Replica Plating

Replica plating: in bacteriology, it is the transfer of isolates from one substrate to other selective media.

Joshua and Esther Lederberg sought to create a technique capable of facilitating the handling and transfer of large numbers of bacterial clones on differing media.

The only method of bacterial transfer that was being employed at the time was the use of single inoculating needles. There had been talk of a device with multiple inoculating needles in a fixed pattern to accelerate the process.

Figure 1. Replica plating for the isolation of auxotrophic colonies (A. Initial plate, B. first replica, C. second replica.) (Lederberg and Lederberg 1952).

The technique is seen to be applicable to classify:

Antibiotic-sensitivity spectra, responses to bacteriophages (phage typing), fermentation characteristics, nutritional requirements, etc. as long as a selective agar medium can be created (Lederberg and Lederberg 1952).

Instead of a device containing multiple inoculating needles, Esther Lederberg and her husband suggested:

Using the surface wooden cork (diameter = 9cm) , and covering it with a piece of velveteen fabric. The velvet acts as a vertical plane for moisture that might smear the impression (Lederberg and Lederberg 1952).

Process (Lederberg and Lederberg 1952)

The velvet is placed in petri dishes and sterilized in the autoclave, after sterilization a 12 cm square of fabric is placed on the wooden cork and secured in place with a metal hoop.
The agar with the initial colonies is inverted onto the fabric with slight digital pressure to transfer the growth, this imprinted fabric now facilitates the transfer of replica-inocula to other plates following the same methods.

The fabric square can be washed, sterilized and used repeatedly.

Limitations (Lederberg and Lederberg 1952)

The resolution of the imprint relies on the conditions of the agar, colonies and fabric (large initial clumps could result in a smearing of the replica colonies). Therefore, there will be some forms of bacteria that cannot effectively be transferred using this technique due to their bacterial morphology.

Clonal occurrence of phage resistant mutants (Lederberg and Lederberg 1952)

E.coli was observed to transfer 10-30 % of initial cells to the fabric and an equal amount to the successive replica plates.
This method would directly demonstrate clonal mutants. The clones on the initial plate would be seen by recurring, resistant colonies on superimposable sites on the replica plate containing the phage.

Joshua and Esther observed half of all of the resistant mutants reappearing on the superimposable sites, therefore agreeing with their above statement.

This clonal occurrence of phage resistant mutants was tested using a W-1 culture derived from E.coli (K-12) and the phage T-1 as well as EMB lactose agar.

Indirect selection of phage resistant mutants (Lederberg and Lederberg 1952)

Joshua and Esther hypothesized that adapted mutants could be isolated in pure culture without direct exposure to a selective agent through replica plating.

If the initial inoculum was diluted enough, there would only be a few clones on a single plate. If the same superimposable sites were chosen for the inoculum of the second tube of broth, the mutants would be concentrated in the same proportion as the cells per plate.
After ~4 stages of this indirect selection, the resistant clones should appear as discrete colonies that can be identified, purified, and maintained by conventional methods. Replica plating thus provides a technique for isolating resistant or otherwise adapted mutants without altering the media in which the bacteria are grown.
This indirect selection of phage-resistant mutants was tested using Streptomycin resistance of the aforementioned W-1 culture, agar containing 200 ug of Streptomycin/mL was used for the replica plates.
This experiment refuted Lamarck’s idea that genetic traits change in response to the environment, because it allowed for the isolation of antibiotic-resistant mutants that had not previously been in contact with the drug. Therefore, it helped establish that such mutants arose spontaneously and not in response to an environmental cue (Schaechter 2016). As such, it was important for modern genetics' understanding of mutation as a random process (Ferrell 2018).

Esther's father, David, ran a print shop, and seeing his press at work may have inspired Esther to invent replica plating, as she needed an efficient way to inoculate bacteria onto different plates of media in order to test their traits (Ferrell 2018). Esther first tried the technique using her powder puff, and then went to different fabric stores, searching for a velvet cloth whose thickness and surface fibers were most suitable for her purpose (Schaechter 2016). She also tried various detergents to find the best one for washing them. Since Esther invested so much time in creating and refining this technique, it is speculated that she was the true inventor of replica plating (Ferrell 2018; Schaechter 2016). However, Joshua was nevertheless listed as the first author on the published article.

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