I want 1000 bacteria (numbers) to begin my experiments .How will i get these numbers? Any specific methods? please give suggestions.
Hello, believe me, if there was any possibility available for receiving exact numbers, you saw the exact numbers in all Certified Reference Materials for microbiology. So we all talking about approximal numbers, nothing else...
Hello, take your stock solution and made decimal dilutions to understand the number of colony forming unit (CFU/ml) in your stock sample. Than after you can calculate the exact dilution for receiving approx. 1000 colonies.
See attached picture for more info.
Good luck!
You can never reach exactly 1000 bacteria in your sample. You must understand that dilution methods can reach given accuracy of the method.- It means you can obatin sample with the number of bacteria from approx. 950 to 1050.
Best reagrds
Vit
Yes. You cant expect the exact number of bacteria. Instead you could calculate the cfu/ml as answered previously or go with McFarland's turbidity standard.
Good luck.
Data from viable plate counts are after the fact and not useful. Counts in dilution will not remain stable in the 24-48 hours incubation required for growth on plates. A standard curve - counts vs. OD is not gong to be sensitive enough to approximate 1000 cfu/ml though you could dilute from a higher count estimated from that curve. You could also use a hemocytometer and dilute appropriately but that's pretty labor intensive.
No such accurate method available to get exact 1000 no. of cells, mostly serial dilution technique is practiced.as mentioned in Karen's answer or you can use McFarland's turbidity standards.
God Luck
Hi
Take the sprcific bacteria you need and culture on selective media of bacteria after incubaton for 24 hours spelect pure colony and put in nutrient broth for 18 hour and take loopful of colony and numerate the number of colony .
Hi Ramiz, The answers above are good practices to gain colony forming units (CFU) on solid agar media by using serial dilutions.
I would like to know more about the experiment, but an easy way to measure growth of a culture is taking the optical density (OD). If you want to go a step further you can do what Phil suggested and combine the optical density with serial dilutions and spread plating for colony forming units. By combining this data, you can find out the number of cells "cfu" per volume in your culture.
Some rercommendations are OK, but not one can give you a result, you are looking for, unfortunately. It is impossible to prepare sample with exactly 1000 bacteria.
Best regards
Vit
Hi Ramiz Raza ,
As Vit Mateju and others point out, it is impossible to dispense an exact number of bacteria. However, I think what you are trying to do is begin with a consistent number of bacteria for each experiment. This is commonly done by finding the 'CFU/mL ratio' for your given bacterium. Once you have this ratio, you could technically calculate for 1000 bacteria but it is impossible to guarantee that it will be that exact number. The better your pipetting the closer (albeit over or under) that number you will be. Yet, it will give you a consistent density of bacteria (colony forming units/milliliter or 'CFU/mL') to use for experiments, which I assume is your goal.
For this purpose, here is a basic starting point (as illustrated in Karen A. Darbinyan answer). You will want to prepare your bacteria as you would for your experiment and measure their 'optical density' or 'OD' at 600 nm in a spectrophotomer. From that, you will do a set of serial dilutions (1-7) of that stock culture. Commonly, 100 uL from the stock culture into 900 uL of PBS or saline (1). Vortex thoroughly and change your tip. Then take 100 uL from tube (1) and dispense into tube (2). Vortex thoroughly and change your tip. Repeat and continue through the 7th tube. From each thoroughly vortexed tube, take 100 uL of the solution and dispense on an LB plate (x3 i.e. 100 uL on each of 3 plates from each dilution. Then add sterile plating beads to the plate, dispense evenly, and incubate overnight. Repeat this for each dilution.
The next day, when colonies are big enough to count on the plates, you can count colonies from each of the 3 plates/dilution and calculate your 'CFU/mL' [(avg colonies of 3 plates/0.1 mL)*dilution]. If the plates have more than 300 colonies or less than 30 colonies they are uncountable and should be excluded. You will probably end up with 1 or 2 sets of dilution plates that are countable. From that you can determine your 'CFU/mL ratio'. Once you have that ratio for your bacteria, at OD600, under your experimental conditions, you can calculate and prepare a specific density of bacteria for your experiment.
Hope this was helpful. If you have any other questions I might be able to help with, feel free to ask. Good luck with your experiments!
Sarah
Hello, believe me, if there was any possibility available for receiving exact numbers, you saw the exact numbers in all Certified Reference Materials for microbiology. So we all talking about approximal numbers, nothing else...
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