In the case of liquid samples, the dilution of the liquid sample with diluent is an easy task. But in the case of the solid sample or a powdered sample, it's a bit tedious work. I would like to know the viable micro organisms (TPC) of chilli powder. Thus, I have taken 1 gram of chili powder in 9mL of diluent (10^-1). And while going from 10^-1 to 10^-2, I am facing issues of transferring 1 mL of a vortexed sample of (10^-1) to (10^-2) dilution, as particulate matter of 10^-1 is coarse and getting struck in the microtip of the pipette leading to inaccurate sucking of sample (less than requisite amount of 1 mL). Thus after vortex of the sample (10^-1), is it advisable to wait till the particulate matter settle down (sedimentation) and to go ahead with pipetting the clear liquid part of the solution to next dilutions. Or is it hard fast rule to even take the particulate matter to next dilutions in a proportionate way (1/10th dilution factor)? Kindly share your views.
I recommend using a thick pipette tip to prevent clogging. and also by sucking and dropping it several times with a pipette would be helpful for homogeneity.
Never centrifuge the samples during microbial dilution preparation, just vortex for 10 to 15 min depend on your solid or powder samples. You may use wide bore micro -pipette tip(Like Tarsons 521017) to prevent clogging.
Thank you for your response Dr. Mondal. Thus, can I conclude that pipetting out sample after sedimentation is wrong protocol? One has to pipette the particulate matter too to the next dilution before sedimentation.
I would rather suggest you to take bit higher amount of chilly powder say 10g and dissolve it with 90 my of distilled water, vortex it and use the supernatant .Now from this you can easily take 1ml of suspension. I hope this will be helpful for you and you need to change the protocol that you are following. Good luck.
Dear Syed, I would agree with the comments above. Vortex for a long period of time followed by an aliquot. Using a slightly higher amount of chilli powder would be useful as I imagine quite a bit might be lost during transfer! Sounds like a fun experiment!
Dear Syed,
Since you are interested in checking/isolating native microbes from the chilli powder, you can just dilute the sample in water (1 g/10 ml may be) and vortex it for few hours to spread the microbial cells evenly. Use this sample directly for plating in a solid nutrient medium. Else, wait for the sedimentation of the undissolved particles to settle down, then filter the supernatant using a filter paper having pore size lager than at least 0.45 micrometer and prepare the serial dilutions without problem of clogging.
I suggest avoiding the particulate matters in the very first step would be wise, as it will not be beneficial for microorganism isolation.
I guess it should be 1g in 10mL for finely powdered solid. Or alternatively final volume should be 10mL if I am not confused. So I doubt adding 1g chili powder in 9mL would make final volume 10mL. For your scenario I would suppose longer vortexing after sample addition and leave for sedimentation. And slight vortex before taking aliquot.
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