It is a mistake to assume that molecular methods alone can give you an answer. There are well-known examples where bugs with very similar molecular results cannot possibly be the same (eg one's a thermophile, the other's a psychophile). If an accurate identification is essenial, the best thing to do is to combine molecular and classical taxonomy techniques.

Your question is not clear. What type of characterization you are planing, is it morphological/biochemical or molecular? Each of these have immense importance in characterizing a bacterial cell.

Each method for characterization has its own limit like molecular characterization is expensive, Biochemical characterization is time consuming and less precised, morphological characterization is not specific.

And usually few characterization techniques together are required to know about an isolate..

If you want to identify an isolate or an organism, it is best to do it at molecular level, 16 s RNA can be done. The main aim behind characterization is important, so first you will have to specify your problem.

characterization of 16S rRNA actually doesn't give you the clear picture during the process of bacterial identification. However, sequencing of other housekeeping genes; viz. rpoB, gyrB etc may be helpful for proper bacterial characterization.

Best method may be dictated by lab capacity level and resources available and the traits you wish to capture. Methods inclusively are : Morphology on staining, Cultural requirements (nutrients substrates / environment) and colony morphology, Antibiograms, Biochemical tests, phage typing, R - plasmids, conjugation experiments, Molecular methods (Nucleic acid sequences -gene based).

Thanks for the answers. I will do culture for the environment samples and then morphological grouping and 16s identification. My question is for chemiccal charactefization. Such as php plate and gen lll.

It is a mistake to assume that molecular methods alone can give you an answer. There are well-known examples where bugs with very similar molecular results cannot possibly be the same (eg one's a thermophile, the other's a psychophile). If an accurate identification is essenial, the best thing to do is to combine molecular and classical taxonomy techniques.

Please consider use the Bible for microbiologist, i.e., Bergey’s Manual of Determinative Bacteriology in addition to 16S rRNA sequencing (with some other genes too, if possible)...However, you said environmental samples....If you are going to do classical tests you get the culturable fractions to work on. It appears so since you mentioned about techniques like PHP plate. You must go for meta-genomics approach to characterize a major (still not all) part of microbial community from your environmental samples.

How about php plate and gen iii biolog?

http://www.biolog.com/products/?product=Microbial%20ID%20%2F%20Characterization&view=Product%20Summary

http://www.phplate.se/

If I have environmental sample: after bacterial culture and isolation and identification of 16S rRNA, I want to identify prominent bacteria. Do you think php and biolog could be good enough for characterization?

If you are doing 16S rRNA, and then php and biolog...it should be enough....In case of rare confusion you can use some biochemical tests (those not covered by your php or biolog) as acid tests...Good luck..:-)

@Mohsen: For characterization of a bacterium you have to all the tests as given above. You have to start with morphological, physiological and biochemical characterizations. Then you have to do Biolog, and 16S rRNA gene sequencing and then if necessary go for additional differential biochemical tests to characterize the bacterium. You may even require to do FAME analysis if the bacterium is novel along with sequence analysis so some addition genes.

Dear Sohrabi,

The basic approach is polyphasic taxonomic approach. Introduced by Rita Colwell towards the beginning of 1970s, This includes morphological, biochemical, physiological, chemotaxonomic (includes whole cell fatty acids what we call FAME analysis, whole cell lipid profile analyses, cell wall type, menaquinone/ubiquinone type etc), mol% G+C content, 16S rRNA gene based phylogenetic analyses (depending up on group you may have to add gyraseB/other gene seq. analysis as well) and to add to these recently added MALDI based profile study, BIOLOG based substrate utilization studies and lastly genome sequence analysis to determine average nucleotide identity value (ANI value). If you are lucky this will end here but if you are unlucky (and your bacterial strain showed greater than 98.65- 99% seq. similarity to closest type strains, wrt 16S rRNA gene sequence analysis), you have to carry out overall genome realtedness and deltaTm value determination. 

This characterization varies depending upon which group of bacteria you are characterizing. For Gram negative: the characterization does not go much deep into cell wall chemistry but for Gram positive, it goes much deep into cell wall chemistry and menaquinones and Gram negatives these are ubiquinones.

Mostly, people starts with 16S rRNA gene seq. analysis to define the phylogenetic group and then carry out this massive job. If you are doing this please do this at EzTaxon site. Because this site provides you option to carry out analyses with type strains of taxonomically valid strains. moreover, you have additional links available at this site to cross-check (these are LPSN, for nomenclature update; link for sequence retrieval in FASTA format; sequence identity in fraction level, pairwise alignment and lot more). I think it is currently best to refer.  

All these analysis can be carried out by outsourcing, if you have money. Look for DSMZ (www.dsmz.de) or KCTC (Korean culture collection), MCC (www.ncbi.mcc.res.in (culture collection at Pune, India), MTCC (www.imtech.mtcc.res.in (another IDA in India). thay all offer these characterization to be carried out by outsourcing.

Please remember, if your strain is taxonomically novel, you have to deposit that culture in two different countries of independent origins (i.e. if your country has a IDA you can deposit there, but in addition to that you have to deposit that in a culture collection centre another country (although IDA is mandetary for patentable cultures, since their culture maintaining regulations and protocols are well estublised, very authentic you can blindly go with them for culture deposition).

Finally you have to name your bacterium (as per subject that is called Etymology, based on Latin, Greek and neolatin and neogreek).

Once you are through, your name must be published in IJSEM (either as complete descriptive or as part of approve list of name which the journal publishes) so as to make your name legitimate. otherwise it will be called as illegitimate and will not be recognised.

I mean this is not all. you please start. this entire process is called taxonomy in local scenario and biosystematics in global scenario. It will give you lots of ideas to plan and look for and with the availability of genome sequences, i think its output is dependent upon human brain and i think even sky is not the limit there is something beyond it.

taxonomy is a dying art and taxonomist are rare specimens. There are not many people who loves taxonomy and there are even fewer people who likes taxonomists. Sometimes, people who loves taxonomy ends up with doing some other science to survive............

Please if you need to know more on taxonomy of prokaryotes and it comes under my domain, i would love to share my knowledge. Feel free to ask me by giving a mail to [email protected]

best wishes

Pradipta

Dear Sohrabi,

Basic steps can be followed for characterization of Bacteria. I am giving you the outline of such characteristics that you can follow.

1) Morphological Characterization (shape, size, color, etc.)

2) Biochemical Characterization (acid production, carbohydrate, amino acid, citrate utilization, starch, gelatin hydrolysis, MR-VP, etc.)

3) Physiological Characterization (salinity, temperature and pH)

4) Phylogenetic analysis (16S rRNA analysis)

These above (Sr. No. 1-4) steps helps in the identification of bacterium upto genus and strain level. One cannot sure about identification upto species level (species level can be assessed if %similarity of the query isolate is below 97 using 16S rRNA technique). 

For determining upto species level, you can also go for additional parameters such as %G+C content, Fatty acid methyl esterase (FAME) analysis, and DNA-DNA hybridization.

Thanks.

Kunal

I think, Mr Kunal's answer is right for characterization of unknown bacteria. Pls try it. regards.

Article A Survey of the Methods for the Characterization of Microbia...

There are several ways to identify or characterize bacteria.The basic followed techniques are physiological,Biochemical morphological identification.

Thanks Mr Kunal.

Dear Huda S. Al-Rawazq, i need the full text of ''A Survey of the Methods for the Characterization of Microbial Consortia and Communities'' publication, thanks.

@Dr. Kunal is absolutely right about the identification and characterization of bacteria at different level.