Penicillin enrichment

A commonly used technique for isolating auxotrophs (mutants).

Now another method for mutant selection is penicillin enrichment all right it’s called penicillin enrichment now the term enrichment is used because uh it increases the percentage or the probability of uh the oxytrophs versus the prototrops right so increases your chances of picking up the mutant versus the wild type that’s why it’s called an enrichment method

And why penicillin enrichment simply because this particular method uses uh the antibiotic uh penicillin so what happens here is um so in a tube of um glucose salt broth so we looked at glucose this is glucose salt broth which means the broth is made up purely of glucose and other dissolved mineral salts now this combination is sufficient for prototroughs but

Not for oxatrops right the wall type the non-mutants can grow on this they can use the glucose to make whatever they need but the oxytrops because they are mutants this is insufficient they need more additional growth factors to be included in the media so the oxatrops can’t grow but the prototrops can grow so what happens is um once you introduce the cells into the

Glucose salt broth you add the and the antibiotic penicillin now penicillin is an is an antibiotic that targets only actively growing cells or rather actively dividing cells now if you remember the mechanism of action or the mode of action of penicillin penicillin interferes with peptidoglycan synthesis particularly at the point of the interbridge formation right

So penicillin uh interferes at that particular stage where the interbridge forms so if the inter bridge doesn’t form it compromises the integrity of the peptidoglycan and which weakens the bacterial cell wall and the bacteria basically dies because the cell wall is no longer resilient enough to give it its protection so that’s how penicillin works it interferes

With peptidoglycan synthesis now when is peptidoglycan synthesized peptidoglycan is only synthesized when the cell is undergoing division cell division because in a cell that doesn’t divide then there is no need to make new peptidoglycan or more peptidoglycan peptidoglycan is only made when the cell divides because it needs to make new components which include

The cell wall so penicillin is an antibiotic that targets actively dividing cells because it targets peptidoglycan synthesis which only happens in actively dividing cells so the oxotrops in this case will not be harmed by penicillin because the oxotrops are not dividing in the glucosalt broth right because the growth conditions are not suitable so the oxytrops

Are not dividing in the glucose salt broth only the prototroughs are dividing in this case right because the prototroughs uh they they can make use of the glucose salt broth and make all the necessary uh nutrients and everything else that they need and so the cells are happily dividing away and therefore penicillin will have an effect on those cells not on the

Oxytrops so once you add penicillin and incubate uh the tubes penicillin will start targeting the oxotrops and it will start killing the uh sorry penicillin will start targeting the prototrophs and it will start killing the prototropics right penicillin will not harm the oxatrops it only kills the prototrophs now after a while you need to add an enzyme called

Penicillinase right so penicillinase is an enzyme right that breaks down penicillin so you need to add these you need to add this enzyme to basically stop the action of penicillin so once the antibiotic has been added and the cells have been incubated for a sufficient duration of time then the action of the antibiotic must be stopped right you can’t let it go

On and on and on forever the amount of action of penicillin has to be stopped now this uh this might be a puzzling to many people as to why do you need to stop the action of penicillin since penicillin is targeting the wild type prototroughs your oxytrops are not harmed by the antibiotic then there is no need for you to stop the action of the antibiotic you

Can just let it go on and just let it kill the wild type prototrops and just let the oxytrops survive right that’s what one would tend to think however there is a trick here right there is a slight snitch here now what happens is when the antibiotic starts killing the prototroughs right what happened the prototrophs will burst open right because peptidoglycan

Synthesis is inhibited so the cells will lyse they would burst open and all the internal contents will spill out into this media right into this particular into this media and the internal contents would also include all the nutrients that the cell has synthesized for itself so the oxotrops which lack one or two nutrient capabilities would start taking these

Nutrients that have been released by the dead cells and then they would start multiplying as well your oxotrops will start multiplying benefiting from the contents that came out from their dead wild-type friends right when the wild-type cells died the cells burst open the internal contents will leak out and these contents can be taken in by the mutant cells and

The mutant cells will start dividing and when these cells start dividing what’s going to happen is penicillin will start killing the oxotrops as well because penicillin targets actively dividing cells so if you let penicillin go on to do its function indefinitely then you would have no cells at all the end the antibiotic will kill the prototroughs and it will

Also end up killing the oxytops because the oxotrops have started to divide as well so for that purpose the enzyme penicillinase has to be added to stop the function of penicillin after a sufficient amount of time so when you take samples from the tubes and if you were to plate them so if you take a sample from tube a before penicillin was added of course you

Would see lots of colonies all right especially because this is nutrient agar so the prototypes would grow the oxytrops would grow but once you do your penicillin enrichment so coming back to why this is called penicillin enrichment so by adding penicillin the antibiotic gets rid of the prototrops gets rid of the wild type and it increases the uh the the numbers

Of the oxotrops right so now the ratio has shifted you will have more oxytrops than the prototrops initially you have more prototropes here if you look at this if you look at this plate you have more prototroughs than oxide troughs right the prototrophs are the blue circles and the oxatrops are the pink circles but after penicillin and richmond now you have more

Oxotrops than prototrophs right you have increased the the ratio the ratio has shifted so that’s a reason why this is called penicillin enrichment so if you compare the colonies between both the plates then you would realize that you will have far fewer colonies on uh on the plate uh after penicillin enrichment and a majority of the colonies that you find here

Would be the mutants would be the oxotrops whereas here on this plate majority of the colonies would be the prototropes now this is a penicillin enrichment

Transcribed from video
Penicillin enrichment By Microbiology