a) FIELD OF INVENTION:
The present invention relates to microbial media solidification agent. It is aimed to replace current expensive agar agar and work at extreme pHs and temperature at low cost.
b) BACKGROUND OF INVENTION:
A culture medium may either be a liquid or gelled substance that supports the growth of microorganisms under laboratory conditions. Various media are used for growing different types of organisms. The use of agar was first proposed by Hesse (1881) for microbiological purpose. By early 1900s, it was widely used as it remains firm at growth temperatures as high as 45°C for many pathogens. Conversely, it melts at approximately 55°C. Moreover, agar is generally resistant to shearing forces but different agars may have different gel strengths or degrees of stiffness (Hesse, 1894; Hitchens and Leikind, 1939). Although, agar is still the most widely used solidifying agent for microbial culture media, the exclusive use of agar may result in over-exploitation of its resources. Moreover, high cost of bacteriological grade agar necessitates that alternatives be sought (Jain et al., 1997; Babbar and Jain, 1998; Jain and Babbar, 2002). A range of gelling agents such as corn flour (Zea mays var. amylacea), isubgol husk (Plantago ovata), coconut powder (Cocos nucifera) and many others are available; arrowroot (derived from the roots of a tropical South American plant Maranta arundinacea) powdery white arrowroot starch resembling corn flour. Coconut powder is obtained by drying the granulated or shredded white, fully mature coconut kernel, by means of a mechanical air drying. Corn flour or cornrneal is flour ground from dried maize or American corn. Gel rite is a water soluble polysaccharide produced by Sphingomonas elodea, a bacterium. Glue which is an adhesive, or glue, is a mixture in a liquid or semi-liquid state that adheres or bonds items together. Adhesives may come from either natural or synthetic sources. Katira gum, which has only been available commercially since 1920, forms an extremely strong adhesive with small amounts of water. Katira gum is the resin extracted from a particular tree. It looks like golden orange tan small balls. Katira gum occurs as tears of variable size and of a somewhat crystalline appearance. Guar gum is a polysaccharide with a straight chain of D-mannopyranose units joined by p (1-4) linkages with a side branching unit of a single D-galactopyronase joined to every other mannose unit by L (1-6) linkages. It is a white to yellowish-white powder, practically odorless and has a bland taste. It will disperse and swell almost completely in hot or cold water and is insoluble in organic solvents. The viscosity depends on temperature, time, pH, agitation rate and particle size of the powder. Pectin was first isolated and described by Henri Braconnot (1825) from Greekpektikos, "congealed, curdled" which is a structural hetero polysaccharide contained in the primary cell walls of terrestrial plants. It is produced commercially as a white to light brown powder, mainly extracted from citrus fruits. Rice powder, also rice flour is a form of flour made from finely milled rice. It is distinct from rice starch, which is usually produced by steeping rice in lye. Rice flour may be made from either white rice or brown rice. To make the flour, the husk of rice or paddy is removed and raw rice is obtained which is then ground to flour.In the recent past, a number of substances namely, agarose (Johansson, 1988), carrageenan (Lines, 1977; Bromke and Furiga, 1991), katira gum (Jain and Babbar, 2002), isubgol (Jain et al., 1997; Babbar and Jain, 1998; Ozel et al., 2008; Atici et al., 2008; Saglam and Ciftci, 2010), kappa-carrageenan (Abbott et al., 1981), starch (Henderson and Kinnersley, 1988; Zimmerman et al., 1995; Nene et al., 1996), guar gum (Babbar et al., 2004), gel rite (Harris, 1985), chickpea dextrose tapioca (CDT) (Nene et

al., 1996) and gellan gum (Shungu et al., 1983) have been used. But could not replace Agar-Agar as microbiological solidification agents. The present studies were undertaken to validate the cheap and easily available substitutes of agar to be used in microbiological culture media.
Solidified media are indispensable in various aspects of microbiological research. For solid cultures of mesophilic microorganisms, agar is commonly used as a solidifying agent. It is anonionic polysaccharide consisting mainly of D-galactose and 3, 6-anhydro-L-galactose and is produced by sea-weeds. Ease of the handling, resistance to enzymatic degradation by most microorganisms, and transparency of solidified plates make agar an ideal solidifying agent, and agar-supported media have been used essentially unchanged since they were first introduced in the late nineteenth century.
The situation is very different when it comes to culturing extremophiles on solidified media. For example, agar media are not suitable for culturing thermophiles and hyperthermophiles because the solidification of agar is thermoreversible at around 50-60°C.
Agar has many other properties that make it desirable as a gelling agent for microbial culture media. In particular, agar remains solid at 37°C (human body temperature) and, after melting during the sterilization process, remains liquid to about 45°C, at which time it can be poured into sterile vessels or Petri dish plates. In addition, unlike gelatin, which many bacteria can degrade, causing the medium to liquefy, agar is not degraded by most bacteria. Agar also renders most solid culture media transparent, making it easier to differentiate bacterial colonies from inanimate particulate matter suspended in the medium. Hence, agar found its place early in the annals of microbiology and is still used today all over the world for obtaining and maintaining pure cultures of bacteria.
Agar, the most commonly used solidifying agent for culture media, sets at about 45 °C and Gelrite, an alternative, has a holding temperature of 60 °C (Shungu et al, 1983). Therefore these substances may not be used to culture heat-sensitive bacteria except by surface inoculation of the pre-set medium. Gelatin sets at about 25 °C but is liquefied by proteolytic bacteria and, of the 'cold setting' agents, silica gel is tedious to prepare. Among other gelling agents which have been used for specialized applications are methyl cellulose, Polycell cellulose paste and alginate gel. Semi-solid gels and the introduction of precipitates such as AIPO4 have also contributed towards the successful isolation of bacteria.
The present innovation aims to replace Agar-Agar and solution to prepare solid media at extreme pHs and high temperatures.

c) SUMMARY OF INVENTION:
Problem to be solved:
1. Microbiological media solidification agent Agar-Agar is expensive and costing around 10 rupees per plate.
2. Agar based solid media does not get solidified at acidic and alkaline pHs.
3. Agar based microbiological solid media shows cracking at 50°C and above temperatures.
Means of solving problem:
1. Matrix formed with silica gel and Agar-Agar is a good solidifying agent.
2. Agar concentration can be reduced from 2% to 0.5%.
EFFECT OF INVENTION:
1. Agar silica gel matrix is hard enough for streaking and spreading.
2. Solid media tolerates extreme pH and temperatures.
3. Microbial degradation is not observed.
d) BRIEF DESCRIPTION OF THE DRAWINGS:
Figure-1:
It is Petri-plate (1) with solid medium (2) prepared with invented solidification medium.

e) DETAILED DESCRIPTION OF THE INVENTION:
1. The current invention relates to solve the problem of expensive agar and to make the media cost efficient utilizing agar agar and silica gel.
2. In another aspect the present invention also relates to process of preparation of microbial solidification agent which is used as supplement for standard agar plates comprising 0.2-0.5% silica gel and 0.2-0.5% Agar-Agar.
3. In another aspect the present invention also relates to the process of preparation of microbial solidification agents nutrients as per requirement added to 900ml of distilled water, 5g of each of Silica gel and Agar-Agar were taken together and autoclaved at 10/15 LBS for 20/30min and cooled to a temperature of about 40-45°C at room temperature and is then poured into sterile Petri plates under sterile conditions and left to solidify.
4. In another aspect, the current invention also relates to preparation of semi-solid media sterilizing the semi solid media by autoclaving and usage by taking 0.25% each of agar-agar and silica gel along with the required nutrients.
Examples:
1. Preparation of solid Nutrient Media:
In a laboratory grade 2L Erlenmeyer flask; 3gms yeast extract, 5gms peptone, 5gm sodium
chloride, 5 gms agar-agar and 5gms silica gel to make 1 Litre with distilled or deionized water.
The suspension is then heated to boiling to dissolve the medium completely. The dissolved
medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes. Once autoclaving is
complete, the flask is taken out and cooled to a temperature of about 40-45°C at room
temperature conditions.
If enrichment is desired, blood or other biological fluids can be added. The media is then poured
into sterile Petri plates under sterile conditions.
Once the media solidifies, the plates can be placed at a lower heat setting for a few minutes to
remove any moisture present on the plates before use, plates can be kept in refrigerator at 4°C
till use.
2. Preparation of Solid Media at extreme pH:
In a laboratory grade 2L Erlenmeyer flask; 3gms yeast extract, 5gms peptone, 5gm sodium chloride, 5 gms agar-agar and 5gms silica gel to make 1 Litre with distilled or deionized water. pH adjusted to acidic 2-5 with Sulphuric acid and alkaline 10-12 with NaOH separately. The suspension is then heated to boiling to dissolve the medium completely. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes. Once autoclaving is complete, the flask is taken out and cooled to a temperature of about 40-45°C at room temperature conditions.
The media is then poured into sterile Petri plates under sterile conditions. Once the media
solidifies, the plates can be placed at a lower heat setting for a few minutes to remove any
moisture present on the plates before use, plates can be kept in refrigerator at 4°C till use.
Proper solidification was observed at acidic and alkaline conditions. »
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3. Preparation of Solid Media and incubation at high temperature:
In a laboratory grade 2L Erlenmeyer flask; 3gms yeast extract, 5gms peptone, 5gms Sodium chloride, 5 gms agar-agar and 5gms silica gel to make ILitre with distilled or deionized water. The suspension is then heated to boiling to dissolve the medium completely. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes. Once autoclaving is complete, the flask is taken out and cooled to a temperature of about 40-45°C at room temperature conditions. The media is then poured into sterile Petri ^plates under sterile conditions. After inoculation with bacteria plates were incubated at 60 to 75°C. At high temperature no breaks were observed in the media.
4. Composition of different solid media with solidifying agents:
a) Nutrient Agar: beef extract-lg, yeast extract- 2g, peptone-5g, sodium chloride (Nacl)-5g, agar- agar-5g, silica gel -5g with distilled water make to 1L.
b) Potato Dextrose Agar: potatoes starch-4g (from 200g potato infusion), dextrose
- 20g, agar-agar- 5g, silica gel - 5g with distilled water make to 1L.
c) Blood Agar: peptone-5g, Nacl-5g, yeast extract-3g, agar-agar-5g, silica gel-5g, 900ml distilled water, mammalian blood-25ml after autoclaving and cooling to 450°C.
d) Chocolate Agar: In 900ml water casein/Peptone digest-15g, cornstarch-lg, potassium phosphate (dibasic)-4g, potassium phosphate (monbasic)-lg, sodium chloride - 5g Agar-Agar-5g, silica gel-5g, 25ml Blood after autoclaving then heating on flame to get chocolate color.
e) MRS Agar: peptone-lOg, beef extract- lOg, yeast extract-4g, glucose-20g, sodium acetate trihydrate-5g, polysorbate 80-1 g, dipotassium hydrogen phosphate-2g, triammonium citrate-2g, magnesium sulfate heptahydrate -0.2g, manganese sulfate tetrahydrate-0.05g, agar-agar-5g, silica gel-5g with distilled water make to 1L.
f) Bristol Agar: NaN03-25g, CaCl2.2H20-2.5g, MgS04.7H20-7.5g, K2HP04 -7.5g, KH2P04 -17.5g, NaCl -2.5g, agar-agar -5g, silica gel-5g with distilled water make to 1L.
g) MacConkey Agar: peptone(pancreatic digest of gelatin)-17g, proteose peptone (meat and casein)-3g, lactose monohydrate-lOg, bile salts-1.5g, sodium chloride
- 5g, neutral red-0.03g, crystal violet-O.OOlg, agar-agar-5g, silica gel-5g with
distilled water make to 1L.
h) SS Agar: beef extract-5g, enzymatic digest of casein-2.50g, enzymatic digest of animal tissue-2.50g, lactose-lO.Og, bile salts-8.50g, sodium citrate-8.50g, sodium thiosulfate-8.50g, ferric citrate-1.00g, brilliant green-0.00033g, neutral red-0.025g, agar-agar-5g, silica gel-5g with distilled water make to 1L.

5. Inoculation and Microbial growth Characteristics on solid media:
Streak plate: E Coli and Bacillus subtillis were streaked on solid nutrient medium
prepared with solidification agent. Normal streaking was observed but shearing was
observed when high pressure is applied. After 18-24hrs normal colony morphology was
observed.
Spread plate: E Coli and Bacillus subtillis were spreaded on solid nutrient medium
prepared with solidification agent. Normal spread was observed. After 18-24hrs normal
colony morphology was observed.
6. Preparation of semi-solid media and Motility checking by semi-solid media:
Semi solid Nutrient Agar: beef extract-O.lg, yeast extract-0.2g, peptone-0.5g, sodium
chloride (Nacl)-0.5g ,agar- agar-0.2g, silica gel-0.2g added & 100ml distilled water.
Heat the above medium till clear suspension is obtained. Transfer 10ml semi solid
medium in a test tube and plug with non absorbant cotton and sterilize. After sterilization
keep tubes upright for cooling. Inoculate bacteria with stroke in tube. Motile bacteria
showed zigzag movement where as non motile bacteria showed single line growth.