Help. Why biochemical identification and molecular give different results? - (Apr/09/2015 )
Hello,
Could you please help me if you have any idea about my results :
I isolated lactic acid bacteria from cattles sample. Then i grew them with MRS agar in anaerob environment. I was very careful about contamination.
I choosed two potential isolates. After that I did biochemical identification using API 50 CHL kit and molecular identification using 16S rRNA.
But then i found my two isolates gave different results.
1st Isolat : API - Lactococcus lactis 78%; 16S - Enterococcus sp. 99%
2nd isolat : API - Lactobacillus fermentum 96% ; 16S - Streptococcus sp. 99%
Just, how come??? I don't understand.
Why it gave different results even though it's from same culture?
The 16S rRNA identification didn't give the information about species but only as sp. The 2nd isolats even more confusing because one is bacils and one is coccus. And i'm not sure either if it's coccus or bacil..i'm only sure if it chains in microscope.
I'm so curious and no one near me could help me about what's actually going on.
Thank you very much.
Looking forward to hear your suggestion.
Almost certainly you have a mixed culture. You should re-streak them on MRS plates and re-grow colonies. You could do that a second time, picking a single colony for re-streaking. Only when you have a single species can you effectively do API or 16S characterization. Your original sample likely has hundreds or thousands of distinct species.
API 50 CHL is ONLY for Lactobacillus and some related genera, following the identification guide should make no sense if your isolate is something outside the identification guide associated to the API kit. How do you know they are lactic acid bacteria?
BTW, which database did you use for the ID? nt from NCBI? That one is not the ideal for ID. The first match doen't have to be the real ID, many times the names are wrong. Select the 16S RNA sequences database or try EzTaxon.
Read the literature. API 50 CH/CHL reportedly have some problems.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1248469/
http://www.ajevonline.org/content/39/4/347.abstract
http://link.springer.com/article/10.1007/s002840010085