Molecular approach is most suitable

Biochemical testing also is ok, but the most advanced one is the molecular one.

for identification and enumeration of bacteria in environmental samples one can also use fluorescence in situ hybridization (FISH or CARD-FISH)

What is the average cost involved in FISH?

FISH is relatively cheap, but you need to make some initial investment. Monolabelled probes (i.e. oligonucleotides with e.g. Cy3 at the 5’ end) are about 60 euros each (I guess it also depends on the company you order). The chemicals are relatively cheap (NaCl, paraformaldehyd, Tris-HCl, EDTA, SDS ethanol etc), but polycarbonate filters may be also costly, again depending on the company (i.e. Whatmann is cheaper that Millipore). So, before you start you need to spend about 250 euros (very roughly) + probes, but then you proceed many samples with no additional spending. From the equipment, hybridization oven, water bath and epifluorescnece microscopy are essential, and this is rather expensive to buy just like that (especially the microscope). CARD-FISH is more expensive, because you need more expensive chemicals and the HRP-probes are 250 each (but you get discount when order in bulk). If you are interested, I can send detailed protocols, and then you may calculate the exact quote yourself.

The detection of microbial pathogens in foodstuffs can be done by different analytical

approaches, among which the molecular methods are useful tools of investigation, particularly because in this way it is possible to establish the presence of the target genes of a bacterial specie, but also the presence/absence of the genes encoding for pathogenic factors.

On the other hand the safety criterions established all over the world, and certainly by the EU legislation in force, are based on the isolation and the biochemical characterization.

Therefore the development of an holistic approach is fundamental, because we need to understand microbial physiology, as much as microbial genetic, and possibly overcome the unsuitability of some common devices. This is the case, for example, of the API (bioMèrieux, France) system, that is largely recognized not suitable for Vibrio species identification.

Classification of bacteria is determined by publication in the International Journal of Systematic Bacteriology, and Bergey's Manual of Systematic Bacteriology. The International Committee on Systematic Bacteriology (ICSB) maintains international rules for the naming of bacteria and taxonomic categories and for the ranking of them in the International Code of Nomenclature of Bacteria.

Of course the molecular techniques are the most accurate but where you don't have access to it or cant afford to get lab. space abroad,, the use of differential media, gram staining & biochemical reactions remains the only option available.

Molecular techniques as 16s r RNA sequencing using universal primers are the most reliable method for the identification of bacteria and for identification of fungi, you can use staining and microscopic methods.

Hybridisation methods (FISH) overcome many of the biases associated with PCR. Yet, as pointed out earlier start-up is not cheap (Nikon is probably a good less expensive option c.f Ziess). CARD-FISH can also be problematic due to cell wall permeabilisation issues, especially in G+ves. If you are alone, then the least difficult and inexpensive path for pathogen and indicator organisms are the many simple biochemical based commercial test kits OR PCR based methods based on 16S rRNA gene as others have suggested. Good Luck

The method used must be suited to what you want i.e. a human microbiological diagnostic lab may at this stage find traditional methods such as culture and Vitek or API systems of greater value. Food labs will follow standardised protocols usually based on chromogenic or selective media and biochemical testing. Both of those type of laboratories will make use of well tried , reproducible and accurate methods, that are relatively easy to do. Automated or semi-automated methods are also preferred in a diagnostic laboratory, especially those with high turnovers. These laboratories are increasingly making use of PCR or gene probes for specified pathogens, especially viral pathogens i.e. cell cultures are rarely done nowadays and are often restricted to producers of vaccines and laboratory diagnostic kits or research where it is necessary to generate lots of virus. Research laboratories are focusing predominantly of genetic-based methods.

The main aim of any diagnostic laboratory is to run the needed tests, do it as quick, as cheap and as accurately as possible.

Yes this are first step for microbial identification thn u cn go further for molecular level

Thanks everybody I have been out of circulation lately.

We use the MicroLog system from Biolog. It's much more accurate than Vitek and you can test many many more organisms at the same time. (Another added bonus is that you don't need to know the gram status of the organism before testing) It's less precise than molecular methods but more affordable if you want to set up your own shop and test in-house.

1st basic level identification for different type media s are available and enrichmant depends on micro arganisams reqirements n suitable conditions. Standard methods are very use full to detection of micro organisams

I am currently investigating the purchase of a BioLog system and found that the initial purchase costs are similar to the Vitek system and of course one can use the manual or semi-automated system which is even cheaper. However, the cost of the consumables are far greater and therefore routine use of this system only becomes more useful for high throughput diagnostic or reference laboratories, where labour is limiting or a high level of identification is desireable. I intend to compromise and only use the system when I have difficultly in identifing bacterial isolates, which in most cases are environmental or commensal bacteria i.e. one step down from 16S or 23S RNA sequencing.

As a primary identification you can perform GRAM STAINING followed by CULTURING,BIOCHEMICAL,still not identified you can go for molecular level .This is sequential way that you can identify a microorganism without much expence

I agree that the Gram's stain whether on culture or directly from the sample will assist you in deciding what tests to implement. Just a few provisos. On direct smears some Gram positive bacteria will stain Gram-negative. Unless found in a thin portion of the direct sear or the bacteria have a thick wall or capsule, Gram-negative bacteria can be difficult to visualise in direct smears as the background tissue will also stain pink. For cultureable bacteria, stained smears are less sensitive than culture. The Gram's stain must also be properly performed as incorrect decolourisation can lead to erroneous results.

Yet another strength of the biolog system. No need to gram stain, in usually 2 days from initial plating to result, you have definitive ID's with gram status, and the database is significantly larger than the Vitek resulting in much fewer indefinitive and incorrect answers in the final ID and much less guesswork. You can also test as many samples simultaneously as you want instead of the limited number allowed by Vitek. (sorry if I sound like a promoter, but I really enjoy this system after dealing with lots of other ID methods) I once figured out the cost per test and it worked out to about $12 per ID including all supplies.