Method of determining maximum concentration of living bacteria

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of determining maximum concentration of living bacteria. A suspension of bacteria with turbidity standard of 5 units is brought to concentration of 500000 mc per 1 ml. Electrical resistance of the suspension is then determined in a direct current field with maximum voltage across open ends of electrodes of 2.8 V. Maximum resistance in the range of 900-1500 kOhm indicates maximum concentration of living bacteria in suspensions of different types of microorganisms.

EFFECT: method provides quality estimation of concentration of living bacteria, which is sufficient for further identification of said bacteria, and cuts analysis time.

1 tbl, 4 ex

 

The method relates to the field of medicine, namely Microbiology, and is designed to determine the concentration of bacteria in nutrient media.

Improvement of methods for determining the concentration of bacteria in various suspensions is up to date. In the process of cultivation of bacteria in nutrient media is a complex biological process associated with the nutrition of bacteria and accumulation of products of their metabolism. Bacteria fails the process of their dying [Growth and methods bacteria. Pp.62-66. In the book: Medical Microbiology, Virology and immunology / edited ABV. - M., 2004. - 691 S.]. The need to determine the concentration of microbial cells (MC) in suspensions occur when you obtain standard diagnostic, therapeutic and preventive drugs. The diagnosis of infectious disease is put on the basis of determining the genus and species of the pathogen. More effective identification of bacteria in terms of the maximum concentration of living microbial cells during bacterial growth on nutrient media.

There is a method of determining the concentration of microbial cells in the turbidity standard, recording the total number of live and dead microbial cells [Patent RU No. 2285257 C2. Bull. No. 28 dated 10.10.2006,]. This method does not allow to determine the concentration of W is o microbial cells and the timing of their maximum accumulation.

A known method of determining the amount of live microbial cells in culture results in a dense nutrient medium and counting the grown colonies of individual microbial cells [Definition total number of bacteria in the water. S-306. In the book: Microbiology techniques in microbiological research. M, Medicine, 1978, 394 S.], we have chosen as a prototype. However, this method has significant drawbacks: 1) it is impossible to determine the concentration of living microbial cells in the process of cultivation of bacteria in nutrient media; 2) the determination of the amount of live microbial cells in the studied samples can only be obtained after 24 hours of growth of isolated microbial cells on solid nutrient medium (in this period in the study population, usually, ends the process of the death of microbial cells); 3) for the implementation of the method requires the additional costs of the nutrient medium and the time spent specialists Microbiology laboratory (titration study population of bacteria, counting the number of grown colonies of individual microbial cells, mathematical calculation of the concentration microbial cells in the test sample).

The present invention is to develop a rapid method of determining the maximum concentration of living microbial cells in the process of their cultivation on pitt the selected environments. The maximum concentration of live bacteria in suspension refers to the concentration of bacteria, sufficient for the identification of selected bacteria from the patient. Based on the above it follows that the present invention is a qualitative rather than a quantitative estimate of the maximum concentration of live bacteria.

The technical result. The method is based on the electrokinetic properties of microbial cells in a liquid medium and their ability in the field of a permanent electric current having a certain electrical resistance.

Theoretical substantiation of the possibility of determining the electrical resistance of biological particles is reflected in theory after h.helmholtz - Jirina, according to which on the surface of biological particles in a liquid medium, there is the electric double layer condenser, one plate of which is charged surface of a biological particle, and the other ions present in the liquid and carrying the opposite charge. It is established that the bacteria on their surface are defined by the size and sign of the electrical charge. Electrokinetic properties of bacteria may change during the process of food microorganisms, which leads to improvement in electrical resistance of bacteria and demonstrates what isresponse. Based on the study of electrokinetic properties of bacteria Adetokunbo proposed device for the detection of bacteria [A.S. No. 288898, 1971. Bull. No. 7].

The technical result of the proposed method is achieved by the fact that the bacteria in liquid medium in the field of a permanent electric current have some electrical resistance. In this regard, a suspension of bacteria on the turbidity standard in 5 units adjusted to a concentration of 500,000 MK 1 ml and determine in the field of a permanent electric current at a voltage of 2.8 V at open ends of the electrodes, the electrical resistance of an index in the range from 900 to 1,500 ohms indicates a concentration in the suspension of live bacteria in sufficient quantities for identification of bacteria.

The method is as follows. The investigated suspension of bacteria in liquid medium was adjusted to the turbidity standard is 5 units to the concentration of 500000 MK 1 ml of suspension. Received a suspension of bacteria in a volume of 2.0 ml, is placed in the hole plexiglass plate, commonly used in laboratory practice for serological reactions. Using a digital multimeter (Mini digital multimeter) brand M in the limit of measurement 2000 ohms with a resolution of 1 K and an accuracy of 0.8% of units of account, at the maximum voltage on the electrodes of 2.8 spend In determining the electrical resistance of the bacterial suspension is. When the measure of electrical resistance in the range from 900 to 1,500 ohms registers the concentration of live microbial cells, sufficient for identification of bacteria.

Examples of usage of the proposed method

Example 1.

After 15 hours of growth sporadic culture of Staphylococcus aureus on blood mesopatamia agar (MPA) conducted flush grown colonies of bacteria physiological solution of sodium chloride and the concentration of bacteria increased to 500000 MK 1 ml of standard turbidity in 5 units. Using a digital multimeter (Mini digital multimeter) brand M in the limit of measurement 2000 ohms with a resolution of 1 K and an accuracy of 0.8% of units of account, when the voltage at the electrodes of 2.8 In the determination of the electrical resistance of the suspended bacteria. Changing the resistance reached 1260 ohms. The concentration of bacteria in the suspension of the results of counting of grown colonies of individual microbial cells (MC) on the solid nutrient medium was 2.41012MK 1 ml.

Example 2.

After 12 hours of growth sporadic culture of Proteus mirabilis on blood MPA held flush grown colonies physiological solution of sodium chloride and the concentration of bacteria increased to 500000 MK 1 ml of standard turbidity in 5 units. Using a digital multimeter (Mini digital multimeter) brand M in the limit of measurement 2000 ohms with a resolution of 1 K, and t is the durability of 0.8% of units of account, when the voltage at the electrodes of 2.8 In the determination of the electrical resistance of the suspended bacteria. Changing the resistance reached 1490 com. The concentration of microbial cells in the suspensions according to the results of calculation of grown colonies on solid culture medium of individual microbial cells was 2.61012MK 1 ml.

Example 3.

After 10 hours of growth of hospital strains of culture of Citrobacter freundii in blood MPA held flush grown bacteria physiological solution of sodium chloride and the concentration of bacteria increased to 500000 MK 1 ml of standard turbidity in 5 units. Using a digital multimeter (Mini digital multimeter) brand M in the limit of measurement 2000 ohms with a resolution of 1 K and an accuracy of 0.8% of units of account, when the voltage at the electrodes of 2.8 In the determination of the electrical resistance of the suspended bacteria. Changing the resistance reached 1115 com. The concentration of bacteria in the suspension of the results of counting of grown colonies of individual microbial cells on solid nutrient medium was 1.4109MK 1 ml.

Example 4.

After 13 hours of growth of hospital strains culture of Pseudomonas aeruginosa in blood MPA held flush grown bacteria physiological solution of sodium chloride and the concentration of bacteria increased to 500000 MK 1 ml of standard turbidity in 5 units. With SIP is using a digital multimeter (Mini digital multimeter) brand M in the limit of measurement 2000 ohms with a resolution of 1 K and an accuracy of 0.8% of units of account, when the voltage at the electrodes of 2.8 In the determination of the electrical resistance of the suspended bacteria. Changing the resistance reached 926 com. The concentration of bacteria in the suspension of the results of counting of grown colonies of individual microbial cells on solid nutrient medium was 6.01010MK 1 ml.

The method of determining the concentration of live bacteria in the process of their cultivation on nutrient media according to their electrical resistance is introduced on the basis of the division of General Microbiology FGUZ "Center of hygiene and epidemiology in the Tyumen region". The electrical resistance was determined from hospital and sporadic strains of the following bacteria: Pseudomonas aeruginosa, Proteus mirabilis, Citrobacter freundii, Hafnia alvei, Staphylococcus aureus, Enterobacter cloacae. Just spent 48 studies.

Assessment of the economic and positive (significant acceleration of the results of the analysis of the effect of the proposed method performed in comparison with the prototype and presented in the table.

Our proposed method allows the concentration of living microbial cells, necessary for the identification of bacteria isolated from patients, reduces the time of research, does not require large expenditures. The total economic effect on the conduct 100 tests was 343980 rubles.

Table
Characteristics of a positive effect determine the maximum concentration of live bacteria on the proposed method and prototype
SignThe placeholderThe proposed method
The duration of one analysis24 hours3-5 minutes
Consumption of nutrient media to conduct a single analysis (in rubles)1250 rublesThe nutrient medium is not required
Consumption of laboratory glassware for holding 1 analysis25 cups for culturing bacteria, 15 germ tubes and 15 pipettes in the amount of 661 RUB1 hole 72 plexiglass plate
Preparation of glassware for analysisWashing dishes, corking, sterilizationNot required
Time spent specialists Microbiology laboratory to conduct 1 analysis9 hours (1530 rubles) 3-5 minutes
The cost of the device for measuring the resistance of suspended bacteriaNot used120 rubles
Total expenses 1 analysis3441 rubles120 rubles
Expenses 100 research344100 rubles120 rubles
Note
Laboratory glassware and apparatus for measuring the resistance of suspended bacteria are used repeatedly.

The method of determining the maximum concentration of live bacteria, characterized in that a suspension of the bacteria on the turbidity standard in 5 units adjusted to a concentration of 500,000 microbial cells in 1 ml and determine in the field of a permanent electric current at the maximum voltage of 2.8 V at open ends of the electrodes, the electrical resistance, the maximum rate of which is in the range from 900 to 1,500 ohms indicates a maximum concentration of live bacteria in suspension.



 

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