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ISOLATION, IDENTIFICATION & PRESERVATION OF INDUSRTIAL MICRO-ORGANISMS.

Presented By,

PRANAV H.NAKHATE M.Tech Biochemical Engg. Amity University, Jaipur

Microbial preservation

POINTS TO BE DISCUSS

Identification of microbes

Isolation of microbes Why microbes are important industrially ?

INTRODUCTION
The microbe will have the last word.

Louis Pasteur
Industrial microbiology associates with the commercial exploitation of microbes for the benefit of mankind. These microbial products may have direct or indirect impact on the economics, environment and social parameters of the society. Microorganisms are used to create a wide variety of products and to assist in maintaining and improving the environment

Microbes have wide scope in different areas. Some of them are, starter cultures in commercial food and beverage production. Products of alcoholic fermentation. Production of Enzymes and other industrial products. Production of Alternative fuels. Production of Pharmaceuticals, like antimicrobials, recombinant hormones, and other cell regulators. Pesticides and agricultural products Biosensors and bioreporters.

INDUSTRIAL STRATEGY FOR USAGE OF MICROBES


Isolation of microbes from natural source.

Identification of desired microbes.


Characterisation of microbes Screening of desired microbes. Inoculum preparation Srain Improvement

Fermentation

ISOLATION OF INDUSTRIAL IMPORTANT MICROBES Isolation is having mainly 2 strategies : 1) from the environment by collecting samples of free living microorganism from natural habitats. 2) By sampling of microbes with desired characteristics from specific sites such as found among the natural micro flora. Hence, its important to isolate that specific microbe from the environment. The site for isolation of microbes is decided by the type of microbe one is interested in.

Sr. No. 1 2 3 4 5 6

PRODUCT Thermophiles Alkalophiles Acidophiles Osmophiles Yeast Phosphate solubilizers

SITE Hot spring, thermophilic vents, compost High salinity soils and wells Pine growing sites, fruits Seas and oceans Wine yards Agricultural soil, rhizosphere

7
8

Protease producers
Enzymes like cellulase, pectinase, xylanase etc.

Dairy industry, meat processing units.


Agricultural soil, paper and pulp industry, waste disposal area of fruit juice plants, grain market. Gober-gas plants, effluent collection sites.

Biogas (methane) producers

Cont.

(Ref. 2)

After sampling of the organism the next step is of enrichment. This increases the quantity of the desired organism prior to isolation and screening. Although numerous media have been devised for the growth of microbes, there is no single media that supports all kinds of microbes. E.g. A soil sample plated on nutrient agar (pH 7.0), incubated at 37C for 24-48 hrs, will grow only aerobic mesophiles, but anaerobes, thermophiles, psychrophiles, alkalophiles and acidophiles will not grow

IDENTIFICATION OF INDUSTRIAL IMPORTANT MICROBES ( Ref 3) After the isolation of bacteria, it is important to identify the specified bacteria. Identification is mostly done with the help of following characteristics 1) Morphology, 2) Selective and diagnostic media, 3) Cultural characteristics, 4) Additional recognized biochemical tests, 5) Profile of microbial stains, and 6) Rapid identification methods.

1) Morphology : It is the science dealing with the structures and forms of organisms. It exclusively trusts confidently upon a number of factors, such as shape, length and breadth, ends of organism, body arrangement, motility, forms spores, capsule 2) Selective and diagnostic media : It specifically favour the growth of

particular microorganisms. Eg. MacConkeys agar medium


for E. coli. Differential media, Enrichment

media, and Characteristic media


are types of this media.

3) Additional recognized biochemical tests : These tests are extensively used for identification purpose. These tests are, Carbohydrate (Sugar) Fermentation,

Litmus Milk, Indole Production, Methyl Red Test [MR-Test], Voges-Proskauer Test [VP-Test], Citrate Utilization, etc.
4) Profile of microbial stains : microbes are extremely too small in size, hence to visualise them, staining is takes place, in which either cell interior or exterior part stains in colour. Different Staining technique includes, Gram staining, Differential staining, Capsule staining ; Endospore staining ; Flagellar staining.

5) Cultural characteristics : Based upon a broad spectrum of intensive and conclusive research carried out during the past few decade These characteristics are, Shape, Size, Elevation Margins,

Surface, Edges ,Colour , Structure, Consistency, etc.

6) Rapid identification methods :These includes advanced techniques like SEM, TEM.

CULTURE PRESERVATION The conventional microbiology is totally dependent on the availability of pure and stable cultures. serial transfer is often the first preservation technique used by microbiologists, but culture is prone to contamination by this. In order to prevent the loss of production of a desired metabolite and to preserve culture, a variety of culture preservation techniques have been developed.

(Ref 4)

1) Culture Transfer :- To avoid contamination, genetic change 2) Refrigeration from 0o to 5oC :short term storage 3) Low Temperature Freezing :ultra low temp. freezer (-80oC), liquid nitrogen (196oC) 4) Lyophilization: freeze with dry ice and acetone sublime off water (dries cells without disruption) use of glycerol, or sucrose to protect cells

5) Mineral Oil
6) Dry Spores

USES OF PROPER ISOLATION, IDENTIFICATION & PRESERVATION

To Improve industrial production. To study proper morphology & physiology of microbes. To understand microbial kinetics. Microbial biosensor. Biopesticides over Chemical pesticides.

REFERENCE
Ref 1 - biotechnology. editor: u. satyanarayanan. publishers: books and allied (p) ltd. calcutta. india. 2005. Ref 2 Pharmaceutical Microbiology,sixth edition edited by w.b.hugo & a.d.russell, professor of pharmaceutical microbiology university of wales cardiff, cardiff Ref 3 - Aamand, J., T. Ahl and E. Spieck. 2006. Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris. Appl.

Environ. Microbiol.
Ref 4 - Becker W.M., Kleinsmith L, and Hardin J. The World of the Cell, 4th ed., Benjamin Cummings, Redwood City, Calif., 2008.