A DISSERTATION THESIS

BIODECOLORIZATION OF ACTIVE ACID YELLOW AZO DYE BY USING

BACTERIA ISOLATED FROM DYE-CONTAMINATED SOIL AND EFFLUENT

Thesis submitted for the award of the degree of

Master of Science
In
MICROBIOLOGY

By

MOTISHA JALANSHI SHITANSHU

[Enrollment No:- 200507041018]

Under the guidance of

Dr. Shital Thacker

Submitted
To
Department of Microbiology
Silver Oak Institute of Science, Silver Oak University,
Ahmedabad- 382481, Gujarat (INDIA)
[Year:2020-2022]
 A DISSERTATION THESIS

BIODECOLORIZATION OF ACTIVE ACID YELLOW AZO DYE BY USING

BACTERIA ISOLATED FROM DYE-CONTAMINATED SOIL AND EFFLUENT

Thesis submitted for the award of the degree of

Master of Science
In
MICROBIOLOGY

By

MOTISHA JALANSHI SHITANSHU

[Enrollment No:- 200507041018]

Under the guidance of

Dr. Shital Thacker

Submitted
To
Department of Microbiology
Silver Oak Institute of Science, Silver Oak University,
Ahmedabad- 382481, Gujarat (INDIA)
[Year:2020-2022]
 Certificate
This is to certify that the data embodied in the Dissertation Thesis entitled “Biodecolorization of
active acid yellow azo dye by using bacteria isolated from dye-contaminated soil and
effluent” represents the bonafide work carried out by MOTISHA JALANSHI SHITANSHU
in the Microbiology laboratory, Department of Microbiology, Silver Oak Institute of Science,
Silver Oak University, Ahmedabad 382 481, Gujarat, India. This work is original and no part of
this work has been submitted for degree or diploma or any other academic award of this
university or any other university.

Dr. Shital Thacker Dr. Rupal K. Shah

Department of Microbiology, Head of Department,
Silver Oak Institute of Science, Department of Microbiology,
Silver Oak University, Silver Oak Institute of Science,
Ahmedabad Silver Oak University,
Ahmedabad
 Declaration

I hereby declare that the dissertation work entitled “Biodecolorization of active

acid yellow azo dye by using bacteria isolated from dye-contaminated soil and
effluent”, is a record of the original investigation and a genuine piece of work.

At the same time we also declare that we would never make any changes in
the name of the collaborators who also equivalently deserve and worked together
in bringing forth this dissertation work to success.
 Dedication

Every hard-work requires self-dedication and the blessing of parents,

siblings, friends and the people who plays most important role in our life.

I would like dedicate my whole work

To
My loving mother, my brother, my friends, my Guide and Professors who
supported me in each step of the path of this work and without their support I could
not have completed my dissertation work.

“All power is within you; you can do anything and everything.”

-Swami Vivekananda
 ACKNOWLEDGEMENT
It is my foremost duty to pay my sincerest thankfulness to numerous people for the infinite
support, encouragement, the essential guidance and co-operation that I have received from them
and various institutions who have walked along with me during my research.

It gives me immense pleasure to express my profound feeling of reverence and heartfelt gratitude
to Dr. Shital Thacker, for her inspiring guidance, dynamic initiation, continuous
encouragement, thoughtful discussions, and untiring supervision throughout the work. she is the
figure without whom it would be quite difficult to shape this work in present format starting from
conceptualizing the thought to achieving the dream. Her ideas, patience and encouragement that
carried me out through difficult times and for her insights and suggestions that helped to shape
my research skills. I would also like to thank her for encouraging participation in National,
International Workshops and Conferences that gave me immense knowledge.

I would like to express my gratitude to Dr. Rupal shah, Head of the Department, Department
of Microbiology, silver oak institute of science, silver oak university who allow me t a project
on decolorization of dyes and for providing such a useful and sophisticated laboratory for
research work for this untiring help and inspirational words. I would like to thanks to Dr.Edwin
Pithawala and Dr. Urja Pandya for their constant support and encouragement to keep my
morale high in times of difficulty. I am thankful to Microbiology department for nontechnical
help. I am also thanks to Prof. Nidhi Purswani, Ms. Nidhi Rana and Ms. Jolly Patel for
providing me help for the completion of my project.

My special thanks to the Dr. Manthan Panchal, Principal, Silver Oak Institute of Science,
Silver Oak University for their constant support and providing cooperation to apply for this
project.

I have very much thankful Ms. Brinda Panchal, Ms. Kruti Doshi, Ms. Yesha Pandya helping
me with laboratory requirement during the work.

I am specially thanking to my friends Mumuksha patel, Nainshee patel, Krupa Bhatt, Nafisa
Pothiawala to always moral support in my thesis work. I shall always cherish the sweet
memories of lively company and sincere corporation of loving friends would like to
acknowledge my friends Tushar sonagara, Sanket Patel, Komal Nathvani, thanks to all my
colleagues from My college.

After thanking to all its my Family who has always been my backbone. A Special thanks to My
Mother Mrs. Zankhana Motisha, who has always been my support system and My Nana Mr.
Arvind Mehta my guiding force, for believe in me and for inspiring me whenever had faced any
tough times during this journey. Special thanks to my younger brother Viraj Motisha for his
constant support, love, care and providing equal motivation on this path. I also bestow my
sincere thank to my best friend Vishal Dhanani for showing his blessings and encouraging on
me every time on this journey.

I would like to thanks to God, for persisting me with the potential, calm, making me capable and
focus on my Project work.

If I missed any names in here, I hereby would like to thank all of them who has been a part of my
Project Work.

- JALANSHI MOTISHA
 INDEX

Chapters Contents Page no.

Abstract I

List of tables II

List of figures III

List of abbreviation IV

Chapter – 1 Introduction 1

Chapter – 2 Literature review 5

Chapter – 3 Material and methods 13

Chapter – 4 Results and Discussions 19

Chapter – 5 Conclusions 32

Chapter – 6 References 34

Appendix – i Media composition 37

Appendix – ii Reagent 40

Appendix – iii Instrument 41

Abstract

Dyes are coloured compounds that, when applied to textiles, provide a permanent colour that is
resistant to fading from perspiration, light, water, and a variety of chemicals. Only natural
sources were used to create dyes in the past, those produced from natural sources such as plant
leaves, roots, bark. However, as requirements and expectations grew, businesses increasingly
reliant on colours made from petrochemicals, or synthetic dyes. They are classed in the colour
index based on the chemical structure of their chromophoric group, colour, and application
technique. The chromogenic chromophore is a collection of atoms found in dye compounds that
are responsible for the colour. Textile dyes are categorised by their chemical structure (Azo dyes,
Nitro dyes, Indigo dyes, Anthraquinone dyes, and so on) or their industrial
applicability.Environmental issues associated with the textile industry are with the water
contamination produced by the discharge of untreated wastewater into the environment and it
impacts visibility inside the receiving waters, reducing the amount of light available to aquatic
plants and animals. Existing dye removal procedures may be divided into three categories:
biological, chemical, and physical therapies. In this project work, discusses the mechanism of
decolorization of textile dyes by microorganisms, as well as classification of dyes and
optimization.
The decolorization of acid Yellow 99 dye by both isolates was investigated using various
environmental and culture factors. Under static conditions, the appropriate starting pH for
decolorization of all dyes was in the range of 5-9. The best temperature for decolorization was 37
degrees Celsius. Bacterial isolates may decolorize up to 100mg/l of dye under ideal conditions,
however increasing the dye concentration reduces the efficacy of decolorization. In order to
produce effective and quicker decolorization, the isolates were evaluated for their ability to
decolorize acid YELLOW dye in the presence of additional carbon sources such as (1 percent
glucose, 1 percent fructose, 1 percent sucrose). Glucose had the highest percentage of
decolorization (84%) whereas fructose and sucrose had modest decolorization (81 and 65%).In
comparison to isolate-1, isolate-4 had a higher percentage of decolorization. This might be
because isolate 4 was more dye-absorbent than isolate 1, allowing it to decolorize faster.

I
 LIST OF TABLES

No. Title Page No.

Table 01 Dye specification 03

Table 02 Advantages and Disadvantages 06

dye removal techniques

Table 03 Application and Properties of 07

Dyes

Table 04 Characteristics of samples 20

collected from industries.

Table 05 Colony characteristics of 24

isolate(C-4)

Table 06 Colony characteristics of 25

isolate(C-5)

Table 07 Gram staining observation of 25

isolate(C-4)

Table 08 Gram staining observation of 26

isolate(C-5)

Table 09 Biochemical characteristics of 26

isolates

Table 10 Decolorization percentage 27

achieved by isolates

II
 LIST OF FIGURES
No. Title Page No.
Fig 01 Dye sample 20
Fig 02 Before and after observation of 21
decolorization of dye from soil and
effluent for screening of isolate
Fig 03 primary Screened of isolate 22
Fig 04 Before the treatment of individual 22
isolate form soil
Fig 05 After the treatment of individual isolate 23
form soil
Fig 06 Before the treatment of individual 23
isolate form effluent
Fig 07 After the treatment of individual isolate 24
form effluent
Fig 08 Gram staining observation 25
Fig 09 Decolorization of dye achieved by 27
selected isolate
Fig 10 Maximum absorption of acid YELLOW 28
azo dye before treatment
Fig 11 Maximum absorption of acid YELLOW 28
azo dye after treatment
Fig 12 Declolorization % at different incubation 29
condition

Fig 13 Declolorization % at different dye 29

concentration

Fig 14 Declolorization % at different incubation 30

Ph

Fig 15 Declolorization % at different incubation 30

temperature

Fig 16 Effect of various carbon source on dye 31

decolorization

III
List of symbols and abbreviations

˚C : Degree centigrade

h : Hours

etc :Etceteras

mg : Milligram

gm : Gram

ml : Mililiter

l : Liter

µg : Microgram

µl : Milliliter

e.g. : Exempli gratia

OD : optical density

Fig : Figure

C : Colony

pH : Negative logarithm of hydrogen ion concentration

% : Percentage

UV : Ultraviolet

Fig : Figure

mol : Mole

% : Percentage

Rpm : Rotation per minute

Conc : concentration

IV
λmax :absorption maxima

ppm :parts per million

NA : Nutrient Agar
CMB : Complete Medium Broth
DW : Distilled Water
EMB : Eosin methylene YELLOW agar
GPB : Glucose phosphate broth

V
 CHAPTER:1
INTRODUCTION

VI
1

VII
 Introduction
Human beings have always been drawn to colour. In various fields, such as paintings, printings,
garments, images, and screens, defining a hue and turning the tint is crucial. Because the human
eye is extremely sensitive to light frequency, this task is difficult. Hundreds of studies have been
conducted throughout the years to develop dyes with specialised qualities for certain uses. The
colour that is most strongly absorbed is the colour that passes through the material's
complement.[1]

Dyes are coloured compounds that, when applied to textiles, provide a permanent colour that is
resistant to fading from perspiration, light, water, and a variety of chemicals, including oxidising
agents and microbial assault[2]. Only natural sources were used to create dyes in the past. Until
the development of the first synthetic dye in 1856, the only dyes accessible for the colouring of
textiles were those produced from natural sources such as plant leaves, roots, bark, insect
secretions, and minerals[3,4]. However, as requirements and expectations grew, businesses
increasingly reliant on colours made from petrochemicals, or synthetic dyes. In comparison to
natural dyes, these dyes are water-soluble, easily absorbed, and colour extremely quickly, and
they offer a wide range of colour options. In the current state of affairs, the global output of dyes
is close to 800,000 tonnes per year. The textile industry consumes a considerable portion of the
dyes produced[4]. Synthetic dyes have industrial benefits in terms of (i)chemical stability over
time, (ii) inertness to physical, chemical, and biological degradation, (iii) ability to deliver colour
to the fibre to be dyed by repeatable methods while preserving colour intensity, and (iv) cheap
cost.[7]

A dye might be characterized as a natural compound containing both an auxochromic group and
a chroophoric group, where chromophoric group is responsible for the property of imparting
color to dye and auxochromic group is responsible for the property of electrolytic dissolution.
The chromogen-chromophore structure is frequently not adequate to bestow solvency and cause
adherence of dye and fiber. The binding affinity groups or auxochromic groups are hydroxyl,
carboxyl,amines,or sulfonic radicals or derivatives.

Azo dyes have (N=N) in its strucyure. azo dyes have different variety of structures as. Monoazo
dyes have just a single N=N double bond, while diazo and triazo dyes contain two and three

1
N=N double bonds respectively. The azo groups are for the most part connected with benzene
and naphthalene rings, however can likewise be connected to aromatic heterocycles or enolizable
aliphatic groups. These side groups are responsible for the color of dye with different intensities
and different colors.[8]

Textile manufacturers use a large amount of water and chemicals in the wet processing of
textiles. Desizing, scouring, bleaching, dyeing, printing, and finishing are some of the processes
in which these chemicals are employed extensively. Inorganic substances and elements, as well
as polymers and organic products, are among them. The Colour Index lists about 8000 chemical
compounds used in the dyeing process, with over 1,00,000 commercially accessible dyes and
over 7 x 105 metric tonnes of dyestuff generated each year. These dyes appear in a range of
structural types, including acidic, reactive, basic, disperse, azo, diazo, anthraquinone-based, and
metal-complex dyes[5].

Textile processing uses a lot of water and generates a lot of waste. Unfortunately, due to the
inadequate depletion of colours onto textile fibre during an aqueous dyeing process, a significant
amount of dyestuff is discharged with the wastewater[4]. The wastewater released contaminates
land and water, causing substantial environmental damage. The ensuing effluent generates
substantial water pollution problems because of the colour concentration and dangerous
components. The introduction of heavy metals into water bodies, a rise in biochemical oxygen
demand (BOD), chemical oxygen demand (COD), pH, and suspended particles are all difficulties
associated with coloured textile effluent. Furthermore, the presence of synthetic colours in
wastewater makes the water smell terrible and appear undesirable, as well as reduces the amount
of sunlight that reaches the water, affecting photosynthesis and entire aquatic ecosystems.
Furthermore, most dyes, as well as their breakdown products, are toxic, carcinogenic, mutagenic,
and teratogenic to humans and other living things when dumped into wastewater. [6].

The firm operates a wastewater treatment facility that processes wastewater in three stages:
biological treatment (using bacteria that feed solely on the cellulose components in the effluent),
ultra filtration, and reverse osmosis (RO). For the decolourization of dye-containing wastewater,
many Physico-chemical processes have been utilised, including adsorption, electro coagulation,
flocculation, ion exchange, membrane filtration, ozonation, and reverse osmosis. However, these
procedures are pricey, and they create enormous volumes of waste after treatments, which must

2
be disposed of safely. Biological treatment processes, on the other hand, have been proven to be
more suited and frequently employed in recent years owing to their cost-effectiveness, capacity
to create less sludge, and environmentally benign nature[6].

Bioremediation is the utilization of natural system (essentially microorganisms and plants) for
the treatment of contaminated air, amphibian or earthly part of climate. Textile industry is the
significant client of water. Decreased water sources because of quick populace development and
modern improvement has set off need to reuse of city and industrial waste water after legitimate
treatment and disposal of expected toxins. Regular treatment processes have for some time been
laid out in eliminating numerous dangerous synthetic compounds of general wellbeing and
ecological concern. These ordinary techniques have their own drawbacks overall scale
Microorganisms can breakdown most mixtures for their development as well as energy need. At
times, metabolic pathways which life forms follow for its own typical development and
improvement may likewise be utilized to break down contaminants. In this cycle microorganisms
don't benefit straightforwardly, however scientists use for the process of bioremediation.[9]

Table: 01 Dye specification

Name Acid Yellow 99

Physical appearance Liquid

Peak absorption 434

Molecular formula
C16H15CrN4NaO9S
Molecular Weight
513.986248
Structure

IUPAC name Sodium;3-[[(Z)-1-anilino-3-hydroxy-1-oxobut-2-en-2-

yl]diazenyl]-2-hydroxy-5-
nitrobenzenesulfonate;chromium;hydrate

3
Name Acid Yellow 99

Melting point 360 ˚C

Uses wool, silk, polyamide fiber dyeing and printing directly,
for leather dyeing, wool carpet and influential dyeing, for
the preparation of solvent dye.

Solubility Soluble in sulfuric acid

Other Name 10343-58-5 Acid Yellow 99, Dye content 40 %

AKOS024319573

4
 CHAPTER:2
REVIEW LITURATURE

5
 2

REVIEW LITERATURE

2.1 Historical background of Textile Dyes

Since the start of humankind, they have been trying to add tone and colour to their surroundings.
They utilized regular make a difference to stain stows away, enrich shells also, plumes, and paint
their story on the dividers of old caverns. Researchers have had the option to date the dark,
white, yellow and ruddy colors produced using ochre involved by crude man in cave artworks to
north of 15,000 B.C. With the advancement of fixed settlements and horticulture around 7,000-
2,000 B.C., man started to create and utilize materials, and would in this manner add colour to
them too. Natural regular colorants have an ageless history of use, particularly as textile dyes

William Henry Perkin found the first synthetic dye, an understudy at the Regal College of
Chemistry. He attempted to make the medication quinine from aniline (a synthetic tracked down
in coal). The investigation delivered a thick dull sludge. Rather than discarding it, Perkin dilutes
it with liquor and tracked down that the arrangement was purple. He found that it would color
silk and that it was a 'quick' color, impervious to washing and to the blurring impacts of
light.[10]

The idea of innovative work in assortment of dye producing was before long followed by others
and new dyes started to show up available. This process was definitely strengthened by Kekule's
disclosure of the sub-atomic design of benzene in 1865.

2.2 Color Removal Techniques

Different physical and chemical methods for dye removal from textile effluent with its
advantages and disadvantages(Table:02).[12]

Table : 02 Advantages and Disadvantages dye removal techniques

Physical/chemical Advantages Disadvantages

Method

6
 Electrochemical Break-down compounds High electricity consumption
destruction are non-hazardous
Fentons reagent Effective decoloration of Sludge generation
soluble and insoluble dyes
Ozonation Applied in gaseous state, no Short half life (20 min)
alteration of volume
Photochemical No sludge production Formation of by-products
Membrane filtration Removal all types of dyes Concentrated sludge production
Sodium Initiates and accelerate Release of aromatic amines
hypochlorite azo bond cleavage

Activated carbon Good removal of wide Very expensive

variety of dyes
Peat Good adsorbent due Specific surface area for adsorption
to cellular Structure are lower than activated carbon

Ion-exchange No adsorbent loss due to Not effective for all dyes

regeneration
Silica gel Effective for basic dye Side reactions prevent
removal
commercial application
2.3 Textile Dyes

Synthetic dyes are widely used in textile dyeing, paper printing, color photography,
pharmaceutical, food, cosmetics and other industries. Significant classes of synthetic dyes
integrate azo, anthraquinone and triaryl-methane dyes, and a large number of them are harmful
or even cancer-causing compounds with long turnover times. With the extended use of a wide
collection of dyes, contamination by dye's wastewater is turning out to be increasingly
disturbing.[13]

Table: 03 Application and Properties of Dyes[11]

Dye
S.N. Category Applications Properties
Class

7
 Dye
S.N. Category Applications Properties
Class

1 Direct Contains sulphonic acid group, however, Cotton, rayon, Water

Dyes these are not the point of attachment. paper, leather, soluble and
and some anionic dyes
amount to nylon.

2 Vat dyes Like sulphur dyes, however, used in Cotton cellulosic Water
reduced form after treatment with fibers and for insoluble
reducing agents rayon and wool

3 Basic Basic amino group protonated under Paper, Watersoluble

acidic condition, formation of salt polyacrylonitrile,
Dyes
linkages modified nylons,
modified
polyesters,
cation dyeable
polyethylene
terephthalate,
and medicine

4 Reactive Forms covalent bond with fibers Cotton and other Watersoluble
possessing hydroxyl or amino groups, cellulosic fibers
Dyes
e.g., dyes with chlorine atom and some extent
wool and nylon
fibers

5 Solvent No water solubilizing group, soluble in Plastics, Solvent

gasoline, soluble
Dyes organic solvent
lubricants, oils, while water
and waxes insoluble
and nonpolar

8
 Dye
S.N. Category Applications Properties
Class

or little polar

6 Acid Sodium salts of color acids that contain Nylon, wool, Watersoluble
sulphonic acid or phenolic group silk, modified
Dyes
acrylics, paper,
leather, ink-jet
printing, and
food

7 Mordant Contains group that can hold metal in Cotton and Watersoluble
chelate groups or coordination complexes rayon
Dyes

8 Disperse Water insoluble dyes, small, and contain Polyester and Water
hydroxyl or amino group to give finite some amount insoluble
Dyes
water solubility at definite temperature nylon, cellulose, and nonionic
cellulose acetate, dyes
and acrylic
fibers

2.4 Textile Effluent

The significant causes of release of dyes into the climate are the dyestuff and textile producing
ventures. These textile effluents are remarkably shaded colors delivered by dye and dye
producing ventures that contain huge measure of combinations of colors These dyes are
challenging to corrupted and present dangerous impact on amphibian life. Notwithstanding dyes,
material effusions likewise contains high ionic strength, salt and high pH values also.[14]

2.5 Environmental concerns in relation to Dyes

Textile dyes have viewed as harmful, genotoxic and mutagenic in different test frameworks.
Dyes with azo bonds nitro-or amino-groups are cancer-causing, causing growths of liver and
urinary bladder in test creatures. Notwithstanding, decrease of azo dyes, for example cleavage of

9
the dye's azo linkages, prompts formation of sweet-smelling amines and a few sweet-smelling
amines are known mutagens and cancer-causing agents. In vertebrates, metabolic actuation
(decrease) of azo dyes is predominantly because of bacterial movement in the anaerobic pieces
of the lower gastrointestinal lot. Different organs, particularly the liver and the kidneys, can,
nonetheless, likewise lessen azo dyes. The harmfulness of sweet-smelling amines relies upon the
nature and area of other substituents. As an illustration the replacement with nitro, methyl or
methoxy groups or on the other hand halogen atoms may lead to increased toxicity; while
replacement with carboxyl or sulphonate groups for the most part bring down the
harmfulness.[15]

Most of dyes represent a potential wellbeing risk to all types of life. These dyes might cause
unfavorably susceptible reactions, skin dermatitis, dermatitis, and may influence the liver, the
lungs, the vasco-circulatory framework, the resistant framework and the conceptive framework
of exploratory creatures as well as people.[16]

2.6 Decolourization by Bacteria

Decolorization of azo dyes by microbes. Reductive cleavage of the - N. N-bond is the underlying
advance of the bacterial decolorization and degradation of azo dyes. Decolorization of azo dyes
happen under anaerobic (methanogenic), anoxic and high-impact aerobic conditions by various
trophical groups of microscopic organism.[17]

2.7 Mechanism of Decolorization and degradation by Azoreductase Enzyme

Azo reductase is an enzyme that decolorizes and degrades azo dye into amines that are colourless
through a reductive cleavage process. It requires low sub-atomic weight lessening comparable,
for example, FADH or NADH as the electron giver as a redox response.[18]

2.8 Application of Genetic Engineering

Cloning, characterization and over expression portrayal has turned into a fundamental device in
reading up genetic engineering to improve any biological interaction. For the overexpression and
characterization of azoreductase, different hereditary designed strains (generally Escherichia
coli) strains were developed.[19], moved flavin reductase (fre) to Sphingomonas sp. Strain BN6
furthermore, found 30 overlay expansion in azo reductase action in the wild-type strain. On the

10
other hand, entire cells of the recombinant Sphingomoas sp. BN6 showed as it were roughly
triple expansion in the decrease rate for Amarnath and Stringent Yellow 3 azo dyes contrasted
with wild sort strain. It has been accounted for by [20] that E. coli strain conveying the
azoreductase quality (azo B) from X. azovorans KF46F, communicated just about 50 times
higher azoreductase movement in its cell separates than that saw in wild type strain. In any case,
the resting recombinant cell suspension showed no distinguishable azoreductase movement.

Aim and Objective

Aim
To study the biodecolorization of active acid yellow azo dye by using bacteria isolated from
dye contaminated soil and effluent.
Objective
1. To Enrichment of microorganisms present in textile Soil and effluent that decolorize dyes.
2. To isolate textile dye decolorizing bacterial strain.
3. To Identify dye decolourizing bacteria.
4. To check the potential of bacteria to decolorization of dyes.

11
12
 CHAPTER:3
MATERIAL AND METHODS

13
 3

MATERIALS AND METHODS

3.1 Sample collection of Soil and Effluent

Effluent Samples were collected from Ahmedabad textile industries research association Atira,
Ahmedabad . Soil samples were collected from areas around the industry. Samples were in the
form of liquid untreated effluent and soil. All the samples were collected in sterile container and
preserved at 4˚C in refrigerator.

3.2 Sample collection of Dye

Dye sample were collected from meghamani dyes, vatva, ahmedabad. Sample were collected in
sterile container and preserved at 4˚C in refrigerator.

3.3 Physico-chemical characterization of samples

The samples, mainly before treatment were tested for its physico-chemical characteristics like,
color, order, pH, Temperature, etc.

3.4 Materials

 Dyes: 10,000 ppm Acid Yellow 99 azo dye

 Chemicals: Kovac’s reagent, Methyl red indicator, KOH reagent, H2O2 , Trtramethyl-
p-Phenylendiamine dihydrochloride
 Medium: Nutrient Broth, Nutrient Agar, Tryptophan broth, Glucose phosphate broth,
Eosin methylene YELLOW agar(EMB), MacConkey’s agar.

3.5 Primary screening and isolation of dye decolorizing bacteria

Samples were used for the primary screening and isolation of dye decolorizing bacterial cultures
by using enrichment culture techniques with nutrient broth supplemented with individual dyes a
concentration of 10 ppm solution. The enrichment was carried out in 50 ml nutrient broth
medium in 100 ml Erlenmeyer flask by adding 10ml of inoculum. The culture flasks were
incubated on orbital shaker with 120 rpm, at 30˚C for 72 hrs. After 72 hrs of incubation (i) 1ml

14
of sample from decolorized flask was serially diluted up to 10-6 dilution and plated on the agar
medium and incubate at 37˚C for 24 hrs. (ii) The colony morphology was noted after incubation
at 37˚ C for a period of 24 hour. Sub-culturing the individual colonies to obtain pure culture .The
pure cultures of individual bacterial strains were maintained by streaking on nutrient agar slant
and stored at 4˚C.
3.6 Enrichment and secondary Screening of dye decolorizing strains

All of the colonies isolated by enrichment technique were individually inoculated into 50 ml of
the Nutrient broth medium with respective dyes with a concentration of 10 ppm (Acid yellow
99).The medium was sterilized through autoclaving. These Flask were inoculated with 1 ml of
active culture of selected organisms and these were incubated in incubator at 37˚C for 72 hrs and
the color change was noted at regular intervals. The organisms that show maximum
decolorization within 72 hrs were selected and used for further studies. biodecolorization studies
of the 10 morphologically different strains isolated from the Soil and effluent, only six were
found from the soil and four were found from the effluent. In that only 2 were found more than
50% decolourization of the respective dye within 48 hrs. These organisms were selected and
were maintained for further studies. The selected bacteria were purified by repeated streaking on
NA and were stored at -4˚C in refrigerator.
3.7 Identification of selected bacterial strains

The identification of dye decolorizing bacterial strains was carried out based on morphological
characteristics, Gram staining characteristics, biochemical characteristics.
3.7.1 Colony characteristics

Colony characteristics of selected bacterial isolates were carried out by streaking of single colony
on NA plate and incubate at 37˚C for 24 hrs. The colony shape, size, color, arrangement and
other colony characteristics of the isolated bacteria were studied after 24 hrs of incubation and
were tabulated.
3.7.2 Staining

The morphological characterizations of the isolates were checked by Gram’s reaction. 24 hr old
cultures of all of the isolates were used to study Gram reaction. At first smear was prepared from
colony and heat the fix the smear. Crystal violet was added on the smear, left for 1 minute and

15
rinsed with water. Then iodine was added and kept for 1 minute, wash with distilled water. After
this alcohol was added to remove the excessesive color of primary stain. Thoroughly washed
with alcohol until color removal occurs. Then saffranine was added and wait for 30 seconds.
Finally wash with water and air dried the slide. Observe microscope under oil immersion lens.
Gram positive cells will purple where Gram negative cells stain pink or red.
3.7.3 Growth on different selective media

Isolated colonies were grown on different selective media namely MacConkeys agar and etc.
Colonies from pure culture were streaked on to the plate and incubate at 37˚C for 24 hrs. After
incubation observe the growth of the colony.
3.7.4 Biochemical Reactions

All required media for biochemical tests were prepared in respective test tubes, flasks and Petri
dishes. All biochemical tests were performed with specific requirements for each test as outlined
in Experimental Microbiology Volume 1(9th Edition).
a. Indole Test
Tryptophan is an essential amino acid, which is oxidized by some bacteria resulting in the
formation of indole, pyrivic acid and ammonia. The organisms were inoculated and grown in
10 ml of sterilized Tryptophan broth dispersed in test tubes and incubate the tubes at 37˚C for
24hours. After incubation, add 1 ml of Kovac’s reagent to all the tubes. The appearance of
fuchsia red color ring in the surface layer indicated a positive reaction.

b. Methyl Red (MR) Test

The Methyl-Red tests for the ability to perform mixed-acid fermentation. MR-VP broth
contains glucose, peptone, and a phosphate buffer. Organisms that perform mixed-acid
fermentation produce enough acid to overcome the buffering capacity of the broth, so a
decrease in pH results. Organisms that perform other kinds of fermentation cannot overcome
the buffering capacity of the broth. Isolates were inoculated to 10 ml of sterilized Glucose
phosphate broth in the test Tubes and incubated for 48-72 hours at 37˚C. Add 5 drops of
Methyl Red indicator was added into each inoculated as well as uninoculated tubes.
Appearance of stable bright red color indicated a positive test.

16
C. Voges-Proskauer Test
VP is a test used to detect acetoin in a bacterial broth culture. Tubes with 10 ml of Glucose
phosphate broth were inoculated with each microorganism, incubated at 37˚C for 24-48
hours. Add 0.6 ml of alfa- naphthol and 0.2 ml of KOH solution. The mixture was shaken
vigorously for few minutes and allowed to stand for 15-16 min and positive reaction was
given by the appearance of red color.

d. Catalase Test
Catalase is a ubiquitous antioxidant enzyme that degrades hydrogen peroxide into water and
oxygen. A few drops of H2O2 were kept on a clean glass slide. A loopful of 24 hrs grown
colonies of isolated bacterial culture was placed in to the drop and the observations were
noted. Bubble production indicate the positive result.

e. Oxidase Test
Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron
containing haemoprotein). Test organism was grow under aerobic condition on NA for 18-24
hours. Add 2 -3 drops of tetramethyl-p-phenylenediamine dihydrochloride reagent directly to
suspect colonies on an agar plate. Observe for color change within 10-15 seconds.

f. EMB
It is a differential microbiological medium, which slightly inhibits the growth of Gram-
positive bacteria and provides a color indicator distinguishing between organisms that
ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella).

g. MacConkey’s
It is a selective and differential medium designed to isolate and differentiate enteric based on
their ability to ferment lactose. Bile salts and crystal violet inhibit the growth of Gram
positive organisms. Lactose provides a source of fermentable carbohydrate, allowing for
differentiation.

17
3.8 Assay of decolorization

Decolorization activity was expressed in terms of percentage decolorization and was determined
by monitoring the decrease in absorbance at absorption maxima (λ max) of respective dye (Acid
yellow 99). The uninoculated CMB supplemented with respective dye was used as reference.
The culture suspension was centrifuged at 6,000 rpm for 15 min for removal of the biomass. The
degree of decolorization of the tested dye was measured at its respective maximum absorbance
wavelength using supernatant by UV-visible spectrophotometer (Easytronics). The
decolorization assay was calculated according to the following formula.

Decolorization activity (%) = (A-B)/A x 100

Where, A = initial absorbance
B = Observed absorbance

18
 CHAPTER:4
RESULT AND DISSCUSION

19
 4

RESULTS AND DISCUSSIONS

4.1 Physico-chemical characterization of collected samples

The samples were collected in sterilized container from respective sites. The color, temperature
and pH of the sample were recorded on the site and samples were transported to the laboratory
by storage at 4°C. All the characteristics were measured on the same day of collection of sample
as per (table : 03).
Table : 04 Characteristics of samples collected from industries
SR. NO. SAMPLE NATURE COLOR pH TEMPERATURE
OF
SAMPLE

1. Soil Sample Brown 6.5 35°C

2. Effluent Liquid Black 6.8 32°C

Sample

3. Acid Yellow Liquid Yellow 6.3 33°C

Azo Dyes

Fig 01: Dye sample

20
4.2 Isolation and primary screening of bacterial isolates for decolorization of
dye samples

Liquid effluent sample collected from waste disposal site of textile industry have decolorization
ability led to the isolation of 5 morphologically distinct isolates and 6 from the soil. This isolate
isolated from the enrichment techniques which are observed in fig 03 and Before and after
observation of decolorization of dye from soil and effluent shows in fig 02.

Fig 02: Before and after observation of decolorization of dye from soil and
effluent for screening of isolate

21
 Fig 03: primary Screened of isolate
4.3 Enrichment and secondary Screening of bacterial strains

To observe the ability of decolorization of dye (Acid yellow), at the concentration of 10 ppm all
the isolates were tested individually. Out of 6 colonies only 1 different colonies showed better
decolorization from soil(fig 05) and out of 5 colonies only 1 from effluent(fig 07).

Fig 04: Before the treatment of individual isolate form soil (Flask1.control, 2.C-1, 3.C-
2, 4.C-3, 5.C-4 ,6.C-5 ,7.C-6)

22
Fig 05: After the treatment of individual isolate form soil (Flask-1.control, 2. C-1, 3.
C-2, 4. C-3, 5. C-4 ,6. C-5 ,7. C-6)

Fig 06: Before the treatment of individual isolate form effluent (Flask1.control, 2.C-
1, 3.C-2, 4.C-3, 5.C-4 )

23
Fig 07: After the treatment of individual isolate form effluent (Flask-1.control, 2. C-
1, 3. C-2, 4. C-3, 5. C-4)
4.4.1 Morphological characteristics of isolates

Isolates those showed better decolorization potential were further grown on Nutrient agar plate
and showed different pigmentation like white, dirty white, yellow orange etc. Size of the colonies
varies from pin head, small to large. Texture of the colony was Shiny and Shiny smooth form.
Gram staining result indicated that selected 2 isolates 1 were Gram positive short
Rods(Table:05), 1 were Gram positive large rods (Table:06).

Table : 05 Colony characteristics of isolates (C-4)

Characteristics Observation
Size Pin head
Shape Round
Margin Entire
Elevation Raised
Surface Smooth
Consistency Moist
Opacity Opaque
Pigment Orange red

24
Table : 06 Colony characteristics of isolates (C-5)
Characteristics Observation
Size Large
Shape Irregular
Margin Undulated
Elevation Flat
Surface Smooth
Consistency Moist
Opacity Opaque
Pigment Creamy white

Table : 07 Gram staining observation of isolates (C-4)

Gram’s Reaction Observation

Gram staining Gm + ve

Shape (Microscopic) Rod

Size (Microscopic) Small

Fig 08: Gram staining observation

25
Table : 08 Gram staining observation of isolates (C-5)
Gram’s Reaction Observation

Gram staining Gm +ve

Shape (Microscopic) Rod

Size (Microscopic) Small

4.4.2 Growth on selective media

Selected bacterial colonies were grown different selective media for their selectivity.

4.4.3 Biochemical characteristics

For further identification of bacterial isolates Oxidase ,Catalase, Indole, MR,VP, Citrate
Utilization Test, were conducted for 2 isolates.

Table : 09 Biochemical characteristics of isolates

TEST Colony-4 Colony-5

Indole test - -

Methyl Red (MR) Test - -

Voges-Proskauer Test - -

Catalase Test + +

Oxidase Test + +

EMB test - +

MacConkey’s - +

26
4.5 Determination of decolorization efficiency

Those isolates namely C-4, C-5 were further assayed for decolorization efficiency.
Decolorization efficiency of 2 isolates were studied and the results were showed in table:10 and
visualized in fig :9.The result indicated that decolorization ability was ranging from 68.2-98.3%
of acid YELLOW dyes. Among the isolates colony C-4 showed better decolorization efficiency
(91.2-98.3%) of the dye.

Table : 10 Decolorization percentage achieved by isolates

ISOLATE NO. ABSORBANCE MAX %OF DECOLORIZATION

Isolate-4 628 68.2-70.3 %

Isolate-5 628 91.2-98.3 %

Fig 09: Decolorization of dye achieved by selected isolate

27
 Fig 10: Maximum absorption of acid yellow azo dye before treatment

Fig 11: Maximum absorption of acid yellow azo dye after treatment

4.6 Effect of environmental and nutritional conditions for dye decolorization

4.6.1 Incubation condition

Bacterial strains were able to decolorized acid yellow dye. Ranging from 27-67% Depending on
the bacterial strain and dye structured decolorization efficiency of the selected bacterial sp was
better under static condition as compared to shaking condition. Decolorization under static and
shaking condition after 72 hrs of incubation time was 67 and 27(Fig 12).

28
 80
70

% of Decolorization
60
50
40
30
20
10
0
Static Shaking
Incubation Condition

Fig 12: Decolorization % at different incubation condition

4.6.2 Decolorization at different dye concentration

Study of the effect of initial dye concentration on the efficiency of color removal of the isolated
strain revealed that, the bacterial strain is capable of decolorizing an acceptable high
concentration of color. Decolorization with different initial dye concentration ranging from 100
mg/L to 2500 mg/L was studied. All the selected two bacterial strains were able to decolorized
acid YELLOW. All two isolates showed maximum decolorization at a concentration 100mg/l of
dye (Fig 13). But further increase of dye concentration isolates showed decrease rate of
decolorization.

Fig 13:Decolorization % at different dye concentration

29
 4.6.3 Effect of pH
It was noticed in study that initial pH had a great influence on the decolourization of textile dyes.
The dye removal by bacterial isolates when studied at different pH values at constant
concentration of dye i.e. 100mg/l and temperature was 37°C. Removal of dye was maximum at
pH 8(Fig 14).Lowest decolorization observed at pH 4.

100
% of decolorization 80
60
40
20
0
4 5 6 7 8 9
Effect of pH

Fig 14: Decolorization % at different pH

4.6.4 Effect of temperature
In order to determine the optimum temperature, decolorization assay were performed at
30°C,37°C and 45°C temperature. Figure 15 shows that the decolorization rate increases with
increase in temperature from 30°C-37°C. At 45°C, the decolorization ability was reduced.
Highest decolorization 78% was noticed at 37°C. So, 37°C temperature was optimum for growth
of bacteria and decolorization of dye. As the isolate was mesophilic so at high temeperature
growth rate was decreased and the decolorization was also low.

100
% of decolorization

80
60
40
20
0
30 37 45
Temperature

Fig 15: Declolorization % at different incubation temperature

4.6.5 Effect of different carbon sources

30
The efficacy of additional carbon sources such as (1%glucose,1% fructose, 1% sucrose) was
tested in order to obtain efficient and faster decolorization. The maximum percentage of
decolorization was observed with glucose (84 %), while fructose and sucrose showed a moderate
decolorization value of 81 % and 65 % respectively (Fig 16).

100
90
% of Decolorization

80
70
60
50
40
30
20
10
0
Glucose Fructose Sucrose
Effect of various carbon source

Fig 16: Effect of various carbon source on dye decolorization

31
CHAPTER:5
CONCLUSION

32
 5

CONCLUSION
Textile effluent is an excellent place for dye-decolorizing bacteria to thrive. Despite the fact that
biodecolorization/bioremediation/degradation is a difficult process for both the textile industry
and wastewater treatment scientists, the findings of this study and others in the literature imply
that bacteria have a lot of promise for removing contaminants from textile effluents. A total of 10
isolates were obtained from the waste effluent and discharge in this investigation. Gram's
reaction, cell morphology, colony features, growth patterns in nutrient broth, growth patterns in
selective medium, and biochemical tests were used to characterise 10 distinct colonies that
demonstrated decolorization capabilities at the screening level. dye decolorization was found in
two major bacterial species.

Various aspects of environmental and cultural parameters were studied for decolorization of acid
yellow 99 dye by all the two isolates. The suitable initial pH for decolorization of all dyes was in
the range of 5-9 under static condition. Optimum temperature for decolorization was 37˚C.
Under optimized condition bacterial isolates can decolorization up to 100mg/l of dye while
further increase of dye concentration causes decrease in efficiency of decolorization.

The efficacy of the isolates to decolorized acid Yellow 99 dye in presence of additional carbon
sources such as(1%glucose,1% fructose,1%sucrose)was tested in order to obtain efficient and
faster decolorization. The maximum percentage of decolorization was observed with glucose (84
%), while fructose and sucrose showed a moderate decolorization value of 81 % and 65 %
respectively.

Decolorization % was better in isolate-5 than isolate-4. This may occur due to the isolate 5 was
more addaptibility to the dye, so it decolorized better than isolate 4.

The study concludes with isolated bacteria could effectively be used as an alternative to physical
and chemical processes used for textile effluent treatment. Further study on azoreductase enzyme
activity of isolates may reveal the mechanism for dye degradation and thus facilitate possible for
biotechnological application of treating dye containing waste water.

33
CHAPTER:6
REFERENCE

34
 6

REFERENCES
1. Chemistry International, C. (2019, April 19). Basics in colors, dyes and pigments chemistry:
A review. https://doi.org/10.5281/zenodo.1470528
2. Saratale, R. G., Saratale, G. D., Chang, J. S., & Govindwar, S. P. (2011). Bacterial
decolorization and degradation of azo dyes: a review. Journal of the Taiwan institute of
Chemical Engineers, 42(1), 138-157.
3. Verma, S., & Gupta, G. (2017). Natural dyes and its applications: A brief
review. International Journal of Research and Analytical Reviews, 4(4), 57-60.
4. Jamee, R., & Siddique, R. (2019). Biodegradation of synthetic dyes of textile effluent by
microorganisms: an environmentally and economically sustainable approach. European
journal of microbiology and immunology, 9(4), 114-118.
https://doi.org/10.1556/1886.2019.00018
5. Banat, I. M., Nigam, P., Singh, D., & Marchant, R. (1996). Microbial decolorization of
textile-dyecontaining effluents: a review. Bioresource technology, 58(3), 217-227.
https://doi.org/10.1016/S0960-8524(96)00113-7
6. Afena, A. S., Boateng, D. K., Darkwah, L., & Adjaottor, A. A. (2021). Decolourisation of
Textile Wastewater by Dye Degrading Microorganisms Isolated from Textile Effluent.
https://doi.org/10.4236/jep.2021.1210046
7. Ardila-Leal, L. D., Poutou-Piñales, R. A., Pedroza-Rodríguez, A. M., & Quevedo-Hidalgo,
B. E. (2021). A brief history of colour, the environmental impact of synthetic dyes and
removal by using laccases. Molecules, 26(13), 3813.
https://doi.org/10.3390/molecules26133813
8. Zollinger, H. (2003). Color chemistry: syntheses, properties, and applications of organic dyes and
pigments. John Wiley & Sons.
9. Keharia, H., & Madamwar, D. (2003). Bioremediation concepts for treatment of dye containing
wastewater: a review. http://nopr.niscair.res.in/handle/123456789/17164
10. Welham, A. (2000). The theory of dyeing (and the secret of life).
11. Kiernan, J. A. (2001). Classification and naming of dyes, stains and fluorochromes. Biotechnic &
histochemistry, 76(5-6), 261-278. https://doi.org/10.1080/bih.76.5-6.261.278

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12. Robinson, T., Chandran, B., & Nigam, P. (2002). Removal of dyes from an artificial textile dye
effluent by two agricultural waste residues, corncob and barley husk. Environment
International, 28(1-2), 29-33. https://doi.org/10.1016/S0160-4120(01)00131-3
13. Rafi, F., Franklin, W. and Cerniglia, C.E. (1990). Azoreductase activity of anaerobic bacteria isolated
from human intestinal microflora. Appl. Environ. Microbiol., 56:2146-2151.
https://doi.org/10.1128/aem.56.7.2146-2151.1990
14. Nigam, P., Banat, I. M., Singh, D., & Marchant, R. (1996). Microbial process for the decolorization
of textile effluent containing azo, diazo and reactive dyes. Process biochemistry, 31(5), 435-442.
https://doi.org/10.1016/0032-9592(95)00085-2
15. Correia, V. M., Stephenson, T., & Judd, S. J. (1994). Characterisation of textile wastewaters‐a
review. Environmental technology, 15(10), 917-929. https://doi.org/10.1080/09593339409385500
16. Su, J. C., & Horton, J. J. (1998). Allergic contact dermatitis from azo dyes. Australasian journal of
dermatology, 39(1), 48-49. https://doi.org/10.1111/j.1440-0960.1998.tb01243.x
17. Zimmermann, T., Gasser, F., Kulla, H. G., & Leisinger, T. (1984). Comparison of two bacterial
azoreductases acquired during adaptation to growth on azo dyes. Archives of Microbiology, 138(1),
37-43. https://doi.org/10.1007/BF00425404
18. Zahran, S.A., Ali-Tammam, M., Hashem, A.M. et al. Azoreductase activity of
dye-decolorizing bacteria isolated from the human gut microbiota. Sci Rep 9, 5508
(2019).https://doi.org/10.1038/s41598-019-41894-8
19. Russ, R., Rau, J., & Stolz, A. (2000). The function of cytoplasmic flavin reductases in the reduction
of azo dyes by bacteria. Applied and Environmental Microbiology, 66(4), 1429-1434.
https://doi.org/10.1128/AEM.66.4.1429-1434.2000
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36
 Appendix - i

Medium composition
Nutrient Agar

Agar 15.00 g
Peptone 05.00 g
Sodium Chloride 05.00 g
Beef extract 1.500 g
Yeast extract 1.500 g
Ph 7.4
Distilled water 1000 ml

Nutrient Broth

Peptic digest of animal tissue 5.00 g

Sodium chloride 5.00 g

Beef extract 1.500 g

Yeast extract 1.500 g

pH 7.4

Distilled water 1000 ml

Eosin methylene YELLOW agar(EMB)

Peptone 10.0 g

Lactose 5.0 g

K2HPO4 2.0 g

37
Eosin Y 0,4 g

Methylene blue 0.065 g

Agar 30.0 g

Distilled water 1000 ml

pH 7.4

Dissolved the ingredient in DW by heating and sterilized by autoclaving. Add lactose, eosin and
methylene blue after autoclaving. Mix it well and steam it for 5 minutes.

Glucose phosphate broth(GPB)

Glucose 5.0 g

K2HPO4 5.0 g

Peptone 5.0 g

Distilled water 1000.0 ml

pH 6.9

Dissolve by heating and adjust the pH and distribute 10 ml in to the test tube. Sterilized by
autoclaving.

MacConkey Agar

Peptone 20.0 g

Lactose 10.0 g

NaCL 5.0 g

Bile salts 3.0- 5.0 g

Neutral red 30.0 mg

38
Crystal violet 10.0 mg

Distilled water 1000.0 ml

Agar 30.0 g

pH 7.4

Dissolved by heating and pH adjust to 7.4 and sterilized by autoclaving.

Tryptone broth

Tryptone 10.0 g

Sodium chloride 5.0 g

Distilled water 1000.0 ml

Ph 7.5

Dissolved by heating and pH was adjusted to 7.5 and was distribute in 5 ml amounts in test tubes
and sterilized by autoclave.

39
 Appendix - ii
Regents
KOVAC’S REAGENT
P-dimethylaminobenzaldehyde 5.0 g
Amyl alcohol 75.0 ml
Concentrated HCl 25.0 Ml
The aldehyde in alcohol by gently warming in a water-bath was dissolved (about 50-55°C) and
was settled down to cool and acid was added with care. It was stored at 4°C in ordered to protect
from light.

METHYL RED INDICATOR

Methyl Red 0.1 g
95% ethanol 300.0 ml
The dye was dissolved in alcohol and was used.

Potassium hydroxide solution(KOH)

KOH 40.0 g
Distilled water 100.0 ml
The KOH was dissolved in 100 ml water to make the final volume.

HYDROGEN PEROXIDE (H2O2)

H2O2 0.0015 mol
Distilled water 100 ml
0.0015 mol hydrogen peroxide was dissolved in 100 ml water to make to make final volume.

40
 Appendix - iii
Instruments

Name of Instrument Company

Autoclave PSI

Light Microscope Lab domed

Weighting balance Wensar

BOD Incubator PSI

Orbital shaking incubator PSI

Centrifuge Machine(Microcentrifuge) Remi

Refrigerator cum deep freezer Samsung

pH meter Digitronics

Vortex (Cyclo) Mixer Remi

UV-VIS Spectrophotometer(UV Double Easytronics

Beam)

SN Name of Glassware and Equipments

1 Measuring cylinders

2 Test Tubes

3 Beaker

4 Spatula

5 Erlenmeyer Flask(100ml,250ml)

6 Eppendorf Tubes

41
7 Petri dishes

8 Slides

9 Inoculating needle, Inoculating loop,Spreader

10 Micropipettes ( 5µl,10 µl,100 µl,1000 µl)

11 Disposable tips

42