CS248402B1 - Method for determining microbial contamination - Google Patents

Abstract

The procedure for the rapid determination of microbial contamination in solutions, atmosphere or on solids is based on the cultivation of the determined germs of the examined samples in a nutrient medium containing, in addition to non-radioactive oligosaccharides and/or polysaccharides and other non-radioactive substances, only monosaccharides labeled with carbon 14 with a specific activity higher than 1.8 TBq/M and the subsequent absorption of the resulting carbon dioxide labeled with -*-4C in an alkaline solution, for example 0.1 M sodium hydroxide, the residual radioactivity of which is measured by a radiometric method, for example liquid scintillation. The usability of the culture medium is indicated by the color of the solution, which changes when the storage period is exceeded. The method can be used in the field of medicine, especially radiopharmaceuticals, in the food industry, in environmental control and in clinical practice, when there is a requirement for rapid and quantitative determination of microbial contamination.

Description

The invention relates to a method for the determination of microbial germs in solutions, the atmosphere and / or on solid surfaces, which can be used in particular in the field of the manufacture of pharmaceuticals, in particular radiopharmaceuticals.

According to the first known method, the detection of microbial germs is carried out by adding the sample to the culture medium and culturing the detected germs under optimized conditions until the germs present have multiplied to the extent that the culture medium becomes cloudy. This procedure requires a test lasting about 14 days, with turbidity being assessed subjectively.

According to a second known method, the nutrient medium is radiolabeled and the microbial germs are detected by radiolabeled metabolic products, the culture requiring a period of several days and the volatile radioactive metabolism products that pass into the gas phase above the culture are transferred to the ionization chamber by carrier gas. which is measured with an electrometer.

The disadvantage of the first procedure lies mainly in the long cultivation time required for sufficient biomass for visual indication and subjective evaluation, so the procedure is poorly sensitive and cannot be applied to turbid samples or samples that form turbidity with the culture medium.

The disadvantage of the latter lies in the relatively long cultivation time, the relatively low sensitivity and the fact that special apparatus is required to measure the radioactivity of gaseous metabolic products serving as an indicator of the presence of microbial germs.

The aforementioned disadvantages are eliminated by the method of determining the microbial contamination according to the invention, which is based on the fact that the sample is cultivated in a culture medium containing besides non-radioactive oligosaccharides and / or polysaccharides and other necessary components of the culture medium. up to 12 TBq / M and the resulting labeled carbon dioxide is absorbed in an alkaline solution, e.g. 0.1 M sodium hydroxide, whose total radioactivity is measured by a radiometric method, e.g. liquid scintillation.

The advantages of this method are extreme sensitivity} *

- 2 24b 4U2 where the cultivation time can be significantly shortened and the evidence of microbial contamination is rapid, further that a conventional measuring device can be used, that the kit and its behavior towards the culture medium do not affect the result and that the results are obtained in a simple and safe way objective record and can be quantified.

The following is an example of a non-limiting application.

Example 1

To the sugar-free culture solution, a 14-labeled monosaccharide having a specific activity of 1.8 to 12 TBq / M, e.g., Ul / c / ribose, is added and the required carbohydrate level is supplemented with a suitable oligosaccharide or polysaccharide such as sucrose and protein. a component such as casein peptone, nutrient salts, vitamin components and a dye serves as a pH indicator such as bromocresol purple and finally the test sample is added, the culture is sealed, the medium is cultured at room temperature for less than 24 hours and then carbon dioxide of high specific activity is eluted by means of a carrier gas stream and introduced into a container containing an alkaline solution, such as an ethanol amine solution, in which it is absorbed, and after addition of scintillation solution is measured as radioactivity using a conventional liquid scintillation counter.

E.g. the culture medium of the composition given in Example 2 is prepared by dissolving the substrates in distilled water and sterilizing the solution in the usual manner.

- 3 Example 2

Substance casein peptone sacfearosa yeast extract sodium chloride sodium citrate medium potassium phosphate tween magnesium sulfate manganese chloride ferrous sulfate vitamin ribose (specific activity 1,8-12 TBq / mol) bromocresol purple

248 402

Concentration 9 g / l g / l

7.5 g / l 5 g / l

2.5 g / l

1.0 g / l 0.2 g / l 1.1 g / l 0.1 g / l 0.01 g / l 5 mg / l

KBq / L 1 mg / L

The pH is 7.2

The blue-colored sterilized broth is airtight and can be stored for at least half a year. To detect microbial plots, aseptically a 10 ml culture medium is aseptically filled into a sterile 25 ml culture vial to which a liquid or solid sample is added, the vial is sealed airtight and cultured with shaking at 37 ° C. A 2 ml solution of 1M ethanolamine in methylcellosolv is then placed in a second vial, which is shaped to serve as a scintillation cuvette. By means of a suitable system consisting of two injection capillaries, which are passed through the piercing plug of the culture vial, a gas flow of 1.5 l / h is slowly passed. e.g., compressed air, the space above the culture medium into an ethanolamine solution.

After 5 minutes, the introduction of the gas into the ethanolamine is stopped, added

- 4,248,402 with 8 ml dioxane scintillation cocktail and measure the radioactivity. Usually, less than 3 Bq is measured in seed-free samples. Doubling of this value or the value of the larger induces microbial contamination.

This procedure can also be used in the food industry, environmental control and clinical practice when there is a need for rapid and quantitative determination of microbial contamination.

Claims (1)

Method for the determination of microbial contamination in solutions, atmosphere or solids _f, characterized in that the test sample is cultivated in a culture medium containing, in addition to non-radioactive oligosaccharides and / or polysaccharides and other necessary nutrient components, only 14-labeled monosaccharides 12 TBq / M and the resulting labeled carbon dioxide is absorbed in an alkaline solution, e.g. 0.1 M sodium hydroxide, whose total radioactivity is measured by a radiometric method, eg liquid scintillation counting.