CS268012B1 - Method of preserving luminescent bacteria by lyophilization - Google Patents

Abstract

Preservation of luminescent bacteria involves lyophilization in a cryoprotective medium with a lower ionic strength (1% NaCl) containing 15% lactose and 5% soluble starch. The dehydrated cultures are stored in an evacuated state in the cold and reconstituted by adding the original volume of 2% NaCl before use.

Description

The invention relates to a method for the concentration of luminescent bacteria by lyophilization for the purposes of a microbial toxicity test or long-term storage in collections.

Some marine bacteria (eg from the genera Photobacterium and Vibrip) are characterized by intense bioluminescence, ie the production of visible light. Various toxic substances interfere with bioluminescence, eg heavy metal ions, pesticides, organic solvents, synthetic fertilizers, etc., which can penetrate into the cells of luminescent bacteria from the external environment. From the changes of the bioluminescence intensity, it is possible to infer the toxicity of unknown samples, or to determine the concentration of known toxicants. The microbial toxicity test is routinely performed using a luminescent photometer and specially prepared dehydrated luminescent bacteria, the luminescence of which is measured in the presence and absence of potential toxicants (so-called Microtox test: Bulich A.A., Process Biochem. 17, 45-47, 1982). The k method is suitable as a primary screening in the control of environmental cleanliness.

Methods have been described in the literature by which it is allegedly possible to lyophilize photobacterial cells so that they not only survive in the dried state for several months, but are also capable of bioluminescence immediately after rehydration. According to one method (Nealson H., Methoda Enzymol. 57, 153-166, 1978k), the luminescent bacterial cells are resuspended in a medium with a lower ionic strength, which contains about 0.75% by weight. NaCl, other inorganic salts, 0.5 wt%, peptone and 0.5 wt%. yeast extract, frozen and lyophilized. The second method (NSR Patent No. 28 41 8964) uses the suspension of photobacteria in 2% by weight, a NaCl solution containing 20% by weight, of skim milk before lyophilization. '

The disadvantage of these methods is that they do not provide reproducible results for a wide range of luminescent bacterial species, the main disadvantage is that the survival rate and intensity of the bioluminescence of preserved cells are low. Commercially produced cans of photobacteria for the Microtax test are very expensive. In the CMEA countries, the technology of mass production of dehydrated photobacteria has not yet been introduced.

The fact that luminescent bacteria cannot be reliably lyophilized by conventional methods is also a major problem for collections of microorganisms. The methods used so far for the long-term preservation of these bacteria are either expensive (storage in liquid nitrogen) or laborious and inconvenient (storage on agar media in a refrigerator and frequent revaccination).

These disadvantages are eliminated by the method for the preservation of luminescent bacteria according to the invention, the essence of which is that the bacterial cells are lyophilized in a lyophilic medium containing 10 to 25% by weight of cryoprotectant and 1 to 10% of soluble starch as a rehydration facilitator. and are rehydrated before use with a solution of a uniform electrolyte, preferably NaCl, the concentration of which before lyophilization is 0.8 to 1.2% by weight, the rehydration being carried out by making up to the original volume by adding 2% NaCl solution. Cells of luminescent bacteria grown in liquid medium or on solid medium are resuspended in lyophilized medium with a lower concentration of NaCl (e.g. 1%), which contains lactose as a cryoprotectant and screw as a substance that facilitates later rehydration; this suspension is further lyophilized in the usual manner. The lyophilisate is stored in an evacuated container in the cold (about 4 ° C). Prior to use, the lyophilized culture is reconstituted to the original volume by adding NaCl solution so that the final NaCl concentration is optimal for bioluminescence (about 3%). Under these conditions, rehydrated cells show intense and stable bioluminescence so that they can be used for the Microtox test. Resuscitation of the collection strains is carried out in the usual way, eg by suspension in growth m = medium.

The method of preserving luminescent bacteria according to the invention ensures: 1. a high degree of survival of bacterial cells even after several months of storage; 2. easy storage of preserved cultures, which allows field toxicity tests to be performed;

3. easy rehydration of the dried material, which is a reproducible condition

CS 268012 81 and the correct distribution of the luminescent culture in an aliquot at the beginning of the test; 4. sufficiently strong and constant luminescence of preserved cells after rehydration.

The following is an example of the use of the method according to the invention. Only one of the possible embodiments is described - the specific values of concentration, temperature, pressure and other quantities cannot be given a limiting importance with respect to the protection of the invention.

Example 1

The luminescent bacteria Photobacterium lelognathi LN-1a were cultured on a rotary shaker at 22 ° C in a nutrient medium containing (g / L) tKHgPO 2, 2.5 ^: NaCl, 30.0 ^: NH 4 Cl, 0.3: MgSO 4. 7 HgO, 0.4: FeCl 2, 0.005: glycerol, 5.0: yeast extract (SERVA, NSR) 1.0: trypcon (0X010, UK) 15.0 (pH 7.1). The late logarithmic phase culture was cooled in ice, the cells were separated by centrifugation and resuspended in 3% w / w. NaCl at a concentration of 1.4 x LO L ^^ ^- ^. Aliquots (0.04 ml) were added to 0.5 mL of lyophilized medium containing in 100 mL: 1 g NaCl, 23 mg sodium pyrophosphate (decahydrates), 0.5 mg yeast extract (SERVA), 15 g lactose and 5 g soluble starch (according to Loulier; LACHEMA).

A portion of this suspension was used to determine bioluminescence intensity and live cell concentration. The number of cells capable of forming colonies was determined by seeding the diluted suspension on Petri dishes containing 2% by weight, agar in nutrient medium of the above composition and incubating at 22 ° C. The luminescence of the bacterial suspension was measured at 15 ° C in a luminescent photometer Microtox model 2055 (Beckman, USA), setting the zi _t SKU photomultiplier 4.00 9.50 0 × 1 (AA Bulich, Process Biochem. 17, 45-47, 1982 ).

Immediately after suspension, the other samples were frozen in ice / ethyl alcohol and lyophilized in a lyophilizer (HETOSICC CO 52-1; HETO, Denmark) under a vacuum of 7 Pa for 3.5 h. at 2 to 4 ° C.

After 90 days of cold storage, the preserved cultures were resuscitated by the addition of 0.5 mL and 2% by weight. NaCl (so the final NaCl concentration was 3%). Bioluminescence and viability of rehydrated cells were tested by the same methods as before lyophilization. The measured values are given in the table.

Example 2

As in Example 1, the preservation of luminescent bacteria Photobacterium phosphoreum MVI 02 was performed.

Example 3

As in Example 1, the procedure for the preservation of luminescent bacteria Vibrio fischer! MO-1.

It is clear from the table that in all the microorganisms studied, the lyophilization according to the invention reduces the ability of the colony to form to values which are completely sufficient for collection purposes. The total bioluminescence of the cultures is reduced to 1/10 to 1/4 of the original value before lyophilization, which very well meets the needs of the microbial toxicity text.

Table

Influence of the preservation method according to the invention on the viability and bioluminescence of photobacteria

Microorganism Ability to form colonies ³ Bioluminescence ⁸

before lyof. 90 days by lyof. before lyof. 90 days after phyof. P. phosphoreum MVI 02 4.28 x 10 ⁹ 1.48 x 10 ⁸ 695 65 P. leiognathi LN - la 2.04 x 10 ⁹ 5.70 x io ⁸ 191 48. V. fiocheri MO-1 4.40 x 10 6.81 x 10 ⁸ 944 243

= converted to the concentration of viable cells in 1 mL of suspension (average of 5 values) = In the arbitration units of the Microtox 2055 luminometer display. The luminescence of 0.01 mL of bacterial suspension in 0.5 mL of 3% w / w was always measured. NaCl (average of 3 values).

Claims (1)

OBJECT OF THE INVENTION A method for preserving luminescent bacteria by lyophilization, characterized in that the bacterial cells are lyophilized in a lyophilized medium containing 10 to 25% by weight of cryoprotectant lactose and 1 to 10% by weight of soluble starch as a rehydration facilitator, and before use. they are rehydrated with a solution of a uniform electrolyte, preferably NaCl, the concentration of which before lyophilization is 0.8 to 1.2% by weight, the rehydration being carried out by making up to the original volume by adding 2% NaCl solution.