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APIACTA 38 (2003) 246-248 MODIFICATION OF MICROBIOLOGICAL DETECTION METHOD OF
TETRACYCLINE IN HONEY.
Rail Khismatoullin1, Rafael Kuzyaev2, Yaroslav Lyapunov2, Elena Elovikova2 1 Tentorium Ltd, 39 Energetikov St, 614065 Perm, Russia ; Tel: +7 3422 260262, Fax: +7 3422 264702 E- mail: [email protected] ; 2 Laboratory of Ecological Monitoring of Bees "Federal", 38 Energetikov, 614065 Perm Russia, Tel: +7 3422 260262 Fax: +7 3422 264702 E-mail: [email protected] Tetracycline derivatives are the main chemotherapeutical means against American foul brood of bee brood. In most developed countries the presence of these antibiotics in food products is not possible according to sanitary code. Among other methods of tetracycline detection in food products the most simple is microbiological detection in agar during antibiotic diffusion from lunes with sample solution. According to the information taken from different sources, when using bacteria Bacillus cereus species as a test specimen the concentration of the antibiotic in honey detected by this method is not less than 40 – 90 ppb (Gordon L., 1989) Sensitivity of the diffusion method is limited by the following factors - value of minimum inhibitory concentration of tested antibiotic for the used - speed balance of germ culture growth and advancement of minimum inhibitory concentration front along the radius from the lune with sample; - volume of sample in the lune. To increase susceptibility in the lune it is necessary to put maximum amount of sample
and the balance moves to the diffusion speed part by means of growth inhibition with
ageing agar with a sample in a frig. Nevertheless the method is absolutely unsuitable for
determination of antibiotics concentration that is less than minimum inhibitory typical for
the used test organism.
For microbiological detection of tetracycline residues we tried a number of standard
method modifications where we made an attempt to take into account physical-chemical
factors that influence its sensitivity.

Materials and methods.
As a test specimen Bacillus subtilis АТСС 6633 culture was used. A standard method of
tetracycline detection consisted of the following stages:
- cultivation of the test specimen in Petri dishes with nutrient agar (0.1% glucose) during 7 days at 350C. - receiving of spore soliquid by heating dredge of the test specimen at 750C during - preparation of Petri dishes and lunes (8 millimeter diameter) for test solutions of antibiotic and tested samples in agar medium sowed with spores of test specimen. - building of calibration curve – the graph of relation between inhibition area diameter and antibiotic concentration in the sample. Citrate buffer рН=5,0-5,2 was used for preparation of tetracycline test solutions and samples growing. Honey with different self antibacterial activity was used in model experiments. APIACTA 38 (2003) 246-248
Results and argument.
Method changing turned out to be the most productive when inhibitory front concentration
is spread from lune much faster against a background of subliminal antibiotic
concentration in agar. For the used test specimen – Bacillus subtilis ATCC 6633 when
carrying out a standard analysis, minimum determined concentration of tetracycline in
honey was 300ppb (diameter of suppression growth zone 13mm). There was no zone at
150ppb antibiotic content. The amount of liminal background concentration of tetracycline,
when only trace growth in agar thickness was observed for the used test organism turned
to be equal to 40ppb. Against a background of tetracycline content in agar 30ppb the
diameter of deficiency growth zones was 15mm at the equal concentration of antibiotic in
the lune.
The usual method of the test specimen inhibition growth is holding of dishes with samples
at 40C during 1-2 hours. We used the method of predrying in Petri dishes at 500C covering
them with heavyweight paper. At this temperature the growth of the test specimen is
inhibited and the speed of diffusion process increased not less than 16 times in
accordance with the general regularity of physical-chemical process double acceleration
when increasing temperature per 100C. During an hour the sample is noticeably absorbed
into agar thickness and it make additional concentration of antibiotic.
For advancing of the default growth zone, the suspension density of the test specimen
matters much when antibiotic concentration is close to minimum inhibitory. Independently
of reference quantity it is increased fast from the moment of growth onset. In every circular
part there is opposition process of antibiotic inflow from the lune and its fixation with target
molecule of the microorganism. In case of cells excess a steadier part of the population
keeps on growing and the zone moves to antibiotic increasing direction up to equilibrium.
And vice versa, in case of light cells density the unbounded with the target molecule
antibiotic is spread from the center enlarging the zone of inhibitory concentration. When
carry out the experiments the diameter of the inhibition growth zone when testing
sensitivity of the test-specimen by a standard disks method fluctuated between 30 and 38
mm depending on bacterial suspension density.
The speed of antibiotic diffusion is determined both its concentration in studied sample
(factor 1) and quantity of the sample in the lune (factor 2). The first amount is sought and
in this case – minimum. The necessity of honey dissolving in buffer for possibility of
accurate volume dosage and lowering its antibacterial activity additionally decreases
sensitivity of the method in 2-4 times. The second amount is limited to the size of the lune
– its diameter. Moreover the small size of agar antibiotic source (in the limit - point source)
the speed of antibiotic diffusion is down because of concentration decrease when moving
away from the center is connected with its displacement volume distribution, which is
proportional to radius square (factor 3). Expansion of the lune diameter at light antibiotic
concentration makes the volume of the sample increase. At the same time if the size of
the lune is bigger (comparatively with the inhibitory zone breadth) then the front of
antibiotic spread can be considered as flat and the factor 3 does not reduce sensitivity of
the method.
When comparing the effectiveness of increasing lunes' volume the evaluation of inhibitory
zone diameter we changed into measuring breadth zone from the lune brim by means of
eyepiece ruler of a stereoscopic loupe. To smooth inequalities connected with precise
estimate, we measured breadth zones in three directions situated at angle of 1200 to each
other calculating average quantity.
APIACTA 38 (2003) 246-248 Expansion of the lune size in 2 times – from 8mm up to 16mm and correspondingly 400 microliters of the sample instead of 100 microliters increases sensitivity of the method in model experiment in buffered solution of tetracycline four times more. Table 1 Depending of the lune size on inhibitory zone breadth (mm) Tetracycline concentration Lune diameter 1,0 microliters/ milliliter 4,0 microliters/ milliliter Thus, modification of the standard microbiological detection method taking into account actual physicochemical regularities makes its sensitivity considerably increase.

Source: http://www.apimondia.com/apiacta/articles/2003/khismatoullin_1.pdf