UA151172U - Method for the determination of minimum inhibitory concentration of iodoform against microorganisms - Google Patents

Abstract

A method for the determination of minimum inhibitory concentration of iodoform against microorganisms involves use of the method of serial dilutions. Cultures of museum strains of microorganisms Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990, Candida albicans ATCC 10231 are exposed to a 5% iodoform bandage, for the production of which 96% ethyl alcohol with a volume of 20 ml is used. 500 mg of fine-crystalline iodoform powder is added, incubation is carried out for 24 hours at a temperature of 35-36°C till complete dissolution of iodoform powder. Sterile bandage segments weighing 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 mg are impregnated with 5% iodoform-alcohol solution, those have the concentration of iodoform 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 mg/ml, respectively, in equivalent of iodoform dry matter content.

Description

A useful model belongs to the field of medicine, in particular microbiology, and can be used to study the minimum inhibitory concentration of Iodoform on strains of microorganisms.

Among those that are known, there are the following methods of testing microorganisms for sensitivity: 1. A method for determining the minimum inhibitory concentration of an antibiotic for the treatment of staphylococcal infection, which includes the assessment of the antimicrobial activity of the antibiotic after adding it to the culture of the infectious agent according to the standard scheme, which differs in that the obtained after adding the antibiotic, the material is examined by polymerase chain reaction (PCR) using primers 5 TA АСА SAT SAT tat TaА СО-3/5

AdSsaTa Adi TOasii TAA TAS ba, the size of the PCR product - 550 pairs of nucleotides; a positive result of the study of the tested sample of material is ascertained if there is a band of the formed amplicon on the track, the size of which is identical to the corresponding size of the control amplicons; a negative result of the tested sample is determined when there is no amplicon of any size on the track or when an amplicon (or several amplicons) is present, the size of which differs from the size of the corresponding control amplicons. (Invention patent 112557,

Ukraine, IPC C20O1M 33/00. The method of determining the minimum inhibitory concentration of an antibiotic for the treatment of staphylococcal infection / Authors: Ivanova Nina Mykolaivna (SHA); Remiz Oleksandr

Mykolayovych (USA); applicant and patent owner: State institution "Institute of Dermatology and Venereology of NAMNU", str. Chernyshevsky, 7/9, Kharkiv, 61057 (OA). - Mo agO1404569; Application 28.04.2014; Publ. 26.09.2016, Bull. May 18/2016. 2. The method of determining the sensitivity of microorganisms of biofilms to disinfectants, which includes the preparation of a solution of a disinfectant, a bacterial culture and a nutrient medium, exposure of a mixture of a suspension of a microorganism culture with a disinfectant in the wells of immunological tablets, adding a neutralizer of a disinfectant, sowing the suspension on a nutrient medium with further evaluation the growth of microorganisms on the nutrient medium by recording the growth in the cultures of control and experimental samples, comparing and determining the lowest bactericidal concentration at which the bactericidal properties of the disinfectant were manifested, which is characterized by the fact that they first form a biofilm on a liquid nutrient medium in the wells of immunological tablets, with

The liquid nutrient medium is removed from the wells of the tablet with the formed biofilm, then the biofilm is treated with a disinfectant after exposure, a part of their contents is taken from the experimental wells and transferred to another tablet to the neutralizer of the disinfectant, then the contents are sown on a solid differential medium; perform a control study of a suspension of these bacteria with the same sequence of actions, the crops are incubated at 37 "C for 24-48 hours, after which the growth of microorganisms is evaluated. (Pat.: on the useful model 89438,

Ukraine, IPC C120 04/31. The method of determining the sensitivity of microorganisms of biofilms to disinfectants of different groups of chemical compounds/ Authors: Marievsky Viktor Fedorovych (OA);

Krolevetska Nadiya Mykhailivna (SHA); Ruban Nadiya Mykhailivna (USA); Galina Viktorivna Matoshko (USA); Bubalo Volodymyr Oleksandrovych (USA); Omelchenko Volodymyr Viktorovych (SHA); applicant and patent owner: State institution Institute of Epidemiology and Infectious Diseases named after

L.V. Gromashevsky National Academy of Medical Sciences of Ukraine, str. M. Amosova, 5, Kyiv, 03680 (OA). - MO and 201311162; Application 09/19/2013; Publ. 25.04.2014, Bull. May 8/2014.

The closest analogue to the stated method is the method of determining the sensitivity of microorganisms to antibacterial drugs (ABP) by the macro method of serial dilutions in nutrient broth, which consists in the fact that the working solution of ABP is prepared from the basic solution using a liquid nutrient medium. The working solution in the amount of 0.5 cubic cm using a micropipette with a sterile tip is introduced into the first test tube containing 0.5 cubic cm of broth. Mix thoroughly and use a new sterile tip to transfer 0.5 cubic cm of the solution of the antibacterial drug in the broth to the next test tube with 0.5 cubic cm of the broth. This procedure is repeated until the entire necessary series of dilutions is prepared. 0.5 cubic cm of broth is removed from the last test tube. Thus, a number of test tubes with a solution are obtained

APB, the concentrations of which differ in neighboring test tubes by a factor of 2. For inoculation, a microbial suspension equivalent to 0.5 McFarland standard is used. 0.5 cubic cm of inoculum is introduced into each test tube containing 0.5 cubic cm of the appropriate dilutions of APB and into one tube with 0.5 cubic cm of nutrient broth without APB as a "negative control". The test tubes are incubated at a temperature of 35 "С for 20-24 hours. To determine the presence of growth of the microorganism, the test tubes with cultures are viewed in transmitted light.

On the approval of methodological instructions "Determining the sensitivity of microorganisms to antibacterial drugs": the order of the Ministry of Health of Ukraine dated April 5, 2007 Mo 167. - access mode:

porz//2аКоп.гада.дом.са/гада/5пом/м0167282-07ЖТехи The disadvantages of this method in the case of using it to determine the sensitivity of microorganisms to Iodoform are that 1) Iodoform is almost not soluble in water and therefore it is impossible to prepare it for conducting research its aqueous solution; 2u Modoform is soluble in ethyl alcohol and it is possible to prepare its basic alcohol solution. However, when preparing a working solution by diluting with a nutrient medium,

Iodoform precipitates.

These shortcomings make it impossible to use the antiseptic Iodoform in the form of a powder for the preparation of its main and working solutions. | therefore, the research becomes impossible.

The basis of the useful model is the task of studying the antimicrobial effect of the antiseptic drug Iodoform on various strains of microorganisms.

The task is solved by the fact that the method of determining the minimum inhibitory concentration of Iodoform for microorganisms, which includes the use of the method of serial dilutions, according to a useful model, of cultures of museum strains of microorganisms earpuiosossiv asheyvz ATS 25923, Etarpuiosossiv ATS erideptidie ATOS 14990, Sapaida aibisape

ATOS 10231 are exposed to the action of 5 96 iodoform bandage, for which 96 95 ethyl alcohol in a volume of 20 ml is used, 500 mg of fine crystal powder is added to it

Iodoform, incubated for 24 hours. at a temperature of 35-36 C, achieving complete dissolution of the Iodoform powder, this 595 iodoform-alcohol solution soaks segments of a sterile bandage weighing 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 mg, which in recalculation of the dry matter content of Iodoform in it, contains concentrations of Iodoform, respectively 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 mg/ml.

The method is carried out as follows.

To obtain 5 95 iodoform bandage, 20 ml of 96 95 ethyl alcohol was used and 500 mg of fine crystalline powder of Iodoform was added to it, this solution was incubated for 24 hours. at a temperature of 35-36 "C. After incubation, complete dissolution of the Iodoform powder was achieved and a working 5 95 iodoform-alcohol solution was obtained. To achieve two-fold successive dilutions of the drug, this solution soaked sterile bandage segments of different weights: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 mg, which, based on the content of dry matter of Iodoform in it, contained the following concentrations of Iodoform: 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2, 5 mg/ml respectively.

To determine the sensitivity of microorganisms to Iodoform, 12 test tubes containing nutrient broth, 595 Iodoform bandage of a certain weight and microbial inoculum were used according to the standard method, taking into account controls with "positive" and "negative" results. 5 95 iodoform bandage was subjected to titration. Thus, a number of test tubes were obtained in which the concentration of Iodoform differed by a factor of 2 between adjacent test tubes. For preparation of standard microbial inoculum equivalent to 0.5 turbidity standard

McFarland, used daily cultures of museum strains of earpuiosossi5 ashteibz ATO 25923, Eiarpuiosossiv eridentidie ATOS 14990, Sapaida airisap5

ATOSS 1023. According to the method, a microbial suspension was used, which was a diluted inoculum and contained 1.5x106 CFU / cm3. Ready microbial suspension no later than 15 min. from the moment of its preparation, it was added to each test tube, which contained the appropriate amount of nutrient broth and the studied antiseptic in the form of a soaked bandage. The test tubes were incubated in a normal atmosphere at a temperature of 35-36 "C for 24-48 hours. After incubation, the results were evaluated and the minimum inhibitory concentration (MIC) of Iodoform was determined.

The delay in the growth of museum cultures under the influence of Iodoform was determined by viewing the contents of the test tubes in transmitted light, comparing them with the "negative" control (table).

The lowest concentration of Iodoform, which caused a delay in the growth of the strain biarpuiosossiv erideptidie ATSS 14990, was 40 mg/ml. In culture tubes, where the concentration

Iodoform was equal to 20-2.5 mg/ml, culture growth was observed. MIC of Iodoform in relation

Ziarpuyusossiv asheiv ATSS 25923 was 160 mg/ml. The least sensitive to Iodoform was the culture Sapaida airisape ATOS 10231. Growth inhibition of Sapaida airisape ATOS 10231 was observed at a concentration of Iodoform of 1280 mg/ml, a further decrease in the concentration of the studied ABP did not delay the growth of the culture compared to the "negative" control.

Table

Determination of the inhibitory effect of Iodoform on museum strains of microorganisms (mg/ml)

Culture | 1280 | 640 | from the 2nd | 160 80 | 40 | tho | then | 5 |(25|kK| ky zZ.eridegtiaiv y y y y

ATSS 14990 at 8.ashev | - | - | - | - 5 | 5 | 5 | i drive 5 | -

Sabrisapya | - | ya | 5 | 5 | 5 | 5 | ya | 5 trip 1 5

Note: growth of microorganisms, - absence of growth of microorganisms, K/K - culture control,

K/I - Iodoform control.

The data of the above-claimed method can be used in the further study of the antimicrobial effect of the antiseptic preparation Iodoform on clinical strains of microorganisms.

Claims (1)

USEFUL MODEL FORMULA The method of determining the minimum inhibitory concentration of iodoform for microorganisms, which includes the use of the method of serial dilutions, which differs in that the cultures of museum strains of microorganisms Earpuiosossiv ashteiz ATS 25923, Arnuiosossiv erideptiaiv ATSOS 14990, Sapaida airbisap5 ATS 10231 are exposed to the action of 595 iodoform bandage, to obtain which use 96 95 ethyl alcohol in a volume of 20 ml, add 500 mg of fine crystalline iodoform powder to it, incubate for 24 hours. at a temperature of 35-36 "С, achieving complete dissolution of the iodoform powder, this 5 96 iodoform-alcohol solution is impregnated with segments of a sterile bandage weighing 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 mg , which, based on the content of dry matter of iodoform, contains iodoform concentrations of 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 mg/ml, respectively.