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PHYTOCHEMICAL ANALYSIS OF PLANT EXTRACTS AND THEIR ACTION AGAINST PATHOGENS ISOLATED FROM
MASTITIS SUSPECTED MILK SAMPLES.
A DISSERTATION SUBMITTED
TO
THE UNIVERSITY OF MUMBAI
FOR THE PARTIAL FULFILMENT OF THE DEGREE OF
MASTER OF SCIENCE IN BIOTECHNOLOGY
BY
SHAIKH AREEBA M. ASLAM
M.Sc. - II
R.D. NATIONAL COLLEGE, BANDRA,
MUMBAI-50
UNDER THE GUIDANCE OF
DR. VIKAS KARANDE,
ASSISTANT PROFESSOR,
DEPARTMENT OF VETERINARY PHARMACOLOGY AND TOXICOLOGY,
BOMBAY VETERINARY COLLEGE, PAREL,
MUMBAI – 400012
2014-2015
Acknowledgements
This research received support and cooperation from Department of Veterinary
Pharmacology and Toxicology, Bombay Veterinary College, and the dairy farms who provided
the milk samples for this study.
I want to use this opportunity to express my gratitude to everyone who supported me
throughout the course of this M.Sc. Dissertation. I am thankful for their aspiring guidance,
invaluably constructive criticism and friendly advice during the project work. I am sincerely
grateful to them for sharing their truthful and illuminating views on a number of issues related to
the project.
I would like to first thank Dr. Vikas Karande, Assistant Professor, and
Dr. Mrs. M. Gatne, Professor, Department of Veterinary Pharmacology and Toxicology for their
constant guidance, personal attention, suggestions and endless encouragement and full support
during the three months of this research.
I would also like to express my sincere appreciation to my co-workers and friends, Mr.
Suhas Mestry and Ms. Sayli Chalke who gave me enormous valuable discussions, technical
support and hands-on help in many aspects of this research program, and my classmate
Mr. Mayur D. Chauhan for his motivation, care and concern about this dissertation.
This dissertation could not have been finished without the help and support of
Dr. Vijendra Shekhawat, Head of Department of Biotechnology, and Prof. Laukik Shetye for his
constant support and guidance.
Finally, I would express my deepest gratitude to my parents. Words cannot express how
grateful I am to my Father and my Mother for all of the sacrifices that they have made on my
behalf. Your prayers for me is what has sustained me this far. I would also like to thank my
younger brothers for their love and support.
INDEX
Sr. No.
Title of Chapter
Page No.
1
INTRODUCTION
1-4
2
OBJECTIVE
5
3
REVIEW OF LITERATURE
6-17
4
MATERIALS
18
5
METHODS
19-23
6
RESULT AND DISCUSSION
24-35
7
SUMMARY AND CONCLUSION
36
8
BIBLIOGRAPHY
37-41
9
ANNEXURE
41-49
LIST OF TABLES
Sr.no. Title of Tables Page no.
1 Location of sample collection and
physical appearance of milk samples.
19
2 Isolation of organisms on their respective
selective media, method of streaking and
Incubation conditions.
20
3 Information of the four medicinal plants
used in the study.
22
4 Preliminary phytochemical screening
procedures.
23
5 Number of samples from which the the
organisms were isolated.
25
6 Observation for presence of
Phytochemicals.
33
LIST OF FIGURES
Sr.no. Title of Figures Page no.
1 Four different plant extracts 22
2 Enrichment broths showing growth 24
3 Media plates showing growth and no
growth
24
4 Grams nature of isolates from MSA 25
5 Grams nature of isolates from EMB 26
6 Grams nature of isolates from MAC 27
7 Biochemical tests for organisms on MSA 28
8 Biochemical tests for organisms on EMB 29
9 Biochemical tests for organisms on MAC 30
10 Influence of plant extracts on isolates
(AST)
31
11 Graph showing ZDI by plant extracts 32
12 Phytochemical analysis of CIN, CLV and
CMN
34-35
ABBREVIATIONS
MSA Mannitol Salt
EMB Eosin Methylene Blue Agar
MAC MacConkey’s Agar
MH Mueller Hinton Agar
CIN Cinnamon
CLV Clove
CMN Cumin
CHI Chirayita
AST Antibiotic Sensitivity Testing
IMViC Indole test, Methyl Red test, Voges
Proskauer test and Citrate utilization test.
MR Methyl Red
VP Voges Proskauer
ZDI Zone Diameter Inhibition
1. INTRODUCTION Milk and milk products are excellent high quality foods providing both nutritional and culinary
values. However, milk is extremely susceptible to spoilage by microorganisms and the
microbiologist plays a major role in the dairy industry in quality control of milk. Cow’s milk
consists of a variety of nutrients such as fats, proteins, minerals, vitamins, carbohydrates and
water and thus it serves as an excellent medium for bacterial growth. Given the appropriate
conditions milk can act as a carrier of disease causing microorganism’s transformation from
cows to human. Bacteria can be introduced into milk from a wide variety of sources such as
workers, infected cows’ udder, faeces and dust in barns, milk containers or other equipment.
Some microbes can serve as disease causing agents when present in milk. [1]
Coliform bacteria include the organism Escherichia coli which is normal inhabitants of the large
intestine. The presence of these organisms in milk therefore indicates fecal contamination. The
milk can be contaminated by unsanitary handling after the completion of the pasteurization
process. E. coli is an important food-borne disease organism and enteropathogenic type which
can cause diarrhea, even cause complications resulting in fatalities. [2]
Coliform contamination ranks high among the most common types of contamination in the dairy
industry. Microorganisms such as Escherichia coli and Klebsiella spp can multiply in the normal
summer temperatures and hence unpasteurized milk has every chance of containing E coli.
Therefore, even nowadays, basic microbiology tests performed on milk or any dairy product are
aimed at detecting coliforms. [3]
Humans and dairy cows are the main carriers of this microbe, presenting mucosal or cutaneous
lesions such as impetigo or cattle mastitis. Therefore, either the udder of cattle or the hands of
milkers can be responsible for passing on the bacteria to milk, and Staphylococcal mastitis is
known to be prevalent in India even nowadays, [4]
Bovine mastitis, defined as `inflammation of the mammary gland', can have an infectious or
noninfectious etiology. Mastitis can be caused by a variety of bacterial pathogens, most
commonly coagulase positive and negative Staphylococcus, species of Streptococcus, and Gram
negative bacteria including Escherichia coli. [5]
The main bovine mastitis pathogens that have been investigated using molecular methods are the
gram-negative species Escherichia coli and Klebsiella pneumoniae and the gram-positive species
Staphylococcus aureus. Most of these organisms also occur as pathogens of humans. [6]
S. aureus mastitis is typically refractory to antibiotic treatment. Prophylactic measures, including
the development of an effective vaccine, have so far proven unsuccessful for the control of the
disease. [7]
Staphylococcus aureus and E. coli can develop drug resistance to many chemical drugs. Thus,
considerable effort has been expanded by investigators in the development of herbal drugs.
Plants and their products have been used by humans for treatments of numerous diseases for
thousands of years. Traditional medicine (also known as indigenous or folk medicine) comprises
knowledge that developed over generations within various societies before the era of modern
medicine. [8]
In recent years, the interest in the study of medicinal plants as a source of pharmacologically
active compounds has increased worldwide. Many plants have not been studied yet for the
claimed biological activities and possible adverse effects. It is estimated that there is
approximately 500,000 species of plants on earth. [9]
Only a relatively small percentage, 1% to 10%, is used as food by humans and other animal
species together. On the other hand, probably more than 10% of plants are used for medicinal
purposes. Consumers demand for “natural” products is also responsible for this renewed interest.
It is estimated that the world is losing one major drug from these plants every two years. [10]
Natural products derived from medicinal plants have proven to be an abundant source of
biologically active compounds, many of which have been the basis for development of new lead
chemicals for pharmaceutical companies. The large number of pharmacologically active
compounds in plant medicines increases the likelihood of interactions taking place. It is possible
that a number of active molecules of plant extracts could act synergistically to produce the
observed therapeutic effects. [11]
The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic
microbial infectious agents has led to the screening of several medicinal plants for potential
antimicrobial activity, and the plant extracts were found to have potential against
microorganisms. The spices that are generally used as food additives in order to provide taste,
smell, and color also exhibited antibacterial activity. Agaoglu et al. reported the antibacterial
activity of different spices against gram-negative and gram-positive bacteria including
Staphylococcus aureus. Khan et al. reported that spices can be used against multidrug resistant
(MDR) microbes causing nosocomial and community acquired infections such as S. aureus.[12,
13,14,15]
Plants have the capacity to synthesize an almost limitless number of aromatic compounds. A
large proportion is constituted of phenols. Most of these compounds are secondary metabolites
serving in many cases as plant defense mechanisms against predation by herbivores, insects,
bacteria, fungi and viruses. Some of these substances are terpenoids responsible for the
characteristic plant odors, other are plant pigments like quinones and tannins. Herbs and spices
used by humans as food seasoning are also used as medicinal compounds. [16]
Many plant products are known to inhibit the growth (bacteriostatic) or kill the bacteria
(bactericidal). Antimicrobial activities of plant products based on bacterial targets unexploited by
actual antibiotics could constitute a breakthrough for treating infections. This approach could be
more relevant for treatment of etiological agents that are resistant to available drugs. In the best
of cases, the plant products identified could be active only against pathogens without affecting
the other microorganisms of the normal flora. Many plant products have been studied for their
antimicrobial activities either bacteriostatic or bactericidal. [17]
For this study, milk samples were collected from three different dairy farms from cattle
exhibiting signs of mastitis. From these animals one sample was collected and the severity of the
clinical case was scored based on physical appearance of the milk and udder and whether the
cow was exhibiting signs of systemic disease.
Clove (Syzygium aromaticum), Cinnamon (Cinnamomum zeylanicum ), Cumin (Cuminum
cyminum) and Chirayita (Swertia chirayaita) were used in this study to observe their
antibacterial activity against S. aureus and E. coli.
2. OBJECTIVE OF STUDY
1. To isolate organisms from mastitis suspected milk samples from
different regions of Mumbai.
2. To identify the organisms using biochemical tests.
3. To check antibiotic resistance of the isolated organisms against four
plant products.
4. To study the phytochemical properties of the given plant products.
3. REVIEW OF LITERATURE
3.1 Milk - A potent vehicle for the transmission of diseases
Milk is one of the most versatile ingredients and a staple in most households. However, as an
animal product full of nutrients, there are several infectious diseases associated with microbe-
contaminated milk and milk products.
Although milk and dairy products are important components of a healthy diet, if consumed
unpasteurized, they can also pose a health hazard due to possible contamination with pathogenic
bacterium. These bacteria can originate even from clinically healthy animals from which milk is
derived. The decreased frequency of bovine carriage in certain zoonotic pathogens and improved
milking hygiene have contributed considerably to decreased contamination of milk, but have not
and cannot fully eliminate the risk of milk borne diseases.
In addition to being a nutritious food for humans, milk provides a favourable environment for the
growth of microorganisms. Yeasts and a broad spectrum of bacteria can grow in milk,
particularly at temperature above 16°C. The temperature of freshly drawn milk is about 38°C.
Bacteria multiply very rapidly in warm milk, and milk sours rapidly if held at this temperature.
The low pH retards growth of lipolytic and proteolytic bacteria and therefore protects the fat and
protein in the milk. The acidity of milk also inhibits the growth of pathogen but does not retard
the growth of moulds (Jayarao, 2006).
Holly in 2006 carried out the bacteriological examination of milk sold in London shops, milk
which normal in appearance, chemical analysis and taste, and was found to contain hundreds to
thousands of bacteria per cubic centimeter. These samples were taken in sterile glass stoppered
bottle from milk churns, sent from country farms to the principle station in London, before being
handled over to the agents. Immediately after filling the bottles were carefully stoppered, sealed
and brought to laboratory, where its bacterial analysis was undertaken chiefly with a view of
seeing whether or not any of milk contained the tubercle bacillus. It was found that 7% of
samples of country milk produced typical true tubercle in the guinea pigs, 8% of samples of
country sample milk produced typical pseudo tuberculosis. 1% of milk samples produced
diphtheria in guinea pigs, yielding the typical true B. diphtheria, 1% of milk samples caused a
chronic disease due to pathogenic torula.
3.2 Organisms found in milk
Milk is supposed to constitute a complex ecosystem for various microorganisms including
bacteria. Milk products like cheese and curd are widely consumed and market for them has
existed in many parts of the world for many generations. There is an increase demand by the
consumer for high quality natural food, free from artificial preservatives, and contaminating
microorganisms. Contamination of milk and milk products, with pathogenic bacteria is largely
due to processing, handling, and unhygienic conditions. E. coli frequently contaminates food
organism and it is a good indicator of fecal pollution. Presence of E. coli in milk products
indicates the presence of enteropathogenic microorganisms, which constitute a public health
hazard. Enteropathogenic E. coli can cause severe diarrhoea and vomiting in infants, and young
children. Of late L. monocytogenes has been recognized as a food born pathogen that can
contaminate dairy products. Its virulent strain can cause a serious disease called listeriosis,
particularly the risk populations including pregnant women, newborns, the very old, and people
who are immune compromised (Fleming et al., 1985; Bille, 1989).
The list of bacteria which can be responsible for milk-borne diseases is long and it includes
Brucella spp, Campylobacter jejuni, Bacillus cereus, Shiga toxin-producing E. coli (E. coli
O157:H7), Coxiella burnetii, Listeria monocytogenes, Mycobacterium tuberculosis,
Mycobacterium bovis, Mycobacterium avium subspecies paratuberculosis, Salmonella spp,
Yersinia enterocolitica, and certain strains of Staphylococcus aureus which are capable of
producing highly heat-stable toxins.
Coliform contamination ranks high among the most common types of contamination in the dairy
industry. Microorganisms such as Escherichia coli, Pseudomonas aeruginosa, Citrobacter spp,
Klebsiella spp and Proteus mirabilis can multiply in the normal summer temperatures and hence
unpasteurized milk has every chance of containing E coli. Therefore, even nowadays, basic
microbiology tests performed on milk or any dairy product are aimed at detecting coliforms.
The mechanism behind staphylococcal enterotoxin gastroenteritis is the production of a heat-
stable enterotoxin by certain strains of Staphylococcus aureus. Humans and dairy cows are the
main carriers of this microbe, presenting mucosal or cutaneous lesions such as impetigo or cattle
mastitis. Therefore, either the udder of cattle or the hands of milkers can be responsible for
passing on the bacteria to milk, and staphylococcal mastitis is known to be prevalent in India
even nowadays, with an older study showing that staphylococci were isolated from 61.97% of
the bacteriologically-positive samples, appearing to be the main etiological agents of bovine
mastitis in India.
3.3 Mastitis by Staphylococcus and E. coli
Mastitis is the first cause of economical loss in milk production worldwide and is a major
concern in milk transformation. The problem is however currently hard to tackle for mastitis in
dairy cows, sheep and goats [18].
Especially, S. aureus mastitis is typically refractory to antibiotic treatment. Prophylactic
measures, including the development of an effective vaccine, have so far proven unsuccessful for
the control of the disease. S. aureus is well-known to produce a large variety of virulence factors
(including numerous proteins like toxins or adhesins). Consequently, it induces a large panel of
infections, and the clinical acuteness of each infection type may also be variable. For example, S.
aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis.
Such variability relies on staphylococcal virulence factors as well as host factors. Until now, no
study has been performed to identify the transcripts and proteins commonly or specifically
produced in vivo by S. aureus strains during mastitis [19].
Mastitis caused by Escherichia coli is common in high-producing cows with low milk somatic
cell count. The severity and outcome of E. coli mastitis vary between cows of the same herd and
between different lactation stages in the same individual. Variation in susceptibility of cows to E.
coli mastitis and disease severity can be caused by differences in infecting bacteria or cows’
immune response. Presence of some virulence factors has previously been reported in mastitis
causing E. coli bacteria, with serum resistance being the most important one. In early lactation,
the decreased immune defense of the cow is regarded as the primary reason for increased
susceptibility to E. coli mastitis [20].
The study was to investigate bacterial and host factors affecting the severity and outcome of E.
coli mastitis in dairy cows. To study the bacterial factors in E. coli mastitis, E. coli isolates of
clinical bovine mastitis from Finland and Israel were studied by polymerase chain reaction to
detect the genes for certain virulence factors. Serum resistance and capsule formation of the
isolates were also examined, as these affect the pathogenicity of the strain. The studied isolates
possessed a variety of different virulence factors, but none of them was common [21].
3.4 Plants and plant products used as antimicrobial agents
Finding healing powers in plants is an ancient idea. People on all continents have long applied
poultices and imbibed infusions of hundreds, if not thousands, of indigenous plants, dating back
to prehistory. There is evidence that Neanderthals living 60,000 years ago in present-day Iraq
used plants such as hollyhock [21]; these plants are still widely used as ethnic medicine around
the world. Historically, therapeutic results have been mixed; quite often cures or symptom relief
resulted. Poisonings occurred at a high rate, also.
Currently, of the one-quarter to one-half of all pharmaceuticals dispensed in the United States
having higher-plant origins, very few are intended for use as antimicrobials, since we have relied
on bacterial and fungal sources for these activities. Since the advent of antibiotics in the 1950s,
the use of plant derivatives as antimicrobials has been virtually nonexistent. Clinical
microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts.
The plants that showed healing powers are referred as medicinal. Historically, these treatments
would cure or relieve symptoms. In western countries, alternative or complementary medicine
refers to traditional medicine used outside its traditional culture. Today, many plant compounds
are readily available as over-the-counter self-medication. These preparations are relatively
unregulated and as a result, herbal suppliers and natural food stores provide the customers with
variable amounts of active substances of more or less controlled purity.
3.5 Major groups of Antimicrobial compounds from plants.
Plants have an almost limitless ability to synthesize aromatic substances, most of which are
phenols or their oxygen-substituted derivatives. Most are secondary metabolites, of which at
least 12,000 have been isolated, a number estimated to be less than 10% of the total 195. In many
cases, these substances serve as plant defense mechanisms against predation by microorganisms,
insects, and herbivores. Some, such as terpenoids, give plants their odors; others like quinones
and tannins, are responsible for plant pigment. Many compounds are responsible for plant flavor,
and some of the same herbs and spices used by humans to season food yield useful medicinal
compounds. [22]
PHENOLS QUINONES FLAVONOIDS TANNINS
COUMARINES TERPENOIDS ALKALOIDS
3.5.1 Phenolics and Polyphenols
Simple phenols and phenolic acids: Some of the simplest bioactive phytochemicals consist of a
single substituted phenolic ring. Cinnamic and caffeic acids are common representatives of a
wide group of phenylpropane-derived compounds which are in the highest oxidation state. The
common herbs tarragon and thyme both contain caffeic acid, which is effective against viruses,
bacteria, and fungi. Catechol and pyrogallol both are hydroxylated phenols, shown to be toxic to
microorganisms. Catechol has two 2OH groups, and pyrogallol has three. [22, 23, 24]
The sites and number of hydroxyl groups on the phenol group are thought to be related to their
relative toxicity to microorganisms, with evidence that increased hydroxylation results in
increased toxicity. In addition, some authors have found that more highly oxidized phenols are
more inhibitory. The mechanisms thought to be responsible for phenolic toxicity to
microorganisms include enzyme inhibition by the oxidized compounds, possibly through
reaction with sulfhydryl groups or through more nonspecific interactions with the proteins.
Phenolic compounds possessing a C3 side chain at a lower level of oxidation and containing no
oxygen are classified as essential oils and often cited as antimicrobial as well. Eugenol is a well-
characterized representative found in clove oil. Eugenol is considered bacteriostatic against both
fungi and bacteria.
3.5.2 Quinones
Quinones are aromatic rings with two ketone substitutions. They are ubiquitous in nature and are
characteristically highly reactive. These compounds, being colored, are responsible for the
browning reaction in cut or injured fruits and vegetables and are an intermediate in the melanin
synthesis pathway in human skin. Quinones provide a source of stable free radicals, and are
known to complex irreversibly with nucleophilic amino acids in proteins, often leading to
inactivation of the protein and loss of function. For that reason, the potential range of quinone
antimicrobial effects is great. Probable targets in the microbial cell are surface-exposed adhesins,
cell wall polypeptides, and membrane-bound enzymes. [25, 26]
3.5.3 Flavones, flavonoids, and flavonols
Flavones are phenolic structures containing one carbonyl group. The addition of a 3-hydroxyl
group yields a flavonol. Flavonoids are also hydroxylated phenolic substances but occur as a C6-
C3 unit linked to an aromatic ring. Since they are known to be synthesized by plants in response
to microbial infection, it should not be surprising that they have been found in vitro to be
effective antimicrobial substances against a wide array of microorganisms. Their activity is
probably due to their ability to complex with extracellular and soluble proteins and to complex
with bacterial cell walls, as described above for quinones. More lipophilic flavonoids may also
disrupt microbial membranes. Flavonoids lacking hydroxyl groups on their b-rings are more
active against microorganisms than are those with the 2OH groups; this finding supports the idea
that their microbial target is the membrane. Lipophilic compounds would be more disruptive of
this structure. However, several authors have also found the opposite effect; i.e., the more
hydroxylation, the greater the antimicrobial activity. [27, 28]
3.5.5 Tannins
Tannin is a general descriptive name for a group of polymeric phenolic substances capable of
tanning leather or precipitating gelatin from solution, a property known as astringency. Their
molecular weights range from 500 to 3,000, and they are found in almost every plant part: bark,
wood, leaves, fruits, and roots. Many human physiological activities, such as stimulation of
phagocytic cells, host-mediated tumor activity, and a wide range of anti-infective actions, have
been assigned to tannins. One of their molecular actions is to complex with proteins through so-
called nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by
covalent bond formation. Thus, their mode of antimicrobial action is related to their ability to
inactivate microbial adhesins, enzymes, cell envelope transport proteins, etc. [29]
3.5.6 Coumarins
Coumarins are phenolic substances made of fused benzene and a-pyrone rings. They are
responsible for the characteristic odor of hay. Coumarins are known to be highly toxic in rodents
and therefore are treated with caution by the medical community. [30]
3.5.7 Terpenoids and Essential Oils
These oils are secondary metabolites that are highly enriched in compounds based on an isoprene
structure. They are called terpenes, their general chemical structure is C10H16, and they occur as
diterpenes, triterpenes, and tetraterpenes (C20, C30, and C40), as well as hemiterpenes (C5) and
sesquiterpenes (C15). When the compounds contain additional elements, usually oxygen, they are
termed terpenoids. Terpenoids are synthesized from acetate units, and as such they share their
origins with fatty acids. They differ from fatty acids in that they contain extensive branching and
are cyclized. Examples of common terpenoids are methanol and camphor (monoterpenes) and
farnesol and artemisin (sesquiterpenoids). The triterpenoid betulinic acid is just one of several
terpenoids which have been shown to inhibit HIV. The mechanism of action of terpenes is not
fully understood but is speculated to involve membrane disruption by the lipophilic compounds.
[31]
3.5.8 Alkaloids
They are heterocyclic nitrogen compounds. The first medically useful example of an alkaloid
was morphine, isolated in 1805 from the opium poppy Papaver somniferum; the name morphine
comes from the Greek Morpheus, god of dreams. Codeine and heroin are both derivatives of
morphine. Diterpenoid alkaloids, commonly isolated from the plants of the Ranunculaceae, or
buttercup family, are commonly found to have antimicrobial properties. Solamargine, a
glycoalkaloid from the berries of Solanum khasianum, and other alkaloids may be useful against
HIV infection as well as intestinal infections associated with AIDS. [27]
While alkaloids have been found to have microbiocidal effects, the major antidiarrheal effect is
probably due to their effects on transit time in the small intestine. Berberine is an important
representative of the alkaloid group. [32, 33]
3.6 EXPERIMENTAL APPROACHES DEALING WITH EXTRACTION METHODS
OF PHYTOCHEMICALS FROM PLANT PRODUCTS
Advice abounds for the amateur herbalist on how to prepare healing compounds from plants and
herbs. Water is almost universally the solvent used to extract activity. At home, dried plants can
be ingested as teas (plants steeped in hot water) or, rarely, tinctures (plants in alcoholic solutions)
or inhaled via steam from boiling suspensions of the parts. Dried plant parts can be added to oils
or petroleum jelly and applied externally. Poultices can also be made from concentrated teas or
tinctures. [34]
Initial screenings of plants for possible antimicrobial activities typically begin by using crude
aqueous or alcohol extractions and can be followed by various organic extraction methods. Since
nearly all of the identified components from plants active against microorganisms are aromatic
or saturated organic compounds, they are most often obtained through initial ethanol or methanol
extraction. [35]
3.6.1 Efficacy of in vitro experiments studying action of Phytochemicals against Bacteria.
Initial screening of potential antibacterial and antifungal compounds from plants may be
performed with pure substances or crude extracts. The methods used for the two types of
organisms are similar. The two most commonly used screens to determine antimicrobial
susceptibility are the broth dilution assay and the disc or agar well diffusion assay; clinical
microbiologists are very familiar with these assays. Adaptations such as the agar overlay method
may also be used. In some cases, the inoculated plates or tubes are exposed to UV light to screen
for the presence of light sensitizing phytochemicals. [36, 37]
Clove oil has biological activities, such as antibacterial, antifungal, insecticidal and antioxidant
properties, and is used traditionally as a savoring agent and antimicrobial material in food. In
addition, clove oil is used as an antiseptic in oral infections. This essential oil has been reported
to inhibit the growth of molds, yeasts and bacteria. The high levels of eugenol contained in clove
essential oil are responsible for its strong biological and antimicrobial activities. It is well know
that both eugenol and clove essential oil have phenolic compounds that can denature proteins and
react with cell membrane phospholipids changing their permeability and inhibiting a great
number of Gram-negative and Gram-positive bacteria as well as different types of yeast. [38, 39,
40]
Cinnamon is a common spice used by different cultures around the world for several centuries.
In addition to its culinary uses, in native Ayurvedic medicine Cinnamon is considered a remedy
for respiratory, digestive and gynaecological ailments. Almost every part of the cinnamon tree
including the bark, leaves, flowers, fruits and roots, has some medicinal or culinary use. The
volatile oils obtained from the bark, leaf, and root barks vary significantly in chemical
composition, which suggests that they might vary in their pharmacological effects as well. The
different parts of the plant possess the same array of hydrocarbons in varying proportions, with
primary constituents such as; cinnamaldehyde (bark), eugenol (leaf) and camphor (root).[39, 41,
42]
Cumin is a widely used spice condiment, and is known for carminative, stimulant, diuretic,
antispasmodic and astringent properties. The aqueous extract of of cumin is reported to inhibit
the growth of many pathogens including E. coli, S. aureus and Salmonella species. [38]
Swertia chirata belongs to family Gentianaceae. It is an erect annual or perennial herb found in
Himalaya and Meghalaya at an altitude of 1200–1300 meters. The plant has been reported to
possess hypoglycemic activity, anti‐inflammatory activity, hepatoprotective activity, wound
healing activity as well as antibacterial activity. On the basis of these wide ranges of therapeutic
uses, this plant is evaluated for its antimicrobial activity. [43, 44, 45, 46, 47]
3.7 CONCLUSIONS AND FUTURE DIRECTIONS
Scientists from divergent fields are investigating plants anew with an eye to their antimicrobial
usefulness. A sense of urgency accompanies the search as the pace of species extinction
continues. Laboratories of the world have found literally thousands of phytochemicals which
have inhibitory effects on all types of microorganisms in vitro. More of these compounds should
be subjected to animal and human studies to determine their effectiveness in whole-organism
systems, including in particular toxicity studies as well as an examination of their effects on
beneficial normal microbiota. It would be advantageous to standardize methods of extraction and
in vitro testing so that the search could be more systematic and interpretation of results would be
facilitated. Also, alternative mechanisms of infection prevention and treatment should be
included in initial activity screenings. Disruption of adhesion is one example of an anti-infection
activity not commonly screened for currently. Attention to these issues could usher in a badly
needed new era of chemotherapeutic treatment of infection by using plant-derived principles.[31,
76]
4. MATERIALS
4.1. Dehydrated media, chemicals and reagents
4.1.1. Selective Media: Nutrient agar, EMB Agar, Mannitol Salt Agar, MacConkey’s Agar,
Bismuth Sulphite Agar, Mueller Hinton Agar, Lactose Broth.
4.1.2. Grams staining: Crystal Violet, Safranin, Grams Iodine, Ethanol, Immersion oil.
4.1.3. IMViC test reagents: Tryptophan Broth, Kovac’s reagent, Xylene, Glucose phosphate
broth, Methyl red indicator, 5% alcoholic alpha-naphthol, 40% KOH Solution, Simmon’s
citrate agar, Hydrogen peroxide, Saline.
4.1.4 Sugar Fermentation Test: Lactose, Glucose, Mannitol, Sucrose, Phenol Red Broth Base.
The dehydrated bacteriological media components, chemicals, reagents and supplements used in
the study were procured from M/s Hi-Media Laboratories Limited, Mumbai (India).
4.2 Glass wares and Plastic wares
Petri plates, Pipettes, Test tubes, beakers, flasks and measuring cylinders.
All the glass wares used in the study were of Class ‘A’ Borosil grade brand, while plastic wares
were procured from M/s . All the glass wares and plastic wares were sterilized before
every use.
4.3 Antibacterial activity:
Plant products: Clove (Syzygium aromaticum), Cinnamon (Cinnamomum zeylanicum ), Cumin
(Cuminum cyminum) and Chirayita (Swertia chirayaita)
Solvents for extraction: Ethanol, Methanol
Clove, Cinnamon and Cumin were brought from a local market, whereas Chirayata was brought
from Rajasthan.
4.4 Miscellaneous: Laminar Air flow hood, Incubator (37℃), Refrigerator, Bunsen burners,
gas cylinder, weighing balance, Autoclave, Microscope.
5. METHOD
5.1 Standard strains Standard strains of E. coli (MTCC-443) and S. aureus (MTCC-3381) were procured.
All the isolates were confirmed through biochemical tests by comparing with the results of
standard strains.
5.2 Collection of samples For this study, 14 milk samples were collected during the period of I month from dairy farms in
3 different regions of Mumbai, namely Andheri, Malad and Marol, from cattle exhibiting signs
of mastitis (Table 1).
From these animals one sample was collected and the severity of the clinical case was scored
based on physical appearance of the milk and udder and whether the cow was exhibiting signs of
systemic disease.
Samples were collected aseptically, transferred to sterile containers and were directly transported
to the laboratory under cold conditions. They were stored at 4 °C and analyzed within 24 hours.
Location of dairy farm
Sample number
Physical appearance pH of milk sample
ANDHERI
1 Normal 7.0 2 Normal 7.0 3 Normal 7.0
MALAD
4 Yellow, watery 5.5 5 Off white in colour, watery 4.0 6 Normal 7.0 7 Yellowish in colour 5.5
MAROL
8 Normal 4.0 9 Off-white, watery 4.0
10 Yellow, watery 5.0 11 Normal 7.0 12 Normal 6.5 13 Normal 6.5 14 Normal 6.0
Table 1: Location of sample collection and physical appearance of milk
These milk samples were examined for the presence of E. coli, S. aureus, K. pneumonia and
Salmonella.
5.3 Enrichment of microorganisms
1 ml of each sample was extracted aseptically and homogenized with 9 ml sterile enrichment
broth (lactose broth for E. coli, K. pneumonia and Salmonella and peptone water for S. aureus)
and incubated at 37 °C for 24 hours, for further analysis.
5.4 Media and growth conditions
The enriched milk samples were cultured on selective media and incubated as mentioned in the
Table 2.
Organism to be Isolated Medium used Method of
streaking
Incubation
conditions
Escherichia coli EMB Agar
Hexagon
Method
37℃ for 24
hours
Staphylococcus aureus MSA
Klebsiella pneumonia MacConkey’s Agar
Salmonella Bismuth Sulphite Agar
Table 2: Isolation of organisms on their respective media, method of streaking and
incubation conditions.
5.5 Physiological and biochemical examination 5.5.1 Colony Characteristics were observed from all the streaked media plates.
5.5.2 Gram Staining was performed for all the isolates in the following manner:
1. Prepare heat-fixed smears of the test cultures.
2. Flood the smear with gram crystal violet primary stain and stain for 1 minute.
3. Wash off the crystal violet with cold water.
4. Flood the slide with Gram’s iodine mordant and let sit for 1 minute.
5. Wash off the mordant with safranin counterstain solution.
6. Then add more 95% Alcohol to the slide and stain for 20 to 50 seconds.
7. Wash off the Alcohol with cold water.
8. Either blot or air dry.
9. Observe the slide under oil immersion lens.
Four to five suspected colonies from each bacterial plate were picked, cultured and then
identified by biochemical tests.
Biochemical tests were performed to confirm E. coli, K. pneumonia and Salmonella using Gram
staining, Indole, Methyl red, Voges- Proskauer test, Simon citrate agar, and various sugar
fermentation tests
Confirmation of the genus, Staphylococcus was done by Gram staining and various biochemical
tests including Catalase test, and different sugar fermentation tests.
5.6 Antibacterial activity of different spices and herbs
5.6.1 Extract preparation of the herbal samples:
Clove (Syzygium aromaticum), Cinnamon (Cinnamomum zeylanicum), Cumin (Cuminum
cyminum) and Chirayita (Swertia chirayaita) (Table 3) were used in this study to observe their
antibacterial activity against S. aureus and E. coli.
Species Family Local Name Part used Ethanolic extract Cumin
(Cuminum cyminum)
Umbelliferae
Jeera
Fruit
CMN
Clove (Syzygium
aromaticum)
Myrtaceae
Labanga
Flower stalk
and bud
CLV
Cinnamon (Cinnamomum
zeylanicum)
Lauraceae
Darchini
Stem bark
CIN
Chiretta (Swertia chirata)
Gentianaceae Chirayita Stem CHI
Table 3: Information of the four herbal medicinal plant parts used.
The herbal samples were ground into a fine powder in a mortar and pestle and an extraction was
made by soaking the 5g of each herb in 50ml of 50% ethanol for 24 hours, and making a final
concentration of 100mg/ml. The extraction was filtered aseptically and sterilized using syringe
filter.
Fig 1: Four different Plant Extracts; Chirayita, Cinnamon, Clove, Cumin.
5.6.2 Antibiotic sensitivity testing using Agar cup method
S. aureus and E. coli were spread on the MH Agar plates, and 4 wells on each plate were made
with the help of a sterile borer. The filtrate of the extraction was then inoculated in the 4 wells
made on MH Agar plate, and kept at 37℃ for 24 hours.
5.7 Preliminary phytochemical screening
Alkaloids, Anthocyanins, Anthraquinones, Flavonoids, Phenols, Saponins, Tannins, were
screened according to the common phytochemical methods.(Table 4)
Plant Secondary metabolites
Method
To observe
Alkaloids
5 mg plant extract in 10 ml methanol; a portion of 2 ml extract + 1% HCl + steam, 1 ml filtrate + 6 drops of Mayor’s reagent.
Creamish precipitate indicates presence of Alkaloids.
Anthocyanins 5 mg plant extract in 10 ml methanol; a portion 2 ml + 1% HCl +heating.
Orange color indicates the presence of Anthocyanins.
Anthraquinones
5 mg plant extract in 10 ml methanol; a portion of 2 ml + 2 ml ether-chloroform 1:1 (v/v) + 4 ml NaOH 10% (w/v).
Red color indicates the presence of Anthraquinones.
Flavonoids
5 mg plant extract in 10 ml methanol; a portion of 2 ml + conc.HCl + magnesium.
Ribbon pink-tomato red color indicates the presence of flavonoids.
Phenols
5 mg plant material in 10 ml methanol; a portion of 2 ml + 2 ml FeCl3.
Violet-blue or greenish color indicates the presence of phenols.
Saponins Frothing test: 0.5 ml filtrate + 5 ml distilled water.
Frothing persistence indicates presence of Saponins.
Tannins
5 mg plant extract in 10 ml distilled water; a portion of 2 ml + 2 ml FeCl3.
Blue-black precipitate indicates the presence of tannin.
Table 4: Preliminary phytochemical screening procedure.
6. RESULTS AND DISCUSSIONS 6.1 Enrichment
A B
Fig. 2: Enrichment broths (A: Lactose Broth; B: MSB) showing growth.
After the samples were inoculated in the Lactose broth and MSB for enrichment of Coliforms
and Staphylococcus respectively, and incubated at 37°𝐶𝐶 for 24 hours, a thick white pellicle like
growth was observed in the enrichment media, along with turbidity.
6.2 Streaking on selective media
Fig. 3: (Clockwise) Negative and Positive plate of MSA; Pink colonies on MAC; Negative and positive plates of EMB; Negative plates of Bismuth Sulphite Agar; Isolates obtained on MSA.
After the enriched organisms were streak inoculated on the selective media of MSA, EMB, MAC
and Bismuth Sulphite Agar plates. 10 Isolates were obtained on MSA, whereas 6 and 5 isolated
colonies were observed on EMB Agar and MAC respectively. The data is summarized in Table
5.
Media used for streaking of culture
Samples showing growth
Total number of
samples
Type of colonies
Mannitol Salt Agar 1, 2, 3, 5, 7, 9, 11, 12, 13, 14
10 Yellow colonies turning the medium yellow
EMB Agar 1, 2, 3, 9, 10, 12 6 Colonies with metallic green sheen
MacConkey’s Agar 1, 2, 3, 9, 11 5 Pink Colonies Bismuth Sulphite Agar None 0 -
Table 5: Number of samples from which the isolates were observed after streaking on respective selective media.
6.3 Colony Characteristics and Grams Nature
10
Isolates found
on MSA
Characteristics Observation Size Small Shape Circular Colour Yellow Margin Round and complete Elevation Slightly elevated Opacity Opaque Consistency Smooth Grams Nature Gram positive cocci in present in chains
6
Isolates found
on EMB
Characteristics Observation Size Small Shape Circular Colour Green and purple Margin Round and complete Elevation Flat Opacity Opaque Consistency Buttery Grams Nature Gram negative rods in chains
5
Isolates found
on MAC
Characteristics Observation Size Medium Shape Circular Colour Pink Margin Round and complete Elevation Slightly elevated Opacity Opaque Consistency Buttery Grams Nature Gram negative bacilli in
clusters
Fig. 4: Grams nature: Gram positive cocci in chains observed at 100x magnification
Fig. 5: Grams Nature: Gram negative rods observed at 100x magnification
Fig. 6: Grams Nature: Gram negative bacilli in clusters observed at 100x magnification.
Grams nature and colony characteristics of all the 3 type of isolates obtained on MSA, EMB and
MAC was carried out efficiently. On MSA, the isolate was found out to be gram positive cocci,
whereas on EMB and MAC, the isolates were both gram negative and rod-shaped and bacilli
respectively (Fig. 4-6)
6.4 Biochemical tests for identification and confirmation of organisms
Biochemical tests were performed for identification of the isolates obtained on MSA, EMB and
MAC. On MSA, the organism was found out to be Mannitol sugar fermenter. Since MSA also
distinguishes bacteria based on the ability to ferment the sugar mannitol, it can be concluded that
the gram positive cocci shaped organism must be Staphylococcus aureus.
Staphylococci can withstand the osmotic pressure created by 7.5% NaCl, while this
concentration will inhibit the growth of most other gram-positive and gram-negative bacteria.
Also, the test for Catalase was found to be positive. Staphylococci live in oxygenated
environments and have the ability to produce enzymes, which neutralizes the toxic forms of
oxygen. Catalase breaks hydrogen peroxide into water and molecular oxygen. Staphylococci
produce this enzyme and bubble when placed in H2O2 due to release of Oxygen.
6.4.1. Identification of Staphylococcus aureus:
TEST TO OBSERVE INFERENCE SUGAR FERMENTATION TEST
Colour of the phenol red broth containing all the three sugars changes from red to yellow indicating acid production(Figure 7A and 7B)
1. Mannitol Acid production 2. Lactose Acid production 3. Sucrose Acid production
CATALASE TEST (Colony + H2O2)
Strong effervescence Positive for Catalase (Figure 7C)
MANNITOL TEST Media turns yellow Positive for Mannitol fermentation (Figure 7D)
A B
C D
Fig. 7: Biochemical tests for S. aureus isolated from MSA
7A- Before culture inoculation; 7B-After fermentation showing yellow colour in Tryptone broth; 7C- Effervescence observed; 7D- Yellow colouration of Mannitol indication
Mannitol fermentation.
6.4.2. Identification of E. coli
TEST TO OBSERVE INFERENCE SUGAR FERMENTATION TEST
Colour of the phenol red broth containing all the three sugars changes from red to yellow indicating acid production(Figure 8A and 8B)
1. Glucose Acid production 2. Lactose Acid production 3. Sucrose Acid production
IMViC TESTS 1. Indole test
(Sample + xylene+10 drops of Kovac’s reagent)
Red layer at the top of the solution
Positive (Fig. 8C)
2. Methyl Red Test (Sample + 10 drops of Methyl Red)
Solution turns red Positive (Fig8C)
3. VP Test (Sample + KOH +α-Naphthol)
Mahogany Red colour (Negative)
Negative (Fig. 8C)
4. Citrate Utilization Test Colour change from green to blue (negative)
Negative (Fig. 8C)
8A 8B 8C
Fig. 8: Biochemical tests for E. coli isolated from EMB
8A- Before culture inoculation; 8B-After fermentation showing yellow colouration of Tryptone broth; 8C- IMViC Tests- Tube-1: Indole ring test, Tube-2: MR test showing red coloration of solution, Tube-3: No change observed in VP Test, Tube-4: Citrate Utilization test showing no colour change in Media.
6.4.3. Identification of organism (K. pneumonia/ E. coli)
TEST TO OBSERVE INFERENCE SUGAR FERMENTATION TEST
Colour of the phenol red broth containing all the three sugars changes from red to yellow indicating acid production(Figure 9A and 9B)
4. Glucose Acid production 5. Lactose Acid production 6. Sucrose Acid production
IMViC TESTS 5. Indole test
(Sample + xylene+10 drops of Kovac’s reagent)
Red layer at the top of the solution
Positive (Fig. 9C)
6. Methyl Red Test (Sample + 10 drops of Methyl Red)
Solution turns red Positive (Fig9C)
7. VP Test (Sample + KOH +α-Naphthol)
Mahogany Red colour (Negative)
Negative (Fig. 9C)
8. Citrate Utilization Test Colour change from green to blue (negative)
Negative (Fig. 9C)
9A 9B 9C
9A- Before culture inoculation; 9B-After fermentation showing yellow colouration of Tryptone broth; 9C- IMViC Tests- Tube-1: Indole ring test, Tube-2: MR test showing red coloration of solution, Tube-3: No change observed in VP Test, Tube-4: Citrate Utilization test showing no colour change in Media.
MAC is a differential medium which differentiates between lactose fermenters and Non-
fermenters on the basis of colour change reaction. Lactose fermenters produce organic acid after
utilizing the Lactose in the medium, which lowers the pH and results in appearance of Pink
colonies. Non-lactose fermenters do not utilize lactose, and use peptone instead, so they end up
forming white or colourless colonies. E. coli is a lactose fermenter and pink colonies were
observed on MAC and metallic green sheen was observed on EMB Agar, which is a selective
media for E. coli.
Grams nature and colony characteristics were studied, and gram negative rods and bacilli were
observed. After the IMVic Tests were performed, the isolate was positive for sugar fermentation,
as well as for methyl red and Indole ring test. So it can be concluded that E. coli was isolated on
EMB as well as MAC, and no Klebsiella was present on MAC, since the VP test and Citrate
Utilization tests were found to be negative, whereas Klebsiella is positive for VP and Citrate
Utilization.
Hence, S. aureus and E. coli were present in the milk samples.
6.5 Antibacterial activity of different plant extracts
10.1 10.2 Fig 10.1: Influence of Plant extracts against S. aureus.(A) No inhibition by CMN; (B) ZDI by CIN=18mm; (C) ZDI by CLV=14mm; (D) No Inhibition by CHI. Fig 10.2: Synergistic action of plant extracts against S. aureus. (A) Ethanol control; (B) Zone diameter inhibition by CIN=26mm; (C) ZDI by CIN=14mm; (D) ZDI by synergistic effect of CIN+CLV=31mm
Figure 11: Zone diameter of inhibition (ZDI) of the four plant extracts for S. aureus CIN= Cinnamomum zeylanicum; CLV= Syzygium aromaticum; CMN= Cuminum cyminum. CHI=Swertia chirayaita
The agar well diffusion test results are represented in Fig. 10. All the S.aureus isolates were
found to be sensitive to the crude ethanolic extracts of the two spices CIN and CLV, while CHI
and CMN were not found to be effective against S. aureus. No antibacterial activity of these four
plant extracts was observed against E. coli isolates. Since CLV and CIN alone gave good results
against S. aureus, they both were checked for synergistic activity. The synergistic effect was
found to be more effective than the activity of the two herbs alone. CIN gave a ZDI of 26mm and
CLV of 14mm. The combined effect of both the herbs gave a ZDI of 31mm. This may mean that
both the plant extracts when used in combination can also prove to be effective against MRSA.
0
5
10
15
20
25
30
35
S. aureus E. coli S. aureusCinnamon 18 0 26
Clove 14 0 14
Cumin 0 0 0
Chirayita 0 0 0
CIN/CLV 31
Zone
Inhi
biti
on D
iam
eter
(m
m)
ZDI of plant extracts against S. aureus and E. coli
6.6 Phytochemical analysis of the plant extracts Plants possess phytochemicals, which have antimicrobial activity. Out of the for plants selected,
two of them showed an effective antimicrobial activity against S. aureus. So there was a need to
identify which phytochemicals were present in CIN, CLV and CMN, so preliminary standard
phytochemical analysis was performed using different chemical reagents as mentioned in
‘Method’ section of this study.
Plant
Secondary
metabolites
To observe
CIN
CLV
CMN
Alkaloids Creamish precipitate - + +
Anthocyanins Orange color - - +
Anthraquinones Red color - + +
Flavonoids Ribbon pink / tomato red color - - -
Phenols Violet-blue or greenish color - + +
Saponins Frothing + - -
Tannins Blue-black precipitate + + +
Table 6: Observations for the presence of phytochemicals in CIN, CLV and CMN; (+) shows positive for test; (-) shows negative for test.
Cinnamon was found to have Saponins and Tannins, while Clove showed positive results for
presence of Alkaloids, Anthraquinones, Phenols and Tannins. Cumin, though did not have any
antibacterial effect against S. aureus, was studied for presence of phytochemicals as well. It
showed positive results for Alkaloids, Anthrocyanins, Anthraquinones, Phenols and Tannins.
Fig. 12 A
Fig. 12 B
Fig. 12 C
Fig. 12: Phytochemical analysis of (A) Cinnamon, (B) Clove and (C) Cumin plant extracts.
7. SUMMARY AND CONCLUSION 14 raw milk samples which were suspected to be affected by Mastitis were collected from
different regions of Mumbai, during the period of 1 month. From these animals one sample was
collected and the severity of the clinical case was scored based on physical appearance of the
milk and udder and whether the cow was exhibiting signs of systemic disease. The samples were
checked for the presence of pathogens S. aureus and E. coli, which mostly dominate during this
clinical case. After enrichment and isolation on selective media, 10 samples showed presence of
S. aureus and 6 of the total milk samples showed presence of E. coli. Morphological
characteristics of these organisms were studied and the organisms were identified by
Biochemical tests which included Sugar fermentation test, IMViC tests and Catalase test.
The isolates were then checked for their susceptibility against plant extracts such as Clove
(Syzygium aromaticum), Cinnamon (Cinnamomum zeylanicum), Cumin (Cuminum cyminum)
and Chirayita (Swertia chirayaita), out of which Cinnamon showed the highest inhibitory activity
against S. aureus followed by Clove. The inhibitory action of Cinnamon was 46.15% higher than
that of Clove. Cumin and Chirayita did not show any inhibition. The synergistic action of of
Cinnamon and Clove together showed a higher inhibitory activity (19.23% of Cinnamon alone
and 121.4 % of Clove alone). No inhibitory activity was seen of these plant extracts against E.
coli isolates.
Since phytochemicals are responsible for the antibacterial activity of plants and plant products,
these samples were then tested for the presence of phytochemicals by basic preliminary
phytochemical screening tests, by which, presence of Alkaloids, Phenols, Tannins, Quinones and
Saponins was observed, which may be the reason for the antibacterial activity of the plants
against S. aureus.
Conclusion: Cinnamon and Clove along with a combination of different herbs with antibacterial
property can be used as traditional herbs in synergy against MDR phenotypes of S. aureus.
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9. ANNEXURE
[1] Nutrient Agar
This media has a relatively simple formulation. It provides the nutrients necessary for the
replication of a large number of non-fastidious microorganisms.
Nutrient Agar is a basic culture medium used for maintenance or to check purity of subcultures
prior to biochemical or serological tests from water and Dairy. This medium may be used as
slants or plates for routine work with non-fastidious organisms.
Nutrient Agar, pH 6.8 has relatively simple formulation which provides the necessary nutrients
for the growth of many microorganisms which are not very fastidious. Many bacteria have the
optimum pH growth range of 6.6 to 7.0.
Beef extract contains vitamins, organic nitrogen compounds, salts and little carbohydrates.
Peptic digest of animal tissue provide amino acids and long chain peptides for the organisms.
Composition
Ingredients Grams/litre Peptone 5.00
Beef extract 1.50
Sodium chloride 5.00
Agar 15.00
Distilled water 1000ml
2] MacConkey’s Agar
This is a selective medium that inhibits the growth of gram positive bacteria due to the presence
of crystal violet and bile salts while gram negative bacteria grow well on it.
This is also a differential medium and it differentiates between lactose fermentors and lactose
non-fermentors on the basis of colour change reaction. This is due to the presence of neutral red
(a pH indicator) and lactose (a disaccharide).
Utilizing the lactose available in the medium, certain bacteria (lactose fermentors) produce
organic acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink
colonies.
Non-lactose fermenting bacteria cannot utilize lactose, and will use peptone instead. This forms
ammonia, which raises the pH of the agar, and leads to the formation of white/colourless
colonies.
Composition
Ingredients Grams/litre Peptone 17.00
Proteose peptone 3.00
Sodium chloride 5.00
Lactose 10.00
Bile salts 1.50
Neutral Red 0.03
Agar 13.5
Distilled water 1000ml
[3] Mannitol Salt Agar
Mannitol salt agar (MSA) is both a selective and differential medium used in the isolation of
staphylococci. It contains 7.5% sodium chloride and thus selects for those bacteria which can
tolerate high salt concentrations. MSA also distinguishes bacteria based on the ability to ferment
the sugar mannitol, the only carbohydrate in the medium. It is selective for Staphylococci.
Staphylococci can withstand the osmotic pressure created by 7.5% NaCl, while this
concentration will inhibit the growth of most other gram-positive and gram-negative bacteria.
Additionally, MSA contains mannitol and uses phenol red as a pH indicator in the medium. At
pH levels below 6.9, the medium is a yellow color. In the neutral pH ranges (6.9 to 8.4) the color
is red; while above pH 8.4, the color of phenol red is pink.
When mannitol is fermented by a bacterium, acid is produced, which lowers the pH and results
in the formation of a yellow area surrounding an isolated colony on MSA. A non-fermenting
bacterium that withstands the high salt concentration would display a red to pink area due to
peptone breakdown.
Composition
Ingredients Grams/litre Beef extract 1.00
Proteose peptone 10.00
Sodium chloride 75.00
D-Mannitol 10.00
Phenol Red 0.025
Agar 15.0
Distilled water 1000ml
[4] Eosin-methylene Blue Agar
Eosin-methylene blue agar is selective for gram-negative bacteria against gram-positive bacteria.
EMB agar contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue; it is
commonly used as both a selective and a differential medium. EMB agar is selective for gram-
negative bacteria. The dye methylene blue in the medium inhibits the growth of gram-positive
bacteria; small amounts of this dye effectively inhibit the growth of most gram-positive bacteria.
Eosin is a dye that responds to changes in pH, going from colorless to black under acidic
conditions.
EMB agar medium contains lactose and sucrose, but not glucose, as energy sources. The sugars
found in the medium are fermentable substrates which encourage growth of some gram-negative
bacteria, especially fecal and nonfecal coliforms. Differentiation of enteric bacteria is possible
due to the presence of the sugars lactose and sucrose in the EMB agar and the ability of certain
bacteria to ferment lactose in the medium. Lactose-fermenting gram-negative bacteria (generally
enteric) acidify the medium, and under acidic conditions the dyes produce a dark purple complex
which is usually associated with a green metallic sheen. This metallic green sheen is an indicator
of vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms.
Composition
Ingredients Grams/litre Peptic Digest of animal tissue 10.00
Dipotassium phosphate 2.00
Lactose 5.00
Sucrose 5.00
EosinY 0.40
Methylene Blue 0.065
Agar 13.5
Distilled water 1000ml
[5] Mueller Hinton Agar
Mueller Hinton Media is recommended for use in the cultivation of a wide variety of
microorganisms. Mueller Hinton Agar is recommended for disk diffusion sensitivity testing of
non-fastidious organisms. This media has been used in standardized antimicrobial disk
susceptibility testing, as described by Bauer, Kirby, et al.
Mueller Hinton Media contains beef infusion, casamino acids, and starch. Starch acts as a colloid
that protects against toxic material in the medium. Beef infusion and casamino acids provide
energy and nutrients.
The Kirby-Bauer antimicrobial disk diffusion procedure is used with Mueller Hinton Agar
plates. It is based on the use of an antimicrobial impregnated filter paper disk. The impregnated
disk is placed on an agar surface, resulting in diffusion of the antimicrobial into the surrounding
medium. Effectiveness of the antimicrobial can be shown by measuring the zone of inhibition for
a pure culture of an organism. Zone diameters established for each antimicrobial determining
resistant, intermediate, and sensitive results for pathogenic microorganisms are listed in the
Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), document M2-A,
Performance Standards for Antimicrobial Disk Susceptibility Tests.
Mueller Hinton Broth is the same formulation, without the added agar. It is used for the
cultivation of microorganisms, and for making dilutions of organisms to be used in the Kirby-
Bauer disk diffusion procedure.
Composition
Ingredients Grams/litre Beef extract 300.00
Casein acid hydrolysate 17.5
Starch 1.50
Agar 17.00
Distilled water 1000ml
[6] Bismuth Sulfite Agar
Bismuth Sulfite Agar is a highly selective medium used for isolating Salmonella spp.,
particularly Salmonella Typhi, from food and clinical specimens.
In Bismuth Sulfite Agar, beef extract and peptone provide nitrogen, vitamins and minerals.
Dextrose is an energy source. Disodium phosphate is a buffering agent. Bismuth sulfite indicator
and brilliant green are complementary in inhibiting gram-positive bacteria and members of the
coliform group, while allowing Salmonella to grow luxuriantly. Ferrous sulfate is included for
detection of H2S production. When H2S is present, the iron in the formula is precipitated, giving
positive cultures the characteristic brown to black color with metallic sheen. Agar is the
solidifying agent.
Composition
Ingredients Grams/litre Beef extract 5.00
Dextrose 5.00
Disodium phosphate 4.00
Ferrous sulphate 0.30
Bismuth sulphite indicator 8.00
Brilliant green 0.025
Agar 20.00
Distilled water 1000ml
[7] IMViC Tests IMViC reactions are a set of four useful reactions that are commonly employed in the
identification of members of family enterobacteriaceae. The four reactions are: Indole test,
Methyl Red test, Voges Proskauer test and Citrate utilization test.
INDOLE TEST
Some bacteria can produce indole from amino acid tryptophan using the enzyme typtophanase.
Production of indole is detected using Ehrlich’s reagent or Kovac’s reagent. Indole reacts with
the aldehyde in the reagent to give a red color. An alcoholic layer concentrates the red color as a
ring at the top.
METHYL RED (MR) TEST
This is to detect the ability of an organism to produce and maintain stable acid end products from
glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation
that they overcome the buffering action of the system. Methyl Red is a pH indicator, which
remains red in color at a pH of 4.4 or less.
VOGES PROSKAUER (VP) TEST
VP test detects butylene glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in
the production of butylene glycol. Two reagents, 40% KOH and alpha-naphthol are added to test
broth after incubation and exposed to atmospheric oxygen. If acetoin is present, it is oxidized in
the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of
peptone, in the presence of alpha-naphthol to produce red color. Role of alpha-naphthol is that of
a catalyst and a color intensifier.
CITRATE UTILIZATION TEST
This test detects the ability of an organism to utilize citrate as the sole source of carbon and
energy. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator
bromothymol blue. The medium also contains inorganic ammonium salts, which is utilized as
sole source of nitrogen. Utilization of citrate involves the enzyme citritase, which breaks down
citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO2.
Production of Na2CO3 as well as NH3 from utilization of sodium citrate and ammonium salt
respectively results in alkaline pH. This results in change of medium’s color from green to blue.
[8] SUGAR FERMENTATION TEST
Phenol red broth is a general purpose fermentation media comprising of trypticase, sodium
chloride, phenol red and a carbohydrate. The trypticase provides amino acids, vitamins, minerals
and other nitrogenous substances making it a nutritious medium for a variety of organisms.
Sodium chloride helps in maintaining the osmotic balance and provides the essential electrolytes
for the transport into the cell while the carbohydrate acts as the energy source. The phenol red is
the pH indicator and is initially neutral (pH 7). It supports the growth of most organisms whether
they are able to ferment sugar or not. When the bacterium is inoculated into the tube, the
bacterium which ferments the sugar will result in the production of acid that will change the
color of phenol red.
Step 1:
1. Using aseptic technique, inoculate each Phenol red sugar tube with the corresponding microbial
culture. Leave the one tube un-inoculated. The tubes may be mixed by rolling them back and
forth between the palms of the hands.
2. Place the tubes in a test-tube rack and incubate at 35°C for 24 to 48 hours.
Step 2:
1. Examine the tubes carefully between 2 to 4 hours, at 8 hours, and 18 hours in order to avoid false
negatives due to reversal of the fermentation reactions that may occur with long incubations.
2. Examine all carbohydrate broth cultures for evidence of acid (A), or acid and gas (A/G)
production. Acid production is detected by the medium turning yellow and gas production by a
gas bubble in the Durham tube.
3. The control tube should be negative for acid and gas production, and should have no turbidity.
4. Based on your observations, determine and record in the report for exercise 20 whether or not
each microorganism was capable of fermenting the carbohydrate substrate with the production of
acid, or acid and gas. Compare your results with other students who used other sugars.
