Primary screening

The presentation gives details about primary screening of micro-organisms and explains varying techniques for isolation of organisms.

1. SCREENING OF MICROORGANISMS Renu NK Jaisinghani Assistant Professor Department of Microbiology Smt.CHM College

2. Screening It is defined as use of highly selective procedure for detection and isolation of only those microorganisms of interest from among large microbial population and test their real capability by qualitative and quantitative analysis.

3. Primary Screening It is defined as those elementary techniques required for isolation and detection of new microbial species exhibiting desirable or industrially important properties.

4. Secondary Screening It is defined as techniques that select only those organisms amongst hundred of isolates having real industrial values and test their capabilities by qualitative and quantitative analysis.

5. General steps involved in primary screening Collection of sample Preparation of sample Carry out 10 fold dilution Isolation on suitable media Using suitable method like streaking, spreading, pour plate or membrane imprints(water sample)

6. Examples of Primary Screening Acid/ Amine producers Antibiotic producers Isolation of enzyme producers Specific C and N Utilizer Degraders of synthetic waste Isolation of Vitamin, aminoacid producer

7. I Isolation of Acid/ Amine producer Microorganisms capable of producing acids or amines from natural sources can be detected using this method by incorporating certain pH indicator dyes such as neutral red or bromothymol blue into nutrient agar medium. The change in the color of a particular dye in the vicinity of a colony will indicate the ability of that colony to produce an organic acid or base. pH Indicator ACIDIC ALKALINE NEUTRAL NEUTRAL RED PINK YELLOW ORANGE BROMOTHYMOL BLUE YELLOW BLUE GREEN

8. Isolation of Acid/ Amine producer contd …. Production of an organic acid can also be detected by an alternative method. In this method calcium carbonate is incorporated into the agar medium. The production of organic acid is indicated by the formation of a clear zone around those colonies which release organic acid into the medium.

9. II Isolation of Antibiotic Producer Crowded plate technique Principle Is different organisms present in the sample are made to grow very close to each other (using LOW DILUTION) so that any antibiotic producer among them can exert antagonistic effect towards other Organisms in its close vicinity. Thus antibiotic producer can be easily detected by visual detection of clearing around the colony

10. Crowded plate technique https://youtu.be/Wf4BBYmQD_g

11. Wilkin’s Agar Overlayer Method Principle Is to allow different organisms present in sample to grow on a suitable media as isolated colonies(using HIGH DILUTIONS) and detection of antibiotic producer among them as colony showing zone of inhibition of test organisms around it after overlayering colonies with sterile molten Wilkins’s agar butt seed inoculated with organisms. Bromothymol blue, a pH indicator in Wilkin’s agar helps in differentiating zone of inhibition due to antibiotic and or acid/alkali produced by the isolate.

12. Wilkin’s Agar Overlayer Method STEP 1 DAY 1 NUTRIENT AGAR COLONIES

13. Wilkin’s Agar Overlayer Method STEP 2 DAY 2 Overlayering with Wilkin’s agar(10ml) butt seed inoculated with test organism

14. Wilkin’s Agar Overlayer Method Step 3 Day 3 Alkali producer Antibiotic producer Acid producer

15. Wilkin’s Agar Overlayer Method Antibiotic Producer

16. III Isolation of vitamin, aminoacid , Growth factor producer

17. ENRICHMENT This technique is generally employed to isolate those microorganisms that are very less in number in a soil sample and possess specific nutrient requirement and are important industrially. They can be isolated if the nutrients required by them is incorporated into the medium or by adjusting the incubation conditions.

18. ENRICHMENT TECHNIQUE Few Examples PROTEASE PRODUCER- MEDIA CONTAINING PROTEIN CELLULASE PRODUCER- MEDIA CONTAINING CELLULOSE LYSINE PRODUCER- MEDIA DEVOID OF LYSINE

19. Auxanography

20. Terminology Auxotroph – Requires a particular growth factor for its growth i.e it cannot synthesize its own growth factor. It is denoted as name of growth factor and –(minus) For Example: Lys - , VitB 12 - Prototroph – produces its own growth factor. It is denoted as name of growth factor and +(plus) For Example: Lys + , VitB 12 +

21. Auxanography technique is employed for detecting microorganisms able to produce growth factors , vitamins , amino acids etc. extracellularly

22. REPLICA PLATE TECHNIQUE Principle Is to transfer large number of colonies from one plate(MASTER PLATE) to another plate(REPLICA PLATE- usually minimal medium), that will support growth of any one type of organisms of interest from number of different types present on master plate, in a single step using sterile replicator block covered with sterile velveteen cloth whose threads serve as needles and pick up colonies from one plate to transfer on another.

23. REPLICA PLATE TECHNIQUE Source: Researchgate.net

24. REPLICA PLATE TECHNIQUE https://youtu.be/uYb-_hYSVYw

25. IV ISOLATION OF ENZYME PRODUCERS Enzymes have huge demand in varying industry pharmaceuticals, brewing, textile ,cheese, meat, baking etc. For isolation of enzyme producer specific substrate is required to be provided Eg : PROTEASE- Any media containing protein AMYLASE- Media containing Starch LIPASE- Media containing oil or lipids

26. V ISOLATION OF DEGRADERS OF SYNTHETIC WASTE Pesticides, detergents and solvents do not get degraded easily. -STARTER CULTURE -CONSORTIUM OF ORGANISMS

27. VI ISOLATION OF SPECIFIC ‘C’ AND ‘N’ UTILIZER This technique is employed for the detection and isolation of microorganisms capable of utilizing carbon source from volatile substrates like hydrocarbons, low molecular weight alcohols and similar carbon sources. Suitable dilution of a microbial source like soil suspension are spread on to the surface of sterile agar medium containing all the nutrients except the one mentioned above.

28. VI ISOLATION OF SPECIFIC ‘C’ AND ‘N’ UTILIZER The required volatile substrate is applied on to the lid of the petri plates, which are incubated by placing them in an inverted position. Enough vapors from the volatile substrate spread to the surface of agar within the closed atmosphere to provide the required specific nutrient to the microorganism, which grows and form colonies by absorbing the supplemented nutrient. The colonies are isolated, purified and stock cultures are made which may be utilized for further screening tests.

29. VI ISOLATION OF SPECIFIC ‘C’ AND ‘N’ UTILIZER Source: ResearchGate.net

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