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Summer Internship – Microbiological Assessment Of Pathogens Present In Airline Food

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TajSATS Air Catering Ltd. is a joint venture of the Indian Hotels Company, popularly known as the Taj Hotels Resorts and Palaces, and SATS (formerly known as Singapore Airport Terminal Services).

The Taj Hotels Resorts and Palaces is part of the Tata Group, India’s largest business conglomerate. The Tata Group has 98 companies spread across 80 countries in 6 continents.

SATS, is a leader in airline catering and ground handling services in Asia.

TajSATS is the market leader in airline catering. The company provides in-flight catering at Mumbai, Delhi, Chennai, Kolkata, Amritsar, Goa and Bangalore. Taj Madras Flight Kitchen is a joint venture of the Indian Hotels, SATS and Malaysian Airlines.

All these facilities are equipped with state-of-the-art technology and advanced kitchen equipment for efficient and hygienic food production and handling.

At the heart of their offering is a commitment to be ‘One with the Customer’. They are focused on creating affinity with their customers every single day. They believe in strengthening this relationship by delighting the customer in every interaction.

TajSATS is also looking at future expansion in select cities. They have kept pace with the developments that have taken place in the Indian aviation industry and are geared to face whatever the future brings. While catering to the ever increasing needs of their demanding business, they have simultaneously expanded from an Indian catering service to an International one.

SERVICES

Airline Services

i. Vistara(UK)

ii. Jet Airways(9W)

iii. Air India(AI)

iv. Indigo(6E)

v. Air India (Domestic IC)

vi. Singapore Airlines(SQ)

vii. Malaysia Airlines(MH)

viii. China Eastern Airlines(MU)

ix. All Nippon Airlines(ANA)

x. Bhutan Airlines

xi. Druk Air

xii. Air Astana(KC)

xiii. Korean Air

xiv. Japan Airlines

Non-Airline Services

i. TATA Starbucks

ii. 24SEVEN

MANUFACTURING PROCESS

1. HACCP PLAN

CRITICAL CONTROL POINTS

OPERATIONAL PREREQUISITES PROGRAMS

VARIOUS WORK STATIONS INSIDE THE PREMISES

i. Bakery Section

ii. Confectionary Room

iii. Decantation Room

iv. Egg Breaking Room

v. Seafood Section

vi. Butchery Shop

vii. Walk In Chiller Room

viii. Storage (Main)

ix. Hot Kitchen

x. Cold Kitchen

QUALITY TEST

1. TYPES OF FOOD ANALYSED IN MICROBIOLOGY LAB FOR CONTAMINATION

Type I – Cooked Food

Type II – Dessert

Type III – Raw and Frozen Vegetables and Fruits

Type IV – Vaccum Packed Items, Smoked Salmon and Fish

Type V – Dairy Product (Cheese and Cream)

Type VI – Raw Meat and Fish

2. MICROBIOLOGY TESTING OF FOODS

Many flights kitchen have inhouse laboratories for quality control and quality assurance purposes. Others will sub-contract such work to other agencies. The laboratories will sample incoming suppliers to ensure that they meet specifications, and complete products to verify that hygiene management systems and production processes are operating correctly. Periodic environmental sampling may also be undertaken to ensure that personal hygiene, cleaning and sanitation regimes are effective.

Items will be sampled, meaning that not every item will be tested, but just a representative number. The propotions of items tested, and the frequency of testing, will depend on the severity of the risk posed by the micro-organism of interest or the process. Sampling should, however, cover all times of the day, as problems could be unique to the actions of a particular operator, which could be missed if samples are taken routinely at one time in the day. Care must be taken when taking samples (including methods of taking samples and lablling of samples) and,where is it necessary, in the transportation of samplesto the laboratory (eg. Temperature control).

Records of testing results over time should be maintained,as these may show a gradual deviation towards unacceptable levels of micro-organisms. This allows action to be taken before unacceptable levels of micro-organisms are reached. As mentioned above, it also allows identification of personnel of processes contributing to rise levels of micro-organisms.

Each product testing is important to verify the safety of products and the effectiveness of hygiene management systems. The preffered approach, however, is to ensure the potential hazards are predicted and controlled prior to them causing a problem. The current method of choice to achieve this, is the application of hazard analysis critical control points (HACCP).

VARIOUS EQUIPMENTS IN QA LAB

i. Weigh balance

ii. Water bath

iii. Stomacher

iv. Autoclave

v. Incubator

vi. Hot air oven

vii. Refrigerator

viii. Laminar air flow bench

ix. Microprocessor colony counter

x. Microscope

xi. Infrared thermometer

xii. Probe thermometer

xiii. Glassware dryer

xiv. Centrifuge

xv. Hand refractometer

xvi. Heating mantle

TESTS PERFORMED FOR FOOD PATHOGENS

TPC- TOTAL PLATE COUNT

It is used for the determination of number of microbes in food, water and waste water.

Agar : total plate count agar

Sterilization

a. Sterilize the laminar airflow with 70% ethanol and open UV light.

b. Sterilize your hands with sanitizer.

c. Take all sanitized petri plates from oven after sterilizer at 171 degree celcius for 3-4 hours.

d. Take sterilized spatula from oven.

Media Preperation

Dissolve 4.5g of media in 200ml of distilled water. Sterilize it under autoclave at 121C for 15mins at 15 psi.

Procedure

a. Prepare 90ml Peptone dilution and sterilize by autoclave.

b. Take 10gm of required sample in a sterilize bag.

c. Add the 90ml Peptone Water in the bag carrying sample, homogenize the sample by stomacher.

d. Carry out the serial dilution like 1/10, 1/100, 1/1000 (as per food sample).

e. Now take 1ml of this serial dilution (10-3) by micro pipette or pipette or pour plate technique.

f. Pour 1ml sample in petri plate and then pour 13-15ml of TPC Agar (Note that media temp. should be 45 C).

g. Mix the sample along with media in a petri plate, carefully (media doesn’t touch the top portion of petri plate during mixing).

h. Allow the media to solidify.

i. After solidified place the plate in the inverted position in the incubator for 48hrs at 37C.

j. After incubation period count the bacterial colonies if present.

k. Mention the colony as per the dilution.

COLIFORM TESTING

This is used to test coliform in food, water, milk samples. For selective isolation, detection and enumeration of coli-aerogens bacteria in milk, water and other dairy product.

Agar – Violet red bile agar

Sterilization

e. Sterilize the laminar airflow with 70% ethanol and open UV light.

f. Sterilize your hands with sanitizer.

g. Take all sanitized petri plates from oven after sterilizer at 171 degree celcius for 3-4 hours.

h. Take sanitized spatula from oven.

Media Preperation

Dissolve 8.3g of media in 200ml of distilled water. Sanitize it under autoclave at 121C for 15 mins at 15 psi.

Procedure

l. Prepare 90ml Peptone dilution and sterilize by autoclave.

m. Take 10gm of required sample in a sterilize bag.

n. Add the 90ml Peptone Water in the bag carrying sample, homogenize the sample by stomacher.

o. Carry out the serial dilution like 1/10, 1/100, 1/1000 (as per food sample).

p. Now take 1ml of this serial dilution (10-2) by micro pipette or pipette or pour plate technique.

q. Pour 1ml sample in petri plate and then pour 13-15ml of VRB Agar (Note that media temp. should be 45 C).

r. Mix the sample along with media in a petri plate, carefully (media doesn’t touch the top portion of petri plate during mixing).

s. Allow the media to solidify.

t. After solidified place the plate in the inverted position in the incubator for 24hrs at 37C.

u. After incubation period count the bacterial colonies if present.

v. Mention the colony as per the dilution.

E.COLI TESTING

For selective enumeration and presumption E.Coli by MPN Technique or selective enumeration of fecal and non fecal coli forms in water, wastewater and shell fish.

Broth – E.coli broth

Sterlization

a. Sterilize test tube in oven at 170 degree Celsius for 3-4 hrs.

Media Preperation

Take 7.4g of EC Broth in 200ml of distilled water, pour 9ml of it into 8 test tubes sterilized. Put Durham test tubes inside test tubes. Sterilize it in autoclave at 121 degree Celsius for 15mins. at 15psi.

Procedure

a. Take 10gm of each sample and add 90ml of peptone water and digest it in stomacher.

b. Add 1ml of 1st dilution by micropipette in above sterilized test tubes containing Durham tubes.

c. Incubate the tubes at 41 degree Celsius for 24hrs. Tubes with gas production are considered positive.

d. Streak onr loopful from the positive tube on EMB Agar (Eosin methylene blue agar).

e. Keep in incubator for 24hrs.

f. One colony is taken in Indol test tube.

g. If pink colour appears, show the presence of E.Coli.

DETECTION OF SALMONELLA

Principle

Salmonella when resent are usually found in low numbers in food and often in the presence of considerably large numbers of other members of enterobacteria. In food which has been heated, refrigerated or frozen or dried, visible salmonella bacteria may be present.

This method is based on giving the chance for the few number of normal or stressed salmonella bacteria to grow first in a non selective liquid medium at 37 degree (re-enrichment) such a medium and a temperature will allow other bacteria to grow as well, therefore this step is followed by sub-culturing the re-enrichment medium into a liquid selective medium and incubating it at 42-43C. The latter is inoculated into a solid selective and differential medium, and after incubation at 37C the plates are examined for the presence of colonies, which by their characteristics are considered presumptive salmonella. These colonies are then examined for biochemical characteristics of salmonella species.

Procedure

a. Take 25g of sample.

b. Mix/ blend with 225 ml buffered peptone water.

c. Incubate at 40 degree C for 24-48hrs for enrichment.

d. Mix 10ml with 100ml of TT broth and incubate at 40degree C for 24-48hrs.

e. Add 10mml to RV media (100ml). Incubate overnight at 40 C.

f. Streak on BSA/ BGA/ XLD/ SS Agar and incubate at 37C for 24-48hrs.

g. Transfer positive colonie to TSI agar slant and incubate overnight at 37C.

h. Finally count the colonies.

DETECTION OF BACILLUS CEREUS

Principle

Bacillus cereus is widely distributed in nature and is commonly found in a variety of foods. When B.Cereus grows to high numbers in a food ( .106/g), sufficient enterotoxin may be produced resulting in food borne illness. This method determines the presence of B.Cereus by plating known quantities of dilution of a food sample onto a selective agar. After incubation, presumptive B.Cereus colonies are selected and subjected to comfirmatory testing. From the results obtained, the number of B.Cereus per gram or ml of the food is calculated.

Procedure

a. Take 25g of sample.

b. Add 225ml of buffered peptone water.

c. Blend the mixture at 15000-20000rpm.

d. Prepare the dilution.

e. Spread plating is done on BCA agar.

f. Incubate at 37C for 72hrs.

g. Finally count the colonies.

ENUMERATION OF STAPHYLOCOCCUS AUREUS

Principle

This method is based on the spreading of 0.25ml of the food homogenate and of the subsequent decimal dilution on the surface BPA. This medium contains various inhibitory substance, which do not interfere with the growth of S.Aureus. The ability of S.Aureus to reduce the potassium telluride and to hydrolyze the egg yolk present in the medium results in the appearance of black colonies surrounded by clear zone characteristics for S.Aureus. This coagulates the substance produced by S.Aureus, which clots the plasma of humans and animals.

Procedure

a. Take 25g of sample.

b. Add 225ml of buffered peptone water.

c. Blend the mixture at 15000-20000rpm.

d. Prepare the dilution and pipette out 1ml from test tube onto the BPA agar.

e. Incubate at 37C for 24-48hrs.

f. Finally count the colonies. (Black body surrounded by clear zone)

ENUMERATION OF YEAST AND MOLD

Principle

This method is based on the assumption that the microbial cells present in a simple mix with PDA each from visible separated colonies. This is obtained by mixing decimal dilution of the food sample and homogenate with the medium. After incubation of the plates at 30C for 72hrs, the number of yeast and mold per gram of the food sample is calculated from the number of the colonies obtained in selected petriplate at levels of dilution giving a significant result. It should be borne in mind that this method as all other methods has some limitations. Microbial cells often occur as clumps, clusters, chains or pairs in fruit and may not be well distributed irrespective of the mixing and dilution of the sample

Consequently, each colony that appears on the agar plate may arise from the single cell or from group of cell and hence the colony count may not reflectthe actual number of the viable bacteria in the food. Moreover some micro-organism may fail to grow and form visible colonies on the agar medium as a result of unfavourable conditions of temperature, oxygen or nutrition or because the cells are weak.

Procedure

a. Take 25g of sample.

b. Add 225ml of buffered peptone water.

c. Blend the mixture at 15000-20000rpm.

d. Prepare the dilution and pipette out 1ml from each test tube onto a sterile Petri plates.

e. Pour Rose Bengal Agar into each Petri plate (15ml).

f. Incubate at 37C for 72hrs.

g. Finally count the colonies.

VIBRIO IDENTIFICATION

Procedure

a. Weight 10 grams of the sample in 90 ml alkaline peptone water.

b. Mix using stomacher.

c. Incubate for 36°c for 6 – 8 hours.

d. Plating: Inoculate loopful on TCBS agar by streaking.

e. Incubate plates 37°c for 24 hours.

f. Yellow colonies cofirm the presence of V.Cholerea on TCBS agar.

IDENTIFICATION OF LISTERIA MONOCYTOGENIS

Procedure

a. Take 1g of Frazer in 225ml of distilled water to prepare half frazer solution.

b. Add 25g of food sample and blend in a stomacher.

c. Incubate for 24hrs at 40C.

d. Prepare 10ml of full frazer broth in a test tube.

e. Transfer 1ml of half frazer solution to this test tube.

f. Incubate for 24hrs at 40C.

g. Transfer the colony from the test tube to the Palcom Agar petri plate.

TESTS PERFORMED ON WATER SAMPLES

ESTIMATION OF TOTAL HARDNESS OF WATER

Reagents

a. Eriochrome Black T-Indicator tablet.

b. Ammonia Buffer Solution.

c. 0.02N Ethylene Diamine Tetra Acid Solution (EDTA)

Procedure

a. Take 50 ml water sample in 250 ml conical flask.

b. Add 2 ml of ammonia buffer solution and crushed indicator tablet.

c. Titrate against 0.02N EDTA solution until red colour disappears.

d. End point will be blue, blue grey depending on the water.

Calculation

Total Hardness (ppm) = 1000X ml of 0.02N EDTA / Vol. of water sample

=1000X Burette Reading /50

=20 X Burette Reading

PROCEDURE FOR CHLORINE ANALYSIS

Determination of free chlorine using chlorine indicator solution.

Reagents

Chlorotex reagent

Apparatus

25 ml measuring cylinder

10ml pipette

Procedure

a. Take 10 ml of water sample in a measuring cylinder

b. Add 1 ml of chlorotex reagent

c. Record the results as per the color chart given.

MOST PROBABLE NUMBER (MPN)

Procedure

a. Take 3 tubes of double strength and 10 tubes of single strength for each water sample to be tested.

b. Using a sterile pipette add 10 ml of water to 3 tubes containing 10 ml double strength medium.

c. Similarly add 1 ml of water to 3 tubes containing 10 ml half strength medium and 0.1 ml water to remaining 3 tubes containing 10 ml half strength medium.

d. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs.

e. Compare the number of tubes giving positive reaction to a standard chart and record the number of bacteria present in it.

INHOUSE FOOD MICROBIOLOGY RECORD

Table 6.1. Inhouse microbiology lab record for various tests

S.NO PRODUCT NAME AIR LINE TYPE TPC COLIFORM E.COLI EMB GRADE

Paneer Tikka Sandwich 6E II 9 18 +ve -ve B

Desi Vada Pao 6E II 2 2 -ve -ve B

Pasta Salad With Thousand Island Dressing UK III TNTC 18 +ve -ve C

Paneer Makhani/ Steamed Rice/ Dal Makhani IC I 55 30 +ve -ve C

Curry P.Chicken Tikka/ Aloo Matar UK I 1 0 -ve -ve B

Lamb Top Side/ L.Jus/ R.Veg/ M.Pot SQ I 12 7 +ve -ve C

Palak Paneer/ Rice/ Dal Makhani AI I 43 23 +ve -ve C

Table 6.2. Inhouse microbiology lab record for various tests

S.NO PRODUCT NAME AIR LINE TYPE TPC COLIFORM E.COLI EMB GRADE

Motichoor Laddoo With Rabdi AI II 3 5 -ve -ve B

Mango Phirni AI II 4 Nil -ve -ve B

Bhaji / Pao / Bonda 9W I 10 3 -ve -ve C

Chocolate Brownie UK II 10 Nil -ve -ve B

Chilli Paneer /

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