TRAINING REPORT ON

FOOD MICROBIOLOGY / FOOD FERMENTATION AND DOWNSTREAM

PROCESSING / FOOD SAFETY AND MICROBIAL ANALYSIS.

FROM
REGIONAL FOOD RESEARCH AND ANALYSIS CENTRE, LUCKNOW.

SUBMITTED TO
INSTITUTE OF FOOD PROCESSING AND FOOD TECHNOLOGY ONGC
CENTRE IN ADVANCE STUDIES
UNIVERSITY OF LUCKNOW, LUCKNOW.

FOR FULFILLMENT OF SUMMER TRAINING

SUBMITTED BY: NEHA SHARMA

ROLL NO: 200014205009

INSTITUTE OF FOOD PROCESSING AND TECHNOLOGY

1
ABOUT RFRAC

2
R-FRAC, Centre of Excellence is a public charitable organization established vide
G.O.No.322/58-2-2002-100 (118) 2000 Dated – 0505-2002 by Govt. of U.P. is registered under
society of registration act 1860 under the department of horticulture & food processing, Uttar
Pradesh. R-FRAC is also declared as centre of excellence in Food safety, testing, consultancy as
well as HRD purposes by the state Govt. vide their letter No.163/58-22014-100(1)/2014 Food
Processing/Lucknow: dated 18.03.2014 followed by state Govt. vide their letter
No.20/2015/58-2-2015-100(1)/2014 Food Processing/Lucknow: Dated 07.05.2015 at the
level of Chief Secretary, Group and also state Govt. vide their letter No.198/58-
22017100(1)/2014 Food Processing/Lucknow: Dated 22.03.2017 at the level of Chief
Secretary, Group.
RFRAC is NABL accredited ISO/IEC 17025:2005 as well as ISO 9001:2008 certified and well
equipped with all latest equipment’s and tests are being done by highly qualified scientists.
We would like to inform that our organization also having a pool of
experts/technocrat/financial adviser/Chartered engineers (mechanical/civil)/subject
specialist in the area of agriculture/horticulture/dairy/food safety/HACCP/ISO others sectors
for the benefit of universities/farmers/entrepreneurs. RFRAC is multidisciplinary and
knowledge resource centre. We are also having a pool of PG Diploma Food Safety expert pass
out from IGNOU (RFRAC as Study Centre, UP) for IGNOU. We offer our experts for Mega Food
Park developmental projects, cold chain infrastructure undertaken and can provide Human
resource, HRD, Skill development training, Food safety Implementation training, Hygiene &
sanitation Training to Food Park workers engaged in food manufacturing and in contact with
food stuff. As per Food Safety Management System (FSMS) Plan is mandatory in FSSAI Act,
RFRAC is a designated nodal agency in UP for it.

ACKNOWLEDGEMENT

3
 It was a great pleasure for me to get an opportunity to work as a one month trainee at
Regional Food and Analysis Research Centre, Lucknow, Uttar Pradesh. It has given me
hands-on experience in lab testing, and it will definitely help me in my future career. I would
like to thank Dr. S.K. Chauhan, Director R-FRAC, for allowing us to work in the laboratory
and provide a platform for getting familiar with the lab testing methods for Quality Control
of food products.
I would like to extend my gratitude to Dr. Priyanka Nayak for advising us on the module
- "Food Standards and Quality Control Lab Testing (FSQCLT)" for our lab training.
I want to express my sincere thanks to Mrs. Jyoti P. Mishra, Mr.

Ashutosh Tiwari, Ms. Srishti Verma from the chemical laboratory and Ms. Archana Kumari
from the microbiology lab for teaching, guiding and encouraging us throughout the one-
month training.
I would also like to express my gratitude to Mr. Govind (microbiology lab) and Mr. Vinod
(chemical lab) for their constant assistance in our training.

Neha Sharma

INDEX

S.NO CONTENTS PAGE NO

4
1. (HACCP), (GMP), (GLP), Food Adulteration, Food 7-11
Fortification.

2. To determine the moisture content of the food sample. 12

3. To determine the ash content of the food sample. 13 -14

4. To determine the fat content of the food sample. 15

5. To determine the total alkalinity of Water. 16

6. To determine the total hardness of the Water. 17-18

7. To determine the pH of tap water. 19

8. To determine the presence of Chloride as CI in Water. 20-21

9. To detect adulteration in milk. 22-23

10. To determine the nitrogen in the food sample by the 24-25

Kjeldahl method.

11. Media Preparation and Making of Slant. 27

12. Streaking of Bacteria. 28

5
 13. Gram Staining. 29

14. Isolation and Enumeration of Total Plate Count of Water. 30-31

15. Isolation and Enumeration of Coliform in Water. 32-33

16. Isolation and Enumeration of E. coli in Water. 34

17. Isolation and Enumeration of Yeast and 35-36

Mould

HAZARD ANALYSIS CRITICAL CONTROL PRACTICE (HACCP)

6
 Development of HACCP over the past 50 year has led to documented improvement in food
safety outcomes. Throughout the evolution of HACCP, there was refinement from the original
idea to the seven principles in practice today. In terms of industry cooperation, GFSI has
allowed multiple food safety management systems to be benchmarked and considered
acceptable for use; all require HACCP.

Today, food safety systems differ between developed and developing countries. Developed
countries all have the core components of reducing foodborne illness, with additional
considerations that include traceability, sustainability, food fraud or food defense.

On the other hand, developing countries still struggle with uniform regulatory
implementation of food safety standards. To continue to decrease foodborne illness
worldwide, focus needs to be expended on increasing implementation of these proven
systems in developing countries.

GOOD LABORATORY PRACTICE (GLP)

The following practices should be followed in the laboratory to maintain hygiene:

1. Always wear personal protective equipment Decide not to run a control sample.

7
2. Update a laboratory notebook with only abbreviated details Use samples before checking
them.
3. Always wear mask and gloves while handling bacteria.
4. Wash your hands before and after working with isopropyl alcohol. Write data and value
right after they happen.
5. Do not falsify information. Keep your working area clean.
6. The laminar should be cleaned with ethanol before and after working on it.
7. The UV of laminar should be kept on and while working visible light and exhaust should be
on. 8. Wear eye protection equipment while handling the bacterial cultures.

GOOD MANUFACTURING PRACTICES (GMP)

Good Manufacturing Practice or GMP is a term that is recognized worldwide for the control
and management of manufacturing and quality control of foods, pharmaceutical products,
and medical devices.

8
It is aimed to ensure that quality and safety are maintained throughout a process and thus
prevent product rejection and financial losses. The pressures for this development came
from two sources:
First, commercial pressures including: increasing competition between companies the need
to access expanding national and international food markets product quality management
systems required by major retailers.
Second, new legislation demanded systems maintaining both quality
and safety, and proving a business in control of these.It is aimed to ensure that quality and
safety are maintained throughout a process and thus prevent product rejection and financial
losses.

FOOD ADULTERATION

Adulteration is a legal term meaning that a food product fails to meet the legal standards.
One form of adulteration is an addition of another substance to a food item in order to
increase the quantity of the food item in raw form or prepared form, which results in the loss
of actual quality of a food item. These substances may be either available food items or
nonfood items.

9
 FOOD FORTIFICATION

Food fortification is defined as the practice of adding vitamins and minerals to commonly
consumed foods during processing to increase their nutritional value. It is a proven, safe and
costeffective strategy for improving diets and for the prevention and control of micronutrient
deficiencies.

10
 EXPERIMENT NO.1

OBJECTIVE: To determine the moisture content of the food sample (Wheat Flour )

APPARATUS & EQUIPMENTS REQUIRED: Hot air oven, weighing balance, Petri plates,
spatula.

PROCEDURE:

1. Take the blank weight of the two Petri dishes (BW). Weigh 5 gm of the sample each (W)

11
2. Keep the Petri plates in the hot air oven at 105°C for 5-6 hrs.

3. Cool the Petri dishes in a desiccator. Take the dry weight of the Petri dishes (DW).

OBSERVATION:
Sample no. Blank weight Weight of sample Dry weight

Wheat flou (A) 58.9459 gm 5.0672 gm 63.5586 gm

Wheat flour (B) 51.6989 gm 5.0357 gm 56.1440 gm

CALCULATION:

Moisture content (%) = [Sample weight-(DW-BW)]X 100

Sample weight

RESULT:
The moisture content in the food sample (atta):
Sample no. Moisture content

Wheat flour A 8.96

Whaet flour B 11.92

EXPERIMENT NO.2

Objective: To determine the ash content of the food sample.

APPARATUS & REQUIRED: Muffle furnace at 550°C, weighing balance, crucibles,

spatula, desiccator.

PROCEDURE:

1. Take the blank weight of the two crucibles (BW).

12
 2. Weigh 5 gm of the sample each (W).

3. Keep the crucibles in the muffle furnace at 550°C for 5-6 hrs.

4. Cool the crucibles in a desiccator.

5. Take the dry weight of the crucibles (DW).

OBSERVATION:

Sample no. Blank weight Sample weight Dry weight

Food sample (A) 20.1848gm 5.0102gm 20.1931gm

Food sample (B) 19.5102gm 5.0306gm 19.5642gm

CALCULATION:

Ash content (%) = [Sample weight-(DW-BW)] X 100

Sample weight
RESULT:

The ash content in the food sample –

Sample no. Ash content

Food sample A 0.99

Food sample B 0.98

13
 EXPERIMENT NO.3

OBJECTIVE: To determine the fat content of the food sample.

APPARATUS & EQUIPMENTS REQUIRED: Soxhlet

apparatus, weighing balance, thimble, spatula, desiccator, conical flask.

PROCEDURE:

1. Rinse the glass apparatus with petroleum ether and dry it in the hot air oven at 105°C
and keep it in the desiccator.

14
 2. Take the blank weight of the conical flask. (W₁) Weigh 5 gm of the sample in the flask
(W). Wrap the sample in a filter sample and put it in the thimble. Place the thimble in
the Soxhlet extractor and fill it with 150 ml of petroleum ether.

3. Connect the water assembly. Place the flask in the heating mantle with a temperature of
30°C. The process continues for 5-6 hrs. After the lipid is extracted, the spirit distills
and collects in the condensing unit. Remove the water assembly. Cool the conical
flasks in a desiccator.

4. Take the dry weight of the conical flasks (W₂).

OBSERVATION:

BLANKWEIGHT * (W_ {1}) = 160.5394gm

WEIGHT OF SAMPLE * (W) = 5.0566gm.

DRY WEIGHT (W_ {2}) = 160.7564gm

CALCULATION:

Fat content (%) = (W2-W1) X 100

= (160.7564-160.5394) X 100

5.0566

= 4.29 %.

RESULT: The fat content in the food sample 4.29 %

EXPERIMENT NO.4
OBJECTIVE: To determine the total alkalinity of Water.

APPARATUS & EQUIPMENTS REQUIRED: Measuring cylinder, conical flask, burette,

Methyl red indicator, bromocresol green indicator, phenolphthalein indicator,
0.02 N H₂SO.
PROCEDURE:

15
 1. Measure 50 ml of water sample in a conical flask Add one drop of phenolphthalein
indicator, if it turns pink titrate with standard H₂SO, till it turns colourless. 2. Then
add two drops of methyl red indicator and two drops of bromocresol green indicator.

3. Titrate the solution with 0.02 N H₂SO till the solution turns from green to apple red.

4. Note the titre value (TV).

OBSERVATION:
Titre Value = 18.35 ml.

CALCULATION:
Total alkalinity = (TV×N × 5000)

Sample weight.

= (18.35 x .0198 x 5000)

50

= 36.33 mg/l.

RESULT:

The total alkalinity in the Water is 36.33 mg/L.

EXPERIMENT NO.5

OBJECTIVE: To determine the total hardness of Water.

APPARATUS & EQUIPMENTS REQUIRED: Measuring cylinder (50ml), conical flask,

burette, ferrochrome black indicator, 1ml pipette, Ammonia buffer, 0.01 N EDTA.

PROCEDURE:
16
 1. Measure 50 ml of water sample in a conical flask Ammonia buffer.

2. Add one pinch of Eriochrome T black indicator and 1 ml solution. Titrate the solution
with 0.01 N EDTA till the solution turns to a sky-blue colour.

3. Note the titre value (TV).

OBSERVATION:

Sample no. Titer value (ml)

Water sample A 10.3

Water sample B 10.9

CALCULATION
Total alkalinity * (A) = (TV ×1000)

Sample volume

= (10.3×1000)

50

=206 mg/l.

Total alkalinity * (B) = (Tv ×1000)

Sample volume

= (10.9 x 1000)

50

= 218 mg/L.

17
RESULT:

The total hardness in the Water is in the range of 206-218 mg/L.

EXPERIMENT NO.6

OBJECTIVE: To determine the pH of tap water.

APPARATUS & equipment REQUIRED: Measuring cylinder
(50ml), pH meter, beaker (100 ml)

PROCEDURE:

1. Measure about 100 ml of water sample in a beaker. Put one of the electrodes of the
pH meter in the beaker containing the water sample while the other electrode
remains in distilled Water.

18
 2. Note the pH.

OBSERVATION:
pH = 7.9
RESULT:

The pH of the Water is 7.9.

EXPERIMENT NO.7
OBJECTIVE: To determine the presence of Chloride as CI in Water.

APPARATUS & MATERIALS REQUIRED: Measuring cylinder (50ml), conical flask,

burette, ml pipette, Potassium chromate indicator, 0.1 N AgNO3

PROCEDURE:

1. Measure 50 ml of water sample in a conical flask. Add 1 ml of Potassium chromate

indicator.

19
2. Titrate the solution with 0.1 N AgNO3 till the solution turns to brick red.

3. Note the titre value (V1).

OBSERVATION:
Sample no. Titer value(ml)

Water sample A 0.4

Water sample B 0.5

CALCULATION:
Chloride as Cl(A)= (V1-V2) × N × 1000 × 35.46

Sample volume where, V2 = 0.15 ml.

= (0.4-0.15) x 1000 x 0.09 x35.46 50

= 15.957 mg/l.

Chloride as Cl(B)= (V1-V2) ×N×1000×35.46

Sample volume

Where, V2= 0.15ml

=(0.5-0.15) x 1000 x 0.09 x35.46

50

=22.34 mg/l.

RESULT:
The presence of Chloride as CI (A) in water is 15.957mg/l.

The presence of chlorine as Cl (B) in water is 22.34mg/l.

20
 EXPERIMENT NO. 8

OBJECTIVE: To detect adulteration in milk (Parag Gold).

APPARATUS REQUIRED: Pipette (5ml and 1ml), beaker, testtube, test-tube holder.

PROCEDURE:

 SAMPLE STARCH;

1. Take a measured amount of milk sample.

21
 2. Add few drops of iodine solution or tincture of iodine.

3. If starch is present, the colour changes to blue.

OBSERVATION: There was no change.

RESULT: Starch in milk was absent.

 SAMPLE DETERGENT;

1. Take 5ml of the milk sample.

2. Add the same volume (5ml) of distilled Water.

3. Shake gently and observe. 4. The formation of soapy lather confirms the presence
of detergent in milk.

OBSERVATION: No soapy lather formed.

RESULT: Detergent was absent in the milk.

 SAMPLE SUGAR;

1. Take 3 ml of milk sample.

2. Add 2ml of concentrated HCI and 1ml of resorcinol to heat it.

3. If the milk turns red, it indicates the presence of sugar.

OBSERVATION: Milk turns red.

22
RESULT: Sugar is present.

 SAMPLE FORMALIN;

1. Take 10 ml of milk sample

2. Add 5ml of concentrated H2S04 from the sides.

3. The formation of violet/blue rings at the intersection of two layers indicates the
presence of formalin.

OBSERVATION: No ring formation is observed.

RESULT: Formalin is absent in the milk.

EXPERIMENT NO. 9

OBJECTIVE- To determine the nitrogen in the food sample by the Kjeldahl method.

PRINCIPLE –The Kjeldahl method consists of three steps, which have to be carefully
carried out in sequence: Digestion, Distillation, Titration.

 DIGESTION
1. We are weighing out approximately 1 gm of the sample containing protein, making a
note of the weight, and placing the sample into a digestion flask, along with 12-15 ml
of concentrated sulfuric acid (H2SO4).
2. We are adding seven grams of potassium sulphate and a catalyst, usually copper.

23
 3. Bringing the digestion tube/flask and mixture to a "rolling boil" (about 370oC to 400oC)
using a heating block.
4. Heating the mixture in the tube/flask until white fumes can be seen, and then
continuing the heating for about 60-90 mins.
5. Cooling the tube/flask and cautiously adding 250 ml of Water.

 DISTILLATION
The purpose of the next step, distillation, is to separate the ammonia (that is, the nitrogen)
from the digestion mixture. This is done by raising the pH of the mixture using sodium
hydroxide (45% NaOH solution). This has the effect of changing the ammonium (NH4+) ions
(which are dissolved in the liquid) to ammonia (NH3), which is a gas—separating the
nitrogen away from the digestion mixture by distilling the ammonia (converting it to a
volatile gas, by raising the temperature to boiling point) and then trapping the distilled
vapours in a special trapping solution of about 15 ml HCl (hydrochloric acid) in 70 ml of
Water and removing the trapping flask and rinsing the condenser with Water so as to make
sure that all the ammonia has been dissolved.

 TITRATION

1. As the ammonia dissolves in the acid trapping solution, it neutralizes some of the HCl
it finds there. What acid is left can then be "back titrated," which is titrated with a
standard, known solution of base (usually NaOH). In this way, the amount of
ammonia distilled off from the digestive solution can be calculated, and hence the
amount of nitrogen in the protein is determined.

2. The quantities of acid, and hence ammonia, are determined by adding an indicator
dye to the acid/ammonia trapping solution. This dye should turn a strong colour,
indicating that a significant amount of the original trapping acid is still present.
Putting a standard solution of NaOH (sodium hydroxide) into the burette (a long tube
with a tap at the end) and slowly, slowly adding small amounts of the sodium
hydroxide solution to the acid solution with the dye.

3. Watching for the point at which the dye turns orange, indicating that the "endpoint"
has been reached and that now all the acid has been neutralized by the base.

4. Recording the volume of the neutralizing base (sodium hydroxide solution) that was
necessary to reach the endpoint.

OBSERVATION
24
 SAMPLE WEIGHT TITER VALUE

0.5061 (vegetable powder) 7.7

0.5075 (fish feed) 17.9

CALCULATION
%N= 14.01*T.V.*N OF 0.1N HCL *6.25*100
Sample weight *1000

RESULT
Percentage nitrogen in sample A – 12.08 %.

Percentage nitrogen in sample B – 28.00 %.

25
 FOOD
MICROBIOLOGY

MEDIA PREPARATION

Media preparation is done in order to prepare a nutrient medium for the growth of bacteria.
There are various media for different bacteria like -

Procedure -

1. The media is prepared by weighing media powder into the flask.

2. Then add hot distilled Water to the flask and make up the volume. The media should
be completely dissolved.

3. Cotton plugs the mouth of the flask and covers it with aluminium foil.
26
 4. Autoclave the media at 121°C for 15 minutes.

5. Allow the media to cool and use before it solidifies.

MAKING OF SLANT

1. Prepare the nutrient media and sterilize it at 121°C for 15 minutes.

2. Take a test tube and test tube rack.

3. Pour 5 ml of the media into the test tubes and plug the mouth of the test tubes with
cotton.

4. Tilt the test tube rack with the help of a book or an object.

5. Allow the media to solidify in this position.

6. Store the tubes in the refrigerator.

STREAKING OF BACTERIA

Principle - Inoculum is diluted by streaking it across the surface of the agar plate while
streaking in successive areas such that there are single bacteria in a few mm so that they
divide and form colonies.

Materials required -Source of bacteria, inoculation loop, lighter, spirit lamp, ethanol, agar
plate.

Procedure-

1. Sterilize the inoculum loop until red hot.

27
 2. Put the inoculation loop in the source of bacteria and spread it over the first
quadrant of the agar plate in a close parallel streak.

3. Flame the loop again and allow it to cool and extend the streaks into the second
quadrant.

4. Flame the loop again and allow it to cool and extend the streaks into the final
quadrant and centre of the plate in a parallel motion.

5. Incubate the plate at 37°C for 24 hrs.

Results -The bacterial colonies grow in the same manner and form a streak.

GRAM STAINING

Principle- Gram-positive bacteria have cell walls having peptidoglycan and lipid content.
When primary staining is done, the stain enters the cell wall pores and does not exit from
the pores on decolorizing, which causes the bacteria to appear blue/purple in colour.

Gram-negative bacteria have cell walls having thin peptidoglycan and a thick lipid layer.
When primary staining is done, the stain enters the cell wall pores and leaches from the
pores on decolourizing due to the dissolution of the lipid layer. When safranin is added, it
retains colour and appears red.

28
Materials required- Crystal violet (primary stain), iodine mordant, Decolourizer, and
Safranin (counter-stain).

Procedure-

1. Take a clean slide.

2. Smear the slide with inoculum.

3. Air dry and heat fix. Pour crystal violet and wait for 30 sec-1 min. Rinse with Water.
Flood the slide with Gram's iodine. Wait for 1 minute and then wash with Water.
Then wash with 95% ethanol for about 10-20 seconds and rinse with Water.

4. Add safranin for about 1 min and wash with Water.

5. Air dry. Blot dry and observe under the microscope.

Interpretation

 Gram-positive: blue/purple colour.

 Gram-negative: red colour.

EXPERIMENT NO. 1
OBJECTIVE: Isolation and Enumeration of Total Plate Count OF Water.

REFERENCE:1S:5402 2012

APPARATUS AND EQUIPMENT REQUIRED: Hot air oven, Petri dishes, pipette
holder, micropipette (1mL and 0.1mL), colony counter, autoclave, incubator (30°C)

PROCEDURE:
 MEDIA PREPARATION –
29
 1. The media used for the detection of Total Plate Count is Plate Count Agar (PCA).

2. Weigh PCA in a calculated amount of hot distilled Water and mix.

3. Plug the neck of the conical flask using cotton and then wrap the foil around the
cotton plug.

4. For sterilizing, autoclave the media at 121°C for 15 minutes at 15 lbs 5. After the
autoclave has released the pressure. Allow it to cool down a little.

 INOCULATION –

1. Take 7sterile Petri dishes and code them accordingly: Sample Code/TPC/date/Sign

2. In one sterile petri dish, add 1ml of water sample with the help of sterile 1ml pipette
and prepare its duplicate.

3. Now add up to 10-15ml of the autoclaved media in the Petri dishes by putting it in
front of the spirit lamp to heat sterilizing the neck and mouth of the petri dish.

4. Prepare one control plate to check for sterility.

5. Allow the steam to escape and keep the Petri dishes in the laminar for solidification.

6. Alter the media has solidified, keep it in the incubator at 30°C for 72 hrs.

OBSERVATION:
Count the no. of colonies after the specified period.

DILUTION

D0 25 18

D2 10 6

EXPRESSION OF RESULTS

30
N = (25+18+10+6) / (2.2010)

= 26.8*10^1

=2.7*10^2

RESULT:
TPC: 2.7*10^2 CFU/ML.

EXPERIMENT NO.2

OBJECTIVE: Isolation and Enumeration of Coliform in Water.

REFERENCE: IS:5401(P-1):2012

APPARATUS & EQUIPMENT REQUIRED: Hot air oven, Petri dishes, pipette holder,
micropipette (1ml and 0.1ml), colony counter, autoclave, incubator (37°C).

PROCEDURE:
 MEDIA PREPARATION -
31
 1. The media used for the detection of Coliform is Violet Red Bile Agar (VRBA).

2. Weigh VRBA for making of the media.

3. Plug the neck of the conical flask using cotton and then wrap the foil around the
cotton plug.

4. For sterilizing, autoclave the media at 121°C – 15 psi for 15.

5. After the autoclave has released the pressure, allow it to cool down a little.

 INOCULATION -
1. Take seven sterile Petri dishes and code them accordingly Sample
Code/Coliform/date/Sign.

2. In one sterile Petridis, add 1ml of water sample with the help of sterile 1ml
pipette and prepare its duplicate and in the other two sterile Petridis, add
1ml from second and third dilution, respectively.

3. Now add up to 10-15ml of the autoclaved media in the Petri dishes by putting
it in front of the spirit lamp to heat sterilizing the neck and mouth of the Petri
dish.

4. Prepare one control plate to check for sterility.

5. Allow the steam to escape and keep the Petri dishes in the laminar for
solidification.

6. After the media has solidified, keep it in the incubator at 37°C for 24 hrs.

 OBSERVATION:
Count the no. of colonies after the specified period.

Control plate – nil.

 EXPRESSION OF RESULTS:

32
 I II

D1 NIL NIL

D2 NIL NIL

D3 NIL NIL

RESULT: <1 CFU/gm.

EXPERIMENT NO. 3

OBJECTIVE: Isolation and Enumeration of E. coli in Water.

REFERENCE IS:5887-1(1976).

APPARATUS & EQUIPMENT REQUIRED: Hot air oven,

Petri dishes, pipette holder, micropipette (1ml), colony counter, autoclave, incubator (37°C)

PROCEDURE:
 MEDIA PREPARATION -

1. The media used for the detection of E. coli is EMB agar.

33
 2. Weigh EMB for preparing the media. Plug the neck of the conical flask using cotton
and then wrap foil around the cotton plug.

3. For sterilizing, autoclave the media at 121°C for 15 minutes. Then after the autoclave
has released the pressure, allow it to cool down a little.

 INOCULATION –

1. Take seven sterile Petri dishes and code them accordingly: Sample
Code/TPC/date/Sign.

2. In one sterile Petridis, add 1ml of 10 with the help of sterile 1ml pipette and prepare
its duplicate and in the other two sterile Petri dish add 1ml ofd²and d³.

3. Now add up to 10-15ml of the autoclaved media in the Petri dishes by putting it in
front of the spirit lamp to heat sterilizing the neck and mouth of the Petri dish.

4. Prepare one control plate to check for sterility.

5. Allow the steam to escape and keep the Petri dishes in the laminar for solidification.

6. . After the media has solidified, keep it in the incubator at 37°C for 24 hrs.

RESULT = NIL >1CFU/M.

EXPERIMENT NO. 4
OBJECTIVE: Isolation and Enumeration of Yeast and Mould.

REFERENCE:IS:5403(1999).

APPARATUS & EQUIPMENT REQUIRED: Hot air oven,

Petri dishes, pipette holder, micropipette (1ml), colony counter, autoclave, incubator (25°C).

PROCEDURE:

 MEDIA PREPARATION

1. The media used for the detection of Total Plate Count is Plate Count Agar (PCA).

34
2. . Weigh YXT (Yeast Extract Dextrose Chloramphenicol Agar Medium). Pour hot
distilled Water into a conical flask and mix well.

3. Plug the neck of the conical flask using cotton and then wrap the foil around the cotton
plug.

4. For sterilizing, autoclave the media at 121°C for 15 minutes. After the autoclave has
released the pressure, allow it to cool down a little.

 SERIAL DILUTION -

1. Take a 100 ml dilution bottle and add 90ml distilled water. Take 2 test tubes and add
9 ml distilled water to each.

2. Autoclave them at 121°C for 15 minutes. Let them cool after sterilization.

3. Weigh 10 gm of sample and add to 90 ml of sterilized water in the dilution tube. This
is called first dilution or (10¹). The ratio of sample to the dilution medium should
always be 1:9.

4. Pipette 1 ml of the dilution from d' and add to the first test tube. This becomes
second dilution or (10¹²).

5. Now pipette 1 ml of the dilution from d² and add to the second test tube. This
becomes the third dilution or d³ (10³).

 INOCULATION –

1. Take seven sterile Petri dishes and code them accordingly: Sample
Code/TPC/date/Sign.

2. In one sterile petri dish, add 1ml of 10 with the help of a sterile 1ml pipette and
prepare its duplicate and in the other two clean petri dish add 1ml of d²and d³ &
Mould of curd.

3. Now add to 10-15ml of the autoclave media in the Petri dishes by putting it in front
of the spirit lamp to heat sterilizing the neck and mouth of the petri dish.

4. Prepare one control plate to check for sterility.

5. Allow the state escape and keep the Petri dishes in the laminar for solidification.

35
 6. After the media has solidified, keep it in the incubator at 25°C for five days.

OBSERVATION

D0 32 25

D1 12 9

EXPRESSION OF RESULTS
N= 32+25+12+9

2.2*10^1

= 35.45 *10 ^1

RESULT = 3.6*10^2CFU/GRM.

CONCLUSION

During one month training period, a lot of experience, knowledge and exposure that I have
handy . All disclosures were awaken myself in a boost of self-confidence to face life more
challenging now. Practical is a complement to the science or theory learned, without practice
will be lost and will not give anything.

36
During my training, there were many changes from the point of learning environments and
discussion among colleagues. It can directly increase the dedication and rational attitude
toward myself.

However, there are still some weaknesses that can be improved in the future. Therefore I
conclude that the training program has provided many benefits to students even if there are
minor flaws that are somewhat disfiguring condition , so that this weakness can be rectified in
the future.

I can conclude that through training I received a lot of exposure in the computing world. I
again express my deepest appreciation to all those who provided me the opportunity and
guidance.

37