I tried to use OD of 0.1 (660nm) as standard. While on plate count it came different than my expectation. Then, I tried OD 0.08, yet I did not get the actual number 5x105 cfu/ml on plate count on 1:200 dilution (It came more than this value). Can anyone clarify this?
Very good question. Well at the onset remember that since the bacteria in suspension behave like any other particles in suspension in most of their movements. Therefor, a suspension of rods will show different OD at different time intervals in the same suspension. That is because the rods, as they show different movements, will orient their position at different time. This not so with cocci. I would suggest that, you take several such OD in a short span of time, check the mean and calculate the Standard deviation . Now you see how much they are within the limits of tolerance as required by you. Then if you find its tolerance is satisfactory you accept the mean to be the required number of cells. Otherwise the plate count might be OK for some experiments too.
Hi Dinesh,
You are working around the right OD600 to yield an equivalent 0.5 McFarland for Ppseudomonas (aeruginosa), but a 0.5 McFarland is not expected to be 5 x 105 cfu (consistent with your results!).
CLSI recommends using a 0.5 McFarland equivalent, then diluting this 1:20 to yield 5 x 106 cfu, and inoculating at a 10-fold dilution to yield ~ 5 x 105 cfu in a given MIC experiment.
We typically use an inoculated control well diluted 1:1000 and plate 100 uL to yield ~ 50 colonies. When back calculated, this will yield a concentration in the MIC plate of ~ 5 x 105 cfu.
Hope that helps and good luck!
Christina
Dear Dinesh Subed
you can find your answer in atached file page 17 clinical and laboratory standards institute (clsi).
OD600 to yield an equivalent 0.5=1x108 ~1.5x108
- 100000000/20=1x1o6 CFU OR 150000000/20=1.5X106CFU
- 1000000/2=5000000 ~ 5x105 CFU OR 1500000/2=7500000~7.5X105 CFU
Many thanks all of you. Just one more query; my lab has been using 660nm to calculate OD for bacteria count. So, which is appropriate for above process 600 or 660? Note that MHB is being used.
Hi Dinesh -
It doesn't matter if you use 600nm or 660nm. You are not measuring absorbance, you are only measuring turbidity. The wavelength is essentially visible light.
You could test this yourself - measure at both and see if you get different results.
Good luck.
Dinesh,
600 nm or 625 nm, depending on the standard used OR you can use BBL™ CrystalSpec™ Nephelometer very easy use and standard
http://www.analisisavanzados.com/modules/mod_tecdata/antibiograma/IFU_McFarland.pdf
http://www.bd.com/ds/productCenter/245009.asp
Dear Colleague,
May I ask to all,
if OD600 = 0.5, is equal to how many bacterial cells ? Many thanks before.
Hi Yayuk,
The optical density of broth depends on many factors including size and shape of of the cells. For example, for same no of CFU, OD may be lesser for cocci than rods. If you want to calculate no of bacterial cells on the basis of OD, it's good to have a standard curve on CFU ( by plate count ) and OD600. In my case 0.5 OD =~ 5x10^8 CFU/ml (for P. aeruginosa).
- Badges
- Science topic