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Any microbiologists around to solve this puzzle (E.coli on XLD plates)


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Ecoli_tinsel_EMBA

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Posted 09 March 2015 - 08:07 AM

Hi,

 

I'm a food microbiologist here in Australia and was spending my (sad) day off googling around for more info regarding a current issue I am having in my lab.

 

We use XLD agar (Xylose lysine deoxycholate) for our Salmonella testing.  As part of our Quality control for new batches coming in, we use a salmonella strain as the positive (no issues) and E.coli as the negative (For our testing, negative used is Citrobacter).  Just wondering if anyone out there uses XLD and if you are finding that you are not getting the <G1+ growth (equivalent to <4 streaks of the 16 streaks).  I am finding that I am getting above and in some cases full growth.

 

I've gone back to the manufacturer who have said they haven't had that issue..... even though we use the same strain of E.coli!  Driving me crazy.

 

Comments from any professionals here would be much appreciated!

 

Regards,

 

Jen



Charles.C

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Posted 09 March 2015 - 09:02 AM

Dear ete,

 

and E.coli as the negative (For our testing, negative used is Citrobacter)

meaning ??

 

g1+

meaning ??

 

not getting the <G1+ growth

 

with respect to what ??

 

I am finding that I am getting above and in some cases full growth.

 

with respect to what ??

 

Sorry for my lack of semantics. Pls clarify.

 

Rgds / Charles.C


Kind Regards,

 

Charles.C


SUSHIL

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Posted 09 March 2015 - 12:14 PM

Hello Jen,

salmonella,shigella ,e coli and citrobacter are a group of organisms belonging to enterobacteriaceae (coliforms) and citrobacter and e coli are different organisms belonging to different genera.

 Citrobacter on XLD will give yellow colonies (due to fermentation of xylose) and e. coli is gennerally inhibited and Red colonies may occur with some Proteus and Pseudomonas species.

 

Colonial Appearances

Organism

Appearance

Salmonella, Edwardsiella

Red colonies with black centres

Shigella, Providencia, H2S-negative Salmonella (e.g. S. paratyphi A)

Red colonies

Escherichia, Enterobacter, Klebsiella, Citrobacter, Proteus, Serratia

Yellow, opaque colonies

 

 

Quality control

Positive control:

Expected Results (48 hours)

Salmonella Typhimurium ATCC® 14028 *

Good growth; red colonies with black centre

Negative control:

 

Escherichia coli ATCC® 25922 *

No growth

 

 

Test Organisms

Inoculation Method*

Incubation

Results

Time

Temperature

Atmosphere

Salmonella enterica
ATCC® 14028

A

24hr

35°C

Aerobic

Growth; red colonies with black centers

               

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

Shigella flexneri
ATCC® 12022

A

24hr

35°C

Aerobic

Growth; red to pink colonies

Enterococcus faecalis
ATCC® 29212

B

24hr

35°C

Aerobic

Partial to complete inhibition; clear, pinpoint colonies

Escherichia coli
ATCC® 25922

B

24hr

35°C

Aerobic

Partial to complete inhibition; yellow to yellow red colonies

 

 

 Attached here are some files of XLD MEDIA-

Attached Files



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Charles.C

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Posted 09 March 2015 - 12:28 PM

Hi ete/Caz,

 

Citrobacter is a genus. E.coli is a species.

 

Citrobacter spp are often related both biochemically and serologically to Salmonella spp. C.freundii is a common example, from memory.

Citrobacter spp are a well-known source of problematic false positives in Salmonella testing for foods.

The mimicry is often exhibited on XLD media so perhaps not a logical negative control for XLD unless traceable to a defined reference strain/species.

 

Attached File  BAX system.pdf   71.26KB   13 downloads

Attached File  Merck testing methods for food relevant microorganisms.pdf   3.57MB   25 downloads

Attached File  Distinguishing Citrobacter from Salmonella.pdf   121.25KB   16 downloads

 

@Sushil, the data in yr post is not necessarily incorrect but is probably incomplete.

 

Rgds / Charles.C


Edited by Charles.C, 09 March 2015 - 12:43 PM.
expanded

Kind Regards,

 

Charles.C


Ecoli_tinsel_EMBA

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Posted 10 March 2015 - 12:06 AM

Hi all,

 

Wow didn't expect to get so many responses so fast :)

 

Firstly, thanks for everyone that replied.  Appreciate them all.

 

Charles-

 

1.  E.coli is the negative control we use to test the QC of the media so it is a once off when we get a new batch of XLD powder in.  Citrobacter is the negative control we use during routine testing with XLD to check that the test is working as expected (so QC of the test).

2. Depending on how you streak your plates, there are 2 ways we do it.   I guess just imagine if the plate is divided into 4 and you streak 4 lines in each and then one small one in the middle.  so saying E.coli should not grow more than G1 means it can only grow in one section for it to pass. 



Ecoli_tinsel_EMBA

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Posted 10 March 2015 - 12:11 AM

Caz,

 

We tried a different batch from a different supplier and have had similar results.  We have since switched over to the water strain of E.coli for general testing so am testing this out today.

 

yes Citrobacter comes up yellow - E.coli has a similar appearance. 

 

We've gone back to the supplier to ask for their advice on preparation and even had them send through some prepoured plates.  incubation, temperature of storage has all been checked.

 

Will probably start from the beginning again :)  Am debating on asking to go to the suppliers QC department and have a view of how things are done there as they are nearby as I keep going in circles. 

 

Thanks for the response :)  Have a good day!  Good to see a forum that is actually active and helpful :)

 

Regards

 

Jen



Charles.C

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Posted 10 March 2015 - 04:22 AM

Dear Ecoli-t-e,

 

Thks yr response.

 

As i understand, yr query relates to (a)  applying species C.x, strain XXXX to brand Y, XLD plates and (b) getting excessive yellow colonies on the plate (ie >>G1) when minimal growth was expected.

 

Or, as in yr No.2 of post 6, perhaps E.coli - strain XXXX should replace the C.x above.?? I guess from title of this thread - probably YES. :smile:

 

I cannot find any refs/media specs using defined strains (or sometimes self-defined :smile: )  of citrobacter spp as a -ve control. As per my previous post, this genus/species can be problematic. Are you using a defined strain, ie one defined to have minimal growth.? or a possibly contaminated self-defined strain. ?

 

I can find refs to defined strains of E.coli, eg -

 

Attached File  xld control.png   79.9KB   3 downloads

 

I have never used quartering so cannot comment much in that respect. IMEX mainly (questionably :smile: ) focussed on  + ve controls / multi-media/multi-broths for Salmonella. More often been hampered by good growth Citrobacter spp.and mimicing Salmonella, even in O-serology

 

Sadly, E.coli I have no experience on XLD. The chart above (partial inhibition) needs quantifying. Other charts are more growth indicative, eg - 

 

Attached File  xld control.png   79.9KB   3 downloads

 

(other refs indicate inoculum size can also be a factor)

 

Rgds / Charles.C

 

 

 

 

 

 

 

.

 

 

 

 


Kind Regards,

 

Charles.C





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