5 replies to this topic

[Ismene]

Hello all,

I've been studying the ISO 11731:2017 (Enumeration of Legionella). And I have a question about the procedure for samples with high concentration of interfering microorganisms.

One of the procedures that is required for these samples, is Plating after dilution (1:10). To be able to count the Legionella colonies, shouldn't the cfu/lt of the legionella be extremely high too, despite the number of the interfering microorganisms?

For example, if I follow the procedure and count 15 cfu (legionella), then it will be 300000 cfu/lt (legionella).

If I count 90 cfu (legionella), then it will be 1800000 cfu/lt (legionella).

Did I understand the procedure correctly? The dilution should occur to the sample (1000ml) itself, or to the concentrated form?

Should we follow this procedure only if we expect high number of Legionella?

 

Regards,
Ismene

[Scampi]

Here's hoping your count isn't extremely high!!  The dilutions are to give you a more robust look at your samples, and to verify that your counts are correct.

 

I would follow the procedure as written (sorry I have not looked at it, $285CDN!!!) 

 

This may be of assistance

http://www2.muw.edu/...enumeration.doc

[Ismene]

Here's hoping your count isn't extremely high!!  The dilutions are to give you a more robust look at your samples, and to verify that your counts are correct.

 

I would follow the procedure as written (sorry I have not looked at it, $285CDN!!!) 

 

This may be of assistance

http://www2.muw.edu/...enumeration.doc

 

Thank you for your answer.

Well, I haven't done the experiment yet, I was just looking at the numbers. If the sample has less than 1000cfu/lt, I won't have any count on my petri dish, correct?  For a 5 cfu on a petri dish, the sample must have at least 10000 cfu/lt. The dilution will help with the interfering microorganisms but i don't know how I'll be able to count any legionella in it.

[Scampi]

www.ncbi.nlm.nih.gov/pmc/articles/PMC2519344/

 

https://www.gov.uk/g...FEPTU169.03.pdf

 

http://www.gov.uk/go...200_1028650.pdf

 

 

Perhaps one of the above shall help

[Ismene]

Thank you for these links!

 

I found this paper:

http://www.wrc.org.z...001_04_1394.pdf

 

In this they used this method:

"For the ISO method, sample concentrates were divided into 3 portions: no pretreatment, acid pretreatment and heat pretreatment. Tenfold serial dilutions of each of these portions were inoculated onto aBCYE and GVPC agar".

So they did the dilution in the concentrated sample.

 

In the ISO 11731 it says:

"Culture samples with a high concentration of interfering microorganisms unconcentrated(direct),concentrated or diluted(1:10)"

"For the detection of Legionella, a concentration by membrane filtration will be required in most cases.If the concentration of Legionella is expected to be greater than 10^4 cfu/l, direct planting of the unconcentrated sample can also be carried out. For highly contaminated samples, dilute and use direct plating..."

[Charles.C]

Hi ismene,

 

the count refers to the weight of original sample actually plated and the number of colonies thereby generated.

 

eg if you plate 1ml of a 1/10 dilution the 1ml will contain 0.1 gram sample.

 

So if the plated 1ml generates 100 colonies after incubation  it means 0.1gram sample generated 100 colonies, ie there are 1000colonies / gram sample

 

For selective agars you also need to assure that counted colonies are the specific target also.

 

For standard plates the minimum number for statistical reasons is usually recommended  in the 100-200 area. In practice there is inevitably flexibility.