Replica plating technique

Though we all know what is replica plating technique practically, theortically it gets under my nerves a lot of times.

The reason being...

• Do I need to isolate mutants or wild type?
• What will I obtain on this plate?
• Will be prototrophs will get selected or auxotrophs??

And many more similar doubts.

So here, we will try to simplify and answer all these doubts, and get the concepts clear for life.

Oh by the way...Did I say a hi to you guys??

I guess no.

So, hello everyone. the topic that we will be discussing today is RPT.

Let's start...5 6 7 8.

Concepts first.

What is RPT?

It is a microbiological technique which involves us to first isolate the culture on a solid media and after growth, it is replicated using a velveteen assembly onto a secong solid media to obtain the similar colony pattern.

Whether these medias will be nutritionally complete or deficient will depend and vary according to the requirement. This technique is very useful and probably the only one to identify the mutants and wild - types from a mixed culture.

What are auxotrophs and phototrophs?

Auxotrophs are the micro - organisms which due to some mutations have lost the ability to synthesize an XYZ compound or utilize and XYZ compound. Prototrophs are the wild - types which can synthesize all the components required for it's growth and also utilize all the nutrients. RPT makes use of this property of auxotrophs and phototrophs for differentiation.  

Is any other property used for differentiation?

Yes, apart from auxotrophy any other mutations can be used for differentiation. Mutations not always result in loss of function. Sometimes due to certain mutation, there can be a gain of function in mutations like resistance to certain antibiotics or chemicals etc. which also helps in classifying them from wild - types.

How should the media be selected?

Now this is a really tricky question.

But let's try making it simpler.

There are two categories of media pairs that you can use as master plate and the replica plate.

1. Nutritionally complete medium and nutritionally complete medium minus one component that the mutant is not able to synthesize.

On the nutritionally complete media, both auxotrophs and prototrophs will grow, But in the medium which is devoid of a particular component which the auxotrophs are unable to synthesize (leu, proline, etc) only prototrophs will grow and auxotrophs will fail to grow. Thus by comparing both the plates you can determine auxotrophic colonies.

An alternative to this medium can be - minimal medium as the master plate and the minimal medium with the component that the auxotroph is not able to synthesize. Here, on the replica plate both auxotrophs and prototrophs will grow whereas on the master plate only phototrophs will grow.

     2.  Nutritionally complete medium and nutritionally complete medium with a selective agent that             the mutant is sensitive or resistant to (antibiotic, disinfectant, etc) (Also known as negative                   selection)

On the nutritionally complete medium, wild types and mutants will grow. But on the plate containing the selective agent, only the mutants will grow. Or vice versa, if the wild type was resistant and mutated to sensitive, then the mutants will fail to grow on the replica plate and only wild types will grow. By comparison of both the plates, you can determine the sensitive and resistant colonies. 

After the replica plate, an efficiency plate which has the medium similar to master plate is also plated to ensure the efficiency of replication. Mostly, the results of replica plate are compared with the efficiency plate and not the master plate.\

Hufff, finally done.

I hope this helps you. 

Feel free to ask any more queries if you have.

Lots of love, high six.

-Staph :D