Shubham pawar.

SY BSC BIOTECHNOLOGY
FS 17027
DEVELOPMENT OF LOW COST MEDIA FOR BACTERIOLOGY
ELPHINSTONE COLLEGE
 INTRODUCTION
To isolate, culture and maintenance of microorganisms culture media are required.
These cultural media are prepared in laboratories. The media and their constituents
are costly and may not be available at remote place laboratories. Therefore, the
research workers are always in search of cheap and alternative source of the
mediumMicroorganisms need nutrients, a source of energy and certain
environmental condition in order to grow and reproduce. In environment, microbes
adapt to the habitatmost suitable for their needs while in laboratories these
requirements is met by a culture medium.
The growth of microorganisms in an artificial medium is influenced by several
physical and chemical factors. A nutrients material prepared for growth of
microorganisms in laboratory is called culture media.
AGAR- AGAR
Agar is complex polysaccharide extractedfrom several types and species of red
seaweed belonging to the Rhodophyceae class.
It is insoluble in cold water but soluble in boiling water 1.5% solution is clear and
when it is cooled to 34-43 degree it form a firm gel which doesn’t melt again below
85 degree. Polysaccharide can be sulphated in very variable degrees but to a lesser
degree than in carrageen. A 5% maximum ash content acceptable for agar although
it is normally maintained between 2.5 to 4%.
HISTORY OF AGAR-AGAR
Agar was accidently discovered in japan in 1600S by an Innkeeprminotarozaemon
who was serving some special guest a very hard- to -make seaweed jelly noodles.
To make this dish the seaweed jelly had to be painstakingly dried boiled for several
days so it was a dish that was reserved only for rich. But sadly there were lot of
leftovers that night and had to be thrown out. But when he woke up the next morning
he saw that jelly noodles had dried up in the snow overnight into a paper like
substance from that he developed “KANTEN” which is our modern day “AGAR-
AGAR.”
Agar was first subjected to chemical analysis in 1859 by the French chemist
Anselempayen who had obtained agar from marine algae Gelidiumcorneum.
Beginning in late 19thcentury agar begin to be used heavily as a solid medium for
growing various microbes. Agar quickly supplanted gelatine as the base of
microbiological media due to it’s higher melting temperature allowing microbes to be
grown at higher temperature without the media liquefying.
SOURCES OF AGAR
Agar-agar is extracted from several types and species of red seaweed belonging
to the Rhodophyceae class. These agar containing seaweeds are called
agarophytes and the major commercial species are Gracilaria, Gelidium and
Pterocladia.
The agar content of seaweeds varies according to the concentration, oxygen
tension, water temperature and intensity of solar radiation can have significant
 influence. Seaweeds are usually harvested manually by fishermen in low depths at
low tides or by diving using appropriate equipment.
PRODUCTION OF AGAR
1. The seaweed treatment prior extraction.
2. The control of molecular weight distribution during the extraction.
3. The removal of undesired products.
4. The need to work with large volumes of diluted extracts.
5. The economics of dehydrating the dilute extracts.

CHINA GRASS
China grass is also known as agar-agar, faluda Japanese moss, Ceylon moss and
Bengal lsinglass. It is obtained from various seaweed has no aroma and has a neutral
taste and are 100% veg.
1. Nutritional information of china grass per unit /8g.
2. Energy per kcal 24.88.
3. Carbohydrates 6.20
4. Protein 0.00
5. Fat 0.00
PRICE DISCRIMINATION BETWEEN CHINA GRASS AND AGAR-AGAR

Agar-Agar is mostly sold in the form of strips or powder. Most of the agar
seaweed are imported from different countries like japan, Taiwan, Philippines, brazil,
south Africa, Madagascar.

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 various according to
it’s concentration.

China grass

China grass is very cheap and can be used as substitute of agar-agar in

microbiology. For example 500g of china grass cost upto 800 to 900 rupees. The
best quality of china grass can cost upto 1800 to 2000 per kg.

One the yearly basis every college spend 40,000 to 50,000 on 5kg agar which is
used in laboratory. Where else in same price 20-25kg of china grass can be
purchased.

DEVELOPMENT OF LOW COST MEDIA FOR BACTERIOLOGY WAS UNDERTAKRN

WITH FOLLOWING OBJECTIVE

 To search low cost medium for bacterial growth and reproduce by replacing agar.
 To search low cost nutrients for bacterial culture media.
 METHODS

AIM :To estimate cheap nutrient media for bacteriology using tang
powder,electrolyte powder and china grass.

MATERIALS

tang powder (lemon flavour), electrolyte powder, oats powder china grass ,pH
paper , distilled water and glasswares.

PROTOCOL

COMPOSITION OF MEDIA COMPONENTS

Tang powder 2.5g

Electrolyte powder 1g
Oats powder 0.2g
China grass 4.5g
Distilled water 100ml

PREPARATION OF MEDIA

1. Weigh all the components according to the composition.

2. Soak the china grass in 80 ml distill water in a conical flask (1st flask).
3. Now dissolve the tang powder, oats powder and electrolyte powder in 20ml of
distilled water in a beaker or another conical flask (2nd flask)8
4. Now put 1stconical flask in microwave until the china. grass gets completely
dissolved in water.
5. Now add the mix dissolved in 20ml of distilled water to the 1st conical flask by
filtering it with. whattman paper 1 so that the heavy particles are removed out.
6. Again dissolve this mixture in until the particles are unseen.
7. Let it cool cotton plug the flask and then autoclave it for 30 mins.
AIM:To rectify whether nutrients of china grass allow bacterial growth.

MATERIALS

china grass, distilled water

PROTOCOL

COMPOSITION OF MEDIA COMPONENTS

China grass 4.5g

Distilled water 100ml

PREPARATION OF MEDIA

1. Soak china grass for 15-20 minutes in 50ml of distilled water in conical flask.
2. Now add 50ml of distilled water in conical flask.
3. Now put the flask in microwave so china grass will. get completely dissolved
in distilled water.
4. Let it cool cotton plug the flask and then autoclave it for 30 mins.
5. Test for bacterial growth :
6. Preparation on bacterial suspension :

7. Withdraw 3 ml of sterile saline in a sterile test tube with sterile pipette in

invitro (Sterile) conditions.
8. Take a loop full of bacterial (E.coli) culture in sterile conditions.

9. Now dip the loop full of culture in sterile saline maintaining sterile conditions
and mix it well. A bacterial suspension is prepared.

STREAK METHOD
MATERIAL
Sterile plain china grass plate, nicrome loop and bacterial suspension.

PROTOCOL

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

2. Streak on the plain china grass plate in sterile conditions.
3. Incubate the plate for 24 hrs at 37 °C and observe it.
AIM : To estimate cheap nutrient media for bacteriology using tang powder
and china grass.

MATERIALS

Tang powder (lemon flavor), china grass , Distilled water and glassware’s.

PROTOCOL

COMPOSITION OF MEDIA COMPONENTS

Tang powder - 3.5g

China grass - 5g

Distilled water - 100 ml

PREPARATION OF MEDIA

1. Weigh all the components according to the composition.

2. Soak the china grass in 80ml of Distilled water in conical flask(1st flask)
3. Now dissolve the tang powder in 20ml of Distilled water in a beaker or another
conical flask(2nd flask)
4. Now put, 1st conical flask in microwave until the china grass gets completely
dissolved in water.
5. Now add the mixture dissolved in 20ml of Distilled water to the 1st conical flask.
6. let it cool cotton plug the flask and then autoclave it for 30 mins.
AIM :To estimate the solubility of china grass in different concentration of Distilled
water.

MATERIALS

China grass , Distilled water, and glasswares.

PROTOCOL

1 CONICAL FLASK

COMPOSITION OF MEDIA COMPONENTS

China grass - 1.5g

Distilled water - 50 ml

PREPARATION OF MEDIA

1. Weigh all the components according to composition.

2. Soak china grass for 15-20 mins in 30ml of Distilled water in conical flask.
3. Now add remaining 20 ml of Distilled water in conical flask.
4. Now put the flask in microwave so china grass will get completely dissolve in
Distilled water.
5. Let it cool, cotton plug the flask and then autoclave it for 30 mins.

2 CONICAL FLASK

COMPOSITION OF MEDIA COMPONENTS

China grass - 2.5g

Distilled water - 100 ml

PREPARATION OF MEDIA

1. Weigh all the components according to composition.

2. Soak china grass for 15-20 mins in 50ml of Distilled water in conical flask.
3. Now add remaining 50 ml of Distilled water in conical flask.
4. Now put the flask in microwave so china grass will get completely dissolve in
Distilled water.
5. Let it cool, cotton plug the flask and then autoclave it for 30 mins.

3 CONICAL FLASK

COMPOSITION OF MEDIA COMPONENTS

 China grass - 5g

Distilled water - 100 ml

PREPARATION OF MEDIA

1. Weigh all the components according to composition.

2. Soak china grass for 15-20 mins in 50ml of Distilled water in conical flask.
3. Now add remaining 50 ml of Distilled water in conical flask.
4. Now put the flask in microwave so china grass will get completely dissolve in
Distilled water.
5. Let it cool, cotton plug the flask and then autoclave it for 30 mins.

AIM :To check the pH of different sources which can be used as cheap nutrients in the
preparation of media.

MATERIALS
pH paper, Distilled water,warm milk, cold milk,cheap nutritional samples
(bournvita n tang powder) and glassware.

METHOD:

TEST FOR PH OF BOURNVITA IN DISTILLED WATER

1. Take a approx amount (10 ml) of distilled water in a beaker and dissolve some
amount of bournvita (0.5 -1.0 g) in it .Stir it properly with glass rod for proper
dilution.
2. Take pH paper and dip in the diluted solution to check the pH range.

TEST FOR PH OF BOURNVITA IN WARM MILK :

1. Take a approx amount of warm milk (10 ml) in a beaker and dissolve some
amount of bournvita (0.5 - 1.0 g) in it.Stir it properly with glass rod for proper
dilution
2. Take pH paper and dip in the diluted solution to check the pH range

TEST FOR PH OF BOURNVITA IN COLD MILK

1. Take aapprox amount of cold milk (10 ml) in a beaker and dissolve some amount
of bournvita.
2. Take pH paper and dip in the diluted solution to check the pH range

TEST FOR PH OF TANG POWER IN DISTILLED WATER

1. Take a approx amount (10 ml) of distill water in a beaker and dissolve some
amount of tang powder (0.5 -1.0 g) in it .Stir it properly with glass rod for proper
dilution.
2. Take pH paper and dip in the diluted solution to check the pH rang
Preparation of nutrient media using china grass (Agar-Agar) by

replacing agar

Materials
Distill water, peptone, yeast or meat extract, NaCl, china grass (Agar - Agar) ,
pH paper and glasswares.

Protocol
COMPOSITION OF MEDIA COMPONENTS :

NaCl - 0.75 g

Peptone - 1.5 g

Meat or yeast extract - 0.5 g

China grass - 5.5 g

Distill water - 150 ml

PREPARATION OF MEDIA

1. Weigh all the components as per the compostion

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. 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
 7. Cotton plug the conical flask and autoclave it for 1hour.

Test for bacteriology using nutrient china grass medium

PREPARATION OF BACTERIAL SUSPENSION
Materials
Sterile saline, nichrome loop, sterile pipette, sterile test tubes and fresh
culture of E.coli
PREPARATION OF SALINE
Weigh 0.85 gm NaCl.

Dissolve it properly in 100 ml of distilled water.

Autoclave it for 1hour

PREPARATION OF SUSPENSION

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 maintaining sterile
conditions and mix it well.
4. Bacterial suspension is prepared.

TEST FOR BACTERIAL GROWTH

Materials
Sterile NG (Nutrient china grass) media plates, bacterial (E.coli) suspension,
nichrome loop, and sterile glassware
ISOLATION OF 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

SPREAD PLATE METHOD

1. Withdrawn 0.1 ml of E.coli suspension by sterile pipette and spray it on

the sterile NG media plate maintaining sterile condition.
2. Take the spreader and dip it in 70 % alcohol and flame it.
3. Spread the suspension sprayed on plate with the help of spreader all
through the edges till the surface feels rough.
4. Incubate the plate for 24 - 48 hours at 37°C.

OBSERVATION.

E.coliwas streaked on plain china grass NO GROWTHwas observed.

E.coli bacteria was grown on nutrient china grass media by T-streak method.

E.coli bacteria was grown on nutrient china grass media by polygonal streak method.
 RESULT AND DISSCUSSION

 CHEAP NUTRIENT MEDIA FOR BACTERIOLOGY USING TANG POWDER,

ELECTROLYTE POWDER AND CHINA GRASS.

No solidification because of acidic nature of tang and electrolyte powder which

causes acid hydrolysis. Tang and eletolyte powder cleave the chemical bond via
nucleophilic substitution reaction with the addition of the element of water.

 CHEAP NUTRIENT MEDIA FOR BACTERIOLOGY USING TANG POWDER

AND CHINA GRASS.

No solidification because of acidic nature of tang powder which causes acid

hydrolysis. Tang powder cleave the chemical bond via nucleophilic substitution reaction
with the addition of the element of water.

 THE SOLUBILITY OF CHINA GRASS IN DIFFERENT CONCENTRATION OF

DISTILLED WATER
 China grass was solidify in different concentration of distilled water. I have observed
that solidification of 5g china grass in 100ml distilled water was one of the best solidify
sample.

 pH OF DIFFERENT SOURCES WHICH CAN BE USED AS CHEAP NUTRIENT

IN THE PREPARATION OF MEDIA

 pH of bournvita in WATER is acidic.

 pH of bournvita in WARM MILK is acidic.
 pH of bournvita in COLD MILK is acidic.
 pH of tang in WATER is also acidic.

 TO CHECK THE STRENGTH OF CHINA GRASS

Media solidifiy and there was no bacterial growth after incubation of 24 hours at
37 degree Celsius.

 PREPARATION OF NUTRIENT MEDIA USING CHINA GRASS BY

REPLACING AGAR

Media was successfully prepared

 TEST FOR BACTERIAL GROWTH

No growth because there was problem in media or bacterial suspension

CONCLUSION

The above tests conclude china grass is eligible for culture medium by replacing agar.
Because properties of china grass allowed it for bacterial growth, reproduce and
contact. It is a cheap source which is available anywhere around the world. Bacteriology
can be perform using china grass as agar substituent.
 FUTURE PERSPECTIVE

The knowledge we get form this research can help in preparation of different types of
culture media and cheap nutrient by using china grass.

Here, is the some examples of media in which we can use china grass instead of agar-
agarin future.

1. Preparation of Drosophila media

2. Preparation of Mac conkeys media
3. Preparation of Gel electrophoresis media
4. Preparation of Gibson media
5. Preparation of Murashige media