DEVEPLOMENT OF LOW COST MEDIA FOR BACTERIA

CULTURE

THESIS SUBMITTED TO THE UNIVERSITY OF MUMBAI

FOR PARTIAL FULFILLMENT OF DEGREE
B.Sc. BIOTECHNOLOGY
SEMESTER VI

BY
MR.SHUBHAM VINAYAK PAWAR

FOR THE ACADEMIC YEAR 2019-20120

UNIVERSITY OF MUMBAI

UNDER THE GUIDANCE OF

MS. NAMRATA MORE
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY
ELPHINSTONE COLLEGE
FORT, MUMBAI-400032

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 CERTIFICATE
Department of Biotechnology
Elphinstone College
University of Mumbai

This is to certify that MRS.SHUBHAM VINAYAK PAWAR, student of Department of

Biotechnology, Elphinstone College, seat no. ________________has completed the thesis titled
‘Development of low cost media for bacteria culture’ towards the partial fulfillment of the
Research Project of B.Sc Biotechnology for the academic year 2019-2020.

PLACE: MUMBAI
DATE:

Ms. Namrata More Dr. Nikita Naik

Assistant Professor Co-ordinator,
Department of Biotechnology Department Of Biotechnology
Elphinstone College Elphinstone Collage

Signature of Examiner

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 Declaration

I hereby, declare that this thesis or a part has not been submitted by me or any other person to
any other university or institute for degree or diploma and this research project entitled
“Development of low cost media for bacteria culture” was carried by me under the direct
supervision of my project guide Ms.Namrata More at the Department of Biotechnology of
Elphinstone College, Mumbai.

PLACE: MUMBAI

DATE:

MR. SHUBHAM PAWAR

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 ACKNOWLEDGEMENT

I, Mr. Pawar Shubham Vinayak, feel to acknowledge my indebtedness of gratitude to my mentor,

Ms. Namrata More Department of Biotechnology, Elphinstone College, for her valuable
guidance and kind supervision throughout the project which shaped the project work as it shows.

I express my sincere thanks to Dr. Madhuri kagalkar, principal of Elphinstone College and Dr.
Nikita Naik, Coordinator, Department of Biotechnology, Elphinstone College, for their constant
support by providing me with all the laboratory facilities.

I cannot express enough thanks to my professors, Ms. Revati Dhayfule and Mr. Mayur Gaikwad
for their much needed suggestions and advice.

My heartfelt thanks to my colleagues Ms.Siddhi Pandit, Ms.Anita Kumawat and Ms Abiha Baig
for lending their hands for countless times.

I sincerely thank Mr. Kashinath Tandel, Mr. Manoj Waghela, and Ms. Manali Raut, the non-
teaching staff of Department of Biotechnology, Elphinstone College, Mumbai, for their
wholehearted co-operation and valuable help.

I am extremely thankful to my fellow classmates for providing a friendly atmosphere during the
course of this work.

And last but not the least I owe my debt of gratitude to my family and friends for their constant
motivation and support without which this project could not been successfully completed.

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 CONTENTS Page No

1. ABSTRACT 8
2. INTRODUCTION 9-10
3. OBJECTIVES 12
4. REVIEW OF LITERATURE 13
4.1 Gelling agents 13-15
4.2 Nutrient substitute 16-18
5. MATERIALS AND METHODS 19
5.1 General procedure 19
5.2 Test culture media 20-26
6. EXPRIMENTAL RESULTS AND DISSCUSION 27-33
7. CONCLUSION 34
8. FUTURE PROSPECTS 35
9. BIBLIOGRAPHY 36-37

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 LIST OF TABLES

Table Title Page

No. No.
1 Effect of solidifying agents and its concentration on 27
the solidification and consistency of the medium
2 Number of colonies grown on nutrient agar plate 31
3 Number of colonies grown on soya china grass plate 32

LIST OF FIGURES

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 Fig Title Page
No. No.
1(a) Sterile plain agar plate 28

(b) Sterile plain china grass plate 28

2 E.coli grown on nutrient china grass plate 29
3 Soya broth 30
Nutrient broth 30
4 (a) Nutrient agar plate 10-4 31
-5
(b) Nutrient agar plate 10 31
5 (a) Soya china grass plate 10-4 32

(b) Soya china grass plate 10-5 32

1. ABSTRACT

To isolate, culture and maintenance of microorganism culture media are required. These culture
media is well formulated and standardized in research laboratories and nowadays readily
available in the market. However the exorbitant cost of culture media has hindered many

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microbiology research programs in developing countries. The media and their constituents are
costly and many not are available at remote place laboratories. Therefore the high cost of culture
media has opened up a way for the production of alternative media using cheap local raw
materials. The replacement of agar and nutrient source are therefore challenging task. To replace
agar gelling agent like china grass was used 5% china grass was a good solidifying agent for
bacterial streaking. Replacement of nutrient source was tried with soya meat powder instead of
standard nutrient source. This nutrient source was added with china grass. Soya meat powder
proved to be cheap nutrient source with 0.5% of china grass supported very good bacterial
growth Thus SCM (soya china grass medium) can be used as a universal medium for working on
bacteria in research laboratory. Most importantly the cost of the medium is half of readymade
medium used in research laboratory.

Keywords: culture media, nutrient agar, bacteria, china grass, soya meat powder

2. INTRODUCTION

Researchers have consistently been eager to enter into new horizons of science, technology and
development. Their aim is for the betterment of human life through their discoveries. The main
concept behind new innovation and discoveries is to first fulfill the basic necessity of mankind

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and then the comfort and finally to replace the traditional things. A similar pattern is seen in the
field of biotechnology and microbiology concerning with the growth of microorganism.

Microorganisms need nutrients, a source of energy and certain environmental conditions to in

order to grow and reproduce. In the environment, microbes adapt to the habitats most suitable for
their needs while in laboratory these requirements is met by a culture medium (simian,
2011).when a medium is being prepared for microbial growth, consideration is given to the
provision of carbon and energy sources and other growth factors that are essential for organism
( Laleye et al., 2007).

For growth of microorganism in laboratory we require artificial medium which is influenced by

several physical and chemical factors. Nutrient medium which is prepared in laboratory for
growth of microorganism is called culture media and the composition of a culture medium plays
a major role in microbial growth. For microbiological studies, commercially available
readymade culture media are used in research laboratories. These culture media are standardized
and they are of different types. Some media are used for general purpose while some others are
used for specific growth of certain micro-organism Nutrient Agar is commonly used for isolation
and growth of wide range of bacteria in laboratories and its composition are well defined

Although in developing countries microbiological research has slow down because of high cost
and scarcity of cultural media. The high cost of microbial culture media has opened up a way
for the production of alternative media. Different media have been formulated by using cheap
resources for growth and isolation of bacteria. Ravathie et al. used edible leguminous seeds such
as green gram, black gram; soya meat and cowpea which serves as a good proteins source were
selected to formulate the media. Even though they successfully cultivated bacteria on the
alternative medium but in all cases they used agar as solidifying agent and the cost of agar is
additional burden.

Agar can be defined as a hydrophilic colloid extracted from certain seaweeds of the
rhodophyceae class and is extensively use in microbiology field all over the world but it is
expensive and not easily available in developing countries. Agar represents one of the most
expensive and commonly used in media components, contributing 70% of total cost production.

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China grass is used as replacement of agar-agar. China grass is also known as Agar-Agar. It is
obtained from various seaweeds. Agar-Agar and China grass are same in nature but the only
difference is agar is well formulated and standardized for scientific use and china grass is much
used in desserts. Essentially like agar china grass is hydrophilic consisting in powder and strips
from energy/kcal 24.88, carbohydrates6.20, protein 0.00 and fat 0.00 (nutritional information of
china grass per unit/8g) .The most important characteristic of china grass is odorless and plastic
clarity and there it is used as alternative of agar in this research.

The readymade nutrient medium which is used in laboratories contains beef extract which is an
aqueous extract of lean beef tissues. It contains water soluble substance of animal tissue which
includes carbohydrates, organic nitrogen compounds, water soluble vitamins and salts. Peptone
is made by digesting by animal tissues which contains organic nitrogen source and amino acids
like serine, glycine, thronine, alanine, methionine, proline, a-aminabutryric acid, leucine, valine
and isoleucine all this components are essential for growth of bacteria.

Soya meat powder is used as replacement of nutrient source in culture media contains carbon
source, nitrogen source, vitamins, minerals and amino acids like histiden, isoleucine, leucine,
lysine, methionine, phenylalanine, thronine, tryptophan and valine. Some of the components
match with readymade nutrient medium and thus can be used as a alternative nutrient source for
growing bacteria.

Agar is extensively used in microbiological media all over the world. But it is expensive and not
easily available in most developing countries. For example cost of 500g agar powder is 6,148
rupees ($88). Cost of agar powder varies according to its concentration.

China grass is very cheap and can be used as substitute of agar-agar in microbiology. For
example 500g of china grass cost up to 800 to 900 rupees. The best quality of china grass can
cost up to 1800 to 2000 per kg.

One the yearly basis every college spend 10,000 to 20,000 on agar which is used in laboratory.
Where else in same price 20-25kg of china grass can be purchased. The cost of Hi Media
laboratories Nutrient broth is Rs 3,733/500g which is very exorbitant and the cost of soya meat
granules is Rs 44/500g.

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Growth of selected bacteria on low cost (soya + china grass) media was observed as compared
to standard media and can be used to isolate the bacteria and for the maintenance and long-term
preservation of bacteria cultures. Nevertheless most of the laboratories in our country and
elsewhere are not using these medium or substrate for bacteria culture. The reason remains
unknown.

“DEVELOPMENT OF LOST MEDIA FOR BACTERIA CULTURE” was undertaken with

Following objectives

Objectives

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 1. TO search low cost ingredients for bacterial isolation by replacing the manufactured
media components.
2. TO search solidifying agent for medium instead of sole agar.

3. REVIEW OF LITERATURE

3.1 Gelling agents

Lines AD (1977) studied the value of K+ salt of carageenan as an agar substitute in

bacteriological media. The K+ salt of carageenan has no distinct advantage as a gelling agent but

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it compared favorably with agar in most of the media tested. The difficulty involved in the
preparation of blood plates and results obtained with this medium prohibit its complete
acceptance as a substitute for agar in the routine solid media However, it could be a suitable
substitute for agar in all other routine bacteriological media.

Bruno J. Bromke and Merewyn Furiga (1991) several gelling agents were tested as potential
substitutes for agar in media growing Trichomonas vaginalis. They were evaluated on several
criteria growth of trichomonads, normal morphology of trichomonads, buoyancy of culture,
clarity of intercellular fluid, proper harvesting of trichomonads and cost-efficiency. Only
carrageenan, used at a 0.3g in 100ml concentration.

Nene and Shiela (1995) proved the use of granulated Tapioca as an agar substitute for the
growth and sporulation of 6 pathogenic fungi isolated from chickpea and pigeon pea on Tapioca-
based media.This cheaper substitute for routine culture of fungi.

Jain et al. (1997) use of Isubgol, the mucilaginous husk derived from seeds of plantago ovata as
a gelling agent from microbial culture media. As illustrative examples, fast growing symbiotic
Rhizobium meliloti and saprophytic fungi, Asprgillus flavus and penicillium chrysogenum were
cultured on media gelled with either isubgol or agar. All the three microbes employed in the
study exhibited normal growth when culture on their respective media gelled with isubgol.
Rather, isubgol gelled medium appears to promote the growth of bacteria cultures as the colonies
on this medium were denser than he corresponding ones on the medium gelled with agar.
Likewise, sporulation in both the fungi took place earlier than on the medium gelled with agar.

Naik and Sakar (2001) used sago, a processed (gelatinized) edible starch as a gelling agent in
culture medium. The efficacy of sago-gelled (80g dm-3) medium was used in ten potato
genotypes during micro propagation and minimal growth conservation. Sago starch provide a
firm gelling surface through the entire culture period, and fostered optimum plantlet growth in
terms of shoot height, number of nodes per plant, number of leaves and fresh mass. No softening
of sago-gelled medium occurred over prolonged storage. The study showed that sago starched
could be used as substitute of agar.

Tewary et al. (2001) reported the effect of some cheap gelling agents on micro propagation of
mullbery.The result showed that 12g/liter china grass was as good as 8g/liter agar as gelling

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agent for shoot formation. All other gelling agents tested were found inferior. For vitro
rhizogenesis, maximum response was elicited by either sago (150g/liter) or china grass
(12g/liter) gelled media. It was concluded that expensive agar could be suitable replaced by
china grass for in vitro shoot development and by sago for in vitro rhizogenesis.

Jain and Babbar (2002) reported the use of Gum katira, an insoluble gum derived from the bark
of Cochlospermum religiosum, as a gelling agent in tissue culture media for in vitro shoot
formation and rooting in Syzygium cuminiand somatic embryogenesis inAlbizzia
lebbeck.The epicotyl segments, excised from in vitro grown seedlings of S. cuminii, developed
shoots when cultured on MS medium (Murashige and Skoog, 1962), supplemented with 4percent
sucrose and 1 mg benzyladenine (BA)/litre. The so-developed shoots were rooted on Knop's
medium, augmented with2 % sucrose and 1 mg IAA/litre. For somatic embryogenes
is, hypocotyl segments derived from in vitro developed seedlings of A. Lebbeckwere cultured on
B5 containing 2 % sucrose. Media were gelled with either 3 % gum or 0.9 % agar. The
quantitative response obtained on media fortified with either of the gelling agents was not
significantly different. The media gelled with gum katira were almost as transparent as the liquid
medium. However, viscosity of gum katira gelled medium was less than one-sixth of the
viscosity of agar-gelled media, and therefore, shaking of the culture vessel often resulted in
submersion of the explants. Nevertheless, even these submerged explants responded positively.
To increase the firmness of the gum katira-gelled medium, various combinations of agar (0.2-0.6
%) and gum (1-3 %) were used. However, the viscosities of the media gelled with 3 % gum
katira as well as different concentrations of agar (0.2-0.6 %) were lower than that of the medium
containing only gum katira (3 %). Moreover, the explant productivity obtained in neither of these
combinations was more than those recorded on the control media, which were gelled either with
0.9 % agar or 3 % gum alone.

Azar Hussain et al. (2003) studied cost effective micropopagation technology for potatoes.
Isubgol husk was evaluated as a gelling agent for their performance in solidification of media for
in vitro micropopagation or production of virus free potatoes. The objective of this study was to
find a cheaper substitute for agar. It was concluded that Isubgol husk might emerge as a cheaper
alternative for agar that could lead to substantial reduction in cost of production per potato
planet. 

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Kumar (2003) studied the effects of eight nutrient media on the growth and sporulation of A. b
rassicaeThe maximum growth of the fungus was observed in both solid and liquid forms of
radish root extract, followed by carrot root extract, potato dextrose and oat meal agar. Poor was
observed on Asthana and Hawker’s, Richards’s, standard nutrient and Czapek (Dox) media. No
sporulation was observed in any of solid or liquid media.

3.2 Nutrient substitute

Adesemoye and Adedire (2005) reported the use of cereals as basal medium for the formulation
of alternative culture media for fungi. The feasibility of developing alternative media to different
culture media particularly potato dextrose agar was assessed using local cereal species as the
basal media. Three cereal meal extracts - corn, sorghum and millet were prepared, using them as

15
substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard
set up with which the performances of the formulated media were compared. Eight genera of
fungi (Aspergillus Niger, Fusarium moniliforme, Penicillium sp, Cercospora sp,
Curvularia palescens, Botryodiplopodia sp, Rhizopus sp. And Rhodotorula rubra) were isolated
and pure cultures of each species aseptically inoculated onto the three different
formulated media including PDA and allowed to grow. Their growths were measured at 24, 48,
72, and 96 hr after inoculation, using diameter of growth as an index. Growth of all the fungal
species were observed to be about the same or sometimes better in the formulated media relative
to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F.
moniliforme had an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at
96 hr while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 +
1.16 mm at 48 hr. Botryodiplopodiasp. Grew through the whole diameter of the plate (covering
the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 hr.

Pandey et al. (2006) studied the effect of various culture media on thegrowth, sporulation and
morphological variations of Alternaria alternate causing leaf spot, rust and anthracnose diseases
on Dolichos lablab.

Omemu et al.(2008) studied Maize Cob as Microbiological growth medium for fungi. Waste
cobs from variously processed (premature, uncooked, roasted,cooked salted, cooked unsalted)
maize (Zea mays) were used as raw materials to prepare growth agar media for six moulds:
Rhizopus nigricans, Aspergillus niger, Trichoderma viride, Alternaria tenuis, Fusarium
sp,Penicillium sp. and one yeast: Debaryomyces sp. All the formulated media supported the
growth of the microorganisms tested. Fusarium sp produced the widest diameter of growth
ranging from 40.0 mm on unsalted cooked cob agar (UCCA) to 54.0 mm on fresh cob agar
(FCA).The least diameter of growth was observed with Debaromyces sp.and it ranged from 0.1
mm on premature cob agar (PCA) to 0.9 mm on UCCA. The addition of dextrose showed that
except with Debaromycessp., the diameter of growth of fungi on media weresignificantly
(p<0.05) wider as compared to the formulated media without dextrose. Correlation between
addition of dextrose and diameter of growth was significant at 95 % confidence interval. The
diameter of growth was increased with increasing in time of incubation for all the media tested.

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For Fusarium sp., there were no differences in the diameter of growth at 48hr ofincubation and at
72hr on all the media tested.

Hassan and Bullerman (2009) studied wheat bran as an alternative substrate for macroconidia
formation by some Fusarium sp. Different plant preparations were evaluated fortheir ability to
support macroconidia formation byFusarium sp in an attempt to find a replacement for the
conventional Carnation Leaf Agar (CLA)media. Carnation leaves, typically require an expensive
and more complex preparation steps were replaced with corn hulls and wheat bran. This
studyillustrated the possibility of replacing CLA plates with a lower-cost simplified media
without affecting the morphological or growth characteristics of the isolated fungi. Sorensen and
Jens Laurids (2009) reported a semi-selective medium for isolation of Alternariaspp., Epicoccum
sp. and Phoma spp. from soil and plant samples where the basal medium was a modified potato
carrot agar (PCA).

Onygeme-okrenta et al. (2009) reported that cassava shavings significantly produced the highest
amount of mycelia weight of Penicillium chrysogen PCL501umthan all the other substrates viz.,
corncob, sawdust, and sugarcane pulp. Cassava shavings appeared to be the best substrate for
cultivating the organism. The substrate was better than glucose in terms of total sugar content
and mycelial production.

Mathew et al. (2010) studied a cheap nutritional liquid medium for enhancement of Trichoderma
harzianum and pseudomonas fluorescenspopulation. The result clearly indicated that 50 %
coconut or 25% coconut water supplemented with sugar @15g/l was cheap nutritional liquid
medium for mass multiplication of trichoderma harzianumand pseudomonas fluorescens.

Iran Zanjan (2010) studied various solidified date syrups as culture media and the effect of date
constituents with/without ultrasound wave’s irradiation using selected natural microflora of date
by agar dilution method and the results were compared with classical culture media containing
PDA for fungi and PCA for bacterial cultures. The results showed that, extracted date syrup by
ultrasound waves was an ideal media for enriching Aspergillus spp and Mucorspp growth.
Furthermore, it was demonstrated that date syrup containing media can also beused as a selective
media due to its inhibition to certain bacteria and fungi.Hence, there was an economical benefit

17
from the use of date syrup in the, preparati one of certain enrichment and selective
microbiological media.

Ravathie Arulanantham, Sevvel Pathmanathan, Nirmala Ravimannan and Kularajany Niranjan

(2012) used different formulation of protein sources for bacteria culture. The exorbitant costs of
culture media have deprived the use of readymade culture media such as nutrient agar in schools
and laboratories with less facility. Generally legume seeds are found to be a good protein source
for nutritional purposes. This study was carried out to find the feasibility of using legume seeds
as an alternative nutrient source to grow bacteria. Cowpea, green gram, black gram and soya
meat (processed soya bean) were used in this study. The test organisms used were E. coli,
Bacillus sp., Klebsiella sp., Staphyllococcus sp. and Pseudomonas sp. Staphyllococcus sp.
grows well but slowly (332 – 356 CFU/0.1ml) and generally Klebsiella sp. (196 - 232 CFU/0.1
ml) grows least in all the protein formulations tested. All the tested bacteria grow least in green
gram. In comparison with the performance on conventional nutrient agar media, the prepared
protein formulations were found to be cheap and good alternative culture media for
bacteriological studies.

4. MATERIALS AND METHODS

4.1General Procedure

 Collection of bacterial sample

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 The bacterial sample which was used was taken from department of biotechnology
elphinstone college Mumbai.

 Subculture and Maintenance of culture

For purification of bacterial culture, single bacterial colony of isolated bacterium was
picked up, streaked on NA slant (nutrient agar slant) to obtain pure. Bacterial cultures
were renewed twice in a week.
 Identification of the bacterial culture
The bacterial cultures were identified based on Gram staining, colour of bacterial
pigmentation and cell shape.

4.2 Test culture media

 Testing of solidifying agent

China grass, Sago and Isubgol were used as solidifying agent by replacing agar.
Materials

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 China grass, Isubgol, Sago, Distilled water, and glassware.

COMPOSITION OF MEDIA

China grass ( gm) Sago ( gm)
1 1
2 2
3 3
4 4
5 5
6 6
7 7
Isubgol (gm) Distilled water (ml)
1 100ml
2 100ml
3 100ml
4 100ml
5 100ml
6 100ml
7 100ml

Protocol

1. Weigh all the components according to composition.

2. Dissolve sago and isubgol in 100ml of dist. water.
3. Soak china grass for 10-15 minutes in distilled water.
4. Melt all the components in microwave.

5. Let it cool, cotton plug the flask and then autoclave it for 20 minutes.

 To rectify whether nutrients of agar-agar and china grass allow bacterial

growth or not

Materials:

Agar powder, china grass, distilled water and glassware’s

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 COMPOSITION OF MEDIA

Agar 3g

China grass – 5g

Distilled water 200ml

PREPARATION OF MEDIA

Protocol

1. Add 3g of agar powder in 100 ml of distilled water.

2. Soak china grass for 10-15 minutes in 50ml of distilled water in another conical flask.
3. Now add 50ml of distilled water in conical flask.
4. Melt china grass and agar in microwave.
5. Let it cool cotton plug the flask and then autoclave it for 20 minutes

 Test for bacterial growth

PREPARATION OF BACTERIAL SUSPENSION

1. Take 3 ml of sterile saline in a sterile test tube with sterile pipette in sterile condition.
2. Take a loop full of bacterial (E.coli) culture in sterile conditions. Now dip the loop full of
culture in sterile saline maintaining sterile conditions and mix it well.
3. Bacterial suspension is prepared.

 Streaking method
Material
Sterile plain agar plate, sterile plain china grass plate, nichrome loop and bacterial
suspension

Protocol
1. Take a loop full of culture in sterile conditions.

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 2. Streak on the plain agar plate in sterile conditions.
3. Incubate the plate at room temperature for 24 hrs.

 Preparation of nutrient media using china grass by replacing agar

Material

Distilled water, peptone, beef extract, NaCl, china grass ph paper and glassware’s

COMPOSITION OF MEDIA

NaCl - 0.75 gm

Peptone - 1.5 gm

Beef extract - 0.5 gm

China grass - 5 gm

Distilled water - 100 ml

PREPARATION OF MEDIA

Protocol

1. Weigh all the components as per the composition

2. Soak the china grass in 50 ml of distilled water for 10 to 15 minutes in a conical flask
(1st flask).
3. Then dissolve the other components in the remaining 50 ml of distilled water in
another conical flask (2nd flask).
4. Check the pH of the components dissolve in the 2nd conical flask.
5. Melt the china grass (1st flask) in microwave
6. Now pour the components of 2nd conical flask into the 1st flask and again melt all the
components again.
7. Cotton plug the conical flask and autoclave it for 20 minutes

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 Testing of bacterial growth using nutrient china grass medium

Materials

Sterile saline, nichrome loop, sterile pipette, sterile test tubes and fresh E.coli culture

PREPARATION OF SUSPENSION

Protocol

1. Take 3 ml of sterile saline in a sterile test tube with sterile pipette in Sterile
conditions.
2. Take a loop full of bacterial (E.coli) culture in sterile conditions.
3. Now dip the loop full of culture in sterile saline and mix it well

 Testing of bacterial growth

Materials
Sterile NG (Nutrient china grass), media plates, bacterial (E.coli)

Suspension , nichrome loop, and sterile glassware

Isolation bacteria

STEAKING METHOD

1. Take a loop full of culture from E.coli suspension in sterile conditions.

2. Now take a sterile NG media plate and streak the bacteria on the media maintaining
sterile conditions
3. Incubate plates at room temperature for 24hrs

 Testing of alternative nutrient sources by replacing the standard

nutrient source
Soya meat powder was used as alternative nutrient source in the routine medium to see

23
 their performance on the growth of bacteria.

 To check the pH of different sources which are used as alternative

nutrient in the preparation of media
Materials
Ph paper, distilled water and alternative nutrient sample (soya meat powder)

TEST FOR PH OF SOYA MEAT POWER IN DISTILLED WATER

1. Take 5ml of distilled water in a beaker and dissolve soya meat powder (0.5 -1.0 g) in
it .Stir it properly with glass rod.
2. Take pH paper and dip in the diluted solution to check the pH range

 Preparation of nutrient media using china grass and using alternative

nutrient source (soya meat)
Materials
Distilled water, china grass, soya meat powder, bacterial sample, sterile saline, sterile
sugar tubes, sterile medium size test tubes, sterile pipettes and other glassware’s
(manufactured nutrient agar was used as control to compare the growth with test culture
medium)
COMPOSITION OF BROTH
NB (NUTRIENT BROTH)
Nutrient broth powder 0.4gm
Distilled water 50ml
SOYA BROTH
Soya meat powder 1.5gm
Distilled water 50ml
PREPARATION OF BROTH CULTURE
Protocol
1. Weigh all the components according to the composition

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2. 1st flask take 50ml of distilled water add 0.4gm of nutrient broth powder
3. 2nd flask take 50ml of distilled water add 1.5gm of soya meat powder
4. Cotton plug the flask and autoclave the broth and cool it at room temperature
5. Transfer approximately 1ml of overnight E.coli culture to the flask
6. Seal the mouth of the flask with cotton plug
7. Incubate it overnight at 37 degree

Plating of E.coli
COMPOSITION OF MEDIUM
Soya meat powder 3gm
China grass 5gm
Distilled water 100ml
PREPARATION OF SOYA MEDIUM
1. Weigh all the components as per the composition
2. Soak the china grass in 120ml of distilled water for 10 to 15 minutes in a conical flask
(1st flask).
3. Then dissolve the other components in the remaining 30ml of distilled water in
another conical flask (2nd flask).
4. Melt the china grass (1st flask) in microwave
5. Now pour the components of 2nd conical flask into the 1st flask and again melt all the
components
6. Add 20ml of liquid medium in 5 sugar tubes
7. Cotton plug the conical flask and autoclave it for 20 minutes

PREPARATION OF SERIAL DILUTION

1. The first step in making a serial dilution is to take a known volume of E.coli bacterial
suspension 1ml from NB and place it into a known volume of sterile saline 4ml.
2. This produces 5ml of dilution solution
3. This makes 10-1 dilution then adds 1ml from 10-2 and repeats the same process for

25
 10-3, 10-4 and 10-5
4. Make 5 such test tubes
5. Take a known volume of E.coli bacterial suspension 1ml from Soya broth and place
it into a known volume of sterile saline 4ml.
6. This produces 5ml of dilution solution
This makes 10-1 dilution then adds 1ml from 10-2 and repeats the same process for
10-3, 10-4 and 10-5

POUR PLATE METHOD

1. Keep the SOYA butt and NA butt in water bath for 20 minutes. Don’t pour the agar
until it has cooled
2. Add the prepared dilutions in the butt
3. And pour the butt in sterile pertiplate in sterile condition
4. Incubate the plates at room temperature for 24hours

5. EXPRIMENTAL RESULTS AND DISSCUSION

The results obtained during present research on “Development of low cost media for bacteria
culture” are described under following sub heading

Testing of solidifying agent

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Three solidifying agent mainly china grass, sago and isubgol with various concentration were
tested for solidifying ability.

The results (Table 1) indicate that 1% to 7% of china grass formed solidified transparent
medium. 1% to 3% of sago formed non solidified opaque medium whereas 4% to 7% of sago
formed semisolid opaque medium.1% to 2% of
isubgol formed non solidified non transparent
medium whereas 3% to 7% formed semisolid
non transparent medium.

Thus the results indicated that china grass

formed solid transparent medium. 5% (5gm) of
china grass shows best solidification and
therefore agar can be replaced by china grass.
Which reduces the 70% cost bacterial medium.

Table 1 Effect of solidifying agents and its concentration on the solidification

and consistency of the medium
Name of solidifying agent with Solidifying ability Consistency and
different concentration in (%) transparency of the medium

China grass Sago Isubgol China Sago Isubgol China Sago Isubgol
grass grass
1% 1% 1% S NS NS TRP OP OP
2% 2% 2% S NS NS TRP OP OP
3% 3% 3% S NS SS TRP OP OP
4% 4% 4% S SS SS TRP OP OP
5% 5% 5% S SS SS TRP OP OP
6% 6% 6% S SS SS TRP OP OP
7% 7% 7% S SS SS TRP OP OP
NS=no solidification SS=semi solid medium S=solidified medium TRP=transparent OP=opaque

To rectify whether nutrients of agar and china grass allow bacterial

growth or not

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 Fig 1(a) sterile plain agar plate
Fig 1(b) sterile plain china grass plate

Loop full E.coli culture was streaked on

sterile plain agar plate and sterile plain
china grass plate and incubated at room
temperature for 24hours. After incubation it
was observed that there was no bacterial
growth on sterile plain china grass medium.

Therefore china grass can be used as solidifying agent by replacing agar.

Preparation of nutrient media using china grass

Media was prepared by replacing agar-agar with china grass and lab nutrients were used as
Nutrient source

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 Fig 2. Nutrient china
grass plate

E.coli streaked on NCG (nutrient china

grass medium) growth was observed in
clumps there must be problem in bacterial suspension or in technique,

To check the pH of different sources which are used as alternative nutrient

in the preparation of media

Test for ph of soya meat powder

Neutral ph 7.0

Efficacy of alternative nutrient source on the performance of china grass

medium
Soya meat powder was used as alternative nutrient source .Instead of readymade nutrient

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source. To compare the growth between two media pour plate method was used. Two sets of
serial dilution were prepared one of NB and other of soya meat powder and 24hour old broth
culture was used. Each dilution was inoculated in five different soya and nutrient agar buts
and plated on sterile perti plate

Fig 3
Soya broth after Nutrient broth after
24hr incubation 24hr incubation

The turbidity in the broth indicates that there is growth of bacteria in broth medium

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 Fig 4 (a) 10-4 Fig 4 (b) 10-5

The number of colonies grown on nutrient agar plate of 10-4 is 98 and on 10-5 is 35 the average
cfu/ml 2.24 x 10-3
Table 2 Number of colonies on nutrient agar plate

Dilution Number of
number colonies
10-1 ++++
10-2 ++++
10-3 ++++
10-4  98
10-5 35

++++=uncountable colonies

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 Fig 5 (a) soya china grass plate Fig 5 (b) soya china grass plate
10-4 10-5
The number of colonies grown on nutrient agar plate of 10-4 is 97 and on 10-5 is 35 the average
cfu/ml 2.23 x 10-5
Table 4 Number of colonies on soya plate

Dilution Number of
number colonies
10-1 ++++
10-2 ++++
10-3 ++++
10-4  97
10-5 35
++++ = uncountable colonies

Thus result (table 3, table 4 and) indicates that there is no difference in the growth of bacteria

32
 on soya china grass plate as compared with plate nutrient agar plate. The average cfu/ml of
NA is 2.24 x 10-3 and average cfu/ml of soya china grass is 2.23 x 10-3 . When E.coli was
grown on SCM (Soya china grass medium) there was no significant variation in colony
morphology.

Therefore soya + china grass medium seems to be ideal medium for growth of bacteria.

6. CONCLUSION

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Based on the findings of this study, it is concluded that china grass is best substitute of agar-agar
and soya meat is best substitute for nutrients in culture medium. The Soya meat + china grass is
an effective alternative culture media source next to nutrient agar to grow bacteria as there is not
much difference on number of colony grown on soya china grass medium as compared to
nutrient agar.

It should be noted that this study has used a common bacteria E.coli .It is important to note that
E.coli is generally used in microbiology studies in almost all the labs.

7. FUTURE PROSPECTS

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It is recommended that further research be conducted with other possible protein sources for
production of bacterial culture medium

Use of domestic waste and flower waste is recommended for further research for production of
bacterial culture medium.

8. BIBLIOGRAPHY

35
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Pandey, B.N., S.P Srivastava and R.K Srivastava 2006 studies on effect of various culture media
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