Title of Invention	A CULTURE MEDIUM COMPOSITION
Abstract	The present invention relates to a culture medium composition for fungi, said composition comprising a) at least one carbohydrate source in an amount of 1% to 5% of the mass of the composition; b) sodium chloride in an amount of about 0.05 % to about 0.3% of the mass of the composition; c) disodium hydrogen orthophosphate dihydrate in an amount of about 0.02 % to about 0.1 % of the mass of the composition ; d) potassium nitrate in an amount of about 0.01 % to about 0.1 % of the mass of the composition; e) ammonium persulfate in an amount of about 0.01% to about 0.2 % of the mass of the composition; f) agar in an amount of about 1 to about 2% of the mass of the composition; and g) distilled water.

Title of Invention

A CULTURE MEDIUM COMPOSITION

Abstract

The present invention relates to a culture medium composition for fungi, said composition comprising a) at least one carbohydrate source in an amount of 1% to 5% of the mass of the composition; b) sodium chloride in an amount of about 0.05 % to about 0.3% of the mass of the composition; c) disodium hydrogen orthophosphate dihydrate in an amount of about 0.02 % to about 0.1 % of the mass of the composition ; d) potassium nitrate in an amount of about 0.01 % to about 0.1 % of the mass of the composition; e) ammonium persulfate in an amount of about 0.01% to about 0.2 % of the mass of the composition; f) agar in an amount of about 1 to about 2% of the mass of the composition; and g) distilled water.

Full Text	FORM-2 THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003 PROVISIONAL SPECIFICATION (See section 10; rule 13) A CULTURE MEDIUM COMPOSITION (a) HARNE SHRIKANT RAMDAS An Indian National of "Rajkamal", Moti Nagar, Pusad, Tal-Pusad, Dist. Yawatmal-445204, Maharashtra, India; (b) LA WARE SHANKAR LAXMAN an Indian National of 86/15, Om Namah Shivay Raj Park, Sairaj Residency, New Sangvi, Pune 411 027, Maharashtra, India; and (c) SECRETARY, DECCAN EDUCATION SOCIETY an Indian National of Deccan Education Society, Fergusson College Campus, F.C.Road, Pune 411 004, Maharashtra, India THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION FIELD OF THE INVENTION The present invention relates to a culture medium composition. BACKGROUND OF THE INVENTION A growth medium or a culture medium is a liquid or gel designed to support the growth of microorganisms. There are two major types of growth media. One is used for cell culture that uses specific cell types derived from plants or animals. Second is microbiological culture media which is used for growing microorganisms, such as bacteria or fungi. Contamination in the microbial cultures is a common problem faced by almost all the pathologists, microbiologists and lab technicians. These contaminants generally come from the environment and are always difficult to eradicate completely. Bacteria are the most common group of contaminants which hamper the processes of isolation of fungi, purification of fungi and sub-culturing of pure colonies of industrially and medically important fungal specimens. A wide range of media are used for growing fungi and a variety of media are available for the primary inoculation and recovery of fungi from clinical specimens. There is not a single specific medium. or combination of media which is adequate for all such specimens. Most mycologists develop preferences for certain types of media based on experience and peculiarities of the type of fungi that are routinely grown. The characteristics such as colony morphology, color, etc. are affected by the medium used. Further, the type of carbohydrate used in the medium determines the growth rate of fungi. There appears to be a 1^-ge number of carbohydrates, but the one which is easy to metabolize is preferred over others, e.g. Glucose (dextrose) is the simplest carbohydrate whic;h can easily be taken up by the microorganisms and hence is the most commonly used in the growth media. Fructose and mannose are the next most commonly utilized sugars by fungi and are found in media from natural sources. Sucrose (table sugar) may also be used in some media. There are certain media which makes use of complex carbohydrates, for example, PDA contains starch, for utilization of which, production of amylase enzyme is a must. Some fungi lack the necessary enzyme(s) to utilize such sources of carbohydrates and hence cannot grow. All fungi require several specific elements for growth and reproduction. The requirements for growth are generally less stringent than for sporulation, so it is often necessary to try several types of media when attempting to identify a fungus in a culture. Most fungi thrive on Potato Dextrose Agar (PDA), but this can be too rich for many fungi, hence excessive mycelial growth is obtained at the expense of sporulation. Similarly, wood-inhabiting fungi and dematiaceous (dark pigmented) fungi often sporulate better on Corn Meal Agar (CMA) or Oat Agar, both of which have less easily digestible carbohydrate than PDA. Most fungi are difficult to isolate from the infected tissues, infested soil or decaying organic material due to the rapid and often antagonistic development of associated bacteria and actinomycetes with faster growth rates. Successful isolation of many of these fungi can be achieved by the use of selective media that either slow down the growth or inhibit the growth of these antagonistic organisms. One approach is to surface sterilize the material to eliminate surface contaminants, and then place the material on Water Agar (WA). Most fungi and bacteria will grow on WA, but at such a slow rate that it is relatively easy to isolate the target fungus. Selective exclusion can be accomplished by several approaches known in the prior art. 1. One approach is by selective inhibition i.e. the use of antimicrobial chemicals, primarily antibiotics such as streptomycin sulfate and penicillin. An Antibiotic Agar (AA) was developed to serve this purpose. Antibiotic agar is a choice of agar when the isolation is done from a material that cannot be surface sterilized and hence have a heavy load of bacteria. However, some bacteria show resistance to antibiotics or some times develop resistance after some days of culture and interfere with the isolation of fungi and during maintenance of pure cultures. 3 2. Another approach is to create an unfavorable environment in the isolation medium such as high pH in which fungus would grow but bacteria would be inhibited. Acidified Commeal Agar (ACMA) works well for the isolation of fungi from various types of plant material. It is best to use surface-sterilized material in conjunction with ACMA. However, under this condition, the possibility of growth of acidophilic and/or acidotolerant bacteria is more. 3. The third approach is the use of a specific medium is the one which allows the growth of only one type of microorganism and inhibits/suppresses the growth of others such as use of high sugar concentration (Saborauds Dextrose Agar), This method suffers a major drawback as a higher sugar concentration may induce excessive mycelia growth which might not be desirable. 4. During the process of isolation and purification of fungi from various sources such as soil, water, air, wood, etc., the possibility of bacterial contamination is much higher. To avoid these problems, continuous sub culturing techniques are used. This generally takes more time, and results into contamination by unwanted organisms and also increases the cost of purification. Accordingly, it is desirable to produce a culture medium which can avoid bacterial contamination and selectively support the growth of fungi only. Furthermore, the medium must be cost effective. OBJECTS OF THE INVENTION It is an object of the present invention to provide a culture medium composition which supports growth of selective microorganism. It is another object of the present invention to provide a culture medium composition which has antibacterial activity. It is still another object of the invention to provide a culture medium composition which is easy to prepare and cost effective. 4 It is yet another object of the invention to provide a culture medium composition which is used for isolation of particular microorganism from clinical samples, soil, sewage water, milk and air. It is a further object of the invention to provide a culture medium composition which is used for production of alcohol, antibiotics, enzymes and preservation of pure culture. SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a culture medium composition for fungi, said composition comprising: D- Glucose, Sodium chloride, Disodium hydrogen orthophosphate dihydrate, Potassium nitrate, Ammonium persulfate, Agar, and Distilled water Typically, the amount of D- glucose in the culture medium in accordance with this invention is in the range of about 1 to about 5 % of the mass of the composition. Preferably, the amount of D- glucose in the culture medium in accordance with this invention is in the range of about 1.5 to about 2 % of the mass of the composition. Typically, the amount of sodium chloride in the culture medium in accordance with this invention is in the range of about 0.05 to about 0.25 % of the mass of the composition. 5 Preferably, the amount of sodium chloride in the culture medium in accordance with this invention is in the range of about 0.075 to about 0.2 % of the mass of the composition. Typically, the amount of disodium hydrogen orthophosphate dihydrate in the culture medium in accordance with this invention is in the range of about 0.025 to about 0.2 % of the mass of the composition. Preferably, the amount of disodium hydrogen orthophosphate dihydrate in the culture medium in accordance with this invention is in the range of about 0.05 to about 0.15 % of the mass of the composition. Typically, the amount of potassium nitrate in the culture medium in accordance with this invention is in the range of about 0.015 to about 0.20 % of the mass of the composition. Preferably, the amount of potassium nitrate in- the culture medium in accordance with this invention is in the range of about 0.03 to about 0.15 % of the mass of the composition. Typically, the amount of ammonium persulfate in the culture medium in accordance with this invention is in the range of about 0.025 to about 0.20 % of the mass of the composition. Preferably, the amount of ammonium persulfate in the culture medium in accordance with this invention is in the range of about 0.05 to about 0.17 % of the mass of the composition. Typically, the amount of agar in the culture medium in accordance with this invention is in the range of about 1 to about 2 % of the mass of the composition. 6 Typically, the pH of the culture medium is in the range of about 5 to about 7. In accordance with another aspect of the present invention, there is provided a process for preparation of culture medium composition, said process comprising the following steps: a. mixing D- glucose, sodium chloride, disodium hydrogen orthophosphate dihydrate, potassium nitrate and agar together in distilled water to form a mixture, b. sterilizing the resultant mixture in an autoclave at a temperature of about 121°C under the pressure of about 1.03421 bar for a time period of about 15-30 minutes. c. sterilizing ammonium persulfate in an autoclave at a temperature of about 12l°C under the pressure of about 1.03421 bar for a time period of about 15-30 minutes. d. adding sterilized ammonium persulfate to sterilized mixture of step b aseptically to form a culture medium composition. The culture medium composition prepared in accordance with the present invention is not only cost effective but has several applications such as: 1. It can be used for isolation of fungi from clinical samples, soil, sewage water, milk, air etc. 2. It can be used to maintain pure fungal cultures without being contaminated by bacteria. 3. It can be used in the fermentation industries e.g it can be used for the production of alcohol. 4. It can further can be used for the production of enzymes such as amylase, protease etc. 5. It can be used in the production of antibiotics (eg. Penicillin). 6. It can be used in large scale production of metabolites. 7 7. It can be used for isolation of pathogenic fungi from plants. 8. It can be used as a basal medium in Plant Tissue Culture technique. While considerable emphasis has been placed herein on the specific ingredients of the preferred composition, it will be appreciated that many additional ingredients can be added and that many changes can be made in the preferred composition without departing from the principles of the invention. These and other changes in the preferred composition of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Full Text

FORM-2
THE PATENTS ACT, 1970
(39 of 1970)
& THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10; rule 13)

A CULTURE MEDIUM COMPOSITION
(a) HARNE SHRIKANT RAMDAS
An Indian National of "Rajkamal", Moti Nagar, Pusad, Tal-Pusad, Dist. Yawatmal-445204,
Maharashtra, India;
(b) LA WARE SHANKAR LAXMAN
an Indian National
of 86/15, Om Namah Shivay Raj Park, Sairaj Residency, New Sangvi, Pune
411 027, Maharashtra, India; and
(c) SECRETARY, DECCAN EDUCATION SOCIETY
an Indian National
of Deccan Education Society, Fergusson College Campus, F.C.Road, Pune
411 004, Maharashtra, India

THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION

FIELD OF THE INVENTION
The present invention relates to a culture medium composition.
BACKGROUND OF THE INVENTION
A growth medium or a culture medium is a liquid or gel designed to support the growth of microorganisms. There are two major types of growth media. One is used for cell culture that uses specific cell types derived from plants or animals. Second is microbiological culture media which is used for growing microorganisms, such as bacteria or fungi. Contamination in the microbial cultures is a common problem faced by almost all the pathologists, microbiologists and lab technicians. These contaminants generally come from the environment and are always difficult to eradicate completely. Bacteria are the most common group of contaminants which hamper the processes of isolation of fungi, purification of fungi and sub-culturing of pure colonies of industrially and medically important fungal specimens.
A wide range of media are used for growing fungi and a variety of media are available for the primary inoculation and recovery of fungi from clinical specimens. There is not a single specific medium. or combination of media which is adequate for all such specimens. Most mycologists develop preferences for certain types of media based on experience and peculiarities of the type of fungi that are routinely grown. The characteristics such as colony morphology, color, etc. are affected by the medium used. Further, the type of carbohydrate used in the medium determines the growth rate of fungi. There appears to be a 1^-ge number of carbohydrates, but the one which is easy to metabolize is preferred over others, e.g. Glucose (dextrose) is the simplest carbohydrate whic;h can easily be taken up by the microorganisms and hence is the most commonly used in the growth media. Fructose and mannose are the next most commonly utilized sugars by fungi and are found in media from natural sources. Sucrose (table sugar) may also be used in some media.

There are certain media which makes use of complex carbohydrates, for example, PDA contains starch, for utilization of which, production of amylase enzyme is a must. Some fungi lack the necessary enzyme(s) to utilize such sources of carbohydrates and hence cannot grow. All fungi require several specific elements for growth and reproduction. The requirements for growth are generally less stringent than for sporulation, so it is often necessary to try several types of media when attempting to identify a fungus in a culture. Most fungi thrive on Potato Dextrose Agar (PDA), but this can be too rich for many fungi, hence excessive mycelial growth is obtained at the expense of sporulation. Similarly, wood-inhabiting fungi and dematiaceous (dark pigmented) fungi often sporulate better on Corn Meal Agar (CMA) or Oat Agar, both of which have less easily digestible carbohydrate than PDA.
Most fungi are difficult to isolate from the infected tissues, infested soil or decaying organic material due to the rapid and often antagonistic development of associated bacteria and actinomycetes with faster growth rates. Successful isolation of many of these fungi can be achieved by the use of selective media that either slow down the growth or inhibit the growth of these antagonistic organisms. One approach is to surface sterilize the material to eliminate surface contaminants, and then place the material on Water Agar (WA). Most fungi and bacteria will grow on WA, but at such a slow rate that it is relatively easy to isolate the target fungus. Selective exclusion can be accomplished by several approaches known in the prior art.
1. One approach is by selective inhibition i.e. the use of antimicrobial chemicals, primarily antibiotics such as streptomycin sulfate and penicillin. An Antibiotic Agar (AA) was developed to serve this purpose. Antibiotic agar is a choice of agar when the isolation is done from a material that cannot be surface sterilized and hence have a heavy load of bacteria. However, some bacteria show resistance to antibiotics or some times develop resistance after some days of culture and interfere with the isolation of fungi and during maintenance of pure cultures.
3

2. Another approach is to create an unfavorable environment in the isolation medium such as high pH in which fungus would grow but bacteria would be inhibited. Acidified Commeal Agar (ACMA) works well for the isolation of fungi from various types of plant material. It is best to use surface-sterilized material in conjunction with ACMA. However, under this condition, the possibility of growth of acidophilic and/or acidotolerant bacteria is more.
3. The third approach is the use of a specific medium is the one which allows the growth of only one type of microorganism and inhibits/suppresses the growth of others such as use of high sugar concentration (Saborauds Dextrose Agar), This method suffers a major drawback as a higher sugar concentration may induce excessive mycelia growth which might not be desirable.
4. During the process of isolation and purification of fungi from various sources such as soil, water, air, wood, etc., the possibility of bacterial contamination is much higher. To avoid these problems, continuous sub culturing techniques are used. This generally takes more time, and results into contamination by unwanted organisms and also increases the cost of purification.
Accordingly, it is desirable to produce a culture medium which can avoid bacterial contamination and selectively support the growth of fungi only. Furthermore, the medium must be cost effective.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide a culture medium composition which supports growth of selective microorganism.
It is another object of the present invention to provide a culture medium composition which has antibacterial activity.
It is still another object of the invention to provide a culture medium composition which is easy to prepare and cost effective.
4

It is yet another object of the invention to provide a culture medium composition which is used for isolation of particular microorganism from clinical samples, soil, sewage water, milk and air.
It is a further object of the invention to provide a culture medium composition which is used for production of alcohol, antibiotics, enzymes and preservation of pure culture.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a culture medium
composition for fungi, said composition comprising:
D- Glucose,
Sodium chloride,
Disodium hydrogen orthophosphate dihydrate,
Potassium nitrate,
Ammonium persulfate,
Agar, and
Distilled water
Typically, the amount of D- glucose in the culture medium in accordance with this invention is in the range of about 1 to about 5 % of the mass of the composition.
Preferably, the amount of D- glucose in the culture medium in accordance with this invention is in the range of about 1.5 to about 2 % of the mass of the composition.
Typically, the amount of sodium chloride in the culture medium in accordance with this invention is in the range of about 0.05 to about 0.25 % of the mass of the composition.
5

Preferably, the amount of sodium chloride in the culture medium in accordance with this invention is in the range of about 0.075 to about 0.2 % of the mass of the composition.
Typically, the amount of disodium hydrogen orthophosphate dihydrate in the culture medium in accordance with this invention is in the range of about 0.025 to about 0.2 % of the mass of the composition.
Preferably, the amount of disodium hydrogen orthophosphate dihydrate in the culture medium in accordance with this invention is in the range of about 0.05 to about 0.15 % of the mass of the composition.
Typically, the amount of potassium nitrate in the culture medium in accordance with this invention is in the range of about 0.015 to about 0.20 % of the mass of the composition.
Preferably, the amount of potassium nitrate in- the culture medium in accordance with this invention is in the range of about 0.03 to about 0.15 % of
the mass of the composition.
Typically, the amount of ammonium persulfate in the culture medium in accordance with this invention is in the range of about 0.025 to about 0.20 % of the mass of the composition.
Preferably, the amount of ammonium persulfate in the culture medium in accordance with this invention is in the range of about 0.05 to about 0.17 % of the mass of the composition.
Typically, the amount of agar in the culture medium in accordance with this invention is in the range of about 1 to about 2 % of the mass of the composition.
6

Typically, the pH of the culture medium is in the range of about 5 to about 7.
In accordance with another aspect of the present invention, there is provided a process for preparation of culture medium composition, said process comprising the following steps:
a. mixing D- glucose, sodium chloride, disodium hydrogen
orthophosphate dihydrate, potassium nitrate and agar together in
distilled water to form a mixture,
b. sterilizing the resultant mixture in an autoclave at a temperature of
about 121°C under the pressure of about 1.03421 bar for a time period
of about 15-30 minutes.
c. sterilizing ammonium persulfate in an autoclave at a temperature of
about 12l°C under the pressure of about 1.03421 bar for a time period
of about 15-30 minutes.
d. adding sterilized ammonium persulfate to sterilized mixture of step b
aseptically to form a culture medium composition.
The culture medium composition prepared in accordance with the present invention is not only cost effective but has several applications such as:
1. It can be used for isolation of fungi from clinical samples, soil, sewage water, milk, air etc.
2. It can be used to maintain pure fungal cultures without being contaminated by bacteria.
3. It can be used in the fermentation industries e.g it can be used for the production of alcohol.
4. It can further can be used for the production of enzymes such as amylase, protease etc.
5. It can be used in the production of antibiotics (eg. Penicillin).
6. It can be used in large scale production of metabolites.
7

7. It can be used for isolation of pathogenic fungi from plants.
8. It can be used as a basal medium in Plant Tissue Culture technique.
While considerable emphasis has been placed herein on the specific ingredients of the preferred composition, it will be appreciated that many additional ingredients can be added and that many changes can be made in the preferred composition without departing from the principles of the invention. These and other changes in the preferred composition of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Documents:

1053-MUM-2009-ABSTRACT(20-04-2010).pdf

1053-MUM-2009-CLAIMS(20-04-2010).pdf

1053-MUM-2009-CLAIMS(AMENDED)-(2-4-2014).pdf

1053-MUM-2009-CLAIMS(AMENDED)-(3-5-2013).pdf

1053-MUM-2009-CLAIMS(MARKED COPY)-(2-4-2014).pdf

1053-MUM-2009-CLAIMS(MARKED COPY)-(3-5-2013).pdf

1053-MUM-2009-CORRESPONDENCE(20-04-2010).pdf

1053-MUM-2009-CORRESPONDENCE(23-11-2010).pdf

1053-MUM-2009-CORRESPONDENCE(IPO)-(6-11-2009).pdf

1053-mum-2009-correspondence.pdf

1053-MUM-2009-DESCRIPTION(COMPLETE)-(20-04-2010).pdf

1053-mum-2009-description(provisional).doc

1053-mum-2009-description(provisional).pdf

1053-MUM-2009-DRAWING(20-04-2010).pdf

1053-mum-2009-form 1.pdf

1053-MUM-2009-FORM 18(23-11-2010).pdf

1053-MUM-2009-FORM 18(23-11-2010).tif

1053-mum-2009-form 2(20-04-2010).pdf

1053-MUM-2009-FORM 2(TITLE PAGE)-(20-04-2010).pdf

1053-MUM-2009-FORM 2(TITLE PAGE)-(COMPLETE)-(20-4-2010).pdf

1053-MUM-2009-FORM 2(TITLE PAGE)-(PROVISIONAL)-(22-4-2009).pdf

1053-mum-2009-form 2(title page).pdf

1053-mum-2009-form 2.doc

1053-mum-2009-form 2.pdf

1053-mum-2009-form 26.pdf

1053-mum-2009-form 3.pdf

1053-MUM-2009-FORM 5(20-04-2010).pdf

1053-MUM-2009-PETITION UNDER RULE-137(2-4-2014).pdf

1053-MUM-2009-POWER OF ATTORNEY(2-4-2014).pdf

1053-MUM-2009-REPLY TO EXAMINATION REPORT(3-5-2013).pdf

1053-MUM-2009-REPLY TO HEARING(2-4-2014).pdf

« Previous Patent

Next Patent »

Patent Number

260402

Indian Patent Application Number

1053/MUM/2009

PG Journal Number

18/2014

Publication Date

02-May-2014

Grant Date

29-Apr-2014

Date of Filing

22-Apr-2009

Name of Patentee

HARNE SHRIKANT RAMDAS

Applicant Address

"RAJKAMAL", MOTI NAGAR, PUSAD, TAL-PUSAD, DIST-YAWATMAL

Inventors:

#	Inventor's Name	Inventor's Address
1	HARNE SHRIKANT RAMDAS	"RAJKAMAL", MOTI NAGAR, PUSAD, TAL-PUSAD, DIST-YAWATMAL-445204,
2	LAWARE SHANKAR LAXMAN	86/15, OM NAMAH SHIVAY RAJ PARK, SAIRAJ RESIDENCY, NEW SANGVI, PUNE-27,

PCT International Classification Number

C05F17/00;C12N1/14

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA