DD226010A1 - METHOD FOR FIXING MICROORGANISMS - Google Patents

Abstract

The invention relates to a method for fixing microorganisms. The goal is a low-loss fixation of microorganisms while maintaining the ability to live and germinate. The object of the invention is to achieve a high binding capacity while maintaining the Lebensfaehigkeit by using a new carrier material. The carrier material used according to the invention consists of cellulose and / or cellulose derivatives with 4,6-dichloro-1,3,5-triazine groups, of 2,4,6-trichloro-1,3,5-triazine, of halides, of cellulose and / or cellulose derivatives and optionally from buffer substances. The carrier material is used in the Hauptausfuehrungsform as Flaechentraeger with papieraehnlicher matrix. The invention is used in technical microbiology, molecular genetics and medical diagnostics.

Description

Field of application of the invention

The method serves to fix microorganisms to a new carrier system. It is used in technical microbiology, molecular genetics and medical diagnostics.

Characteristic of the known technical solutions

Fixation of DNA from lysed bacteria and viruses on nitrocellulose is known (see Schleicher and Schull, Prospectus 366).

The nucleic acid is fixed to the filters after lysis of the microorganisms.

There is a covalent bond of the nucleic acid. Nitrocellulose shows low mechanical stability, limited shelf life. Due to low carrier stability, the repeatability of the hybridization reaction is limited.

Fixation of viable microorganisms to a surface carrier has hitherto only been described by introducing gelable suspensions of these organisms onto surface carriers (eg paper / agarose). There is no covalent bond.

The number of fixable microorganisms is limited by the suspension capacity.

Desirable physical properties (such as filterability) of the surface carrier are impaired.

Microorganisms were applied to replicate or paddle on paper filters for replica plating procedures. There is no covalent bond and the microorganisms are relatively easy to wash out.

Object of the invention

The aim of the invention is a low-loss fixation of microorganisms while maintaining the vitality and germination.

Explanation of the essence of the invention

The object of the invention is to achieve a high binding capacity of microorganisms while maintaining viability by using a new carrier.

The object is achieved by a carrier composed of cellulose and / or cellulose derivatives with 4,6-dichloro-1,3,5-triazine groups in an amount of 1-80% by volume, from 2,4,6- Trichloro-1,3,5-triazine in an amount of 0.5-50 vol .-%, of chlorides in an amount up to 20VoI .-%, of cellulose and / or cellulose derivatives in an amount of 5-80 vol. -% and optionally from buffer substances up to 5 vol .-% consists. An advantageous design of the carrier is the surface form with paper-like matrix.

By virtue of this structure, the support material is mechanically extremely stable and thus well suited for storage: the fixation process does not affect the mechanical properties (e.g., filterability).

The following microorganisms can be bound to the carrier, for example:

- Bacteria such as Bacillus subtiiis, Clostridium, E.coli

- mushrooms like Saccharomyces

- Cells that can be treated like microorganisms (cell cultures).

The carrier used according to the invention exhibits an extraordinarily high binding capacity, whereby surprisingly the life and germinability of the objects are preserved.

Ausführungsbeispieie

The surface carrier was cut into 4 χ 4cm squares.

Per unit area 5μΙΙ buffer (0.1 M phosphate 0.9% NaCl) with various dilutions of Bacillus subtiiis (168M) were added dropwise in ordered order.

Dilution 1 = 100,000 germs

2 = 10 000 germs

3 = 1 000 germs

4 = 100 germs

5 = 10 germs

  6 = 1 germ

In this case, phosphate buffer of pH range 5-8 were tested. As a control, the analogous plots were made on plain filter paper.

After application, incubate 10-20 min at room temperature.

Thereafter, the carrier is placed with the dripping side down on a frit and washed with 10 ml of phosphate buffer pH 7.6.

The support is then placed with the Auftropfseite upwards in Petri dishes on agar and with 10 ml of soft agar at 42 ⁰ C overcoated (9cm petri dish, glucose-bouillon agar pH 7.2 ± 0.2). The incubation was carried out at 37 ° C in the incubator. After 36 hours, a colony growth at the place of dripping is evident.

The reference paper showed colony growth to dilution 3 only at pH 7.0, whereas the used substrate showed colony growth to pH 4 at pH 6.5. At pH 7.0, the area carrier shows a colony growth to dilution of 6. At pH 7.6, only the area carrier shows colony growth at dilution 1.

The results show that the surface support at pH 7.0 has complete binding at full viability. The bond is between 6.5-7.0 at least 1,000 times increased compared to comparison paper.

Claims (3)

-1 - ZbZ ZbU ö Invention claims: 1. A method for fixing microorganisms and cells, characterized in that a support material is used which consists of cellulose and / or cellulose derivatives with 4,6-dichloro-1,3,5-triazine groups in an amount of 1-80VoI. -%, from 2,4,6-trichloro-1,3,5-triazine in an amount of 0.5-50Vol .-%, of chlorides in an amount up to 20VoI .-%, of cellulose and / or cellulose derivatives in an amount of 5-80Vol .-% and optionally of buffer substances up to 5 VoI .-% consists. 2. The method according to item 1, characterized in that the carrier material has a paper-like matrix. 3. The method according to item 1 and 2, characterized in that the cellulose and / or the cellulose derivatives consist partially or completely ausfaserförmigen particles.