Strain improvement : Techniques used for Isolation of mutants

Techniques that will help you how the mutants are being isolated...

1. STRAIN IMPROVEMENT Part III Isolation of Mutants Mentor: Ms. Renu NK Jaisinghani Prepared By : Ms. Shruti Jawale Assistant Professor Ms.Akshta Desai Department of Microbiology Smt. CHM College

2. Techniques Used To Isolate Mutants

3. Direct Method : Replica plate Technique Sandwich Technique Indirect Method : By using Penicillin By Filtration- enrichment technique.

4. a) Replica Plate Technique It was invented by Lederberg in 1952 . Method : 24 hrs young cell suspension with cell density 7 × 10 8 cells/ml Expose to sublethal dose of mutagenic agent at predetermined concentration & time (or dose) Wash the cells 2 to 3 times to make them free of mutagenic agent (if chemical mutagen is used ) Surviving population is inoculated in st. Complete medium broth. Incubate at R.T for 8-24 hrs (i.e. Atleast for 3-4 generation for expression of mutation phenotypically )

5. Surviving population is inoculated in St.Complete medium broth Incubate at R.T for 8-24 hrs (i.e. Atleast for 3-4 generation for expression of mutation phenotypically) Separate cells by centrifugation & give washings to remove any nutrients sticking to the cells. Cell suspension is diluted serially using St. Saline 0.1ml of diluted sample is spread on St. Complete medium( e.g St. Nutrient Agar plate). Plates incubated at optimum temperature & time. Select a plate with well isolated colonies (3 to 30) as master plate.

6. Replicate it on St. Replica plate (St. Minimal agar plate lacking any one critical nutrient whose auxotroph's are to be isolated) Incubate replica plate at RT for optimum time & preserve master plate in fridge By comparing master plate with replica plate, detect colonies of auxotrophic mutants on master plate but lacking on replica plate. Such colonies are picked up & tested further.

7. b) Sandwich Technique : Principle : Is to grow mixture of prototroph & auxotroph under condition that allows only prototroph to grow initially since of minimal medium and form distinct big colonies & suppress growth of auxotrophs. Later on allow auxotrophs to grow (by providing complete medium) for very short time period, so that they form very tiny colonies & can be easily distinguished from that of prototrophs. Advantage/ Significance/ Importance : i) Visual identification of auxotrophs. ii) Simple, 2 steps, doesn’t require skill or sophasticated equipments.

8. Method : Indrustrial strain to be improved Inoculate in complete medium & incubate at RT for 24 hrs. Young cell suspension with cell density 7 × 10 8 to 1.1 × 10 9 cells/ml Expose to sublethal dose of mutagen for specific time. Wash surviving population 2-3 times. Inoculate in complete medium. Incubate at 30˚C for 8 hrs. Separate cells by centrifugation & wash for 2 to 3 times. Seed inoculate 1ml of washed cell suspension in St. Molten minimal agar medium, mix & pour in st.plate & allow to settle. Cover this with another layer of St. Minimal medium

9. Incubate plate at 30˚C for 24 hrs. Colonies that appear are of prototrophs, mark at the base of the plate. Now cover the plate with another layer of St. Molten complete medium & allow it to solidify. Incubate plate at 30˚C for 24 hrs. Minute colonies appear on plate & they are of auxotrophic mutant Initially medium being minimal only prototrophs grow & form colonies. Then, when medium is supplemented with nutrients (by covering with complete medium) auxotrophs can grow but their colonies remain very minute, as amount of nutrients available is less & also they are just 24 hrs old colonies compared to prototrophs which are 48 to 72 hrs old.

10. C) Penicillin Method : Principle : Is to enrich culture under conditions that adversly affects prototrophs but do not damage auxotrophs. Such condition include, growing mixture of prototrophs & auxotrophs on minimal medium in presence of antibiotic Penicillin that affects & kills only growing young cells of prototrophs without damaging nondividing auxotrophs. This technique was used first time by Davis in 1949. Advantage / Significance : a) Simple,2 step technique. b) Doesn’t require skill & sophisticated equipments. Disadvantage : a) No direct visual detection of auxotrophs. b) Auxotrophs obtained should be tested for their auxotrophic nature

11. Method : Cells/ spores (that are to be improved by mutation) Inoculate in St.Complete medium Incubate at 30˚C for 24 hrs. Cell suspension/ spore suspension is prepared with density 7×10 8 to 1.1×10 9 cells/ml. Expose suspension to sublethal dose of mutagen for specific time. Wash the survivals for 2 to 3 times to make them free of chemical mutagen if used. Inoculate in complete medium & incubate at 30˚C for 8 hrs. Separate cells by centrifugation & wash them 2 to 3 times to make them free of nutrients. Inoculate cells in St. Minimal medium with 5 to 500U/ml of penicillin

12. Incubate at 30˚C for 24 hrs. Harvest cells by centrifugation & wash them to make free & Penicillin. Inoculate in complete medium & incubate at 30˚C for 24 hrs. For testing & confirming auxotrophic nature St. Minimal St. Complete Medium Medium Incubate at 30˚C for 24 hrs. No growth Growth (Confirms auxotrophic nature of isolated mutant) On incubating mixture of prototrophs & auxotrophs in minimal medium with penicillin, only prototrophs can grow & divide & get killed by penicillin present in the medium. Whereas auxotrophs cannot grow & divide on minimal medium & escape action of penicillin and survive

13. This technique is sucessfully used to isolate auxotrophic spores of Pen.chrysogenum, Strep.aurefaciens , Strep.olivaceous , B.subtilis . Here auxotrophic spores germinate & get killed. Instead of Penicillin, ‘Na- pentachlorophenate ’ can be used. d) Filteration enrichment method : Method is deviced by Catcheside in 1954 . And is used specially to separate auxotrophic & prototrophic spores of filamentous fungi. Principle : Is to enrich spores of prototroph & auxotroph under condition that will allow only prototroph to grow & form long filaments but auxotroph can’t grow by use of minimal medium. Then spores of auxotroph's can be easily separate from filamentous prototroph by filtration. Method : Cells/Spores that are to be improved by mutation Inoculate in complete medium

14. Incubate at 30˚C for 24 hrs. Cell suspension/spore suspension is prepared with density 7 × 10 8 to 1.1 × 10 9 cells/ml Expose suspension to sublethal dose of mutagen for specific time. Wash the survivals for 2 to 3 times to make them free of chemical mutagen if used. Inoculate in complete medium & incubate at 30˚C for 8hrs for mutation to express phenotypically. Separate spores by centrifugation & wash them 2 to 3 times to make them free from nutrients Inoculate spores in st . Minimal medium Incubate at 30˚C for 48 hrs till prototroph germinate.

15. Filter the broth through sintered glass so as to retain filamentous fungi Collect filterate containing spores of auxotroph.

16. Isolation of Analogue Resistant Mutants Gradient Plate Technique

17. Gradient plate Technique : Principle : is to detect resistant mutants by exposing cells to concentration gradient of drug/antibiotics/analogue at the surface of medium generated by allowing diffusion of drug from lower slanting layer of medium with it to upper layer of medium without it. • Advantage /significance /Important : To isolate resistant mutant that can give higher yield. To measure degree of resistance, indicated by the position of colonies on the gradient plate. Technique saves time and labor of isolating resistant mutant and different organisms on different plates with different conversation.

18. • Method : This technique is carried out in two steps. A) Determination of MIC of toxic analogue. B) Isolation of resistant mutant.

19. Inoculation cells in medium. incubate 30 o C for 24h Cell suspension with density 7 x 10 6 - 1.1 x 10 cells/ ml 0.1ml of this cell suspension is inoculated in various concentration of analogue prepared in total 10ml of St. Medium . Observe inhibition &determine the lowest (minimum) concentration of toxic analogue required to inhibit growth of test organisms ie . MIC of analogue A) Determination of Minimum Inhibitory Concentration [MIC]:

20. B)Isolation of analogue resistant mutant . Inoculate cells in complete medium Incubate 30 o C for24h Cell suspension with density 7 x 10 6 - 1.1 x 10 cells/ ml Expose cells to sublethal dose of mutagen for specific time period . Separate cells & wash them 2-3times to make them free of chemical mutagen if used. Inoculate cells in St. Complete medium (for expression of mutation) & incubate at RT for 8hrs.

21. Cells are washed &used for isolation of analogue resistant mutant by gradient plate technique . Preparation of gradient plate –plate having concentration gradient at the surface of medium. Surface spread cells exposed to mutagenic agent over surface of gradient plate. Incubate plates at 30 o C for 24 hrs Resistant mutants are the one growing at high concentration of analogue.

22. Gradient Plate Technique

23. Reference Stanbury, P.F., Whitaker, A. Principles of Fermentation Technology 2E.

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