Nutrient medium for growing filamentous fungi-dermatophytes from clinical material

FIELD: biotechnology.

SUBSTANCE: nutrient medium for growing filamentous fungi-dermatophytes from clinical material comprises glucose, bacteriological agar, meat peptone, casein hydrolyzate, yeast extract, sodium chloride, sodium carbonate, L-cysteine, thioglycolic acid and distilled water in a predetermined ratio of components.

EFFECT: invention enables to reduce time of growing filamentous fungi-dermatophytes from clinical material.

1 tbl, 2 dwg, 4 ex

 

The invention relates to medical Microbiology and can be used for the selection of clinical material is the most important pathogens - pathogenic fungi-dermatomitsetov, agents of fungal diseases of humans and animals.

The essence of the invention is to design an efficient dense semi-synthetic nutrient medium to obtain primary cultures of fungi-dermatomitsetov from clinical material, taking into account their needs batteries and physiological characteristics.

For isolation and identification of fungi currently in clinical Microbiology, they use solid and liquid nutrient media Saburo, approved by the USSR Ministry of health order No. 535 of April 22, 1985, "On the unification of microbiologic methods used in clinical diagnostic laboratories medical institutions". It relates to semi-synthetic environments, which, along with the substances of uncertain composition includes compounds of known chemical nature. Wednesday Saburo: peptone - 10 g, glucose or maltose) - 40 g, agar - 18... 20 g, distilled water 1 l, pH 5,6 5,8..., broth prepared similarly, but without agar [2].

Today, however, the percentage of positive results of culture experiments is of dermatomitsetov on the environment Saburo if positive direct microscopy and undoubted clinical picture remains low. Reportedly, A. Sergeev, to highlight the culture of the pathogen in 1994-1996 he succeeded in 36% of cases [3], which cannot be considered optimal for laboratory diagnosis. There are foreign corporate environment for Trichophyton (Mycosel, Mycobiotic) with similar indices in Visavuori mushrooms from primary clinical material. This is probably related to a number of features of this kind of fungi. First, the relatively slow growth - unlike most mizelialnah fungi, the growth of Trichophyton begins 5-7 days after sowing (fungi on day 2), and secondly, higher nutrient needs of Trichophyton. Most species dermatomitsetov to start growth requires the presence in the medium of a complete set of vitamins and proteins of animal origin.

The purpose of the claimed invention is to develop a dense semi-synthetic nutrient medium to obtain primary cultures of fungi-dermatomitsetov from clinical material meeting the established performance criteria, which is a necessary prerequisite for reliable results of any clinical, microbiological, and providing accurate and objective results.

The technical result of the invention is to obtain a dense semi-synthetic nutrient medium is La obtain primary cultures of fungi-dermatomitsetov from clinical material.

The implementation of the invention is achieved by preparing a nutrient medium containing 1 liter of distilled water: glucose - 30,0 g bacteriological agar - 20,0 g, peptone meat - 5.0 g, casein hydrolysate - 2.5 g yeast extract, 0.8 g sodium chloride 0.4 g sodium carbonate is 0.16 g, L-cystine - 0.12 g, acid thioglycolate - 0.08 g, pH of the medium is 6.5.

Components are successively dissolved in water, make agar and heated to melt the agar. If necessary, the medium is filtered. Then add the glucose, mix thoroughly and set a pH of 6.5. Then poured into a sterile vessel and sterilized for 60 minutes at 120°C.

Example 1. The method of obtaining the composition. In 1 l of distilled water was dissolved sample: meat peptone - 5.0 g, casein hydrolysate - 2.5 g yeast extract, 0.8 g sodium chloride 0.4 g sodium carbonate is 0.16 g, L-cystine - 0.12 g, acid thioglycolate to 0.08, Stirred the resulting solution. To the dissolved components were added to the agar bacteriological - 20,0 g and heated to melt the agar. Filtered if necessary (in case of presence of insoluble particles). Then add the glucose - 30,0 g and thoroughly mixed. Established a pH of 6.5.

The prepared medium was poured into sterile dishes and sterilized for 60 minutes at 120°C.

Freshly prepared medium was poured into Petri dishes in an amount not IU the 20 ml and the same day produced planting material.

The difference between the proposed composition of the medium is that to improve the effectiveness of seeding dermatomitsetov on Wednesday added L-cystine and sodium carbonate to neutralize free radicals and reduce the redox potential of the environment and thioglycolic acid that helps to release elements of the fungus from keratin masses due to the destruction of disulfide bonds in the keratin of pathogenic material (skin, scales, nails, hair), which provides the contact elements of the fungus with nutrients environment and a more rapid onset of growth dermatomitsetov.

For a better understanding of the invention examples of effectiveness of the proposed environment.

Study of the effectiveness of the proposed environment, in contrast to the environment Saburo, was performed by the method of comparative analysis of fungal growth-dermatomitsetov as in model experiments and clinical material. In model experiments took into account the speed and pattern of growth of the colony and manifestations of morphological characteristics of fungi dermatomitsetov. The study was conducted for 21 days with 3 species of fungi (Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum). These types of fungi are the most frequently isolated from patients with causative agents of fungal infections. The strains were obtained from the collection model and pathogenic cultures mycological laboratory Kazanskogo the scientific research Institute of epidemiology and Microbiology of the CPS. Were used as Museum strains and isolated from patients with a diagnosis of mycosis.

Example 2. Nourishing environment conducive to accelerating the growth of fungi dermatomitsetov, allows you to achieve greater efficiency in identifying dermatomitsetov, since the rate of growth dermatomitsetov is an important factor in the efficiency of the environment due to frequent contamination of clinical material growing mold fungi. It is known that mushrooms dermatomitsety grow in 2, and sometimes 4 times slower fungi [4]. For typical signs dermatomitsetov - branching, sporulation, pigmentation currently recommended to maintain the incubation period crops up to 1 month [6].

To study the effect of medium composition on the linear growth of colonies Museum of test strains of Trichophyton rubrum and Epidermophyton floccosum were sown into the center of a dense nutrient medium small one AMF inoculum density. The colony diameter was measured in two mutually perpendicular directions in two or three repeats every 24 hours [5]. The sowing of the test strains on our proposed environment showed an increase in the rate of growth in 1,5-2 times in comparison with the crops on the environment Saburo (figure 1, 2). However, given that these species of fungi on the surface of some limiting growth in dense environments form a widely rasprostranen the Yu, but thin filmy colony, and with the growth of dense optimal environment colony of the fungus can reach the same diameter, but can be high, fluffy, have a richly developed aerial mycelium, in addition to the growth rate of colonies was determined the density of hyphae colony [5]. The density of hyphae was determined by their total length, representing the total length of the main hyphae and branches of all orders in the same field of view under a microscope at magnification 20x20. The density was determined in different parts of a growing colony. On the sixth day the length of hyphae (H) of Trichophyton rubrum on the environment Saburo and the environment amounted to 648 and 817 μm, respectively. The length of the hyphae Epidermophyton floccosum 547 μm on the environment Saburo and 651 μm on the environment.

Example 3. Getting the nutrient medium on which the fungus would give a typical colony and more differentiated fruiting bodies, is an important task to identify dermatomitsetov. Analysis of differential diagnostic features of strains of fungi Trichophyton rubrum, Trichophyton mentagrophytes, grown on the environment and the environment comparison Saburo, showed the effectiveness of the proposed environment. In ten cases out of ten strains of Trichophyton rubrum was isolated in an environment of bright pigment rich red color, which is one of the main diagnostic characters of this species (table 1). While strains, wirawan the e on the environment Saburo, allocated in the environment pigment pale pink color, and in 3 cases the pigment was absent.

Microscopic picture of the different strains of Trichophyton mentagrophytes grown on medium Saburo, differed from those grown in the proposed environment. Seven out of ten strains were not fertile hyphae, microconidii was absent.

Example 4. Evaluation of the effectiveness of the proposed environment on clinical material obtained from patients with limited ringworm-onychomycosis. Criteria for inclusion in the study: patients with onychomycosis confirmed by the positive results of mycological examination (microscopy of the nail plates in 20% KOH or fluorescent microscopy with calcofluor white).

Large pieces of nails cut into several pieces under sterile conditions. Then an equal amount of material at the same time planted on the studied environment. On the seventh day after inoculation of the 25 samples, sown on Wednesday, Saburo, growth dermatomitsetov was detected in 44% (11 samples). Culture the culture on offer Wednesday revealed mushrooms dermatomitsety in 18 samples, which accounted for 72%. Thus the contamination of mold fungi is not hindered identification dermatomitsetov.

Thus, we offer nutritional environment allows you to complete the task to get rapid growth dermatomitsetov, vladusic characteristic features (pigmentation, the sporulation), allowing their detection and identification in primary culture.

LITERATURE

1. Order of the Ministry of health of the USSR No. 535 of April 22, 1985, "On the unification of microbiologic methods used in clinical diagnostic laboratories medical institutions".

2. Semenov S.M. Laboratory environment for actinomycetes and fungi. The Handbook. - M.: Agropromizdat, 1990. - 240 S.

3. Sergeev, A., Sergeev, Y.V. Fungal infections. A guide for physicians. 2 ed. - M.: Publishing house of the BINOMIAL, 2008. - 480 S.

4. Sergeev, A. Fungal infections of the nails. 2 ed. - M.: national Academy of Mycology, 2007. - 164 S.

5. Methods experimental Mycology. Reference (Dudka I.A., Vaser S. p., Elanskaya I.A. and others). - Kiev.: Naukova Dumka, 1982. - 550 C.

6. Medical Mycology: management / V.A. Andreev, A.V. Zachinaeva, A. Moskalev, V.B. have been Konstantin; Ed. by V.B. have been Sboychakov. - M.: GEOTAR-Media, 2008. - 208 S.

Table 1
Formation of differential diagnostic elements in fungi dermatomitsetov under cultivation in the investigated environments.
StrainsGrowth of strains is of fungal on the environment Saburo, dayGrowth of the strains on the environment, the day
12345671234567
T. rubrumPPM And
T. rubrumPPM
T. rubrumPPM And
T. rubrumPP
T. rubrumP
T. rubrumP
T. rubrumPP
T. rubrumP
T. rubrumPPM
T. rubrumPPM And
T. mentagrophytesP, CPC
T. mentagrophytesPP, C
T. mentagrophytesPCM
T. mentagrophytesPC
T. mentagrophytes PCPC
T. mentagrophytesP, CP, C
T. mentagrophytesP, C
T. mentagrophytes PPC, M
T. mentagrophytesPC
T. mentagrophytesPC
P - education pigment M - microconidii And - arthroconidia, C - crimps or knots.

Nutrient medium for you is asiania filamentous fungi-dermatomitsetov from clinical material, containing in one liter of distilled water: glucose - 30,0 g bacteriological agar - 20,0 g, peptone meat - 5.0 g, casein hydrolysate - 2.5 g yeast extract, 0.8 g sodium chloride 0.4 g sodium carbonate is 0.16 g, L-cystine - 0.12 g, acid thioglycolate - 0.08 g, pH of the medium is 6.5.



 

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