EXPERIMENT NO 1

(1a)

TO ANALYSE THE MICROBIAL QUALITY AND ASSURANCE OF

RAW MILK FROM DIFFERENT LOCALITIES OF KARACHI.

THEORY
Milk is regarded as a complete food because of its rich content of protein, fat, carbohydrates, all
known vitamins and various minerals essential for sustaining life and maintaining good health.
But this complete food is also a very good medium for bacterial growth. So, there is an
increasing focus on milk quality and hygiene in the dairy industry. Milk and the products derived
from milk of cows can harbor a variety of microorganisms and can be important sources of food
borne pathogens. The safety of milk is an important attribute for consumers of milk and dairy
products.

A major factor determining milk quality is its microbial load. It indicates the hygiene practiced
during milking, like cleanliness of milking utensils, condition of storage, manner of transport as
well as the cleanliness of udder of individual animal.

We have designed an experiment to analyze the microbial load of different types of milk
collected from different areas of Karachi. The experiment is divided into 5 different experiments
which includes testing of raw, pasteurized, UHT, boiled and powdered milk and compare all of
them for microbial load they possess. In this particular experiment “1a” we have analyzed raw
milk for microbial quality and assurance.

REQUIREMENTS:
⮚ Raw milk
⮚ MRD containing test tubes(6)
⮚ Alcohol(10ml) in 50ml flask
⮚ 1ml pipettes and fillers
⮚ Nutrient agar plates(3)
⮚ MacConkey’s agar plates(3)
⮚ Spreader

1
MAXIMUM RECOVERY DILUENT (MRD)

This is a saline peptone water which is an isotonic diluent used for maximum recovery of
microorganisms, and for the growth of bacterial cultures.

Recipe of MRD

Dissolve 0.5g of peptone in 1 liter of normal saline. Dispense into the final containers and
sterilize by autoclaving at 121°C for 15 minutes.

Group no. Locality

1 Malir
2 gulshan
3 Gulistan e johar
4 Orangi town
5 Nazimabad
6 North Karachi
7 Gulshan-e-iqbal
8 Sultanabad
9 Gulistan e johar

PROCEDURE
⮚ Disinfect the table top and light the flame.
⮚ Take 1 ml of milk sample and add into 9 ml MRD blank this will give 10-1 dilution.
⮚ Serially dilute the sample up to 10-6 dilution.

⮚ Take 0.1 ml from 10-4, 10-5 and 10-6 dilutions and spread on its respective Nutrient Agar
plate.
⮚ For MacConkey’s agar take sample only from first three dilutions 10-1, 10-2 and 10-3

⮚ Incubate at 37 0 C for 24 hours.

⮚ Observe the results next day.
⮚ Calculate colony forming units per ml of Nutrient Agar plates.
COLONIAL CHARACTERISTICS : (NA)

2
Group No. 10-4 10-5 10-6
Group 01 (uc) (66) (uc)
Creamy, small raised Small, off white, Raised, creamy, small
and mucoid colonies irregular and and mucoid colonies.
mucoid colonies
Group 02 (71 colonies) (51 colonies) (18 colonies)
Smooth, rounded, small Circular, small, Small, elevated ,round
and creamy creamy and raised and creamy.
Group 03 UC UC UC
White, elevated, small White, elevated, White, elevated, small
and circular, entire, small and and circular, entire,
translucent circular, entire, translucent
translucent
Group 04 UC UC UC
Circular, pin pointed, off Circular, Circular, pin pointed, off
white, entire, pinpointed, white, entire,
translucent offwhite, entire, translucent
translucent
Group 05 UC UC UC
Small,elevated,creamy Small,elevated,cre Pinpointed,transparent,
white,circular amy white,circular flat ,circular
Group 06 (Uncountable colonies) (Uncountable (Uncountable colonies)
White, elevated, small colonies) Small, circular, white
and circular Small, white, and elevated
elevated and
circular

Group 07 (98 colonies) (74 colonies) (43 colonies)

Circular, entire, convex, Circular, entire, Circular, entire, convex,
moderate, shiny, convex, moderate, shiny,
transparent and off- moderate, shiny, translucent and off-
white translucent and white
off-white
Group 08 UN, small, white 16 colonies small, 21 colonies small, white,
raised, mucoid colonies white raised, raised, mucoid

3
 mucoid colonies Colonies
Group 09 UN, off white, small, 108 colonies, 105 colonies, small,
circular, regular, raised, small, off white, cream, regular, raised,
smooth creamy, smooth, smooth
regular, raised

Key:

UC =Uncountable

COLONIAL CHARACTERISTICS ON MacConkey’s AGAR

GROUP NUMBER 10-1 10-2 10-3

1 (UC) (27)
Pink, small, raised, Pinpointed,pink and Pin pointed pink
circular colonies circular colonies circular colonies
2 Circular, smooth, Irregular, pink, small Pink, small , smooth
small, pink and raised colonies colonies.
colonies
3 UC UC UC
Pink colored, Pink colored, Pink colored,
elevated, circular elevated, circular elevated, circular
entire and small, entire and small, entire and small,
translucent translucent translucent

4 UC UC UC
Small, pink, circular, Small, pink, circular, Small, pink, circular,
entire, mucoid, entire, mucoid, entire, mucoid,
smooth, elevated, smooth, elevated, smooth, elevated,
translucent translucent translucent
5 UC UC UC
Small,pink,circular, flat or slightly flat or slightly
convex with convex with
elevated
irregular irregular
edges ,spreaded, edges ,spreaded,
cream cream
6 (Uncountable (Uncountable (Uncountable
colonies) colonies) colonies)

4
 Pink colored, Small, elevated, Entire, pink colored,
elevated, entire and entire and pink small and elevated
small colored
7 (uncountable (uncountable (178 colonies)
colonies) colonies) Circular, irregular,
Circular, entire, Circular, entire, raised, large, smooth,
convex, pinpoint, convex, moderate, shiny, opaque and
smooth, shiny, smooth, shiny, mucoid
transparent and transparent and
colorless colorless
8 UN, white small UN, white small 221 colonies white
,raised, mucoid ,raised, mucoid small ,raised, mucoid
colonies Colonies colonies

9 UN, small, smooth, UN, medium sized, Small, pink, mucoid,

regular, raised, pink raised, pink, regular raised, smooth,
regular, UN

Gram Staining

Group Media Dilution Gram Shape Arrangement

No. Reaction

10-4 Gram positive Cocci Scattered

Group 01 Nutrient agar 10-5 Gram positive Cocci Scattered
10-6 Gram positive Cocci Scattered
MacConkey 10-1 Gram negative Coccobacilli Scattered
agar
10-2 Gram negative Coccobacilli Scattered
10-3 Gram negative Coccobacilli Bunches
10-4 Gram positive Bacilli Scattered
Group 02 Nutrient agar 10-5 Gram positive Diplobacilli Scattered
10-6 Gram negative Cocci Scattered
MacConkey 10-1 Gram negative Rods Scattered

5
 agar 10-2 Gram negative Short rods Scattered
10-3 Gram negative Short rods Scattered

10-4 Gram positive Bacilli Scattered

Group 03
Nutrient agar 10-5 Gram positive Bacilli Scattered
10-6 Gram positive Bacilli Scattered
MacConkey 10-1 Gram negative Bacilli Scattered
agar
10-2 Gram negative Bacilli Scattered
10-3 Gram negative Cocco bacilli Scattered
10-4 Gram positive Bacilli Scattered
Group 04 Nutrient agar 10-5 Gram positive Bacilli Scattered
10-6 Gram positive Cocco bacilli Scattered
MacConkey 10-1 Gram negative Bacilli Scattered
agar
10-2 Gram negative Bacilli Scattered
10-3 Gram negative Bacilli Scattered
10-4 Gram positive Cocco bacilli Scattered
Group 05 Nutrient agar 10-5 Gram positive Cocco bacilli Scattered
10-6 Gram positive Bacilli Scattered
MacConkey 10-1 Gram positive Bacilli Scattered
agar
10-2 Gram positive Bacilli Scattered
10-3 Gram positive Bacilli Scattered
10-4 Gram positive Bacilli Scattered
Group 06 Nutrient agar 10-5 Gram positive Bacilli Scattered
10-6 Gram positive Bacilli Scattered
MacConkey 10-1 Gram positive Bacilli Scattered
agar
10-2 Gram positive Bacilli Scattered
10-3 Gram positive Bacilli Scattered
Nutrient agar 10-4 Gram-negative Cocci Scattered and bunches
Group 07 10-5 Gram-positive Bacilli Scattered

6
 10-6 Gram-negative Cocci Scattered
MacConkey 10-1 Gram-positive Diplobacilli and Scattered
agar cocci
10-2 Gram-negative Bacilli Scattered
scattered bacilli
10-3 Gram-positive Cocci Scattered and in
bunches
10-4 Gram positive Cocci Scattered in bunches
Group 08 10-5 Gram positive Cocci Scattered in bunches
Nutrient agar
10-6 Gram positive Cocci Scattered in
bunches
MacConkey 10-1 Gram negative Short rods Scattered
agar
10-2 Gram negative Short rods Scattered
10-3 Gram negative Short rods Scattered
10-4 Gram positive Cocci diplococci
Group 09 Nutrient agar 10-5 Gram negative Rods chains
10-6 Gram positive Short rods Chains/scattered
MacConkey 10-1 Gram negative Rods Chains/scattered
agar
10-2 Gram negative Long/short rods Scattered
10-3 Gram negative Short rods Chains

7
Figures of NA:

Gram positive
scattered

10-1
10-2

Gram positive
scattered
cocci

10-3

8
Figures on MacConkey agar:

Gram
negative
scattered
coccobacilli

10-4 10-5

Gram
negative
coccobacilli in
bunches

10-6

9
RESULTS:
On Nutrient agar Gram positive bacilli were observed with scattered arrangement and small, white ,
elevated and uncountable colonies were observed. And Gram negative bacilli with scattered
arrangement were also observed on MacConkey agar with uncountable, elevated ,small and pink
colored colonies were observed.

DISCUSSION:
The Lactic acid bacteria, the most abundant microorganism found in milk that can cause
fermentation and promote health. The normal flora of milk contain lactobacillus which are
Gram positive and low G-C rods.

The possible reason of uncountable colonies may be because of contamination of bacteria from
equipment , utensils, udder etc.

REPORT:
In all samples uncountable colonies with different morphology were observed and Gram
positive and Gram negative rods and cocci were also observed may be because of contamination
of udder, utensils, water supply and more than standard count of bacteria due to higher
temperature during transportation and long period of time taken from farm to fork

10
 EXPERIMENT NO 1
(1b and 1c)

TO ANALYSE THE MICROBIAL QUALITY AND ASSURANCE OF

POWDERED MILK AND UHT MILK

THEORY
Powdered milk or dried milk is a manufactured dairy product made by
evaporating milk to dryness. One purpose of drying milk is to preserve it; milk powder has a far
longer shelf life than liquid milk and does not need to be refrigerated, due to its low moisture
content. Another purpose is to reduce its bulk for economy of transportation. Powdered milk and
dairy products include such items as dry whole milk, nonfat (skimmed) dry milk, dry buttermilk,
dry whey products and dry dairy blends. 
Ultra-high temperature processing (UHT), ultra-heat treatment, or ultra-pasteurization is a food
processing technology that sterilizes liquid food, chiefly milk, by heating it above 135 °C
(275 °F) – the temperature required to kill spores in milk – for 1 to 2 seconds. UHT is most
commonly used in milk production, but the process is also used for fruit juices, cream, soy milk,
yogurt, wine, soups, honey, and stews. UHT milk was first developed in the 1960s and became
generally available for consumption in the 1970s.
The heat used during the UHT process can cause  browning and change the taste and smell of
dairy products. An alternative process is HTST pasteurization (high temperature/short time), in
which the milk is heated to 72 °C (162 °F) for at least 15 seconds.
UHT milk packaged in a sterile container, if not opened, has a typical unrefrigerated shelf life of
six to nine months. In contrast, HTST pasteurized milk has a shelf life of about two weeks from
processing, or about one week from being put on sale.

REQUIREMENTS:

11
⮚ Powder and UHT milk sample
⮚ MRD containing test tubes(6)
⮚ Alcohol(10ml) in 50ml flask
⮚ 1ml pipettes and fillers
⮚ Nutrient agar plates(3)
⮚ MacConkey’s agar plates(3)
⮚ Spreader

Group no. POWDER UHT

1 Ensure -
2 Nido -
3 Comel -
4 Nestle bunyad -
5 - Provided by media room
6 Everyday -
7 - Provided by media room
8 Everyday -
9 - Provided by media room

PROCEDURE
⮚ Disinfect the table top and light the flame.
⮚ Take 1 g/ml of milk sample and add into 9 ml MRD blank this will give 10-1 dilution.
⮚ Serially dilute the sample (Powder or UHT) up to 10-6 dilution.

⮚ Take 0.1 ml from 10-4, 10-5 and 10-6 dilutions and spread on its respective Nutrient Agar
plate.
⮚ For MacConkey’s agar take sample only from first three dilutions 10-1, 10-2 and 10-3.

⮚ Incubate at 37 0 C for 24 hours.

⮚ Observe the results next day.
⮚ Calculate colony forming units per ml of Nutrient Agar plates.

OBSERVATIONS :
12
Group No. Media Dilution Colonial Colonial
characteristics for characteristics for
powder milk UHT milk

Group 01 Nutrient agar 10-4 Off white, irregular, _

flat colonies
10-5 Pinpointed, off white, _
convex colonies
10-6 No colonies _
MacConkey 10-1 No colonies _
agar
10-2 No colonies _
10-3 No colonies _
Group 02 Nutrient agar 10-4 No colonies _
10-5 No colonies _
10-6 No colonies _
MacConkey 10-1 No colonies _
agar
10-2 No colonies _
10-3 No colonies _
Group 03 Nutrient agar 10-4 No growth -
10-5 No growth -
10-6 No growth -
MacConkey 10-1 No growth -
agar
10-2 No growth -
10-3 No growth -
Group 04 Nutrient agar 10-4 No growth -
10-5 No growth -
10-6 No growth -
MacConkey 10-1 Less than 30 -
agar Pink, small, entire,

13
 circular,smooth,
translucent
10-2 Less than 30 -
Pink, small, entire,
circular,smooth,
translucent
10-3 Less than 30 -
Pink, small, entire,
circular,smooth,
translucent
Group 05 Nutrient agar 10-4 Less than 30
Large, creamy
white,circular,flat
10-5 Less than 30
Large, creamy
white,circular,flat
10-6 Less than 30
Large, creamy
white,circular,flat
MacConkey 10-1 Pink,small,circular,smo
agar oth,opaque
10-2 No growth
10-3 No growth
Group 06 Nutrient agar 10-4 Irregular, raised, -
spreaded edge, large,
white colony
10-5 Irregular, flat raised -
elevation, white, large
colony
10-6 No growth -
MacConkey 10-1 No growth -
agar
10-2 No growth -
10-3 No growth -
Group 07 Nutrient agar 10-4 - No growth
10-5 - Circular, entire, off-
14
 white, smooth, flat
pinpoint, dull and
opaque.
10-6 - Circular, entire, off-
white, smooth raised,
pinpoint, dull and
opaque.
MacConkey 10-1 - No growth
agar
10-2 - No growth
10-3 - Circular, entire, pink,
smooth, flat, pinpoint,
dull, and opaque.
Group 08 Nutrient agar 10-4 No colonies
10-5 No colonies
10-6 No colonies
MacConkey 10-1 No colonies
agar
10-2 No colonies
10-3 No colonies
Group 09 Nutrient agar 10-4 - 34 colonies, white
centred, off white, flat,
rhizoid, dry
10-5 - 31 colonies, same as
above
10-6 - 65 colonies, same as
above
MacConkey 10-1 - No growth
agar -
10-2 UN, flat, lobate,
mucoid
10-3 - 1 colony, circular,
regular, pink, raised

GRAM REACTION :

milk powder sample

15
Group No. MEDIA DILUTIONS MICROSCOPIC CHARACTERS

Group 1 Nutrient agar 10-4 Gram negative rods scattered

10-5 Gram negative rods scattered
10-6 No characteristics
Group 2 Nutrient agar 10-4 No characteristics
10-5 No characteristics
10-6 No characteristics
Group 3 Nutrient agar 10-4 No characteristics
10-5 No characteristics
10-6 No characteristics
Group 4 Nutrient agar 10-4 No characteristics
10-5 No characteristics
10-6 No characteristics
Group 6 Nutrient agar 10-4 Gram positive, diplobacilli, streptobacilli, scattered
bacilli with endospores
10-5 Gram positive, scattered bacilli with endospores
10-6 -
Group 8 Nutrient agar 10-4 No characteristics
10-5 No characteristics
10-6 No characteristics

UHT milk sample:

Group No. MEDIA DILUTIONS MICROSCOPIC CHARACTERS

Group 1 Nutrient agar 10-4

10-5

16
 10-6
Group 2 Nutrient agar 10-4
10-5
10-6
Group 07 Nutrient agar 10-4 _
10-5 Gram-negative scattered coccobacilli
10-6 Gram-negative scattered cocci
Group 09 Nutrient agar 10-4 Gram positive, long rods, scattered
10-5
Gram positive, long rods, scattered
10-6
Gram positive, long rods, scattered

FIGURES

17
 Gram
negative Gram
short rods negative
short rods

10-4

RESULT:
On Nutrient agar irregular, raised, spreaded, pinpointed, white colony was observed. No colony
was observed on Nutrient agar plate of dilution 10-6. Similarly, on MacConkey agar for dilution
10-1,10-2 and 10-3 growth was absent. Upon microscopy Gram negative scattered rods were
observed.

DISCUSSION:
The presence of bacteria might indicate that sample was already contaminated with bacteria
which under vacuum conditions formed spores as can be seen upon microscopy. Other reason
might be aseptic conditions were not maintained, pack was kept open for a long time which lead
to improper results.

REPORT:
Overall improper results were observed i.e. presence of bacteria in both dry milk powder and
UHT milk sample. The reasons may include that from weighing of the sample till the spreading
of dilution aseptic conditions were not maintained, the pack was already cut upon for a longer
period. Hence the presence of microbes in both the samples indicate improper handling, no
aseptic conditions were maintained

EXPERIMENT NO 1
(1d)

TO ANALYSE THE MICROBIAL QUALITY AND ASSURANCE OF 18

BOILED MILK FROM DIFFERENT LOCALITIES OF KARACHI.
THEORY
A major factor determining milk quality is its microbial load. It indicates the hygiene practiced
during milking, like cleanliness of milking utensils, condition of storage, manner of transport as
well as the cleanliness of udder of individual animal. So to reduce this microbial load boiling is
done which if done properly kills vegetative cells but spores do survive boiling.

 Boiling of milk would mean raising the temperature of milk to its boiling point under
atmospheric temperature and pressure. The boiling point of milk is l00.17°C which is decidedly
higher than the usual temperature adopted in holding method of HTST pasteurization process.
Boiling milk is a common practice at home and is an easy way to decrease the microbial load of
raw milk. Boiling also increases the shelf life of raw milk.

REQUIREMENTS:
⮚ Boiled milk
⮚ MRD containing test tubes(6)
⮚ Alcohol(10ml) in 50ml flask
⮚ 1ml pipettes and fillers
⮚ Nutrient agar plates(3)
⮚ MacConkey’s agar plates(3)
⮚ Spreader

Group no. Locality

1 Malir
2 Gulshan
3 Gulistan e Johar
4 Orangi town
5 Gulshan
6 Gulistan-e-Johar
7 Gulshan-e-iqbal

19
 8 Orangi town
9 Gulistan e johar

PROCEDURE
⮚ Disinfect the table top and light the flame.
⮚ Take 1 ml of milk sample and add into 9 ml MRD blank this will give 10-1 dilution.
⮚ Serially dilute the sample up to 10-6 dilution.
⮚ Take 0.1 ml from 10-4, 10-5 and 10-6 dilutions and spread on its respective Nutrient Agar
plate.
⮚ For MacConkey’s agar take sample only from first three dilutions 10-1, 10-2 and 10-3.
⮚ Incubate at 37 0 C for 24 hours.
⮚ Observe the results next day.
⮚ Calculate colony forming units per ml of Nutrient Agar plates.

OBSERVATIONS

Number Of Colonies
Grou Cfu/ml
p No (No of colonies x
Dilution Factor x
Volume Factor)

10-4 10-5 10-6

1 UC UC UC -
2 UC UC UC -
3 UC UC UC -
4 UC UC UC -
5 Less than 30 Less than 30 Less than 30 -
6 TNTC TNTC TNTC -
7 40 65 TNTC 3.75x108 cfu/ml
8

20
 9

OBSERVATIONS :
COLONIAL CHARACTERISTICS (NA)

GROUP 10-4 10-5 10-6

NO.
1 Rough, raised, off- Small,off white and Yellowish white, small,
white,small and mucoid irregular colonies raised and mucoid
colonies colonies
2 Pin pointed, flat, white Small, white, raised and Circular, off white, large
colonies mucoid colonies and opaque colonies.
3 Pinpointed, off white, Irregular, white, elevated, Small, smooth, elevated,
circular, entire, smooth, large colony white, entire colony
transparent
4 Large, white, entire, Pinpointed, off white, Small, white, circular,
elevated, smooth, circular, entire, smooth, entire, irregular, opaque
opaque transparent
5 Less than 30 Less than 30 No growth
Creamy white with dark Circular,large,white,
centered,large elevated
6 Small, gummy, regular, Irregular, white, elevated, Small, smooth, elevated,
smooth, circular, large colony white, entire colony
slightly elevated, white
colony
7 Irregular, undulate, off- Filamentous, irregular, Circular, entire, off-
white, rough, flat, large, white, moist center with white, smooth, flat,
dull and opaque dull sides, flat, large, pinpoint, dull and
opaque sides with opaque
translucent center.
8 UN, small, UN, small, UN, small,

21
 white,raised ,mucoid white,raised ,mucoid white,raised ,mucoid
9 UN, Pin pointed, UN, Pin head, circular, UN, large, mucoid,
colourless, flat, translucent, flat,small white, irregular, flat
irregular,

Key:

UC =Uncountable

COLONIAL CHARACTERISTICS ON MacConkey’s AGAR

GROUP 10-1 10-2 10-3

No.
1 No growth No growth No growth
2 Circular, pink, smooth No colonies No colonies
and raised colonies
3 Elevated, medium, Elevated, medium, entire, Elevated, medium,
entire, regular, pink regular, pink colony entire, regular, pink
colony colony
4 Pin point, entire, off Pin point, entire, off Pin point, entire, off
white, elevated white, elevated white, elevated
5 Less than 30 Greyish,flat,large, No growth
Greyish,small,flat spreaded
,circular
6 Medium, elevated, pink, Elevated, medium, entire, Pinpointed, entire,
regular, entire colony regular, pink colony regular, flat, pink colony
7 Circular, entire, pink, Circular, entire, off- Circular, entire,
smooth, raised, medium, white, smooth, raised, colorless, smooth,
shiny and opaque medium, shiny and raised, pinpoint, shiny
opaque and translucent.
8 UN, white, small, rasied, UN, white, small, rasied, 296, white, small,
mucoid colonies mucoid colonies rasied, mucoid colonies
9 UN, Pink, pinhead, No growth 1 colony, white, large,
smooth, flat, yellow, smooth, flat
small, circular

22
KEY - Symbolized for no growth

RESULTS
Nutrient agar:

Small raised off white and mucoid colonies were observed on nutrient agar.

Upon microscopy Gram positive scattered bacilli were observed.

MacConkey agar:

No growth was observed on MacConkey plate.

DISCUSSION:
Overall bacteria were observed along with some yeast cells in boiled milk sample.

Reasons for presence of microorganisms in milk are:Use of unsterilized tubes for transporting
the sample, improper handling, not maintaining aseptic conditions.

REPORT:
The presence of increase number of microbes in the boiled milk sample might be since the milk
was brought from local milk shop where the milk comes from local milk man who does not
maintain GHP during milking of the cow.

EXPERIMENT NO 2
TO ANALYSE THE MICROBIAL QUALITY AND ASSURANCE OF
FROZEN PARATHA OF DIFFERENT BRANDS.

23
THEORY:

Microorganisms differ significantly in their sensitivity to freezing, thus the main concern
about the microbiological aspects of freezing is the growth of organisms during thawing
rather than during freezing. A number of microorganisms are present even at frozen
temperatures but most occurring organisms including Psychrotrophic microorganisms &
Listeria monocytogenes. Frozen food items may contains psychrotrophic organisms.
Psychrotrophic microorganisms have the ability to grow at 0°C. Psychrotrophic
microorganisms have a maximum temperature for growth above 20°C and are widespread in
natural environments and in foods. Listeria monocytogenes is a regular nonsporing gram-
positive rods. It belongs from the genus Lactobacilli. L.monocytogenes is a pathogen that can
contaminate food, especially dairy products and processed delicate meats. This organism
survives within phagocytic cell and is capable of growth at refrigeration temperatures. It is
capable to increase in its number during a food’s shelf life. A number of reasons are possible
for microbial load in frozen items. Some reasons are as follows;

1. Fluctuation in storage temperature is one of the main reasons for microbial deterioration
of frozen products during storage.
⮚ A careful inspection of frozen products is essential to ensure proper freezing storage with
constant temperatures.
⮚ A good quality freezer container should be used to maintain the quality of frozen

MATERIALS:

⮚ Sterile saline 1000 ml

⮚ Nutrient agar 400 ml
⮚ EMB 400 ml
⮚ MacConkey’s agar 400 ml
⮚ SDA 400 ml
⮚ Spreader + 70% alcohol 02
⮚ Sterile flasks (200 ml) 07
⮚ Sterile McCarty’s tubes 36
⮚ Sterile empty plates 50
⮚ Slants 30
⮚ Juster + tips (sterile) 02

GROUP NO PARATHA BRAND NAME

1 dawn

24
 2 Man o salwa
3 Dawn
4 Man o salwa
5 Lal Qila
6 Lal Qila
7 Dawn
8 Ponam paratha
9 Khatir tawaza

METHOD:

⮚ Disinfect the table top with disinfectant then lighten up the flames.
⮚ Then take out one paratha from packet and cut (2x2) piece of that paratha.
⮚ Then add it in flask containing 100 ml of saline.
⮚ Tease that piece with the help of wire loop so that it will break into many small
pieces.
⮚ Then leave that flask undisturbed for at least 5 min so that solid particles will settle
down.

⮚ Then make serial dilution of that mixture till .

⮚ Transfer 0.1 ml from and diluted tubes on SDA plates and then
spread it with spreader.
⮚ Transfer 0.1 ml from 10-1 and 10-2 diluted tubes on EMB, MacConkey and 10-5, 10-6
on Nutrient agar plates and then spread it with spreader.
⮚ Incubate SDA at room temperature whereas incubate MacConkey, EMB and Nutrient
agar plates at 37°C in incubator.
⮚ Next day observe results on MacConkey, EMB and Nutrient agar plates.
⮚ Count cfu/ml of Nutrient agar plates.

OBSERVATIONS:

25
NUMBER OF COLONIES

Number Of Colonies
Group No Cfu/ml
(No of colonies x Dilution Factor x Volume
10-5 10-6 Factor)
1 TNTC TNTC -
2 TNTC TNTC TNTC _
3 TNTC TNTC -
4 Less than 30 Less than 30 -
5 Less than 30 Less than 30
6 08 TNTC -
7 TNTC TNTC _
8
9

COLONIAL MORPHOLOGY

Group No. MEDIA DILUTION COLONIAL

CHARACTERSTICS
Group 01 Nutrient agar 10-5 Mucoid, circular,
offwhite colonies
10-6 Pinpointed,
translucent, circular
and regular colony
MacConkey’s agar 10-1 No growth
10-2 No growth

EMB agar 10-1 No growth

10-2 No growth
SDA 10-2 No growth
10-3 No growth

26
Group 02 Nutrient agar 10-5 Pin pointed, smooth, off
white colonies
10-6 Small, circular, off
white and raised
colonies
MacConkey’s agar 10-1 Pink coloured, small
and smooth colonies
10-2 Circular, pink and
small colonies
EMB agar 10-1 No growth
10-2 No growth
SDA 10-2 No growth
-3
10 No growth
Group 03 Nutrient agar 10-5 Large, creamy white,
raised, regular, circular
colony
10-6 Flat, white,
filamentous, irregular
MacConkey’s agar 10-1 No growth
10-2 No growth
EMB agar 10-1 No growth
10- No growth
SDA 10-2 No growth
10-3 No growth
-5
Group 04 Nutrient agar 10 Small, circular, entire,
white, elevated,
smooth, translucent
10-6 Pin pointed, circular,
entire, white, elevated,
smooth, translucent

27
 MacConkey’s agar 10-1 No growth
10-2 No growth
EMB agar 10-1 No growth
-2
10 No growth
SDA 10-2 No growth
10-3 No growth
Group 05 Nutrient agar 10-5 UC

Circular,creamy
white,small,elevated
10-6 Circular,creamy
white,small,elevated
MacConkey’s agar 10-1 No growth
10-2 NO growth
EMB agar 10-1 NO growth
10-2 NO growth
SDA 10-2 NO growth
10-3 NO growth
Group 06 Nutrient agar 10-5 Large, creamy white,
raised, regular, circular
colony
10-6 Flat, white,
filamentous, irregular
colony
MacConkey’s agar 10-1 No growth
10-2 No growth
EMB agar 10-1 No growth
10-2 No growth
SDA 10-2 No growth
10-3 No growth
Group 07 Nutrient agar 10-5 Circular, entire,
elevated, pinpoint,

28
 smooth, shiny, off-
white and opaque
-6
10 circular, entire,
elevated, moderate,
smooth, shiny off-
white and opaque
MacConkey’s agar 10-1 Circular, entire,
elevated, small,
smooth, shiny, off-
white and opaque
10-2 Circular, entire, flat,
pinpointed, smooth,
shiny, pink and opaque
EMB agar 10-1 No growth
10-2 No growth
SDA 10-2 No growth
10-3 No growth
Group 08 Nutrient agar 10-5 Small,white circular
,raised colonies
10-6 Small,white circular
,raised colonies
MacConkey’s agar 10-1 Small, pink ,circular
raised colonies
10-2 Small, pink ,circular
raised colonies
EMB agar 10-1 No growth
10-2 No growth
SDA 10-2 No growth
10-3 No growth
Group 09 Nutrient agar 10-5 UN, small, yellowish,

29
 elevated, smooth,
10-6 UN, Small, yellow,
elevated, smooth
MacConkey’s agar 10-1 No growth
10-2 Pink, smooth, flat,
regular, small
EMB agar 10-1 No growth
10-2 No growth
SDA 10-2 Olive green, large
cottony
10-3 Clear white, cottony

GRAM REACTION

GROUP# MEDIA DILUTION GRAM SHAPE ARRANGEMENT

STAINING
1 10-5 Gram rods Scattered
NUTRIENT AGAR positive
10-6 Gram Short rods scattered
positive
MAC’CONKEY’S AGAR 10-1 _ _ _

10-2 _ _ _
2 10-5 Gram Cocci Bunches
NUTRIENT AGAR positive
10-6 Gram cocci bunches
positive
MAC’CONKEY’S AGAR 10-1 Gram rods svattered
negative
10-2 Gram rods scattered
negative

30
3 10-5 Gram bacilli chain
NUTRIENT AGAR positive
10-6 Gram bacilli chain
positive
MAC’CONKEY’S AGAR 10-1 - - -

10-2 - - -
4 10-5 Gram bacilli chain
NUTRIENT AGAR positive
10-6 Gram bacilli chain
positive
MAC’CONKEY’S AGAR 10-1 - - -

10-2 - - -
5 10-5 Gram bacilli chain
NUTRIENT AGAR positive
10-6 Gram bacilli chain
positive
MAC’CONKEY’S AGAR 10-1 - _

10-2 - _
6 10-5 Gram Bacilli Scattered
NUTRIENT AGAR positive
10-6 Gram cocci In chains
positive
MAC’CONKEY’S 10-1 - - -
AGAR
10-2 - - -
7 10-5 Gram- Diplobacilli Scattered
NUTRIENT AGAR positive
10-6 Gram- short bacilli Scattered
positive
MAC’CONKEY’S 10-1 Gram- Diplococci Scattered
AGAR positive
10-2 Gram- Coccobacilli Scattered

31
 positive
8 10-5 Gram Cocci In chains
NUTRIENT AGAR positive
10-6 Gram cocci In chains
positive
MAC’CONKEY’S AGAR 10-1 Gram Short rods Scattered
negative
10-2 Gram Short rods Scattered
negative
9 10-5 Gram cocci Tetrads
NUTRIENT AGAR positive
10-6 Gram Short bacilli Chains
positive
MAC’CONKEY’S AGAR 10-1 Gram rods Scattered
negative
10-2 - - -

Wet Mounting

Group No. Media Dilution Microscopic characteristics

1 SDA 10-2 ___
10-3 ___
NUTRIENT 10-5 ___
AGAR
10-6 ___
2 SDA 10-2 ___
10-3 ____
NUTRIENT 10-5 ____
AGAR
10-6 ____
3 SDA 10-2 Aseptate hyphae
10-3 Aseptate hyphae
NUTRIENT 10-5 Septate hyphae

32
 AGAR 10-6 -
4 SDA 10-2 -
10-3 -
NUTRIENT 10-5 Gram positive rods in chain
AGAR
10-6 Gram positive rods in chain
5 SDA 10-2 -
10-3 -
NUTRIENT 10-5 Gram positive rods in chain
AGAR
10-6 Gram positive rods in chain
6 SDA 10-2 -
10-3 -
NUTRIENT 10-5 Gram positive, scattered bacilli
AGAR
10-6 Gram positive, cocci in chains
7 SDA 10-2 -
10-3 -
NUTRIENT 10-5 Gram-positive diplobacilli and scattered
AGAR short bacilli
10-6 Gram-positive scattered short bacilli
8 SDA 10-2 -------
10-3 -----
NUTRIENT 10-5 Gram positive cocci in chains
AGAR
10-6 Gram positive cocci in chains
9 SDA 10-2 Aseptate hyphae
10-3 Conidium with septate hyphae
NUTRIENT 10-5 Aseptate hyphae
AGAR
10-6 Septate hypha

Figures:

33
 Gram positive
Strptococci

Gram positive
scattered
Bacilli

Nutrient Agar(10-5) Nutrient Agar (10-6)

RESULTS:
On nutrient agar small pinpointed circular mucoid colonies were observed upon microscopy Gram
positive scattered bacilli were observed. MacConkey plate and SDA plates were observed
negative.

DISCUSSIONS:

Growth observed on the plates of nutrient agar was due to already contaminated sample or maybe
due temperature fluctuation during the bring of the sample which allow other bacteria to grow on
the sample. Another reason could be due to poor handling during weighing , dilution preparation
or spreading. No growth on the MacConkey, EMB and SDA plates could be due to the spreading
with the hot spreader so that all microbes in plated are killed.
REPORT:
In majority of the groups no colonies were seen on the MacConkey, EMD and SDA which
indicates poor handling or may be some of the plates were old ,there may be some problem in the
media preparation.

EXPERIMENT NO 3
34
 TO ANALYSE THE MICROBIAL QUALITY AND ASSURANCE OF
FROZEN CHICKEN NUGGETS OF DIFFERENT BRANDS.

THEORY:
The purpose of this experiment is to check the sterility of the frozen food products. The food
products that are available in the market should be of the quality that it’s fit for human
consumption. There are a large variety of frozen food items available in the market from frozen
meats to vegetables to snacks of all kind. All the frozen food commercially available should be
able to meet the quality standards set by the government. Not all producers strictly follow the
rules and regulations regarding the quality control of their products. So we will assess a number
of products of different companies to see which product is the most safe regarding its microbial
count. We used frozen chicken nuggets in this practical and as the product is essentially poultry
based we can suspect some coliforms, staph and some fungal spores. By comparing the different
products we will determine which of the product is the most sterilized and acceptable for human
consumption. The most common contaminants of frozen meats are Listeria monocytogenes.
There are also some psychotrophic organisms that are responsible for contamination. The chance
of contamination can appear at different stages of processing i.e. production, transportation,
distribution etc. Also the conditions at which the products are kept i.e. optimum temperature and
environment (clean and sterile) should also be controlled.

REQUIREMENTS:
1- Frozen product (kids chicken nuggets K&Ns)
2- 100ml saline in flask
3- 6 tubes with 9 ml saline in each.
4- 2 EMB agar plates
5- 2 Nutrient agar plates
6- 2 MacConkey agar plates
7- Jester pipette with sterile tips

GROUP NO NUGGETS BRAND NAMES

35
 1 Menu
2 Mon salwa
3 K&Ns
4 Man o salwa
5 K and Ns
6 Dawn
7 Menu
8 K and Ns
9 K and Ns

PROCEDURE:
1- Disinfect the table top and light the flame.
2- Measure 1 gram of the frozen food in sterile conditions and add it to the flask containing
100 ml saline. Distort the piece of food in the flask with a sterile wire loop or a needle so
that the meat is exposed.
3- Shake the flask well for 5 minutes then let it rest for 20 minutes so that all the solid
particles settle down at the bottom.
4- Make serial dilution of the mixture till10−6 .
5- Now transfer 0.1 ml from the dilution tubes 10-1 and 10−2 to EMB and MacConkey’s
agar. Spread with the help of a spreader.
6- Transfer 0.1 ml from dilutions tubes 10−5 and 10−6 into two Nutrient agar plates.
7- Incubate the plates at 37˚C for 24 hours.
8- The next day observe the results on the plates.
9- Count CFU per ml of Nutrient Agar plates.

OBSERVATIONS:

36
NUMBER OF COLONIES

Number Of Colonies
Group No Cfu/ml
10-5 10-6 (No of coloniesxDilutionFactorxVolume
Factor)
1 UC UCUU UC _
2 UC UCUU UC _
3
4 Less than 30 Less than 30 -
5 Less than 30 Less than 30 -
6 07 05 -
7 TLTC 56 5.6x108 cfu/mL
8 UC UC -
9

COLONIAL MORPHOLOGY

Group No. MEDIA DILUTION COLONIAL

CHARACTERSTICS
-5
Group 01 Nutrient agar 10 Elevated, small and off
white colonies
-6
10 Shiny, off white,
circular colonies
MacConkey’s agar 10-1 Pink, irregular, flat and
medium colonies
10-2 No growth
EMB agar 10-1 No growth
10-2 No growth
-5
Group 02 Nutrient agar 10 Irregular, white, small,
raised colonies
10-6 White, pin
pointed,circular and

37
 elevated colony.
MacConkey’s agar 10-1 Pink, large, elevated,
colonies
10-2 No growth
EMB agar 10-1 No growth
10-2 No growth
Group 03 Nutrient agar 10-5 White,spreaded large
colonies,irregular edges
10-6 White,spreaded large
colonies,irregular
edges
-1
MacConkey’s agar 10 Pink,circular,large,elev
ated,regular margin
-2
10 Yellow,Small,circular,el
evated,regular
EMB agar 10-1 No growth
10- No growth
-5
Group 04 Nutrient agar 10 Pin pointed, white,
entire, circular,
elevated, transparent
10-6 Pin pointed, white,
entire, circular,
elevated, transparent
-1
MacConkey’s agar 10 No growth
10-2 No growth
EMB agar 10-1 No growth
10-2 No growth

Group 05 Nutrient agar 10-5 White,spreaded large

colonies,irregular
edges
-6
10 White,spreaded large

38
 colonies,irregular
edges
-1
MacConkey’s agar 10 Pink,circular,large,elev
ated,regular margin
-2
10 Yellow,Small,circular,el
evated,regular
EMB agar 10-1 No growth
-2
10 No growth
-5
Group 06 Nutrient agar 10 Irregular, large, white,
lobate margin, centre
elevated and rough
colony
10-6 Irregular, lobate,
translucent, flat,
opaque and large
colony
MacConkey’s agar 10-1 Gummy, irregular,
elevated andlarge
colony
10-2 No growth
EMB agar 10-1 No growth
10-2 No growth
Group 07 Nutrient agar 10-5 Circular, entire,
elevated, small,
smooth, shiny, off-
white, opaque.
10-6 Circular, entire,
elevated, medium,
smooth, shiny, off-
white and opaque
MacConkey’s agar 10-1 No growth
10 -2 Irregular, lobular,
flat, medium,
smooth, shiny, off-

39
 white and
translucent.

EMB agar 10-1 No growth

10-2 No growth
Group 08 Nutrient agar 10-5 Off white, flat, small,
shiny, raised colonies.
10-6 Large, shiny, raised,
mucoid, off white
colonies.
-1
MacConkey’s agar 10 No growth
-2
10 Pink, Large, mucoid,
raised, shiny colonies.
-1
EMB agar 10 No growth
-2
10 No growth
Group 09 Nutrient agar 10-5 UN, yellow, flat,
irregular, smooth,
small
10-6 UN, irregular, off white,
large, flat
MacConkey’s agar 10-1 UN, pink, small,
circular, elevated
10-2 UN, same as above
EMB agar 10-1 No growth
10-2 No growth

GRAM REACTION

GROUP# MEDIA DILUTION GRAM SHAPE ARRANGEMENT

STAINI
NG
1 10-5 Gram coccobacilli scattered
NUTRIENT AGAR positive

40
 10-6 Gram baciilli scattered
postive
MAC’CONKEY’S AGAR 10-1 Gram rods scattered
negative
10-2 _ _ _
2 10-5 Gram bacilli Scattered
NUTRIENT AGAR positive
10-6 Gram bacilli Scattered
positive
MAC’CONKEY’S AGAR 10-1 Gram cocci bunches
negative
10-2
3 10-5 Gram Bacilli scattered
NUTRIENT AGAR positive
10-6 Gram Bacilli scattered
positive
MAC’CONKEY’S AGAR 10-1 - -

10-2 - -
4 10-5 Gram Bacilli scattered
NUTRIENT AGAR positive
10-6 Gram Bacilli scattered
positive
MAC’CONKEY’S AGAR 10-1 - - -

10-2 - - -
5 10-5 Gram Bacilli scattered
NUTRIENT AGAR positive
10-6 Gram Baclli scattered
positive
MAC’CONKEY’S AGAR 10-1 Gram rods scattred
negative
10-2 Gram cocci bunches
negative
6 10-5 Gram Bacilli and Scattered
NUTRIENT AGAR positive coccobacilli

41
 10-6 Gram Bacilli Scattered, bunches,
positive diplobacilli
MAC’CONKEY’S AGAR 10-1 GramB bacilli,Scattered, tetrad, bunches
negative cocci,
coccobacilli
10-2 - - -
7 10-5 Gram- Bacilli Scattered
NUTRIENT AGAR negative
10-6 Gram- Cocco- Scattered
positive bacilli
MAC’CONKEY’S AGAR 10-1 - - -

10-2 Gram- cocci Scattered

positive
8 10-5 Gram Cocci Chain
NUTRIENT AGAR positive
10-6 Gram Rods Chain
positive
MAC’CONKEY’S AGAR 10-1 - - -

10-2 Gram Rods Scattered

negative
9 10-5 Gram Rods Scattered
NUTRIENT AGAR negative
10-6 Gram Rods Pairs
positive
MAC’CONKEY’S AGAR 10-1 Gram Short rods Scattered
negative
10-2 Gram Short rods Scattered
negative

42
FIGURE:

Gram positive
Scattered
coccobacilli

Gram positive
Scattered
Bacilli

Nutrient Agar (10-5) Nutrient Agar (10-6)

Gram negative
Coccobacilli in
bunches

Gram negative
Bacilli in tetrad

Mac Conkey’s Agar (10-1)

43
RESULT:

Uncountable colonies were observed for cfu/ml. Pink irregular flat and medium sized colonies
were observed on MacConkey plate and Gram negative bacilli were observed on MacConkey’s
agar and elevated small off-white colonies observed on NA plates Gram positive bacilli were
observed on Nutrient Agar and no growth was observed on EMB agar.

DISCUSSSION:

Numerous microbial growth was observed may be because of improper handling and poor and
insufficient hygienic condition in production and processing of chicken meat products.

REPORT:

Results indicated contamination and showed that the nuggets prepared from these brands are not
suitable for human consumption. It is ensured to implement good manufacturing practices and
proper guidance and trainings for workers about hygiene safety and quality assurance during
manufacturing and handlings of the products.

44
 EXPERIMENT NO 4
EXPERIMENT NO 3
ANALYSIS OF MICROBIAL QUALITY ASSURANCE OF FROZEN
CHOCOLATES.

THEORY:
Chocolate is a raw or processed food produced from the seed of the tropical Theobroma cacoa
tree. The seeds of the cacao tree have an intense bitter taste, and must be fermented to develop
the flavor. After fermentation, the beans are dried, then cleaned, and then roasted, and the shell is
removed to produce cacao nibs. The nibs are then ground to cocoa mass, pure chocolate in rough
form. Because this cocoa mass usually is liquefied then molded with or without other
ingredients, it is called chocolate liquor. The liquor also may be processed into two components:
cocoa solids and cocoa butter. Cocoa solids contain alkaloids such as theobromine and
phenethylamine, which have physiological effects on the body. It has been linked to serotonin
levels in the brain. Some research found that chocolate, eaten in moderation, can lower blood
pressure. The presence of theobromine renders chocolate toxic to some animals. Chocolate has
become one of the most popular food types and flavors in the world.Chocolate is commonly used
as a coating for various fruits and fillings, such as cherries.

REQUIREMENTS:
1- Chocolate sample

2- Nutrient agar plates 3 per group)

3- E.M.B agar (3 per group)

4- MacConkey’sagar (3 per group)

5- 100ml flask, 6 blanks (containing 9ml distill water)

45
6- Sample dairy milk chocolate

Group no. Chocolate brand

1 perk
2 Dairy milk
3 Dairy milk
4 Perk
5 Dairy milk
6 Sonnet
7 Whispy
8 Dairy milk
9

PROCEDURE:
2. Take a small piece of chocolate put it in flask(containing 100ml sterile distill water)
3. Shake the flask during shaking break the piece of chocolate.
4. Take 1ml from the flask and add it in the blanks (containing 9ml distill water).
5. Make serial dilutions (10-1 to 10-6) respectively.
6. Take 0.1 ml from 10-4, 10-5, 10-6 and spread on Nutrient agar plates while take 0.1 ml
from 10-1, 10-2 and 10-3 and spread on EMB and MacConkeys plate
7. Incubate all the plate of NA, EMB, and MacConkey’s agar at 37°C for 24 hours.
8. Note down the colonial characteristics, cultural characteristics and morphology by
performing the microscopy.

46
OBSERVATION:

Group MEDIA DILUTION CULTURAL MICROSCOPY

No. CHARACTERISTICS
Group Nutrient agar 10-4 Small, white, elevated Gram positive
01 colonies scattered bacilli
10-5 No growth _
10-6 No growth _
MacConkey’s 10-1 No growth _
agar 10-2 No growth _

10-3 No growth _

EMB agar 10-1 No growth _

10-2 No growth _
10-3 No growth _
Group Nutrient agar 10-4 Pinpointed ,circular, Gram positive
02 mucoid and off white cocci in chain
colonies
10-5 Circular, large ,white Gram positive
and rough colonies diplococcic
10-6 Small, white, mucoid Gram positive
colonies cocci in bunches
MacConkey’s 10-1 No growth _
agar 10-2 No growth _

10-3 No growth _

EMB agar 10-1 No growth _

47
 10-2 No growth _

10-3 No growth _

Group Nutrient agar 10-4 Circular, large, creamy

03 white colony
10-5 Circular, large, creamy
white colony
10-6 Circular, large, creamy
white colony
-1
MacConkey’s 10 No growth
agar 10-2 No growth
10-3 No growth

EMB agar 10-1 No growth

10- No growth
10-3 No growth

Group Nutrient agar 10-4 Small, circular, white, Gram positive

04 regular, elevated scattered bacilli
-5
10 Small, circular, white, Gram positive
regular, elevated scattered bacilli
10-6 Small, circular, white, Gram positive
regular, elevated scattered bacilli
MacConkey’s 10-1 No growth -
agar 10-2 No growth -
10-3 No growth -
EMB agar 10-1 No growth -
10-2 No growth -
10-3 No growth -
Group Nutrient agar 10-4 Flat, regular, large, Gram postive
05 raised,white colony scattered bacilli
10-5 Flat, regular, large, Gram postive
raised, white colony scattered bacilli

48
 10-6 Flat, regular, large, Gram postive
white colony scattered bacilli
-1
MacConkey’s 10 No growth -
agar 10-2 No growth -
10-3 No growth -
EMB agar 10-1 No growth -
10-2 No growth -
10-3 No growth -
Group Nutrient agar 10-4 Flat, regular, large, Gram-positive,
06 white colony scattered bacilli,
diplobacilli, in
bunches and in
palisade
arrangement
-5
10 Circular, large, creamy Gram positive,
white colony streptobacilli,
diplobacilli and
coccobacilli in
chains
-6
10 Circular, medium, Gram positive,
regular, raised, convex, scattered cocci,
creamy white colony diplococci,
streptococci,
staphlococci
-1
MacConkey’s 10 No growth -
agar 10-2 No growth -
10-3 No growth -
-1
EMB agar 10 No growth -
-2
10 No growth -
-3
10 No growth -
Group Nutrient agar 10-4 Circular, entire, Gram-positive

49
07 moderate, flat, opaque, scattered bacilli.
creamy white, smooth
and dull.
10 -5
Filamentous, rhizoid, Gram-negative
small, raised, opaque, scattered
white, rough and dull. coccobacilli
10 -6
Filamentous, rhizoid, Gram-positive
small, convex, diplococci.
translucent, off-white,
smooth and shiny.
MacConkey’s 10-1 No growth -
agar 10-2 No growth -
10-3 No growth -
EMB agar 10-1 No growth -
10-2 No growth -
10-3 No growth -
Group Nutrient agar 10-4 UC, pinpointed, Gram positive
08 offwhite, circular and cocci in
entire scattered.
10-5 UC, pinpointed, Gram positive
offwhite, circular and cocci in chain
entire and scattered.
10-6 UC, small, colorless, Gram positive
circular, rough and cocci in
entire bunches.
-1
MacConkey’s 10 No growth -
-2
agar 10 No growth -
10-3 No growth -
EMB agar 10-1 No growth -
10-2 No growth -

50
 10-3 No growth -

Group Nutrient agar 10-4 UN, light yellow, Gram positive

09 creamy, pinpoint, cocci in bunches
elevated, circular
-5
10 UN, round, circular, Gram positive
smooth, creamy white, cocci in
pin head, elevated scattered
10-6 UN, pin head, off white, Gram positive
creamy, elevated, cocci in bunches
regular, smooth
-1
MacConkey’s 10 No growth -
agar 10-2 No growth -
10-3 No growth -
EMB agar 10-1 No growth -
-2
10 No growth -
-3
10 No growth -

Figure:

Gram positive
scattered bacilli

Nutrient Agar (10-4)

51
RESULT:
On the nutrient agar dilution 10-4 small white elevated colonies were seen. On Gram staining
Gram positive scattered bacilli were observed. While rest of the plates showed no growth.
DISCUSSON:
Many plates showed no growth and on only one plate colonies were observed on the nutrient
agar which indicates that maybe the chocolate was not upto the mark it may contains some
organisms, Good manufacturing practices may be not adopted during processing. On more
reason could be that during the experiment aseptic condition was not maintained and during
weighing sample was openly placed in the machine which maybe a source of contamination. No
growth on MacConkey and EMB may be due to the usage of the spreader or not shaking the tube
during making dilution so that the organisms may settled down
REPORT:
On plates of all groups no colonies were observed in MacConkey and EMB agar which may
indicated that there was may be some problem in the media. Otherwise on NA many colonies
were obtained in whole class.

52
 EXPERIMENT NO 5

TO CHECK THE VALIDITY OF AUTOCLAVES OF DIFFERENT LABS

OF MICROBIOLOGY DEPARTMENT.

THEORY:

An autoclave is a device used to sterilize equipment and supplies by subjecting

them to high pressure saturated steam at 121 °C for around 15–20 minutes
depending on the size of the load and the contents. It was invented by Charles
Chamber land in 1879.

USES:

Autoclaves are widely used in microbiology, medicine, tattooing, body piercing,

veterinary science, mycology, dentistry, chiropody and prosthetics fabrication.
They vary in size and function depending on the media to be sterilized.

53
A new generation of waste converters is capable of achieving the same effect
without a pressure vessel to sterilize culture media, rubber material, gowns,
dressing, gloves, etc. It is particularly useful for materials which cannot withstand
the higher temperature of a hot air oven.

Autoclave quality assurance:

Sterilization bags often have a "sterilization indicator mark" that typically darkens
when the bag and its contents have been adequately processed. Comparing the
marks on an unprocessed bag (L) and on a bag that has been properly cycled (R)
will reveal an obvious visual difference. There are physical, chemical, and
biological indicators that can be used to ensure that an autoclave reaches the
correct temperature for the correct amount of time.

Chemical indicators on medical packaging and autoclave tape change color once
the correct conditions have been met, indicating that the object inside the package,
or under the tape, has been appropriately processed. Biological indicators contain
spores of a heat-resistant bacterium, Geobacillus stearothermophilus. If the
autoclave does not reach the right temperature, the spores will germinate when
incubated and their metabolism will change the color of a pH-sensitive chemical.
Some physical indicators consist of an alloy designed to melt only after being
subjected to a given temperature for the relevant holding time. If the alloy melts,
the change will be visible.

MATERIALS:

⮚ BHI broth 60ml

⮚ Nutrient agar 1400ml
⮚ Bacillus subtilis slants 2
⮚ Mccarty’s tubes 6 per group

54
 ⮚ Sterile saline 1000ml
⮚ Sterile plates 14 per group
⮚ Spreader with 70% Alcohol 2
⮚ Micropipettes.

METHOD:

1. The culture of Bacillus subtilis was inoculated in given 60ml BHI broth in
aseptically conditions.
2. The flask was incubated for 48 hours.
3. 5 tubes were taken which contained 6ml BHI broth.
4. Mccarty’s tubes were marked as according their dilution factors (10-1 to 10-6).
5. Serial dilution was performed from BHI broth contain culture by transferring
1ml from the broth to the tube marked as 10-1.
6. Nutrient agar plates were marked respected to the dilution factor.
7. 0.1ml from each dilution was transferred at their respected Nutrient agar
plates.
8. Incubate all the plates at 37C for 24 hours.
9. Remaining BHI broth was divided into three fractions (12ml in each flask).
10.Each flask was autoclaved in different labs.
11.Same process was performed and the microbial colonies on Nutrient agar
plates were counted.
55
 12.Cfu for each dilution was calculated.

OBSERVATIONS:

Grou DILUTION NO. OF COLONIES

p No.
BEFORE AUTOCLAVES AFTER AUTOCLAVES
1 10-2 UC UC
-3
10 UC UC
10-4 UC UC
10-5 UC No growth
2 10-2 UC UC
10-3 UC UC
-4
10 UC No growth
-5
10 UC UC
3 10-2 UC UC
10-3 UC UC
10-4 UC UC
10-5 UC UC
-2
4 10 UC UC

56
 10-3 UC UC
10-4 UC UC
10-5 UC UC
-2
5 10 UC UC
-3
10 UC UC
10-4 UC UC
10-5 UC UC
6 10-2 UC UC
10-3 UC UC
-4
10 UC UC
-5
10 UC UC
7 10-2 UC UC
10-3 UC UC
10-4 UC UC
10-5 UC UC
-2
8 10 UC UC
-3
10 UC UC
10-4 UC No growth
10-5 UC No growth
9 10-2 UN UN
10-3 UN UN
-4
10 No growth UN
-5
10 UN UN

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RESULTS:
From all dilution 10-2,10-3, 10-4 , uncountable colonies were present in both cases i.e before and
after autoclave. While 10-5 plate after autoclave no growth was observed.

DISCUSSION:
For testing autoclave bacillus spores were used which is supposed to be most resistant. The
amount of colonies present on the plates indicate the inefficiency of the autoclave. As we know
spores are heat resistant, so colonies on the plate is showing that the autoclave didn’t reach the
maximum temperature required to kill the spores hence after autoclave they survive and
germinate when grown in nutrient agar plate.
On other reason for the growth is that during the transfer of the autoclaved culture, aseptic
condition was not maintained and contamination results in the large no of colonies in the plates

REPORT:
Out of nine group, 4 groups have no growth in their plates after autoclave which shows that
maybe the handling error was done in rest of the plates hence growth appeared.

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 EXPERIMENT NO 6
EXPERIMENT NO 3
TO OBSERVE THE MICROBIAL LOAD OF DIFFERENT
HOUSEHOLDS AND ELECTRONIC GADGETS.

THEORY:

Contamination of different objects by potential pathogenic microorganisms is of public health

importance as contaminated materials can be possible sources of from hand to hand are of
considerable likelihood to be contaminated with disease-causing microorganisms especially if
handled with unclean hands or kept in dirty or contaminated surroundings. The environment
plays important role in transmission of microbial agents to humans, with many environmental
materials serving as vehicles.

Microorganisms are ubiquitous in nature. It is equally important to sanitize the places, and items
commonly used within a typical house. Variety of microorganisms (e.g. bacteria and fungi)
would be found from common household surfaces, even when those surfaces are regularly
cleaned, and that a strong disinfecting agent, such as ethanol (70%) should eliminate the majority
of these microorganisms. The appearance of microorganisms was observed on nutrient agar
plates which were swabbed.

REQUIREMENT:

1. Sterile test tubes with 5ml P.B.S buffer solution.

2. Nutrient agar plates, MacConkey’s agar plates.

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PREPARATION OF PBS BUFFER:

REAGENTS USE:

1. NaCl 8grams
2. KCl 0.2grams
3. Na2HPO4 1.44grams
4. KH2PO4 0.24grams

To prepare 1 liter solution of PBS,add above reagents in 800 ml of distilled water.Adjust P H at

7.4-7.2 with the help of NaOH then add 200 ml of distilled water to make the volume up to 1
liter. Check the PH again and adjust in between 7.4---7.2 by adding NaOH if necessary.Store PBS
at room temperature after Autoclaving.

PROCEDURE:
1. Collect the samples with the help of sterile cotton swabs from different places and
gadgets.
2. Make a lawn on the given Media plates.
3. Incubate the plates at 37C for 24 hrs

.OBSERVATIONS:

Group MEDIA Plate COLONIAL MICROSCOP

No. No. CHARACTERISTICS Y
1 NUTRIENT Plate 1 White, large and opaque Gram positive
AGAR colonies rods
Plate 2 White, large opaque colonies Gram positive
rods
Plate 3 Large, irregular and white Gram positive
colonies rods in chains
MacConkey’s Plate 1 No growth _
AGAR Plate 2 No growth _
2 NUTRIENT Plate 1 White, large opaque colonies Gram positive
AGAR rods
Plate 2 Irregular,flat and large Gram positive

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 colonies rods
Plate 3 Pinpointed, offwhite and Gram positive
convex colony diplococci
MacConkey’s Plate 1 No growth _
AGAR Plate 2 No growth _
3 NUTRIENT Plate 1 Pin pointed, white, entire, Gram positive
AGAR circular, elevated, rods
transparent
Plate 2 Pin pointed, white, entire, Gram positive
circular, elevated, rods
transparent
Plate 3 Pin pointed, white, entire, Gram positive
circular, elevated, rods
transparent
MacConkey’s Plate 1 No growth -
AGAR Plate 2 No growth -
4 NUTRIENT Plate 1 Large, white, flat, irregular Gram positive
AGAR colony bacilli in chains
Plate 2 Large, white, flat, irregular Gram positive
colony bacilli in chains
Plate 3 Large, white, flat, irregular Gram positive
colony bacilli in chains
MacConkey’s Plate 1 No growth -
AGAR Plate 2 No growth -
5 NUTRIENT Plate 1 White large flower like Gram positive
AGAR colonies rods
Plate 2 White large flower like Gram positive
colonies rods
Plate 3 White large flower like Gram positive
colonies rods
MacConkey’s Plate 1 No growth
AGAR Plate 2 No growth

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6 NUTRIENT Plate 1 Large, irregular ,white and Gram positive,
AGAR flat colony bacilli in chains
and in scattered
arrangement
Gram negative
coccobacilli with
polar ends
Plate 2 Medium, circular, smooth, Gram positive,
slightly elevated, yellow bacilli in palisade
colored colony and scattered
arrangement
Plate 3 Small, pink, circular, Gram positive,
elevated, smooth colony coccobacilli with
swollen ends
MacConkey’s Plate 1 Medium, smooth, regular, Gram positive,
AGAR red colored, circular, slightly diplococci, cocci
elevated colony in bunches,
scattered and
tetrad
arrangement
Plate 2 Medium, pink, irregular Gram positive,
margin, centered circular, diplobacilli,
slightly elevated colony bacilli in chains,
palisade and
scattered
arrangement
7 NUTRIENT Plate 1 White, circular, entire, Gram-positive
AGAR convex, shiny, smooth, threads.
opaque and moderate
Plate 2 White, circular, entire, Gram-positive
convex, shiny, smooth, scattered cocci.

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 opaque and moderate.
Plate 3 Irregular, undulate, flat, Gram-positive
shiny, skin color, rough, scattered cocci.
translucent and large.
MacConkey’s Plate 1 No growth -
AGAR Plate 2 No growth -
8 NUTRIENT Plate 1 Large, round, creamy, Gram positive
AGAR convex, entire colonies bacilli in chains
Plate 2 Large, round, creamy, Gram positive
convex, entire colonies. cocci in chains
Plate 3 No growth -
MacConkey’s Plate 1 Pink, Large, raised, mucoid, Gram negative
AGAR shiny colonies. rods, scattered.
Plate 2 No growth -
9 NUTRIENT Plate 1 UN, Off white, large, Gram negative
AGAR regular, flat, white boundry diplo bacilli
scattered
Plate 2 UN, Same as above same as above
Plate 3 UN, Same as above Same as above
MacConkey’s Plate 1 Gummy mucoid, irregular, Gram positive,
AGAR light pink, elevated large scattered, cocci
Plate 2 Yellow, pin head, regular, Gram positive
elevated diplococci

63
FIGURE:

Gram positive
Scattered
Bacilli

Gram
positive
Streptobacilli

Plate 01 (N.A) Plate 02 (N.A)

Gram positive bacilli

Plate 03 (N.A)

64
RESULTS:
White large opaque colonies were observed on all nutrient agar plates while no growth was observed on
MacConkey plates.

DISCUSSION:
Microorganisms are ubiquitous that is they are found on everywhere as normal flora on environment
and on human skin as well some are pathogenic and some are nonpathogenic but mostly gram positive
organisms were found. Reason why no growth observed on MacConkey’s plate is may be because
sampling was done from refrigerator and that environment is not suitable for gram negative microbes.

REPORT:

Microorganism diversity and abundance are determined by the biogeographical habitat they
occupy . In this study all the gadgets were contaminated with microorganisms so there should be
use of disinfect to clean the surface and laboratories as well as limited use of sanitizer is also a
good hygienic practice to keep away from microbes.

65