Four-plate test

Although some European countries still accept the results of the four plate test as confirming the presence of antibiotic residues in samples ( ), other work indicates that FPT test is not necessarily reliable. The occurrence of natural microbial inhibitors in tissues has frequently been noted (4,9,49,82), It has also been frequently observed that the results obtained by microbial and physicochemical procedures sometimes differ considerably (9,10,45,82,86), Results obtained in our laboratory suggest that even inactivation by penicillinase may not be totally specific for B-lactam antibiotics (W), The specificity of immunoassay procedures depends on the specificity of the antibody used in the test (95), Specific antisera are not widely available at present. Physicochemical procedures are therefore essential for identification and confirmation of suspect residues detected by microbiological tests. 

In cases where quantitative analyses give concentration values much lower than the specified MRL for a certain analyte, positive quantitative errors do not play a role in the no answer. However, negative quantitative errors may be important if a screening test such as the four-plate test for antibiotics or an enzyme-immunosorbent assay for -agonists is used, some specific analytes for which the test has no or very low response may slip through the holes (43). 

In Belgium, 0.1% of the slaughtered cattle and swine are screened for antibiotic residues each year. In the analytical strategy applied, meat samples are screened with a modified four-plate test followed by screening with a group-specific ELISA for the identification of the antibiotics and confirmation by specific LC methods. 

The four-plate test was initially based on the German Hemmstoff-test with an additional plate of Sarcina lulea at pH 8.0, designed for the detection of lower levels of macrolides, and a fourth plate of Escherichia coli at pH 7.2 for the detection of sulfonamides (74,75). The modified version adopted by the European Community for screening carcasses is based on three plates with Bacillus subtilis BGA at pH values of 6.0, 8.0, and 7.2 with added trimethoprim, respectively, and a fourth plate with Micrococcus luteus NCTC 8340 at pH 8.0 (74). This test as described elsewhere (76) is intended to detect residues of -lactams, tetracyclines, aminoglycosides, sulfonamides, and macrolides in muscle tissue of slaughtered animals, without any prior extraction or cleanup. 

In the four-plate test, the sample is applied in the form of a sliced deep-frozen tissue disk directly on each of the four plates, and incubation is carried out at 30 C for 18-24 h. The test is sensitive and reasonably easy to standardize but it is time-consuming and thus rather expensive. Its performance has been found to be affected, at least in part, by the composition and the properties of the test medium, and the nature of antibiotics being tested (77-80). 

However, recent investigations on the effect of the tissue matrix on the detection limits attained by this test have indicated that ceftiofur, sulfonamides, streptomycin, and some macrolide antibiotics cannot be detected in intact meat with the plates and the bacterial strains prescribed in the European four-plate test (81, 82). Two plates of this system were not found suitable for screening sulfamethazine or streptomycin at levels far above the MRL the third plate detected tetracyclines and -lactams up to the MRL levels whereas the fourth was sensitive to -lactams and some but not all macrolides. Detection, on the other hand, of the fluoroquinolones enrofloxacin and ciprofloxacin could only be made possible by an additional Escherichia coli plate not included in the four-plate test. 

Antibacterial German three-plate test EEC four-plate test CHARM II test New Dutch kidney test STOP CAST FAST CHARM farm test ATP test... 

The NKDT test is sensitive to residues of -lactams, tetracyclines, and macrolides. It is more sensitive to sulfonamides than the German three-plate test or the EU four-plate test, but it is relatively insensitive to aminoglycosides. With respect to MRLs set for liver or kidney, the NKDT is too insensitive for aminoglycosides, sulfonamides, and macrolides. One analyst can complete 150-200 tests per day. Due to the high ratio of residue levels in preurine and muscle, a negative result of this test implies that residue levels in muscle are below the limit of detection. 

The Four-Plate Test and the New Dutch Kidney Test... 

Despite the limitations associated with plate-based tests, the majority of routine antimicrobial screening is carried out using in-house prepared plate-based assays. Commonly used examples of such plate-based tests are the four-plate test (FPT) and the modified four-plate test (mFPT) and are widely used as official screening tests within the EU. ... 

Chang CS, Tai TF, Li HP, Evaluating the apphcabhity of the modified four-plate test on the determination of antimicrobial agent residues in pork, J. Food Drug Anal. 2000 8 25-34. 

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