ISOLATION, IDENTIFICATION & PRESERVATION OF

INDUSRTIAL MICRO-ORGANISMS.

Presented By,

PRANAV H.NAKHATEM.Tech Biochemical Engg.Amity University, Jaipur

POINTS TO BE DISCUSS

Why microbes are important industrially ?

Isolation of microbes

Identification of microbes

Microbialpreservation

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, it’s 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. PRODUCT SITE

1 Thermophiles Hot spring, thermophilic vents, compost

2 Alkalophiles High salinity soils and wells

3 Acidophiles Pine growing sites, fruits

4 Osmophiles Seas and oceans

5 Yeast Wine yards

6 Phosphate solubilizers Agricultural soil, rhizosphere

7 Protease producers Dairy industry, meat processing units.

8 Enzymes like cellulase,pectinase, xylanase etc.

Agricultural soil, paper and pulp industry, waste

disposal area of fruit juice plants, grain market.

9 Biogas (methane) producers

Gober-gas plants, effluent collection sites.

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 37°C 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. MacConkey’s 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 (…Ref 4)• 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.

1) Culture Transfer :- To avoid contamination, genetic change

2) Refrigeration from 0o to 5oC :-

short term storage3) 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 cells5) 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.