Please explain 16s rrna in simple terms

[Thorstein]

If you have multiple bacterial strains on an agarplate, how do you isolate them using simple techniques at home?

Second, if you try to isolate one strain, do you put it on a new agarplate and then do the 16s rrna test from the agarplate. Or how it is done.

Sorry if the questions are too basic for you experts. Would appreciate if someone took the time. Very curious.

[JennYeap]

firstly, for isolation of bacterial strains, streak plate will do to get single colony.
secondly, u should streak them onto new agar plate to prevent contamination and use 1 colony from streaked plate as template to perform PCR of 16S rRNA then sequence for identity. generally, 16S rRNA= subunit of bacterial ribosomal RNA, they are quite conserved which can be used for bacterial identification.

[Thorstein]

Thank you Jennyeap I am very grateful.

How difficult is it to perform a successful streaking of a colony to a new agar plate? That is a successful isolation. If I gently touch a colony with a loop and then move it to the new agar plate, how likely will it be to have only one kind of bacteria on the new agar plate? I mean roughly, how hard is it?

Should the new agar plate with the single colony be cultivated in an incubator before being sent off for 16s rrna? How long?

Would you send the agar plate for processing or should it be sent in any other type of container, or what ever word is appropriate to use? I don't know.

[Jon Moulton]

Hi Thorstein,

Simply touching a colony with a sterile loop and moving it to a new plate will not get you isolated clones. You need to use the streak-plate method, which is a several step dilution of the original inoculum you transferred to the plate. Be sure to re-sterilize your loop (likely in flame) between each set of streaks.

Here are some sites which discuss the technique, with images and figures.

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This animation is nice. You can take the initial inoculum from a plate instead of the broth they show (they mention that technique at the end of the narrated animation)
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[JennYeap]

hi, streaking plate is easy, Jon is correct, u'll need to touch the colony and streak it across one corner of agar, then re-sterilize each time when u repeat another streak (5 streaks).

for Q1, yes, u need to incubate your plates overnight for the bacteria to grow.
for Q2, normally we send PCR products for sequencing. 1st, you will need to isolate the DNA from bacteria as PCR template. 2 options for DNA isolation; 1= small scale, pick 1 colony and dilute it in 50uL sterile water in a sterile PCR tube, heat it at 95C for 5 mins, cool it down and spin down the cell debris. The DNA will be in the solution. 2= you scale up the culture by growing 1 colony in suitable broth, perform a colony PCR using 1uL culture as template. Anyway, perhaps some company does provide services that include PCR and sequencing, then you can just send the DNA to them.