LV11869B - Culture medium for trichomonas vaginalis - Google Patents

Abstract

This culture medium is being used for cultivation of Trichomonas vaginalis, which basically contains egg powder after lysozyme extraction as a source of nitrogen, dextran, streptomycin, penicillin, serum of animals blood and distilled water. It also contains ascorbic acid, orotic acid and calcium lactate. This culture medium increases the speed of culture growth and it is technologically easy to produce.

Description

The invention relates to medicine, including medical microbiology and laboratory diagnostics, but specifically to gynecology, venerology and urology. The invention can be used for laboratory diagnosis of diseases caused by Trichomonas vaginalis.

Inflammatory diseases of the urinary tract and genital organs among many viruses and bacteria are also caused by a single-celled organism, Trichomonas vaginalis, which is pathogenic to humans and localizes in the urogenital organs. Various exogenous and endogenous factors, as well as virulence of the disease agent, contribute to the aggressiveness of the clinical course in macro-cancer. Trichomonas is found in up to 32% of women and 53% of men in patients with inflammatory diseases of the urinary tract and genital organs of the sexually transmitted diseases.

Trichomonas are optional anaerobic microorganisms that feed endosmatically by absorbing nutrients from the environment through the surface of the cell envelope. They multiply at 35-37 ° C at pH 5.5 - 7.5.

In clinical practice, it is important to know the relationship between Trichomonas vaginalis and the associated microflora in the genitourinary organs, such as gonococci, chlamydia, mycoplasma, ureaplasma, candidiasis, etc. Different combinations of microorganisms influence the clinical course of the disease, the choice of treatment method and the anti-epidemiological measures. Urogenital diseases such as microbial infections occur in 80-35% of cases. Trichomonas can also serve as a reservoir for other microorganisms, such as gonococci, which can live and reproduce.

In order to optimize the treatment of urogenital Trichomoniasis diseases, it is particularly important to use sensitive and specific laboratory diagnostic methods for urogenital irichomoniasis. The most specific and sufficiently sensitive method of laboratory diagnosis of urogenital trichoraoniasis is the cultivation of Trichomonas vaginalis in culture medium or culture diagnostics, which allows the isolation of pure culture of Trichomonas vaginalis by subsequent microscopy. Surface material of the mucous membrane of the urogenital tract and of the anus of the anus is used as examination material

-2pa »samples taken (1,2). The use of a special instrument, the tubular organ removal device (3), is recommended for clinical examination.

A culture is required to obtain a pure culture of Trichomonas vaginalis. Culture diagnosis can be achieved by seeding the test substance in a trichomonous selective medium (Feinberg's modification), in which the nitrogen source is the liver substrate. Dextrase, sodium chloride and agar are added to the medium as well as antibiotics (penicillin and streptomycin) which inhibit the growth of side-germs; This medium is a prototype of the invention (4).

Composition of one liter of prototype medium:

hepatic substrate dextrase sodium chloride g 5 g

6.5 g 1 g to 1000 ml agar distilled water

After autoclaving the medium (20 min at 0.5 atmospheres) and cooling to 50 ° C, 30 ml of horse blood serum, 1000 units of pencillin and 500 mcg of streptomycin per ml of medium are added before use.

This medium is widely used in the cultural diagnosis of urinary trichomoniasis. Growth of Trichomonas in culture indicates that Trichomonas vaginalis is a causative factor for the disease caused.

The object of the invention is to create a new medium which would allow to accelerate the growth of Trichomonas vaginalis culture and to simplify the technology of its preparation before use of the medium under laboratory conditions.

The stated objective is achieved by using a culture medium containing nitrogen source, dextrase, streptomycin, pencillin, animal blood serum and distilled water for growth of Trichomonas vaginalis, additionally containing ascorbic acid, citric acid, orotonic acid, lactic acid calcium, source uses egg powder after lysozyme extraction, in the following component ratios, g / 1:

egg powder after lysozyme extraction 13-15 ascorbic acid 0,5-0,7 citric acid 0,11-0,13

-3orotinic acid lactic acid calcium dextrase streptomycin pencillin amphoglucamine animal blood serum distilled water to pH

0.07 - 0.08 0.9 - 1.1 5.0 - 7.0 0.9 - 1.1

900,000 - 1,100,000 alone 39,000 - 41,000 alone

199-201 ml

- 1 liter

6.0 - 6.5

A new culture allows for accelerated growth of Trichomonas vaginalis culture.

Media: Prototype media and three variants of the new medium are placed under sterile conditions in 30 bacteriological tubes (5 ml each). Equal volumes of test material (0.5 ml of the culture medium of the frichomons) are placed in all tubes in the culture medium. The culture is grown at 34-35 ° C. The inocula are listed in culture sections 1-3, 5-7. and 9-11. around the clock.

Trichomon onset, growth rate, Trichomon mobility and morphology are noted and evaluated. Trichomon studies are performed under the microscope of the microscope in the dark field of vision.

Trichomon growth rate is evaluated by a scoring system:

Trichomonas per ml medium,

1 bail. - up to 200 thousands 2 bail. - up to 450 thousands, 3 bail. - up to 1700 000, 4 bail. - up to 3000 000, 5 bail. - above 3000 000.

Trichomon mobility assessed by 4+ system:

+ - does not move in the field of view of Trichomonas, only their pennants move;

+ - The body of the Trichomons and their scabs are actively moving;

+ - Trichomon puppets are actively moving and their bodies are moving;

+ - Trichomonas actively moves in and out of the microscope's field of view.

Trichomon morphology was evaluated microscopically. Using microscopy in the dark field of vision. Trichomonous forms were observed, their reproduction and movement characteristics were observed.

-4Trichomone growth in culture medium - prototype.

Evaluation. Trichomonas multiplied (positive result) on day 1, 5 media, second day on 10 media, third day on 9 media, fourth day on 2 media, fifth day on media 1, seventh day on media 1, day 1 and day 1, feed.

Trichomon growth rate:

bail. - 3, bail. - 7, bail. - 5, bail. - 10, bail. - 5.

Trichomon Mobility:

1+ to 2;

2+ - 7;

3+ - 12;

4+ - 9.

Trichomon morphology was typical.

Example 1 of a newly created medium.

The medium shall be prepared in the following proportions of its components, in grams per liter:

egg powder after lysozyme excretion - 13.0 ascorbic acid - 0.5 citric acid - 0.11 orotic acid - 0.07 calcium in lactic acid - 0.9 dextrase - 5.0 streptomycin - 0.9 pencillin - 900,000 alone amphoglucamine 39,000 units horse or bovine serum (ml) - 199 ml distilled water to - 1 liter

pH 6.0

Evaluation. Positive result on the first day in 28 media and on the second day in 2 media.

Trichomon growth rate:

bail. - 2;

2 bail. - 2; 3 bail. - 5; 4 bail. - 14; 5 bail. - 7.

Trichomon Mobility:

2+ to 5;

3+ - 17;

4+ - 8.

The morphology of Trichomons in the medium - prototype and in the newly created medium was typical.

Example 2 of a newly created medium.

The medium is prepared with the following ingredients, g / l:

egg powder after lysozyme excretion - 14.0 ascorbic acid - 0.6 citric acid - 0.12 orotonic acid - 0.075 lactic acid calcium - 1.0 dextrase - 6.0 streptomycin -1.0 pencillin - 1000 000 units.

amphoglucomine - 40,000 units.

horse or bovine serum - 200 ml distilled water to - 1 liter pH 6.3

Evaluation. First round positive results in 30 tubes. Growth intensity:

2 bail. - 3; 3 bail. - 4; 4 bail. - 15; 5 bail. - 8.

Mobility: 2+ - 1;

3+ to 20;

4+ - 9.

Morphology: typical.

6Examples of Newly Created Feeds 3.

The medium is prepared with the following ingredients, g / 1:

egg powder after lysozyme excretion ascorbic acid citric acid orotic acid lactic acid calcium dextrase streptomycin pencillin amphoglucamine horse or bovine serum distilled water to

- 15.0

- 0.7

- 0.13

- 0.08 - 1.1

- 7.0

- 1.1

1,100,000 alone 41,000 units

201 ml per liter ptt medium 6.5

Evaluation. In the first 24 hours positive results in 30 stobriaos.

Growth intensity:

bail. - 4;

bail. - 3;

bail. - 15;

bail. - 8.

Mobility:

2+ to 2;

3+ - 21;

4+ - 7.

Morphology: typical.

The newly developed culture medium for Trichomonas vaginalis improves the detection of infected patients:

Investigation of 100 patients with medium - 12 infected patients were detected in the prototype, and 17 infected patients with the newly created medium for the same group of patients.

The newly created medium allows for improved growth of Trichomonas vaginalis culture, which increases the positive result in the diagnosis of the disease.

In addition, the culture does not require autoclaving, which simplifies its preparation technology under laboratory conditions.

The medium is economical because it uses waste from lysozyme production.

LITERATURE USED

1. U.F. Haaustin Sexuell ubertragbare Kronkheitena - Gustav Fischer Verlag Jena, 1990.-S.257-263.

2. P.Vasariaš, A.Miltiuš - Dermatovenerology for Family Physicians - Riga: Zvaigzne, 1993- 4.p. 366 - 368.

3. A.Miltiash - Device for determining the content of tubular organs II Latvian patent for the invention EN 10905 B, int.Cl ^

A 61 B 10/00. Date of application: June 2, 1994, Date of publication of the patent: April 20, 1996.

4. E. Van Dyck, P.Piot, A. Meheus. Bench - Ievel laboratory manual for sexunally transmitted diseases - Geneva:

WH0 / VOT / 89, 443. - P.55-56.

Claims (1)

Culture medium for Trichomonas vagmalis, containing slate source, dextrase, sodium chloride, agar, streptomycin, pencillin, animal blood serum and distilled water, characterized in that it additionally contains ascorbic acid, citric acid, orotonic acid, lactic acid, source contains egg powder after lysozyme release in the following component ratios, g / 1: egg powder after lysozyme excretion 13-15 ascorbic acid citric acid orotic acid lactic acid calcium dextrase streptomycin pencillin amphoglucamine animal blood serum distilled water to Medium pH 6.0-6.5 0.5 - 0.7 0.11 - 0.13 0.07 - 0.08 0.9 - 1.1 5.0 - 7.0 0.9 - 1.1 900,000 - 1,100,000 alone 39,000 - 41,000 alone 199 - 201 ml - per liter