Fresenius J Anal Chem (1995) 352 : 240-245

Fresenius' Journal of

© Springer-Verlag 1995

Development, production and certification of microbiological reference materials B. Janning, P. H. in 't Veld, K. A. Mooijman, A. H. Havelaar RIVM, National Institute of Public Health and Environmental Protection, P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands Received: 12 September 1994 / Accepted: 24 November 1994

Abstract. Since 1986 the National Institute of Public Health and Environmental Protection (RIVM) in the Netherlands has been developing and testing reference materials (RMs) for use in the field of water and food microbiology within the framework of the 'Measurements and Testing Programme' (former BCR) of the European Union. The materials consist of gelatin capsules filled with artificially contaminated spray dried milk. Individual and collaborative studies on stability and homogeneity of the materials are described. The positive results obtained are followed by certification studies on certain reference materials. The results of two certification studies have now satisfactorily been completed, and certified values, related to the methods used, have been given for two certified reference materials (CRMs) containing the bacterial strains Salmonella typhimurium (5 cfp/capsule) and Enterococcus faecium (500 cfp, capsule), respectively. Two other RMs containing Bacillus cereus and Enterobacter cloacae will be certified by the end of 1994.

Introduction The 'Measurements and Testing Programme' (former BCR) of the Commission of the European Union is certifying a variety of reference materials to be used in different fields of analysis: environment, food and agriculture, biomedicine, physical properties, industrial raw materials and products, water and food microbiology [1]. Especially in the field of microbiological analysis of food and water the implementation of quality assurance schemes is becoming increasingly important. Efforts are made to develop and agree on reference methods. But the use of standard methods for the microbiological analyses of water and food, e.g. ISO methods, cannot guarantee correct results in an individual laboratory and equal results in different laboratories. Depending on the circumstances, such as the type of sample or the presence of competing microorganisms, even (agreed) reference methods can lead to systematic Correspondence to: B. Janning

errors. Results which are not trustworthy as well as poor performance by (microbiological) laboratories can create public health problems, mis-interpretation of environmental processes and economic losses. Therefore, there is a growing need for readily available microbiological reference materials (RMs) to compare the performance of individual laboratories and to define the limits of inter-laboratory variability.

Development of microbiological reference materials The development of microbiological reference materials has long been hampered by the instable concentrations of viable organisms in the materials during longer periods of storage. In addition, the quality of some materials was not satisfying because of insufficient homogeneity. These two critical aspects are due to the fact that in this case viable (prokaryotic) cells form the basis of the reference materials. The viability of these cells is strongly affected by the conditions during the production of the RM, as e.g. high temperature and desiccational stress. Hence, viable microorganisms are sublethally damaged and this causes varying survival rates. In cooperation with the European Union's 'Measurements and Testing Programme' (the former BCR) the Netherlands' National Institute of Public Health and Environmental Protection (RIVM) carried out investigations to demonstrate that the production of sufficiently stable and also homogeneous microbiological reference materials is possible [2]. The method used to produce the materials is based on the stabilization of the target microorganisms by spray-drying them in a preservation medium based on sterile evaporation milk [3]. The resulting highly contaminated milk powder has, depending on the test strain and the contamination level of the milk suspension, a final contamination level of 10 4 to 10 7 colony forming particles per gram (cfp/g). After a period of stabilization while stored at a temperature of +5 ° C or -20 ° C, the highly contaminated milk powder is mixed with sterile milk powder to achieve the desired contamination level depending on the application of the final material. Gelatin capsules are

241

[Cultivation of bacterial

strain~

=

V

i Suspension in evaporated

veloped. The first statistical test examines the counts of the subsamples from the same capsule solution and is performed by calculating:

milk 1

I

v

lSpray drying ! --~

~ghiy cootarninated milk powder I

iMixing

with sterile milk

powder I

~Fillingo=f gelatine capsules ]

where J indicates the number of subsamples, usually 2, Zij the counts per subsample and zi+ the total count of all subsamples from one capsule. If the subsamples follow a Poisson distribution, then the statistic T 1 follows a Chisquare distribution with I degrees of freedom if J = 2. As a measure of dispersion between total counts of different capsules of one batch, the Cochran's dispersion test statistics is also calculated for the sum of the replicate counts:

iStorage at -20°C ] Fig. 1. Production of microbiological reference materials: flow diagram then filled with 0.2-0.3 g of the mixed powder with the desired contamination level. These capsules, the final RMs, are stored at a temperature o f - 2 0 ° C. Figure 1 presents the most important steps of this production process in a flow diagram.

r2=2,[(z,+-z++/z)2/(z++/t)] where the symbol I stands for the number of capsules and z++ stands for the total count over all capsules and subsamples. The statistic T2 is approximately distributed as a constant times a Chi-square distribution with I-1 degrees of freedom. In case of a Poisson distribution the expected values of T 1 and T 2 a r e the same as the degrees of freedom. In that case, T1/{I(J-1)} and Tz/(I-1) are expected to be equal to one.

Requirements of microbiological reference materials The most important requirements, which a reference material must fulfil, are representativity, homogeneity and stability [2].

R epresentativity For microbiological reference materials this implies that the microorganism present in the material should represent the target organism as expected in a typical water- or food sample. This means that the organism should show the same typical (e.g. biochemical and immunological) properties as the target organism. These properties are of basic interest regarding the identification of the target microorganism and may not be changed or lost during the production process of the reference material.

Homogeneity In order to obtain reproducible results, the distribution of the organisms within the material must be homogeneous. The distribution of microbes in liquids or powders follows certain statistical rules, in this case the number of bacteria within a sample should approach a Poisson distribution. Data from different batches of a RM do not necessarily have equal variances [4]: if the mean contamination levels are different, the variances are also different. Instead of directly comparing variances, it is therefore more appropriate to look at ratios between variances and means. Cochran's dispersion test statistics [5] is based on this principle. To examine whether the viable count results follow a Poisson distribution, two statistical tests were de-

At the moment two types of reference materials are distinguished on the basis of their contamination level: 'high level' and 'low level' capsules: 'High level' capsules. In these materials, the expected number of colony forming particles (cfp) per capsule is at least 300. These materials are used for quantitative procedures, such as membrane filtration, pour plate and spread plate techniques. Before use, a capsule is reconstituted and subsamples from the resulting capsule solution can be analysed. ' L o w level' capsules. In these materials, the number of colony forming particles (cfp) per capsule is approximately 5. These materials are used for qualitative procedures (presence/absence tests). The capsules are analysed as a whole; so no subsamples can be analysed. In case of 'low level' capsules only the T 2 test statistic is performed to test the distribution of the bacteria between different capsules of one batch. The T 1 test statistic is not performed here, because no subsamples of the same dissolved capsule can be examined. In this case the whole volume of a dissolved capsule is inoculated. In case of an application of 'high level' capsules both test statistics are performed. Figure 2 shows the results of a homogeneity test carried out on a batch of the C R M containing Enterococcus faecium [6]. Here T2/(I-1), with I = 20, is plotted against the time. The figure clearly shows that T2/(I-1) never exceeded the m a x i m u m accepted value of 2 and that no trend in time could be detected.

Stability During storage and transport the contamination level of microbiological reference materials must remain stable, that means that the contamination level should neither be

242 100 90 i

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[days]

Fig. 2. Results of the homogeneity test of a batch of capsules containing the test strain Enterococcusfaecium

. . . .

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[d]

time

Fig. 4. Challenge test on Bacillus cereus. Storage temperatures: - 2 0 ° C A ; + 2 2 ° C O ; +30 ° C +; +37 ° C [ ]

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Fig. 3. Improvement of the desiccation resistance of Escherichia coli. Cultivation in: BHI + ; BHI + 5% NaC1 A, desiccation in evaporated milk with: 0.5 mol/1 sucrose O; 1.0 mol/1 sucrose @; 2.0 mol/1 sucrose []

decreasing nor increasing within the precision limits of the method of determination within a specified period. A certain decrease as a result of sublethal damages of the bacterial cells during spray drying is inevitable. This decrease should be kept within certain limits by controlling several factors having an influence on the stability of the contamination level of the RMs. The stability of the contamination level of microbiological reference materials is directly influenced by: The type of the test organism. Bacterial strains show a wide range of resistance against different kinds of stress including desiccation. Culture conditions have an influence on the desiccation resistance. Investigations carried out at our laboratory revealed that cultivation in a liquid medium with an elevated salt concentration of 5% NaC1 can improve the survival rates of E. coli after desiccation on silica gel and storage at a temperature of +22 ° C (Fig. 3). This desiccation method was used as a model system to study the survival of various bacterial strains after drying [7]. By exposure to an increased osmotic pressure during cultivation

storage

time

[d]

Fig. 5. Challenge test on Escherichia coli. Storage temperatures: -20 ° C A; +22 ° C O; +30 ° C + ; +37 ° C []

the cells are prepared against the extreme osmotic shock during desiccation. According to HPLC-analysis (results not presented), under these culture conditions the cells accumulate osmoprotectants, so-called compatible solutes [8], e.g. glycine betaine, up to a concentration of 3.3% of the cells' dry weight. These solutes protect the cells against damage caused by the extreme osmotic stress during desiccation without impeding cellular enzymatic activities. Spray drying is, similar to silica gel drying, a very fast desiccation procedure. Therefore, there is no time left for the organisms to activate physiological procedures to adapt to the stressfull treatment. Hence, the cells must be prepared for the stress before it occurs. Also the composition of the desiccation medium is important with regard to the desiccation resistance of bacteria. The addition of a 0.5, 1.0 or 2.0 mol/1 concentration of sucrose to evaporated milk as the basic desiccation medium leads to improved survival rates of the bacteria strain E. coli after desiccation on silica gel, storage at + 2 2 ° C and reconstitution, as also presented in Fig. 3. HPLC-analyses of the cytoplasmatic content of these bacterial cells revealed an sucrose-accumulation of up to

243 6.8% of the ceils' dry weight, leading to osmoprotection against cell damage caused by desiccation (results not presented). T h e a g e o f t h e h i g h l y c o n t a m i n a t e d m i l k p o w d e r has an influence on the stability of a microbiological reference material [9]. Several experiments with different microbiological RMs revealed that after a period of storage at +5 ° C or - 2 0 ° C the stability of the contamination level of a milk powder improves. The time point of stabilization and the stabilized contamination level depend on the test strain. S t o r a g e c o n d i t i o n s , especially the storage temperature, have a very significant influence on the stability of the contamination level. This temperature dependence was evaluated in stability tests at +5 ° C and - 2 0 ° C and in so-called challenge tests at higher temperatures [6]. During a challenge test some capsules of one batch are stored for at least 4 weeks at +22 ° C, +30 ° C and +37 ° C. After regular time intervals samples are taken to determine the contamination level of the material and its decrease by carrying out regression analyses on the received data. Figure 4 presents the results of a challenge test carried out with a reference material containing the strain B a c i l l u s c e r e u s . In Fig. 5 the survival of E . c o l i during a challenge test with storage temperatures of - 2 0 ° C, +22 ° C, +30 ° C, + 3 7 ° C is described. The results of these two challenge tests clearly demonstrate that the stability of a RM depends on the storage temperature as well as on the test strain.

Table 1. Collaborative studies and certification studies organized by the RIVM and the 'Measurements and Testing Programme' between 1987 and 1994 Year

Bacterial strain

Type of analysis

International standardmethod used during the study

1987

Salmonella typhimurium

F°

ISOb 6579

1988

Salmonella typhimurium

F

1988

Escherichia coli

Wd

-

Enterobacter cloacae

W

-

Listeria monocytogenes

F

-

1989 1989 1989

Escherichia coli

W

-

Enterobacter cloacae

W

-

Enterococcus f a e c i u m

W

-

Staphylococcus warneri

W

-

1990

Listeria monocytogenes

F

-

1991

Escherichia coli

W

-

Enterobacter cloacae

W

-

Bacillus cereus Staphylococcus aureus

F F

ISO 6391 ISO 6888

1992

Salmonella typhimurium a

F

1992

Enterococcus f a e c i u m

W

-

Staphylococcus warneri

W

-

1992

Enterococcus f a e c i u m a

W

-

Collaborative studies

1993

Escherichia coli

F

ISO 7932/ ISO 7402

The suitability of a microbiological RM is tested at the R I V M as well as in collaborative studies, involving 2 5 40 laboratories throughout the European Union. These laboratories were invited to participate by the 'Measurements and Testing Programme'. The performance of these collaborative studies has two basic aims; first of all to test and evaluate the reference materials and accompanying protocols in laboratories in all EU member states and to incorporate comments made by participants into the development work. Furtheron, it is very important to bring together European laboratories in order to exchange experiences and information on quality assurance in water and food microbiology and to stimulate harmonization of the methods and principles being used. Table 1 gives an overview about all international collaborative studies and certification studies (see sect. Certification) which have been performed by the R I V M between 1987 and 1994.

1993

Bacillus cereus a

F

-

1993

Clostridiumpelfringens

F

ISO 6461/2

1993

Enterobacter cloacae a

W

-

1994

Clostridium pelfringens

F

ISO 6649

1994

Salmonella p a n a m a

F

ISO 6579

t994

Eseherichia coli a

W

1994

Listeria monocytogenes a

F

Certification After it has been successfully tested in an international interlaboratory study, a microbiological RM can be used for a certification study leading to a certified RM (CRM). During these studies a group of 11 or 12 qualified European laboratories simultaneously examines the materials using standardized methods and following a very strict

1992

IDFe, revised provisional standard 143

a Certification study b ISO = International Organization for Standardization c F = food analysis d W = water analysis e IDF = International Dairy Federation

analytical protocol. In 1993 the first two microbiological reference materials have been certified by the Commission of the European Union. They contain the bacterial strains S a l m o n e l l a t y p h i m u r i u m (CRM 507) [10] and E n t e r o c o c c u s f a e c i u m (CRM 506) [6], respectively. Two other materials containing E n t e r o b a c t e r c l o a c a e and B a c i l l u s c e r e u s will follow by the end of 1994. Tables 2 and 3 show the data on certified contamination levels, 95% confidence intervals and the ISO methods to which the

244 Table 2. Certified values of the CRM 506 containing Enterococcus faecium; CRM 506, Certified number concentration of colony forming particles of Enterococcus faecium (WR63) from artificially contaminated milk powder Method"

Certified 95% Confidence Number of valueb, c limitsd accepted CE, cfp/m1-1 CE.cfp/m1-1 sets (n) of datae

ISO 7899/2, 1984 KFA

79

70-88

7

ISO 7899/2, 1984 m-EA

76

70-83

7

ISO 6222, 1988 YA

110

r

60

50

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12/8 18/11 27/01 29•06 19/08 02/12 13/12 17/02 23•03 31/03 05•04 12/04 date

100-121

8

KFA: Kenner Fecal Streptococcus Agar m-EA: membrane Enterococcus Agar, according to Slanetz and Bartley YA: Yeast extract Agar b This value is file geometric mean of n accepted sets of data, independently obtained by 7 (KFA and m-EA) or 8 (YA) laboratories c C~.cfp: Number concentration of colony forming particles (cfp) of Enterococcus faecium (E) determined in one analytical portion. Analytical portion: A volume of (I.00 + 0.02) ml from 10 ml peptone saline solution in which one capsule has been reconstituted d These values are the 95 % confidence limits, belonging to the certified values of the specified method e Total number of individual results was 206 for KFA, 197 for mEA and 220 for YA

certified data refer. Each get of 10 capsules of a C R M is provided with a report c o n t a i n i n g data on (amongst others) type, concentration, h o m o g e n e i t y and stability of the test organism.

P o s s i b l e fields

.

of application

The m i c r o b i o l o g i c a l R M s and C R M s as prepared by the R I V M are available at the S V M Sales Department, Bilthoven, The Netherlands, (RMs), and the Institute for

Table 3. Certified values of the CRM 507 containing Salmonella typhimurium; CRM 507, Number fraction of negative capsules and number content of cfp's of Salmonella typhimurium from artificially contaminated milk powder

Fig. 6. Control chart with data resulting from analyses of a microbiological RM containing Eschericha coli. X: geometric mean of the contamination level; + 2s: twofold standard deviation; + 3s: threefold standard deviation; Ccfp: number concentration of colony forming particles in one analytical portion (discrete portion of a sample which will be subjected to examination; in this case the analytical portion means a volume of (1.00 + 0.02) ml taken from 10 ml peptone saline solution in which one capsule has been reconstituted)

Reference M e a s u r e m e n t s and Materials, Geel, Belgium, (CRMs). They can be used by laboratories performing quality assurance on food- and water-analyses. Therefore R M s should regularly be used while C R M s are proposed for incidental use. The materials can be used for the imp l e m e n t a t i o n of quality control schemes, to prepare laboratory accreditation, and for any approach to m u t u a l r e c o g n i t i o n of laboratory results. Practical examples are the d e v e l o p m e n t and validation of methods and m e d i a and the d e t e r m i n a t i o n of the i n f l u e n c e of matrix ingredients and competitive flora [11]. M i c r o b i o l o g i c a l collaborative studies can be based o n the use of these materials. For a p p l y i n g the R M s in first line quality control it is very useful to record the data in control charts, a procedure which is well established in the field of quality assurance of analytical chemistry, with the data resulting from a n a l y s i n g a certain R M with a specific (ISO) method. Figure 6 shows a control chart as established in our laboratory of an E. c o l i - R M analysed to determine total coliforms.

Quantity (test procedure)

Certified value

Confidence limit(s)

Sets of accepted results

Number of Salmonella colony forming particles in one capsule (Ns,cfp) (enumeration procedure) Fraction of capsules in which no Salmonella could be detected (enumeration procedure) Fraction of capsules in which no Salmonella could be detected (presence/absence procedure)

5.9

5.3-6.4 a

10¢

0,61%

0-1.6% b

10d

2.7%

0-44% b

9e

a Two-sided 95% confidence interval b One-sided 95% confidence upper limit ° Based on the results of 485 capsules

d Based on the results of 492 capsules e Based on the results of 414 capsules

245

Further investigations The R M s and C R M s as d e v e l o p e d up to n o w b y the R I V M d e m o n s t r a t e d v e r y satisfying stabilities during storage at - 2 0 ° C. Short time challenge tests (see Fig. 5) s h o w e d that for m a t e r i a l s containing certain bacterial strains losses in v i a b i l i t y occur at t e m p e r a t u r e s o f +22 ° C and above. This is o f i m p o r t a n c e w h e n c o n s i d e r i n g the transport o f reference m a t e r i a l s to laboratories t h r o u g h o u t the EU. W h e n n o r m a l (air) m a i l is used, transport d e l a y s v a r y f r o m two days to m o r e than two weeks. Hence, norm a l (air) m a i l without further p r e c a u t i o n will not a l w a y s be sufficient to guarantee the stability o f the reference materials. E v e n if transport times c o u l d be c o n t r o l l e d to a m a x i m u m o f e.g. one week, this guarantee c o u l d not be g i v e n for all materials. T h e b e s t solution to solve this p r o b l e m w o u l d be to i m p r o v e the stability o f the m a t e r i a l s at h i g h e r temperatures. A s m e n t i o n e d before, a m o d e l system has b e e n d e v e l o p e d to screen the d e s i c c a t i o n resistance and stability at a storage t e m p e r a t u r e of +22 ° C for bacterial strains b e i n g c o n s i d e r e d as c a n d i d a t e strains for the d e v e l o p m e n t o f R M s / C R M s . F u r t h e r investigations need to be carried out to i m p r o v e the d e s i c c a t i o n resistance o f those strains m a r k e d b y an insufficient stability after d e s i c c a t i o n and storage at +22 ° C (see Fig. 3).

Acknowledgements. The work described here was carried out within and on behalf of the 'Measurements and Testing Programme' (former BCR), EU. The authors wish to thank Dr. E. Maier as coordinator of the project for his support on the development of microbiological RMs and CRMs, and for his chairmanship during the meetings on the collaborative studies. The European laboratories

who participated in the collaborative studies within this project are acknowledged for their contributions.

References 1. Commission of the European Community (1994) Community Bureau of Reference, BCR, BCR Reference Materials 2. Mooijman KA, in 't Veld PH, Hoekstra JA, Heisterkamp SH, Havelaar AH, Notermans SHW, Roberts D, Griepink B, Maier E (1992) Commission of the European Community, BCR Information, Report EUR 14375 EN 3. Beckers HJ, van Leusden FM, Roberts D, et al (1985 a) J Appl Bacteriol 59 : 35-40 4. Heisterkamp SH, Hoekstra JA, van Strijp-Lockefeer NGWM, Havelaar AH, Mooijman KA, in 't Veld PH, Notermans SHW, Maier E, Griepink B (1993) Commission of the European Community, Community Bureau of Reference, BCR Information, Report EUR 15008 EN 5. Cochran WG (1954) Biometrics 10:417-451 6. Mooijman KA, van Strijp-Lockefeer NGWM, van der Heide R, Heisterkamp SH, Havelaar AH, Griepink B, Maier E (1994) Commission of the European Community, Community Bureau of Reference, BCR Information, Report EUR 15061 EN 7. Janning B, in 't Veld PH (1994) J Appl Bacteriol 7 7 : 3 1 9 324 8. Brown AD (1976) Bact Rev 40:803-846 9. Mooijman KA, Havelaar AH, Hoekstra JA, van Strijp-Lockefeer NGWN (1991) War Sci Tech 24:53-56 10. in 't Veld PH, Hoekstra JA, Heisterkamp SH, van Strijp-Lockefeer NGWM, de Winter ME, Hobers J, Havelaar AH, Notermans SHW, Maier E, Griepink B (1994) Commission of the European Community, Community Bureau of Reference, BCR Information, Report EUR 15062 EN 11. in 't Veld PH, Soentoro PSS, Notermans SHW (1993) Int J Food Microbiol 20:23-36