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1
Synthesizing and digitization of a Microbial Culture Collection
Centre
Dissertation submitted to
NATIONAL COLLEGE (AUTONOMOUS)
affiliated to
Bharathidasan University,
Tiruchirapalli
in partial fulfillment of the requirements for the award of the degree of
MASTER OF SCIENCE IN BIOTECHNOLOGY
By
J.ARULPRINCY
Admission No. BTPS20011
Under the Guidance of
Dr. Chinnamani Prasannakumar
Associate Professor,
DEPARTMENT OF BIOTECHNOLOGY & MICROBIOLOGY
DEPARTMENT OF BIOTECHNOLOGY & MICROBIOLOGY
(Supported under DBT-PG TEACHING & DBT-STAR COLLEGE Schemes)
NATIONAL COLLEGE (AUTONOMOUS)
(Nationally Re-accredited with A+ Grade by NAAC)
(College with Potential for Excellence)
TIRUCHIRAPALLI-620001
AUGUST 2022
Estd.1919
NATIONAL COLLEGE (Autonomous)
(Nationally Re-accredited at ‘A+’ Grade with 3.61 CGPA by NAAC)
(College with Potential for Excellence)
TIRUCHIRAPALLI - 620 001. TAMILNADU
email: b.m@nct.ac.in
Website :www.nct.ac.in/b.m
DEPARTMENT OF BIOTECHNOLOGY AND MICROBIOLOGY
(Supported under the DBT-PG Teaching & DBT-STAR College Schemes)
Date: ……….……….
BONAFIDE CERTIFICATE
This is to certify that the Dissertation, titled entitled ‘‘Synthesizing and digitization of a
microbial culture collection centre’’ submitted to National College (Autonomous),
Tiruchirappalli 620001, in partial fulfillment of the requirements for the award of degree
of Master of Science in Biotechnology is a record of original research work done by Ms.
ARULPRINCY J, bearing the Admission No. BTPS20011 during the period 2020-2022 of
his/her study in the PG & Research Department of Biotechnology & Microbiology, National
College (Autonomous), Tiruchirappalli 620 001, under the guidance of, Dr. Chinnamani
Prasannakumar, Associate Professor, Dept. of Biotechnology & Microbiology, National
College (Autonomous), Tiruchirapalli; and the dissertation has not formed the basis for the
award of any Degree/ Diploma / Associateship/ Fellowship.
Project Guide Head of the Department
Thesis evaluated and candidate subjected to presentation & viva-voce examination held on ……………….
1. Signature of the External Examiner 2. Signature of the Internal Examiner
NATIONAL COLLEGE
Dr. CHINNAMANI PRASANNAKUMAR, M. Phil. Ph. D. (DST-INSPIRE fellow)
Associate Professor
PG & Research Department of Biotechnology and Microbiology
National College (Autonomous) (Estd. 1919)
(Nationally Re-Accredited at "A+" Grade by NAAC)
(A College with Potential for Excellence)
Tiruchirapalli - 620001, Tamil Nadu, India.
https://www. nct. ac. in/dept-biotech. html
+91-9865929945
micropras@gmail. com, cprasanna@nct. ac. in
CERTIFICATE
This is to certify that the thesis entitled “Synthesizing and digitization of a Microbial
Culture Collection Centre” submitted by Ms. Arulprincy J. , for the award of the Degree
of Master of Science in Biotechnology is based on original studies carried out by her under
mysupervision.
The thesis or any part thereof has not been submitted for any other degree or diploma in
any university or institution.
Place: National College
Date:
(CHINNAMANI PRASANNAKUMAR)
DECLARATION
I, ARULPRINCY J hereby declare that the Dissertation, titled “Synthesizing and
digitization of a Microbial Culture Collection Centre” submitted to the National College
(Autonomous), Tiruchirapalli in partial fulfillment of the requirements for the award of the
Degree of Master of Science in Biotechnology, is my original work executed between
March, 2022 & August, 2022, under the supervision and guidance of Dr. Chinnamani
Prasannakumar, Associate Professor, Department of Biotechnology & Microbiology,
National College (Autonomous), Tiruchirapalli, and it has not formed the basis for the
award of any Degree / Diploma / Associateship / Fellowship.
Signature
ARULPRINCY J
ACKNOWLEDGMENT
Foremost, I would like to thank the most almighty god without whom nothing is possible, for the
wisdom he bestowed upon us, the strength, peace of mind, and good health in order to complete
our research in good manner.
First and foremost, I would like to thank the Almighty for his kind blessings that sustained me
and helped me to carry out this project. I express my thanks to Dr. Chinnamani
Prasannakumar, Associate Professor, Department of Biotechnology & Microbiology,
National College (Autonomous), Tiruchirappalli, for being a perfect mentor and guiding me
during the course of the project work.
I would like to thank Dr. M. S. Mohamed Jaabir, Head, Department of Biotechnology
&Microbiology, National College (Autonomous), Tiruchirappalli, for his valuable support.
Barcode Biotechnologies Pvt. Ltd. Vasco DaGamma, Goa, India, is acknowledged for their
active role in providing cost effective sequencing services.
It is impossible to mention ever ribution to my career till
date, I know I have missed many of them; I thank all of you individually for the love, support
and encouragement.
I would also like to thank my friends Mr. Yoganadhan, Ms. Kalaiyarasi who supported me
throught my project work.
Thanking my parents is too small a word for them for their deep love, constant encouragement
and guidance which kept me going. I love a lot to my parents, who encouraged and helped me
at every stage of my personal and academic life and longed to see this achievement come true.
ARULPRINCY J
TABLE OF CONTENT
S. No.
Topic
Page No.
*
Abstract
6
*
Abbreviations
7
*
List of Figures
8
*
List of Tables
11
1
Chapter-1: Introduction
12
2
Chapter-2: Review of Literature
18
3
Chapter-3: Scope of the work
23
4
Chapter-4: Material & Methods
24
5
Chapter-5: Results
46
6
Chapter-6: Discussion
89
7
Chapter-7: Summary
94
8
References
96
5
Synthesizing and digitization of a Microbial Culture Collection Centre
Abstract
The present study was under taken to with an objective of synthesizing and digitizing a microbial
culture collection. To achieve the objective, microorganisms including bacteria (Proteobacteria,
actinobacteria, cyanobacteria), molds, yeast, higher fungi (mushrooms), and algae were obtained
from a various sample sources, including AC condensate water, human microflora, fish gut
microorganisms, marine waters, soil, and sewage, etc. Despite the fact that all microbial species
were at varying phases of culture characterization in the making of culture collection centre,
biochemical and molecular characterization techniques used has identified 107 different
microbial species taxonomically resolved up to species level. The 16S rRNA (for bacteria,
actinobacteria, and cyanobacteria), ITS (for mold, yeast, and mushrooms), and 18S rRNA (for
microalgae) gene sequences shared more than 98 percent sequence similarity with the reference
database (NCBI-GenBank), implying the species level resolution of the respective species
(supported with biochemical characterizations). All 107 species in the culture collection is
could be accessed through www.ncccc.in. Besides culture collection, NCCCC supports the
services related to DNA sequencing, sequence analysis and biochemical characterization of
microbial strains.
Keywords: Microbial culture collection, Biochemical characterization, Molecular
identification, Bioinformatics analysis, Construction of culture collection Database.
6
ABBREVIATIONS
Abbreviation
Definition
µg/L
- Microgram per Liter
mg/L
- Milligram per Liter
Mm
- Millimeter
ATTC
- American type culture collection
BLAST
- Basic Local Alignment Search Tool
BRC
- Biological resource centre
CaCl2
- Calcium chloride
CFU
- Colony forming unit
CTAB
- cetyltrimethylammonium bromide
CuSO4. 5H2O
- Copper (II) sulfate pentahydrate
Co (NO3)2. 6H2O
- Cobalt nitrate hexahydrate
DNA
- Deoxyribonucleic acid
EDTA
- Ethylenediaminetetraacetic acid
EtBR
- Ethidium bromide
dNTPs
- Deoxynucleotide triphosphates
HCL
- Hydrochloric acid
H3BO3
- Boric acid
KCL
- Potassium chloride K2HPO4.
3H2O
-Dipotassium hydrogen phosphate
KNO3
- Potassium nitrate
7
MTCC
- Microbial Type Culture Collection
MgSO4. 7H2O
-Magnesium sulphate heptahydrate
MRVP
- Methyl Red Voges-Proskauer
Mncl2. 4H2O
- Manganese (II) Chloride
mRNA
- messenger RNA
NaCl
- Sodium chloride
NaOH
- Sodium hydroxide
Na2HPO4
- Sodium Phosphate
PBS
- Phosphate-buffered saline
PCR
- Polymerase chain reaction
RNA
- Ribonucleic acid
SDS-PAGE
- Sodium dodecyl-sulfate polyacrylamide gel electrophophoresis
WDCM
- World data centre for microorganism
8
LIST OF FIGURES
Fig
number
Title of the figure
Page no
Fig 1
Pie chart representing diversity of samples used
for building the culture collection.
46
Fig 2
Spread plate method of the different listed microbial samples
47
Fig 3
Isolates pure culture of bacteria on nutrient agar medium
48
Fig 4
Isolates pure culture of yeast on glucose yeast mannitol agar
medium
49
Fig 5
Purification of molds in potato dextrose plate
49
Fig 6
Purification of cyanobacteria NCT201 (Chlorella vulgaris),
NCT65 (Synechococcus elongatus) on MN Agar medium
50
Fig 7
Three flask containing pure culture of NCT201 (Chlorella vulgaris),
NCT65 (Synechococcus elongatus), NCT 51
(Tenebriella curviceps)
50
Fig 8
Slide pictures of various Gram positive and negative strains of NCCCC.
51
Fig 9
Microscopic examination of spore forming bacteria
52
Fig 10
Microscopic examination of fingal isolates
52
Fig 11
Microscopic examination of Yeast isolates
53
Fig 12
Microscopic examination of NCT51 (Tenebriella curviceps),
NCT65 (Synechococcus elongates), NCT201 (Chlorella vulgaris)
53
Fig 13
The microbial culture glycerol stocks were stored in -
54
Fig 14
The Negative result NCT34 (Enterococcus faecalis) is indicated
by the catalase negative. The positive result NCT45 is
indicated by catalase negative
54
9
Fig 15
The Positive result NCT25 (Vibrio cholerae) is indicated by the
Oxidase positive. The Negative result NCT38 is indicated by
Oxidase negative.
54
Fig 16
The positive result (A) is indicated by the red layer at the top of
the tube after the addition of Kovacs reagent. The negative
result (B) is indicated by the lack of color change at the top of
the tube after the addition of Kovacs reagent.
55
Fig 17
Positive methyl red test (A) is indicated by the development of
red color after the addition of methyl red reagent. The negative
methyl red test (B) is indicated by no color change after the
addition of methyl red reagent
55
Fig 18
A positive Voges-Proskauer test (A) is indicated by the
development of red-
lack of color change after the addition of A and
B reagents.
56
Fig 19
A positive citrate result (A) is indicated by growth and a blue
color change in the tube. A Negative citrate utilization test (B) is
indicated by the lack of growth and color change in the tube
56
Fig 20
Muller Hinton agar plate showing zones of inhibition due to various
antibiotic discs against the NCCCC bacterial isolates
60
Fig 21
Positive (A) Presence of clear halos surrounding colonies for
their ability to digest the starch and thus indicates presence of
alpha-amylase. Negative (B) No clearing; only a blue/black area.
64
Fig 22
Positive (A) clear zone around the bacterial growth. Negative
(B) No zone of clearing
64
Fig 23
Negative lipase enzyme test is indicated by the development of
pink color. The yellow colour was indcated as positive lipase
enzyme.
65
10
Fig 24
SDS-PAGE Fingerprinting of whole cell proteins of
(A)NCT265, NCT266, NCT267, NCT268. (B)NCT243,
NCT24 4.1, NCT244.2, NCT245, NCT246. (C)NCT258,
NCT259, NCT260, NCT261, NCT127.2, NCT130.
(D) NCT204.1, NCT205, NCT207, NCT210, NCT212 (E)
NCT121, NCT122, NCT123, NCT124, NCT125, NCT126. The
diverse pattern of bands in the SDS PAGE
fingerprinting denotes the distinct bacterial species
66
Fig 25
Genomic DNA isolation visualized in agarose gel electrophoresis.
66
Fig 25
Phylogenetic analysis
67
11
List of Tables
Table
No.
Title of the table
Page
No
Table. 1
Biochemical test for identification of the isolates
57
Table. 2
Response pattern of isolates for different antibiotics on
(Tetracycline, Rifambicin, Gentamycin, Streptomycin, Penicilin,
Ampicillin, Amoxilin)
61
Table. 4
Results of BLAST analysis including percentage of similarity, query
coverage and alignment scores were tabulated.
116
Table 3
List of strains being explored Foy bio-prospecting by various PG
Students of Department of Biotechnology & Microbiology, National
college, Trichy.
94
12
Chapter 1
1. General Introduction
Background
Microbes constitute the largest biomass on the earth and comprise three domains of life (bacteria,
archaea and eukaryotes). Almost 90 % of this diversity is still unexplored. These microbes play
an integral and unique role in the functioning of the ecosystems in maintaining a sustainable
biosphere and productivity (Whitman et al., 1998). Culture collections are valuable resources for
the long-term use and conservation of microbial diversity. Biotechnology advancements have
increased their importance, and some of them have been designated as International Depositary
Authorities (IDA) for the deposit of patent cultures (Sharma et al., 2004). Culture
collections are critical resources for biodiversity conservation, and collaboration among
collections is essential to ensuring their long-term viability. DNA sequencing technique plays an
important role in culture collection by providing correctly identified strains and associated
metadata, as well as by accessioning strains from sequencing projects. Group efforts for
developing legal frameworks, databases, and quality control standards and protocols for culture
collections are being developed in other parts of the world. Validating and tracking the purity,
identity, and provenance of microbial strains are among the most important functions of a culture
collection and are required for a collection to be considered a Biological Resource Center (BRC)
according to the guidelines published by the Organization for Economic Cooperation and
Development (OCED) (Boundy-Mills et al., 2015). Culture collections are important store
houses of information on cultures that can be accessed by direct contact with individual
collections or contacting microorganism database organizations (Stacey et al., 2007).
Microorganisms have enormous ecological, medical, and practical importance. They are
beneficial to the environment, the food industry, biofuel production, bioremediation, industrial
microbiology, biotechnology, and human welfare (Madigan et al., 2015). Agriculture benefits
from the cycling of nutrients by microorganisms. Nitrogen fixing bacteria convert atmospheric
nitrogen into ammonia that the plants can use as a nitrogen source (Berersen and Turner 1968).
Microbial world is invisible to unaided human eyes.
13
Thus what goes on in their domain is not easily perceived, unless some direct measurements and
analyses are carried out. Consisting of bacteria, fungi, yeasts, protozoans, phytoplankton and
sizes, microbial communities perform immense tasks mainly to
keep themselves perpetuating in their ecosystems (Barkay et al., 2003).
Microbes are in charge of nutrient recycling and detoxification. They function as biological
control agents, biocatalysts, and producers of a wide range of pharmaceutical and industrial
products. They are also thought to be potential solutions to the world's looming food and energy
crises. We also understand that they would play a critical role in improving the quality of life,
alleviating poverty, malnutrition, and other issues. The advent of the metagenomics era and
projects such as metagenomic analysis of the human intestine, the Human Microbiome Project,
and soil metagenome (Terragenome) have highlighted their critical role on the planet and in
human health. However, events such as global warming, lifestyle changes, and anthropogenic
activities are causing a loss of microbial diversity, but because microbes are not visible, the loss
is not being noticed. Microbiology's advancement required the establishment of collections of
microbial cultures for the study and ex situ preservation of biodiversity in ecosystems, as well as
the distribution of promising microbial strains for the production of goods and services. As a
result of these advances in molecular biology, intensive bioprospecting programmes for all of
these microorganisms have been developed (Díaz-Rodríguez et al., 2021). Culture collections
are responsible for stewardship of microbial resources of vital importance to science and society.
Basic and applied research depends upon the availability of suitable biological material
(Sigler, 2004). Microbial culture collections aim at collecting, maintaining and distributing
microbial strains among microbiologists, and are considered to be a means to preserve microbial
diversity ex situ. Culture collections are services dedicated to supporting a wide variety of
microbiological work. Their primary function is to collect, store, and distribute microbial strains
requested by microbiological laboratories for use in teaching, research, quality control assays,
biotechnology, and other applications. Culture collections are similar to libraries, except that
instead of books, they house living material, such as microorganisms. Usually, culture
collections are considered to be a means to preserve microbial diversity ex situ (Uruburu, 2003).
Microbial culture collections have a crucial role to play in the maintaining, understanding and
utilization of microbial diversities (Caktu et al., 2011). Implementation of quality measures,
14
compliance with the Convention on Biological Diversity (CBD), and adoption of latest
bioinformatics tools are among the main steps to be taken by microbial culture collections in
order to provide resources for the emerging area of the knowledge-based bioeconomy
(Stackebrandt, 2010). Culture collections are responsible for stewardship of microbial resources
of vital importance to science and society. Basic and applied research depends upon the
availability of suitable biological material (Sigler, 2004). Culture collections are heavily utilized
as sources of biological materials and standards by academia, industry, agriculture, medicine and
research. The first public service bacterial culture collection was established by Prof. Frantisek
Kral at the German University of Prague, Czech Republic. He worked as a technician in 1890 at
the institute of Hygiene; German University of Prague (Sharma et al., 2014). Their primary
function is to collect, maintain, and distribute microbial strains ordered by microbiological
laboratories for use in teaching, research, quality control assays, biotechnology, and other
applications. Culture collections are similar to libraries, but instead of books, they house living
material, such as microorganisms. Microbial culture collections are considered as libraries, but
instead of books they hold microorganisms (Caktu et al., 2011). Since microbial culture
collections are now engaged in meeting high quality operational standards, they are facing the
challenge of establishing quality control criteria to certify their biological materials. Culture
collections worldwide have been gathering efforts to develop standardized guidelines applicable
for their products and services (Simoes et al., 2016).
Bacteria are diverse organisms; therefore generalizations cannot be made regarding pH values,
media, and incubation temperatures that will support optimum growth. However, the majority of
organisms will grow at pH values near neutrality, at temperatures between 20 to 37°C and in a
medium containing an energy source (e. g., glucose) and an organic nitrogen source (e. g.
peptone). Many UK collections supply information on media and growth conditions in their
catalogs or with the strains supplied and will provide advice on an individual.
Fungi, in general, grow best on mediums made from the natural materials from which they were
separated. CABI Bioscience utilizes soil and plant extracts such as leaves, stems, and seeds that
are deposited on solid agar. It is critical to optimize growth conditions. Avoiding variation
selection from within the population, strain degradation, and contamination are critical for
establishing strains. Medium, temperature, light, aeration, pH, and water activity are the most
important elements of growth. Mycologists face the daunting task of characterizing the very large
15
and unwieldy fungal kingdom in a taxonomic context. Estimated at 1.5 million species and
reported from little short of all biota on Earth, fungi are thought to be responsible for many key
ecological functions such as wood and litter decomposition, mycorrhizal associations, and other
forms of nutrient recycling. Inconspicuous by default, fungi are typically noticed only when they
form above-ground fruiting bodies or other propagules (Nilsson et al., 2009).
Chlorella vulgaris is of great biotechnological potential for producing valuable substances for
the food, cosmetics, and nutraceutical, pharmaceutical industries (Muller et al., 2005). The first
collections of laboratory-maintained algae were established at the end of the 19th/beginning of
the 20th century, due to increasing scientific interest in the study of algae, especially arising from
the need to perform experiments on defined strains (Day et al., 2004).
After a microbial strain is isolated, the subsequent investigations, publications and the deposit in
a culture collection result in an increasing amount of information that typically is broadly
scattered over many different scientific journals, books, culture collection catalogs and life
science databases around the world. Since these data are an important basis for future research in
the life sciences it is essential to mobilize, harmonize and match scattered scientific information
about microbial strains in order to foster a seamlessly structured database content and provide an
easy and reliable access to the data (Sohngen et al., 2015).
Collecting examples of different types of organisms has been the pursuit of scientists and
amateur collectors for centuries. This activity was originally
regarding the natural dive
rsi
ty of
his
environment, but for
w
ell over a century
s
cientis
ts
have
been collecting strains of animals, plants, and microorganisms with specific scientific and
technical aims relating to taxonomy, infectious disease, and biochemistry. Today, culture
collections, or more broadly, biological resource centers (BRCs), are a mixture of academic,
public service, private, government and commercial activities that deliver important
characterized culture
s
a
s
se
e
d
st
oc
ks:
1. For the development of industrial processes.
2. As reference strains for biological assays and published scientific literature.
3. As type strains for taxonomic studies.
16
4. As centers for conservation of biodiversity
Conservation of biodiversity
Microbial culture collections, viral repositories, herbaria, botanical gardens, zoos, and ex situ
plant and animal genetic resource collections all contribute to the preservation of biodiversity,
which is under threat from unsustainable economic development, natural disasters, and climate
change. The benefits of biological resource conservation are emphasized by the Convention on
Biological Diversity (CBD), which emphasizes the need for BRCs to act as ex situ conservation
of biodiversity.
Towards the establishment of national BRCs in Japan: Japan maintains several collections of
micro-organisms and other biological resources that could become national BRCs. In addition to
the existing culture collections, Japan is establishing a new national biological resource center of
industrially useful microorganisms to meet the requirements of the life sciences and
biotechnology in the 21st century.
Phenotypic characterization a basis for species identification:
This method measures various morphological, metabolic, physiological, and environmental
parameters that can lead to a better understanding of how the microorganism functions
(O'Connell et al., 2007). Culturing a microorganism remains the only way to fully characterize
its properties and predict its impact on an environment (Madigan et al., 2015). One of the
shortcomings of culture-based techniques is that there is a large discrepancy between the number
of bacterial colonies that form on solid media and the total number of bacterial cells actually
present in the sample (Joseph et al., 2003). It has been estimated that only 0. 1-1% of soil
bacteria are accessible by conventional culture-dependent techniques, which leaves most of the
phylogenetic diversity unstudied (Zhang and Xu, 2008). However, the utilization of secondary
metabolites (such asorganic acids and antibiotics), industrial enzymes (amylases, lipases) lies in
the ability to culture the microorganism. Even though phenotypic variations are the basis of
species differentiation, molecular characterization forms a basis for species identification.
DNA sequencing and phylo analysis
Carl Woese proposed a radical new taxonomic scheme based on phylogenetic relationships
rather than visible morphological similarities in 1977. He used the small subunit rRNA
17
gene (16S rRNA in bacteria and 18S rRNA in eukaryotes) as a universal phylogenetic marker
(Albers et al., 2013; Fox et al., 1977). The 16S rRNA gene is a section of prokaryotic DNA
found in all bacteria and archaea. This gene codes for an rRNA which makes up part of the
ribosome. The ribosome is composed of two subunits, the large subunit (LSU) and the small
subunit (SSU). These two subunits sandwich the mRNA as it feeds through the ribosome for
translation. Woese realized that rRNA genes make excellent candidates for phylogenetic
analysis because they are found in all known life forms, functionally constant, and highly
conserved (Madigan et al., 2015). Their highly conserved nature is due to their important
function of translating mRNA into proteins. However, there are portions of the genes that are
more conserved than others. Hence 16S rRNA gene sequences are universally used for
prokaryotic species identification and 18S rRNA gene sequences for eukaryote identification.
Biotechnological potential
The present study synthesized a microbial culture collections through isolation, pure culturing
and identifying the microorganisms (via DNA sequencing) collected from various ecosystems .
Bioresource potential of few strains in enzyme production and dye degradation was explored.
18
Chapter 2
2. Review of Literature
2.1. United States culture collection network:
The US National Science Foundation has supported select living microbe collections for many
years through the Division of Biological Infrastructure, and in 2012 funded a research
coordination network (RC
N) proposa
l for
a
community of ex
si
tu microbial germpla
s
m
repositories and a exchange of best practices during meetings, workshops. Living microbe
collections are an integral but often overlooked aspect of a mature research and development
infrastructure and have been described as the foundations of the modern bioeconomy . Living
microbe collections, sometimes called culture collections or biological resource centers, hold and
distribute authenticated living microbial cultures for a broad range of basic and applied research
applications (McCluskey et al., 2014).
2.2. Culture Collections in Australia
A review of culture collections in Australia concluded that there was an urgent need for a
network of adequately funded culture collections to conserve and supply Australian microbial
cultures for use in science, industry and education, and that electronic access to information on
many countries have developed national collections to meet their scientific and industrial needs.
Australia, on the other hand, has depended on institutional collections to meet the needs of their
host institutions, with little national perspective and coordination, except for some plant
pathology collections and herbaria. Australia has approximately 50 culture collections listed
with the World Federation for Culture Collections World Data Center for Microorganisms
(WDCM) (Wu et al., 2016).
Some 10 of these could form the core of a coordinated Australian Collections of Microorganisms
(ACM), covering a broad range of microbial diversity (Sly, 2010). In addition, many specialized
research collections also contribute to valuable resources but often are difficult to locate. Access
to information on culture held in Australian culture collections is extremely limited. Few
collections have the resources to publish catalogs and those which exist are often out of date. A
few collections have developed web accessible catalogs which is the way of the future.
19
2.3. Biological Resources and Information (CABRI): An European network
Common Access to Biological Resources and Information (CABRI) is a regional network
linking the major European ex situ collections (Romano et al., 2005). It provides a federated
database system accessible through the World Wide Web. The centers taking part in this project
currently hold 21 collections covering human and animal cells, bacteria, fungi, yeasts, plasmids,
animal and plant viruses and DNA probes. Instead of having to examine a large number of
databases.
2.4. Cabi culture collection: Center for Agriculture and Bioscience International
The CABI collection is just one of several global culture collections each with such history and
holdings as a result of researchers depositing microorganisms for future study and use in
biotechnology. CABI is an international not-for-profit organization, owned by 49 member
countries which applying scientific
expertise to solve problems in agriculture and the environment. The CABI culture collection
began as a series of individual collections held by institute mycologists. The CABI culture
collection has UK isolates of more than 1300 species which could be sequenced over the full 10
years of this study. As CABI also holds a number of UK strains of Oomycete species, we will
also feed into the Protist part of the tree. Not all species, genera or even families are represented
by culture collection holdings indeed, not all described taxa can be cultured (Smith et al., 2020).
2.5. Culture collection in India
India is recognized as one of the hotspots of mega biodiversity encompassing enormous endemic
microbial diversity. Extensive bio-prospecting projects in the past funded by the Department of
Biotechnology revealed the immense microbial diversity which could potentially be exploited for
medicinal, industrial and agricultural purposes (Sharma et al., 2017). Biological Resource
Centers (BRCs) are the next generation culture collections and are a key element of the scientific
and technological infrastructure for the life sciences and biotechnology. The microorganisms
they store, characterize and supply provide microbial solutions to food production, health care
and environmental problems. There is an ever increasing demand to demonstrate that such
reference materials are authentic and remain unchanged as more and more bio-industries are
adopting certification or accreditation as a means to demonstrate quality and competence. This
20
may be the s strategy for long-term
sustainability, but it is also an increasing requirement to satisfy the funders of research who seek
high quality science and solutions (Smith et al., 2008). The establishment and maintenance of
biological resource centers (BRCs) requires careful attention to implementation of reliable
preservation technologies and appropriate quality control to ensure that recovered cultures and
other biological materials perform in the same way as the originally isolated culture or material.
There are many types of BRC that vary both in the kinds of material they hold and in the
purposes for which the materials are provided (Stacey et al., 2007).
Although 27 culture collections in India are registered with WDCM, only a few provide regular
services to the scientific community. Major among them are Microbial Culture Collection
(MCC, Pune), Microbial Type Culture Collection (IMTECH, Chandigarh), National Fungal
Culture Collection of India (Pune), National Collection of Industrial Microorganisms (Pune),
agriculturally important National Bureau of Microorganisms and National Collection of Dairy
Cultures (Karnal), etc. Coordination, collaboration, and discussion on approaches to microbial
resource collection establishment and organization take place at numerous levels. On a national,
regional, and worldwide scale, organizations exist to assist collection activities. These include
national federations such as the
United Kingdom Federation for Culture Collections (UKFCC)
United States Federation for Culture Collections (USFCC),
Japanese Federation for Culture Collections (JFCC).
European Culture Collection Organization (ECCO), and at the international level, the World
Federation for Culture Collections (WFCC), Microbial Strain Data Network (MSDN), and
Microbial Resource Centers (MIRCENs) operate.
MCC
Thus the Microbial culture collection at National Centre for Cell Science (NCCS) Pune was
funded by DBT and mandated that all the isolates generated in this project would be made
available at this collection for future use by others who wish to exploit them for biotechnological
purposes. MCC is presently affiliated with NCCS, Pune. MCC got recognition as IDA by the
21
WIPO, Geneva, Switzerland under Budapest Treaty on 9th April 2011, and recently MCC is also
recognized as Designated National Repository for Microorganisms on 8 July 2013 by Ministry of
Environment and Forests (MoEF), New Delhi, India under Biological Diversity Act 2002. MCC
accepts microorganisms for deposit under general, safe, patent, and IDA deposits. MCC holds
more than 150,000 bacterial strains isolated project (Sharma
et al., 2014)
ATCC
The American Type culture collection historically received public support, but became self
supporting many years ago through modern marketing and intellectual property management.
The American Type Culture Collection (ATCC), a non-
diverse biological resource center (BRC). ATCC supports public health initiatives for the US
government, academia, pharmaceutical industry, and research foundations. ATCC has a variety
of collections spanning the biological spectrum. The ATCC Microbiology Collection contains
bacteria, viruses, fungi, yeasts, protozoa, and parasites, as well as molecular biology resources
for studying these. ATCC Cell Biology Collection is the most comprehensive bioresource in the
world, consisting of over 3600 cell lines from over 150 different species, including continuous,
hTERT-immortalized, and iPS cell lines as well as culture media and associated reagents. One
newest departments is the Cell Derivation Unit (Bens et al., 1996).
The World Federation for Culture Collections (WFCC) is a federation of the International Union
of Microbiological Societies (IUMS) and a commission of the International Union of Biological
Sciences (IUBS) with responsibility for the promotion and development of collections of cultures
of microorganisms and cultured cells. The WFCC must review how best it can provide services
to members as the members in turn evaluate carefully the activities they become involved in and
how they get value for money as society membership subscriptions increase. The WFCC is
developing a strategic plan to define its future activities and to put in place a sound financial plan
in order to take forward key initiatives in the interest of the organization. The World
Federation of Culture Collections (WFCC), created in 1971, has more than 500 members,
including culture collections in more than 60 countries. The WFCC is a member of international
scientific organizations affiliated to the International Council of Scientific Unions (ICSU). It
played an instrumental role in creating the regulatory framework under which the Budapest
22
Treaty was implemented. It promotes the activities of traditional microbial culture collections by
providing venues for the exchange of information about microbial collections and taxonomy.
JFCC
The Japanese Federation of Culture Collections (JFCC) The JFCC was established in 1951
following the recommendation of the Japanese Ministry of Education and Science Council of
Japan. The aim of the federation is to encourage research on microorganisms and exchange
information on microbial cultures. The federation currently consists of 23 Japanese culture
collections and encompasses collections in the fields of general microbiology, medical
microbiology, applied microbiology, and environmental microbiology. The JFCC was the first
"network" of culture collections and data banks in Japan.
Nowadays, microbial diversity is viewed as available yet invisible, resource for science and
industry with implications for economy and finance. Two major events in the beginning of the
vance to
biotechnology, healthcare, agriculture, industry and other services. The first was the kral
symposium in 1990, for the commemoration of 100 years of culture collections. since the
establishment of the first culture collection of yeast and filamentous fungi, in prague, in 1980 by
Frantisek kral, for the preservation and distribution of microorganisms for research, industry,
diagnosis, and teaching.
23
Chapter 3
3. Scope of the Study
Culture collections play a vital role in the conservation and sustainable use of microbial
resources. They also provide the authentic biological material for high quality research and
teaching in the form of reference strains, reagents for quality control, etc. micro-organisms have
played a specific role in human and animal disease and in the production and spoilage of food
and drinks. Microorganisms are an essential part of human wellbeing, participating in medicine,
agriculture, aquaculture, food industry, and biotechnology, among others. Since microbiota is
found in every place on the planet, it plays vital roles in ecosystems, such as; (i) social and
ecological sustainability, (ii) adaptation and mitigation of climate change, (iii) as a
biotechnological resource for humanity, (iv) water cycling and nutrients, and (v) the increase of
food production. However, the agro-ecosystems are undergoing accelerated deterioration
worldwide due to erosion, loss of organic carbon, nutrient depletion, soil sealing, climate change,
and other threats, generating a loss of those promising PGPM genera (FAO, 2015a). Therefore,
the conservation of this biological diversity is essential for its re-incorporation into agro-
ecosystems. Thus, the role of MCC of a culture collection is crucial in achieving this goal.
3.1. Objective
Isolation and identification of microorganisms from various niches.
Preservation and supply of microbial species colleted.
Bio-prospecting of microbial strains collected.
Digitization of culture catalog for end users.
24
4. MATERIALS AND METHODS
4.1. SAMPLE COLLECTION
Since the synthesis of microbial culture collection involves the handling of diverse samples,
sample processing, depends on the nature of the sample collected.
4.1.1. CLINICAL SAMPLE
Clinical samples were collected from the hospital environment. The sample was sealed
in sterile condition. were transported to the
laboratory for further study and samples were stored in the refrigerator until use (Dinis-
Oliveira et al., 2016).
4.1.2. GARDEN SOIL SAMPLE
Soil samples were collected from different gardens, and plant pots. Soil samples were
collected at the depth of 5-6 cm from the top using a sterile spatula in sterile polythene bags,
which were sealed tightly to prevent external microbial contamination during the transport.
Soil samples were sieved to get rid of pebbles, rocks and leaves. They were labeled specifying
the serial number of the soil sample and place (Rahman et al., 2020). Soil samples are serially
diluted before plating. About 10g of Garden soil sample was dissolved into 100ml sterile
distilled water. The slurry was thoroughly mixed the solution. The serial dilution was done in
test tubes containing 9ml of sterile distilled water each. Spread plate method was performed
to isolate and enumerate the organism of interest present in different samples. A control
plate was made by putting 1 ml of distilled water. Inoculated (NA) plates were incubated for
24 hours
4.1.3. AGRICULTURE SAMPLE
Soil samples were also collected from different agricultural fields. Soil samples were
collected at the depth of 5-6 cm from the top using a sterile spatula in sterile Polythene
bags, which were sealed tightly to prevent external microbial contamination during the
transport. Soil Samples were sieved to get rid of pebbles, rocks and leaves. They were
25
labeled specifying the serial number of the soil sample and place (Rahman et al., 2020). Soil
samples are serially diluted before plating. About 10 gm of Agricultural soil sample was
dissolved into 100 ml sterile distilled water. The serial dilution was done in test tubes
containing 9 ml of sterile distilled water each. The soil samples were serially diluted from
10-3 to 10-7 dilutions using sterile distilled water as a blank and were inoculated on the
nutrient agar medium by Spread plate technique using L- Rod. Inoculated plates were
incubated for 24 hours .
4.1.4. PLANT PATHOGEN SAMPLES
Infected plant tissues were selected and after washed and surface sterilized with 0.1%
mercuric chloride for 1 min and rinsed in sterile distilled water. A small piece of tissue part
was dispensed into sterile distilled water and teased apart with sterile needle . The samples
were stored in sterile condition (Senanayake, 2020). Until it was plated in an appropriate
media. The Collected infected parts of the plant were collectd and sliced into small
pieces. After washing the tissues thoroughly in sterile water. The infected tissues along with
adjacent small unaffected tissue are cut into small pieces (25 mm squares) and by using
flame-sterilized forceps, they are transferred to sterile petri dishes containing 0.1% mercuric
chloride solution used for surface sterilization of plant tissues. The plant parts were
transferred to PDA plates and Incubated for 5- 7 d for the complete growth of fungi. The
resulting fungi were purified and then subculture of each isolated fungus on a slant medium
for future studies (Thilagam et al., 2018).
4.1.5. PLANT ENDOPHYTE
A segment of 5-cm was cut from the middle of each collected plant entophyte. The outer
layers 1 mm of the segments were peeled to remove fungal and bacterial contaminants on the
surface. The part of the segments were collected by sterile polythene bag to avoid
contamination (Taylor et al., 1999). The plant leaves and stems were washed with running tap
water; leaf segments were equally cut by sterilized scalpel from the mid portions of healthy
leaves to include the midrib. The cut segments were surface sterilized by immersing into the
following series of solutions: sterile H2O for 60 s, 70% ethanol for 60 s, 2. 5% sodium
26
hypochlorite for 4 min, 70% ethanol for 30 s, and a final rinsing in sterile distilled water three
times. The sterilized plant leaves were cut into five segments (5 mm), and 20 leaf segments
per individual plant were placed on the surface of PDA plate (five segment for each plate),
supplemented with 0. 05 g of streptomycin sulfate per 100 mL of medium to inhibit bacterial
growth and incubated at 28 °C ± 2 °C. The plates were checked daily for any fungal growth;
single isolates grown out from the tissues were re- inoculated on fresh PDA plates and
maintained at 4 °C in PDA slants (Khalil et al., 2021).
4.1.6. AIR SAMPLE
The Isolation of microorganism from air is performed by using the settle-plate technique. In
this method, a suitable medium is poured over a sterile petri dish and then allowed it to
solidify. After that the plate is exposed to the open air for a few minutes. The plates are
incubated.
4.1.7. HUMAN MICROFLORA
4.1.7.1. HUMAN EAR
The specimen was collected by swabbing the human external auditory canal with a sterile
cotton swab. Swab was into the ear canal and rotated it gently along the walls of the canal.
The swab, was withdrawn carefully not to touch any other surfaces and swabbed on an
appropriate media (Senanayake, 2020).
4.1.7.2. SPECTACLE
Cotton swab moistened in sterile distilled water was swabbed to spectacle on its inside lens
and the part touching the nose. The swab was withdrawn carefully not to touch any other
surfaces and plated in nutrient agar (NA) plates (Landers et al (2010).
4.1.7.3. BUCCAL CAVITY
A sterile cotton swab was inserted into the mouth and swabbed several times across the inner
side of the left cheek. The cotton swab was then placed the tube containing sterile saline and
sealed. Serially diluted and plated on to NA plates.
27
4.1.8. FISH GUT SAMPLE
Fishes were anesthetized in an ice bath for 5-10 min or fresh fishes were collected and each
individuals were surface sterilized by immersion for 30 seconds in 70% ethanol. The gut was
musculature. Gut was weighed and placed into a
10ml sterile double strength phosphate- buffered saline (PBS) solution (disodium
phosphate,2-3%,sodium phosphate 0-6%,and sodium chloride,1-2% ) (Sivasubramanian et
al., 2012). These slurry samples were serial diluted and plated in NA plates.
4.1.9. MARINE WATER SAMPLE
in sterile plastic bottles which are rinsed several times with sample before collection.
Subsequently, all the samples were transported to the laboratory for further study and samples
were stored in the refrigerator until use. Samples were serially diluted and plated in Zobell
Marine Agar. About 10ml of marine water sample was mixed into 100ml sterile seawater. The
serial dilution was done in test tubes containing 9 ml of sterile seawater each. The marine water
samples were serially diluted from 10-3 to 10-7 dilutions using sterile sewwater as a blank.
4.1.10. RABBIT FECAL SAMPLES
Rabbit fecal samples were collected in sterile polythene bags, which were sealed tightly to
prevent external microbial contamination during transport. NA plates were used for the
bacterial isolation. About 0.8 % (0.8g in 100ml) saline was prepared in a conical flask and
sterilized in an pressure. One gram of rabbit
fecal material in the saline solution and incubated for 30 minutes in shaking incubator at
Flow Chamber, incubated sample was serially diluted up to 10-4
dilution. 100 µl of diluted sample from 10-4 tube was spread plated in Nutrient Agar medium
using L-Rod. Inoculated plates were incubated for 24 hours at incubator.
4.1.11. SPOILED FRUIT SAMPLE
Spoiled Fruits were collected in separate sterile polythene bags from supermarkets. The
samples include tomato and apple. They were immediately carried to the laboratory for
analysis at room temperature. The freshly collected samples were subjected to microbial
28
isolation. Fruit sample was homogenized in tissue homogenizer. One ml aliquot of the fruit
homogenate was aseptically spread with 9ml sterile double strength PBS onto potato
dextrose and Nutrient agar medium by spread plate technique using L-Rod. Inoculated plates
were incubated for 24 hours
Zulkahar., 2018
4.1.12. AlGAE & CYANOBACTERIA SAMPLE
Water sample collected from marine & fresh water ecosystems were (minimal nutrient
solution) inoculated in Erlenmeyer flask having algal culture medium and incubated at room
temperature under continuous dark and sunlight period for 15-20 days. Growth from the
incubated flask were spreaded on algal culture plate and incubated at room temperature
under continuous dark and light period for 15-20 days as described by (patil et al., 2015).
4.2. MEDIA PREPARATION, INOCULATION, INCUBATION
4.2.1. MEDIA PREPARATION
4.2.1.1. NUTRIENT AGAR MEDIA FOR BACTERIA
Nutrient Agar (Himedia) Powder was weighed in a conical flask and properly mixed by
dissolving it in 100 ml distilled water. The pH of the media was adjusted to near nutrients.
Conical flask Conical flask was covered properly with the help of a cotton plug and autoclaved
at 121°C for 15 min. Nutrient agar media was poured in each of the sterile petri plates inside
the laminar airflow hood. About 20ml of agar was poured on each plate. The plates were
allowed to set for inoculation.
4.2.1.2. POTATO DEXTROSE AGAR MEDIA FOR FUNGUS
Potato dextrose Agar Powder (Himedia) was weighed in a conical flask and properly mixed by
dissolving it in 100 ml distilled water. The pH of the media was set near to 7. Conical flask
was covered properly with the help of a cotton plug and autoclaved at 121°C for 15 min. Potato
dextrose media was poured in each of the sterile petri plates inside the laminar airflow hood.
About 20ml of Agar was poured in each plate. The plates were allowed to set for incubation.
29
4.2.1.3. YEAST MANNITOL AGAR MEDIUM FOR AGROBACTERIUM
TUMEFACIENS
Yeast mannitol Agar Powder was weighed in a conical flask and properly mixed by dissolving
it in 100 ml distilled water. Conical flask was covered properly with the help of a cotton plug
and autoclaved at 121°C for 15 min. Once the nutrient agar has been autoclaved, allow it to
cool but not solidify.Yeast mannitol agar media was poured in each of the sterile petri plates
inside the laminar airflow hood. About 20ml of agar was poured on each plate. The plates were
allowed to set for incubation.
4.2.1.4. ZOBEL MARINE AGAR MEDIUM FOR MARINE BACTERIA
Zobel marine Agar Powder (Himedia) was weighed in a conical flask and properly mixed by
dissolving it in 100 ml distilled water. The was pH adjusted to 7. Conical flask was covered
properly with the help of a cotton plug and autoclaved at 121°C for 15 min. Zobel agar media
was poured in each of the sterile petri plates inside the laminar airflow hood. About 20ml of
agar was poured on each plate. The plates were allowed to set for incubation.
4.2.1.5. GLUCOSE AGAR MEDIUM FOR YEAST
Glucose Agar Powder was weighed in a conical flask and properly mixed by dissolving it in
100 ml distilled water. The was pH adjusted to 7. Conical flask was covered properly with the
help of a cotton plug and autoclaved at 121°C for 15 min. Yeast mannitol agar media was
poured in each of the sterile petri plates inside the laminar airflow hood. About 20ml of agar
was poured on each plate. The plates were allowed to set for incubation
4.2.1.6. MINERAL NUTREIENT MEDIA FOR CYANOBACTERIA
MN Agar (Himedia) was weighed in a conical flask and properly mixed by dissolving it in 100
ml distilled water. Conical flask was covered properly with the help of a cotton plug and
autoclaved at 121°C for 15 min. Once the MN agar has been autoclaved, allow it to cool and
then trace metal mix was added to the media. MN agar media was poured in each of the sterile
petri plates inside the laminar airflow hood. About 20ml of agar was poured on each plate.
30
4.2.1.7. NMS MEDIA FOR METHANOBACTERIA
NMS Agar was weighed in a conical flask and properly mixed by dissolving it in 100 ml
distilled water. The was pH adjusted to near neutral. Conical flask was covered properly with
the help of a cotton plug and autoclaved at 121°C for 15 min. Once the NMS agar has been
autoclaved, allow it to cool and then trace metal mix and iron solution was added to the media.
NMS agar media was poured in each of the sterile petri plates inside the laminar airflow hood.
About 20ml of agar was poured on each plate. The plates were allowed to set for incubation.
The plates were incubated in methane environment.
4.2.3. STREAK PLATE METHOD
Quardrant streak plate method was used to purify the bacteria. Using a sterile inoculation loop,
a suspension of bacterial culture was first made as a small spot in the fresh nutrient plate.
From that spot 4- 5 streaks were made as a straight line. Again the loop was sterilized and
from each end of the line, streaking was made from it. Similarly the streaking was made
covering four sides of the plate. The plates were incubated at 37oC for 24 hours incubation.
After incubation, the plates were observed for the isolated colonies.
4.2.4. PURE CULTURE OF MOLDS
Sterilize the forceps and let it cool in air. Place a mold collectedin the forceps at the centre of
the potato dextrose agar plate. Incubate the plate at 25-(Boyle et al., 2003).
4.2.5. PRESERVATION TECHNIQUE
4.2.5.1. MAINTENANCE OF PURIFIED MICROBIAL CULTURES
Selected strains were stored at -80°C in glycerol stock. For preparation of the glycerol stock,
(50%) 10ml of glycerol in 10ml of distilled water. This solution (1:1 ratio) was prepared in
sterile Test tube and autoclaved at 121°C for 15 min. 1ml or 2ml of 50% glycerol solution
was collected in an eppendorf tube and loopful of bacteria, mold, yeast, and microalgae
cultures was added and stored. For further experiments, the strains were revived in the growth
medium.
31
4.2.5.2. RE-VIABILITY TEST
The glycerol stocks previously was stored was then taken and used for re-viability test. The
inoculation loop was sterilized in Bunsen burner flame. A loop of culture was then picked and
streaked in parallel as the first quadrant part. The loop was then flamed gently, cooled and
then streaked from the end of first streaking. Again the loop was flamed gently, cooled and
streaked from the end of 2nd streaking. The same step was done again. Take note that it
should not touch thestarting point of first streaking. The plate was then sealed and kept for
incubation.
4.2.6. GRAM STAINING
4.2.6.1. Procedure
Smear preparation
Slides were properly cleaned with the help of ethanol so that any kind of dirt is removed. One
drop of distilled water was placed on the slide at the center. Bacterial colonies of each isolate
were picked from the plate with the help of a sterilized loop and dispersed well into the water.
In order to prepare a thin smear, the emulsion was spread evenly over the center of the slides.
They were further allowed to dry completely and the slides were repeatedly passed over the
flame to heat fix the bacterial isolates.
Staining procedure
The crystal violet was poured on the slides over the smear and allowed to stay for one minute.
The stain was washed with water and the excess water was drained off. Now, Gram's
iodine solution is added on the smear and slides kept aside for one minute. The stained slides
were washed with distilled water and for decolorization 95% ethyl alcohol was used for about
30s, the slides were thoroughly agitated in the alcohol till no color came out from the
smear. Safranin was used as counterstain for 20s. The slides were further washed with
distilled water for a few seconds and allowed to dry at room temperature. All slides were
examined under oil immersion microscope at 100X.
32
4.2.7. SPORE STAINING
Slides were properly cleaned with the help of ethanol so that any kind of dirt is removed. One
drop of distilled water was placed on the slide at the center. Bacterial colonies of each isolate
were picked from the plate with the help of a sterilized loop and dispersed well into the water.
In order to prepare a thin smear, the emulsion was spread evenly over the center of the slides. .
They were further allowed to dry completely and the slides were repeatedly passed over the
flame to heat fix the bacterial isolates. The malachite green solution was added on the slide. .
Steam for 3-6 minutes, and rinse under running tap water Safranin was used as counterstain for
30s. The slides were further washed with distilled water for a few seconds and allowed to dry
at room temperature. All slides were examined under oil immersion microscope at 100X.
4.2.8. LACTOPHENOL COTTON BLUE STAINING
A clean glass slide was taken sterilize with 70% ethanol. few drop of lactophenol cotton blue
dye was added on a slide with the help of a dropper. loofull of f7ungi with the help of needle
and forceps both from periphery of colony and from center; do not pick excess culture. Spread
the culture in drops of lactophenol cotton blue (Leck A 1999). Place the needle at 30 degrees
to the surface of the slide. Pick up the coverslip with the help of forceps from the other hand,
and put it over the needle. Slowly move the needle out of stain such that the coverslip touches
the dye and no air bubbles form. Examine slide under the microscope 40x and oil immersion
100x magnification.
4.2.9. METHYLENE BLUE STAINING
A clean glass slide was taken sterilize with 70% ethanol . One drop of methylene blue dye was
added on a slide with the help of a dropper . A quarter loops of cells were taken from an
isolated colony and smeared from the center of the droplet . The slide washed under tap water.
Blot dried with tissue paper. The slide was observed under oil immersion 100x magnification.
4.2.10. IDENTIFICATION OF MICROALGAE &CYANOBACTERIA
Isolated colonies were observed in a microscope for morphological characterization.
Identification of Blue green algae Microscopic observation was done by spreading isolated
culture on glass slide using forceps. Culture were covered with glass cover slips and observed
33
under low (10X) and high power (100X) objective lens of compound light microscope.
4.2.6. MOTILITY BY HANGING DROP METHOD
A clean coverslip was held by its edges and vaseline was carefully dabbed on its corners using
a toothpick. Each bacterial isolate culture was placed in the center of prepared coverslip with
the help of a loop. A clean concavity slide was turned upside down (Concavity down) over the
drop on the coverslip so that the vaseline seals the coverslip to the slide around the concavity.
The slide was turned over so that the coverslip was on top and the drop could be observed
hanging from the coverslip over the concavity. The slide preparation was placed under the
microscope (100X) for examination.
4.2.7. BIOCHEMICAL CHARACTERIZATION OF THE BACTERIAL ISOLATES
Biochemical test for all the isolates performed, following the protocols given in
Manual of Determinative Bacteriology (Holt et al., 1994)
4.2.7.1 CATALASE TEST
SLIDE METHOD
Using a sterile inoculation loop or Pasteur pipette, few drops of bacterial culture were placed
on a clean, dry glass slide. Over the sample,2-3 drops of 3% hydrogen peroxide solution
(H2O2) was treated and mixed well. The evolution of oxygen bubbles was observed.
TUBE METHOD
1-2 ml of hydrogen peroxide solution was taken in a test tube. Using a sterile inoculation
loop, an 18 to 24 hours test organism was inoculated in the hydrogen peroxide solution. The
formationof bubbles was observed.
4.2.7.2. OXIDASE TEXT
A clean glass slide was taken and an aseptically oxidase disc was placed. Over the disc
aseptically few drops of bacterial culture was placed. The change in the color of the disc from
white to violet was observed. The change in the color indicates oxidase positive.
34
4.2.7.3. INDOLE TEST
About 5ml of Tryptone broth was prepared and transferred to test tubes, sterilized by
autoclaving at 121°C for 15 minutes. After sterilization, tubes were cooled, then the test
cultures were inoculated to the respective test tubes except control tube. All the tubes were
incubated 24 hours
the formation of a red layer at the top of the culture was observed indicating the positive
reaction.
4.2.7.4. METHYL RED TEST
The broth was prepared by mixing all the contents and the sterilization process. 5 mL MR VP
medium was transferred to the test tubes and sterilized by autoclaving at 121°C for 15 minutes.
After sterilization, the tubes were inoculated with the test culture (except for the control
tube). All tubes were incubated at 37°C for 24 hrs. After incubation, 3-4 drops of methyl red
indicator was added into each tube. A distinct red color indicates the positive test; yellow color
indicates a negative test.
4.2.7.5. VOGES PROSKAUER METHOD
About 5ml MR VP medium was transferred to the test tubes and sterilized by autoclaving at
121°C for 15 minutes. After sterilization, the tubes were inoculated with the test culture
(except for the control tube). All tubes were incubated at 37°C for 24 hrs. After incubation, 0.5
ml of A
pink color formation indicates the positive test; yellow color indicates a negative test
4.2.7. 6. CITRATE TEST
Simmons Citrate agar was prepared and sterilized at 121°C for 15 minutes. After sterilization ,
the media was cooled and transferred to test tube forming slants. After solidification, test
tubes were inoculated with the bacterial culture except the control tube. The tubes were
incubated at 37°C for 2448 hrs. After incubation, the color change in the media from green to
blue is regarded as positive.
35
4.3. ANTIBIOTIC SENSITIVITY ASSAY
Preparation of inoculum in broth
Nutrient broth was prepared in sterile Test tubes and properly mixed by dissolving it in 10 ml
distilled water. Test tube was covered properly with the help of a cotton plug and autoclaved at
121°C for 15 min. Once the nutrient broth has been autoclaved, allow it to cool but not
solidify. The pure isolated colonies were inoculated in the autoclave nutrient broth. The broth
was incubated 24hrs
Preparation of Muller Hinton plate
Muller-Hinton Agar Powder was weighed in a conical flask and properly mixed by dissolving
it in 100 ml distilled water. Conical flask was covered properly with the help of a cotton plug
and autoclaved at 121°C for 15 min. Once the Muller-Hinton agar has been autoclaved, allow
it to cool but not solidify. Mueller hinton agar media was poured in each of the sterile petri
plates inside laminar air flow hood. About 20ml of agar was poured on each plate. The plates
were allowed to set.
Inoculation in the MH plate
A sterile cotton swab was dipped into the known concentration of bacterial inoculums and
excess medium was removed by pressing the swab onto the wall of the tube. The surface areas
of the Muller-Hinton agar plates were swabbed completely by rotating the plate to produce
lawn culture. The plates were allowed to dry for 2 mints for the proper absorption of the
inoculums.
Placement of the antibiotic discs
The forceps was sterilized with alcohol before picking up antibiotic disks. The antibiotic disks
were placed at a distance of about 24mm from each other. Each disk was slightly pressed with
forceps to ensure that it is in good contact to avoid misplacement. The plates were incubated
upside down for 24 hours ( Murray et al., 1995).
4.4. PROTEIN FINGERPRINTING USING SDS PAGE
SDS-PAGE
Completely dry and clean glass plates, combs, and spacers were taken. Gel cassette was
36
assembled by using Paraffin wax. Gloves were used while preparing the gel.Separating gel
was prepared. TEMED was added in the last, mixed well and the gel solution was quickly
transferred to the casting chamber between the glass plates with the help of 1ml pipette and
filled up to a required level, leaving an area of approximately 1cm from the top where the
comb was to be positioned.To straighten the layer of gel and to remove any air bubble, a small
layer of isopropanol is added at the top of the gel. After approximately 30 minutes, when the
gel has polymerized, this layer of isopropanol was completely removed with the help of filter
paper.Stacking gel was prepared. TEMED was added and the solution was mixed well. This
stacking gel solution was then quickly transferred by using a 1 ml pipette until the space was
completely full. The comb was then inserted appropriately. Once the top was solidified, the
comb was removed carefully.
Sample preparation
About10 of the protein sample was added to 10 of 2 x protein sample buffer, mixed and
kept in a boiling water bath at 95°C for 10 min. The sample was kept at room temperature until
the gel is ready for loading the sample.
Gel electrophoresis
The gel cassette was removed from the casting stand and placed in the electrode assembly
keeping the face of the short plate towards the inner side.The electrode assembly was pressed
down while clamping the frame to secure the electrode assembly and the clamping frame was
put into the electrophoresis tank.1xelectrophoresis running buffer was poured into the opening
of the casting frame between the gel cassettes to fill the wells of the gel. The outside region of
the frame was also filled with a 1x running buffer.The prepared protein sample was slowly
loaded into each well. protein MW marker was also loaded. The lid was
closed and the electrophoresis tank was connected to the power supply. 30mA current was
allowed to pass through it for approximately 3.5 hours.
Protein band detection
The gel was stained with Coomassie staining solution , which contains methanol, acetic acid,
and Coomassie brilliant blue R250 dye. After overnight staining, the gel was de-stained with a
destaining solution, containing methanol and acetic acid mixture to visualize the protein bands
37
only.
4.5. ENZYME SCREENING
4.5.1. AMYLASE
An inoculum from a pure culture was taken by using an inoculation loop and it was streaked
on a sterile plate of starch agar under the laminar airflow hood. The inoculated plate was
incubated at 37°C for 24-48 hr. Iodine reagent was then added to flood the growth. The test
result was recorded.
4.5.2. LIPASE
Phenol red agar was prepared to adjust Ph to 7. 4 in distilled water, sterilized for 15 min by
autoclaving with 15 lb pressure at 121 °C and cooled to 60°C. 10 ml/L phenol red dye (1
mg/ml) and 10ml/L substrate (coconut oil) were added. Aliquots were transferred to Petri
dishes and allowed to solidify. A loopful of each pure culture was streaked onto phenol red
agar and incubated at 37 °C For 48h. After incubation, plates were analyzed for the change of
color. This indicated the release of fatty acids due to lipolysis.
4.5.3. CASEINASE
Skim milk agar was prepared . All the contents were mixed and autoclaved to make sterile
media. Plates were poured to allow setting properly. A single streak of the sample strain
was made by the inoculation loop on a skim milk agar plate from the pure culture.
Plateswere incubated at 37°C for 24-48 hr. Test result was then noted.
4.6. DYE DEGRADATION
4.6.1. DYE DEGRADATION:BACTERIA
The culture to be tested for dye degradation was revived from glycerol stock and inoculated
into 10 ml nutrient broth.Following incubation for 24hrs at 37ºC, 0. 2ml of culture was
transferred into 100 ml of decolorization broth (glucose 10g/L, yeast extract 5g/L, NaCl
5g/L) and incubated in shaker incubator at 37ºC for 6hrs (until log phase of bacterial
growth).After 6hrs, the cells were harvested by centrifugation at 3000g for 5 min.Following
38
centrifugation, supernatant was discarded and the cells were suspended in 10mL of 0.8%
NaCl solution.When required the OD was adjusted to ~0.6 to 0.8 (~6x 108 cells/ml) at 600
nm using 0.8% NaCl solution.200 µl of bacterial cell suspension were inoculated into fresh
decolorization broth with different concentrations (1% to 20%) of congo red dye (20 ml
each) and incubated at 37ºC for 48 hrs against a negative control (without bacterial
suspension). 5.2 ml of the solution was withdrawn at the interval of 3 hrs and centrifuged at
12000 rpm for 1 minute.
4.6.2. DYE DEGRADATION:FUNGI
The culture to be tested for dye degradation was revived from glycerol stock and inoculated
into 10 ml Potato dextrose broth.Following incubation for 24hrs at 30ºC, 2ml of culture was
transferred into 100 ml of decolorization broth (glucose 10g/L, yeast extract 5g/L, NaCl 5g/L)
and incubated in shaker incubator at 30ºC for 12hrs (until log phase of fungal growth).After
sterilization, broth was brought to the laminar air flow chamber.After 6hrs, the cells were
harvested by centrifugation at 3000g for 5 min.Following centrifugation, supernatant was
discarded and the cells were suspended in 10mL of 0.8% NaCl solution.When required the
OD was adjusted to ~0.6 to 0.8 (~6x 108 cells/ml) at 600 nm using 0.8% NaCl solution.1ml of
fungal suspension were inoculated into fresh decolorization broth with different concentrations
(1% to 20%) of congo red dye (20 ml each) and incubated at 37ºC for 48 hrs against a negative
control (without bacterial suspension). 5.2 ml of the solution was withdrawn at the interval of
3 hrs and centrifuged at 12000 rpm for 1 minute.The cell free supernatant was measured at
490 nm using UV-Vis spectrophotometer against the control.Decolorization percentage was
calculated using the formula: Decolorization % = (initial absorbance final absorbance) /
initial absorbance x 100 10. Control and decolorized solution were further subjected to spectral
analysis where the decolorized solution was screened at 200 700 nm in UV
spectrophotometer and the spectral peaks were cross-matched for comparison.
4.7. MOLECULAR CHARACTERIZATION
4.7.1. DNA ISOLATION
4.7.1.1. DNA ISOLATION OF BACTERIA
For bacterial isolates from solid media, A loop full of bacterial colony was dispensed into 1ml
of phosphate buffer saline (PBS) (FOR 100 ml volume : 300 mM Nacl (1.75g), 2. 7 mM KCL
39
(0.02g), 10 mM Na2HP04 (0.14g), 1.7 mM KH2PO4 (0.022G), pH 7. 4) in 1. 5 ml centrifuge
tube under clean room condition. The culture in PBS was well agitated or flash spinned to
dispense most of the bacterial cells into the PBS solution. The inoculation loop was then flame
sterilized and kept aside.1. 5ml tube was closed and inverted several times for dispensing the
bacterial cells and centrifuged at 6000 g for 5 minutes. For broth cultures, 1. 5ml of culture
broth was taken in the 1. 5ml centrifuge tube and centrifuged at 6000 g for 5 minutes. The
supernatant was carefully discarded and 600 ml of lysis buffer (for 500 ml volume:50 mM
Tris-HCL (3.028g), 20mM EDTA (2.922g). 2% SDS (10g),pH 8 ) was added to the pellet. The
vortexed foe 30
seconds every 10 minutes throughout the water, both incubating an equal volume (600 ml) of
phenol chloroform Isoamyl alcohol (PCI) (25:24:1) reagent was added and gently mixed by
inverting the tube. The tubes were centrifuged at 6000rpm for 10 minutes and the supernatant
was transferred to a fresh 1.5 ml tube. Equel volume of 100% ethanol was added and DNA
was precipitated (overnight incubation at- could be followed after ethanol addition for
enhanced precipitation). Tubes were centrifuged at 10000 rpm for 10 minutes and the pellets
were air drie. 20- 50ml of TE buffer (for 100ml TE buffer: 1M Tris HCl (0.121g), 0.5M
EDTA(0.02g), (pH8) was added and the DNA was stored at
4.7.1.2. DNA ISOLATION OF FUNGI
Fungal cultures were grown on PDA plates for 5-20 days and total DNA was extracted from
fresh mycelium. The Mycelium was directly scraped off from culture plates and transferred
into a 1.5 µl centrifuge tube. Mycelium was mixed with 0. 2 g of sterile white quartz sand
and 600 µl of preheated (60°C) 2X CTAB buffer [2% v/w CTAB, 100 mM Tris-HCl, 1.4 M
NaCl, 20 mM EDTA, pH 8. The Mycelium was ground with a plastic pestle for 5-10 min and
incubated at 60 °C for 40 min with occasional gentle swirling every 10 min. Then 600 µl of
phenol:chloroform (1:1) was added into each tube and gently mixed. The mixture was
centrifuge at 13000 rpm for 30 min and the aqueous extraction layer was transferred into a new
1.5ml tube. Phenol:chloroform (1:1) extraction was repeated 2-3 times until no interface was
visible. Two volumes of 100% cold ethanol were added and the tube was inverted gently and
stored overnight at -20 °C to precipitate DNA. Contents were then centrifuged at 11000 rpm
for 30 min at 4 °C. The DNA pellet obtained was washed with 70% cold ethanol twice and
dried using. The pellet was suspended in 100 µl of TE buffer [10 mM Tris-HCl, 1 mM EDTA,
40
pH 8] containing 20 µg/ml RNase. DNA samples were checked for purity by electrophoresis in
1% (w/v) agarose stained with ethidium bromide (10 mg/ml).
4.7.2. AGAROSE GEL ELECTROPHORESIS:
The amplified products were recognized by agarose gel electrophoresis. Electrophoresis was
carried out by preparing 0. 8% agarose gel (Sigma Aldrich). For this 0.8 g of agarose was
mixed in 100 ml of 1X TAE buffer (40 mM Tris-acetate, 1 mM ethylene diamine tetra acetic
acid (EDTA) (SRL); pH 7. 4) was boiled till it become transparent. The boiling liquid agarose
was cooled down and then 4 µl of Ethidium Bromide (EtBr) was added. After mixing it
properly, the mixture was poured into a gel casting tray. After the solidification of gel the
combs were removed and the gel was placed into an electrophoresis unit containing 1X TAE
buffer. 2 µl of DNA sample was mixed with 2 µl of 6X loading dye (Thermo Scientific).
Mixture was loaded into agarose gel. 3 µl of 1kb DNA ladder (Invitrogen) was also added in
the first well of the agarose gel. Assembly was connected to a power supply that ran at 100 V
for 1 hour. Gel was observed under UV in Gel doc.
4.7.3. POLYMERASE CHAIN REACTION
4.7.3.1. PCR- 16S rRNA gene
16S rRNA genes of the bacterial isolates were amplified using a set of bacterial universal
forward primer 8-27F (5'-AGAGTTTGATCCTGGCTCAG- erse primer 1492R
(5'- TACACCTTGGCGACGACT T- (Turner et al., 1999). For polymerase chain
reaction
polymerase buffer , 1 of 10 mM deoxynucleotides mix , 0.2 of each forward and
reverse primer, 1.5U Taq DNA polymerase and 30 ng of genomic DNA. PCR amplification
was carried out using Applied Biosystem Veriti 96 well Thermal cycler. The PCR program
involved an initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at
95°C for 1 min, annealing at 54°C for 45s, primer extension at 7C for 1min for 35 cycles
with a final extension step at 72°C for 10 min.
4.7.3.2. PCR- ITS gene
PCR amplification Primer pair ITS4 and ITS5 (White et al. , 1990) were used to amplify the
5. 8S gene and flanking ITS1 and ITS2 regions. Amplification was performed in a 50 µl
reaction volume containing 5 µl of 10X Mg free PCR buffer, 3 µl of 25 mm mgcl2, 4 µl of
41
2.5 mm deoxyribonucleotide triphosphate (dntps), 171 µl of 10 µm primers (ITS4 and
ITS5)ITS1:TCCGTAGGTGAACCTGCGG,ITS4: TCCTCCGCTTATTGATATGC, 3 µl of
DNA Template,0.3 µl of 2.5 units of Taq DNA polymerase. The thermal cycle 30 cycles with
an initial denaturation at 95°C for 5 min, cyclic denaturation at 95°C for 30 s, annealing at
60°C For 40 s, and extension at 72°C for 1 min with the final extension of 7 min at 72°C.
The PCR products were examined by electrophoresis in 1% (w/v) agarose gel with Ethidium
bromide (10 mg/ml) and check for size and purity.
4.7.5. DNA SEQUENCING
4.7.3.3. 16S rRNA SEQUENCING
16S rDNA gene sequencing is a powerful tool that has been used to trace phylogenetic
relationships between bacteria, and to identify bacteria from various sources, such as
environmental or clinical specimens. 16S rDNA sequencing is also used as a method of
detecting pathogens in normally sterile clinical specimens, or for detecting species that
cannot be cultured. Other authors have also reported its use as a tool for bacterial
identification.The purified 16S rRNA gene was used for sequencing. It includes 4 steps: PCR
(84 min), PCR clean up and cycle sequencing (90 min.), sample purification (40 min.) and
capillary electrophoresis (75 min)
Methodology
The amplifying of the template DNA with PCR was and two tubes were taken in which 10
PCR master mix was added. The tubes were placed in a thermal cycler and
performed PCR for 64 min.The sequencing reaction was prepared by adding of
sequencing master mix to both the tubes. of the forward primer was added to one tube
and l of the reverse primer in the other tube. Sequencing was performed by putting the
tubes in a thermal cycler for 80 min.Purification reagents from Big dye x terminator kit
were added into both the tubes and vortexed for 20 min. The cycle sequenced DNA
sample was then analyzed by Genetic analyzer system. The sample was run in capillary
electrophoresis. In the CE sequencing machine, the DNA fragments are passed through a
long and thin capillary of acrylic fiber instead of gel electrophoresis.The capillary containing the
DNA fragment is dipped in an electrode and applied with the electric current, which
42
causes the movement of DNA fragments towards the end of the capillary. A fluorescence
detector laser present in the machine shoots through the capillary fiber. When it falls on the
DNA fragment which has different colored base terminators moving in the capillary, they
show fluorescence. (A-Green, T-Red, G- Yellow, C-Blue). The fluorescence is captured by a
charged coupling device and recorded by the machine and displayed on the computer in the
form of graphical peaks of different colors. It is a fast method and gives a greater resolution.
The data showing peaks was viewed with the help of user-
friendly free online software for viewing the chromatograms obtained from automated
sequencers.
4.7.6.2. ITS SEQUENCING
The purified PCR products were directly sequenced in an automated sequencer, Primer
pair ITS4 and ITS5 (White et al., 1990) were used in the sequencing reaction. DNA
sequencing was commercially out sourced to Barcode Biotechnologies Private Limited,
Vasco DaGamma, Goa, India.
4.8. BIOINFORMATICS ANALYSIS
4.8.1. CHROMOTOGRAM ANALYSIS
Automated DNA Sequencers generate a four-color chromatogram showing the results of the
sequencing run, as well as a computer program's best guess at interpreting that data --- a text
file of sequence data. That computer program, however, does make mistakes and it is the
responsibility to manually double-check the interpretation of the primary data.
Predictable errors occur near the beginning and again at the end of any sequencing run. Other
errors can show up inthe middle, invalidating individual base calls or entire swaths of data
4.8.2. BLAST ANALYSIS
The Isolated bacteria, actinobacteria, and Molds,Yeast, Microalgae were sent to sequences of
16S rDNA of bacteria, actinomycetes and 18S rDNA of fungal isolate were obtained and
compared with similar sequences were obtained from GenBank, compared using the BLAST
program (Altschul et al. , 1990).
4.8.3. GENETIC DISTANCE ANALYSIS
43
Genetic distance is a genetic divergence measurement between either species or populations
within a species. For autosomal DNA comparisons, genetic distance refers to the length of
the shared DNA segment in centiMorgans (cM)]. A centiMorgan (also genetic map unit
(mu)is a unit of measure used to approximate genetic distance along chromosomes. Genetic
distance analysis is a powerful tool for assorting the unknown genotypes to the groups within
the germplasm. (e. g. , heterotic groups in maize) (Lübberstedt et al. , 2000)
4.8.4. PHYLOGENY
Phylogenetic analysis of DNA or protein sequences has become an important tool for
studying the evolutionary history of organisms from bacteria to humans.
NEIGHBOR JOINING (NJ) METHOD
This method is a simplified version of the ME method for inferring a bifurcating tree. In this
method, the S value is not computed for all or many different topologies, but the examination
of different topologies is embedded in the algorithm, so that only one final tree is produced.
FELSENSTEIN’S BOOTSTRAP TEST
One of the most commonly used tests of the reliability of an inferred
bootstrap test. In this test, the reliability of an inferred tree is examined by using E
bootstrap resampling technique. A set of nucleotide sites is randomly sampled with
replacement from the original set, and this random set is used for constructing a new
phylogenetic tree. This process is repeated many times, and the proportion of replications in
which a given sequence cluster appears is computed.
MEGA
The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain
a large collection of methods and tools of computational molecular evolution. Here, we
describe new additions that make MEGA a more comprehensive tool for building time
trees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods
for estimating divergence times and confidence intervals are implemented to use
probability densities for calibration constraints for node-dating and sequence sampling
dates for tip-dating analyses.
44
CLUSTALX
Clustal X were the most widely used multiple alignment programs. They were able to
align medium-sized data sets very quickly and were easy to use. The alignments were of
sufficient quality not to require manual editing or adjustment very often. This situation
changed greatly with the appearance of the first custom made benchmark test set for
multiple alignment programs (Larkin et al. , 2007).
4.15.1. METHODOLOGY
BLAST
Access the NCBI home page by clicking the above URL. Paste the query sequence in the
workspace of the blast homepage.Run the blast program with default parameters. To collect and
download the similar sequence of a particular organism using the BLAST tool. Retrieve the
several nucleic acid sequences from the NCBI Genbank database in the FASTA format.
Download and install ClustalX on your local machine . To Load sequences in ClustalX. Select
Load Sequences from the File menu in the ClustalWwindow.After Selecting the file containing
the unaligned sequences. highlight the filename in the file selection window with the cursor and
then click the OK button at bottom of window By default, the output file where the program is
produced is in the Clustal format of clustal x which can be further used for the many other
sequence-analysis packages.
MEGA
Open the MEGA software and upload the downloaded file from the ToolEdit or build alignment
option is used to align the sequenceThen the downloaded sequences were pasted on the MEGA
format by exporting the alignment data.The neighbor joining method is adapted to construct the
phylogenetic tree by evaluating tree topologies and also by performing bootstrap analysis and
the kimura-2 parameter method.Phylogenetic tree were constructed. It is the best method to
find the evolutionary distance ofthe organism.
4.9. CONSTRUCTION OF CULTURE COLLECTION DATABASE
The Ncccc database contains information from a variety of sources:Information provided by
culture collection staff . Data from public data sources such as the US National Library of
45
Medicine (PubMed) and the Patent database. Links to external databases. Tools for
bioinformatics analysis including a search engine to enhance exploration of Ncccc data. By the
end of August 2022, the Ncccc contains strain information from 100 collections (Table 1)
located in National College, trichy. While the project is still in its construction phase,
preliminary statistics describing the participating collections are unique and informative Since
ncccc maintains nucleotide sequences data associated with individual strains, a sequence
alignment tool based on the Basic Local Alignment Search Tool (BLASTN) is included.
Results are ordered by similarity. Search results are listed as strain numbers, strain names,
publication abstracts and titles and can be exported in text file format. With the advanced
search tools, the system can perform the following searches
Searching for type strains for some taxa in certain culture collections
Searching for strains with specific characteristics in the list of Culture
Collection, such as range of growth temperature, transfer history, collected
location and others
Searching for strains with specific properties
Searching strains isolated from various substrates, including sludge or
wastewater, soils, sediment, fermentation products. Results are listed in table
format, with the types of organism type used as column name.
46
5. Results
5. RESULTS
5.1. SAMPLE COLLECTION
Among various environments explored for the sample collection, most of the samples (>15%)
were contributed from the marine environment (fig. 1). Clinical samplings were kept minimal to
reduce the opportunistic isolation of risk group 3 & 4 organisms. Based on the nature of the
sample, sample processing protocols were optimized.
Figure 1: Pie chart representing diversity of samples used for building the culture
collection.
47
5.2. SPREAD PLATE METHOD
Spread plate method was adopted for samples like soil, waste water, etc. For
samples of higher bacterial load (such as seawage and agriculture soil), serial
dilution was introduced before spread plating. Also nature of the media was varied based
on the target organism of isolation.
Figure 2: Mother cultures observed following spread plate. Absence of growth in the control
plate and mixed colonies in mother plate was considered as the organisms isolated from the
target samples.
48
5. 3. PURE CULTURE METHOD:
Pure culture method was done by quadrant streak plate technique. Usually, the target
colonies in the mother plate was marked and targeted. In order to purify from mother plate, same
media was prepared and the target colony was quadrant streaked into the plated and incubated at
same conditions. Purity of the colony was confirmed following the observation of single type of
colony (closely resembling to mother plate) in the last quadrant of the plate, same conditions.
Figure 3: Purification of target colonies using quadrant plate.
49
Figure 4: Purification of yeast colonies by quadrant streak technique on glucose yeast mannitol
agar medium
Figure 5: Purification of molds in potato dextrose plate.
50
Figure 6: Purification of cyanobacteria NCT201 (Chlorella vulgaris), NCT65
(Synechococcus elongatus) on MN Agar medium
Figure 7: Three flask containing pure culture of NCT201 (Chlorella vulgaris), NCT65
(Synechococcus elongatus), NCT 51 (Tenebriella curviceps)
5.4. GRAM STAINING
Gram stain was performed to study the cell wall composition of the bacterial isolates (fig. 8).
With the help of Gram stain, bacteria were classified into Gram Positive Bacteria (relatively
less lipopolysaccharides) and Gram Negative Bacteria.
51
Figure 8: Slide pictures of various Gram positive and negative strains of NCCCC.
5.5. SPORE STAINING
Spore staining was performed to study the endospore of bacterial isolates using malachite green and
saffraine stains. Spores are stained green were as vegetative cells are stained pink (fig. 9).
52
Figure 9: Microscopic examination of spore forming bacteria
5. 6. LACTOPHENOL COTTON BLUE STAINING
Lactophenol cotton blue staining was performed to study the morphology of fungal isolates.
Mycelial structures, hyphal filaments, sporulating structures and spores were stained and observed
(fig. 10).
Figure 10: Microscopic examination of fungal isolates
53
5.7. METHYLENE BLUE STAINING
Methylene blue staining was performed to study the morphology of yeast isolates (fig. 11).
Figure 11: Microscopic examination of yeast isolates
5.8. IDENTIFICATION OF CYANOBACTERIA AND MICROALGAE
Microscopic observation was done by spreading isolated culture on glass slides using forceps.
Cultures were covered with glass cover slips and observed under low (10X) and high power
(45X) objective lens of compound light microscope (fig. 12).
Figure 12: Microscopic examination of NCT51 (Tenebriella curviceps), NCT65
(Synechococcus elongatus ), NCT201 (Chlorella vulgaris)
5.9. PRESERVATION OF MICROBIAL CULTURES
The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes
and keeping the cells alive (fig. 13). A glycerol stock of bacteria can be stored stably at -80°C for
many years.
54
Fig 13:The microbial culture glycerol stocks were stored in -
5.10. BIOCHEMICAL CHARACTERIZATION OF THE BACTERIAL ISOLATES
5.10.1. CALATASE TEST
Figure 14: The Negative result NCT34 (Enterococcus faecalis) is indicated by the catalase
negative. The positive result NCT45 is indicated by catalase negative.
5.10.2. OXIDASE TEST
Figure 15: The Positive result NCT25 is indicated by the Oxidase positive. The Negative result
55
NCT38 is indicated by Oxidase negative.
5.10.3. IMVIC TEST
IMVIC Test was done on the basis of following characteristics such as Indole test, Methyl red
test, Voges-proskauer (VP) test, Citrate test.
5.10.3.1. INDOLE TEST
Figure 16: The positive result (A) is indicated by the red layer at the top of the tube after the
addition of Kovacs reagent. The negative result (B) is indicated by the lack of color change at the
top of the tube after the addition of Kovacs reagent.
5.10.3.2. METHYL RED TEST
Figure 17: Positive methyl red test (A) is indicated by the development of red color after the
addition of methyl red reagent. The negative methyl red test (B) is indicated by no color change
56
after the addition of methyl red reagent.
5.10.3.3. VOGES-PROSKAUER(VP) TEST
Figure 18: A positive Voges-Proskauer test (A) is indicated by the development of red- brown
color after th A negative VP test (B) is indicated
reagents.
5.10.3.4. CITRATE UTILIZATION TEST
Figure 19: A positive citrate result (A) is indicated by growth and a blue color change in the
tube. A Negative citrate utilization test (B) is indicated by the lack of growth and color change in
the tube
57
Table 1: Biochemical test for identification of the various bacterial isolates colleted
STRAIN
NAME
SPECIES NAME
CATA
LASE
OXIDA
SE
IND
OLE
M R
VP
CITRA
TE
NCT1
Escherichia coli
+
-
+
+
-
-
NCT4
Bacillus mycoides
+
-
-
+
V
NCT11
Bacillus cereus
+
-
-
-
+
+
NCT24
Micrococcus luteus
+
-
+
-
NCT25
Vibrio cholera
+
+
-
V
+
NCT32
Pseudomonas mendocina
+
+
-
+
NCT33
Shigella sonnei
-
-
-
-
NCT34
Enterococcus faecalis
-
-
-
-
+
-
NCT38
Citrobacter koseri
+
-
+
+
-
+
NCT40
Klebsiella pneumonia
+
-
-
-
+
+
NCT41
Proteus vulgaris
+
-
+
+
-
-
NCT43
Enterobacter aerogenes
+
-
-
-
+
+
NCT45
Staphylococcus aureus
+
-
-
+
+
+
NCT91
Streptococcus thoraltensis
+
NCT94
Serratia fonticola
+
-
-
+
-
+
NCT99
Bacillus subtilis
+
V
-
-
+
+
NCT45
Staphylococcus aureus
+
-
-
+
-
-
58
NCT108
Staphylococcus epidermis
+
-
-
-
+
-
NCT112
Streptococcus mitis
+
NCT114
Pseudomonas aeroginosa
+
-
NCT121
Preista megaterium
NCT122
Bacillus safensis
+
+
-
+
NCT124
Pseudomonas asplenii
-
NCT127
Bacillus pumilus
V
-
NCT128
Bacillus velezensis
NCT129
Bacillus mojevensis
+
+
-
+
NCT255
Solibacillus silvestris
+
-
-
-
-
NCT256
Jeotgalibacillus marinus
+
+
-
-
-
NCT257
Bacillus atrophaeus
+
-
NCT260
Cellulomonas composti
-
NCT261
Enterobacter asburiae
-
NCT262
Lactobacillus delbrueckii
-
NCT263
Bacillus
amyloliquefaciens
+
+
-
+
V
NCT265
Bacillus pseudomycoides
+
-
-
+
NCT266
Bacillus licheniformis
+
+
+
+
NCT267
Neisseria mucosa
+
+
NCT270
Methylosarcina fibrata
+
-
59
NCT271
Methylomicrobium album
+
D
NCT272
Methylomonas methanica
+
+
5. 11. ANTIBIOTIC SENSITIVITY ASSAY
Antibiotic sensitivity assay was performed by the Kirby-Buer method to study the presence or
absence of a zone of inhibition of growth, without regard to its size, is used to determine
susceptibility. For example, a definite zone of inhibition around the lowest concentration disk
indicates that the organism is sensitive, whereas absence of a zone with the highest-concentration
disk indicates resistance
60
Figure 20: Muller Hinton agar plate showing zones of inhibition due to various antibiotic discs
against the NCCCC bacterial isolates
61
Table 2: Response pattern of isolates for different antibiotics (Tetracyclin, Rifambicin,
Gentamycin, Streptomycin, Penicilin, Ampicilin, Amoxilin)
STRAIN
NO
TET
RIF
GEN
STERP
P
AMP
AMX
AMC
Zone of inhibition(cm)
NCT1
-
-
1. 9cm
-
-
-
R
2cm
NCT11
1. 9cm
1cm
2cm
-
-
-
R
-
NCT24
2. 4cm
1. 4cm
2. 5cm
-
-
-
R
-
NCT25
1. 5cm
-
1. 7cm
-
R
-
R
-
NCT32
2. 4cm
1. 2cm
2cm
-
-
-
R
-
NCT33
2. 4cm
1. 4cm
2. 2cm
-
R
-
R
-
NCT38
2. 4cm
1cm
2. 1cm
-
R
-
-
-
NCT43
2. 2cm
1. 5cm
2. 4cm
-
R
-
-
-
NCT99
2. 5cm
1. 5cm
-
-
1. 2c
m
2cm
-
2cm
NCT114
2. 2cm
1. 2cm
-
-
1. 5c
m
2cm
1,1c
m
-
NCT121
2. 3cm
1. 3cm
2. 4cm
2. 6cm
R
R
-
-
NCT122
1cm
1. 5cm
2. 4cm
-
R
R
-
-
NCT123
-
1. 3cm
2. 6cm
2. 6cm
R
R
-
-
NCT124
-
R
2cm
1. 7cm
R
R
-
-
NCT125
-
-
2. 6cm
2. 8cm
R
R
-
-
-
62
-NCT126
2. 2cm
1. 2cm
2cm
2. 3cm
R
-
-
-
NCT127
2. 6cm
1. 4cm
2. 6cm
2. 7cm
1. 9c
m
1. 7c
m
-
2. 2cm
NCT128
1. 6cm
1. 1cm
2. 7cm
2. 8cm
-
R
-
-
NCT129
2. 1cm
1. 4cm
1. 8cm
2. 7cm
-
R
-
-
NCT130
-
1. 4cm
2. 5cm
2. 8cm
R
R
-
-
NCT131
2. 9cm
1. 5cm
2. 6cm
2. 9cm
R
R
-
-
NCT204. 1
-
2. 9cm
2. 2cm
2. 3cm
R
-
-
-
NCT204. 2
-
2. 9cm
2. 2cm
2. 3cm
R
-
-
-
NCT204. 3
-
2. 9cm
2. 2cm
2. 3cm
R
-
-
-
NCT205
-
1. 6cm
2. 4cm
1. 9cm
R
-
-
-
NCT207
-
0. 4cm
2cm
1. 3cm
R
-
-
-
NCT208
-
1cm
1. 9cm
1. 6cm
R
-
-
-
NCT209
-
2. 1cm
1. 5cm
1. 4cm
R
-
-
-
NCT243
-
R
2cm
1cm
-
R
-
-
NCT244. 1
-
R
1. 9cm
1cm
-
R
-
-
NCT244. 2
-
1. 5cm
1cm
-
R
-
-
NCT244. 3
-
R
2cm
1cm
-
R
-
-
NCT245
-
R
2. 4cm
1cm
-
R
-
-
NCT251
2cm
1. 1cm
2. 4cm
1. 5cm
1. 4c
m
1. 5c
m
-
2cm
63
NCT252
2. 4cm
2. 8cm
2cm
1. 7cm
R
1. cm
R
1. 4cm
NCT253
1. 2cm
1. 1cm
2. 5cm
1. 7cm
R
R
R
1. 2cm
NCT254
-
-
1. 8cm
1. 9cm
-
R
R
-
NCT255
2. 2cm
1cm
1. 6cm
1. 5cm
1cm
1cm
R
-
NCT256
-
-
1. 8cm
1. 5cm
-
R
R
-
NCT257
-
-
2cm
-
-
R
R
-
NCT258
-
1. 1cm
1. 5cm
R
-
R
R
-
NCT259
-
1cm
1. 5cm
1. 2cm
-
R
R
-
NCT260
-
R
1. 9cm
1. 2cm
-
R
R
-
NCT261
-
1. 1cm
2cm
1. 4cm
-
R
R
-
NCT262
-
R
1. 9cm
R
-
R
R
-
NCT263
-
1. 2cm
1. 9cm
1. 3cm
-
R
R
-
NCT265
-
-
2. 4cm
R
-
-
-
-
NCT266
-
-
2. 3cm
1cm
-
-
-
-
NCT267
-
-
1. 8cm
R
-
-
-
-
NCT268
-
-
1. 9cm
R
-
-
-
-
5.12. ENZYME SCREENING
5.12.1. AMYLASE ENZYME SCREENING
This test is used to identify bacteria that can hydrolyze starch using the enzymes alpha-amylase. .
The enzyme amylase breaks down starch into smaller components like maltose, glucose, sucrose
and dextrins that are more easily metabolized by the organism.
64
Figure 21: Positive (A) Presence of clear halos surrounding colonies for their ability to
digest the starch and thus indicates presence of alpha-amylase. Negative (B) No clearing;
only a blue/black area.
5.12.2. CASEINASE ENZYME SCREENING
Some microorganisms have the ability to degrade the casein protein by producing a proteolytic
exoenzyme, called proteinase (caseinase). For demonstration of such an activity, in the lab, milk
agar is used.
Figure 22: Positive (A) clear zone around the bacterial growth. Negative (B) No zone of
clearing.
5.12.3. LIPASE ENZYME SCREENING
Pure isolated bacterial cultures were screened for lipase activity using Phenol red agar medium.
The results were observed.
65
Figure 23: Negative lipase enzyme test is indicated by the development of pink color . the
yellow colour was incated as positive lipase enzyme.
5.13. DYE DEGRADATION
Fungal species were isolated (Fusarium solani) from the developed fungal colonies, identified
and tested for their ability to decolorize the five reactive dyes. Strains were maintained and
subcultured on potato dextrose agar (PDA) slants or plates. Analysis of cell free supernatant of
the 2ml of the output decolourized samples were scanned in the range of 200-800 nm usin UV-
Spectometer to observe spectral shifts of The congo red dye.
FIG 24: (A) Dye decolorization of Fusarium Solani with congo red at 0 hr. (B) dye
decolorization of Fusarium solani with congo red at 72hrs
66
5.14. Protein fingerprinting using SDS-PAGE
Page Contents. SDS-PAGE is a reliable method for determining the molecular weight (MW) of
an unknown protein, since the migration rate of a protein coated with SDS is inversely
proportional to the logarithm of its MW. The results of whole cell proteins profile was obtained.
It was possible to generate protein fingerprints in different strains . All strain numbers are NCT
numbers. The comparision of protein electrophoretic patterns provides a reliable measure of
taxonomic relatednessbof the different strains.
Fig 25:SDS-PAGE Fingerprinting of whole cell proteins of (A)NCT265, NCT266, NCT267,
NCT268. (B)NCT243, NCT244.1, NCT244.2, NCT245, NCT246. (C)NCT258, NCT259,
NCT260, NCT261, NCT127.2, NCT130.2 (D)NCT204.1, NCT205, NCT207, NCT210,
NCT212. (E)NCT121, NCT122, NCT123, NCT124, NCT125, NCT126. The diverse pattern of
67
bands in the SDS PAGE fingerprinting denotes the distinct bacterial species.
5.15. DNA ISOLATION
For most of the samples (>40%), DNA was isolated and sent for PCR and sequencing (fig. 26).
For remaining samples, either live microbes in slant culture or glycerol stocks were
commercially outsourced for sequencing.
Fig. 26: Genomic DNA isolation visualized in agarose gel electrophoresis.
5.16. BLAST ANALYSIS
The blast analysis reveals an average of 99.36% similarity to the sequences in the Genbank
(Supplementary file 1).
5.17.GENETIC DISTANCE ANALYSIS
5.17.1. GENETIC DISTANCE ANALYSIS FOR BACTERA
The number of base substitutions per site from averaging over all sequence pairs is shown.
Standard error estimate(s) are shown in the second column and were obtained by a bootstrap
procedure (500 replicates). Analyses were conducted using the Kimura 2-parameter model. This
analysis involved 63 nucleotide sequences. All ambiguous positions were removed for each
sequence pair (pair wise deletion option). There were a total of 1257 positions in the
finaldataset. Overall Genetic distance value =0. 20
5.17.1.2. GENETIC DISTANCE ANALYSIS FOR FUNGI
The number of base substitutions per site from averaging over all sequence pairs is shown.
Standard error estimate(s) are shown in the second column and were obtained by a bootstrap
68
procedure (500 replicates). Analyses were conducted using the Kimura 2-parameter model.
This analysis involved 10 nucleotide sequences. All ambiguous positions were removed for each
sequence pair (pairwise deletion option). There were a total of 1374 positions in the final
dataset. Overall Genetic distance value =0. 08
5.17.1.3. GENETIC DISTANCE ANALYSIS FOR YEAST
The number of base substitutions per site from averaging over all sequence pairs is shown.
Standard error estimate(s) are shown in the second column and were obtained by a bootstrap
procedure (500 replicates). Analyses were conducted using the Kimura 2-parameter model. This
analysis involved 10 nucleotide sequences. All ambiguous positions were removed for each
sequence pair (pairwise deletion option). There were a total of 1374 positions in the final dataset.
Overall Genetic distance value = 0.08.
69
5.18.PHYLOGENETIC ANALYSIS
Phylograms were constructed to study the variation and similarity among the sequenced species
with that of the reference sequences collected from Ganbank (fig. 27 - 45).
Fig 27:The percentage of replicate Neighbor-Joining trees in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
toscale, with branch lengths in the same units as those of the evolutionary distances used to
inferthe phylogenetic tree. This analysis involved a minimum of 10 nucleotide sequences.
There werea total of at least 1250 positions in the final dataset. The species names placed at the
branch tipswere prefixed with GenBank accession numbers and the sequences produced in the
present studywere mentioned with strain number prefixed with .
70
Fig 28 :The percentage of replicate Neighbor-Joining trees in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 10 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with .
71
Fig 29:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with .
72
Fig 30:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to
scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present
study were mentioned with strain number prefixed with
73
final dataset. The species names placed at the branch tips were prefixed with GenBank accession
numbers and the sequences produced in the present study were mentioned with strain number
prefixed
Fig 31:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered together in
the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. This
analysis involved minimal of 11 nucleotide sequences. There were a total of at least 1250 positions in the
74
Fig 32:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered together in
the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. This
analysis involved minimal of 11 nucleotide sequences. There were a total of at least 1250 positions in the
75
Fig 33:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with .
76
Fig 34:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 10 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with .
77
Fig 35:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with
78
Fig 36:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered together in
the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. This
analysis involved minimal of 11 nucleotide sequences. There were a total of at least 1250 positions in the
final dataset. The species names placed at the branch tips were prefixed with GenBank accession
numbers and the sequences produced in the present study were mentioned with strain number
prefixed .
79
Fig 37 :The percentage of replicate Neighbor-Joining tree in which associated taxa clustered together in
the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. This
analysis involved minimal of 11 nucleotide sequences. There were a total of at least 1250 positions in the
final dataset. The species names placed at the branch tips were prefixed with GenBank accession
numbers and the sequences produced in the present study were mentioned with strain number
prefixed .
80
Fig 38:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with .
81
Fig 39:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present
study were mentioned with strain number prefixed with
82
Fig 40:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered
together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. This analysis involved minimal of 11 nucleotide sequences. There were a
total of at least 1250 positions in the final dataset. The species names placed at the branch tips
were prefixed with GenBank accession numbers and the sequences produced in the present study
were mentioned with strain number prefixed with
83
Fig 41:The percentage of replicate Neighbor-Joining tree in which associated taxa clustered together in
the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. This
analysis involved minimal of 11 nucleotide sequences. There were a total of at least 1250 positions in the
final dataset. The species names placed at the branch tips were prefixed with GenBank accession
numbers and the sequences produced in the present study were mentioned with strain number
prefixed .
84
Fig 42:The evolutionary history was inferred using the Neighbor-Joining method. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is
shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of
the evolutionary distances used to infer the phylogenetic tree. These analyses involved 7 to 11 nucleotide
sequences. There were a total of atleast 500 positions in the final dataset. The species names of the
reference sequences collected from the GenBank were prefixed with GenBank accession numbers. The
sequences produced in the present study were mentioned with strain number prefixed with .
85
Fig 43:The evolutionary history was inferred using the Neighbor-Joining method. The
percentage of replicate trees in which the associated taxa clustered together in the bootstrap test
(500 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in
the same units as those of the evolutionary distances used to infer the phylogenetic tree. These
analyses involved 11 nucleotide sequences. There were a total of atleast 500 positions in the
final dataset. The species names of the reference sequences collected from the GenBank were
prefixed with GenBank accession numbers. The sequences produced in the present study were
mentioned with strain number prefixed with .
86
Fig 44:The evolutionary history was inferred using the Neighbor-Joining method. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is
shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of
the evolutionary distances used to infer the phylogenetic tree. These analyses involved 11 nucleotide
sequences. There were a total of at least 500 positions in the final dataset. The species names of the
reference sequences collected from the GenBank were prefixed with GenBank accession numbers. The
sequences produced in the present study were mentioned with strain number prefixed with .
87
Fig 45: Neighbor-;Joining tree in which the associated taxa clustered together in the bootstrap
test (500 replicates) are shown next to the branches . The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the phylogenetic
tree. There were a total of 1018 positions in the final dataset. 18S rRNA gene sequences produced
in the present work were indicated by a strain number NCT201.
5.19. Degitization of NCCCC
The present work has led to the synthesis of a culture collection with 107 microbial species. The
culture catalog was digitized and a web database was synthesized for global accession to the
culture. The database could be accessed here www.ncccc.in
Microorganisms (WDCM) which promotes culture and other data exchanges between culture
collection giants such as ATCC and MTCC has recognized the existence of NCCCC under a
register number WDCM1266. Now NCCCC is actively supplying cultures to the consumers after
88
signing the material transfer agreement (supplementary file 2) as mandated by WDCM.
89
6. Discussion
Parallel to the development of biotechnology, there has been an increase in the demand for
authenticated, reliable biological material and related information from culture collections. The
Organization for Economic Co-operation and Development (OECD) has more recently
acknowledged the significance of raising the quality and supply of culture collections in order to
support biotechnology through the adoption of best practices, for which the WFCC guidelines
provided the framework (OECD, 2007). Culture collections play a vital role in the conservation
and sustainable use of microbial resources . They also provide authentic biological material for
high quality research and teaching. in the form of reference strains, reagents for quality control,
etc. The advances in molecular biology have resulted in the continued discovery of new
microbial taxa and strains and there is a need to preserve these so as to make them accessible to
other researchers for research, teaching and for biotechnological exploitation. Individual
laboratories are unable to do this due to lack of financial support and manpower. This role is thus
played by a culture collection.
Culture collections are valuable resources for the sustainable use of microbial diversity and its
conservation. Advances in biotechnology have further increased their importance. Microbial
Culture Collection at National College was established by the Department of Biotechnology and
Microbiology, is the college's newest culture collection with largest holdings. It is recognized as
a Designated Regional Repository by the World Federation of Culture Collection.
The increasing demands on culture collections for authenticated, reliable biological material and
associated information have paralleled the growth of biotechnology. More recently, the
organisation for Economic Co-operation and Development (OECD) have recognised the
importance of taking culture collections to a higher level of quality and delivery to underpin
biotechnology. One key element of this development is the introduction of best practice (OECD,
2007), for which the WFCC guidelines laid the foundation. These guidelines have been updated
to include recent developments and changes to provide basic quality management guidance for
culture collections.The OECD Best Practice Guidelines for Biological Resource Centres
(OECD, 2007) set the standard for quality management and also covers biosecurity, building
capacity, preservation of biological resources and data management.
90
The ever decreasing investment in traditional taxonomy, the increasing demand for a molecular
approach, the continued depletion of natural resources and concerns over biosecurity and climate
change brings a heightened awareness of the value of collections of microorganisms.
Conservation of genetic resources and biodiversity provides the essential underpinning for
emerging biotechnologically based eco-efficient products and industries in both the developed
and the developing world (OECD, 2001); an essential element in the development of a
knowledge-based bioeconomy (OECD, 2009).
In 2021, Department of Biotechnology and Microbiology at National College took major
initiative in microbial prospecting and undertook a screening program involving diverse samples
across various ecosystems including but not limited to human microbiome, water (AC drain,
fresh and marine), soil (garden, agriculture lands), fish guts (marine & freshwater fin/shell
fishes), vegetables, fruits, etc. This effort collected more than 300 strains among which 100
strains were identified to species level from these different ecological niches. NCCCC was
designated as Designated Regional Repository for Microorganisms on 27th July 2022 by World
Federation of Culture Collection (WFCC). NCCCC supplies microorganisms for various
academic and research purposes upon a request to the coordinator and signing the material
transfer agreement as recommended by WFCC.
NCCCC holds more than 100 species of identified microbial strains and are provided to the
academicians and researchers who wish to undertake screening programs under Material
Transfer Agreement. Each strain is preserved in triplicates in two different forms (-80 ºC
glycerol stocks and lyophilized form). The cultures are supplied to the users either as lyophilized
ampoule or glycerol stocks or as growing culture on the slant. NCCC has expertise to handle all
the major groups of bacteria including anoxygenic, phototrophic bacteria and anaerobes.
Currently it can handle BSL-1 and BSL-2 category organisms. The details of the NCCCC
cultures could be accessed through www. ncccc. in. A schematic diagram of the operations at
NCCCC is shown in Fig. 1. In forthcoming years NCCCC strives to build infrastructures for
handling BSL-3 category organisms and provides various services to institutes/universities and
industries like identification services (taxonomic marker gene sequencing, phylogenetic tree
construction, MALDI-TOF, FAME, DNADNA hybridization and Biochemical
91
characterization, etc. ) and educational services (workshops in colleges and universities, hands
ontrainings, etc).
Fig. Flow chart describing the steps in NCCCC, functioning.
th by the World Federation of
Culture Collections (WFCC), a global monitor of collection standards and practices
(https://wfcc. info/static/pdf/Guidelines_e. pdf). The immediate objective of NCCCC is to
provide culture services to various academic institutes to support their in-house research
programmes. The overarching goal of NCCCC will be to harness the technological potential
associated with the microbial biodiversity in the less-studied habitats of India, using a
crowdsourcing model (Suryanarayanan et al. , 2015) that exploits a collaborative network of
educational institutions, national research institutes and industries. Skill development and
curricular enhancement will be a high priority of NCCCC; human resources development in the
various processes involved such as isolation, culturing and identification of microorganisms, and
screening the cultures forspecific bioactivities will add a heuristic approach to higher education.
92
The cultures were screened for bioactive metabolites and enzymes (of clinical importance and
industrial applications) prioritized based on social needs. We intend to expand the culture
collection to 1000 microbial species collection by exploring less explored plants, animals and
extreme
The collaboration between a newly created NCCCC, national laboratories, other culture
collection centres, academia and industry has the potential to contribute to overall research and
economic development in India. In addition to developing its in-house collections, NCCCC will
also provide a service by integrating a decentralized network of experts, research scholars and
existing facilities towards the common objective of microbial culture collections and usage. An
example of what can be discovered by such an enterprise is shown by (Liaud et al. 2014), who
screened fungal isolates housed in the International Centre of Microbial Resources in France for
the production of organic acids, the basis of a huge industry.
From 2012，World Data Centre for Microorganisms (WDCM) started the initiative to construct
an effective information environment called Global Catalogue of Microorganisms (GCM) which
provided a database management system to culture collections and collect strain catalogue
information to form an integrated database. The current version of GCM contains information
93
of over 453，340 strains, which includes 55,038 bacterial, fungal and archaea species from 133
collections in 50 countries and regions, including the famous collections such as DSMZ, CBS,
BCCM, NBRC and so on. GCM as a culture collection management system could help
administrate culture collections by automatically generating homepage as well as strain online
catalog for organizing, sharing and exploring of the strain data resources. What is more, to
improve the accuracy of the catalog information, GCM system also has the Name Check Service
function which could check the names contained in the collection's catalog with the widely used
international taxonomy databases and nomenclature database and then give the advice of
modifying. WDCM has recognized NCCCC and approved its functioning under register number
WDCM1266 https://ccinfo. wdcm. org/details?regnum=1266.
94
7. Summary and future trust
The present work has created a microbial culture collection centre called National
College Culture Collection Centre (NCCCC) consisting of a 107 microbial species
identified to species level.
NCCCC was digitized and could be globally accessed through www. ncccc. in
NCCCC has been registered under WDCM with a register number WDCM1266
https://ccinfo. wdcm. org/details?regnum=1266
Since 2022, NCCCC has being used for various bio-prospecting purposes as listed
below,
Table 3:List of strains being explored by various PG Students at the Department of
Biotechnology & Microbiology, National College, Trichy.
No
Strains
Consumers
Purpose
1
NCT260
Ms. Priya
Screening for plant growth promoters
2
NCT130. 2
Ms. Priya
Screening for plant growth promoters
3
NCT262
Ms. Priya
Screening for plant growth promoters
4
NCT263
Ms. Priya
Screening for plant growth promoters
95
5
NCT1
Ms. Prachi
Antibacterial activity for nanoparticles
6
NCT45. 2
Ms. Prachi
Antibacterial activity for nanoparticles
7
NCT25
Ms. Prachi
Antibacterial activity for nanoparticles
8
NCT114
Ms. Prachi
Antibacterial activity for nanoparticles
9
NCT265
Ms. Akshaya
Aquatic probiotic screening
10
NCT266
MsAkshaya
Aquatic probiotic screening
11
NCT267
Ms. Akshaya
Aquatic probiotic screening
12
NCT268
Ms. Akshaya
Aquatic probiotic screening
13
NCT24
Ms. Akshaya
Enzymatic assay
14
NCT121
Ms. Preethi
Antibacterial activity of plant extract
15
NCT122
Ms. Preethi
Antibacterial activity of plant extract
16
NCT123
Ms. Preethi
Antibacterial activity of plant extract
17
NCT124
Ms. Preethi
Antibacterial activity of plant extract
18
NCT125
Ms. Preethi
Antibacterial activity of plant extract
19
NCT126
Ms. Preethi
Antibacterial activity of plant extract
20
NCT127
Ms. Karthika
Antibacterial activity of plant extract
21
NCT128
Ms. Karthika
Antibacterial activity of plant extract
22
NCT122
Ms. Karthika
Antibacterial activity of plant extract
23
NCT261
Ms. Sowntharya
Fumic acid production
In forthcoming years (before Dec, 2023), NCCCC aims to become a 1000 species culture
collection.
With continued bio-prospecting efforts (such as screening for nano-particle synthesizing
ability, various enzyme and metabolites production, genomic library constructions, etc.),
NCCCC will upgrade from a microbial culture collection centre to full-fledged bio-resource
facility before 2024.
96
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5. Bacteriological Analytical Manual 8th ed. , Revision A (1998)
6. Bauer, A. W. , D. 8M. Perry, and W. M. M. Kirby. 1959. Single disc antibiotic sensitivity
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APPENDIX
1.
ASN 11 MEDIUM
REAGENTS
QUANTITY
NaCl
25g
MgCl2. 6H2O
2g
KCl
0. 5g
NaNO3
0. 75g
K2HPO4. 3H2O
0. 02g
MgSO4 . 7H2O
3. 5g
CaCl2. 2H2O
0. 5g
Citric acid
0. 003g
Ferric ammonium citrate
0. 003g
EDTA (disodium salt)
0. 00055g
Na2CO3
0. 02g
Trace metal mix
1ml
Distilled water
1000ml
Ph
7. 5
Trace metal mix
H3BO3
2. 86g
MnCl2. 4H2O
1. 81g
ZnSO4. 7H2O
0. 222g
106
Na2MoO4. 2H2O
0. 39g
CuSO4. 5H2O
0. 079g
Co (No3)2. 6H2O
0. 0494g
2.
MN AGAR MEDIA
REAGENTS
WEIGH
NaNO3
0. 75g
K2HPO4. 3H2O
0. 02g
MgSO4. 7H2O
0. 038g
CaCl2. 2H2O
0. 018g
Ferric ammonium citrate
0. 003g
EDTA
0. 0005g
Na2CO3
0. 02g
Trace metal mix
1ml
Sea water
750ml
Deionized water
250ml
H3BO3
2. 86g
MnCl2. 4H2O
1. 81g
ZnSO4. 7H2O
0. 22g
Na2MoO4. 2H2O
0. 39g
CuSO4. 5H2O
0. 079g
107
CO(NO3)2. 6H2O
0. 0494g
108
Deionized water
1000ml
3.
Nutrient Agar media
Peptone
0. 5g
Yeast extract
0. 3g
Nacl
0. 25g
Agar
1. 5g
Distilled water
100ml
4.
Potato dextrose agar media
Potato dextrose agar
3. 9g
Agar
1. 5g
Distilled water
100ml
5.
NMS Agar media
REAGENTS
WEIGH
MgSO4. 7H2O
0. 3g
CaCl2. CH2O
0. 06g
KNO3
0. 3g
KH2PO4
0. 0816g
Na2HPO4. 12H2O
0. 2151g
109
Agar
6. 3g
Sea water
300ml
Iron solution
0. 6ml
Trace element
0. 15ml
Ph
8. 5g
6.
Muller Hinton agar media
REAGENTS
WEIGH
Muller hinton agar
4. 13g
Agar agar
1. 5g
Distilled water
100ml
7.
Starch agar media
Peptone
5g
Yeast extract
1. 5g
Sodium chloride
5g
Beef extract
1. 5g
Starch soluble
2g
Agar
15g
Distilled water
1000ml Final pH ( at 25°C) 7. 4±0. 2
8.
Skim milk agar media
Skim milk powder
28g
Tryptone
5g
110
Yeast extract
2. 5g
Dextrose (Glucose)
1g
Agar
15g
Distilled water
1000ml
9.
Phenol Red agar media
Peptone
5g
Yeast extract
3g
Agar agar
15g
CaCl2
1g
0. 1M NaOH
4g
Phenol red dye
1mg/ml
Coconut oil
10ml
Distilled water
1000ml (pH 7. 4)
10.
Indole Test:
PREPARATION OF TRYPTONE BROTH
Tryptone
10gms
Distilled water
1000ml
PREPARATION OF REAGENT
N-amyl alcohol
75ml
Concentrated HCL
25ml
P-dimethylaminebenzaldehyde
5g
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11.
Methyl Red Test:
Methyl red
0. 2g
95% Ethyl alcohol
500ml
Distilled water
500ml
Dipotassium hydrogen phosphate (K2HPO4)
5gm
Peptone
5gm
Glucose
5gm
Distilled water
1000ml
12.
Preparation of MRVP medium or glucose phosphate broth for VP Test
Dipotassium hydrogen phosphate
(K2HPO4
5gm
Peptone
5gm
Glucose
5gm
Distilled water
1000ml
Preparation of reagent:
reagent consists of 2 solutions, i. e. , solutions A and B.
Solution A is prepared by dissolving 6 gms of alpha naphthol in 100 mL of 95% ethyl alcohol.
Solution B is prepared by dissolving 16 gms of potassium hydroxide in 100 mL of water.
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13.
Simmon citrate agar for citrate test
Bromothymol blue
0. 08 g
Magnesium sulfate
0. 2 g
Dihydrogen ammonium phosphate
1. 0 g
Dipotassium hydrogen phosphate
1. 0 g
Sodium citrate
2. 0 g
Sodium chloride
5. 0 g
Agar agar
15g
Distilled water
1000ml
Bromothymol blue
40ml
pH
6. 8-7. 0
13. Buffer preparation for DNA Isolation
Lysis Buffer(500ml)
50mM Tris-HCL =3. 028g
20mM EDTA =2. 922g
2%SDS =10g pH =8. 0
TE buffer (100ml)
Tris-HCl 1M in 1 ml =0. 121g
EDTA 0. 5M in 0. 2ml=0. 0229g
Distilled water =98. 8ml
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Final volume =100ml
TAE Buffer(50x)(100ml)
Tris Base =24. 2g
Acetic acid =5. 71ml
(10 ml of 50mM EDTA=0. 146g,pH =8. 0)
Final volume=100ml
PBS Buffer(100ml)
Nacl(300mM)=1. 75g
Kcl(2. 7mM) =0. 02g
Na2HPO4(10mM)=0. 14g
KH2PO4(1. 7mM)=0.
022g
✔ Saturated phenol:chloroform:isoamyl alcohol(25:24:1)
✔ Absolute Ethanol and 70% Ethanol
Agarose Gel Electrophoresis:
0. 8% Agarose gel is prepared by dissolving 800mg Agarose in 100ml 1x TAE Buffer. Boil the
solution to dissolve agarose . When the solution cools down a bit 2µl ethidium bromide is added
to it and casted in an electrophphoretic casting plate and an end of the gel in a way that the legs
of the comb remain inside the liquefied gel. it is allowed to solidify.
DNA Sample for loading:
12µl DNA Sample in 6x Gel loading dye
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14. SDS-PAGE
STOCK SOLUTION
1. 5M Tris-base (pH8. 8)
0. 5M Tris-base (pH6. 4)
30% Acrylamide: 1% Bis-acrylamide. Stored at
10% SDS
10% APS Freshly prepared
20% Acetic acid
4X SEPERATING GEL BUFFER
1. 5M Tris-base (pH 8. 8)
SEPARATING GEL
Distilled water
4ml
4X Separating gel buffer
2. 50ml
10% SDS
100 µl
30% Acrylamide: 1% Bis- acrylamide
3. 33ml
TEMED
20 µl
10%APS
200µl
STACKING GEL
Distilled water
3ml
Stacking gel buffer
1. 25ml
10% SDS
25 µl
30% Acrylamide: 1% Bis-acrylamide
0. 8ml
TEMED
6 µl
10%APS
60 µl
5X SAMPLE BUFFER
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Stacking gel buffer
2. 5ml
Glycerol
2. 0ml
10% SDS
4. 0ml
10% Bromophenol blue
300µl
- Mercapto Ethanol
400µl
Distilled water
0. 8ml
RUNNING BUFFER
Tris-base
6. 05g
Glycine
28. 8g
10%SDS
10ml
Distilled water
1000ml
GEL FIXING SOLUTION
Ethanol
500ml
Distilled water
400ml
Acetic acid
100ml
STAINING SOLUTION
Coomassie Brilliant Blue R250
0. 24g
50%Methanol
80ml
20%Acetic acid
20ml
Distilled water
100ml
DESTAINING SOLUTION
Methanol(Carbinol)
80ml
Acetic acid
20ml
Distilled water
100ml
116
Supplementary file1: Results of BLAST analysis including percentage of similarity, query coverage
and alignment scores were tabulated.
117
118
Supplementary file 2
National College Culture Collection Centre Material Transfer Agreement for the supply
of samples of biological material from the public collection
Scope of agreement
This Agreement applies to the use, handling, distribution and any disposition of the MATERIAL
supplied by the COLLECTION, and addresses the identified key points
• Traceability
• Fair and Equitable Benefit Sharing
• Intellectual Property Rights
• Quality
• Safety and Security
Definitions
a. The COLLECTION –
National College Culture Collection Centre (N4C)
PG and Research Department of Biotechnology and Microbiology
National College (Autonomous)
Dindigul road, Karumandappam
Tiruchirappalli, Tamil Nadu- 620001
India
b. AGREEMENT: This document.
c. RECIPIENT: (end user)
In case this is not the END-USER but an INTERMEDIARY, this INTERMEDIARY agrees
(i) to forward to the END-USER the present MTA and the MATERIAL in unchanged form and
quantity as received from the COLLECTION, and
(ii) to use for this further shipping the proper packaging, a trained shipper, and an authorized
carrier, according to the applicable laws and regulations.
d. END-USER: Scientist working with the supplied MATERIAL.
e. INTERMEDIARY: Third party, different and independent from the END-USER, that makes an order
119
on behalf of the END-USER, and to which the COLLECTION addresses the MATERIAL. These can be
whole-salers, importers, or other type of intermediary agents, unrelated to the END-USER’s institution.
f. DEPOSITOR: Person(s) or entity that provided the COLLECTION with the ORIGINAL MATERIAL.
g. MATERIAL: ORIGINAL MATERIAL, PROGENY and UNMODIFIED DERIVATIVES. The MATERIAL shall
not include MODIFICATIONS.
h. ORIGINAL MATERIAL: That which was originally supplied to the COLLECTION by the DEPOSITOR.
i. PROGENY: Unmodified descendant (e.g. sub-culture or replicate) from the ORIGINAL MATERIAL.
j. UNMODIFIED DERIVATIVES: Replicates or substances which constitute an unmodified functional
subunit or product expressed by the MATERIAL, such as, but not limited to, purified or fractionated
subsets of the MATERIAL, including expressed proteins or extracted or amplified DNA/RNA.
k. MODIFICATIONS: Substances produced by the RECIPIENT by using the MATERIAL, which are not
the ORIGINAL MATERIAL, PROGENY, or UNMODIFIED DERIVATIVES, and which have new properties.
MODIFICATIONS include, but are not limited to, recombinant DNA clones.
l. COMMERCIAL PURPOSES: The use of the MATERIAL for the purpose of profit.
m. LEGITIMATE EXCHANGE: The transfer of the MATERIAL between scientists working in the same
Laboratory, or between partners in different Institutions collaborating on a defined joint project, for
non-commercial purposes. This also includes the transfer of MATERIAL between public service culture
collections/BRCs for accession purposes, provided the further distribution by the receiving
collection/BRC is under MTA conditions equivalent and compatible to those in place at the supplying
collection.
THE COLLECTION WILL TRANSFER THE MATERIAL UNDER THE TERMS AND CONDITIONS
SPECIFIED IN THIS MATERIAL TRANSFER AGREEMENT.
THE RECIPIENT – BEING END-USER, INTERMEDIARY OR CULTURE COLLECTION / BRC –ACCEPTS
THE TERMS AND CONDITIONS OF THIS MATERIAL TRANSFER AGREEMENT BY PLACING AN ORDER
WITH THE COLLECTION.
Following AGREEMENT is between the COLLECTION and the RECIPIENT of the MATERIAL:
1. RECIPIENT agrees that all information provided to the COLLECTION in connection with any order
for MATERIAL is accurate and complete, and otherwise complying with applicable laws and
regulations.
2. RECIPIENT agrees that MATERIAL designated Risk Group 2 or above (as defined by the national
regulations of the country where the Collection is located) may cause human disease, and that
MODIFICATIONS, or other MATERIAL, not so designated, may cause human disease under certain
conditions.
120
3. RECIPIENT agrees that any handling or other activity undertaken in their laboratory with the
MATERIAL will be conducted under their responsibility and in compliance with all applicable laws and
regulations.
4. RECIPIENT therefore assures that within their laboratory
(i) access to the MATERIAL will be restricted to personnel capable and qualified to safely handle said
MATERIAL and
(ii) RECIPIENT shall exercise the necessary care, taking into account the specific characteristics of the
MATERIAL, to maintain and use it with appropriate precautions to minimize any risk of harm to
persons, property, and the environment, and to safeguard it from theft or misuse.
5. Unless agreed in writing with the COLLECTION, RECIPIENT shall not sell, distribute or propagate for
distribution, lend, or otherwise transfer the MATERIAL to any others, except those RECIPIENT that
acts as INTERMEDIARY and those RECIPIENT involved in LEGITIMATE EXCHANGES as defined above.
6. Subject to the terms and conditions of this AGREEMENT and any statutory, regulatory or other
restriction imposed by law or any third party interest, RECIPIENT may use the MATERIAL in any
lawful manner for non-commercial purposes.
7. If the RECIPIENT desires to use the MATERIAL or MODIFICATIONS for COMMERCIAL PURPOSE(S),
it is the responsibility of the RECIPIENT, in advance of such use, to negotiate in good faith the terms of
any benefit sharing with the appropriate authority in the country of origin of the MATERIAL, as
indicated by the COLLECTION’s documentation.
8. Nothing in this AGREEMENT grants RECIPIENT any rights under any patents, propriety, intellectual
property, or other rights with respect to the MATERIAL.
9. RECIPIENT agrees to acknowledge the COLLECTION as the source of the MATERIAL in any and all
publications that reference the MATERIAL.
10. Warranty: The COLLECTION hereby assures within the scope of its quality system and as far as can
be determined through the COLLECTION’s test regimes, that the MATERIAL shall be viable and pure
upon shipment from the COLLECTION. Any claim against the warranty will have to be communicated
to the COLLECTION within a period of 15 days from the COLLECTION’s shipment, and will have to be
justified to the COLLECTION’s satisfaction. The primary remedy for breach of this warranty is
replacement by the COLLECTION of the MATERIAL free of charge.
11. Disclaimer of warranties. Except as expressly provided in this AGREEMENT and within the limits of
the scope of the COLLECTION’s quality system, there are no representations or warranties by the
COLLECTION with respect to the MATERIAL, express or implied, including without limitation, any
implied warranty of authenticity, typicality, safety, fitness for a particular purpose, or of the accuracy
or completeness of the data.
