“ISOLATION AND IDENTIFICATION OF MICROBES RESPONCIBLE

FOR FOOD SPOILAGE.”

PROJECT REPORT SUBMITTED TO

DR. B.R. AMBEDKAR UNIVERSITY, AGRA

IN PARTIAL FULLFILLMENT OF

MASTER OF SCIENCE DEGREE IN MICROBIOLOGY

UNDER SUPERVISION

DR. SUDEEP TIWARI

HEAD DEPARTMENT OF LIFESCIENCE

IIMT ALIGARH

SUBMITTED BY

Shruti Saxena

Roll No.161556251020

M.Sc. IV Sem. Biotechnology

INSTITUTE OF INFORMATION MANAGEMANT AND TECHNOLOGY, ALIGARH.

 DECLARATION

I am Shruti Saxena student of M.Sc. biotechnology final year hereby declare the
dissertation work report on ISOLATION AND IDENTIFICATION OF
MICROBES RSPONSIBLE FOR SPOILAGE FOOD is original and no part of
this work has been submitted for any other degree or diploma . all the given
information and works are true to my sense and knowledge

DATE:

PLACE
ACKNOWLEDGMENT

I would like to express my deep sense of gratitude and indeptedness to Dr. Sudeep
kumar Tiwari Head department of Lifesciences for introducing the present topic
and for his inspiring guidance , constructive and valuable suggestion throughout
this work .his able knowledge and expert supervision with unanswerving patience
fathered my wrok at every stage ,for without his warm affection and
encouregment , the fulfilment of the task would have been very difficult .

I am genuinely appreciative of all my Friends for their suggestions and

moral support during my work.

Last, but not the least, I would like to thank the Almighty GOD and my
parents, whose dedicated and untiring efforts towards me has brought me at
this stage of my life.

CONTENTS
SL. NO PARTICULARS PAGE NO.

A Abstract I

1 Introduction 1-6

2 Review of literature 7-16

3 Aims and objectives 17

4 Material and methods 18-22

5 Results 23-34

6 Discussion 35-36

7 Conclusion 37

8 References 38-41
 INTRODCTION

There is potential for a wide range of vegetables and fruits products to become
contaminated with microorganisms. The range of microorganisms associated with
outbreaks linked to fresh produce encompasses bacteria, viruses and
parasites. The products of most concern are sprouted seeds and unpasteurized
juices. Most of the reported outbreaks have been associated with bacterial
contamination, particularly members of the Enterobacteriaceae. Of these,
Salmonella and Escherichia coli in sprouted seeds and fruit juices are
of particular concern. Outbreaks linked to protozoa e.g. Cryptosporidium,
Cyclospora, Giardiaetc have been associated more with fruits than with
vegetables. Protozoa and viruses are most often associated with
contaminated water or food handlers. Fruits and vegetables normally carry a
non-pathogenic epiphytic microflora (Ray, 2004).

There is rapid progress in the field of chemical detection technology; little of this
technology appears to have found application in estimation of the remaining
shelf life of foods and early detection of spoilage. Predictive microbiology aims
to summaries the probable behavior of specific spoilage organisms and the
progression of spoilage processes in foods. The quantitative knowledge
generated in the field of predictive microbiology provides a sound basis for
the rational development of devices with which to monitor loss of product shelf
life during storage, distribution and retail sale. To predict remaining shelf life
accurately it is necessary, however, to consider the microbial ecology of the
food system (McMeekin et al., 1996).

A Bacillus strain was isolated from spoiled apple juice. This strain was
acidophilic with a growth range between pH 2.5 and 5.5. Lipid analysis
demonstrated the occurrence of omega clohexane fatty acids and hopanoids. As
these cell constituents have among bacilli been found only in
Bacillus acidocaldarius strains, isolated microorganism seems to be related to
this species. The organism could be a threat to fruit juices during storage at
elevated temperatures, greater than or equal to 26˚C because its spores were able
to survive pasteurization conditions (Cerny et al., 1984). Species most commonly
implicated in fruit and fruit product disintegration are Byssochlamy sfulva,
Byssochlamys nivea, Neosartorya fischeri, Talaromyces flavus and
Eupenicillium brefeldianum. They can survive heat treatments used for fruit
processing and can grow and spoil the products during storage at
room temperature, which results in great economic losses. Besides spoilage, the
heat-resistant molds produce a number of toxic secondary metabolites, such as
byssotoxin A; byssochlamic acid; the carcinogen, patulin, the tremorgenic
substances, fumitremorgin A and C, and verruculogen; fischerin, which caused
fatal peritonitis in mice; and eupenifeldin, a compound possessing cytotoxicity
as well as in vivo antitumor activity (Tournas., 1994). Strain A. acidoterrestris
was identified as the causative agent in spoilage of commercially pasteurized
apple juice.

Alicyclobacillus sp. is soil borne bacteria, and do not strictly require thermophilic
and acidic environments. Alicyclobacillus sp. possesses several distinct
characteristics; the major one is their ability to survive
commercial pasteurization processes and produce off-flavours in fruit juices.
Guaiacol and halophenols were identified as the offensive smelling agent in
many Alicyclobacillus sp. related spoilage. The Alicyclobacillus sp was
identified as potential spoilage bacteria in fruit juices (Chang et al.,
2004). Spoilage of milk products by Pseudomonas fragi is characterized by the
production of a strawberry like odour. Ethyl esters of butyric, hexanoic, and 3-
methylbutanoic acid were shown to be the major contributors to the odour on
the basis of their relative concentration in the headspace (Cormier et al., 1991).
Proteolysis during storage of ultra high temperature skim and whole milks
processed by either direct or indirect systems has been studied.

All the proteolysis indices determined increased activities of both native milk
proteinase and proteinases of bacterial origin were observed in skim ultra high
temperature milks. The different behavior of ultra high temperature skim and
whole milks on storage would have to be taken into account in establishing
the process conditions (Lopez et al., 1993). Proteolysis of milk proteins is
attributed to both native proteases and the proteases produced by psychrotrophic
bacteria during storage of fresh raw milk. These proteases cause beneficial
or detrimental changes, depending on the specific milk product. Plasmin, the
major native protease in milk, is important for cheese ripening.

A microbial protease from a psychrotrophic microorganism can indirectly

increase plasmin levels in the casein curd. This relationship between the plasmin
system and microbial proteases in milk provides a means to control levels of
plasmin to benefit the quality of dairy products (Nielsen, 2002).

The reason for the reported difference in spoilage behaviour of skim and whole
pasteurized milks was investigated. The rates of growth of psychrotrophic
bacteria were not significantly different in the two milk and the bacterial types,
all Pseudomonads, present at spoilage were also similar. However, when
representative spoilage organisms were cultured into freshly pasteurized skim
and whole milks, skim milks exhibited predominantly bitter flavours while whole
milk showed mostly sour flavours. The different spoilage behaviors can be
largely explained by greater proteolysis in skim milk than in whole milk, caused
by higher production of protease and greater susceptibility of the protein to
protease attack (Deeth et al.,2002).

It is estimated one-fourth of the harvested fruits and vegetables is spoiled before

consumption. Spoilage of fresh fruits and vegetables usually occurs during storage
and transport. Vegetables and fruits reach the consumer as fresh, dried, frozen,
fermented, pasteurized, or canned. Contamination may take place during
harvesting, handling, transportation or storage unless proper hygienic conditions
were not maintained. Mechanical damage may increase the susceptibility to decay
and the growth of microorganisms may take place. Washing process in
contaminated water may moisten surfaces enough to permit entry and growth of
organisms. Storage in contaminated containers, use of contaminated dressing
materials, possible contact with decayed products, unhygienic handling, fly
infestation etc. will also cause an accelerated rate of spoilage.

The deterioration of raw vegetables and fruits may result from physical factors,
action of their enzymes, microbial action, or combinations of all these.
Microbial spoilage in fruits and vegetables varies not only with the kind of fruit
or vegetables but also to some extent with the variety. Microbial spoilage may
due to (1) plant pathogens acting on stems, leaves, flowers, or root of the plant,
on the fruit or other special parts used as foods; (2) saprophytic organisms,
which may be secondary invaders after the action of plant pathogen or may
enter a healthy fruit or vegetable, as in the case of various ?rots? or grow on its
surface, as when bacteria multiply on moist piled vegetables At times a
saprophyte may succeed the pathogen or a succession of saprophytes may be
involved in the spoilage. The most commonly occurring types of microbial
spoilage are as follows:

1. Bacterial soft rot, caused by Erwinia crtatowa and related species, which
are fermenters of pectins, Pseudomonas marginalis, Clostridium and
Bacillus spp. Have also been associated with these rots. It results water-
soaked appearance, a soft, mushy consistency, and often a bad odor.
 2. Gray mold rot: caused by species of Botrytis, eg: B.cinerea, which is
favored by high humidity and warm temperature.
3. Rhizopus soft rot: caused by species Rhizopus, eg R.stolonifer. A rot results
that often is soften and mushy. The cottony growth of the mold with small,
black dots of sporangia often covers masses of the foods.
4. Anthracnose, usually caused by Colletotrichum lindemuthianum, C.
coccodes and other species. The defect is a spotting of leaves and fruit or
seedpods.
5. Alternaria rot, caused by Alternaria tenuis and other species. Areas become
greenish-brown early in the growth of the mold and later turn to brown or
black spots.
6. Blue mold rot: caused by species of Penicilfium digitatum and other
species. The bluish-green color that gives the rot its name results from the
masses of spores of the mold.
7. Downy mildew, caused by species of Phytophthora, Bremia, and other
genera. The molds grow in white, woolly masses.
8. Watery soft rot caused chiefly by Sclerotinia sclerotiorum, is found mostly
in vegetables.
9. Stem-end rots, caused by species of molds of several genera, e.g., Diplodia,
Alternaria, Phomopsis, Fusarium, and others, involve the stem ends of
fruits.
10. Black mold rot, caused by Aspergillus niger. The rot gets its name from the
dark-brown to black masses of spores of the mold, termed "smut".
11. Black rot, often caused by species of Alternaria but sometimes of
Cera?tostomella, Physalospora, and other genera.
12. Pink mold rot, caused by pink-spored Trichothecium roseum.
13. Fusarium rots, a variety of types of rots caused by species of Fusarium.
14. Green mold rot, caused usually by species of Cladosporium but some?times
by other green-spored molds, e.g., Trichoderma.
15. Brown rot, caused chiefly by Sclerotinia (Monilinia fructicola) species.
16. Sliminess or souring, caused by saprophytic bacteria in piled, wet, heating
vegetables.

Bacteriocins are the proteinaceous toxins by bacteria to inhibit the growth of

similar or closely related bacterial strain. They are typically considered to be the
narrow spectrum antibiotics through this has been debated. They are
phenomenologically ana,logous to yeast and paramecium killing factors and are
structurally functionally and ecologically divers.
Bacteriocins were first discovered bt A.Gratia in 1925. He was involve in process
of searching for the ways to kill the bacteria ,which also resulted in the
development of antibiotics and discovery of bacteriophage,al within span of few
years he called his first discovery a colicin because it killed E.coli.

Bacetriocins is small molecule produced by bacteria that inhibits closely related

strains. Usually a peptide or a protein varying sized chains of Amino Acids. These
compounds are of interest both in studies of basic microbiology and for the
preservation of food and enhancement of human health.

These toxins are produced by a wide range of bacteria. The first to be studied was
that Escherichia coli, bacterium that lives in human intestines and is frequently
used in laboratory work. The bectriocins fron E.Coli are called colicins (formerly
called as colicines, meaning coli killers). They are longest studied bacteriocins.

They are several ways in which bacteriocins can affect human health. Our
intestine are teeming with a whole microbial world that helps digestion and affects
our immune system. Many of these bacteria produce a bacteriocin to help them
gain a foothold amongst the competition for resources. When one takes antibiotics
that can kill the beneficial bacteria allowing pathogenic organisms to take over.

One way to prevent this from happening is to take food with probiotics, such as
enhanced yogurt. Probiotics are beneficial microorganisms introduced into food
so that they can re-cognioize our intestinal tract frequently these bacteria are in a
group known as lactic acid bacteria,particularly species of Lactobacillus. lactic
Acid bacteria convert sugars to lactic acid and other compounds in absence of
oxygen.

Lactic acid bacteria produce a number of different bacteriocin types known as

lantibiotics.Some of these have been shown to inhibit the growth of bacteria that
can cause disease.Another type of lactic acid bacterium that lives in our intestine
is Enterococcus faecalis. Although tsese bacterium can be human pathogen,
several strains produced a bacteriocins with activity against the pneumococcal
bacteria thart caus pneumonia. Research has been done using non pathogenic
strains that produc the bacteriocins to inoculate the noses of children, in order to
protect them from getting pneumococcal pneumonia.

Fermented foods are another area in which bacteriocins contribute to efforts to

keep pathogens out of humans. They are one of several reasons for the anti
microbial activity of of lactic acid bacteria in these types of foods. Other reasons
include the production of antifungal compounds and organic acids. Starter
cultures that produce bacteriocins have beeb tested with fermented sausage and
cheese and found to protect against the potentially deadly pathogens Listeria in
both cases and Clostridium in the latter.

As research continues into the industrial potential of bacteriocins there is likely to

be greater use of them in the food industry. The use of probiotics is a greatly
expandeing market as 2010.microbiological research is likely to continue to
identify new bacteriocins with novel specificities that can be used to protect
human health.

Although bacteriocins could be categorized as antibiotic they are not. The major
difference between bacteriocins and antibiotics is that bacteriocins restrict the
activity or strains of species related to the producing species particularly to strains
of the same species. Antibiotics on other hand have a wider activity spectrum and
even if their activity is restricted this does not show any preferential effect on
closely related strains. In addition, bacteriocins are ribosomally synthesized and
produced during the primary phase of growth though antibiotics are usually
secondary metabolites.

Lactobacillus also called bacillus is a gram positive facultative anaerobic rod

shaped bacteria. They are major part of lactic acid bacteria group named as such
because most of its members convert lactose an d other sugars to lactic acid. In
human they are present in vagina and gastrointestinal tract where they make a
small portion of gut flora. They are usually benign , except in mouth where they
are associated with activity of tooth decay. Many specie are prominent in decaying
plant material . The production lactic adid make its environment t acidic,which
inhibits the growth of some harmful bacteria. Several members of this genus have
had their genome sequenced.

Some Lactobacillus species like Lactobacillusacidophilus are active in the

production of yogurt, cheese, sauerkraut, pickle, beer, wine, cocoa and other
fermented foods, as well as animal feeds such as silage. Sourdungh bread is made
using a “starter culture”, which is a symbiotic culture of yeast and lactic acid
bacteria grown in water and flour medium. The bacteria metabolize sugars into
lactic acid, which lowers the pH of their environment, creating a signature
“sourness’ associated with yogurt, sauerkraut etc.

In many traditional pickling processes , vegetables are submerged in brine and

salt tolerant Lactobacillus feed on natural sugars found in the vegetables. The
resulting ,mix of salt and lactic acid is a hostile environment for other microbes
and such as fungi , and the vegetables are thus remaining edible foe long periods.

Lactobacillus acidophilus and other lactic acid bacteria are important in the
fermentation of many foods from the dairy products to fruits and vegetables.
Fermentation occurs when bacteria break down sugars and carbohydrates to
produce alcohols ,carbon di ocide and lactic acid . These bi-products are
responsible for the un ique taste of the fermented foods and help preserve an
increase palatability.

Currently new research suggests that there are alternative ways of using lactic acid
bacteria, most notable the species the. The research shows that L.acidophilus can
also be used as a probiotic or living organism, which upon ingestion in certain
numbers , exert health benefits beyond inherent basic nutrition. There is still need
for more research in this area , but L. acidophilus is linked to decrease instances
of vaginal yeast infection, gastrointestinal dysfunction and even boosting immune
function.

Test Organisms

Bacillus

Scientific Classification

Domain Bactria
Division Firmicutes
 Class Bacilli
Order Bacillales
Family Bacillaceae
Genus Bacillus

Bacillus (Latin "stick") is a genus of Gram-positive, rod-shaped bacteria, a

member of the phylum Firmicutes, Bacillus and parasitic pathogenic species.
Under stressful environmental conditions, the bacteria can produce oval
endospores that are not true spores but which the bacteria can reduce themselves
to and remain in a dormant state for very long period. These characteristics
originally defined the genius , but not such species are closely related and many
have been move to other genera of Firmicutes. Many species of Bacillus can
produce copious amount of enzymes which are made use of in different industries.
Some Bacillus species cam form intra cellur inclusion of polyhyroxy-alkanoates
under certain adverse environmental conditions, as in a lack of elements such as
phosphorus Nitrogen or Oxygen combined with an excessive supply of carbon
sources. B.subtalis has proved as a valuable model for research.

Industrial significance

Many Bacillus species are able to secrete large quantities of enzymes. Bacillus
amiloliquefaciens is the source of an natural antibiotic protein barnase
(ribonuclease),α- amylase used in starch hydrolysis the protease is used with
detergent and the Bam H I restriction enzyme used in DNA research. A portion
of the Bacillus thuringiensis genome was incorporated into corn and cotton crops.
The resulting GMOs are therefore resistant to some insect pests.

Use as model organism

Bacillus subtilis is one of the best understood prokaryotes in terms of molecular

biology and cell biology. Its superb genetic amenability and relatively large size
have provided the powerful tools required to investigate a bacterium from all
possible aspects.recent improvements in fluorescent microscopic techniques
have provided novel and amazing insight into the dynamic structure of a single
cell organisms. Research on Bacillus subtilis has been at forefront of bacterial
molecular biology and cytology and the organisms is a model for differentiation
, gene protein regulation and cell cycle events in bacteria. Lactic acid bacteria
produce a number of different bacteriocin types, known as lantibiotics. Some
of these have been shown to inhibit the growth of bacteria that can cause
disease. Another type of lactic acid bacterium can be a human pathogen,
several Enterococcus faecalis. Although this bacterium can be a human
pathogen, several strains produce a bacteriocin with activity against the
pneumococcal bacteria that cause pneumonia. Research has been done using
non-pathogenic strains that produce this bacteriocin to inoculate the nose of
children, in order to protect them from getting pneumococcal pneumonia.

Fermented foods are another area in whichbacteriocins contribute to efforts to

keep pathogens out of humans. They are one of several reasons for the anti-
microbial activity of lactic acid bacteria in these types of foods. Other reasons
include the production of anti-fungal compounds and organic acids. Starter
cultures that produce bacteriocin have been tasted with fermented sausageand
cheese, and found to protect against the potentially deadly pathogensListeria
in both cases, and Clostridium in the latter.

As research continues into the industrial potential of bacteriocins, there is likely

to be greater use of them in the food industry. The use of probiotics is a greatly
expanding market as of 2010.Microbiological research is likely to continue to
identify new bacteriocins with novel specificities that can be used to protect
human health.

Although bacteriocins could be categorized as antibiotics, they are not. The

major difference between bacteriocins and antibiotics is that bacteriocins
restrict their activity to strains of species related to the producing species and
particularly to strains of the same species. Antibiotics on the other hand have a
wider activity spectrum and even if their activity is restricted this does not
show any preferential effect on closely related strains. In addition, bacteriocins
are ribosomally synthesized and produced duringthe primary phaseof growth,
though antibiotics are usually secondary metabolites.
Lactobacillus, also called bacillus, is a genus of Gram-positive facultative
anaerobic rod-shaped bacteria. They are a major part of the lactic acid bacteria
group, named as such because most of its members convert lactose and other
sugars to lactic acid. In humans they are present in the vagina and the
gastrointestinal tract, where they make up a small portion of the gut flora. They
are usually benign, except in the mouth where they have been associated with
cavities and tooth decay. Many species are prominent to decaying plant
material. The production of lactic acid makes its environment acidic, which
inhibits the growth of some harmful bacteria. Several members of the genus
have had their genome sequenced.

Some Lactobacillus specieslike Lactobacillus acidophilus are active in the

production of yogurt, cheese, sauerkraut, pickles, beer, wine, cocoa, and other
fermented foods, as well as animal feeds, such as silage.Sourdough bread is
made using a ‘’starter culture,’’ which is a symbiotic culture of yeast and lactic
acid bacteria growing in a water and flour medium. The bacteria metabolize
sugars into lactic acid, which lowers the pH of their environment, creating a
signature ‘’sourness’’ associated with yogurt, sauerkraut, etc.

In many traditional pickling processes, vegetables are submerged in brine, and

salt-tolerant Lactobacillusspecies feed on natural sugars found in the
vegetables. The resulting mix of salt and lactic acid is hostile environment for
other microbes, such as fungi, and the vegetables are thus preserved remaining
edible for long periods.

Lactobacillusacidophilus and other lactic acid bacteria are important in the

fermentation of many foods from dairy products to fruits and vegetables.
Fermentation occurs when bacteria break down sugars and carbohydrates to
produce alcohol, carbon dioxide and lactic acid. These by-products are
responsible for the unique taste of fermented foods and help preserve and
increase palatability.

Currently new research suggests that there are alternative ways of using lactic
acid bacteria, most notably the species L. acidophilus. The research shows that
L. acidophilus can also be used as a probiotics or living organism, which upon
ingestion in certain numbers, exert health benefits beyond inherent basic
nutrition. These is still need for more researchin this area, but L.acidophilus is
linked to decrease instances of vaginal yeast infection, gastrointestinal
dysfunction and even boosting immune function.

Staphylococcus aureus

Scientific classification

Domain Bacteria
Kingdom Eubacteria
Phylum Firmicutes
Class Bacili
Order Bacillales
Family Staphyloccccaceae
Genus Staphylococcus
Species Aureus

Staphylococcus aureus is a Gram-positive coccai bacterium that is a member of

the Firmicutes, and is frequently found in the human respiratory tract and on the
skin. It is positive for catalase and nitratereduction. Although S.aureus is not
always pathogenic, it is a common cause of skin infection boils), respiratory
disease, and food poisoning. Disease-associated strains often promote infection
by producing potent protein toxins, and expressing cell- surface proteins that
bind and inactivate antibodies. The emergence of antibiotic-resistant forms of
pathogenic S.aureus (e.g. MRSA) is a worldwide problem in clinical medicine.
Staphylococcus was first identified in 1880in Aberdeen, United Kingdom, by
the surgeon Sir Alexander Ogston in pus from a surgical abscess in a knee joint.
This name was later appended to Staphylococcus aureus by Rosenbach who was
credited by the official system of nomenclature at the time. It is estimated that
20% of the human population are long –term carries of S. aureus which can be
found as part of the normal skin flora and in anterior nares of the nasal passage.
S.aureus is the most common species of Staphylococcus to cause Staph
infections and is successful pathogen due to a combination of nasal carriage and
bacterial immuno –evasive strategies. S.aureus can cause a range of illnesses,
from minor skin infections, such as pimples, impetigo, boils (furuncles),
cellulites folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life
threatening disease such as pneumonia, meningitis, osteomyelitis, endocarditis,
toxic shock syndrome (TSS), bacteraemia, and sepsis. Its incidence ranges from
skin, soft tissue, respiratory, bone, and joint Endovascularto wound infections.

REVIEW OF LITRATURE

A study was carried out to isolate and identify the bacteria species that are
associated with fish spoilage using standard bacteriological techniques. The
results of the bacteriological quality of the cat fish showed variation in the total
bacterial and coliform counts to different anatomical parts (skins, gills and
intestine). The highest total bacterial counts was recorded from gills (83 x
105cfu/ml) and lowest in skin (53 x 105cfu/ml) from cat fish. The total coliform
counts of the cat fish ranges from 16 x 103cfu/ml, 36 x 103cfu/ml and 43 x
103cfu/ml in skin, intestine and gills respectively. A total of 288 colonies
belonging to eleven genera where identified after comparing the morphological
characteristics, gram staining reaction, biochemical tests and sugar utilization
with those of known taxa. The identified genera were Streptococcus sp.,
Staphylococcus sp., Salmonella sp., Shigella sp., Pseudomonas sp., E. coli,
Klebsiella sp., Enterobacter sp., Enterococcus sp., Campylobacter sp., and
Proteus sp. The prevalence of these genera shows that Pseudomonas sp ( 96.95%
) was the most prevalent on all the anatomical parts followed by E. coli (14.64%)
Enterococcus sp (9.74%), Klebsiella sp., (6.90%), Enterobacter sp (6.72%). The
presence of these bacteria genera could pose serious health problem if consumed,
it is therefore advised that proper care must be taken to prevent spoilage of the
fish through major preservation techniques (Ibrahim 2013). This study was carried
to isolate and identify the bacteria species that are associated with fish spoilage
using standard bacteriological techniques. The results of the bacteriological
quality of the cat fish showed variation in the total bacterial and coliform counts
to different anatomical parts (skins, gills and intestine). The highest total bacterial
counts was recorded from gills (83 x 105cfu/ml) and lowest in skin (53 x
105cfu/ml) from cat fish. The total coliform counts of the cat fish ranges from 16
x 103cfu/ml, 36 x 103cfu/ml and 43 x 103cfu/ml in skin, intestine and gills
respectively. A total of 288 colonies belonging to eleven genera where identified
after comparing the morphological characteristics, gram staining reaction,
biochemical tests and sugar utilization with those of known taxa. The identified
genera were Streptococcus sp., Staphylococcus sp., Salmonella sp., Shigella sp.,
Pseudomonas sp., E. coli, Klebsiella sp., Enterobacter sp., Enterococcus sp.,
Campylobacter sp., and Proteus sp. The prevalence of these genera shows that
Pseudomonas sp ( 96.95% ) was the most prevalent on all the anatomical parts
followed by E. coli (14.64%) Enterococcus sp (9.74%), Klebsiella sp., (6.90%),
Enterobacter sp (6.72%). The presence of these bacteria genera could pose serious
health problem if consumed, it is therefore advised that proper care must be taken.

food poisoning and diarreheal illness are among the leading causes for world wide
morbidity and mortality (WHO,1998) food safty was identified as a high priority
area in the years 2001-2005 member countries of the OIE considred yhat the
orginisation should be more active as far as public healthe an dconsumer
protection is concerned and this should imclude more involvement in the area of
deseas or pathogen transmissible through food (Droppers ,2006) meat and meat
products are particular important regarding food –born disease food born
pathogen can be transfferd to food during the processing , distribution and
storage from infected human who handle the food or by cross-contamination from
some other raw agricultural products (Headberg et al ,1994)

In recent years ,the Vietnam government has shon considerable attention to

reducing the risk to food safety . the most significant event was the creation of
the food administration within the mininstry of health in 1991 (Kim,2002)
according to the statistics of the foods safety Administration in 200-2006 there
were 174 food poisoning cases at collective kitchen with 1,4650 patients ,97 cases
at industrial the export proceesing zones with 9,900 patients,58 cases in schools
with 3,790 victimes (tow died) and 161 street food poisoning cases with 7,688
victims (7 died).

Vietnam the ministry of health , which i9s responsible or food inspection of the
domestic and imported food , reported over 4 million cases of sever enteric
disease ,eg typhoid , cholera and shigellosis from 1997 to 2000. There were 1,391
involving over ,2,5000 cases and 217 death (Kim and Phoung 2001) there is
aderamatic increase in those disease form the last year , which saw nearly 8,000
cases of food poisoning with 61death .in the first quarter of 2009 vietnam
reported 229 cases of food poisoning ,including two fatalities according to the
food safety and hygien department .

Pork is a popular meat and it is produced under a wide variety of production

system ranging from simple backyard pigs to large- scale integrated pig. Most of
them pork is predominantly produced fro domestic demand and consumption . it
os estimated that 95% of the households consume pork in their daily diets (Dinh
et al.2005.) therefore pigs are sloughtred on adaily basis and most of the pork is
consumed as fresh meat with a day .according to master plan of MARD the aim
to raise the animal production ( including the development in the pig sector ) to
30% of the total agricultural vakue by 2010 and to 35% by 2015 (GAIN 206) .
pig production exhibit strong comparative advanteges in both the north and the
south of the country together with the income of the pork consumption is
increasing very fast (12.3%from 2000 to 2005).

Food spoilage is a metabolic process that causes foods to be undesirable or

unacceptable for human consumption due to changes in sensory characteristics.
Spoiled foods may be safe to eat, i.e. they may not cause illness because there are
no pathogens or toxins present, but changes in texture, smell, taste, or appearance
cause them to be rejected. Some ecologists have suggested these noxious smells
are produced by microbes to repulse large animals, thereby keeping the food
resource for themselves (Sherratt et al., 2006). Food loss, from farm to fork,
causes considerable environmental andeconomic effects. The USDA Economic
Research Service estimated that more than ninety-six billion pounds of food in
the U.S. were lost by retailers, foodservice and consumers in 1995. Fresh produce
and fluid milk eachaccounted for nearly 20% of this loss while lower percentages
wereaccounted for by grain products (15.2%), caloric sweeteners (12.4%)
processed fruitsand vegetables (Kantor et al., 1997). Some of this food would
have been considered still edible but was discarded because it was perishable, past
its sell-by date, or in excess of needs. There are also environmental and resource
costs associated with food spoilage and loss. If 20%of a crop is lost, then 20% of
the fertilizer and irrigation water used to grow that crop was also lost.
Lactic acid bacteria (LAB) are a group of Gram-positive bacteria, including
species of Lactobacillus, Pediococcus, Leuconostoc some of which are useful in
producing fermented foods such as yogurt and pickles. However, under low
oxygen, low temperature, and acidic conditions, these bacteria become the
predominant spoilage organisms on a variety of foods. Other thermophiles
(Bacillus and Geobacillus spp.) cause a flat sour spoilage of high or low pH
canned foods with little or no gas production, and one species causes ropiness in
bread held at high ambient temperatures. Mesophilic anaerobes, growing at
ambient temperatures, cause several types of spoilage of vegetables (Bacillus
spp.); putrefaction of canned products, early blowing of cheeses, and butyric acid
production in canned vegetables and fruits. While others produce off-odors and
gas in vacuum-packed, chilled foods and milk (Bacillusspp.).

Listeria monocytogens is a small, non-spores forming rod gaining public

awareness because of its presence in food products. Contamination of food
products with Listeria monocytogens has resulted in the closing of business or
discontinued production of profitable food products (Kellum et al.,
2002).Listeria is widely found on foods and most raw foods are likely to be
contaminated.
Listeria is easily killed by heat although cooked foods can easily become
decontaminated through poor handling .This is one of the few pathogen that can
grow in the refrigerator. Although it can grow in the fridge, it will do so only
vary slowly so make sure your refrigerator is keeping your food at or less than
5ºC (Bunning et al., 1986). Listeria is wide spread in nature, living closely
associated with soil and plant matter. This organism has been found in feces of
humans and animals, soil leafy vegetables decaying corn and soybeans, raw and
treated sewage, effluent from poultry and meat processing facilities, normal and
mastitis milk and improperly fermented silage (Catherine, 1987).Listeriosis is
rare form food borne illness, but it may be a serious diseases in a small group of
individuals, those who pregnant, immune-compromised and young children and
every year a few people die. Due to this problem it has also caused occasional
outbreak of mild gastroenteritis in healthy people. The symptoms are usually
described as „flu-like‟, although vomiting and discovered urine can occur.
Miscarriage can result if pregnant women is infected, even she does not show
the symptoms. The time from infection to symptoms can be anywhere 8 to 90
days (Dalton et al., 1977).Pseudomonas and related genera are aerobic, Gram-
negative soil bacteria, some of which can degrade a wide variety of unusual
compounds. They generally require a high water activity for growth (0.95 or
higher) and are inhibited by pH values less than 5.4. Some species grow at
refrigeration temperatures (psychrophilic) while other are adapted for growth at
warmer, ambient temperatures.

Bakery products are subjected to spoilage problems. These include physical,

chemical and microbial spoilage. Since the most common factor of bakery
products is water activity, microbiological spoilage, in particular mould growth
is the major economic importance of bakery products. Mould spoilage is a
serious and costly problem for bakeries. Bacterial contamination is more
dangerous because very often the food does not look bad, even though severely
infected, it may appear quite normal. The presence of highly dangerous toxins
and bacterial spores is often not detected until after an outbreak of food
poisoning (Sockett, 1991).

Cakes undergo bacterial spoilage due to their usually high concentration of

sugars, which restrict the availability of water. The baking process is generally
sufficient to destroy microorganisms. Microorganism may enter baked cakes
from handling also from air. Growth of microorganisms on the surface of cakes
is favoured by high humidity and growth of moulds on cakes results in a
hardening of the product (Bamford, 1973). Food spoilage can be defined as
disagreeable changes in food‟s normal state; such changes can be detected by
smell, taste, touch or sight. These changes are due to number of reasons such as
air, moisture, light, microbial growth and temperature. Main single cause of
food spoilage is invasion by microorganisms such as bacteria, yeasts and
moulds. Molds are the most important contaminants because of the low moisture
levels in grains, but molds do require some moisture so efficient drying and
good storage facilities are necessary to prevent their growth.Microbial
populations decrease during milling and storage of grain. Molds cause spoilage
by altering the appearance of grains and flours, and some species also synthesize
toxic secondary metabolites called mycotoxins. Molds are also the primary
spoilage organisms in baked goods, with Aspergillus, Penicillium, and Eurotium
being the most commonly isolated genera. Penicillium tends to be the more
important in sourdough breads and in breads stored at cooler temperatures.
Freshly baked breads do not contain viable molds but soon become
contaminated upon exposure to air and surfaces (Smith et.al., 2004). Bacillus
spores are strongly heat resistant and can survive baking in the interior of bread
loaves and then germinate and start growing as the bread cools. Some strains
cause a defect called ropiness, a soft sticky texture caused by starch degradation
and slimy exopolysaccharides often accompanied by a fruity odor (Pepe et al.,
2003). Yeasts may also be involved in spoilage of some breads and fruitcakes,
causing a chalky appearance on surfaces and off odors. High sugar content and
low water activity of cakes also favors molds over other spoilage microbes but
some species of yeasts and bacteria (Bacillus and Pseudomonas) may also attack
cakes. Bakery products containing cream, custard or fruit filling are targets of
additional spoilage organisms.
severely infected, it may appear quite normal. The presence of highly dangerous
toxins and bacterial spores is often not detected until after an outbreak of food
poisoning (Sockett, 1991).
Cakes undergo bacterial spoilage due to their usually high concentration of
sugars, which restrict the availability of water. The baking process is generally
sufficient to destroy microorganisms. Microorganism may enter baked cakes
from handling also from air. Growth of microorganisms on the surface of cakes
is favoured by high humidity and growth of moulds on cakes results in a
hardening of the product (Bamford, 1973). Food spoilage can be defined as
disagreeable changes in food‟s normal state; such changes can be detected by
smell, taste, touch or sight. These changes are due to number of reasons such as
air, moisture, light, microbial growth and temperature.
Main single cause of food spoilage is invasion by microorganisms such as
bacteria, yeasts and moulds. Molds are the most important contaminants because
of the low moisture levels in grains, but molds do require some moisture so
efficient drying and good storage facilities are necessary to prevent their growth.
Microbial populations decrease during milling and storage of grain. Molds cause
spoilage by altering the appearance of grains and flours, and some species also
synthesize toxic secondary metabolites called mycotoxins. Molds are also the
primary spoilage organisms in baked goods, with Aspergillus, Penicillium, and
Eurotium being the most commonly isolated genera. Penicillium tends to be the
more important in sourdough breads and in breads stored at cooler temperatures.
Freshly baked breads do not contain viable molds but soon become contaminated
upon exposure to air and surfaces (Smith et.al., 2004). Bacillus spores are strongly
heat resistant and can survive baking in the interior of bread loaves and then
germinate and start growing as the bread cools. Some strains cause a defect called
ropiness, a soft sticky texture caused by starch degradation and slimy
exopolysaccharides often accompanied by a fruity odor (Pepe et al., 2003). Yeasts
may also be involved in spoilage of some breads and fruitcakes, causing a chalky
appearance on surfaces and off odors. High sugar content and low water activity
of cakes also favors molds over other spoilage microbes but some species of
yeasts and bacteria (Bacillus and Pseudomonas) may also attack cakes. Bakery
products containing cream, custard or fruit filling are targets of additional spoilage
organisms
 MATARIALS & METHOD

SAMPLE COLLECTION

Sample of curry vegetable were collected from diuffrent sources in noida like
hotle and houses

The samples were collected in sterile containers and transported to the laboratory
of Helix Biogenesis, NOIDA for analysis within 24 hours.

SERIAL DILUTION

Isolation of microorganism from Soil by the serial dilution agar plating

method

The serial dilution-agar plating method or viable plate method is one of the
commonly used procedures for the isolation and enumeration of Bacteria.

This method is based upon the principle that when material containing
microorganism is culture each viable microorganism will develop into a colony,
hence the number of colonies appearing on the plates represent the number of
living organism present in the sample.

No of colonies x Dilution factor

𝑁𝑜 𝑜𝑓𝑐𝑒𝑙𝑙𝑠/𝑚𝑙 =
Dry weight of soil

Dilution factor = Reciprocal of the dilution.

colony count (CFU)on agar

𝑇ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑓𝑜𝑟𝑚𝑢𝑙𝑎 =
Total dilution g tube x Volume plated

CFU =colony formed unit

Requirements: -
• Milk & Curd sample
• Weighing machine
 • Test tube
• Pipette and tips
• Distilled water
• Bunsen burner
• Petri plate
• Nutrient agar media
Autoclave

Procedure: -
1. Collect the sample at random, minimum five, from filed, mix thoroughly to
make a composition sample for microbiological analysis.
2. Label 5 ml sterile water blank as 1, 2, 3, 4 and sterile Petri dishes as for
each dilution test tube.
3. Add 5 gm sample of finely pulverized, air dry soil into numbered 1 water
blank to make 1:10 dilution (10-1).
4. Vigorously shake the dilution on gently for 1-2 min to obtain uniform
suspension of microorganism.
5. Transfer 500 µl suspensions from test tube number 1 into water blank 2
with a sterile pipette under aseptic condition to make 1:100 (10-2) and shake
it well for about 2 min.
6. Prepare another dilution 1:1000 (10-3) by pipetting 500µl of the suspension
into water blank number 3, using sterile pipette and shake it.
7. Transfer 1 ml aliquots each from dilution into each Petri plate.
Observation:-
1. Observe the plate for number and distribution of colonies of bacillus.
2. Select plates from the appropriate dilution which contain colonies.
3. Since the dilution plates are replicates of each other, determine the average
of the triplicate microbial counts.
 SPREAD PLATE TECHNIQUE
The spread plate technique is used for the separation of a dilute, mixed
population of microorganism so that individual colonies can be isolate. In
this technique microorganism are spread over the solidified agar medium
with a sterile L-shaped glass rod while the Petri dish is spun on a turntable

Requirement: -
• 24 hour nutrient broth culture
• Nutrient agar media
• L-shaped glass rod
• Ethyl alcohol
• Bunsen burner
• Autoclave, Marker
Procedure: -
1. Prepare the nutrient agar media and covered the petriplate and autoclave at
121 lb pressure for 15 min.
2. Pour the agar media in petriplate and placed for solidify.
3. Label on nutrient agar plate.
4. Take ethyl alcohol into a beaker and dip the bent glass rod in it.
5. Aseptically transfer a loopful culture in the center of appropriately labeled
nutrient agar plate.
6. Remove the glass rod from the beaker and sterile the bent portion in the
Bunsen burner flame.
7. Cool the rod for 10 -15 seconds.
8. Remove the cover of Petri plate and spin the turntable.
9. Lightly touch the sterile bent rod to the agar surface and move it back and
forth while the turntable is spinning for spreading the culture over the agar
surface.
10. Replace the Petri dish cover when the turntable stops spinning.
 11. Immerse the bent rode in alcohol and reflame to sterilize it.
12. Repeat the step all of the Petri plate.
13. Incubate all plates is an invert position at 35˚𝑐, for 24-48 hours.

Observation: -
Observe all incubate plates as to the distribution of colonies on each of the
agar plate, some of the colonies will be free from each other .select a
describe colony each from first and second plate and record form, elevation,
pigmentation and size of the colony.
 STREAK-PLATE METHOD

The streak-plate method offers a most practical method of obtaining

discrete colonies and pure culture. The aim of this exercise is to obtain
colonies of microorganism that are pure growth derived from a single cell
/ spore.

Requirement

• 24-48 hours nutrient broth culture

• Nutrient agar media
• Inoculation loop
• Bunsen burner
• LB agar media
• Autoclave

Procedure
1. Prepare the L.B agar media and covered the Petri plate, then autoclave at
121 lb at 15 min.
2. After autoclave label the all plate, on the bottom, with name of the organism
to be inoculated.
3. Pour the L.B agar media in each Petri plate and take place for solidifying
at room temperature.
4. Sterilize the loop holding in the right hand, remove the cotton plug and
immediately flame the mouth of the tube.
5. Introduce the loop into the broth and withdraw one loopful of culture.
6. Lift the petriplate cover with the left hand and hold it at angle of 60˚.place
the inoculums on the agar surface at the edge farthest from you and streak
the inoculums from side to side in parallel lines across the surface of area.
7. Reflame and cool the loop and turn the petriplate to 90˚. Touch the loop to
a corner of the culture in area 1 and streak the inoculation across the agar
in area 2.and again and again 4 times.
8. The rest of the agar surface is now used to complete the streaking.
9. Replace the lid of the Petri plate, after completing the streaking, and
sterilize the loop by framing.
10. Incubate all plate at 35˚c, in an inverted position, for 24-48 hours.
Observation
 After incubation, examine each of the all plate for the growth of all
colonies.

BROTH CULTURES

Broth is a liquid formulation (medium) used for growing microorganism

in the laboratory on a small scale or in an industrial fermenter on a large
scale.

Requirement
• Nutrient broth culture and colony
• Nutrient broth in test tube
• Bunsen burner
• Inoculation loop
• Autoclave
Procedure
1. Prepare the nutrient media.
2. Pour the media into test tube and plug it with cotton plug, autoclave at 121
lb for 15 min.
3. Label each nutrient broth tube with the name of the organism.
4. Hold both culture/ colony and broth tube.
5. Sterilize inoculation loop and remove the plugs from these tubes alternately
flame the mouth of both tube.
6. Transfer a loopful of culture into the sterile broth with the sterile loop.
7. Alternately flame the mouth of both tubes and replace both caps to
respective tubes.
 8. Flame sterilized the inoculating loop immediately after transfer.
9. Inoculate each organism into a separate tube sterile nutrient broth.
10. Incubate all the tubes, in incubator at 30˚c for 24-48 hours.
Observation
Observe the broth culture for growth of microorganism, without disturbing
them. Record any change in appearance (growth) from those observed 24 hours
after incubation
 BIO CHEMICAL TEST

1. Gram staining

2 Ureas test

3. VP test

4. Nitrate

5. 7.5% Nacl

6. 10% Nacl

7. Diameter >5mm in 48 hour

8. Test growth at 4500

9. Glucose

10. Lactose

11. Sucrose

12. Glycerole

13. Manitole

GRAM STAINING

It is a very useful stain for identifying and classifying bacteria into two
major groups: the gram-positive and gram negative. In this process, the
fixed bacterial smear is subjected to four different reagents in the order
listed: crystal violet (primary stain), iodine solution (mordent), alcohol
(decolorizing agent) and Saffranin (counter stain).the bacteria which retain
the primary stain (appear dark blue or violet) are called gram positive,
whereas those that lose the crystal violet and counter stained by Safranin
(appear red) are referred to as gram negative.
Requirement

• 24 -36 hr. Growing culture.

• Gram staining reagent:
• Crystal violet
• Gram’s iodine solution
• Ethyl alcohol
• Safranin
• Staining tray, Wash bottle of distilled water
• Droppers inoculating tube
• Glass slides, Blotting paper
• Lens (Microscopy)
• Bunsen burner/spirit lamp
• Microscope

Procedure

• Make thin smear of bacillus on separate glass slides.

• Let the smear air dry.
• Heats fix the smear.
• Hold the smear using slide rack.
• Cover each smear with crystal violet for 30 second.
• Wash each slide with distilled water for a few second, using wash bottles.
• Cover each smear with gram’s iodine solution for 60 second.
• Wash of the iodine solution with ethyl alcohol. Add ethyl alcohol drop by
drop, until no more color flows from the smear.
• Wash the slide with distilled water and drain.
• Apply Safranin to smear for 30 second.
• Wash with distilled water and blot dry with absorbent paper.
 • Let the stained slides air dry.

Observations

• Examine the slides microscopically.

• Identify the gram reaction of both the culture and classify them.
• Make sketches for morphology of the culture.
• Describe the morphology and arrangement of the cell.

1. UREASE TEST
Urea is a major organic waste product of protein digestion in most vertebrates and
is excreted in the urine. Some microorganism has the ability to produce the
enzyme Urease.
Requirements
• Fresh culture
• Urease agar medium /Urease agar broth
• Inoculating loop
• Bunsen burner
• Phenol red (reagent)

Procedure

• Preparation of agar medium in the 100 ml distilled water, adjust the pH to

6.8 and autoclave at 121˚c for 15 min and cool to 50˚c.
• Add Glucose (0.1 gm), phenol red 6-8 drop.
• Keep this boiling water both for 30 min at 100˚c and then cool to 50˚c.
• Add Urease in 2 gm.
 • Label the test tube of urea broth tube with the name of the bacterial
organism to be inoculated.
• Incubate inoculated broth for 24-48 hours at 37˚c.

Observation
Examine the broth as to their color for the presence of Urease (Red or cerise
color) and for no Urease (yellow color).
 2. VOGES-PROSKAUER TEST

The methyl test (MR) and Voges-Proskauer test are used to differentiate two
major types of facultatively anaerobic enteric bacteria that produce large amounts
of acid and those that produce the neutral product action as end product.

Requirement

• Broth culture
• MRVP broth
• MRVP broth test tube -5ml/5 (tube)
• Methyl red pH indicator (dropper bottle)
• V-P reagent I (Napthol solution)
• V-P reagent II (40% potassium hydroxide)
• Clean empty test tube
• Bunsen burner, Inoculating loop
• methyl red indicator solution

Procedure

• Preparation of MRVP broth (pH 6.9) tube of the.

• Pour the 5 ml broth in each test tube and sterilize by autoclaving at 15 lb
psi for 15 min.
• Inoculate two tubes containing MR-VP Broth with a pure culture of the
microorganisms under investigation.
• Incubate at 35 °C for up to 48 hours.
• Add about 5 drops of the methyl red indicator solution to the first tube and
for VP test Napthol solution reagent is added to another tube.
• A positive reaction is indicated, if the color of the medium changes to red
within a few minutes.
Observation

• Observe the change in color of methyl red for MR test.

• Add 12 drops of VP reagent and 2-3 drops of VP reagent II to the other set
of two tube as well as.
• Shake the tube gently for 30 seconds with the caps off to expose the media
to oxygen.
• Allow the reaction to complete for 15-30 min.
• Observe the tube for change in color for the VP test.
 NITRATE TEST

Principle:Heavy inoculum of test organism is incubated in nitrate broth. After 4

hrs incubation, the broth is tested for reduction of nitrate (NO3–) to nitrite (NO2–
) by adding sulfanilic acid reagent and α- naphthylamine.

1 .If the organism has reduced nitrate to nitrite, the nitrites in the medium will
form nitrous acid. When sulfanilic acid is added, it will react with the nitrous
acid to produce diazotized sulfanilic acid. This reacts with the α-
naphthylamine to form a red-colored compound. Therefore, if the medium
turns red after the addition of the nitrate reagents, it is considered a positive
result for nitrate reduction.

2 .If the medium does not turn red after the addition of the reagents, it can mean
that the organism was unable to reduce the nitrate, or the organism was able to
denitrify the nitrate or nitrite to produce ammonia or molecular
nitrogen. Therefore, another step is needed in the test. Add a small amount of
powdered zinc. If the tube turns red after the addition of the zinc, it means that
unreduced nitrate was present*.Therefore, a red color on the second step is a
negative result

Requirements:

1. Media: Nitrate Broth with inverted Durhams tube

2. Reagents: Sulphalinic acid reagent, Alpha napthylamine reagent, zinc dust
3. Others: Inoculating loop, burner, dropper

Procedure;

1. Inoculate nitrate broth with a heavy growth of test organism using aseptic
technique.
2. Incubate at an appropriate temperature for 24 to 48 hours
3. Add one dropperfull of sulfanilic acid and one dropperfull of a α-
naphthylamine to each broth.
1. At this point, a color change to RED indicates a POSITIVE nitrate reduction
test. If you get a red color, then you can stop at this point.
 2. No color change indicates the absence of nitrite. This can happen either
because nitrate was not reduced or because nitrate was reduced to nitrite,
then nitrite was further reduced to some other molecule. If you DO NOT get
a red color, then you must proceed to the next step.
4. Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the
reduction of nitrate to nitrite.
1. At this point, a color change to RED indicates a NEGATIVE nitrate
reduction test because this means that nitrate must have been present and
must have been reduced to form nitrite.
2. No color change means that no nitrate was present. Thus no color change at
this point is a POSITIVE resul

Result and Interpretation;

1. Nitrate Reduction Positive: (Red after sulfanilic acid + alpha-naphthylamine;

no color after zinc)
2. Nitrate Reduction Negative: (No color after sulfanilic acid + alpha-
naphthylamine followed by Red after zinc)

3.Growth at 10% NaCl

The salt tolerance test is performed using Tryptic Soy Broth with added
sodium chloride (regular table salt) to create an overall salt concentration
of 7%. It is a selective medium which tests the ability of an organism to
survive in a salt-rich environment. Most organisms cannot survive in such
an environment.
Requirement

• Fresh culture
• NaCl agar media
• Inoculation broth
• Bunsen burner
• Petri plate

Procedure
 • Preparation of agar NaCl broth media and autoclave at 121˚c pressure for
15 min and cool to 50˚c.
• Pour the media in Petri plate and take place for solidification.
• After solidifying inoculate the culture.
• Incubate the Petri plate for 24-48 hours at 37˚c

4.CARBOHYDRATE TEST

Fermentation degradation of various carbohydrates such as lactose, dextrose and

sucrose (disaccharide) by microbes, under anaerobic condition is carried out in
fermentation tube. A fermentation tube is the culture tube that contains a Durham
tube for the detection of gas production, as an end product of metabolism.

GLUCOSE Lactose, Sucrose, Dextrose

Requirement

• Lactose broth media.

• Fresh and pure broth culture.
• Durham tube and sterile fermentation Test tube of: -
Dextrose broth
Sucrose broth
Lactose broth
Glucose broth
• Inoculation tube
• Bunsen burner
• Wax marking pencil.
• Autoclave
• Phenol red
Procedure: -

• Preparation of fermentation medium whose constituent are a specific

carbohydrate –such as dextrose or sucrose or lactose is added .broth taken
into fermentation tube is autoclave at 12 lb pressure for 15 min.
• Label each of specified fermentation tube of media with the name of the
organism to be inoculated.
• Inoculate the three type of sugar fermentation broth with each bacterium
• Add the Durham tube.
• Take place the fermentation tube in the shaker for 24-36 hour’s.

Observation

Observe the reaction that develop in the three fermentation media change in
color (due to production of acid) or change in color and appearance of bubble
(due to production of acid and gas) and record your finding in the result’
section in a tabular form.

3. MANNITOL TEST

Mannitol salt agar or MSA is a commonly used growth medium in microbiology.

It encourages the growth of a group of certain bacteria while inhibiting the growth
of others. This medium is important in medical laboratories by distinguishing
pathogenic microbes in a short period of time.

Requirement

• Mannitol agar culture

• Fresh culture colonies
• Inoculation loop
 • Petri plate
• Bunsen burner
• Phenol red

Procedure

• Preparation of Mannitol agar medium in the 100 ml distilled water, adjust

the pH and autoclave at 121˚c for 15 min and cool to 50˚c.
• Pour in the Petri plate and place it for solidification.
• After solidification streak the culture and incubate the plate for 24-36
hour’s at 37˚c for growth.
• After growth fill the plate by phenol red.

Observation
After growing the culture use phenol red observes the change of color. The
yellow color is a positive test and red-pink color is negative test.

COMPOSITION

L.B Agar media: -

Yeast Extract 5 gm
Trypton 10 gm
NaCl 10 gm
Agar 15 gm
Distilled water 1000 ml

L.B Broth media: -

 Yeast Extract 5 gm
Trypton 10 gm
NaCl 10 gm
Distilled water 1000 ml

Urea agar (pH 6.8): -

Peptone 1.0 gm
Sodium chloride 5.0 gm
Potassium dihydrogen phosphate 2.0 gm
Glucose 1.0 gm
Phenol red 6.0 ml
Urea (20 %aqueous solution) 100.0 ml
Distilled water 1000.0 ml

Lactose fermentation broth (pH 6.9): -

Lactose 5.0 gm
Peptone 5.0 gm
Beef extract 3.0 gm
Distilled water 1000 ml

Sucrose fermentation broth (pH 6.9): -

Sucrose 5.0 gm
Peptone 5.0 gm
 Beef extract 3.0 gm
Distilled water 1000 ml

Glucose fermentation broth (ph 6.0)

Glucose 5.0gm
Peptone 5.0gm
Beef extract 3.0gm
Distilled water 1000ml

Mannitol test: -

Mannitol 10.0 g
Peptone 10.0 g
Sodium chloride (NaCl) 15.0 g
Beef extract 1.0 g
Phenol red 0.025 g
Agar 15.0 g
Distilled water 1000 ml

Nitrate broth
Peptic digest of animal tissue 5.00
Meat extract 3.00
Potassium nitrate 1.00
Sodium chloride 30.00
Finale ph 7.0

Reagent

1. Gram Stain reagent:-

Crystal violet

Solution A4
 Crystal violet (90% dye content) 2.0 g

Ethyl alcohol (95%) 20.0 ml

Solution B

Ammonium oxalate 0.8 g

Distilled water 80.o ml

Gram’s iodine

Iodine 1.0 g

Potassium iodine 2.0 g

Distilled water 300.0 ml

Ethyl alcohol (95%)

Ethyl alcohol (100%) 95.0 ml

Distilled water 5.0 ml

Safranin

Safranin (2.5% solution in 95% ethyl alcohol) 10.0 ml

Distilled water 100 ml

2. Voges - Proskauer test reagent

α- Napthol 5.0 g

Ethyl alcohol 95.0 ml

 Result & Discussion

REFRENCE

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