PL100028B1 - PREPARATION FOR THE DETERMINATION OF ANTIBIOTICS, ESPECIALLY IN FOODSTUFFS - Google Patents

PREPARATION FOR THE DETERMINATION OF ANTIBIOTICS, ESPECIALLY IN FOODSTUFFS Download PDF

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Publication number
PL100028B1
PL100028B1 PL19261076A PL19261076A PL100028B1 PL 100028 B1 PL100028 B1 PL 100028B1 PL 19261076 A PL19261076 A PL 19261076A PL 19261076 A PL19261076 A PL 19261076A PL 100028 B1 PL100028 B1 PL 100028B1
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preparation
antibiotics
determination
test
amount
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PL19261076A
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Polish (pl)
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Przedmiotem wynalazku jest preparat sluzacy do wykrywania i oznaczania antybiotyków, który moze byc stosowany w przemysle spozywczym, medycynie, lecznictwie weterynaryjnym itp. Wprowadzenie antybiotyków do leczenia chorób zakaznych ludzi i zwierzat wplynelo zasadniczo na poprawe zdrowotnosci czlowieka i zwierzat hodowlanych. Jednoczesnie powszechne naduzywanie antybiotyków sprawilo, ze w coraz wiekszym stopniu przedostaja sie one do surowców rolnych szczególnie do miesa i mleka, powodujac szkody w produkcji, szczególnie przy wytwarzaniu fermentowanych artykulów spozywczych, jak: sery, maslo, twarogi, niektóre wedliny stwarzajac niebezpieczenstwo dla konsumentów. Stad tez wynika potrzeba badania obecnosci antybioty¬ ków w medycynie weterynaryjnej i przemysle spozywczym.The subject of the invention is a preparation for the detection and determination of antibiotics, which may be used in the food industry, medicine, veterinary medicine, etc. The introduction of antibiotics for the treatment of infectious diseases in humans and animals, it significantly improved human health and farm animals. At the same time, the widespread abuse of antibiotics has made it increasingly more the extent to which they get into agricultural raw materials, especially into meat and milk, causing damage to production, especially in the production of fermented food products, such as: cheese, butter, cottage cheese, some meats posing a danger to consumers. Therefore, there is a need to test the presence of antibiotics in the veterinary medicine and food industry.

Biologiczne metody oznaczania antybiotyków, polegaja na okreslaniu wzrostu mikroorganizmu wrazliwego na antybiotyki w obecnosci badanego materialu. Najczesciej stosowana metoda mikrobiologiczna jest metoda dyfuzji na stalym podlozu, w której jako podstawe ukladu testowego stosuje sie podloze odzywcze zawierajace odpowiednie drobnoustroje testowe. Znane i stosowane jest tylko podloze odzywcze i kazdorazowo osobno przygotowuje sie kulture bakterii testowej i dodaje do uprzednio przygotowanego podloza odzywczego w temperaturze nieszkodliwej dla drobnoustrojów testowych.The biological methods of antibiotic determination are based on determining the growth of a sensitive microorganism to antibiotics in the presence of the test material. The most commonly used microbiological method is the method diffusion onto a solid support where a nutrient medium containing suitable test organisms. Only the nutrient medium is known and used, and each time separately a test bacterium culture is prepared and added to a previously prepared nutrient medium at a temperature harmless to test microorganisms.

Natomiast nie znany jest preparat w formie suszonej sluzacy do bezposredniego oznaczania antybiotyków, który zawiera w swoim skladzie równoczesnie podloze odzywcze i utrwalona kulture testowa. Przy stosowaniu Bacillus stearothermophilusjako bakterii testowej stosowane jest podloze odzywcze o skladzie: ekstrakt miesny 0,15% ekstrakt drozdzowy 0,3% pepton z kazeiny 0,6% pepton z miesa 0,1% glukoza 0,1% pH = 7,02 100028 Niedogodnoscia znanych i stosowanych metod dyfuzyjnych oznaczania antybiotyków jest to, ze zakladaja one prowadzenie w kazdym laboratorium hodowli mikroorganizmu testowego: przygotowania jego zawiesiny, jej standaryzacja, odrebne przygotowanie podloza testowego i dodatek do niego mikroorganizmu. W ten sposób przygotowana kulture testowa dodaje sie do uprzednio uplynnionego i sterylizowanego podloza przed zastygnie¬ ciem agaru, co wplywa na wydluzenie czasu analizy i zwiekszenie jej pracochlonnosci wynikajacej z koniecznos¬ ci osobnego kazdorazowego przygotowania kultur testowych.However, there is no known dried preparation for the direct determination of antibiotics, which contains both a nutrient substrate and a well-established test culture. When applying Bacillus stearothermophilus as a test bacterium is a nutrient medium composed of: meat extract 0.15% yeast extract 0.3% casein peptone 0.6% meat peptone 0.1% glucose 0.1% pH = 7.02 100028 The disadvantage of the known and used diffusion methods for the determination of antibiotics is that they assume they carry out in each laboratory the cultivation of the test microorganism: preparation of its suspension, her standardization, separate preparation of the test medium and addition of the microorganism to it. In this way the prepared test culture is added to a previously liquefied and sterilized medium before it solidifies agar, which extends the time of analysis and increases its laboriousness resulting from the necessity to separate preparation of test cultures each time.

Celem wynalazku jest uzyskanie trwalego standaryzowanego preparatu w stanie suchym o odpowiednich wlasciwosciach dyfuzyjnych, ze wskaznikiem ulatwiajacym, obiektywizujacym i przyspieszajacym odczyt, który eliminuje czynnosci wstepne wystepujace przy wykrywaniu antybiotyków metoda dyfuzyjna, zwiazanym z przygotowaniem podloza, roztworów wskaznikowych, otrzymania zawiesiny bakterii testowych itp.The aim of the invention is to obtain a stable, standardized formulation in a dry state with suitable diffusion properties, with an indicator that facilitates, objectifies and accelerates the reading, which eliminates the preliminary steps involved in the detection of antibiotics by the diffusion method, related to with the preparation of the substrate, indicator solutions, obtaining a suspension of test bacteria, etc.

Wedlug wynalazku preparat do oznaczania antybiotyków zawiera w swoim skladzie szereg substancji wzrostowych i odzywczych, które po rozpuszczeniu w wodzie wystepuja w nastepujacych stezeniach: hydrolizat bialek 0,5-5% autolizat drozdzowy 0,1—1% glukoza 0,1-1% skrobia rozpuszczalna 0,4% zelatyna 0,4% agar-agar 1% Liofilizat przetrwalników Bacillus stearothermophilus w takiej ilosci, aby 1 mililitr uplynnionego preparatu zawieral nie mniej niz 1X104 sporów, wskaznik zmiany pH w granicach 5—8.According to the invention, the preparation for the determination of antibiotics contains a number of substances for growth and nutrients which, when dissolved in water, are present in the following concentrations: protein hydrolyzate 0.5-5% yeast autolysate 0.1-1% glucose 0.1-1% soluble starch 0.4% gelatin 0.4% agar-agar 1% Bacillus stearothermophilus spores lyophilisate in such an amount that 1 milliliter of liquid preparation it contained not less than 1X104 spores, the index of pH change in the range of 5-8.

Preparat wedlug wynalazku, który zawiera w swoim skladzie lacznie: podloze dyfuzyjne, bakterie testowe i wskaznik pH umozliwia latwe i szybkie w czasie do 1,5 godzin wykonanie analizy wykrywania antybiotyków metoda dyfuzji na stalym podlozu w laboratoriach kontrolnych. Zwalnia przez to uzytkownika od klopotliwej i pracochlonnej hodowli szczepów testowych, zmniejsza ilosc uzywanego szkla laboratoryjnego, obniza praco¬ chlonnosc analizy, skraca jej czas zachowujac wysoka czulosc, a nawet przewyzszajac w tym wzgledzie znane metody biologiczne. Preparat posiadajacy w swoim skladzie liofilizowany koncentrat sporów Bacillus stearother¬ mophilus umozliwia równoczesne przygotowanie, uplynnianie i jalowienie podloza. Bacillus stearothermophilus w temperaturze 55—60°C rosnie z szybkoscia wystarczajaca do wystapienia widocznych stref zahamowania wzrostu w czasie do 2 godzin.The preparation according to the invention, which includes in its composition: diffusion medium, test bacteria and the pH indicator enables an easy and quick analysis of antibiotics detection up to 1.5 hours solid-support diffusion method in control laboratories. Thus, the user is relieved of the troublesome situation and labor-intensive cultivation of test strains, reduces the amount of laboratory glass used, reduces labor the absorption capacity of the analysis, shortens its time while maintaining high sensitivity, and even exceeding the known biological methods. Preparation containing in its composition the lyophilized Bacillus stearother spore concentrate mophilus enables simultaneous preparation, fluidization and sterilization of the substrate. Bacillus stearothermophilus at a temperature of 55-60 ° C it grows at a sufficient rate for the appearance of visible zones of inhibition growth time up to 2 hours.

Przyklad preparatu wedlug wynalazku. Jeden litr podloza testowego zawiera: bulionu miesnego autolizatu drozdzowego glukozy skrobi rozp. zelatyny agar-agar purpury bromokrezolowej g 3g Ig 4g 4g g 0,016 g zmielony liofilizat przetrwalników Bacillus stearothermophilus varietes calidolactis, w takiej ilosci, by 1 ml gotowego podlpza zawieral nie mniej niz 105 przetrwalników.An example of a formulation according to the invention. One liter of test medium contains: meat broth yeast autolysate glucose starch dissolution gelatine agar-agar bromocresol purple g 3g Ig 4g 4g g 0.016 g ground lyophilisate of Bacillus stearothermophilus varietes calidolactis spores, in such an amount that 1 ml of the finished substrate contained no less than 105 spores.

Sposób stosowania preparatu wedlug wynalazku do szybkiego oznaczania antybiotyków jest nastepujacy.The method of using the preparation according to the invention for the rapid determination of antibiotics is as follows.

Porcje suszonego podloza rozpuszcza sie w odpowiedniej ilosci wody i wstawia do wrzacej lazni wodnej (nie autoklawuje) na 15 minut, czesto mieszajac. Po rozpuszczeniu sprawdza sie pH i w razie koniecznosci ustala na 7,0—7,2. Nastepnie rozlewa na sterylne plytki Petriego i wstawia je do termostatu o temperaturze 62—65°C na 3,5 godzin. Tak przygotowane plytki gotowe do uzycia mozna przechowywac w temperaturze 4—6°C przez 10 dni. Analize obecnosci substancji hamujacych mozna wykonywac metoda: a) studzienkowa b) krazkowa Plytki z naniesionym materialem badanym inkubuje sie w temperaturze 62-65°C w czasie 1,5 godzin. Nie zachodzi koniecznosc pasteryzacji badanego materialu, nalezy jednak ustawic pH na 7,0 w przypadku badania artykulów kwasnych. Obecnosc substancji hamujacych uwidacznia sie strefa zahamowania wzrostu bakterii i równoczesnie strefa zahamowania ma kolor sinoniebieski, a reszta podloza kolor zólty. Substrat badany winien byc w postaci plynnej. W przypadku badania substratu o konsystencji stalej nalezy przeprowadzic jego rozdrobnienie i homogenizacje w srodowisku jalowej wody, w znanym stosunku, homogenizat przefiltrowac i badac filtrat. Czulosc preparatu jest wysoka wykrywalne sa ilosci 0,003 j.m. penicyliny.Dissolve portions of the dried substrate in an appropriate amount of water and put it in a boiling water bath (not autoclaved) for 15 minutes, stirring frequently. After dissolution, the pH is checked and, if necessary, adjusted to 7.0-7.2. Then he pours them on sterile petri dishes and puts them in a thermostat at a temperature of 62-65 ° C for 3.5 hours. Ready-to-use plates in this way can be stored at 4-6 ° C for 10 days. The analysis for the presence of inhibitory substances can be performed by: a) well b) disc The plates with the test material applied are incubated at 62-65 ° C for 1.5 hours. No it is necessary to pasteurize the test material, however, the pH should be adjusted to 7.0 in the case of testing acidic articles. The presence of inhibitory substances shows the zone of inhibition of bacterial growth and at the same time the inhibition zone is blue-blue and the rest of the base is yellow. The test substrate is guilty be in liquid form. In the case of testing a substrate with a solid consistency, it should be carried out grinding and homogenization in a sterile water environment, in a known ratio, filter the homogenate and examine the filtrate. The preparation sensitivity is high, the detectable amounts are 0.003 IU. penicillins.

Antybiogram dla Bacillus stearothermophilus varietes calidolactis okreslony przy zastosowaniu preparatu.Antibiogram for Bacillus stearothermophilus varietes calidolactis determined using the preparation.

Stezenie antybiotyków podano w u/ml.100 028 3 Neomycyna Tetracyklina Chlorotetracyklina Chloramfenikol Streptomycyna Óxyvet Syfttarpen Oxyterracyna Erytromycyna Penicylina 0,2 0,6 0,7 1,0 1,0 0,1 0,1 0,1 0,3 0,003 j.m.Antibiotic concentrations are given in u / ml. 100 028 3 Neomycin Tetracycline Chlorotetracycline Chloramphenicol Streptomycin Óxyvet Syfttarpen Oxyterracin Erythromycin Penicillin 0.2 0.6 0.7 1.0 1.0 0.1 0.1 0.1 0.3 0.003 IU

Claims (1)

1. Zastrzezenie patentowe Preparat do oznaczania antybiotyków zwlaszcza w artykulach spozywczych, znamienny tym, ze w sklad uplynnionego w wodzie podloza wchodzi: hydrolizat bialek w ilosci 1,5%, autolizat drozdzowy w ilosci 0,3%, glukoza w ilosci 0,1%, skrobia rozpuszczalna w ilosci 0,4%, zelatyna w ilosci 0,4%, agar-agar w ilosci 1%, purpura bromokrezolowa 0,0016%, jako wskaznik pH oraz liofilizat przetrwalników Bacillus stearothermophilus w takiej ilosci, aby 1 mililitr uplynnionego podloza zawieral nie mniej niz IX104 sporów.1. Patent claim Preparation for the determination of antibiotics, especially in foodstuffs, characterized by the fact that the base liquefied in water includes: protein hydrolyzate at 1.5%, yeast autolysate at 0.3%, glucose at 0.1% , starch soluble in the amount of 0.4%, gelatin in the amount of 0.4%, agar-agar in the amount of 1%, bromocresol purple 0.0016% as a pH indicator and a lyophilisate of Bacillus stearothermophilus spores in an amount to make 1 ml of liquid substrate contained no less than IX104 disputes.
PL19261076A 1976-09-22 1976-09-22 PREPARATION FOR THE DETERMINATION OF ANTIBIOTICS, ESPECIALLY IN FOODSTUFFS PL100028B1 (en)

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PL19261076A PL100028B1 (en) 1976-09-22 1976-09-22 PREPARATION FOR THE DETERMINATION OF ANTIBIOTICS, ESPECIALLY IN FOODSTUFFS

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PL19261076A PL100028B1 (en) 1976-09-22 1976-09-22 PREPARATION FOR THE DETERMINATION OF ANTIBIOTICS, ESPECIALLY IN FOODSTUFFS

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PL100028B1 true PL100028B1 (en) 1978-08-31

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