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Subject:
From:
David Carlberg <[log in to unmask]>
Reply To:
The Pharmaceutical Microbiology Forum Email List <[log in to unmask]>
Date:
Fri, 6 May 2011 07:48:12 -0700
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Hello Alessandra,

Determining bacterial populations by turbidometry is simple, but you have to realize it is not very accurate and is only reliable for estimating cell densities over about 10E7 cells per ml.   A few rules should be followed:

1. Use the same organism for both the viable count and the turbidometric measurements. I assume you'll be using a plate count for the viable count.
2. Make sure cells are well dispersed and there are no clumps in the suspension when doing both turbidometric and viable counts. 
3. Do the turbidometric measurements at the same time you do your viable count. If the suspension stands for any length of time, its turbidity will change due to growth. Put the suspension in an ice bath if there is going to be a delay (more than 10-15 minutes)  before doing the readings. 
4 The lower the wavelength you use, the more accurate the reading, BUT if your cells are in broth, you should use 650-660 nm. If they are in saline or buffer, then you can use 450 nm. That is because broth contains substances that absorb at the lower wavelengths and their presence would greatly reduce the sensitivity of the measurement.  
5. Even though you will be using Absorbance (O.D.) vs. viable population for your standard curve, read the %T scale and convert to Absorbance mathematically (A = -log(%T/100). The %T scale is much more accurate and easier to read than the A scale on the Spec 20. 
6. Use clean, preferably new, tubes or cuvettes in the spectrophotometer. Avoid tubes with scratches or hazy sides. It's important the tubes are all of uniform inside diameter. Put marks on the tubes and place them in the spectrophotometer the same way each time.
7. Zero the Spec 20 with the fluid your cells will be suspended in.
8. Don't use readings that are less than 10%T (over 1.0A). Accuracy drops off steeply below 10%T. Make  quantitative dilutions of your suspensions if necessary. 
9. If you are using organisms that are pigmented, such as Serratia marcescens, you can do an absorption curve by plotting absorption vs. wavelength and use the wavelength at the absorbance peak. This works best if the cells are in saline or buffer. 


Here is a reference to an article that might be helpful to you. I tried to attached a copy of it, but your network does not allow attachments.

Environmental factors influencing the relationship between optical density and cell count for Listeria monocytogenes

Journal of Applied Microbiology  99(6), 1503–1515 (2005).

Buena Suerte!
.

David Carlberg, PhD
On May 3, 2011, at 2:37 PM, alessandra garces wrote:

> Dear Forum:
> 
> I need your help on the Measurement of the concentration of bacterial
> suspensions by turbidimetry assay.I want to do a job of equivalence of the
> technique by turbidimetry and viable count, but I need information on recent
> articles, I am not going  to work with McFarlan scala, I am goint to work
> Spectronic 20, I will do estimations by turbidimetry and the same bacterial
> suspension by viable count......
> 
> Please if anyone have information, recent articules or any information about
> this topic, please I would greatly appreciate it, that statistical
> techniques applied to the results..please where I can find it. I am in
> Venezuela and it is hard for me to pay in dollars for some papers......
> 
> waiting for their responses and comments
> 
> Alessandra Garcés
> 
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The PMFList (http://microbiol.org/PMFList_info.htm) is operated from
The Microbiology Network (http://microbiol.org) and supported by
our sponsors (http://microbiol.org/sponsor.htm) as a service to
the scientific community.

Please take a second to visit our sponsors' web sites and say thank you for their support of this service.

Accugenix - http://www.accugenix.com

American Type Culture Collection - http://www.atcc.org

BD Diagnostic Systems - http://www.bd.com/ds/

Biolog - http://www.biolog.com

Biomerieux - http://industry.biomerieux-usa.com

Biotest - http://www.BiotestUSA.com/micro

MicroBioLogics, Inc. - http://www.microbiologics.com

MODA Technology Partners: now a LONZA company http://lonza.com/moda

Pall - http://www.pall.com

NovaTek International - http://www.ntint.com

Rapid Micro Biosystems - http://www.rapidmicrobio.com  (formerly GPS)

Veltek Associates, Inc - http://www.sterile.com

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The nature of this service is to provide a medium for communication.  The specific statements and endorsements of individuals participating in the discussions are not necessarily those of The Microbiology Network, Inc., the PMF, or the sponsors of the list.

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