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Broth Microdilution

Broth microdilution video
We are now going to set up a broth microdilution, which is used when you would like to know the minimum inhibitory concentration of an antibiotic. So, the first step is to make up a series of serial dilutions of your antibiotic of choice. So today we are going to test meropenem. And I have a Klebsiella bacterial isolate ready to be tested. The first step is to label a tube of PBS as we did in the antimicrobial susceptibility disc testing. So you need an overnight growth of your bacteria that you wish to test, preferably with single colonies visible because this way you’ll be able to tell if there is any contamination present.
So take approximately one colony of the bacteria and suspend in the PBS. Discard the swab and mixed thoroughly. We are looking for a suspension equivalent to 0.5. McFarland. So take a known McFarland standard and compare this to the suspension you have made. It’s quite often easier to hold something with black print on behind the tubes. You are looking for similar consistency in terms of turbidity or cloudiness of the liquid. And keep adjusting your suspension until the turbidity or cloudiness is the same in both tubes. So once you have the correct suspension, we want to dilute this 1 in 100 in sterile PBS Once your final suspension is ready, this should be used within 30 minutes.
We will now take a microtitre plate and label on the side. Each row will be used for each isolate to be tested. The first 10 wells will contain increasing concentrations of your antibiotic that you are to test. The 11th well will be a sterility control, so it will contain only the broth. The 12th well will be your growth control, which will contain your bacteria. Label the row you are going to use with your bacterial number.
You should have 10 dilutions.
Double the final dilution that you are looking for. So starting with the lowest in well 10, take 50 microliters of your dilution and add to the well. You do this with each dilution you are testing because we’re going to add the same volume of your bacterial suspension, which will give you the actual concentration that you wish to be tested.
So once you have added 50 microliters of your antibiotic concentrations into the first 10 wells, take 50 microliters of your bacterial suspension and add this to each well.
Finally, take 100 microliters of the broth you have used, in this case Mueller-Hinton broth, and add to well 11. This is a sterility control well and we expect to see no growth in this well.
Take 50 microliters of sterile broth into well 12 and also 50 microliters of your bacterial suspension.
The final bacterial concentration in your test wells and in the growth control well number 12 is 5 times 10 to the 5 Colony Forming Units, or CFU, per microliter. So incubate your plate overnight in the temperature and incubation conditions recommended by EUCAST for your bacteria being tested. So after overnight incubation, first thing we will want to check is the control wells. In well 12 you should have visible turbidity or a pellet of bacteria visible at the bottom of the well. And in control while 11, which was the sterility control, we would expect to see no turbidity or visible pellet in the well.
If you can see turbidity or a pellet in control well 11, then the test will need to be repeated from scratch. If your controls have passed, then the next thing we do is to look along the wells of your test isolate and find the first well where there is no visible growth of the bacteria, so no cloudiness or no turbidity. And this is the well that contains the minimum inhibitory concentration of antibiotic for that bacteria. So I tested two isolates and isolate number three only has growth in wells nine and 10. So the first well with no visible growth is well number 8, which contains 0.25 milligrammes per litre of meropenem.
This is the minimum inhibitory concentration for this bacteria of meropenem. Isolate number four, however, has grown in all the wells of the tests, so– has even grown in the well which contains 32 milligrammes per litre of meropenem. So this is a fully resistant isolate, which is resistant to meropenem, so is, therefore, a carbapenemase producing bacteria. So, in today’s session, we have done broth microdilution in which we have tested a variety of concentrations of an antibiotic on a bacterial isolate. This is often done in the case where disc diffusion tested is not supported by EUCAST or when you need a more accurate minimum inhibitory concentration value for your research.

Whilst disc testing is relatively cheap and simple to do, occasionally a more accurate minimum inhibitory concentration (MIC) value is needed. The EUCAST gold standard method of MIC determination is the broth micro dilution method.

Principles of the procedure: The aim of this procedure is to determine the minimum inhibitory concentration (MIC) of a bacterial strain to a particular antimicrobial. This is done by making several dilutions of an antimicrobial in a 96-well plate format. The MIC is the dilution at which there is no visible growth of bacteria. In this practical we will be following the EUCAST ( -European Committee on Antimicrobial Susceptibility Testing) guidelines used in previous steps. Make sure you are using the most updated version.

The choice of dilutions to test will depend on your reason for testing. If for clinical reasons, you will want to test a therapeutic range of dilutions. If for research reasons, you may want to test a wider range. It is normal to test 8-10 dilutions, each one double the dilution of the last.

Notes on the video

A flame curtain is not normally used in the UK

Safety cabinets are not routinely used for non-respiratory CL2 pathogens so long as aerosols are not being generated

It is not normally advisable to talk while inoculating media, and was only done in this video for the purposes of demonstrating

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Bacterial Genomes: Antimicrobial Resistance in Bacterial Pathogens

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