SU652216A1 - Nutritive medium for obtaining microorganism cultures - Google Patents

Description

, one . The invention relates to veterinary and medical microbiology, in particular to the preparation of a nutrient medium for the cultivation of anaerobic microorganisms in the manufacture of bio npehapatOB. It is known that anaerobic MikroortaniizIyI cultivated on nutrient media, made on the basis of open-hearth broth and Hottinger broth, enzymatic and acidic hydrolyzates of saa, casein and liver. The Clastridium perfringens type C and D microorganism in the manufacture of a full-fold hydroxyl alumina permafrost, infectious enterotoxemia, ovarian malignant edema and dysentery, as well as in the manufacture of alum concentrated anatoxins against ovine enterotoxemia, culture, ovine, and in the manufacture of alum concentrated anatoxins against infectious ovine enterotoxaemia, and in the manufacture of aluminous ointment toxins against infectious ovine enterotoxemia, and in the manufacture of alum concentrated anatoxins against ovarian infection of ovine, and in the manufacture of aluminous toxins against infectious ovine enterotoxemia, culture and ovarian tissue. (IPC) containing hydrolyzed meat, meat, casein and liver, taken in a ratio of 2: 2: 1 (h). 1 prepared alum concentrated toxoids on this nutrient medium cause the production of antitoxin in the serum of immunized sheep in titers of 0.10, 76 AU / ml in type C and 0.2-1.4 AU / ml in type D. The disadvantage of the known nutrient medium is 1ts the antigenic activity of toxoids produced on it is weak. In addition, most of the nutrient media used are prepared from top-grade skimmed meat. minced meat and liver, which need 8-15 kg per 100 liters of medium. Manufacturing techniques prevent them from seeing incomplete hydrolysis within 0, an hour. Or, water extracts - decoctions of components, the contents of which in different series of nutrient media prepared according to one recipe, differ sharply and largely depend on the quality of the raw materials. A disadvantage of existing nutrient media is the lack of visual control of the end of the process of cultivation of the culture. This is especially necessary during high temperature regimes of CI incubation, perfringens 40-42 C. Microscopic culture, activation of prototoxin and crude titration on white mice take up to 1 hour and 5-10 white Mijuieft for each determination. Such a method for determining the period of maximum accumulation of the toxin in the culture fluid often leads to re-grafting of the culture, decomposition of the toxin and loss of immunogenic properties, since the optimum time of planting out at the indicated mode is 4-8 hours.

The aim of the invention is a semi "1; 1; cheap, stable nutrient medium, upon cultivation on which it is possible to continuously obtain culture liquids with immunogenic properties, and to monitor the end of the culture growing process visually.

To achieve this goal, yeast water 190210 was introduced into the medium (g / l); peptone (dry) 3.5-5.5; Shieno 8.0-12.0; Wednesday Endo (dry) 1,5-2,5; Gypsy; natr {until pH 8.0-8.3} 1.0-1.5; hydrolyzed boiled minced meat and casein in equal ratios for amine nitrogen (250 mg%) to 1 liter. V-; :: ;; - - -; .-. / - /.

Casein and digested meat file hydrolates are FULLED by enzymatic hydrolysis for 510 days. Additionally, growing materials are introduced. Tyvae substances from drbzhzhy. The Endo medium contains whale or fish meal hydrolkeeta, lactose, sodium sulfate, agar-agar and fuchsin indicator, i.e. substances that contribute to good growth, reproduction and indications of microbes. A significant content of protein and amino-azyTysty substances creates buffering and stability during cultivation. .

Grow up C1. perfrlngens HD THIS nutrient medium allows you to visually monitor the incubation process, to consistently produce toxoids with immunogenic properties / 10-20 times higher than the activity data provided by the instructions. The consumption of white coos reduces, the cost of the nutrient medium decreases by 30%, the manufacturing process of the preparation is simplified.

PR RI er. To prepare the proposed nutrient medium, hydrolyzates are first prepared from casein and boiled minced meat (waste products after the meat pillow has been obtained). The hydrolysates are prepared separately for the following prescription: for 1 kg of dry ka.zein (GOST 1211-41) and 3 kg of boiled minced meat, 100-150 g of pancreatin (MPTU-.42 W5046-65) are added, 10 ml of demineralized water and 100-200 ml of chloroform. The contents of the horse are mixed, tightly closed (so that chloroform does not evaporate) and hydrolyze at 3740 C for 5-10 days, stirring occasionally 2-3 times a day for the first three days and once for the next. At the end of the hydrolysis at the bottom is formed

loose grayish sediment. Amine nitrogen 360-600 mg%, peptone G-2% in meat hydrolyzate of meat and 4-8% in casein hydrolyzate. The supernatant supernatant was decanted, filtered through a cotton-gauze filter, basic biochemical parameters were determined and used as needed. Hydrolysates are suitable for use during the year when stored in a hermetically sealed container at 10-15 ° C.

Yeast water is prepared on the eve or on the day of preparation of the nutrient medium: 20 g of dry baker's yeast is poured over 1 liter of water, boiled for 20 minutes, settled and filtered through a cotton-gauze, paper filter or filter plates F.

Drafting environment. Take 2 parts of casein and boiled hydrolysates. minced meat with amine nitrogen 56 mg% and add 1 h. yeast water. An LH of 8.2-8.35 (1030% alkali solution) is established, the amine nitrogen is determined, which should be 210-230 mg% and peptone (1.5, 5%). Dbvod to boil and poured into containers, depending on the volume of 0.5% dry enzymatic peptone, rpQT 13805-68 (if it is less than 3.5%), 0.2% dry (Hey, Endo and 0 , 8-1% of millet (about ikfcJTof j can also be sacks). The prepared medium is sterilized by autoclaving at 115-120 C for 35-45 minutes.

After sterilization, the medium acquires a crimson-red color and has the following characteristics:

mg%

420-570, 210-230

300-406 0.9-1.5 1.5-3.5.

60-80 g% 8.15-8.35

The prepared medium is cooled to 3740 s and sown with a matrix culture. Cultivation was carried out at 37-42 ° C until the medium began to discolor (a crimson-red to yellow transition). Growth is suspended by cooling and adjusting the pH.

The indicator of the medium, in the process of metabolism of the microorganism, contributes to the growth and division of cells, increasing the concentration of the bacterial mass by 2-2.5 times in comparison with the bacterial mass obtained on known nutrient media, and the immunogenic properties of the antigens manufactured on it are also enhanced.

The toxicity of the culture fluid tested on white mice with intravenous l administration is, on average, 4000 DLM / ml.

Toxoid activity was determined by the accumulation of a titer of epsilon antitoxin (AE / ml) in the serum of vaccinated sheep and rabbits. Sheep are vaccinated twice at an interval of 20 days at a dose of 5.0 ml, while after the first vaccination, the formation of antitoxin in serum was observed in a titer of 0.5-2.0 AU / ml, after re-vaccination 3.3-8.0 and more than AE / ml.

In RABBITS vaccinated at a dose of 2, O and 3.0 ml with an interval of 14 days, the average serum antitoxin titer after re-vaccination is an average of 2.1 AU / ml As control, toxoids made on well-known nutrient media are used. pancreatic meat preparations (PPM); 5 series of anatoxin type P in the amount of 39 liters were manufactured; 4 series in the amount of 26.5 liters were prepared on the nutrient medium IPC (M-hepatocellular casein).

Manufactured toxoids. On these nutrient media led to the production of epsilon antitoxin in the serum of vaccinated sheep by eating double vaccination at a dose of 5.0 ml, respectively, on average, 0.4 and 1.5 AU / ml; in rabbits, 0.2 and 1.7 AU / ml.

The advantage of the nutrient medium in comparison with the nutrient medium, nt Prepared on the basis of Hottin grager, currently used in the bioindustrial industry, is used for growing C1. perfringens type C and D in the manufacture of polyvalent hydroxyaluminum vaccine against bradzot, malignant edema; Infectious enterotoxemia in sheep and dysentery is adverse, tested in the manufacture of alumina

toxoids against infectious enterotoxemia of sheep, industrial reactor method.

The proposed nutrient medium for cultivating CI perfringens of type C and D is stable in production according to biochemical parameters as compared with the known nutrient media, increases the activity of the preparations made on it, allows visual control of the cultivation process (by changing the color of the medium), and simplifies the technology of manufacturing antigens.

Claims (1)

1. Urguev K.R. Veterinary medicine, 1974, p. 4b-47.