Method of detecting and counting viable legionella pneumophila microorganisms

FIELD: chemistry.

SUBSTANCE: present invention relates to microbiology and a method of detecting and counting viable Legionella pneumophila microorganisms in a sample. The described method involves: (1) contacting said microorganisms of said sample with at least one reducing compound which contains glutamate and pyruvate, and a culture medium which is a buffered charcoal yeast (BCYE) or GVPC agar culture medium, (2) incubating the product of step (1), and (3) detecting and determining the number of viable microorganisms. The reducing compound used directly or indirectly affects metabolism, reducing oxidative stress of the microorganism by reducing formation and/or breaking down reactive forms of oxygen.

EFFECT: disclosed method enables to accurately count Legionella pneumophila microorganisms in a stressed condition.

7 cl, 5 dwg, 5 ex

 

The present invention relates to a method for detection and enumeration of viable microorganisms of the species Legionella pneumophila in the sample. The invention also includes a kit suitable for use in this method. This method and set allow us to more quickly determine the number of viable microorganisms

Legionella bacteria are ubiquitous in wet or damp environments, such as soil and nonmarine aquatic habitat. They can also be found in devices for warm and cold water risers cooling in air conditioning systems and humidifiers.

Legionella, particularly Legionella pneumophila, are pathogens that can cause acute bacterial pneumonia, usually known under the name of "Legionnaires ' disease", which often has a fatal outcome for infected individuals.

Traditionally, detection and enumeration of Legionella pneumophila is performed by culturing cells. This method can be performed by determining the number of cultured bacteria by culture on Petri dishes or count microcolonies using the method of membrane filtration. These methods are evaluated viable bacteria by their ability to form a colony or microcolony. Unfortunately, such methods usually require 3 to 10 days for it to form a Colo is AI or micro-colonies. Where water installation are located in the work, there is an unacceptable risk of human infection during this period of time.

Other ways of determining microorganism Legionella generally include PCR (polymerase chain reaction). In the method of PCR uses DNA polymerase to amplify regions of DNA by enzymatic replication in vitro. In the course of the method generated DNA used as a template for replication, which leads to a chain reaction in which the DNA matrix amplificates exponentially. PCR allows a single or few copies of DNA to increase up to a million or more copies of a given stretch of DNA. Basically this method is described Diederen et al., J Environ Med. 2007 Jan; 56 (Pt 1):94-101.

However, a disadvantage of PCR is that the samples may contain inhibitors of the polymerization reaction and therefore do not always provide quantitative results. In addition, the method uses the pre-cleaning stage DNA, which can lead to loss of DNA with subsequent underestimation of the presence of Legionella. To some extent, these shortcomings are overcome by using a panorama in real time, which provides a quantitative assessment. However, this method does not allow to distinguish viable cells from nonviable cells.

Another method is a fluorescence is orescent the in situ hybridization (FISH), in which oligonucleotide probe labeled with a fluorescent substance penetrates into the cells of bacteria. If ribosomal nucleic acid (rRNA) have the proper sequence in relation to the probe, recognizable as a target, the probe will join its target, and will not be removed at a later stage washing. Bacteria, in which the fixed probe, then will emit a fluorescent signal. This fluorescent signal can then be quantified by methods such as flow cytometry, solid-phase cytometry, EPI-fluorescence microscopy. Typical FISH method described Dutil S et al J Appl Environ. 2006 May; 100(5):955-63. However, using only FISH the way, we could determine the total number of viable Legionella pneumophila, but, unfortunately, this method cannot directly determine only those bacteria Legionella pneumophila, who are able to share and to subsequently create a colony.

Another method for the numerical determination of viable Legionella pneumophila includes ChemChrome V6 and described Delgado-Viscogliosi et al Appl Environ Environ. 2005 Jul; 71 (7):4086-96. This method allows for quantification of Legionella pneumophila, as well as to establish the difference between viable and nonviable bacteria. It combines specific detection of Legionella cells, through the use and the antibodies and marker of bacterial viability (ChemChrome V6), and the use of EPI-fluorescence microscopy to count. However, although this method distinguishes between viable and nonviable cells, it is not able to separately identify those bacteria that form colonies.

In the US 20070218522 described methods and compositions for detecting and quantifying viable Legionella and other heterotrophic aerobic bacteria, and a method for the rapid detection and counting of micro-colonies of Legionella includes applying dipslide that include absorbent medium, substances that stimulate growth, and agents for selective growth. In this way not registered damaged bacteria.

European patent EP 1329515 relates to a method for testing for the presence of microorganisms in a gaseous environment containing hydrogen peroxide by contacting a gaseous medium with agar nutrient medium containing a salt of pyruvic acid, and allowing for the development of colonies of microorganisms.

Methods that include growing colonies on nutrient medium, such as a Cup with a nutrient agar, usually considered to be more accurate. Therefore, the plating method of calculation remains the preferred choice of the way to get the total number of viable microorganisms. It usually means the location of the sample, which site is positive, contains microorganisms, the Cup containing solid nutrient medium or culture medium. Such a method generally referred to as culture Petri dishes. Under the total number of viable microorganisms we mean the total number of bacteria that can form the population, visible to the observer. This usually means a visible colony on the surface of the medium such as nutrient agar Petri dish.

However, microorganisms such as Legionella pneumophila in the environment may be exposed to one or more stress influences that impede the growth of the microorganism and reproduction in the environment. Such are in a stressful state, the microorganisms will not share at all, or to form a visible colony under normal cultivation conditions. In the environment part of the cells of the microorganisms are usually under stress due to environmental conditions, such as starvation, the presence of the biocide, heat shock and dehydration. In addition, these cells can be vulnerable physiological condition in which the method of inoculation of microorganisms on cups can increase the stress on these already in a stressful condition of the cells of microorganisms, due to the presence of atmospheric oxygen. In addition, it can lead to artifact destruction bacter the th, under stress, and lead to an underestimation of the total number of viable microorganisms.

In addition, the underestimation of the number of viable Legionella pneumophila when the Cup method can be dangerous due to their pathogenicity.

Since the 1970's, it was reported that it is desirable to use the absorber, reactive oxygen species (RFC), in order to limit the influence of oxidative stress during the procedure of seeding on the Cup. It was published by Speck et al., repair and enumeration of injured colifonns by a plating procedure, Appl Environ 29, 549-50 (1975); Martin et al., Catalase: its effect on microbial enumeration. Appl Environ Environ 32, 731-4 (1976); Brewer et al. Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus. Appl Environ Environ 34, 797-800 (1977); McDonald et al. Enhanced recovery of injured Escherichia coli by compounds that the degrade hydrogen peroxide or block its formation. Appl Environ Environ 45, 360-5 (1983); Marthi et al., Resuscitation effects of catalase on airborne bacteria. Appl Environ Environ 57, 2775-6 (1991); Busch and Donnelly, Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl Environ Environ 58, 14-20 (1992); and (Dukan et al., Oxidative stress defense and deterioration of growth-arrested Escherichia coli cells. J Biol Chem 274, 26027-32 (1999).

However, the inventors of the present invention believe that in all the above cases, we would be reduced by a direct method in which the compound chemically reacts with the RFC.

Berube et al., "Rapid detection and identification of Legionella pneumophila by membrane immunoassay", Applied and Environmental Microbiology, 1989, 55, 1640-1641 describe the detection and Ident is the classification of Legionella pneumophila by immunoblot analysis, using a monoclonal antibody. It is not provided any means to solve damaged bacteria.

In article Pine et al. (Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species. J Clin Environ 23, 33-42 (1986)) shows the need to add ketoacids and absorbing restored the oxygen of the enzyme, in order to optimize the cultivation of Legionella pneumophila, and encouraged to apply these materials in the environment used for the standard calculation of this organism.

However, the application only ketoacids and absorbing the recovered oxygen enzyme is insufficient for recovery under stress of Legionella pneumophila cells, so that cells were recovered and allowed to conduct an accurate count. This is especially true in the case of application specific nutrient medium for Legionella pneumophila, such as buffered charcoal-yeast agar (BCYE). In fact, there is no data concerning the optimization of the standard environment, suitable for accurate estimate of Legionella pneumophila.

Thus, the present invention is to find how accurate estimate of Legionella pneumophila. This is especially important in relation to the standard method, which uses the method of sowing on the Cup.

Thus, according to the present invention we provide methods for the detection and enumeration of viable microorganisms in a sample, which is assumed to contain these microorganisms, and provides:

(1) contacting these microorganisms specified sample with at least one restoring connection and a nutrient medium and

(2) incubation of the product of stage (1) and

(3) detection and identification number of said viable microorganisms, and the microorganisms are of the species Legionella pneumophila, and regenerating connection directly or indirectly affects the metabolism, reducing oxidative stress of the organism.

Under oxidative stress, we mean an imbalance between the concentration RFC (endogenous education or egzogenne cast) and the ability of microorganisms to easily neutralize the reactive intermediates or to effectively repair the resulting damage. This disruption of normal metabolic processes of the organism may cause toxic effects as a result of formation of free radicals and oxidative substances such as peroxides, which can cause damage to components of the cells of the microorganisms, for example, DNA, proteins or lipids.

Effects on the metabolism of the microorganism leads to changes in the natural internal chemical processes in the cell of the microorganism.

The link to the endogeneity oznacza is t, what changes occur within the cells of the organism and leads to reduced oxidative stress. This could be, for example, changing metabolic processes in the organism. It may also include removal of RFC from the cells of the organism.

It is desirable that restores the connection could constitute or include at least one compound that inhibits the formation and/or destroys the RFC. In General this could be achieved through changes in the metabolism.

Thus regenerating the connection may represent or include at least one connection, which indirectly inhibits the formation and/or destroys the RFC. This compound, which has an indirect impact on we can do this by interfering with the metabolism of the microorganism. Such a connection can be considered as indirectly reducing RFC endogenous, for example, during aerobic respiration.

We found that this method causes recovery under stress cells Legionella pneumophila and thus gives a more accurate total number of viable microorganisms. Unexpectedly, we also found that this method reduces the number required for incubation time. In General, we found that the method can reduce the time of incubation for a few hours, and not is which cases, at least for one day. In some cases, the method according to the present invention allows to reduce the time of incubation for several days, for example, for five days, compared with the conventional method.

Unexpectedly, we also found that the method according to the invention can lead to a reduction in nuisance organisms, i.e. organisms other than Legionella pneumophila.

The method according to the present invention preferably includes the contacting of the cells of the microorganism Legionella pneumophila, under stress, at least one compound that inhibits the formation and/or reduces and/or removes the RFC, and it works in such a way that leads to the recovery of cells under stress.

The microorganism Legionella pneumophila can be brought into direct contact with regenerating connection with the selection of the sample. Thus, the container in which is collected the water sample suspected of containing the microorganism, may already contain restorative connection. Alternatively, after he was selected sample of water containing Legionella pneumophila, for the purposes of analysis, it can be diluted with water containing a reducing compound. In another alternative embodiment, the sample is not necessarily diluted, can be brought into contact with a nutrient medium containing vos is Tamaulipas connection, or reducing compound may be applied after contact of the microorganism with a nutrient medium.

One variant of this invention, preferably, includes contacting the specified sample with a reducing environment, preferably non-selective restores the environment containing the specified regenerating compound, and then bringing it into contact with a nutrient medium, preferably, a selective nutrient medium. Preferably, the regenerating medium is a liquid, more preferably, the broth. When regenerating medium is a liquid, it is thus called as liquid recovery method. Generally, when the liquid recovery method the sample is first introduced into the liquid medium containing regenerating connection. Theoretically, the liquid recovery method allows you to recover under stress bacteria in non-selective liquid medium. Preferably, when the liquid recovery method as a liquid medium is applied to the broth. Typically, the liquid medium containing the microorganisms, and then transferred to a nutrient medium. Microorganisms, under stress, or recovered before migrating to breeding grounds or before recovering what are stated in contact with a nutrient medium. More preferably, the culture medium is a selective nutrient medium. Typically, the liquid medium containing the microorganisms is applied to the Cup with a selective nutrient medium, such as a Cup with selective agar nutrient medium.

In an alternative preferred embodiment, step (1) includes contacting the specified pattern with a nutrient medium, preferably, non-selective nutrient medium containing the specified regenerating compound, and then bringing it into contact with a reducing environment containing named restores the connection. Preferably, healing environment is a non-selective restoring the environment, more preferably, a solid environment, and particularly preferably, a selective agar medium. If healing environment is solid, this case should call the repair method on solid medium. Typically, the restore method on solid medium may include contacting the sample with non-selective nutrient medium containing regenerating connection. Then it can be brought into contact with a selective nutrient medium containing regenerating connection. In this embodiment, the selective ingredients and the compound or compounds that prevent the up education, reduce or remove RFK, will diffuse in non-selective medium. It is desirable that non-selective nutrient medium can be non-selective agar nutrient medium. Accordingly, in this embodiment, the sample can do it on a Cup of any non-selective agar, and then selective agar nutrient medium containing the compound or compounds that prevent the formation of, reduce or remove RFK, is imposed on non-selective agar medium.

In another alternative embodiment, the sample can be applied on a selective nutrient medium that already contains restorative connection. Such selective nutrient medium may be a selective agar medium. Seeding of the sample Cup can be performed as described previously.

In another alternative embodiment, the sample may be selected from water in the form of an aerosol. Typically, the aerosol can be localized in the cooling riser or air conditioning. It is desirable that the water are condensed from the spray before the test according to the method of the present invention. In an alternative preferred embodiment, step (1) contains the contacts of the specified sample from the aerosol dilution with water containing restoring the environment, preferably non-selective restoring the redu, contains named regenerating compound, and then bringing it into contact with a nutrient medium, containing named restores the connection.

In all the above embodiments of the invention, the nutrient medium should be suitable for cultivation of Legionella pneumophila. Suitable types of nutrient media described in the literature and well known to specialists in this field. Usually nutrient medium should contain activated charcoal and cysteine.

Preferably, the selective culture medium consisted of a selective agar nutrient medium and, more preferably, represented buffered charcoal-yeast agar (BCYE). BCYE medium becomes selective when adding antibiotic. Extremely desirable BCYE medium with the antibiotic known as GVPC (glycine, vancomycin, polymyxin b, cycloheximide.

Method of sowing on the Cup described in the literature and well known to specialists in this field. Typically, the method comprises applying a quantity of water samples on agar gel, which is placed in a Petri dish. This can be called as a method on a Petri dish or a way of sowing to plates with agar. The purpose of sowing to plates with agar is the application of an aliquot, typically 100 ál of water suspected of containing the microorganism, called emeu bacterial suspension, on solid medium in a Petri dish. Can use glass beads or cell scraper to spread the bacterial suspension on the agar in the Cup. After the distribution of most of the fluid is absorbed by the agar, and a thin layer of bacteria remains on the surface of the agar. During incubation on the surface of the agar was the growth of bacteria in the form of colonies. Incubation usually takes place at a temperature best suited for a microorganism that is well reflected in the literature and known in the art. Usually the temperature is in between 30°C and 50°C, for example, about 37°C.

Regenerating the connection must be added in amounts effective to reduce oxidative stress of the organism. Preferably, it is the amount effective to reduce or substantially remove the RFC of the cells of the organism.

In one preferred embodiment of the invention restores the connection includes at least thioglycolic acid or its salts. Preferably, thioglycolate acid is in the form of thioglycolate and usually in the form of sodium salt. Thioglycolate acid or its salts agogino absorb RFK. Preferably, the concentration present in the environment thioglycolic or its salts is in the range between 0.01 and 1% by weight (calculated as eaglequest).

In another preferred embodiment of the invention restores the connection includes at least one compound from the group consisting of catalase, ascorbic acid (or its salts), proserity acid (or its salts), dimethyl-sulfoxide (DMSO), 3,3'-thiodipropionic acid (TDPA) (or its salts) and pyruvic acid (or its salts). It has been found that all these compounds reduce or remove the RFC. When applied ascorbic acid and pyruvic acid, they are preferably present in the medium at a concentration of from 0.01 to 1% by weight, based on sodium salt. DMSO is preferably used at a concentration of between 0.01 and 0.1% by weight, and catalase, preferably, is present in a concentration ranging from 0.001 to 0.1% by weight. Pyruvic acid, especially the sodium salt is particularly preferred.

In a more preferred embodiment, a healing environment or culture medium may contain as thioglycolic acid (or salt), and at least one compound selected from the group consisting of catalase, ascorbic acid (or its salts), proserity acid (or its salts), dimethyl sulfoxide (DMSO), 3,3'-thiodipropionic acid (TDPA) (or its salts) and pyruvic acid (or its salts). The combination of thioglycolate and pyruvate sodium is especially before occhialini.

If renewing the connection may represent or include at least one connection, which indirectly prevents the formation and/or destroys the RFC, then the named connection can cause a decrease in levels of RFK that interfere with the metabolism of the microorganism. Usually, this connection comprises amino acids or their salts. Especially preferred compound is glutamic acid or glutamate.

In another preferred embodiment of the invention restores the connection may include glutamic acid or glutamate, especially the sodium salt. In General, the amount of glutamic acid or glutamate is in the range between 0.01 and 5% by weight calculated on the sodium salt.

Particularly preferably, regenerating connection included pyruvic acid or its salt (particularly sodium salt) together with glutamic acid or its salt (particularly sodium salt). This combination of pyruvic acid or pyruvate with glutamic acid or glutamate apparently has a synergistic effect, which provides a higher score (and therefore a more accurate estimate) cultivated Legionella, respectively than any of these compounds used alone. In addition, we found that this combination stage is niteline reduces the latent phase during the development of Legionella pneumophila, in particular, in a liquid medium. Such reduction in the latent phase in liquid medium leads to a decrease in the time required to obtain visible colonies on agar Cup.

It is desirable that the amount of pyruvate and glutamate was, as stated earlier. Particularly preferably, the ratio of glutamate to pyruvate were in the range between 1:1 and 50:1, especially between 5:1 and 20:1 and, more preferably, between 7:1 and 15:1.

It is unknown whether glutamate antioxidant. However, it appears that glutamate may indirectly reduce endogenous education RFK, formed naturally during cultivation, or their subsequent effects on macromolecules (oxidation).

Without being limited by theory, believe that glutamic acid alters the metabolism of Legionella, increasing the effect of pyruvate, and the intervention in the metabolism of Legionella indirectly inhibits the formation and/or destroys intracellular RFC.

Also may be desirable to enable a keto acid and/or enzyme-absorbing restored oxygen, restoring the environment and/or culture medium. The ketoacid and/or enzyme, absorbing the recovered oxygen is not considered to restore the connection according to the present invention. However, it may be useful to include one or both of these the compounds with any of the above-mentioned reducing compounds or their combinations.

Detection and counting of viable microorganisms can be performed by any known method, presented in the literature. Usually this means the count of visible colonies on the surface of the medium, such as a Cup with a nutrient agar.

The method according to the present invention facilitates accurate quantitative detection of Legionella pneumophila. In addition, can be greatly reduced incubation time. The method is suitable for determination of Legionella pneumophila in the sample, selected from any of groups selected from industrial cooling water, potable water and natural waters.

The present invention also includes a kit for a more precise definition and counting of viable microorganisms of the species Legionella pneumophila in the sample, which presumably contains the mentioned microorganisms, containing:

(1) at least one reducing compound,

(2) a nutrient medium,

(3) a device for incubation

(4) a device for the detection and counting of microorganisms, which restores the connection directly or indirectly affects the metabolism, reducing oxidative stress of an organism

in which the microorganisms are of the species Legionella pneumophila, and which restores the connection directly or indirectly affects the metabolism, reducing oxidative shooting is from a microorganism.

The kit may also contain any of the embodiments described in relation to the first aspect of the invention

The set is suitable for use in the method of the present invention and enables a more accurate estimate of Legionella pneumophila.

The invention is illustrated by the following examples.

Example 1.

5 flasks containing 50 ml of sterile phosphate buffer (PBS), was added a suspension of Legionella pneumophila to a final concentration of 108bacteria/ml was Added biocidal solution to obtain final concentrations ranging from 10 to 30 mg/L. One flask was prepared in parallel and served as a control without biocide. Used biocide was a THPS (tetrakis(hydroxymethyl)phosphonium sulfate).

After homogenization, all suspensions were incubated at a temperature of 37±1°C in the dark and under stirring for 60 minutes Biocide was removed by 2 times washing in PBS (5000 × g, 10 min) before counting bacteria. Was carried out by serial dilution, and 2 aliquots of 100 μl from the same breeding were sown on BCYE agar Cup and BCYE with the addition of 0.1% pyruvate.

The results are shown in figure 1. Figure 1 shows the calculation of cultivated Legionella Pneumophila after treatment with biocide on BCYE medium (squares) and BCYE medium plus 0.1% pyruvate (circles). The presence of the biocide should lead organisms to stress. The result of the show what if, when organisms are under stress, in the presence of pyruvate, there are a greater number of microorganisms. In the absence of biocide microorganisms are not subjected to stress. In this case, you can see that the presence and absence of pyruvate gives the same result. This shows that in the presence of pyruvate under stress microorganism Legionella pneumophila is restored, and thus provided a more accurate reading.

Example 2.

In flask 1 containing 50 ml of sterile phosphate buffer (PBS), was added a suspension of Legionella pneumophila to a final concentration of 108bacteria/ml was Added biocidal solution to obtain a final concentration of 15 mg/L. Used biocide was a THPS (tetrakis(hydroxymethyl)phosphonium sulfate).

After homogenization, the suspension is incubated at a temperature of 37±1°C in the dark and under stirring for 60 minutes Biocide was removed by 2 times washing in PBS (5000 × g, 10 min) before counting bacteria. Was carried out by serial dilution, and 2 aliquots of 100 μl from the same breeding were sown on BCYE agar Cup, BCYE with the addition of 0.1% pyruvate, BCYE with the addition of 0.1% pyruvate and 1% glutamic acid and BCYE with the addition of 1% glutamic acid. The results are shown in figure 2. Figure 2 shows the compared between the number of cultivated Legionella, received on standard medium (BCYE) and the number of cultivated Legionella obtained on medium with addition of 2 compounds described in this patent (pyruvate and glutamic acid).

The addition of pyruvate to the standard medium (BCYE) leads to an increase in cultivated Legionella detected after treatment with biocide (number of cultivated Legionella "BCYE + Pyruvate" 45 times higher than the number of cultivated Legionella standard environment). Adding one glutamic acid to the standard medium (BCYE) leads to a decrease of cultivated Legionella detected after biocide treatment (×0.2). Surprisingly, the addition of pyruvate and glutamic acid leads to a greater increase in cultivated Legionella, compared to that observed with the compounds separately. This shows that in the presence of pyruvate under stress microorganism Legionella pneumophila is restored to a greater degree when adding glutamic acid, and thus provided a more accurate reading.

Example 3.

1 flask containing 1 l of sterile phosphate buffer (PBS), was added a suspension of Legionella pneumophila to a final concentration of 3×102bacteria /L. After concentration by filtration 2 aliquots of 100 l of the same suspensions were sown on GVPC agar Cup (GVPC), GVPC with the addition of 0.1% pyruvate and 1% glutamine is th acid (GVPC+X). The number of colonies was counted at 0, 3, 5 and 10 days after incubation at 37°C. These results are shown in Figure 3. On the 3rd day of incubation in the medium GVPC no visible colonies were not detected, while on the GVPC medium with additives you could count 300 colonies. At 5 and 10 days of incubation on the environment GVPC you could count 100 colonies, while on the GVPC medium with additives you could count 300 colonies. In the case of medium with additives, colonies could be detected, at least 2 days before colonies counted in a standard environment GVPC.

Example 4.

A sample from the environment containing cultivated Legionella, concentrated by filtration. From concentrate 2 aliquots of 100 μl from the same suspensions were cultured on GVPC agar Cup (GVPC), GVPC with the addition of 0.1% pyruvate and 1% glutamic acid (GVPC+X). The number of colonies was counted after 5 days of incubation at 37°C. In this case, no colonies of Legionella in a standard environment GVPC count could not, at the same time could consist of 17 colonies that were not Legionella. In contrast, at least 10 colonies of Legionella can be counted on medium with additives (GVPC+X), and only 2 colonies that were not Legionella. This is shown in Figure 4.

Example 5.

1 flask containing 50 ml of sterile phosphate buffer (PBS), dobavleniyami Legionella pneumophila to a final concentration of 10 8bacteria/ml was Added biocidal solution to obtain a final concentration of 15 mg/L. Used biocide was a THPS (tetrakis(hydroxymethyl)phosphonium sulfate).

After homogenization, the suspension is incubated at a temperature of 37±1°C in the dark and under stirring for 60 minutes Biocide was removed by 2 times washing in PBS (5000 × g, 10 min) before counting bacteria. Was carried out by serial dilution, either in PBS or in PBS with the addition of 0.5% pyruvate. From each dilution buffer (with the addition of pyruvate or without it) 2 aliquots of 100 μl from the same breeding were sown on BCYE agar Cup, BCYE with the addition of 0.1% pyruvate. The results are shown in Figure 5. Figure 5 shows the number of cultivated Legionella pneumophila obtained in a standard environment, and the number of cultivated Legionella pneumophila obtained in a standard environment with the addition of pyruvate after dilution in PBS (hatched bars) or PBS + Pyruvate (black bars).

As already observed, the addition of pyruvate in standard medium (BCYE) leads to an increase in cultivated Legionella pneumophila detected after treatment with biocide, when for cultivation solution is used only PBS. Surprisingly, the addition of pyruvate in the buffer cultivation leads to an increase in cultivated Legionella pneumophila detected on standard medium (BCYE), but also on the standard sides the addition of pyruvate. This shows that the presence of pyruvate in the buffer cultivation allows for a greater degree of repair under stress Legionella pneumophila.

1. Method detection and enumeration of viable microorganisms of the species Legionella pneumophila in a sample suspected of containing these microorganisms, providing
(1) contacting these microorganisms specified sample with at least one
- regenerating compound containing glutamate and pyruvate, and
- a nutrient medium, which is a buffered charcoal yeast (BCYE) or GVPC agar nutrient medium, and
(2) incubation of the product of stage (1) and
(3) detection and identification number of said viable microorganisms,
in which the microorganisms are of the species Legionella pneumophila and which restores the connection directly or indirectly affects the metabolism, reducing oxidative stress of the organism.

2. The method according to claim 1, wherein regenerating the compound is a compound that inhibits the formation and/or destroys the RFC.

3. The method according to claim 1, in which step (1) includes contacting the specified pattern with a specified regenerating compound contained in the reducing environment, preferably in a non-selective restoring the environment, and for the eating bringing it into contact with a nutrient medium, preferably selective nutrient medium.

4. The method according to claim 3, in which the regenerating medium is a liquid medium, preferably broth.

5. The method according to claim 1, in which step (1) includes contacting the specified pattern with a nutrient medium, preferably non-selective nutrient medium, and then bringing it into contact with the specified regenerating compound present in a healing environment.

6. The method according to claim 3, in which the regenerating medium is a selective restoring the environment, preferably a solid medium and more preferably selective agar nutrient medium.

7. The method according to claim 1, in which step (1) includes contacting the specified pattern with a nutrient medium containing the specified restorative connection.



 

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EFFECT: use of the presented invention provides the prediction of the recurrence-free survival period in local cervical cancer.

FIELD: biotechnology.

SUBSTANCE: invention relates to determining the activity of pyruvate dehydrogenase complex (PDH complex) by 13C-MP detection (magnetic resonance detection based on the isotope 13C). The essence of the method consists in that the change in activity of PDH complex in the subject to be examined by 13C-MR detection (magnetic resonance detection based on the isotope 13C) is determined using the medium of visualisation comprising hyperpolarised 13C-pyruvate, and when detecting a signal of 13C-bicarbonate or the signal of 13C-bicarbonate and the signal of 13C-pyruvate where the said hyperpolarised 13C-pyruvate is selected from the group consisting of hyperpolarized 13C1-pyruvate, 13C1,2-pyruvate, 13C1,3-pyruvate or 13C1,2,3-pyruvate or any of their combination, the activity of PDH complex is determined.

EFFECT: use of the claimed method enables to determine reliably changes in activity of PDH complex in the subject to be examined.

11 cl, 7 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: method for describing indications for choosing a conservative therapeutic approach to the patients suffered recurrent myocardial infarction involves pre-therapeutic patient's blood examination to analyse blood serum for oxidation-resistant lipoproteids (ORLs), to analyse erythrocytes for the pyruvic acid concentration (PAC), and if the ORL value is equal to 1.89 nmole MDA/mg of protein of β-lipoprotein and lower, while the PAC value is equal to 1.81 mmole/l and higher, the conservative therapy is added with the preparations of lipoic acid.

EFFECT: method is simple, has a broad information value and allows a more objective assessment of the patient's state and identifying a group of the patients in need of the conservative treatment and suffered recurrent myocardial infarction by the preparations of lipoic acid.

3 ex

FIELD: medicine.

SUBSTANCE: taken venous blood is separated into two samples. The first sample is stabilised with a solution of sodium citrate, the second one - with ethylene diamine tetraacetate. The first sample of whole blood is added with adenosine diphosphate as an aggregation inducer and tested for a peak amplitude of thrombocyte aggregation and a peak amplitude of adenosine triphosphate release profile by impedance method. The second sample is used to measure a fraction of thrombocytes and a fraction of blood corpuscles. It is followed by calculating a thrombocyte aggregation potential index by formula I=LmaxPTCΩmaxHTC100%; wherein Lmax is the peak amplitude of adenosine triphosphate release profile, Ωmax is the peak amplitude of thrombocyte aggregation, PCT is the fraction of thrombocytes, HTC is the fraction of blood corpuscles. If the value I is less than 0.5%, the low clinical effectiveness of the antiaggregant therapy is stated, and the value I being 1.5-2.5% shows the high effectiveness thereof.

EFFECT: improving the objective estimation of the clinical effectiveness of the antiaggregant therapy in the patients with acute coronary syndrome, and providing an opportunity for predicting the clinical course of the disease.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. A composition for stimulating the skin stem cell production containing interleukin-1 alpha and a dermatologically acceptable diluent or carrier.

EFFECT: invention provides improving the stem cell stimulation.

2 ex

FIELD: chemistry.

SUBSTANCE: method involves dissolving 855 mg of a crystalline hydrate of copper chloride (CuCl2·2H2O) in 100 ml of distilled water (concentration of Cu2+ ions in the prepared solution is 50 mmol/l) and adding 1 ml of the prepared solution to 100 ml of a standard reagent used in glucose oxidase test. The ascorbic acid oxidant used is copper chloride solution in end concentration in the glucose oxidase reagent of 500 mcmol/l.

EFFECT: method enables correct determination of glucose content.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: workers' blood serum is analysed for interleukin 4, protein S-100β, protein S-100 autoantibodies, voltage-dependent Ca-channel autoantibodies, glutamate receptor autoantibodies, γ-aminobutyrate receptor autoantibodies, dopamine receptor autoantibodies; diagnostic coefficients F1 and F2 are calculated; if the value F1 is less than F2, the early changes of the nervous system are diagnosed for the chronic exposure to vinyl chloride; F1 more or equal to F2 enables stating the absence of any signs of the chronic exposure to vinyl chloride. The developed method may be used in the periodic medical screenings, medical examinations of workers to diagnose some occupational diseases.

EFFECT: use of the invention improves higher accuracy of identifying the various signs of the chronic exposure to vinyl chloride through the use of a complex of the immunological structures of nerve tissue in the chronic exposure to vinyl chloride.

1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: atoxigenic strains of cholera vibrios 01 and 0139 serogroups are differentiated from toxigenic strains on presence of hydrolase activity. The substrate used is glycerin, which is introduced into molten Marten's agar at 2.5/100 ml of the medium with pH 7.8±0.1 and Nile blue sulphate indicator 1.5 mg/100 ml of medium, which is poured into petri dishes. Analysed broth cultures grown in a thermostat for 4 hours are deposited in volume of 20 mcl on a sector of developed agar and inoculations are incubated at 37°C for 36 to 48 hours. Strains are differentiated from their results of hydrolase activity: of the colonies grown on the medium are in form of soft semitransparent films, merging with the colour of the medium, then the analysed strain of cholera vibrio is toxigenic and epidermic. The colonies are a thick yellow or whitish deposit, then the strain is non-toxigenic haemolytic and is not epidemic.

EFFECT: method is disclosed for differentiating atoxigenic strains of cholera vibrios 01 an 0139 sergroups from toxigenic strains on hydrolase activity.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently taking Scotch pine needle samples growing on case and control test areas, determining chlorophyll concentration and catalase activity therein. Metabolism intensity coefficient is calculated next in case and control samples as ratio of chlorophyll concentration and catalase activity. The comparison results are interpreted in terms of anthropogenic influence degree.

EFFECT: high result data reliability; low costs and labor inputs level.

3 cl, 5 tbl

FIELD: medicine, oncology.

SUBSTANCE: one should study the activity of catalase in the tissue of malignant mammary tumor and its perifocal area, and at ratio coefficient of catalase activity in the tissue of malignant mammary tumor to that in the tissue of perifocal area being equal to 1.0 ± 0.2 one should predict the chance for the development of new foci of lesion before their clinical manifestation, that provides necessary treatment in due time.

EFFECT: higher efficiency of prediction.

1 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of altering immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which involves obtaining preparations of lipopolysaccharides (LPS) and mouse toxin (MT) Yersinia pestis with subsequent formation of a LPS-MT complex thereof. A modified form of LPS-MT is used as an inducer of synthesis of cytotoxins TNF-α and IFN-γ. To this end, a test sample is prepared, to which LPS is added in amount of 5 mcg (50 mcl from working dilution of 100 mcl/ml) and MT is added in amount of 50 ng (5 mcl from working dilution of 10 mcg/ml); the sample is then incubated for 30 min at 37°C. The volume of the sample in eppendorfs is then brought to 100 mcl with sterile buffered physiological solution of NaCl and the obtained mixture is added a tray dimple containing a culture of human monocyte cell line U-937 (1×106 cells in a dimple); the latter is cultured in a medium of PRMI 1640 with simultaneous double control. Further, 1, 4, 20 hours after the beginning of combined incubation of the preparations of LPS with monocytes, quantitative accounting of the synthesised cytotoxins is carried out, wherein change in the immunomodulating properties of LPS of plague bacteria in vitro is determined from the amount of cytotoxins produced and the dynamics of their synthesis.

EFFECT: invention enables to alter immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which enables to realise toxic potential of the endotoxin of plague bacteria.

2 cl, 8 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: method includes the following stages: contact of a sample with a source of nutrition for cells, containing antioxidant, representing pyroracemic acid or its salt, and an inhibitor of cell proliferation, which is selected from ciprofloxacin and cefalexin; contact of the specified sample with fluorescent-marked oligonucleotide probes, capable of specific hybridisation at least with one section of ribosomal nucleic acids, which belong to microorganisms of Legionella pneumophila kind and type; and detection and quantitative determination of a fluorescent signal.

EFFECT: provided method and set make it possible to more accurately and reliably detect and calculate viable microorganisms of Legionella pneumophila type, having excluded natural fluorescence of microorganisms from calculation.

6 cl, 2 dwg, 6 tbl, 2 ex

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

FIELD: medicine.

SUBSTANCE: method for detection of neutrophil extracellular traps in mucosal secretions provides staining of a native preparation 0.1 ml with an equally component mixture 0.1 ml of the Evans blue dye in the concentration of 1:8000 and the Sytox Green dye in the concentration of 1:500, incubation for 5 minutes in the dark. Luminescent microscopy with using filters generating excitation light at wave length max. 490 nm and emission at wave length 520 nm is used to detect neutrophil extracellular traps, living bacterial cells, apoptotic bacterial cells, green cork bacterial cells. The number of neutrophil extracellular traps (%) containing bacteria in 100 counted traps is evaluated.

EFFECT: method enables visualising chromantin, assessing cell viability and accelerating the study.

2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method involves telomere length measurement by flow-FISH method in cells differentiated by a number of population divisions with a vital stain without preliminary recovery of proliferative cells with using Bis(sulfosuccinimidyl)suberat (BS3) amino group cross-linker.

EFFECT: invention provides the single-step telomere length measurement in cells with a common division quantity.

3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: sample is prepared: an additional weight of nanocarbon forms is dispersed in 1 ml of organic solvents with a degree of polarity smaller than that of water - dimethyl sulphoxide or ethanol. Then it is mixed and exposed to ultrasound for 30 minutes. The prepared nanocarbon suspension is transferred in an aqueous medium to the final concentration of the used solvent 2.5 %. The produced and control samples are added with a viable sensor recombinant luminescent Escherichia coli K12 strain with cloned luxCDABE genes of luminescent Photobacterium leiognathi system. It is followed by incubation for 60 - 180 minutes, measuring luminous intensity and evaluating optical properties of the analysed suspension simultaneously. A toxicity index (T) is calculated with evaluating an actual luminous intensity of the strain (Iact) in comparison with the control of the same concentration of the solvent, considering light absorbing properties of the analysed suspension (D) and an experimental luminescence level of the bacterial luminescent biosensor (Iexp).

EFFECT: invention allows providing higher accuracy and sensitivity of nanocarbon biotoxicity analysis ensured by the introduction of a correction value - the actual luminous intensity of the strain Iact, considering a common factor of emitted light distribution in the analysed suspension.

1 ex

FIELD: chemistry.

SUBSTANCE: method involves collecting dust samples with a sterile cotton wool wad from 10-20 cm2 of any surface or the sterile cotton wool filter of a vacuum cleaner. The dust samples are put into a sterile physiological solution, followed by extraction for 30-60 minutes at room temperature while stirring. The supernatant liquid is collected and multiple cultures of the obtained extract are prepared from the liquid. The obtained cultures are sown in a semi-liquid thioglycol medium and modified thioglycol medium obtained by adding defibrinated blood to the thioglycol medium in a given amount. Culturing is performed for 10 days at temperature 37°C and daily visual monitoring, followed by determining the number of microbial cells which are calculated from the McCready probability table, which is compiled based on data on growth properties of microorganisms and tinctorial properties of the bacterial community of the dust sample are determined through microscopic examination of smears of the mixed culture mass which is gram-stained.

EFFECT: simultaneous quantitative determination of the total bacteria number and obtaining cultural, morphological, tinctorial and haemolytic characteristics of the bacterial community of dust samples from buildings.

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