Hi all
i want to ask you that how can i determine exponential phase of klebsiella pneumoniae? i will try to isolate rna from bacteria and i dont know how many hours the exponential phase takes and harvest klebsiella from petri dishes?
You should grow your strain in liquid medium (eg: TSB -Tryptic Soy Broth - or NB - Nutrient Broth -) to determine the growth curve and hence the exponential phase. You inoculate klebsiella pneumoniae in your broth and you start measuring the optical density (OD) every half an hour (or 15 min). When the OD starts to increase, the exponential phase begins and it lasts until the OD starts to decrease. You don't need to harvest your bacteria from the dish, you already have the suspension in broth, so centrifuge and extract the RNA :)
You should grow your strain in liquid medium (eg: TSB -Tryptic Soy Broth - or NB - Nutrient Broth -) to determine the growth curve and hence the exponential phase. You inoculate klebsiella pneumoniae in your broth and you start measuring the optical density (OD) every half an hour (or 15 min). When the OD starts to increase, the exponential phase begins and it lasts until the OD starts to decrease. You don't need to harvest your bacteria from the dish, you already have the suspension in broth, so centrifuge and extract the RNA :)
Elisa's answer is correct , use both culture.As other Enterobacteriaceae exponential phase of Klebsiella starts 3-4 hours of inoculation and then riches the stationary phase after 24 hours in 37C. so an overnight culture would be perfect for isolation of RNA. Be careful of cross-contamination.
Elisa is correct in how to determine the exponential phase. It very much depends upon what you intend to study with your RNA sample. For studying expression of genes primarily expressed during active growth then it is important to harvest your cells during exponential growth. The is going to be when the optical density of the culture is quite low, for example around 20% of the maximal density your strain will grow to. If you are just looking at overall RNA or at RNA molecules that are present at all times, then you can use an overnight culture. But at 24 hours the cells are likely to be long past exponential phase and well into stationary phase, so it depends whether that is important for you or not.
If you inoculate your culture from a fresh streak plate then the cells will begin exponential growth nearly immediately, you may not see visible turbidity early on but they will nonetheless be in exponential growth. In rich medium like LB growth is likely to slow down and the cells leave exponential by 4-6 hours assuming optimal temperature and aeration.
Thanks all for answers.
First of all, i will study on gene expression, so i think overnight culture not useful for me. i ll try to harvest mid exponential phase of bacteria. also my rna purification kit offers me "do not use overnight culture".
Elisa's answer is the correct one. But if you really need to harvest bacteria from petri, use Blood agar and incubate them at 37°C no more than 8 hours, and no less than 4. That should be your exponential phase for Klebsiella. But it's an "arranged methood". I repeat, the correct one is what Elisa said.
Elisa´s answer is the correct one, be carefully whith the inicial concentration of the your bacterial suspension in the medium, dont forget this point, it´s most be enough to reduce the latency phase, and a big inoculum produce overgroth and a fast reductión of the nutrients in de medium, therefore the death phase apear quickly.
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