Spore staining

The Spore Staining Method-Schaeffer & Fulton's Method

AIM: To perform spore staining technique to demonstrate the spore in a given culture

Principle

A spore is a dormant form of the bacterium that allows it to survive in drastic environmental conditions. Spores have a tough outer covering made of the protein keratin and are resistant to heat and chemicals. The keratin also resists staining, so extreme measures must be taken to stain the spore. In the Schaeffer-Fulton's method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells may be decolourized with water. Vegetative cells are then counterstained with safranin. Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical. Members of the genus Corynebacterium may exhibit club shaped swellings that might be confused with spores. Spore staining distinguishes between true spores and these structures.

Procedure:

Results

Vegetative cells: red

Spores: green

PHB staining

Polyhydroxybutyrate (PHB) granules staining

A number of the micro-organisms are capable of producing polyhydroxyalkanoates (PHA’s) as storage food reserve under unbalanced growth conditions. It is similar to of synthetic plastics like polyethylene,polypropylene etc. The advantage of using PHA’s over synthetic plastic is that PHA’s are completely mineralized into carbon dioxide and water through the action of various microorganisms. PHB are produced intracellularly by various organisms such as Bacillus megaterium, Rhizobium spp., Azotobacter spp., Pseudomonas spp., etc under physiological stress conditions. These bacteria can accumulate up to 60-80% of their weight as PHB under limiting nitrogen substrate and in the presence of an abundant source of carbon (Anderson and Dawes, 1990). The major limitation in the use of biodegradable plastic is their high cost as compared to the synthetic plastic. Rhizobium species are symbiotically associated with several leguminous plants like Pisum sativam, Glycine max, Alfa alfa etc. These are Gram negative, motile, non-endospore forming bacteria. These bacteria are generally cultured in Yeast Mannitol Agar medium. Rhizobium gives colorless gummy appearance when grown on YEMA medium supplemented with congo red. The gummy appearance is because extracellular polysaccharide production. Most importantly,they are able to accumulate a high amount of PHB intracellularly.

There are two methods to achieve PHB granules staining

I. Carbol Fuchsin staining:

Carbol fuchsin staining is performed to determine the intracellular production of PHB by the isolate.

A thin smear was stained with carbol fuchsin stain for 45 seconds.

Slide washed with water and air dries the slide

Observed at 100X magnification.

The isolates capable of producing PHB showed dark colored granules of PHB intracellularly (Aneja, 2001).

II. Sudan black B staining:

 Sudan black B staining PHB producing bacteria was further confirmed using Sudan black B staining method (Schlegel et al., 1970) with some minor modifications.

 Sudan black B stain was prepared as 0.3% solution (w/v) in 60% ethanol.

The smear was prepared on glass slides and heat fixed.

            The samples were stained for 10 min with Sudan black solution, rinsed with water

 Counter stained with 0.5% safranin for 5min.

Observed at 100X magnification.

PHB granules are dark purple granules stained with Sudan black B dye

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biodiversity and IPR

patent law and 3 amendment

PATENT LAW AND AMENDMENTS

The word patent originates from the Latin patere, which means "to lay open" (i.e., to make available for public inspection).

The Act VI of 1856 on Protection of Inventions in India enacted on 28 February 1856. Based on the British Patent Law of 1852

Patent law is a specific area of law that encompasses the legal regulation, jurisprudence (the philosophy, science and study of law) and enforcement of specific intellectual property rights known as patent rights.

A patent law is a government issued right granted to individuals or groups

It protects their original inventions from being made, used, or sold by others without their permission for a set period of time.

Objectives:

 Encouragement of inventions of new and useful manufacture.

 To secure the knowledge of the invention from being lost, and … to enable any person … to avail himself of the invention on expiry of the exclusive privilege

THREE AMENDMENTS

First Amendment in 1 999

 Introduced transitional facility to receive and hold patent applications of pharmaceutical and agricultural chemical products (mail box) till 1January 2005 and for grant of Exclusive Marketing Rights for 5 years or till grant of patent.

Second Amendment

 Bill introduced on 20 December 1 999

 Referred to a JPC (Joint Parliamentary Committee) on 22 December 1 999

 Report submitted on 1 9 December 2001

 Bill passed in 2002

 Major changes

 20 year patent period

 Reversal of burden of proof on the infringer

 Establishment of an Appellate Board

 Public interest safeguards and measures for protecting Traditional Knowledge.

Third Amendment  2005

 Based on Observations of JPC

 Wide spread consultations through country wide interactive sessions with interest groups

 Extensive inter ministerial consultations

 Removed transitory provisions

 introduced various flexibilities provided in the TRIPS Agreement including the Doha Round.

Patent Law - Salient Features

 Both product and process patent provided.

 Term of patent – 20 years.

 Examination on request.

 Both pre-grant and post-grant opposition.

 Fast track mechanism for disposal of appeals.

 Provision for protection of bio-diversity and traditional knowledge.

 Publication of applications after 18 months with facility for early publication.

Safeguards in the Patent Law

 Compulsory licences to ensure availability of drugs at reasonable prices

 Bolar Provision for early manufacture of generics

 Parallel, import to check prices.

 Provision to deal with public health emergency.

 Revocation of patent in public interest and also on security considerations.

 Provisions to prevent grant of patents based on frivolous or trivial inventions.

 Provisions to prevent misappropriation of Genetic Resources and Traditional Knowledge.

NOTE:WEB Referred notes.

gram staining and air sampling plate at ngp college,coimbatore-48

Aminoacid symbols

Single, 3-letter and ambiguity codes for Amino Acids

The 20 naturally occuring amino acids:

Alanine         Ala     A

Cysteine        Cys     C

Aspartic AciD   Asp     D

Glutamic Acid   Glu     E

Phenylalanine   Phe     F

Glycine         Gly     G

Histidine       His     H

Isoleucine      Ile     I

Lysine          Lys     K

Leucine         Leu     L

Methionine      Met     M

AsparagiNe      Asn     N

Proline         Pro     P      

Glutamine       Gln     Q

ARginine        Arg     R

Serine          Ser     S

Threonine       Thr     T

Valine          Val     V

Tryptophan      Trp     W

TYrosine        Tyr     Y

Amino acids ambiguity codes:

Asparagine/Aspartic Acid    Asx   B

Glutamine/Glutamic Acid     Glx   Z

Not assigned to amino acids:

J O U

Symbols for sequence analysis:

deletion or gap    . (dot)

End or Terminator  * (star)