It should be remembered that methods need to be accredited by bodies such as AOAC. Simply using one's own invented technique is not good practice.
- Conventional methods
- ISO 6579 Salmonella 2002 method
- Rapid detection techniques
- Separation and concentration techniques
- Immunomagnetic separation
- Impedance (conductance) microbiology
- Enzyme immunoassays and latex agglutination tests
- Nucleic acid probes and PCR
- ATP bioluminescence techniques and hygiene monitoring
Oxoid Ltd), see also fluorogenic and chromogenic media . These methods require little capital investment and expenditure on consumables is not excessive. However they are labour intensive with regard to media preparation and recording the results. Additionally as a period of incubation is required the procedure requires a time period measured in days. Since the target organism may be in the minority of the total microflora, or sublethally injured, recovery stages may be incorporated prior to selective procedures. The limits of detection are determined by the procedure and appropriate legislation. Hence an approved protocol for Salmonella detection in processed foods must be able to detect one Salmonella cell in 25 grams of material. Limitations of traditional detection techniques have hampered epidemiological studies of food poisoning outbreaks, for example until relatively recently Norovirus (small round structured viruses or SRSV) could only be detected by electron microscopy within 48 hours of illness. Since these conventional procedures are relatively laborious and time-consuming various rapid methods have been developed. Principal rapid methods include immunoassays (ELISA), latex agglutination, impedance microbiology, immunomagnetic separation, luminescence and gene probes linked to the polymerase chain reaction. These procedures either separate and concentrate the target cell or cell fragment to a detectable level by a non-growth step (i.e. immunomagnetic separation). Alternatively the end-detection method does not rely upon an incubation period for colony formation (i.e. luminescence). These rapid methods will be considered later. For detailed conventional protocols there are many standard reference sources;
- Bacteriological Analytical Manual ,
- Microbiological Laboratory Guidebook.
- Compendium of methods for the microbiological examination of foods
- Practical Food Microbiology (HPA - formerly PHLS, UK).
The ISO 6579:2002 standard for the detection of Salmonella in food was published in October (2002) and has three changes compared with the 1993 version
- Selenite cystine (SC) broth is replaced by Muller Kauffmann tetrathionate novobiocin broth (MKTTn) which shows superior selection to SC for Salmonella Typhi and Paratyphi (in addition selenite is hazardous to human health).
- Rappaport Vassiliadis (RV) broth has been replaced by Rappaport Vassiliadis Soya (RVS) broth.
- XLD is the first isolation medium rather than BGA. Nevertheless BGA is a standard alternative medium if its use is appropriate for the type of sample being tested.
Due to the prolonged and laborious nature of traditional methods, numerous rapid and automated methods have been developed and marketed. The definition of `rapid' has not been formally defined, but it generally means any method which yields results quicker than the standard method. AOAC validated methods can be accessed here.
The most frequently used rapid and automated methods in industry are Enzyme Linked Immunosorbent Assays (ELISA), impedance (or conductance), immunomagnetic separation (IMS) and bioluminescence. These techniques may be used on there own or in combination. Therefore a rapid separation method such as immunomagnetic separation can be used in conjunction with a rapid end detection method such as ELISA. These methods have been developed either to (a) replace the enrichment step (which requires a prolonged growth period) with a concentration step, i.e. immunomagnetic separation or (b) to replace the end-detection method, which is usually colony development and hence requires a prolonged incubation period, i.e. impedance microbiology and bioluminescence.
Current methods require approximately 105 organisms/ml for reliable detection. Since the regulatory requirement is the ability to detect 1 cell in 25 gram of food a concentration factor of 107 is necessary. This is equivalent to 2 hours of polymerase chain reaction (PCR) amplification (5 minute cycle) and 2 hours of infection period for Salmonella phages carrying the lux gene. Hence current rapid methods have a minimum period measured in hours. The ideal is an instantaneous or `real time' in vitro method. Potentially the bioluminescence technique coupled with ultra sensitive luminometers offers such a truly rapid technique.
In order to discriminate the target organism from other cells (procaryotic and eucaryotic) a separation step is normally required. This subsequently generates a large quantity of material of which only a portion is used for further analysis unless a concentration step is also used. For example homogenising a food sample in a blender (Stomacher, etc) dilutes the material ten-fold, generating large volume of material (250 ml), yet the detection procedure may only require a few millilitres. By concentrating the target organism the detection period should be shortened and more efficient.
Membrane filtration - Direct Epifluorescent Technique (DEFT) and Hydrophobic Grid MembraneMembranes can be made from nitrocellulose, cellulose acetate esters, nylon, polyvinyl chloride and polyester (Sharpe, 1994). They are very thin and hence can be directly mounted on a microscope. Membrane filters are used in modified conventional techniques for a variety of purposes:
- Concentration of target organism from a large volume to improve detection limits.
- Remove growth inhibitors
- Transfer organism between growth media without physical injury through resuspension
. The sensitivity of the DEFT results from the concentration of cells by membrane filtration before staining. Its ability to distinguish live and dead bacteria comes from the use of the nucleophilic fluorochrome acridine orange, which fluoresces at different colours in cells during different phases of growth. The dye fluoresces red with RNA and green with DNA. Generally, viable cells fluoresce orange-red while dead cells fluoresce green. In 1991 the ISO-GRID