RU1781300C - Method for bacteriological diagnosis of infections ecterotoxemia in animals - Google Patents

Abstract

Usage: veterinary microbiologists. The inventive method of bacteriological diagnosis, infectious enterotoxemia of animals consists in sowing the test material in a liquid nutrient medium of the following composition: vitamin preparation of feed yeast extract 4.5-5.0 g, glucose, 40% solution of 11-13 ml, sodium sulfate 1.0-1.2 g, hemin 0.004-0.005 g, sodium chloride 4.5-5.0 g, gentamicin 0.0075-0.008 g, erythromycin 0.00004-0.000045 g, enzymatic casein hydrolyzate with amine content - Nitrogen 90-100 mg% to 1 l, in the incubation of crops, in reseeding on a solid nutrient medium, containing for example: vitamin preparation of fodder yeast extract 4.5-5.0 g glucose, 40% solution 20.0-25.0 ml, sodium sulfate 1.0-1.2 g, hemin 0.004-0.005 g, sodium chloride 4.5-5.0 g. triphenyltetrazole chloride 0.025-0.03 g, agar 13.0-15.0 g, casein enzymatic hydrolyzate with amine nitrogen content of 170-190 mg% to 1 l, followed by incubation of crops and taking into account the results of the growth of microorganisms. 6 tab. (L C

Description

The invention relates to veterinary microbiology and can be used to increase the effectiveness of timely diagnosis of infectious enterotoxemia in animals and the organization of antiepizootic measures.

Known methods for the diagnosis of infectious enterotoxemia of animals with the release of the causative agent of infection - Cl. perfringens.

In this case, Kitt-Tarozzi, glucose-blood MPA, etc. were used as nutrient media for the cultivation of the infectious agent.

isolating the pathogen requires nutrient media for the manufacture of meat products, animal blood, labor-consuming to manufacture, non-standard in quality composition and do not provide for inhibition of extraneous microflora.

A known method, the diagnosis of which is based on the detection of toxin Cl. perfringens in the test material from sick and dead animals using a bioassay on white mice or isolation of a pathogen culture, followed by a study of its toxigenicity. The implementation of this technique is largely

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It is limited by its laboriousness (multiple transfers) and the use of expensive nutrient media that do not have selective and differentiating properties.

An object of the invention is to provide a method for accelerating the isolation of an infectious enterotoxemia pathogen culture using engineered culture media having selective and differentiating properties.

The goal is achieved in that the selection of the culture of Cl. Perfringens from the test material (feces, intestinal contents, feed, environmental objects) are carried out by plating casein-based accumulations containing glucose, feed yeast extract (ECD), hemin, sodium sulfite, growth inhibitors of concomitant microflora on a liquid nutrient medium - gentamicin and erythromycin in the following ratio of components, g / l:

Vitamin preparation

ECD 4.5-5.0

Glucose,

40% solution 11-13 ml

Sodium sulfite 1.0-1.2

Gemin 0.004-0.005

Sodium

. chloride 4.5-5.0

Gentamicin 0.0075-0.008

Erythromycin 0.00004-0.000045

Enzymatic

casein hydrolyzate (amine nitrogen

90-100 mg%) Up to 1l

Subsequently, the isolation and differentiation of the pure culture of Cl. Perfrin-gene is carried out by reseeding on a solid diagnostic medium containing EKD, glucose, sodium sulfite, hemin, sodium chloride, 2,3,5-triphenyltetrazole chloride (TTX) and agar in the following ratio of components, g / l: Vitamin

drug ECD 4.5-5.0

Glucose (40% solution) 20-25 ml

Sodium sulfite 1-1.2

Gemin 0.004-0.005

Sodium chloride 4.5-5.0

TTX0.025-0.030

Agar 13-15

Enzymatic

casein hydrolyzate (amine nitrogen

170-190 mg%) Up to 1 L The method is carried out as follows.

The basis of casein culture media for the isolation of Cl. perfringens is an enzymatic casein hydrolyzate of medium cleavage (amine nitrogen 450-500 mg%).

Food casein (OST 4970-74 and others) is used to make casein enzymatic hydrolyzate. For accelerated hydrolysis, 1 kg casein is crushed using a RT-1 tissue grinder or some other method and 10 l of distilled water is poured. The mixture is alkalinized with a 20% sodium hydroxide solution (GOST 4320-77) to a pH of 8.0-8.2, heated to 80-85 ° C and, with constant stirring, maintaining the temperature and pH in these parameters, casein is adjusted to complete dissolution. After this, the mixture is cooled to 45-47 ° C and 1 kg of twice minced meat of the pancreas of cattle or pigs is introduced (GOST 11285-73). The mixture is adjusted to pH 8.0-8.2 with a 20% sodium hydroxide solution and canned with chloroform (GOST 20015-74) in an amount of 1.0-1.5% to the total volume. Casein hydrolysis is carried out at 37-42 ° C for 16-20 hours until an amine nitrogen concentration of 450-500 mg% is reached. After this, the hydrolysis is stopped by acidification with glacial acetic acid (GOST 61-75) to pH 4.5-5.0 and subsequent boiling for 10 minutes. Then, the hydrolyzate is left for 2-3 days at 8-10 ° C for settling. After this period, the clear casein hydrolyzate is drained, made basic with a 20% sodium hydroxide solution to pH 7.0-7.2, filtered through a cotton-gauze filter and sterilized at 120 ° C for 30 minutes. Casein sterile hydrolyzate is stored at 8-10 ° C and used for 5-6 months.

Indicators of casein hydrolyzate: amine nitrogen 450-500 mg%, peptone 2-3%, tryptophan 180-200 mg%.

The manufacture of casein storage media.

In order to prepare a growth media, the basic casein hydrolyzate (OGK) is diluted with distilled water to an amine nitrogen concentration of 90-100 mg%. The required amount of OGK is determined by the formula:

.

where X is the required amount of OGK for preparing the nutrient medium (l);

A is the required amount of amine nitrogen in the nutrient medium (mg%);

B — concentration of amine nitrogen in OGK (mg%);

K is the amount of culture medium that needs to be prepared (l),

 Diluted to amine nitrogen 90-100 mg% casein hydrolyzate is heated to boiling, dry extract of fodder yeast is added at the rate of 5 g / l of medium, sodium sulfite (GOST 195-77) - 1 g / l, hemin GGU-09-10-10- 504-7) - 0.005 g / l and sodium chloride (GOST 4233-77) - 5 g / l.

A hemin solution in water is prepared separately, for which a 10 mg sample of the preparation is previously dissolved in a flask with a capacity of 50-100 ml in 1 ml of a sterile 1-normal sodium hydroxide solution and 19 ml of sterile distilled water is added. A hemin solution was added to the culture medium at the rate of 10 ml / l. The sterilized boiling solution of hemin is suitable for storage and subsequent use.

After all components are dissolved with a 20% sodium hydroxide solution, the pH of the medium is adjusted to 7.6-7.8, boiled for 10 minutes, filtered through a cotton-gauze filter, poured into 0.250 or 0.5 L flasks and sterilized with 120 ° C for 30 minutes.

The nutrient medium is transparent, has a reddish tint and a pH of 7.4-7.6, and the concentration of amine nitrogen is 100-110 mg%. The medium, when stored under conditions of 4-8 ° C, is suitable for use in work for 3-4 months.

Before use, glucose (GOST 975-75) is added to a nutrient medium to accelerate the growth of C. perfringens in the form of a 40% sterile solution at the rate of 12.5 ml (0.5% in terms of dry matter) and to inhibit extraneous microflora - gentamicin antibiotics at the rate of 0.008 g / l (8 mg) and erythromycin - 0.000045 g / l (45 μg), after which the medium is poured into sterile 10 ml tubes and layered sterile vaseline oil. The prepared medium is stored at 4-8 ° C and used for 2 months.

The calculation of the required amount of gentamicin from the need of 0.008 g / l is carried out as follows.

Gentamicin in ampoules in the form of a 4% solution:

1 ml of the drug contains 0.04 g of active substance

X ml - 0.008 g,

Y 0.008 1 p, X W ° 2 ML Consequently, 0.2 ml of the drug must be taken per 1 liter of culture medium.

Gentamicin powder in vials containing 0.08 g (80 mg) of active substance:

the contents of the vial are dissolved in 2 ml of sterile distilled water. Therefore, 0.04 g of active substance is also contained in 1 ml of the solution. Subsequently, the calculation is performed as described above. An aqueous solution of gentamicin can be used in the work for 14 days.

Erythromycin is convenient and appropriate to use in the form of disks containing 0.000015 g (15 μg) of active substance. Calculation of the required amount of erythromycin with the need of 0.000045 g / l (45 μg):

1 erythromycin disk contains 0.000015 g of substance X0.000045 g

At 0.000045 1, A 0.000015

Thus, for 1 liter of culture medium, you must take an eluate of 3 disks or directly apply sterile 3 disks.

If erythromycin (erythromycin ascorbate or erythromycin phosphate) is in powder in vials containing 0.05 g of the drug, then the calculation for the same need is as follows.

The contents of the vial are dissolved in 5 ml of sterile distilled water. 0.1 ml of this solution (0.001 g) was taken and transferred into a test tube with 4.9 ml of sterile distilled water. Thus, in 1 ml of solution in a test tube contains 0.0002 g of substance.

1 ml contains 0.0002 g in X ml - - 0.000045 g

40 y 0.000045 0.0002

1

 0.225 ml.

Therefore, per 1 liter of medium, 0.23 ml of an erythromycin solution should be added from a test tube. (An aqueous erythromycin solution is unsuitable for storage).

The final structure of the accumulation medium, g / l:

Vitamin

drug ECD 4.5-5.0

Glucose, 40%

solution 11-1 Zml

Gemin 0.004-0.005

Sodium sulfite 1-1.2.

Sodium chloride 4.5-5.0

Gentamicin 0.0075-0.008

Erythromycin 0.00004-0.000045

Enzymatic

casein hydrolyzate

(amine nitrogen 90-100mg%) Up to 1l

Production of a dense diagnostic environment.

In order to make a solid diagnostic medium, the main casein hydrolyzate is diluted with water to an amine nitrogen concentration of 170-190 mg% according to the formula given above. The diluted hydrolyzate is heated to boiling, agar is added sequentially per 1 liter of medium (GOST 16280-70} - 15 g, sodium sulfite - 1 g, hemin solution - 10 ml (for the preparation of hemin solution see above), sodium chloride - 5 g. After dissolving all the components and adjusting the pH of the medium with a 20% sodium hydroxide solution to 7.6-7.8, boil for 10 minutes, filter through a cotton gauze filter and sterilize at 120 ° C for 30 minutes.

The medium has a reddish tint, pH 7.4-7.6, amine nitrogen concentration 190-210 mg%.

Before pouring into Petri dishes, glucose from a 40% solution in an amount of 25 ml / l (1% in terms of dry matter) and a solution of triphenyltetrazole chloride (TTX) from sterile are added to molten and cooled to 50-60 ° C nutrient agar. calculation of 5 ml / l (0.025 g / l in terms of dry matter).

A TTX solution was prepared separately, for which a weighed portion of the drug in 0.05 g was dissolved in 10 ml of sterile distilled water (or 0.02 g in 4 ml, depending on need), brought to a boil. The TTX solution is suitable for use within 7-10 days.

The final composition of the dense diagnostic medium is as follows, g / l:

Vitamin preparation

ECD 4.5-5.0

Glucose, 40%

solution 20-25 ml

Gemin 0.004-0.005

Sodium sulfite 1-1.2

Sodium chloride 4.5-5.0

TTX, 0.5%

solution 4-5 ml

Agar 13-15

Enzymatic

casein hydrolyzate

(amine nitrogen

170-190 mg%) Up to 1l

EXAMPLE 1. Evaluation of the effectiveness of various casein-based constructs for the growth of C. perfringens.

Inoculum culture C. perfringens represented a suspension of an agar 18-20 hour culture at a concentration of 1 billion cubic meters / ml, which was added to test tubes with media at a rate of 1 ml / 10 ml. Crops

cultured at 37 ° C. The culture growth of C. perfringens was evaluated in units of optical density using FEK-M. As a result, maximum performance

Growth C. perfringens were established in a medium containing glucose, EKD, hemin and sodium sulfite (Table 1).

PRI me R 2. The speed and nature of the growth of microbial cells Kl.perfringens.

0 In a comparative study of the speed and nature of growth m.k. Cl. perfringens reference strains used 17-18-hour agar cultures, which were initially bred to 10 units. according to the optical standard of turbidity, then a 10 dilution was prepared from them followed by reseeding at the rate of 0.5 ml / 10 ml into the experimental and control accumulation media.

The media were evaluated by time according to the influence of visually noticeable growth and its typical manifestation (Table 2). In casein medium, visually noticeable growth was established after 3 hours, in the control medium - 1 hour later. 5.5 hours after seeding, intensive growth was recorded in the experimental medium (severe turbidity with rapid gas formation), in the control - growth signs were less pronounced (slight turbidity and lack of gas formation).

Example 3. Sensitivity of liquid casein medium for microbial cells. Cl. Perfringens.

A 17-18-hour agar of the reference culture strain of C. perfringens type C 392 was used, which was initially diluted to 10 units. according to the optical turbidity standard, then ten times serial dilutions from

0 10 to. From each dilution, inoculation into test tubes with media at the rate of 1 ml / 10 ml was made. Crops were incubated at 37 ° С in microaerostats for 20 h. Media efficiency was assessed by the effect of growth with appropriate dilution (Table 3).

On casein medium, C. peofringens growth was recorded when plating from dilution 10; control growth showed

0 when sowing from a 1CG6 dilution.

PRI me R 4. Sensitivity of dense media for C. perfringens.

In this case, both 16-18-hour agar and 6-hour broth were used

5 cultures Cl. Perfringens C 392, of which 10 dilutions. , and 10 did inoculation of 0.05 ml on Petri dishes with an experimental dense diagnostic medium and a prototype medium (Zeissler agar). Cups were incubated in microaerostats

at 37 ° C for 17-18 hours. The environment was evaluated by the number and size of colonies (table.

4).

Experienced dense diagnostic environment, not inferior to the environment - the prototype in sensitivity, exceeded it in the size of the colonies.

Example 5. Efficiency of a scheme for isolating Cl. Perfringens from a material.

When isolating Cl. Perfringens culture, the initial material was feces and the contents of the small intestine of animals. A sample of feces in 0.8-1 g was suspended in 10 ml of physiological sodium chloride solution containing 0.05% sodium thioglycolate. Suspensions were thoroughly mixed and kept for 30-35 minutes at room temperature to sediment coarse particles, after which they were sown abundantly in experimental casein medium and prototype medium. Crops were incubated for 16-17 hours at 37 ° C. Then, the cultures were microscopically scattered and copious plated onto dense diagnostic casein agar and Zeissler agar (prototype) in Petri dishes. Crops were thermostated in microaerostats for 16-17 hours at 37 ° C. The growth and differentiating properties of the media were evaluated visually and using a MBS-9 microscope at a 12-fold increase.

When 22 samples of material were sown, 19 Cl. Perfringens cultures were isolated in the casein accumulation medium, and Kitta-Tarozzi-9 was isolated on Wednesday (Table 6).

PRI me R 6. Assessment of the growth and differentiating properties of dense media (table. 5).

When replanting cultures from a casein accumulation medium both on a dense casein diagnostic and on a prototype medium, abundant growth of homogeneous colonies corresponding to C. perfringens characteristics was established. When replanting from Kitta-Tarozzi medium in 2 cases out of 3, a small number of Cl. Perfringens colonies were noted against the background of the abundant growth of colonies of concomitant intestinal microflora. Colonies of C. perfringens in the experimental dense diagnostic medium sharply differed from the colonies of other representatives of the intestinal microflora due to their inherent attributes in this medium (shape, size, color, surface and edge character, relief, structure, texture, inclusions).

Isolation of Cl. Perfringens culture.

Isolation of Cl. Perfringens culture from feces and contents of the small intestine.

A sample of feces 0.8-1 g is placed in a physiological solution of sodium chloride with 0.05% (0.5 g / l) sodium thioglycolate (TU 6-09-08-837-74) with a ratio of 1:10 . The suspension is thoroughly mixed and kept at room temperature for 30-35 minutes to precipitate coarse particles. After that, a plentiful inoculation (at least 0.5 ml) is made from the upper liquid layer into a test tube with accumulation medium. Crops are incubated at 36-37 ° C for 16-17 hours, microscopic, and if large polymorphic gram-positive rods with rounded ends are detected, a fractional culture is made on casein diagnostic agar in Petri dishes. Cups with crops are incubated in microaerostats at 36-37 ° C for 16-17 hours. Inspection and selection of Cl. Perfringens colonies is carried out under a microscope MBS-9 (or similar) at 12-fold magnification.

Morphological characteristic of colonies Cl. Perfringens: Size 1-2 mm

Formally oval or

wrong

The edges are flat or wavy.

The surface is smooth with a slight sheen.

Convex, often conical

Opaque

The consistency is pasty, the peel is easily removed

loop

Unevenly brown or reddish brown

Inclusions in the form of red crystals over the entire surface of the colony; on the periphery, there may be less

Isolation of Cl. Perfringens from organs of animal tissue.

Pieces of parenchymal organs (liver, kidneys, spleen) weighing 1-1.5 g are ground in sterile conditions in porcelain mortars, a physiological solution of sodium chloride in a ratio of 1:10 is added. The suspension is thoroughly mixed, sedimented for 20-30 minutes, and from the top layer, plentiful inoculation (at least 0.5 ml) is carried out in a test tube with accumulation medium. The sequence of further work is described above.

Isolation of Cl. Perfringens from peritoneal fluid.

Sterile peritoneal fluid in a volume of not less than 0.5 ml is introduced into a test tube with an accumulation medium. The sequence of further work is given above.

Isolation of Cl. Perfringens from feed.

A portion of feeds of 4-5 g in 0.250 or 0.5 L flasks is poured into 50 ml of physiological sodium chloride solution with 0.05 sodium thioglycolate, shaken thoroughly, kept at room temperature for 20-30 minutes and from the upper liquid layer, make T abundant inoculation into test tube with accumulation medium. Further work is carried out as described above. Instead of sodium thioglycolate, cysteine hydrochloride in an amount of 0.1 g / l can be used.

Thus, the proposed method for the accelerated bacteriological diagnosis of infectious enterotoxemia in animals compared with the prototype has the following advantages: the designed nutrient media have stable selective and differentiating properties, do not require expensive meat products for manufacturing, are standard in quality, and use inhibitors for extraneous microflora, exclude repeated reseeding during isolation and thereby increase labor productivity ists.

The technical and economic effectiveness of the invention consists in increasing the efficiency, reliability and availability of a method for diagnosing infectious enterotoxemia in animals.

Claims (1)

The claims A method for the bacteriological diagnosis of infectious ziterotoxemia of animals, including sowing the test material in a liquid nutrient medium, incubating the crops, followed by reseeding on a solid nutrient medium containing a nitrogen source, chloride 4,5-5,0 11-13 1.0-1.2 0.004-0.005 4.5-5 O o o075-0.008 0.00004-0.000045 Up to 1l triium, glucose, agar, and incubation, followed by the results of the growth of microorganisms, characterized in that, in order to increase the accuracy and speed up the method, the test material is seeded in a liquid medium of the following composition, g / l: Vitamin preparation feed extract yeast Glucose, 40% solution, ml Sodium sulfite Gemin Sodium Chloride Gentamicin Erythromycin Enzymatic casein hydrolyzate with amine nitrogen 90-100 mg% and reseeding is carried out on a solid medium, additionally containing vitamin extract of fodder yeast, sodium sulfite, hemin, triphenyltetrazolium chloride, and as a source of nitrogen, enzymatic casein hydrolyzate with an amine nitrogen content of 170-190 mg% in the following ratio of components, g / l: Vitamin preparation feed extract yeast Glucose, 40% solution, ml Sodium sulfite Gemin Sodium Chloride Triphenyltetrazoli chloride Agar Enzymatic casein hydrolyzate with content amic nitrogen 170-190 mg% Up to 1l Table 1 4,5-5,0 20.0-25.0 1.0-1.2 0.004-0.005 4.5-5.0 0.025-0.03 13.0-15.0 table 2 Table 3 Table 4 Table 5 Note. In the numerator - indicators of the experimental environment. In the denominator cpefla-npQTomrv Editor Compiled by L. Koltsova Tehred M. Morgenthal Corrector O. Yurkovetska Continuation of the table. 5 Table