My clinical isolates are approximately 100 isolates in a total and I need to perform agar dilution at least 7 antibiotics with them. I spent a few week to learn how to do it because no one in my lab has experience.
When my isolates were ready to use for adjusting 0.5 McFarland, I will pump them using replicating device onto penicillin-containing media with different concentration. After that I also have tetracycline-containing media that needed to pump too, how can I make the replicating pin sterile, not contain the previous antibiotic and use it for the next batch of different antibiotic-containing media. Or I need to have multiple replicating pin, flame the pin, whatever.
Please someone suggest me how people actually perform agar dilution in practical way.
Thank you so much.
I did this type of research year ago. Maybe this will be helpful for you.
Isolation of antibiotic producing bacteria from soil sample
A. Diluent preparation
Test tubes were filled with (9 mL and 10 mL portions) distilled water for the preparation of a dilution series. The tubes were autoclaved at15 p.s.i for 15 minutes.
B. Sample preparation
10.0g of the soil sample was aseptically measured in to a sterilized watch glass. Then it was added to a 100.0mL volumetric flask (10 0 dilution) and mixed. 1.0 mL from the volumetric flask was pipetted out and it was aseptically added to a test tube with 9.0 mL of sterile water to get the 10 -1 dilution. Using the same procedure, a serial dilution was prepared using the appropriate diluent, up to 10 -5 . 1.0 mL of each diluted sample was pipetted out and added to a sterilized petri plate. Then 15 mL of molten nutrient agar, which was cooled to about 45 0 C was poured in to the plate and mixed. After the medium was solidified the plates were covered with a polythene bag and incubated at room temperature in an inverted position for 24 hours.
C. Primary screening (spot test) for antibiotic production
Morphologically different colonies on nutrient agar were selected. Given, test bacterial cultures were inoculated on to solidified agar surfaces using the spread plate technique. Small amounts of inocula from the selected colonies were introduced onto the agar surface as spots. The plates were covered with a polythene bag and were incubated at room temperature in an inverted position for 24 hours.
D. Obtaining pure cultures of antibiotic producing isolates
The isolation streak method on nutrient agar plates was used to obtain separated colonies in order to get pure cultures of the antibiotic producing isolates.
Dear Nokchan,
There is some more information required. What actually you mean? is it about agar dilution or different antibiotic concentration (for resistance dose analysis). So far I understood that the replicating pin you are using may be a pipette/similar apparatus you are using to pour media in petri-dishes. If I am write the three possible approach is :
1. use different tips/pipettes for each solutions/media [the ideal case].
2. Alternatively, you may keep an additional flask containing same media i.e. blank media (without agar and antibiotics). After first pouring you can immerse your pipette in blank media (hot ~ 45 degree or so), rinse the pipette and through the liquid in 'Discard' (repeat 2-3 times). Now you can use the same pipette.
3. If you want to check for sensitivity against various concentrations of antibiotic you may use antibiotics-discs pre-loaded with defined quantity of antibiotics. In one Plate you may check 4-6 different concentrations [HiMedia laboratories or similar companies in your area may have these antibiotic discs].
I hope this would be of help.
Let me know if you have specific questions.
Regards
Prasun
In such large number of isolates or organisms it is recommended to use agar dilution assay by using multi-inoculator which inoculates up to 32 isolates on single 90 mm plates. For detail procedure see the attached articles:
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