A better microbiologist then me can probably help more but do consider 16S PCR amplification and sequencing. This is done routinely in any microbial ecology lab these days and will give an answer.

Yoram 

In environmental Microbiology biochemical characterization could just hint you the genus of the bacteria. Your savior would be as  Prof. Yoram says is 16srRNA amplification, sequencing followed by matching the sequence with GenBank. 

Dear Shubha,

I suggest to you the 16S RNA PCR with universal primers and sequencing. 

It is a fast and available in many labs. Be careful with the quality and replicates.

Best regards.

One more point, for useful 16S you will axnic  bacteria (single species), so do at least 5 transfers on agar plates to make sure. See a review here:

Janda and  Abbott (2007) , 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045242/

Thank you all for your suggestions. 

When isolating lactic acid bacteria from curd samples(household not commercial) using MRS media I got this culture. As it was one of perfect rod shaped bacteria ( 100X magnification) in comparision with others I was very much interested in it. But after  5-6 sub-culturing now I am having difficulty in subculturing that bacteria also. Its not growing in broth onmy in agar media its growing but very little growth is seen even after 3-days( earlier 48hr full growth). even i tried aerobic as well as anaerobic I am not getting good growth. Can anyone hepl me with this.

I have seen this phenomena before with with many environmental bacteria but still not sure about the explanation. Nevertheless, since it was cultured from curd, and assuming milk based curd, how about trying milk based medium?  I would suggest low NaCl LB or even milk Agar. 

Do 16S rDNA sequencing

I go with the suggestion of Ruchi Tiwari on further routine biochemical testing. You could also use some kit system of identification to complement the routine biochemicals. After this you must have a narrower possiblity of the identity of the organism. 16S sequencing can be done thereafter and lucky for you Hudson have indicated to help you on this.

Dear: I am in agreement with all the suggestions given to you. Cultivation of isolated bacteria in your given condition may not be suitable for survival and thus you are getting different results at different time.s As your isolate is Gram positive rod with catalase negative, oxidase negative property, it appears to be Lactobacillus spp.  However, before getting this presumption, you need to carry on O-F test (should be +ve), spore staining (should be -ve), raffinose fermentation. Above all, 16S rDNA is the best way of identification.

Survival of the isolate is critical. You may try with 0.6% yeast extract supplement to MRSB/MRSA , it may work. Good Luck

I agree that 16S rDNA identification is the best and more accurate and gives you final answer

You are right Ms. Shubha on subsequent subculture we tend to lost the strains many a times. I am sure if you are still interested in identifying that isolate, you could isolate the DNA from non-revivable culture which you have and perform 16S rRNA PCR followed by sequencing. 

Cld nt edit the upper comment: In subsequent subculture we tend to lose the strains many a times :) Cheers. 

Please use the molecular marker like 16S rRNA  as authentic identification tool.

If you have frozen glycerol stock of the original isolated bacterium, I would suggest that you perform only one subculture (preferably in broth), perform 16S rRNA gene sequencing (it is better to identify more than 1300 bases of the gene). After identification of the Genus & possibly species of the bacterium, you will be able to know which media is best suitable for growth of this bacterium from the literature.

Try to culture your bacterium in minimal medium and see if it grows. Of course the best way is to do 16S rDNA sequencing  to identify unknown. Your bacterium if isolated from curd, and having branched phenotype and other characters as you described earlier, it might be a Bifidobacterium. Please confirm it with 16SrDNA sequencing.

You can go through 16S rRNA and/or MALDI-TOF (molecular weight) and/or other several biochemical tests.

16SrRNA identification will give the final answer. Universal primers are available eg: 27F & 1492R

Morphologically looks like a Lactobacillus sp.  Maldi-Tof MS should be able to identify this organism.

I agree Shubha, 

The bacteria is gram positive, catalase and oxidase negative rod shaped bacteria, which are in chains. that means it might be Lactobacillus sp. But Couldnot identified the bacteria with morphological and physiological conditions. Apart from that genotypic character is must to confirm the name of the bacteria. So, you can perform 16S rRNA gene sequencing method with Universal primers ( 8 F or 27F & 1492R). Good luck. 

As already suggested you need to perform 16SRNA amplification and gene sequencing. Please link up with Andre O Hudson since he has offered to help. The search for substances with antibacterial activities is necessary and you should not spare any effort to determine the identity of your isolate. Good luck 

thank you al so much. I will take up your suggestions.

i am trying to subculture that bacteria, in milk agar also i did not get any growth. once it is done i will go for identification

Some times 16S will only give you genus level identification and you will to sequence some other genes as well. For lactobacilli see this paper by Naser et al 2007: Identification of lactobacilli by pheS and rpoA gene sequence analyses. Available here http://www.ncbi.nlm.nih.gov/pubmed/18048724

You can isolate your DNA even from your non-revivable recent culture too.

Thé morphology is close to bifidobacterium try thé 16S sequencing or try thé pcr for identification of the genus if m'y memory is good thé référence paper was published un 1996 by kok 

Thé 16S sequencing is always thé best thing

I suggested you to use 16 S rRNA or 16Sr DNA or you can use API-20. 

For Lactobacillus spp., you will need a microaerobic environment.  Use a jar with microaerobic sachet or if not use a candle jar.

Rene, are you sure about the MALDI TOF? My understanding is that it works only for known bacteria as you compare the results to a library.

sequencing of 16s RNA amplicon, and also API test to determine the species of bacteria based on the biochemical reactions. 

Hi, you can use sugar fermentation for identification, bio-gram and molecular characterization with the aid of a PCR machine.(polymerase chain reaction)

Hi, you can use sugar fermentation for identification, biogram , the the use of antibiotic subcetibility and PCR machine. Polymerase chain reaction. 

For bacterial identification you can download the following software and by putting all details you can identify it sitting at home. but you have to do many biochemical tests as given in software. This s/w can be used offline... for online identification you can go for ''ABIS bacterial identification online''...I think it will help you a lot... All d best... 

http://www.som.soton.ac.uk/research/sites/pibwin/

http://www.tgw1916.net/bacteria_logare.html

Thank you all

The organism  initially used to grow well at 37 degree in MRS broth and agar.

I tried sub-culturing it again now all possible medias (MRS, Bifido agar, Milk agar) but i couldnt . Let me try it again.

Thank you all 

Regards

Shubha

Best way to figure out is to carry out 16S rRNA gene sequence analysis. FAME analysis, MALDI-TOF with library match will definitely be helpful as well. But to determine phylogenetic affiliation, go for 16S rRNA gene sequencing (to get > 1400 nucleotide long continuous stretch) followed by its analysis at EzTaxon site.

Hope it helps

Pradipta