RU2553549C1 - Lyophilised nutrient medium for visual detection of mycoplasma hominis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: urogenital mycoplasmosis is diagnosed by detecting Mycoplasma hominis in the clinical material, as well as for semi-quantitative determining the titre of the pathogen. The nutrient medium comprises PPLO broth, phenol red, brilliant blue, L-arginine hydrochloride, yeast extract, ceftriaxone, amoxiclav (sodium salt of amoxicillin/clavulanic acid potassium salt), vancomycin hydrochloride, clarithromycin, fluconazole and distilled water in a predetermined ratio of components.

EFFECT: invention enables to improve the accuracy and to reduce the time of detection of Mycoplasma hominis.

4 tbl, 3 ex

Description

The invention relates to medical microbiology, can be used for the cultivation and identification of urogenital mycoplasmas, in particular Mycoplasma hominis, for the diagnosis of urogenital mycoplasmoses by detecting Mycoplasma hominis in the clinical material, as well as for the semi-quantitative determination of the pathogen titer.

In recent years, there has been an increase in urogenital infections. Along with the causative agents of “classical” sexually transmitted infections (STIs), such as gonorrhea, syphilis, trichomoniasis, urogenital chlamydia, the proportion of diseases caused by opportunistic microorganisms, including urogenital mycoplasmas, increases.

According to the data of modern researchers, more than 40% of patients with inflammatory diseases of the pelvic organs have urogenital mycoplasmas, the three types of mycoplasmas being of greatest clinical importance: Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma urealyticum.

Mycoplasma hominis can cause a number of diseases of the urogenital tract: non-gonococcal urethritis, nonspecific vaginitis, prostatitis and epidermitis, the development of cervicitis and colpitis, including endometritis and salpingitis. Mycoplasma hominis can be an etiological factor in miscarriage, preterm birth, impaired reproductive function, cases of stillbirth. According to various studies, the frequency of colonization of urogenital mycoplasmas of the lower parts of the genitourinary system in children varies from 2.9 to 22% for Mycoplasma hominis. Thus, the need for a special examination of all pregnant women to identify cases of urogenital mycoplasmosis and subsequent rehabilitation is obvious.

To identify Mycoplasma hominis, various laboratory diagnostic methods are used. The most reliable and simple is the cultural method for the diagnosis of urogenital infections, including the detection of Mycoplasma hominis in a selective liquid nutrient medium. Using this method allows the identification and identification of Mycoplasma hominis, as well as a semi-quantitative assessment of the titer. The latter is especially important for the clinical assessment of the etiology of inflammatory processes, since for mycoplasma infection the etiological value is the concentration of Mycoplasma hominis in the test material at least 10,000 CFU / ml. The culture method can determine the sensitivity of the isolated cultures to antibiotics, which distinguishes the cultural method from other diagnostic methods for urogenital mycoplasmoses (polymerase chain reaction (PCR) and immunofluorescence (RIF)). The cultural method for diagnosing urogenital mycoplasmoses is more reliable in comparison with RIF, whose lower sensitivity and specificity is determined by the small linear dimensions of these microorganisms, expressed by their heterogeneity and variability, often encountered by a low concentration of mycoplasmas in the primary material and, accordingly, the need for their accumulation to identify, the difficulty of taking a quality scraping for this reaction.

From the patent literature known "nutrient medium for the allocation of mycoplasma" (see RF patent No. 1397481, 1988). To prepare the medium, add to the volume of distilled water, g: nutrient broth 19.21, erythritol 0.01, thiamine bromide 0.004, arginine 0.08, cystine 0.4, glucose 0.8, ECD 4.0, agar 8, 96, Na ₂ CO ₃ 0.64. The medium is thoroughly mixed and brought to a boil, then autoclaved. After cooling, 200 ml of horse serum and penicillin are added to the medium. At a sowing dose of 10 million / cell / ml, 120 colonies grow.

However, this nutrient medium is not lyophilized, which reduces its shelf life, and is also associated with strict adherence to the principles of the cold chain, which complicates its long-term storage, transportation and widespread use. The medium does not contain antimycotics that inhibit the growth of fungi, and the medium does not contain an antimicrobial drug that inhibits the growth of other mycoplasmas, and some representatives of the bacterial flora have acquired resistance to penicillin, which does not fully ensure the selectivity of the medium.

NUTRITIONAL MEDIA FOR CULTIVATION OF MYCOPLASMS is also known. To prepare the medium, a digestion of human blood clots (tryptic digestion) is used as a nutrient base. As a growth promoter, human blood serum of group 0 (1) is used, depleted in mechanisms of 10-20 wt.%. The composition of the medium includes, wt.%: 50% yeast extract 8-15; glucose 0.5-1; sodium chloride 0.5-1. All components are mixed and the volume of the medium is adjusted to 100 ml by digestion of blood clots. pH of the medium is 7.4-7.8. The growth of mycoplasmas on the 3-4th day (See RF patent No. 1527256, C12N 1/20, 1989).

This nutrient medium has similar disadvantages as in the medium described above (see RF patent No. 1397481, 1988).

Closest to the declared one is "NUTRIENT ENVIRONMENT FOR IDENTIFICATION OF MYCOPLASMA HOMINIS AND METHOD FOR DIAGNOSTICS OF UROGENITAL MYCOPLASMS". Nutrient medium for the detection of Mycoplasma hominis, as a nutrient base, it contains an 18-22 cardiac extract and tryptose of 8-12 g / l, yeast extract 3-4 and medium 199 4.7-5.2 g as growth stimulants / l, also contains arginine 5-10 g / l, phenol red 0.04-0.07 g / l, horse serum 0.25-0.30 g / l and distilled water. As selective additives, antibiotics are 0.64-1.27 g / l, with the following ratio of ingredients: ampicillin sodium salt 0.50-1.00 g / l, vancomycin hydrochloride 0.10-0.20 g / l, polymyxin B sulfate 0.01-0.02 g / l, amphotericin B 0.01-0.02 g / l, erythromycin 0.02-0.03 g / l (See patent RU No. 2261903, 2005).

This nutrient medium is not lyophilized, which reduces its shelf life, and is also associated with strict adherence to the principles of the cold chain, which complicates its long-term storage, transportation and widespread use.

A lyophilized selective nutrient medium was developed for the visual detection of Mycoplasma hominis, which possesses the necessary sensitivity and specificity. This nutrient medium makes it possible to identify the selected cultures of mycoplasmas and assess their quantity (titer) in the samples. The optimal proportions of the components of the selective nutrient medium for the detection of Mycoplasma hominis were obtained.

A selective culture medium for the detection of Mycoplasma hominis provides optimal conditions for the growth of this pathogen while suppressing the growth of other mycoplasmas, yeast-like fungi, and most representatives of the bacterial flora that are potentially contained in the test sample. The presence of a pH indicator in the medium allows visual assessment of the results of the study on the color change of the nutrient medium during the cultivation of Mycoplasma hominis due to the manifestation of enzymatic activity.

The nutrient medium for visual detection of Mycoplasma hominis contains a nutrient base, arginine, growth stimulants, selective components, horse serum, a pH indicator and distilled water. PPLO broth serves as a nutritional base, yeast extract serves as growth stimulants, antibiotics and an antifungal drug as selective additives, and phenol red and brilliant blue as a pH indicator.

Yeast extract is prepared from fresh baker's yeast (Du-Le LLC, Yast Balagoet, Sweden, cat. No. 00462, 2014). Yeast is placed in a pan in boiling distilled water at the rate of 250 g / l, boiled for 40 minutes. The resulting extract is clarified and sterilized by autoclaving.

The content of the nutrient medium has the following ratio of ingredients, g / l:

1. PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaCon, cat. No. 1262, 2014) 32.00-35.00 2. Phenol red (Neva Reagent, PANREAC-131615, 2014) 0.10-0.14 3. Diamond Blue (Neva Reagent, ACROS-22973, 2014) 0.04-0.08 4. L-arginine hydrochloride (imp, NTK "Diaem", cat. No. 1119-34-2, 2014) 18.0-22.0 5. Yeast extract 150.00-250.00 ml 6. Horse serum (BioloT LLC, code: 1.1.5., 2014) 150.00-250.00 ml 7. Selective additives 0.199-0.232 8. Distilled water rest

The nutrient medium contains selective additives - antibiotics and an antifungal drug, in the following ratio of ingredients, g / l:

9. Ceftriaxone 0.10-0.14 10. Amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) 0.025-0.035 11. Vancomycin hydrochloride 0.003-0.005 12. Clarithromycin 0.006-0.010 13. Fluconazole 0.038-0.042

The nutrient medium is poured into 100 ml 25 ml vials and freeze-dried. Before use, a vial with lyophilic growth medium to detect Mycoplasma hominis is dissolved in 50 ml of distilled water. The storage conditions of the nutrient medium, see table 1.

The proposed composition of the nutrient medium presents the optimal quantitative ratio of components, concentrations of antibiotics and indicator dye.

The technical result achieved is a simplification of the composition of the formulation, an increase in the speed and accuracy of diagnosis of mycoplasma infection by increasing the sensitivity and selectivity of the medium. In addition, the medium provides high growth properties, allowing for accounting for growth of M. hominis after 24 hours, a clear visual recording of the results. The latter is achieved through the use of a mixture of two dyes, one of which is pH-dependent. At pH 6.7-6.9 (the beginning of M. hominis growth), the color of the medium is purple, which is visually more distinguishable than the yellow or crimson colors of the medium in the prototype. High selectivity of the claimed medium is ensured through the use of an optimal set of antibacterial and antifungal drugs, such as fluconazole, which even in high concentrations does not inhibit the growth of M. Hominis, in contrast to amphotorecin B used in the prototype. The use of newer antibiotics of ceftriaxone, amoxiclav instead of ampicillin and polymyxin B provides high selectivity for most representatives of gram-positive and gram-negative bacterial flora, potentially contained in the test sample.

Table 2 presents a comparative analysis of the components of the claimed medium with the closest analogue. The table on one line shows the components that perform the same function. The analysis of the growth and selective properties of the claimed medium and the closest analogue are presented in table 3.

To prepare the basis of the medium in 400 ml of distilled water dissolve PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaKon, cat. No. 1262, 2014) - 32.00-35.00 g, L-arginine hydrochloride ( Imp, NTK Diaem, cat. No. 1119-34-2, 2014) - 18.0-22.0 g, phenol red (Neva Reaktiv, PANREAC-131615, 2014) - 0.10-0 , 14 g and brilliant blue (Neva Reagent, ACROS-22973, 2014) - 0.04-0.08 g. Bring the solution with distilled water to 700 ml, 600 ml or 500 ml, in accordance with each example below. The resulting medium base is sterilized by autoclaving.

To prepare the medium under sterile conditions, mix 700 ml, 600 ml or 500 ml of the basis of the nutrient medium with yeast extract - 150.00-250.00 ml, horse serum (BioloT LLC, code: 1.1.5., 2014) - 150.00-250.00 ml. The mixture is thoroughly mixed, then ceftriaxone - 0.10-0.14 g, amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) - 0.025-0.035 g, vancomycin hydrochloride - 0.003-0.005 g, fluconazole - 0.038-0.042 g, clarithromycin are added - 0.006-0.010 g. The pH was adjusted to 6.7-6.9 by the addition of 1N hydrochloric acid dropwise. The final volume of the nutrient medium is 1000 ml.

Example 1

The composition of the medium:

1. PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaCon, cat. No. 1262, 2014) 32.00 2. Phenol red (Neva Reagent, PANREAC-131615, 2014) 0.10 3. Diamond Blue (Neva Reagent, ACROS-22973, 2014) 0.04 4. L-arginine hydrochloride (imp, NTK "Diaem", cat. No. 1119-34-2, 2014) 18.0 5. Yeast extract 150.00 ml 6. Horse serum (BioloT LLC, code: 1.1.5., 2014) 150.00 ml 7. Ceftriaxone 0.10 8. Amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) 0,025 9. Vancomycin hydrochloride 0.003 10. Clarithromycin 0.006 11. Fluconazole 0,038 12. Distilled water rest

To prepare the basis of the medium in 400 ml of distilled water dissolve PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaKon, cat. No. 1262, 2014) - 32.00 g, L-arginine hydrochloride (imp, NTK " Diaem ", cat. No. 1119-34-2, 2014) - 18.0 g, phenol red (Neva Reaktiv, PANREAC-131615, 2014) - 0.10 g and brilliant blue (Neva Reaktiv, ACROS- 22973, 2014) - 0.04 g. Bring the solution with distilled water to 700 ml. The resulting medium base is sterilized by autoclaving.

To prepare the medium under sterile conditions, 700 ml of the basis of the nutrient medium is mixed with yeast extract - 150.00 ml, horse serum (BioloT LLC, code: 1.1.5., 2014) - 150.00 ml. The mixture is thoroughly mixed, then ceftriaxone - 0.10 g, amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) - 0.025 g, vancomycin hydrochloride - 0.003 g, fluconazole - 0.038 g, clarithromycin - 0.006 g are added. The pH is adjusted to 6.7- 6.9 by adding dropwise 1N hydrochloric acid. The final volume of the nutrient medium is 1000 ml.

Example 2 (optimal).

The composition of the medium:

1. PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaCon, cat. No. 1262, 2014) 34.00 2. Phenol red (Neva Reagent, PANREAC-131615, 2014) 0.12 3. Diamond Blue (Neva Reagent, ACROS-22973, 2014) 0.06 4. L-arginine hydrochloride (imp, NTK "Diaem", cat. No. 1119-34-2, 2014) 20,0 5. Yeast extract 200.00 ml 6. Horse serum (BioloT LLC, code: 1.1.5., 2014) 200.00 ml 7. Ceftriaxone 0.12 8. Amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) 0,032 9. Vancomycin hydrochloride 0.004 10. Clarithromycin 0.008 11. Fluconazole 0,040 12. Distilled water rest

To prepare the basis of the medium in 400 ml of distilled water, PPLO broth bf is dissolved (Pronadisa, Laboratorios CONDA, CJSC DiaKon, cat. No. 1262, 2014) - 34.00 g, L-arginine hydrochloride (imp, NTK " Diaem ", cat. No. 1119-34-2, 2014) - 20.0 g, phenol red (Neva Reaktiv, PANREAC-131615, 2014) - 0.12 g and brilliant blue (Neva Reaktiv, ACROS- 22973, 2014) - 0.06 g. Bring the solution with distilled water to 600 ml. The resulting medium base is sterilized by autoclaving.

To prepare the medium under sterile conditions, 60 ml of the basis of the nutrient medium is mixed with yeast extract - 200.00 ml, horse serum (BioloT LLC, code: 1.1.5., 2014) - 200.00 ml. The mixture is thoroughly mixed, then ceftriaxone - 0.12 g, amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) - 0.032 g, vancomycin hydrochloride - 0.004 g, fluconazole - 0.040 g, clarithromycin - 0.008 g are added. The pH is adjusted to 6.7- 6.9 by adding dropwise 1N hydrochloric acid.

The final volume of the nutrient medium is 1000 ml.

Example 3

The composition of the medium:

1. PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaCon, cat. No. 1262, 2014) 35.00 2. Phenol red (Neva Reagent, PANREAC-131615, 2014) 0.14 3. Diamond Blue (Neva Reagent, ACROS-22973, 2014) 0.08 4. L-arginine hydrochloride (imp, NTK "Diaem", cat. No. 1119-34-2, 2014) 22.0 5. Yeast extract 250.00 ml 6. Horse serum (BioloT LLC, code: 1.1.5., 2014) 250.00 ml 7. Ceftriaxone 0.14 8. Amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) 0,035 9. Vancomycin hydrochloride 0.005 10. Clarithromycin 0.010 11. Fluconazole 0,042 12. Distilled water rest

To prepare the basis of the medium in 400 ml of distilled water dissolve PPLO b / kf broth (Pronadisa, Laboratorios CONDA, CJSC DiaKon, cat. No. 1262, 2014) - 35.00 g, L-arginine hydrochloride (imp, NTK " Diaem ", cat. No. 1119-34-2, 2014) - 22.0 g, phenol red (Neva Reaktiv, PANREAC-131615, 2014) - 0.14 g and brilliant blue (Neva Reaktiv, ACROS- 22973, 2014) - 0.08 g. Bring the solution with distilled water to 500 ml. The resulting medium base is sterilized by autoclaving.

To prepare the medium under sterile conditions, 500 ml of the basis of the nutrient medium is mixed with yeast extract - 250.00 ml, horse serum (BioloT LLC, code: 1.1.5., 2014) - 250.00 ml. The mixture is thoroughly mixed, then ceftriaxone - 0.14 g, amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) - 0.035 g, vancomycin hydrochloride - 0.005 g, fluconazole - 0.042 g, clarithromycin - 0.010 g are added. The pH is adjusted to 6.7- 6.9 by adding dropwise 1N hydrochloric acid. The final volume of the nutrient medium is 1000 ml.

A selective nutrient medium should ensure the growth of Mycoplasma hominis, which is accompanied by a change in the color of the medium from green to violet due to the manifestation of enzymatic activity, and inhibit the growth of related microorganisms (gram-positive and gram-negative bacteria, fungi and other mycoplasmas).

Diagnosis of infections caused by Mycoplasma hominis using a nutrient medium for visual detection of Mycoplasma hominis includes introducing test material into microtest tubes containing culture medium, conducting two serial dilutions of the test sample in the medium in steps of 10, and assessing the color change of the culture medium in the tubes. As a nutrient medium use:

nutrient medium containing a nutrient base, arginine, growth stimulants, selective components, horse serum, a pH indicator and distilled water. The medium contains PPLO broth as a nutrient base, yeast extract as growth stimulants, antibiotics and an antifungal drug as selective additives, and phenol red and brilliant blue as a pH indicator.

The diagnostic method is simple to set up, does not require special equipment, allows both the identification of the pathogen and the semi-quantitative determination of its titer.

Diagnosis of infections caused by Mycoplasma hominis using a culture medium for the detection of Mycoplasma hominis includes:

1. In a lyophilized growth medium for the detection of Mycoplasma hominis add a double volume of distilled water and mix until completely dissolved (within 1 min).

2. The resulting transparent green medium is poured into 0.9 ml into micro-test tubes, closed and stored until use at a temperature of 2-8 ° C for no more than 7 days or at a temperature of minus 7 ° C and below no more than 2 months. Before analysis, the tubes with the medium are kept at room temperature (18-25 ° C) for 1 h.

3. Depending on the objectives of the study, further determination can be made in the variant of a qualitative or semi-quantitative analysis.

3.1. Conducting a qualitative analysis.

The test sample is introduced in a volume of 100 μl of the solution from the test tube with the sample in the transport medium into the test tube designated K (+++) and containing 0.9 ml of liquid nutrient medium for detection of Mycoplasma hominis.

3.2. Conducting a semi-quantitative analysis.

Two serial dilutions of the test sample are done in steps 10. To do this, shake the initial test tube with the sample, designated K (+++), and transfer 100 μl of the solution to another test tube, designated K (++) and containing 0.9 ml of nutrient environment for the detection of Mycoplasma hominis, which corresponds to a dilution of 10 times. Then, 100 μl of the solution was transferred from the K (++) tube to the tube designated K (+) and containing 0.9 ml of culture medium for detection of Mycoplasma hominis, which corresponds to a 100-fold dilution of the test sample.

4. Test tubes with test samples and one test tube without sample (control of the nutrient medium K (-)) are placed in a thermostat at a temperature of 37 ± 1 ° C.

5. The analysis of the results is carried out after 24 hours. The final analysis of the results is carried out after 72 hours. The growth of Mycoplasma hominis is accompanied by a change in the color of the nutrient medium from green to purple, without turbidity, due to the manifestation of enzymatic activity, hydrolysis of arginine and a pH shift. In the case of low contamination of the material, the color change of the medium occurs on the third day.

The interpretation of the analysis results, see table. 2.

5.1. Interpretation of qualitative analysis.

A positive result “+” is the appearance of a violet color in the test tube with the test sample K (+++) while maintaining the green (original) color in the control test tube K (-). The absence of a change in the color of the medium in the test sample K (+++) compared with the color of the medium in the control test tube K (-) is evaluated as a negative result “-”.

5.2. Interpretation of semiquantitative analysis.

The appearance of a violet color of the medium only in test tube K (+++) in the absence of changes in the color of the medium in test tubes K (++), K (+) and K (-) indicates that the titer of Mycoplasma hominis is not more than 10 ² colony forming units in ml (CFU / ml). The appearance of a violet color in two K (+++) and K (++) tubes in the absence of changes in the color of the medium in K (+) and K (-) tubes indicates that the titer of Mycoplasma hominis is not more than 10 ³ CFU / ml The appearance of a violet color of the medium in three tubes K (+++), K (++) and K (+) in the absence of a change in the color of the medium in the K (-) tube indicates that the titer of Mycoplasma hominis is not less than 10 ⁴ CFU / ml

The absence of a change in the color of the medium in three tubes with a sample of K (+++), K (++) and K (+) compared with the color of the medium in the control tube K (-) is considered a negative result “-”.

5.3. Note.

Turbidity of the medium during cultivation (with or without discoloration in test tubes with test samples) indicates the growth of extraneous microflora. The results of the study of such samples are not subject to accounting and require repeated analysis or additional seeding on a solid nutrient medium for morphological identification of colonies of Mycoplasma hominis.

As a transport medium using the medium in the following ratio of ingredients, g / l:

Placental broth 350.0-450.0 ml Enzymatic peptone (HiMedia Laboratorios Pvt. Limited, India, RM 1892, 2014) 7.0-9.0 Sodium chloride (chemical grade, Neva Reagent, GOST 4233-77, 2014) 3.5-4.5 Yeast extract 80.0-120.0 ml Horse serum (BioloT LLC, code: 1.1.5., 2014) 50.0-70.0 ml Ceftriaxone 0.05-0.07 Amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) 0.01-0.02 Vancomycin hydrochloride 0.0015-0.0025 Amphotericin B 0.004-0.006 Distilled water rest

For the diagnosis, the following clinical material is used:

- scraping of the cells of the vagina, cervical canal, urethral mucosa;

- secretion of the prostate gland (0.50-0.10 ml), ejaculate (0.50-0.10 ml);

- the first portion of morning urine (sediment obtained by centrifugation of 10 ml of the first portion of morning urine).

For reliable diagnosis of pathogens of urogenital mycoplasmosis, it is necessary to take a standard test material and observe its storage conditions:

1. The procedure for taking material should be standard. Samples should be taken using a Volkman spoon or a disposable swab (brush);

2. Do not use local antiseptics when taking material;

3. It is necessary to take material before antibiotic therapy;

4. It is important to obtain a sufficient number of cells, since mycoplasma is a microorganism that colonizes the cell surface;

5. Careful removal of mucus from the cervical canal is necessary;

6. Taking urethral samples should be carried out no earlier than 2-3 hours after urination;

7. Obtaining the secretion of the prostate gland and ejaculate immediately after urination;

8. The test material should be placed in a special transport medium that best ensures the preservation of the viability of mycoplasmas.

9. Close test tubes with samples in the transport medium, mark and deliver to the laboratory. The time and conditions of transportation of the studied samples, see table 1.

The sensitivity and specificity of this nutrient medium are standard values for the cultural diagnostic method.

Claims (1)

Nutrient medium for visual detection of Mycoplasma hominis , containing a nutrient base, arginine, growth stimulants, selective components, horse serum, a pH indicator and distilled water, characterized in that PPLO broth serves as a nutrient base, and yeast extract serves as growth stimulants, as selective additives - antibiotics and an antifungal drug, as a pH indicator - phenol red and brilliant blue, with the following ratio of ingredients, g / l:
PPLO broth 32.00-35.00 Phenol red 0.10-0.14 Brilliant blue 0.04-0.08 L-Arginine Hydrochloride 18.0-22.0 Yeast extract 150.00-250.00 ml Horse serum 150.00-250.00 ml Ceftriaxone 0.10-0.14 Amoxiclav (amoxicillin sodium salt / clavulanic acid potassium salt) 0.025-0.035 Vancomycin hydrochloride 0.003-0.005 Clarithromycin 0.006-0.010 Fluconazole 0.038-0.042 Distilled water rest