Edition 14 - November, 2003

Testing the suitability of microbial cultures for use as reference material in international norms and standardised methods

According to national and international norms and standardised methods, accredited or certified laboratories and companies active in fields related to microbiology have to use well-determined strains of micro-organisms as references. However, there are no criteria for checking the identity and the performances of these reference strains by the strain provider. For that reason, BCCM/IHEM (biomedical fungi and yeasts), BCCM/LMG (bacteria), and BCCM/MUCL (agro-industrial fungi and yeasts), in cooperation with the Belgian accreditation body (Beltest) and the Belgian Institute for Standardisation (BIN-IBN), established a project to study the suitability of these microbial cultures. This project was financed by the Belgian Federal Science Policy Office. The standardised methods and correlated strains were selected in function of the specialisation of each partner collection.
The aims were (i) to test the performances of the reference strains imposed by the standards according to the standardised test methods and to compare them with those of alternative strains, (ii) to test and propose potential reference strains when standards did not specify strains, and (iii) to assess the possibility of distributing the strains in a user-friendly form with properties checked by means of standard operating procedures.
The BCCM/IHEM collection, which is specialised in medical fungi and yeasts, selected three European and one French standard related to disinfectants and antiseptics, each requiring two to four reference strains. In total, two fungal strains and four yeasts were involved. The performances of these strains and those of four other strains belonging to species particularly important in hospitals were first studied following the procedure of the norm EN 1275 using three commercial disinfectants. As the results were too dependant on the quality of the commercial products chosen, it was decided to determine the sensitivities of the strains to increasing concentrations of hypochlorite as the reference disinfectant.
This showed that Aspergillus niger IHEM 3766, listed in most standards in that field, has the same sensitivity than Aspergillus flavus IHEM 5903 but is more resistant than Aspergillus fumigatus IHEM 10045, strains from two species more important from a medical point of view. A. niger was also more resistant to hypochlorite than Penicillium aurantiogriseum IHEM 18001, which is listed in one French standard. A. niger IHEM 3766 is thus a suitable strain for testing antiseptics or disinfectants.
Concerning yeasts, Candida albicans and Saccharomyces cerevisiae (two different strains each), mandatory in standards, were compared to strains of C. tropicalis and C. glabrata, species of growing importance in hospitals. C. tropicalis IHEM 15905 was more resistant than the strains of the three other species, indicating that it could be a good optional or additional reference strain for norms concerning the use of antiseptics and disinfectants in hospitals.
Two of the strains studied and four bacterial strains tested by BCCM/LMG are listed in methods from the European Pharmacopoeia. Their performance tests were satisfactory.
BCCM/LMG selected 37 bacterial strains, currently available in freeze-dried form from the public collection, to be checked for identity and performance (as positive or negative control) according to 48 standardised methods used in the food sector. For each strain, an experimental cell stock was prepared in cryovials with 25 cryobeads and preserved at -20° C for 1-2 years in order to simulate the sub-optimum preservation conditions used by the customer. The performance tests carried out at the beginning and the end of the preservation period were situated on three different levels: detection, enumeration and confirmation. The tests were clustered in 16 groups: Bacillus cereus, Campylobacter, Clostridium perfringens, Coliforms, Enterobacteriaceae, Enterococci, Escherichia coli, Escherichia coli O157:H7, Lactic acid bacteria, Listeria, Pseudomonas, Salmonella, Shigella, Staphylococcus aureus, Vibrio, Yersinia enterocolitica. A total of 373 performance tests were carried out with the 37 selected reference strains in relation to the 48 selected standards. Of these, 358 (96%) gave results that conformed to those described in the standards. Consequently, 33 bacterial reference strains will be available in freeze-dried form with a certificate of proven identity and performance. Storage for 1-2 years at -20° C on cryobeads did not affect performance and maintained good viability for 26 of the 33 reference strains.
BCCM/MUCL selected 20 fungal strains involved in wood degradation to check their performance with respect to four European standards concerning wood preservation products and to standard EN 113 in particular. Wood test specimens were exposed to the strains for determining the mass loss that would allow the virulence of the strains to be evaluated and the assays to be validated. The assay can be accepted if the mean mass loss of the virulence control test specimens is equal to or higher than the respective values for the minimum mass loss (20% (m/m)). The following species of fungi were used: i) Coniophora puteana, Gloeophyllum trabeum, Lentinus lepideus, Poria placenta and Serpula lacrymans on Scots pine (brown-rot) and ii) Coriolus versicolor and Pleurotus ostreatus on beech (white-rot). The virulence of the mandatory reference strains was compared to that of the more recently isolated alternative strains. The virulence of these strains was also determined by the screening test method. The principle of this method is similar to the EN 113 method, except that smaller test pieces of wood are used (30 x 10 x 5 mm as opposed to 50 x 25 x 15 mm) and that the exposure time is shorter (6 weeks as opposed to 16).

Fig. 1 EN 113 test. A, exposure to C. puteana MUCL 11662 (1), C. puteana MUCL 31046 (2), P. placenta MUCL 20569 (3), P. placenta MUCL 30789 (4); B, corresponding specimens of wood after 16 weeks of incubation (0 = control).

Fig. 2 Screening test. A, exposure to C. puteana MUCL 11662 (1), C. puteana MUCL 31046 (2), P. placenta MUCL 20569 (3), P. placenta MUCL 30789 (4); B, corresponding specimens of wood after 6 weeks of incubation (0 = control).

Among the four mandatory reference strains tested, C. versicolor MUCL 11665 was the only one that had virulence greater than the minimum recommended in the EN 113 standard and that showed reproducible results of virulence. Therefore, alternative strains were tested under the EN 113 conditions. The virulence of these alternative strains was greater than the minimum value established in the standard, except for the strains of the species G. trabeum. The use of alternative strains proved to be judicious. For example, the virulence of the mandatory reference strain C. puteana MUCL 11662 was far under the 20% limit while the alternative strain MUCL 31046 showed mean mass loss higher than 30%. The alternative strain P. placenta MUCL 30789 was much more virulent than the mandatory reference strain MUCL 20569
(Fig. 1). Compared to the EN 113 method, the screening test method was more rapid (6 weeks instead of 16) and provided similar range of virulence (Fig. 2).
Each of the three collections has selected representative strains to assess the form of supply on cryobeads at -20°C in order to meet the need of the clients for strains in a user-friendly form. To test the viability, counts were performed on 18 strains (2 from BCCM/IHEM, 7 from BCCM/LMG and 9 from BCCM/MUCL) during a period of 14 to 18 months.
For most of the strains, the recovery from cryobeads was satisfactory. However, the number of colony forming units recovered from different beads with the same strain could vary considerably because of the non-standardized beads, so a quantitative guarantee is not possible.
Twelve clients from BCCM/LMG and BCCM/MUCL agreed to test the strains on cryobeads and to give their opinion in a detailed questionnaire; demonstrating that this form of supply was highly appreciated.
Following this project, 26 out of the 33 BCCM/LMG reference strains will be available with certificate as ready-to-use controlled working stocks in frozen cryovials with 25 cryobeads, which can be preserved at -20°C during 1-2 years at the user’s location.

Authors: Suzanne Heinemann, Tine Ryckoort, Véronique Simons

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