Hello, I first need to select antibiotic resistant bacteria from a bacterial solution so I will use an agar plate with the target antibiotic to do so , then I need to dilute the resistant bacteria to get countable colonies how can I do this? should I inoculate the bacteria then perform serial dilution? Or can I use the selective media directly when performing the serial dilution? is there any other way ?
I'm slightly confused about what you're asking but:
Are there both resistant and non-resistant bacteria in your sample?
What is the goal of this? Is it to count the number of resistant vs. non-resistant bacteria?
Alexandra Johnson Yes the sample have both resistant and non-resistant bacteria and I need to identify the resistant bacteria and dilute them into a countable number of colonies
Better to follow the first scenario, since you are likely to lose some isolates that got inhabited by the selective media. Additionally, presence of selective agent in the medium may also interfere with the growth of the desired resistant isolates.
Since the agar was used with the targeted antibiotic, so the bacteria growing on this agar is resistant (this agar act as a selectable agar for it), to dilute the method is according to the type of bacteria, there are fastidious bacteria that are insulated and shaken by vortex and grown on agar either selective or normal for me I planted it on both and it succeeded. This is called the direct inoculation method...And there are types that need incubation after the inoculation, but in general, the inoculation is with nutritious media. After dilution, it can be planted on selective media or not, according to what is available. Miles and Mazra because they are so accurate. For me, I use the Miles and Mazra method to count the colonies because it is a very accurate method.
Menna Moussa
If you need to count the number of resistant bacterial CFUs in the sample then you would need to do serial dilutions straight from your sample onto agar with the antibiotic, including an plate for undiluted sample, in case their counts are low. If you inoculate them into anything they can grow in prior to plating and dilute from that, you won't get an accurate count of the number of resistant bacteria in your original sample.
You can also always double plate your serial dilutions on agar with and without antibiotic to get countable numbers of resistant vs. non-resistant bacteria.
Per Jameel M. Abduljalil 's point, if you're really worried about the antibiotic interfering with initial growth (if this is important or not depends a lot on the antibiotic, the concentration and the mechanism of resistance) then you can do serial dilutions onto unselective agar, let them grow up visible colonies, then you can do replica plating onto selective media: https://en.wikipedia.org/wiki/Replica_plating
The caveat here is that in my experience, replica plating is a finicky technique to get to work correctly, will only work well with higher dilutions (thus losing precision), and it's difficult to keep both the equipment sterile and not contaminate your lab space.
Thank you all, I don't really care about the number of the resistant bacteria in the original sample, I just need to have a countable number of colonies because I'll further work with them. So from what I understood , it's better to identify the bacteria first using selective agar, then inoculate them and perform serial dilution right?
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