Replica plating

The technique originated from the need to isolate bacterial mutants without prior knowledge of their phenotype. Joshua and Esther Lederberg devised a method where a velveteen cloth is pressed onto a master plate containing colonies grown on a non-selective medium, then transferred to a series of selective plates. Colonies that fail to grow on a selective plate but are present on the master plate can be retrieved from the master for further study.¹ The key innovation is the use of a sterile velvet pad that acts as a stamp, replicating the pattern of colonies with high fidelity. This allowed for indirect selection: for example, to find an antibiotic-sensitive mutant, the master plate is stamped onto a plate containing the antibiotic; colonies absent on the antibiotic plate are identified on the master and isolated.

Replica plating has been a cornerstone of bacterial genetics, enabling the isolation of auxotrophic mutants—those requiring specific nutrients—by comparing growth on complete and minimal media. It is also used to screen for antibiotic resistance or sensitivity, to study metabolic pathways, and to map genes through co-transduction and conjugation experiments.³ In environmental microbiology, replica plating facilitates the isolation of bacteria with specific metabolic capabilities, such as degradation of pollutants. The technique remains valuable in teaching laboratories for demonstrating mutation and selection.

Several modifications have improved the efficiency and scope of replica plating. The use of sterile velveteen or filter paper disks reduces cross-contamination. Automated robotic systems now perform high-throughput replication using multi-pin inoculators, essential for large-scale mutant libraries.⁴ For anaerobic or fastidious organisms, the process can be performed in an anaerobic chamber. Replica plating has also been adapted for yeast and filamentous fungi, using similar stamping methods. In metagenomics, replica plating of environmental samples onto selective media helps isolate novel microbial strains.⁵

Although often attributed solely to Joshua Lederberg, Esther Lederberg performed the majority of the experimental work and is credited as co-inventor. The original 1952 paper was published in the Journal of Bacteriology and was initially met with skepticism; the technique was later nominated for a Nobel Prize, though it did not win.¹ A lesser-known fact is that the Lederbergs used a piece of velvet from a laboratory coat as the first stamp. The method also inadvertently led to the discovery of the bacterial sex pilus, as it facilitated the isolation of F⁺ and F⁻ strains. Today, replica plating is still used in the search for antibiotic-producing bacteria from soil, where it enables the detection of inhibition zones.⁶

Master plate

The original agar plate containing bacterial colonies to be replicated.

Velvet pad

A sterile velveteen cloth used to transfer colonies from the master plate to replica plates.

Auxotroph

A mutant strain that requires a specific nutrient not needed by the wild type.

Selective medium

A growth medium that favors the growth of certain organisms while inhibiting others.