Q.68 An auxotrophic mutant arises spontaneously in a wild type E.coli culture growing in a rich medium.
Which ONE of the following techniques will ensure the isolation of the auxotrophic mutant?
(A) Replica plating (B) Streaking for single colonies
(C) Pour plating method (D) Direct microscopic observation
Replica plating ensures isolation of auxotrophic mutants from wild-type E. coli in rich medium.
Question Breakdown
Auxotrophic mutants require specific nutrients (e.g., amino acids) absent in minimal medium but present in rich medium. In rich medium, both wild-type (prototrophic) and auxotrophs grow. Replica plating screens colonies by comparing growth on rich vs minimal media.
Option Analysis
(A) Replica plating
Correct. Lederbergs’ technique: velvet transfers colonies from master plate (rich medium) to replica plates (minimal medium). Auxotrophs grow on master but not replicas → identified by absence.
(B) Streaking for single colonies
Isolates pure cultures but doesn’t distinguish auxotrophs (both grow on rich medium). No selective screening.
(C) Pour plating method
Embeds bacteria in agar for counting; no differential screening for auxotrophy vs prototrophy.
(D) Direct microscopic observation
Morphologically identical; cannot differentiate metabolic requirements visually.
Correct Choice
(A) Replica plating
Isolation auxotrophic mutant rich medium requires replica plating to screen growth differences between complete and minimal media.
Replica Plating Protocol
-
Master plate: Rich medium (LB) → all colonies grow
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Velvet replica: Transfer identical colony pattern to minimal plates
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Screening: Auxotrophs missing on minimal replicas
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Pick: Return to master plate coordinates
Rich plate: WT ○○○○○○○○○○ aux○○○○○○○○○○
Minimal replica: WT ○○○○○○○○○○ ---○○○○○○○○○○
↑ Auxotrophs identified
Technique Comparison
| Method | Screens Auxotrophs? | Principle |
|---|---|---|
| (A) Replica plating | Yes | Growth comparison rich/minimal |
| (B) Streaking | No | Pure isolation only |
| (C) Pour plating | No | Viable count |
| (D) Microscopy | No | Morphology |
GATE Exam Relevance
Microbiology PYQ tests mutant screening fundamentals. Key principle: Replica plating = phenotypic screening. Alternatives: Penicillin enrichment (kills growing WT), nystatin (kills non-growing auxotrophs on minimal).
