gently tap
the plate dry on tissue.

Add 100µl of TMB substrate. Incubate the plate 20 minutes at temperature (20-25 0c) in the dark.

After incubation add 100µl of stop buffer to stop the enzyme reaction

Read the plate as soon as possible following the addition of stop buffer on a plate reader with 450 nm
wavelength.

STANDARDS CONCENTRATION DILUTION CUT OFF DETECTION LIMIT

FACTOR VALUE µgm/kg /ppb
AOZ 0 2 0.8 0.05

0.02
 0.06
0.18
0.54
1.62
AMOZ 0 2 0.8 0.05

0.025
0.1
0.4
1.6
6.4
SEM 0 2 0.8 0.05

0.025
0.1
0.4
1.6
6.4
AHD 0 2 0.8 0.05

0.025
0.1
0.4
1.6
6.4
CAP 0 0.5 0.2 0.025

0.05
0.15
0.5
1.5
4.5

SOP FOR MICROBIOLOGY (water & ice)

Microbiological parameters are tested for swabs from food contact surfaces, swabs from
worker’s hand, Water & ice samples

1. Total Plate Count

2. MPN for Coliform

3. Vibrio cholerae

Ref: USFDA Bacteriological Analytical Manual

1. TOTAL PLATE COUNT

Aerobic plate count (APC) or total plate count (TPC) is intended to indicate the level of microorganisms in
product or water & Ice or on surface. pour plate and spread plate methods are used to determine APC.

Media required:
1. PCA
2. PHOSPHATE BUFFER

Collection of water sample

a) Sterile bottle/Flask
b) Select the tap.
c) Wipe the tap opening with spirit.
d) Flame it spirit lamp.
e) Leave the tap running for a few minutes.
f) Collect water sterile bottle/flash.

PROCEDURE
Take 4 sterile Petri plates

Transfer 1ml of water sample to each Petri plates

Add approximately 15-18ml of plate count agar (cooled to 40-45 0c each plate)

Immediately mix water sample and agar medium thoroughly and uniformly by alternate
rotation and back-and –forth motion of plates on flat level surface.

Let agar solidify, invert solidified plates

Incubate 2 plates at 370c for 48 hours and other 2 plates at 220c for 48 hours

After incubation the colonies are counted.

RESULT & CALCULATION:

TPC (cfu/ml): Average count

LIMIT:
TPC at 370c: 20cfu/ml

TPC at 220c: 100cfu/ml

Swab sample:
Prepare sterile swabs (swab should be made with absorbent cotton only). Use a sterile template
(inner open area 25 cm2).
PROCEDURE:
Take a flask containing 100ml of sterile diluents (phosphate buffer)
 Dip the swab in the diluents. Drain excess liquid by pressing the swab to the inner walls of
the flask.

Using the moist swab, thoroughly swab 25 cm2 of the surface (worker’s hand, freezing tray,
processing tables or crate).

Place the swab back in the original diluent’s flask, mix thoroughly.

Using a sterile pipette transfer 1ml into two plates.

Add approximately 15ml of PCA (cooled to 40-450c) to each plate.

Immediately mix sample and agar medium thoroughly and uniformly by alternate rotation and
back-and-forth motion of plates on flat level surface.

Let agar solidify, invert solidified plates and incubate plates at 37 0c for 48 hours.

RESULT & CALCULATION:

TPC (cfu/sq. cm): Average count x 100

25
LIMIT:

TPC at 370c: 100 cfu/sq. cm

2. MPN METHOD FOR total COLIFORM and faecal

coliforms in water & ice sample
MPN method for water is a three-step process and all the media used are liquid media.

Media required:
1. Double strength MacConkey purple broth (one flask with 50ml broth and 5 large test tubes with 10ml
broth)
2. Single strength MacConkey purple broth (5 test tubes with 10 ml broth)

STEP I-TEST FOR PRESUMPTIVE TOTAL COLIFORMS

PROCEDURE:

50ml of water sample is added to the flask containing 50ml of double strength MacConkey purple
broth.

10ml of water sample is added to each of the 5 large tubes containing 10ml of double strength
MacConkey purple broth.

1ml of water sample is added to each of the 5 test tubes containing 10ml of single strength
MacConkey purple broth. Label appropriately (either as 50ml or 10ml or 1ml).

Incubate at 370c for 24hours.

After 24h observe the flask and tubes for acid and gas production.

If the colour of MacConkey broth has turned to yellow with gas bubbles in Durham’s tube the
reaction is noted as positive.

Note: the result as number of positives in each set of 50ml, 10ml &1ml tubes. Compare the result
with MPN table (5tube MPN table for water samples) and note the result as MPN presumptive
TOTAL COLIFORMS per 100ml of water.
(Note: incubate all negative tubes for an additional 24h)

STEP II-TEST FOR CONFIRMED TOTAL COLIFORMS

Media required:

1. Brilliant green lactose bile (BGLB 2%) broth.

 PROCEDURE:

Inoculate one loopful of culture from the positive tubes of step I to BGLB 2% broth and mark the
corresponding label (either 50ml or 10ml or 1ml).

Incubate at 370c for 24hours

after 24hour note growth and gas production.

Note: results as positive if there is growth and gas production. Compare with 5 tubes MPN table for
water samples and give the result as MPN CONFIRMED TOTAL COLIFORMS per 100ml of water .

(Note: incubate all negative tubes for an additional 24h)

STEP III-TEST FOR FAECAL COLIFORMS

Media required:

1. EC Broth.

PROCEDURE:
 Inoculate one loopful of culture from the positive tubes of step II t (BGLB 2%) to EC broth and mark
the corresponding label (either 50ml or 10ml or 1ml).

Incubate at 45.50c for 24hours

after 24hour note growth and gas production.

Note: results as positive if there is growth and gas production. Compare with 5 tubes MPN table for
water samples and give the result as MPN FAECAL COLIFORMS per 100ml of water .

(Note: incubate all negative tubes for an additional 24h)

3. VIBRIO CHOLERAE isolation procedure for water and ice

samples
Collection of Water sample:
a) water sample should collect from taps (tap number shall mention in the sample
covering note) in a sterile bottle / conical flask of 1 litre capacity.
 b) Flasks are sterilized at 1600c for 1 hour after being covered by Kraft paper. The opening
and closing of the sterile bottle must be done with meticulous care to avoid any
contamination.
c) When water sample is drawn from a tap, flame the tip of the tap using spirit and allow
water to flow for 5 minutes before collection.

Collection of Ice sample:

A minimum of 1kg. of ice used for processing shall collect aseptically in a sterile stainless-steel
container and transported to the laboratory. If there is considerable delay from the time from drawl
of samples and actual analysis (more than 4 hrs), the sample shall be kept in cool condition.

Membrane filter technique:

Filter 300ml of sample water through a 0.22 membrane (Millipore) filter paper.

Place the filter paper in 100ml of APW in a sterile conical flask and incubate at 35 0c to 370c for
overnight.

after incubation a loopful of culture from APW is streak onto TCBS agar plate and icubate the plates
for 18 to 24 hours at 350 c to 370c for yield isolated colonies round ,opeque, yellow ,2 to 3 mm in
diameter.

Select single isolated typical colonies for biochemical and serological test for confirmation.

SOME IMPORTANT POINTS

1. Bacteria are prokaryotic microorganisms that they don’t contain chlorophyll. Size- 0.2
to1.5 micrometre in diameter and 3 to 5 micrometres in length. It is unicellular.
2. Sterilization- it is a process of removing or killing all microorganism on or in a material
or object.
3. Disinfection – it is a process of sufficiently reducing the no. of pathogenic
microorganism (not bacterial spore) so that they no longer represent a hazard.
4. Culture medium- it is a mixture of chemicals that is able to support the growth of
microorganism. (Contain a source of carbon and energy for organism to be grown.)
5. Agar – it is a complex sulphated polysaccharide, usually extracted from red algae i.e.
used as a solidifying agent in preparation of culture media.
6. Coliform bacteria (gram -ve), non-spore forming bacteria. Found in aquatic
environment, soil. It is in large no in faeces of warm blooded animal.
7. Salmonella (gram -ve), rod shaped, motile enterobacteria.
8. E. coli (gram -ve), rod shaped, facultative anaerobic.
9. Vibrio cholerae (gram -ve), facultative, comma shaped.
10. Vibrio parahaemolyticus (gram -ve), rod shaped, found in salt water.
11. Ppm- parts per million
12. Hot air oven- dry heat sterilization
13. Laminar air flow- sterile air environment
14. Micro pipette- pick up fluid volume
15. Autoclave- steam sterilization
16. Quality control- it is a part of GMP which is concerned with sampling specifications,
testing and within the organization, documentation and release procedures which
ensure that the necessary and relevant test are carried out. It is a lab based.
17. Quality assurance- it is a company based.
18. QA is sum of organized arrangements made with the object of ensuring that the
product will be of the quality reqd. by their intended use.
19. MPN- most probable no technique.