A

Project Report
“ Detection of salmonella and E.coli in the juices.”

Department of Microbiology
JANTA VEDIC COLLEGE, BARAUT (BAGHPAT) 250611

Supervisor: Submitted By :-

Name:-Roli Sharma

Internal : Dr Varun Tomar Roll No:-200867254207

External : Mr. Rajesh kumar Enroll.no :- M17140285

CHAUDHARY CHARAN UNIVERSITY, MEERUT

2022

1
Dr. Varun Tomar Assistant Professor
M.sc, M.phil.,ph.D Deptt of microbiology
Janta Vedic College Baraut
Distt..- Baghpat UP india

Dated……………..

CERTIFICATE
This is to certify that thesis entitled”Detection of Salmonella and E.
Coli’in the juices” in the fulfillment for the award of the degree of M.Sc.
Microbiology in Department of Microbiology, J.V. College, Baraut-
250601, (U.P) is an orginal record of bonafide research work carried out
by Roli Sharma under my supervision and no part of this thesis has been
submitted for any other degree or diploma to any other institute or
university. Roli Sharma worked here 17 may 2022 to 31 Aug 2022

I wish her all the success in future endeavors.

Dated
Place :- Baraut Dr. varun Tomar
Internal Supervisor

2
3
 CANDIDATE’S DECLARATION

I hereby declare that the work being presented in project on Detection

of Salmonella and E. coli in the juices” for partial fulfillment for degree
of Master of Science in Microbiology submitted to Janta Vedic College,
Baraut, Baghpat (CCS University, Meerut) is an authentic work carried
out by me during the period 01st Feb, 2021 to 31st July., 2021 at Sarita
Diagnostic Centre, Shamliunder supervision of Dr. Unendra Kumar.

The matter embodied in this project as not submitted by me for the

award of any other degree.

Dated Roli Sharma

4
 ACKNOWLEDGEMENT
First and foremost, I would like to praise and thank God, the almighty,
who has granted countless blessing, opportunity and knowledge to me,
so that I have been finally able to accomplish the thesis. I would like to
extend my sincere and hearfelt obligation towards all the personages
who have helped me in this endeavor. Without their active guidance,
help, coopartion and encouragement; I would not have made headway
in this project. I would like to express my sincere gratude to my advisor
and mentor. Dr. Mahesh kumar, Dr. Reena Tomar, Dr, Varun Tomer
department of Microbiology, JV College, Baraut for their suggestions,
motivation, enthusiasm and perpetual encouragement throughout this
research. Their invaluable guidance helped me all the time during this
research work.
Besides my advisor, I am deply thankful to Mr. Rajesh Kumar (euruka
analytical service, Sonipat for his guidce, supervison and valuable
suggestions. It was a great privilege and honor to work and study under
his guidance.
I would also like to extend my gratitude to my dearest friends Gautam ,
Monika, Parul, For helping me in completing this work, The countless
time you guys helped me throughout this project and give all your
support, is really appreciable, a big thanks to you cronies.
Last but not the least,my deep and sincere gratitude to my family for
their continous and unparalleled love, care and constant support, Your
encouragement, when the time got rough ar much appreciated.
Finally, thanks also to anybody, I forgot to mention who was involved in
this work and contributed to its
Success.

Roli Sharma

5
 ABBREVIATIONS

BPW : BUFFERED PEPTONE WATER

MKTTN : MULLER KAUFFMAN TETRATHIONATE

NOVOBIOCIN BROTH BASE

RVM : RAPPAPORT VASSILIADIS MEDIUM

XLDA : XYLOSE-LYSINE DEOXYCHOLATE AGAR

BGA : BRILLIANT GREEN AGAR BASE

NA : NUTRIENT AGAR

MCB : MACCONCKEY BROTH

6
INTRODUCTION

7
 INTRODUCTION
Fruit are important in human diets due to their contributions in vital
nutrients particularly vitamin c. Though they are very low in fats &
protiens , they are rich in sugar content as they content as they
content large amount of glucose, fructose of sucrose.

Fruit juice are well recognised for their nutritive value , mineral and
vitamin content. In many tropical countries they are common. Fresh
fruits are essential components of the human diet and there is
considerable evidence of the health and nutriential benefits
associated with the consumption of fresh fruits or their juices.

Nowdays, the demant for freshly squeezed juices in comparison to

battled or canned juices has increased as unpasteurized juices are
preffered by the consumer because of the fresh flavore and no
addiction of presentatives. Traditionally , juice is consumed more in
the morning at breakfast time.Consumption of fresh fruits continues
to increase in many countries during to consumer preferences for
fresher, more nutritious foods that also happen to meet the needs of
busier lifestyles. Fresh fruit juices have no artificial colour, seetness is
natural , and that is why they are preferred over battled or canned
juices.

Freshly squeezed juices are simply prepared by extracting the liquid

and pulp of mature fruit.

8
usually by mechanical means or Blenders. Priar preparation of fruit to
avaid bitterness of skin
or to remove large stone such as mango avocado and pulp by blender.
The final product is an infreated unclarified ,untreated juice ,ready for
consumption .
However , It is well known fact that food serves as very good mediym
for growth of Microorganisms especially when the principles of hygiene
and sanitation are not met the food becomes contaminated by
pathogens from humans or from the environment during producation
processing or preparation specially local preparation of fruit juices has
no process that reduce pathogens
levels, if contaminated, such as microbe killing step. Pathogenic
organisms can enter fruits
becomes contaminated by pathogens from humans or from the
environment during producation processing or preparation specially
local preparation of fruit juices has no process that reduce pathogens
levels, if contaminated, such as microbe killing step. Pathogenic
organisms can enter fruits
through damaged surfaces. such as punctures, waunds,cuts and splits.
This damage can
occur during maturation or during harvesting and processing. A
pathogen that has become internaised within a fruit
must be able to survive in the product until is reaches the consumer in
order to become a
public health hazard. Most fruit juice are sufficiently acidic to inhibit
themgrowth of pathogenic
organisms.

9
 Therefore, this study was aimed at determining the microbiological safty
of fruit juices consumed in our daily life, school of college, restaurants
etc,
fruts juice is a pupular soft drink made of the pulp of different types of
fresh juice" fruit imports natural flavour to fruit juices contain many
components which are beneficial for our health. Though the actual
compnents varies from fruits to fruits, in general they contain flavonaid
glycosides; dietyary fiber, calcium, vitaminc carotenaids. lutin, lycopene,
caratene, phenalic acid, stibenes, ellagic acid, amino acid, aroma
compaunds, anithocyanin, flavanals, polyphenals, potassiu, vitamin D,
low amount of sodium, cholesaeral, fat ec, comonents present in fruits
juices has been proved to help in preeventing heart diseases, certain
conerns, diabetss' catarects, alzheimer';s diseras, asthuma and helps in
the formation of collagen, cartitage, blood vessets and musells
considering those beneficial inparts in human health, frutis jices became
population wordwide.

10
REVIEW OF LITERATURE

11
 REVIEW OF LITERATURE

The purpose of this review article is to introduce the importance of

fruit juices for human health living in the country and in a broad, and
to develop awareness among the people about the diseases caused by
pathogens associated with fruit juice. Health benefits of juices have
been included in this article and how the same juice can cause
problems among people of different ages have been discussed.
Contamination sources and the ways to prevent them is very
important issue in protecting public health Some future
recommendations for fruit juices have also been added in this article.
Different diseases caused by various microbial agents and the
associated symptoms after consuming contaminated fruit juices
worldwide are discussed in this review. This review was aimed at the
possible sources of microbial contamination, disease caused by them
and determining some ways to avoid such phenomena. From the
information provided here, it was noticeable that commercial fruit
juice can also harbour pathogenic microorganisms which can cause
serious disease outbreaks. The contamination can also be initiated
during in house consumption if lack of awareness prevails among the
consumers. Manufacturing process should be much more strict in this
regard to assure the public health safety. Commercially available fruit
juice are consumed worldwide among different ages of people and if
not processed properly, this healthy drink may be hazardous for
human health.

Keywords: Fruit juice, Contamination, Microbial spoilage, Public health

safety, Good Manufacturing practice (GMP).

12
 INTRODUCTION TO FRUIT JUICES AND THE NUTRITIONAL VALUES

Fruit juice is a popular soft drink made of the pulp of different types of
fresh fruits . Fruit imparts natural flavour to fruit Fruit juices contain
many components which are beneficial for our health. Though the actual
components varies from fruits
to fruits, in general they contain flavonoid glycosdides, dietary fiber,
calcium, vitamin C, carotenoids, lutein, lycopene, 2- carotene, phenolic
acids, stilbenes, ellagic acid, amino acids, aroma compounds,
anthocyanin, flavonols, polyphenols, potassium, vitamin D, low amount
of sodium, diabetes, cataracts, Alzheimer’s disease, asthma and helps in
the formation of collagen, cartilage, blood vessels and muscles 5-23.
Considering those beneficial impacts on human health, fruit juices
become popular worldwide.
though most of the case they have better health conditions, better
immune system comparing those who don’t drink fruit juices. Fresh fruit
juices without additives and extra sugar are more healthy and added
ingredient and high sugar contents may damage our health.
High earning and educated people like to drink fruit juice as a
supplement of vitamins and other essential nutrients for health, but low
earning people unable to afford money to buy fruit juices. School going
children also prefer juices due to their attractive freshness and many
flavors as well as the attractive packaging specially manufactured for
them

Global availability of packed juices and the consumers

People of all ages like to drink fruits juices. Young children who drink
fresh fruits or juices regularly, can maintain the habit till the age of
adolescence. Regular fruit juice drinkers have been shown to suffer less
chronic illnesses.Due to worldwide available transportation systems
commercially product fruit juices have been transported from country to
country for marking the juice products available everywhere. Fruit juices
are more popular among children,. but adults used other carbonated
soft drink and/or other energy drinks rather than fruit juices
13
 Microbiological quality perspective of packed juice

Commercial fruit juices can be prepared either by pasteurization or by

adding chemical preservatives. Processed fresh tastes. In the industry
fruit juice is processed by automatic machine (collection, cleaning,
extraction of juice and packaging). There is en every possibility of
contamination of processed fruit juice at any stage of processing.
Cotamination may be occurred by spoilage organisms
and/or by food borne pathogens. Food borne Disease outbreaks have
been documented in different countries. Specially Yeast and molds are
the dominant microorganisms in juice because they can thrive the high
acidic conditions of the juice. Some examples of them are species of the
genera Cladosporium, Candiada , Dekkera , Pichia , Saccharomyces,
Aspergillus, Zygosaccharomyces, Penicillium, Byssochlamys,
Hanseniaspora, Paccilomyces, Mucor, Fusarium, Botrytis, and
Neosartorya Talaromyces etc. Some lactic acid and acetic acid bacteria
may be present in fruit juices. Some pathogenic bacteria like Escherichia
coli O157, Salmonella, and Cryptosporidium, fecal Streptococci and
some spore formers like Clostridium Pasteurianum and some spore
formers like Clostridium Pasteurianum and Bacillus Coagulans may be
present in fruit juice if the juice is not processed adequately.
Bacterial growth in fruit juice depends on pH, storage temperature,
types of packaging material, humidity, water activity, concentration of
preservatives, application of UV treatments during production, sugar
contents, quality of water used in juice, quality of the machines used in
the juice industries, raw materials, quality of fruits etc. Mainly improper
washing of fruits with poor quality water used in the juice preparation
are the major source of contaminating microbes found in the fruits
juices. Some fruit juice spoiling organisms are listed in Table 1.
Fruits juices are stored in cold temperature and or in normal
temperatures. When packed juices are kept in humid condition and at
ambient temperatures (for example during summer seasons), spoiling
microorganisms often get stimulated to grow and cause spoilage and
change in order, taste, visual change etc. If the packaging is not enough
protective, they may invite the spoilage causing microbes to get
14
entrance and spoil the drinks. Smaller packages, juice is often stored
in normal temperature on refrigerators depending on the users, which
may be more vulnerable for contamination.
High pressure, UV radiation, Gamma radiation, ozonisation electron
beam radiation, pasteurization, pulsed electric ofield processing etc. and
chemical preservatives such as organic acids hydrogen peroxide,
dimethyl dicarbonate, vanillin, monocaprylin, essential oils, vanillic acid,
nisin and cinnamon etc are to be applied to preserve fruit juice

What is the difference between pasteurized and unpasteurized juice?

Pasteurized juice have been heat treated to destroy pathogens (germs) and
microbes that can make us sick. This also allows the juice to keep longer as
it destroys many of the microbes that can cause spoilage. Raw freshly
pressed or squeezed juices are not heat treated and are described here as
unpasteurized. These products have short shelf life of a few days. They
must be kept refrigerated and consumed by the best before date.

What is the risk?

In Canada and the US, there were 29 recorded juice and cidere outbreaks. These
outbreaks caused foodborne illness in 1,700 people and 2 deaths over a 20 year
period . Most of these outbreaks involved unpasteurized juices and ciders such as
apple cider, orange juice and lemonades. Other fresh fruit juices such as pineapple,
carrot, cocount. cane sugar, banana, acai and mixed fruit juice have also made
people ill. The most common pathogens in unpasteurized juice afer E. coli O157
and Olll. SalmoneLLA. Cryptospordidlum and novorius. A few others include Vibrio
cholerae. Clastridium botulinum..

How serious is the problem ?

This problem is very serous since these pathogens can cause more than
just short-term diarrhea. E. coli O157:H7 can cause permanent kidney
damage or, in some cases, death Hepatitis can cause liver damage
Botulism impairs nerve signals and, in severe cases, cases death
Cryptosporidilum causes long term diarrhea.

15
 Who is at greatest risk?

People at higher risk of getting sick are:

Infants Young children (5 Years of age and under) Older adults Pregnant
women People with weakened immmune systems (such as those with
HIV or those being treated for cancer) These vunerable groups should
not drink unpasturized juice.
Schools, child and adult day cares, hospitals and other facilities serving
valnerable groups should nto sell or serve inpasteruized juice. Children
on field trips to farms or farm markets should not be offered
unpasteurized juice.
Where do pathogens like E. coli O157:H7 come from ?
These pathogens are commonly found in cattle feces. Most E. Coli
O157:H7 outbreaks are linked to food or water that has been
contaminated with cattle and animal faces. Fruit ands vegetables can
hervesting, storage or processing.

16
How do I know if a juice is pasteurized?

Most juices sold in stories are pasteurized and will have the word "
Pasteurized" on the product label Freshly pressed or squeezed juice sold at
juice bars or at roadside stands and farmers' markets are likely
unpasteurized. The labelling of unpasteurized juice is voluntary. Cheek if the
word" unpastecurized" is on the product label. If n doubt, ask the seller
before deciding to buy and drink the juice.

Does pasteurization reduce the nutrients in juice?

Most commercially pasteurized juices are healted to about 85.C for about
16 seconds to destroy the pathogens that may be present. These products
are just as nutritius as if they were not heate. They taste good and last
much longer than untreated juice.

Will refrigeration make the juice safe?

No. Refrigeration does not destroy pathogens, it only slows their growth.
Unpasteurized juice have a short shelf life of only a few day. Refrigerate
unpasteurized juices and consume them promptly.

How do i reduce the risk of illness?

The best way to kill pathogens like E. coli O157:H7 and other bacteria is
through pasteurization Boil or pasteurize raw ruice and cider before
consumming. To pasteurize juice at home, heat to 700c for at least i minute
A void serving unpasteurized juice and cide products to those most at risk
(Young children 5 years of age or under, pregnant women, the elderly and
people with weakened immune system)
Ensure freshness and quality by refrigerating juice and cide products. Do
not use them after the best before date
If you think drinking unpasteurized juice or cider may have made you ill, see
a health care provider immediately and notify our local health authority.
You can also call 8-1-1 to speak to a registered nurse or registered dietitian.

17
 MATE
RIALS & METHODS

18
 Materials

Equipment
 Autoclave
 Bio safety cabinet
 Water bth
 Weighing balance
 Incubater
 Laminar air flow
 Ph meter
2 CONSU MABLES
 Mask
 Gloves
 Head cap
 Lap coots
 Tissue paper
 Micropipette
 Burner
 Incoculation loop
 Durham’s tube
 Test tube stand
 Disposable sterile petripleates. Etc

19
 About instruments

Autoclave :-

Autoclave is a pressure chamber used to carry out industrial processes

requiring elevated temperature and pressure different from ambient air
pressure. Many autoclaves are uses to wqipment and supplies by subjecting
them to high-pressure saturated steam at 121c (249f) for around 15-20
minutes depending on the size of the load and the contensts. The autoclave
was invented by in 1879, although a precursor known as the was created by
in 1679.

20
 Hot ari ovens are electrical
devices which use dry heat to
sterilize. They were originally
developed by Pasteur.
Generally, they can be
operated from 50 to 300 using
a thermostat to control the
temperature.
It is used for dry heat
sterilization. Glassware’s, Petrit
plates and pipettes are packed
in stainless steel containers
and kept 170c 2 hours.
Th
e standart settings for a hot air
oven are
1.5 to 2 hours at 160c (320f)

Incubator is a device used to grown and

maintain microbiological cultures or cell
cultures. The incubator maintains optimal
temperature, humidity and other conditions
such as the carbon dioxide (co2) and oygen
content of the atmosphere inside.
It is used for providing favourable
temperature condition for the growth of
culture organisms. Generally, the
temperature of the incubator is operated at
30-35 c for growth of bacteria.

21
 Laminar air flow chamber

A laminar air flow chamber is a carefully enclosed bench designed to

prevent contamination of semiconductor wafers, biological samples, or any
particle sensitive materials. Air is drawn through a HEPA filter and blown in
a very smooth, laminar flow towards the user. The cabinet is usually made
of stainless steel with no gape or joints where spores might collect.

Ph meter is a scientific instrument that measures the hydrogen in activity is

water-based solution,s indicating its acidity or alkalinity expressed as ph.
The pH meter measures the difference in electrical potential between a pH
electride and a reference electrode, and so the pH meter is sometimes
referred to as a “potentionmetric pH meter”. The difference in electrical
potential relates to the acidity or pH of the solution. The pH meter is used in
many applications ranging from laboratory experimentation to quality
control.

22
 Microscope

A microscope is an instrument used to see objects that are too small to be

seen by the naked eye. Microscopy is the science of investigating small
objects and structures using such an instrument. Microscopic means
invisible to the eye unless aided by a microscope. There are many types of
microscopes, and they may be grouped in different ways. One way is to
describe the way the instruments interqact with a sample to create images,
either by sending a beam of light or electrons to a sample in ites optical
path, or by scanning across, and a short distance from, the surface of a
sample using a probe. The most common microscope (and the first to be
invented) is the optical microscope, which use light to pass through a
sample to produce an image. Other major types of microscopes are the
fluocresence microscope, the electron microscope (both, the transmission
electron microscope and the scanning electron microscope) and the various
types of scanning probe microscopes.

23
 METHODS

 IS0659-1:2017SAMD2020
 IS 5887Part1.1976RA2018

24
 Method of salmoella

 Add 25 ml sample into 225 ml BPW. Homogenanised and mixed the

sample well with BPW. And intubate at 37 for 20 to 24 hourse.
 After 24 hrs transfer 1 lm culture in MKTTN and 0.1 in RVM.
incubate the RVM tubes at 42’c for 24 hours and MKTTN at 37’c for 24 hrs.
 After 24 hours, streak on XLDA AND BGA FROM MKTTN AND RVM.

Slightly bin-white red surrounding on BGA.

 Salmonella give black shiny colonies with black XLDA and incubate
37’c for 24 hrs.
 Now o select colonies then streak on NA plate. And incubate at 37’c
for 18 hrs.

25
Apple + beetroot + carrot Real juice

26
MKTTN & RVM tubes

27
 XLDA & BGA
XLDA & BGA

28
 NA plates

Method of E. Coli
 Add 25 ml sample into 200 ml of 0.1% peptan water.
Homogenized and mixed the sample sample well with pepton water.

 Then take 10ml of supernatant of suspension into the 10 ml double

of strength and 1 ml single strength of macconkey broth with
Durham’s tube 9MCB). And incub ateat 370c for 24 hours.
 After 24 hour If bubble is observe in the drurham’s tube then streak
on MCA & EMB agar plates with inoculation loop.

 Now incubate all plates at 370c for 24 hours.

 Afte4r 24 hour, observe the pink colar colonies on MCA if netallie

plates.

 Now transfer the colonies in the NA plate. Incubate NA at 37 0 c for

24 hours.

29
Real juice

30
MC btubes

31
32
NA plates

33
 BIO CHEMICAL TEST

Triple Sugar-Iron Agar Medium

Triple sugar Iron Agar Medium is recommended for identification of

gram-negative entric bacilli on the basis of dextrose, lactose and
sucrose fermentation and hydrogen sulphide production in
accordance with India Pharmacopoeia.

Compostion

Ingredients Gms/Litre
Beef extract 3.000
Peptone 20.000
Yeast extract 3.000
Lactose 10.000
Sucrose 0.000
Dextrose monohydrate 1.000
Ferrous Sulphate 0.200
Sodium chloride 5.000
Sodium thiosulphate 0.300
Phenol red 0.024
Agar 12.000
**Formula adjusted, standardized to suit performance parameters

Direction
Suspend 64.42grams (the equivalent weight of dehydrated medium
per litre)in 100ml purified/ distilled water. Heat to boiling to dissolve
the medium competely. Mix well and distribute into test tubes and
Sterilize by maintaining at 101bs pressure(115degree celcuis) for 30
minutesor as per validated cycle. Allow the medium to set in sloped
from with a butt about 2.5cm long.
Note:- Directions specified are as per the concurrent edition of
pharmacopoeia in force. Specified expiry period corresponds th this.

34
Principle and Interpretation

Triple Sugar Iron Agar was originally proposed by Sulkin and Willett
(1) and modified by Hajna(2) for identifying Enterobacteriaceae. This
medium complies with the recommendation of Indian
Pharmacopoeia(3) for the inditification of gram-negative bacilli.

Peptone, yeast-extract abd beef extract provide nitrogenous

compounds, sulphar, trace elements and vitamin B complex etc.
Sodium chloride maintains osmotic quilibrium. Lactose, sucrose and
dextrose monohydrate are the fermentable carbohydrates. Sodium
thiosulphate and ferric or ferrous ions make 112S indicator system.
Sodium thiosulphate is also an inactivator of halogen and can
minimize its toxicity in the testing sample, if any during microbial limit
tests. Phenol red is the pH indicator.

Organisms that ferment dextrose monohdyrate produce a variety of

acids, varying the colour of the medium from red and yellow. More
amounts of acids are liberated in butt region(fermentation) than in the
slant(respiration). Growing bacteria also form alkaline products form
the oxidative decarboxylation of peptone and these alkaline products
neutralize the large amounts of acid present in the butt. Thus the
appearance of an alkaline(red) slant and an acid(yellow) butt after
incubation indicates that the organism is a dextrose fermenter but is
unable to ferment lactose and/or sucrose. Bacteria that ferment
lactose or sucrose (or both), in addition to dextrose, produce large
amounts of acid enables no reversion of pH in that region and thus
bacteria exibhit an acid slant and acid butt. Gas production (Co2) is
detected by the presence of cracks or bubbles in the medium. when
the accumulated gas escape. Thisosulphate is reduced to hydrogen
sulphide by several species of bacteria and H2S combines with ferric
ions of ferric salts to produce the insolube black precipitate of ferrous
sulphide. Reduction of thiodulphate proceeds only in an acid
environment and blackening usually occurs in the butt of the tube.

35
 Triple Sugar Iron Agar should be used in parallel with Urea Agar/
Broth (M112/MM111) to distinguish between Salmonella Alkaline
slant/acid butt-only glucose fermented.
Acid slant/acid butt-dextrose and sucrose fermented or dextrose and
lactose fermented or all the three sugars, dextrose, lactose and
sucrose fermented.
Bubbles or cracks present - gas production
Black precipitate present - H2S gas production

Some members of the Enterobacteriacea and H2S producing

Salmonella may not be H2S positive on TSI Agar. Some bacteria may
show H2S production on Kligler Iron Agar but not on TSI Agar. This
can happen because utilization of sucrose in TSi Agar suppresses the
enzymic pathway that result in H2S production.

36
 Lysine Decarboxylase Broth

Intended Use:

Recommended for differentiating Salmonella arizonae from the Bethesda

Ballerup group of Enterobacteriaceae.
Compoition***

Ingredients Gms/Litre
Peptone 5.000
Yeast extract 3.000
Dextrose(glucose) 1.000
L-Lysine hydrochloride 5.000
Bromocresol purple 0.020
Final pH(at 25 degree celcuis) 6.8+0.2

**Fomula adjusted, standardized to suit performance parameters.

Dirctions

Suspend 14.02grams in 1000ml purified/distilled water. Heat, if necessary

to dissolve the medium completely. Dispense 5ml amount into screw-
capped test tubes. Sterilize by autoclaving at 15lbs pressure(121 degree
celcuis) for 15 minutes. Cool the tubed medium in an upright position and
overlay with 2-3 ml of sterile mineral oil.

Principle and Interpretaion

Decarboxylase media were first described y Moellor (7,8,9) for detecting

and ornithine decarboxylase and arginine dihydrolase Falkow developed a
lysine decarboxylase medium for the identification ad differentiation of
Salmonella and Shigella (4). Lysine Decaboxylase Broth is especially suited
to study the decarboxylase reactions for member of ebnterobacteriaceae.
Lysine Decarboxylase Broth is also reomended by APHA (5,6) and other
standard methods.

37
 Urea Agar base (Christensen)(Autoclavable)

Intended Use:-
Urea Agar Base with the addition of Urea is recommended for the detection
of urease production, particularly by members of the genus Proteus.

Composition***
Ingredients Gms/Litre
Peptone 1.000
Dextrose(glucose) 1.000
Sodium Chloride 5.000
Disodium hydrogen phosphate 1.200
Potassium dihydrogen phosphate 0.800
Phenol red 0.012
Agar 15.000
Final pH(at 25 degree C) 6.8+0.2
**Formula adjusted, standardized to suit performance parameters

Directions
Saspend 24.01 grams in 950ml purifies/distilled water. Heat to boiling to
dissolve the medium completely. Sterilize by autoclaving at 10 lbs
pressure(115 degree Celcius) for 20minutes. Cool to 45-50 degree celcuis
and aseptically add 50ml of sterile 40% Urea Solution(FD048) and mix well.
Dispense into sterile tubes and allow to set in the slanting position. Do not
overheat or reheat the medium as urea decomposes very easily.

Principle And Interpretation

Urea Agar is used to detect urease production. Urea Agar described by

Christensen(3,7) detected urease activity by all rapidly urease-positive
Proteus organisms and also by other members of Enterobacteriaceae (3)
that exhibited a delayed urease reaction (8). This was accomplished by:
a) Adding glucose to the medium
b) Decreasing the peptone concentration and
c) Decreasing the buffering system, as a less buffered medium detects
even smaller amount of alkali(4).

38
Peptone is the source of essential nutrients. Dextrose is the energy source.
Sodium chloride maintains the osmotic equilibrium of the medium whereas
phosphates serve to buffer the medium. Urea is hydrolyzed to librate
ammonia. Phenol red indicator detects the alkalinity generated by visible
color change from orange to pink.

Prolonged incubation may cause alkaline reaction in the medium. A

medium without urea as negative control to rule out false positive results.
Also, all urea test media rely on the alkalinity formation and so they are not
specific for determining the absolute rate of urease activity(8). Thr
utilization of proteins may rise the pH to alkalinity due to proteins
hydrolysis and excess of amino acids liberation results in false positive
reaction.

39
 Malonate Broth

Malonate Broth is recommended for the difference of Enterobacter and

Escherichia on the basis of malonate utilization.

Compostition**
Ingredients Gms/Litre
Ammonium Sulphate 2.000
Dipotassium phosphate 0.600
Monopotassium phosphate 0.400
Sodium chloride 2.000
Sodium malonate 3.000
Bromothymol blue 0.025
Final pH(at 25 degree Celcuis) 6.7+0.2
**Formula adjusted, standardized to suit performance parameters

Direction
Dissolve 8.02 grams in 1000ml distilled water. Dispense and sterilize by
autoclaving at 15lbs pressure(121Degree Celcuis) for 15minutes Avoid the
addition of carbon and nitrogen from other sources.

Principle And Interpretation

Leitson developed a synthetic liquid medium, which differentiated
Aerobacter(now Enterobacter) from Escherichia species based on their
ability to utilize malonate(1) where Enterobacter utilizes manolate and
Escherichia does not.

An organisms that can simultaneously utilize sodium manolate as its carbon

source ammonium sulfate as its nitrogen source produces alkalinity due to
the formation of sodium hydroxide(2). The alkali changes the color of the
bromothymol blue indicator in the medium to light blue and finally to
Prussian blue. The color of the medium remains unchanged in the presence
of an organisms that cannot utilize these substances. Also some malonate-
positive organisms produce only a slight alkalinity that causes the result to
be difficult to interpret. Therefore these tubes should tubes should be
compared with an un-inoculated malonate tube.
40
 Peptone Water
intended Use :
Peptone Water is used as a growth medium and as a base for carbohydrate
fementation media.
composition
ingredients Gms/unit
peptone 10.000
sodium chloride 5.000
Final pH (at 25'c) 7.2+0.2
** Formula adjusted, standardized to suit performance parameters
Directions
Suspend 15.0 grams in 1000 ml distilled water. Add the test carbohydrate in
desired quantity and dissolve completely. Dispense in tube with or without
inverted Durhams tubes and sterilize by autoclaving at 15 ibs
pressure(121'c) for 15 minutes.
Principle and interpretation
Peptone Water is particularly suitable as a substrate in the study of indole
production. Peptone used in Peptone ? Water is rich in tryptophan content.
Presence of indole can be demonstrated using either Kovacs ro Ehlrich
reagent. Peptone Water is also utilized as a base for carbohydrate
fermentation studies with the addtion of sugar and indicators such as
bromocresol purple, phenol red or bromthyml blue.
Peptone water is recommended (3.6.7) for studying the ability of an
organism to ferment a specific carbohydrate which aid in differentation of
genera and species Peptone water is formulated as per Shread. Donovan
and Lee(9). Peptone Water with pH adjusted to 8.4 is suitable for the
cultivation and enrichment of Vibrio species Peptone provides nitrogenous
and cargonaceous compounds. long chain amino acids. vitamins provides
essential nutrients. Sodium chloride maintains the osmotic balance fo the
medium. To study the fermentationj ability of carbohydrates. saccharose,
rhmmnose. salicin are generally added in 0.5% amount seprately to the
basal medium before or after sterlization. ?The acidity formed during
dementation can be deected by addition of phenol red indicator. which
shows a colour change of the medium from red to yellow under acidic
conditions. If desired. Durhan's tube may be used to detect the gas
production if produced.

41
 MR-VP MEDIUM (GLUCOSE PHOSPHATE BROTH)
mr-vp medium (glucose phosphate broth)is recommended for the
perfomance of the methy red and voges proskauer tests in differentiation
of the coli acogenes group
compositon
Ingredients Gms Litre
buffered peptone 7.000
dextrose 5.000
dipotssium phosphate 5.000
final ph (at 25c) 6.9 0.2
**formula adjusted standardized to suit performance prameters
Directions
suspend 17 grams in 1000 ml of distilled water heat if necessary to dissolve
the medium completely. Distribute in lest tubes in 10 ml amounts and
sterilize by autoclaving at 15 Ibs perssure (121c) for 15 minutes
Principal And Interpretation
methy Red and voges Proskauer test are among the two
various tests in the biochemical identification of bacterial species.These
were origially studied by Voges. prokauer(1) and subsequently by clark and
club (2) to diffrentiate between members of the coli acrogens group both
the are based on the detection of specific break down product of
carbohydrate metabolism
all members of enterobacteriacaea are by defination glucose fermenters in
mr vp broth after 18-24 hours of incubation fermention produces acidic
metabolic by products therfore initially all enterics will give a positive mr
reaction if tested (3,4,5) hower after further incubation required by the test
procedure (2-5 days) MR-positive organisms continue to produce acids
resulting in a low Ph (acidic )that overcome the phosphate buffering system
and maintains an acidic environment in the medium (Ph 4.2or less )MR
negative organism furter metabolize the initial fermentation products by
decarboxylation to produce neutral acetyl methylcarbinol(acetoin), which
result in decreased acidity in the medium and raises the pH towards
neutrality(pH 6.0 or above) (6). In the presence of atmospheric oxygen and
alkali, the neutral end products, acetion and 2-3 butanedoil are oxidized to
diacetyl, which creatine to produce a red color.
42
 The Methyl red(MR) test is performed after 5 days of incubation at 30
degree Celcuis(8). The Voges-Proskauer test(VP) cultures are incubated at
30 degree celcuis for 24-48 hrs. Various test procedures have been
suggested for performing the VP test by Werkman (10). OMeara (11),
Levine, et al (12) and Voughn et al.
Werkmans Test. Add 2 drops of a 2% solution of ferric chloride to 50ml
concentrated (40%) sodium hydroxide. Red colour development in a few
minutes after shaking the tube well is a positive reaction.
Levine, Epstein and Voughn modified OMeara technique by dissolving the
creatine in a concentrated solution of potassium hydroxide(R031, OMeara
Reagent). Voughn, Mitvheli and Levine recommended the method of Barritt
as, addition of 1ml of Barritt Reagent B(R030-40%) potassium hydroxide and
3ml of Barritt Reagent A(R029-5% a-naphtol in absolute ethanol) to 5ml
culture. Positive test is indicated by eosin pink color within 2-5 minutes.
The MR and VP tests should not be relied upon as the only means of
differentiating E.coli from the Klebsiella Enterobacter groups. Also
occasionally a known acetoin-positive organism fails to give a positive VP
reaction. TO overcome this possibility, gently heat the culture containig the
VP reagents.

43
 mr-vp medium (glucose phosphate broth)is recommended for the
perfomance of the methy red and voges proskauer tests in differentiation
of the coli acogenes group
compositon
Ingredients Gms Litre
buffered peptone 7.000
dextrose 5.000
dipotssium phosphate 5.000
final ph (at 25c) 6.9 0.2
**formula adjusted standardized to suit performance prameters
Directions
suspend 17 grams in 1000 ml of distilled water heat if necessary to dissolve
the medium completely. Distribute in lest tubes in 10 ml amounts and
sterilize by autoclaving at 15 Ibs perssure (121c) for 15 minutes
Principal And Interpretation
methy Red and voges Proskauer test are among the two
various tests in the biochemical identification of bacterial species.These
were origially studied by Voges. prokauer(1) and subsequently by clark and
club (2) to diffrentiate between members of the coli acrogens group both
the are based on the detection of specific break down product of
carbohydrate metabolism
all members of enterobacteriacaea are by defination glucose fermenters in
mr vp broth after 18-24 hours of incubation fermention produces acidic
metabolic by products therfore initially all enterics will give a positive mr
reaction if tested (3,4,5) hower after further incubation required by the test
procedure (2-5 days) MR-positive organisms continue to produce acids
resulting in a low Ph (acidic )that overcome the phosphate buffering system
and maintains an acidic environment in the medium (Ph 4.2or less )MR
negative organism furter metabolize the initial fermentation products by
decarboxylation to produce neutral acetyl methylcarbinol(acetoin), which
result in decreased acidity in the medium and raises the pH towards
neutrality(pH 6.0 or above) (6). In the presence of atmospheric oxygen and
alkali, the neutral end products, acetion and 2-3 butanedoil are oxidized to
diacetyl, which creatine to produce a red color.

44
 The Methyl red(MR) test is performed after 5 days of incubation at 30
degree Celcuis(8). The Voges-Proskauer test(VP) cultures are incubated at
30 degree celcuis for 24-48 hrs. Various 10). OMeara (11), Levine, et al (12)
and Voughn et al.
Werkmans Test. Add 2 drops of a 2% solution of ferric chloride to 50ml
concentrated (40%) sodium hydroxide. Red colour development in a few
minutes after shaking the tube well is a positive reaction.
Levine, Epstein and Voughn modified OMeara technique by dissolving the
creatine in a concentrated solution of potassium hydroxide(R031, OMeara
Reagent). Voughn, Mitvheli and Levine recommended the method of Barritt
as, addition of 1ml of Barritt Reagent B(R030-40%) potassium hydroxide and
3ml of Barritt Reagent A(R029-5% a-naphtol in absolute ethanol) to 5ml
culture. Positive test is indicated by eosin pink color within 2-5 minutes.
The MR and VP tests should not be relied upon as the only means of
differentiating E.coli from the Klebsiella Enterobacter groups. Also
occasionally a known acetoin-positive organism fails to give a positive VP
reaction. TO overcome this possibility, gently heat the culture containig the
VP reagents.

Simmons Citrate Agar

Intended Use:-
Recommended for differentation the members of Enterobacteriaceae on
the basis of citrate utilization from clinical and non-clinical samples.
Ingredients Gms/Litre
Magnesium Sulphate 0.200
Ammonium dilhydrogen phosphate 1.000
Dipotassium phosphate 1.000
Sodium citrate 2.000
Sodium chloride 5.000
Bromothymol blue 0.800
Agar 15.000
Final pH(at 25 degree celcuis) 6.8+0.2
**Formula adjusted, standardized to suit performance parameters

Direction
Suspend 24.28 grams in 100 ml purified/distilled water. Heat, to dissolve
the medium completely, mix well and distribute in tubes or flasks. Sterlize
by autoclaving at 15lbs pressure(121 Celcuis) for 15 minutes.
45
 Principle and Interpretation
These media are used for the differentiation between Enterobacteriaceae
and the members of aerogenes group on the basis of citrate utilization as
sole carbon source. Initially the citrate medium was developed by Koser
containing ammonium salt as the only nirtogen source and citrarte as the
only carbon source for differntiating Escherichia coli and Enterobacter
aerogenes by IMViC tests. Later on Simmons modified Kosers formulation
by adding sugar and bromothymol blue. It is recommended by APHA.
Ammonium dihydrogen phosphate and sodium citrate serve as the sole
nitrogen and carbon source respectively. Microorganisms also use inorganic
ammonium salts as their sole nitrogen source. Metabolism of these salts
causes the medium to become alkaline, indicated by a change in color of
the pH indicator from green to blue. Bromothymol blue is the pH indicator.
The medium should be freshly prepared because in dry conditions, changes
in colour may appear even before inoculation, especially at the bottom of
the slant.

46
 Andrade Peptone Water
Intended Use:
A basal medium which with carbohydrate addition is used to study
fermentation reactions.

Compositions**
Ingredients Gms/Litre
Peptone 10.000
Sodium chloride 5.000
Andrade indicator 0.100
Final pH (at 25 degree celcuis) 7.4+0.2
**Formula adjusted, standardized to suit performance parameters

Directions
Suspend 15.1 grams in 1000ml purified/distilled water. Heat if necessary to
dissolve the medium completely and dispense in test tube containing
inverted Durhams tubes. Sterilize by autoclaving at 15 lbs pressure
(121degree celcuis) for 15 minutes. Cool to room temperature and
aseptically and sterile stock solution of carbohydrates to a final
concentration of 0.5%.

Principle and Interpretation

Bacteria differ widely in their ability to metabolize carbohydrates and
related compounds. Carbohydrates fermentation reactions aids in the
differentiation and identification of various bacteria. Andrade Peptone
Water is the most commonly used media for carbohydrates fermentation.
Desired carbohydrates is added to the medium, which is inoculated with the
test organisms. This causes a subsequent colour change of the indicator,
from colorless to pink and red. If the added carbohydrates is not
metabolized, the medium remains, pale tan to straw coloured. Gas
produced during fermentation is collected in the Durhams tube.
The peptone used in the medium is free from fermentable carbohydrates
(1,5) and the medium is also free from nitrates which may interfare with gas
production. Andrade indicator is a solution of acid fuchsin which when
triated with sodium hydroxide: changes color from pink to yellow. The
Andrade indicator changes colour from yellow to pink as the pH decreases.
The medium is pink whn hot but becomes straw coloured on cooling. Test
carbohydrates solutions should be sterilized separately and aseptically
47
added to sterile Andrade Peptone Water. Use fresh cultures of organisms
only which have been presumptively identification by Gram staining an
colony morphology. The biochemical identification of organisms capable of
growing in this medium is made by various sugar fermentation results.

48
 ONPG DISCES
ONPG Dises are use for the rapid detection of b-galactosidase activity in
microorganisms, specially to identify late lactose fementers quickly.
Directions
Place one ONPG dise in a sterile test tube. Add. 0.1 ml of sterlie 0.85% w/v
sodium chloride solution(physiological usaline). Pick up the colony undeer
test with a sterlie loop and emulsify it in physiological saline in the tube
containing the dise. incubate at 35-37'c To dect active lactose fermeters
observe the tube at an interval of one hour, for upto 6 hourse. to detect
late lactose fermenters, incubate the tubes for upto 24 hourse.
Precautions
The reaction speed depends upon the size of inoculukum. Use known
postive and negative bets-galactosidase producing organisms to monitor
the dise reactions.
Principle And Interpretation
ONPG(Ortho-nitrophenyl beta-D-galactopy ranoside0 is a synthetic
colurless compound (galactoside) structurally similar to lactors(1).
beta-galactosidase cleaves ONPG to galactose and o-nitrophenyl, a yellow
compund. The ONPG test is specially useful in the repid indentification of
cryptic lactose fermenters (late fermenters). Since members of family
Enterobacteriaceae are rountiely grouped according to their lactose
fermeting ability the ONPG. ONPG is similar in structure to lactose. The
presence of two enzymes is required to demonstrate lactose fementation in
a conventional test. The first eyzyme permease, facilitates the entry of
lactose molecules into the bacteral cell while the second enzyme, beta-
galactosidase, hydrolyzes the lactose to yield glucose and galactose. True
non-lactose fermenters lack both enzymes; however some organisms lack
permease but posses betabalactosidase. These organisms are late lactose
fermenters.

49
GRAM STAINING

50
Gram’s staining
The Gram staining method is named after the Danish bacteriologist Hans
Christian Gram (1853-1938) who originally devised it in 1882 (but published
in 1884), to discriminate between pneumococci and Klebiella pneumonia
bacteria in lung tissue. It is a differential staining method fo differentiating
bacterial species into two large groups(Gram-positive and Gram-negative)
based on the chemical and physical properties of their cell walls. This
reachtion divides the eubacteria into two fundamental groups according to
their stainability and is one of the basic foundations on which bacterial
identification is built. Gram staining is not used to classify archaea, since
these microorganisms give very variable responses.
Gram staining consists of four components:
Primary stain (Crystal violet, methyl violet or Gentian violet)
Mordant (Gram’s lodine)
Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)
Counterstain (dilute carbol fuchsin, safranin or neutral red)
The orginal description of staining technique by Christian Gram in a
publication titled “The differential staining of Schizomycetes in tissue
sections and in dried preparations” in Fortschitte der Medicin; 1884. Vol. 2,
pages 185-189 was slightly different from what we use today. The primary
stain used was aniline gentian violet, mordant was Lugol’s iodine (iodine-
potassium iodide in water), decolorizer was absolute alcohol and bismark
brown was the counterstain.
Procedure:
The smear on a glass slide is covered with few drops of one of the primary
stains. Gentian violet is a mixture or methyl violet and crystal violet. The
primary stain renders all the bacteria uniformly violet. After a minute of
exposure to the staining solution, the slide is washed in water. The smear is
treated will few drop fo Gram’s lodine and allowed to act for a munue. This
results in formation of a dye-iodine complex in the cytoplasm. Gram’s
lodine serves as a mordant. The slide is again washed in water and then
decolorized in absolute ethy; alcohol or acetone. A mixture of ecentone-
ethy; alcohol(1:1) can also be used for descolozation. The process of
decolorization is fairly quick and should not exceed 30 seons for thin
smear.s Acetone is a potent decolorizer and when used alone can

51
decolorize the smear in 2-3 second, A mixture of ethnd and acetone acts
more slowly than pure acetone. Acetone. Decolorization is the most crucial
part of Gram staing and error can occur here. Prolonged decolorization can
lead to over- decolorized smear and a very short deolorization period may
lead to under-decolorized smear.
After the smear is decolorized. It is washed in water without any delay. The
smer is finally treated with few frops of counterstain such as dilute carbol
fuchsin, neutral red or safrain.
Those bacteria that hold on to primary dyeiodline complex and remain
vloet are called Gram positive and those which get decolorized and
subsequently take up counterstain (pink/red) are called Gram negative.
Basic fuchsin (present in dilute carbol fuchsin) stains many Gram negative
bacteria more intensely than does safranin, making them easier to see.
Some bacteria which are poorly stained by safranin, such as Haemophilus
spp., Legionell spp.. and some anaerobic bacteria, are readily stained by
basic fuchsin.
In order to ascertain if the staining procedure was satisfactorily conducted,
a control smar of known Gram positive organism (e.g,. Staphylococcus
aureus) and a known gram negative organism (Esherichia coli) must be
stained simultaneously. May appear gram positive, the pus cells and
epithelial cells are always gram negative.

Mechanism of Gram reaction:

Various theories have been proposed to explain why some bacteria retain
the dye and some don’t. Theories such as differences in cytoplasmic pH (2 in
case of Gran positive bacteria and 3 in case of Gram negative bacteria). And
presence of Magnesium ribonucleate in Gram positive bacteria and its
absence in Gram negative bacteria have not recived widespread
aceptanace. The thickness of Gram positive cell wall and presence of more
lipids in Gram negative cell walls have been more acceptable easons for
Gram stain reactions.
It is believed that the positively charged crystal violet pass through the cell
wall and cell membrane and binds to negatively chard components inside
the cell. Addition of negatively charged iodine (in the mordant) binds of the
positively charged dye and forms a large dye-iodline complex within the
cell. Crystal violet (hexamethy-para-rosaniline

52
 Chloriode). Interacts with aqueous Kl-l2 via a simple anion exchange to
produce a chemical precipitate. The small chloride anion is replaced by the
bulkir iodide, and the complex thus formed becomes insoluble in water.
During decolorization, alcohol dissolves the lipid present in the outer
membrane of Gram negative bacteria and it leaches the dye-lodine complex
out of the cell. A thin layer of peptidoglycan does not offter much resistance
either. The dye-iodline complexes are washed from the Gram negative cell
along with the outer membrance. Hence Gram negative cells readily get
decolorized. On the other hand Gram positive cells become dehydrated
from the ethanol treatment, closing the pores as the cell wall shrinks during
dehydration. The dyeiodine complex gets trapped inside the thick
peptidoglycan layer and does not get decolorized.

Limitations of Gram staining:

Some Gram-positive bacteria may lose the stain easily and therefore
appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-
variable). When over-decolorized, even Gram positive bacteria may appear
pink and when under-decolorized gram negative becteria may appear Gram
positive.
The Gram reaction also depends on the age of the cell. Old cultures of
Gram posiive bacteria (where cell walls may be weakened) may readily get
decorized. Gram positive cells affected by cell wall active agents such as
lysozyme or antibiotics may become Gram negative. Gram-positive bacteria
such Actinoyces, Arthobacter, Corynebacterum, Mycobacterium, and
Propionibacterium have cell walls particularly sensitive to brekage during
cell division, resulting in Gram-negative staining of these cells. In cultures of
Bacillus, and Clostrudium a decrease in peoptidoglycan thickness during cell
growth may cause some of them to appear Gram negative.
Certain group of bacteria can display variable response to the stain, which
can be due to growth stress(e.g,. unsuitable nutrients, temperatures, pHs,
or electrolytes) that results in a number of nonviable, gram-negaive cells in
a gram positive culture, but certain bacterial species are known for their
gram variability even under optimal growth conditions. Some bacteria tend
to appear Gram negative when grown in acidic medium,.
Loss of cell walls in Gram positive bacteria may render them Gram negaivce
(L-forms). Bacteria totally devoid of cell wall (Mycoplasma) are always
Gram negative. Bacteria such as Mycobacterium that have extra waxy
53
content in their cell wall are difficult to stain. Small and slender bacteria
such as Treponema, Chlamydla. Rickettsla are often difficult to stain by
Gram’s method. Gram positive bacteria that have been phagocytosed by
plymorphs may also appear Gram negative.

Modifications of Gram stain:

There have been several modifications of Gram’s stain. Theese are:

1. Kopeloff and Beerman’s modification: Primary stain solution consists
of freshly constituted methyl violet with sodium bicarbonate in
distilled water. Mordant consists of lidline dissolved in 4% NaoH
solution. Decolorization is either using acetone alone or a mixture of
acetone and ethano. Basis fuccsin is used to counteristain the smear.
This method may be modified to stain tissue sections.
2. Jensen’s modification : This method involves use to methyl violet as
primary stain, iodine and potassium iodide in water as mordant,
absolute alcohol as decoorizer and neutral red as counterstain, For
Neisserieria spp, Sandiford’s counterstain is useful
3. Weiger’s modification: This modification is particularly useful for
staining tissue sections. The primary stain carbol gentian violet is
prepared ushing saturate alchlic solution of gentian violet and 5%
phenol solution. Gram’s lodine is used as a mordant and aniline-xylol is
used as a decolorizer. The counterstain carnalum (carminic acid
potasslum alum in water), however is used ahead of primary stain.
This method may be used to stain Pneumocystis cysts.
4. Preston and Morrell’s modification: The primary stain used in this
modification is ammonium oxalate-crystal violet. The smear is washed
in Lugol’s iodine and further treated with iodine solution. The smear is
decolorized using iodine-acetone decolorizer and counterstained using
dilute carbol fuchsin solution. This method has been further modified
to overcome the irritating lodine in aerosols by reducing the lodine
concentration to one-tenth and shortening the duration of
decolorization to ten seconds.
Applications of Gram staining;
1. 1 differentiation of bacteria into Gram positive and Gram negative is
the first step towards classification of bacteria.
54
2. It also the first step towards identification of bacteria in cultures.
3. Observation of bacteria in cinical specimens provides a vital clue in the
diagnosis of infectious diseases.
4. Useful in estimation of total count of bacteria.

5. Empirical choice of antibiotics can be made on the basic of Gram tain’s

report.
6. Choice of culture media for incoculation can be made empirically
based on Gram’s stain report.

Miscellanea :

1. Although Gram stain is useful in staining bacteria, certain fungi such as

Candida and Cyptococcus are observed as Gram positive yeasts.
2. Half-Gram stain refers to modified staining technique, where the
smear is neither decolorized nor counterstained. It is useful to stain a
known Gram positive bacterium.
3. Rapid Gram stain refers to quickened lechnique where the smear is
exposed to only 30 secounds instead of one minute.
4. In specimen such as sputum capsulated bacteria may stand out as
clear spaces between the bacterium and the pink (mucus) background.
5. The spores may stand out as
6. clear, unstained region in sporing bacteria.

55
56
57
 Discussion :-

Fruit jiices are an important part of the diet of all groups due to the
associated health benefits. Freshly squeezed juices are simply
prepared by extracting the lqiquid and pulp of mature fruit usally by
mechanical means or blender.s
Improperly prepared friesh and vegetable juices are recognized as an
emerging cause of food bome illnesses. Such juices haven been found
to be potential sources of bacterial pathogens.
This experiment aimed at ot evaluate the microbiological safety of
avocado and mango juices consumed in cafes and or restaurants and
exmine the hygienic conditions of the fruit juice processing quipment
and sites.

58
59
60
 Summary
Generally, the results in the present studay clearly indicate the poor
hygienic conditions of these juices and the consumers are at risk of
contacting food bome infections.
Based on these data of the assed fruit avocado was found to be
heavily contanminated with bacteria that could pose health problems.
Lack of training on food hygiene and safety; improper storage and
processing of fruit jiices may attributed to contamination of fruit
during harvesting or poor processing and handling of fruit juices

61
62