Quality Control Methods ISO 9308 And ISO 7899 - 2 In Sanitary Microbiology In Center For Public Health Bitola,

, Macedonia
Angela Delova, Vladimir Kakurinov, Olivera Jankova, Dijana Blaekovi, Vladimir Trajkovski Center for public health Bitola, Macedonia

Abstract
Application of the standard ISO/IEC 17025 becomes imperative in ensuring system quality in laboratories. The ISO method 9308 used bases: Lactose TTC Agar, Triptic soy Agar, DEV Tryptophan Broth, Oxydaza test and reference type E. coli ATCC 25922. The ISO 7899-2 method used bases: Slanetz and Bartley Agar, Bille Aesculine Agar and reference type Enterococcus faecalis ATCC 19433. Standard ISO 17025 facilitates cooperation among laboratories in exchanging experience and harmonize standards and procedures. Key words: quality control method, drinking water, ISO 9308, ISO 7899- 2

Introduction
Application of the standard ISO/IEC 17025 becomes imperative in ensuring system quality in laboratories. Among the other procedures required by this standard is the procedure for evaluation of vagueness in the microbiological laboratory testing. It defined procedures that are carried out in the introduction of new method, equipment or changing working conditions.

Aim
Aim of this work is to display the vagueness in carrying out the measurements methods ISO 9308 (detection and counting the bacteria E. coli and coliforms with MF method and ISO 7899-2 (Detection and counting of intestinal enterococcus with MF method in drinking water).

Materials and methods

The ISO method 9308 used bases: Lactose TTC Agar, Triptic soy Agar, DEV Tryptophan Broth, Oxydaza test and reference type E. coli ATCC 25922. The ISO 7899-2 method used bases: Slanetz and Bartley Agar, Bille Aesculine Agar and reference type Enterococcus faecalis ATCC 19433. As criteria for the verification method is selected the following parameters: 1. Quality control of bases which are used in the methods per stages with reference types; 2. Determination of method precision. In quantitative methods the precision is determined by the terms: standard deviation, relative standard deviation. For qualitative methods are defined other performance characteristics of accuracy (sensitivity, specificity), acordation, concordation.

Results
Ten samples of drinking water contaminated with reference type E. coli and 10 samples of water without the presence of bacteria (distilled water) were examinated. Identical results were obtained between the two trials of the same sample in the same laboratory conditions as well as between laboratory comparisons. When enumerating colonies of E. coli (in 20 tests on packed samples), obtained values are within the available range 2 standard deviations (Shewart scheme) and the relative standard deviation 1,42% which indicates very little deviation from the average value. The same approach is shown for the method ISO 7899-2.

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Quality control of bases (E. coli)

Detection and counting of Escerichia coli bacteria and koliformn bacteria with the method of membrane filtration by ISO 9308-1 2000

Sample size is 100ml or more (for the bottled water is 250ml). The size of the sample should be so selected to the filter membranes to retain 20-80 colonies

Subculture (> from 10 colonies) is sowing on: Jump to membrane filter of Laktose TTC Agar with Tergitol
Membrane filtration 1 day

Standard test

Preparation Incubate at 36 2 C in a duration of 21 3 hours If the test is negative reincubate of 44 4 hours

Positive finding of Laktose TTC Agar with Tergitol
2-3 day

Probable result:: Coliform bacteria Yellow lactose positive colonies are counting

Oxidaza tests

Indol tests

Final identification:

Final identification:

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, coli Coliform bacteria E. 29 May 2010 Oxidaza (-) colonies are counting on (TSA) as coliform bacteria Oxidaza (-) and Indol (+) colonies are counting as E. coli colonies

4 day

Prooving - a certificate

Tryptic Soy Agar (TSA) Incubate at 36 2 C in a duration of 21 3 hours

Tryptophan Broth Agar Incubate at 440.5 C in a duration of 21 3 hours

Detection

Figure 1. Lactose TTC Agar

Figure 2. Triptic Soy Agar

Figure 3. DEV Trytophan Broth agar (positive indol reaction)

Figure 4. Oxydaza test

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Determination of measurement uncertainty (E. coli)

Number of measurement 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total Value 74 73 72 74 74 76 73 76 72 74 76 73 72 74 74 76 73 74 72 74 1472

( xi x )
0,4 -0,6 -1,6 0,4 0,4 1,4 -0,6 1,4 -1,6 0,4 1,4 -0,6 -1,6 0,4 0,4 1,4 -0,6 0,4 -1,6 0,4 /

( xi x ) 2
0,16 0,36 2,56 0,16 0,16 1,96 0,36 1,96 2,56 0,16 1,96 0,36 2,56 0,16 0,16 1,96 0,36 0,16 2,56 0,16 20,8

Average value

x=

1 n 1 xi = 20 1472 = 73,6 n i =1

Standard deviation

S=

1 n 20,8 ( xi x ) 2 = 19 = 1,095 = 1,046 n 1 i =1

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Kontrolna Shewart-ova {ema

78 77 76 75 74 73 72 71 70 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5. Control Shewart scheme

Shewart scheme: Average value: 73,6 Standard deviation S: 1,046 Warning limit ( 2SD): 73,6 2,09 Action limit ( 3SD): 73,6 3,14 Variance = S2= 1,095 Relative standard deviation RSD

RSD(%) =

S 1,046 100 = 100 = 1,42% x 73,6

Measurement uncertainty MU:

MU = x (k * S / n ) = 1,046 73,6 (2,01* ) = 73,6 0,47 20

k constant of confidence level of 95% and for 20 measurements is 2,01

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Determination of bases quality (Enterococcus)

Detection and counting of intestinal enterococcus with the method of membrane filtration by ISO 7899 -2 2000

Sample size is 100ml or more. The size of the sample should be so selected to the filter membranes to retain 20-80 colonies

Membrane filter is jumping on Bile Aesculine Azide Agar,which is previously tempered at 44 C Incubation at 440.5C in duration of 2 hours
2-3 day Membrane filtration 1 day

Jump membrane filter on Slanetz Bartley Agar

Probably result: Enterococcus Count red, brown or rose colored typical colonies

Stop with testing if there are no typical red rose colonies

Finaly result: Intestinal enterococcus Counting of yellow browni to black colonies BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010
4 day

Prooving - a certificate

Detection

Incubate on 362C in duration of 44 4 hours

Preparation

Figure 6. Tipical red colonies of enterococcus on Slanetz and Bartley Agar

Figure 7. Yellow brown to black colonies intestinal enterococcus on Bile Aesculine Azide Agar (incubation on 440.5C in duration of 2 h)

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Determination of measurement uncertainty (Enterococcus)

Number of measurement 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total Value 55 58 57 55 56 54 56 57 58 54 55 58 57 55 56 54 56 57 58 54 1120

( xi x )
-1 2 1 -1 0 -2 0 1 2 -2 -1 2 1 -1 0 -2 0 1 2 -2 /

( xi x ) 2
1 4 1 1 0 4 0 1 4 4 1 4 1 1 0 4 0 1 4 4 40

Average value

x=

1 n 1 xi = 20 1120 = 56 n i =1
1 n 40 ( xi x ) 2 = 19 = 2,105 = 1,45 n 1 i =1

Standard deviation

S=

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Kontrolna Shewart-ova {ema

62

60

58

56

54

52

50

48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 8. Control Shewart scheme

Shewart scheme: Average value: 56 Standard deviationS: 1,45 Warning limit ( 2SD): 56 2,9 Action limit ( 3SD): 56 4,35 Variance = S2= 2,105 Relative standard deviation RSD:

RSD(%) =

S 1,45 100 = 100 = 2,6% x 56

Measurement uncertainty MU:

MU = x (k * S / n ) = 1,45 56 (2,01* ) = 56 0,65 20

k constant of confidence level of 95% and for 20 measurements is 2,01

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010

Conclusion
Based on the results obtained from examinated parameters we noticed that with the methods described in our laboratory we obtain reliable results. ISO application methods used in daily routine examinations, does not exclude the need for constant application of internal quality control methods and the results obtained trials among laboratories. Standard ISO 17025 facilitates cooperation among laboratories in exchanging experience and harmonize standards and procedures.

References
Brodsky M., 2003: CAEAL National Standard method, 2005: Uncertainty of measurements in testing, Guidance note, QSOP4, Issued by Standards Unit, Evaluations and Standards Laboratory, Issue no: 5, pp. 1-21 Standards ISO IEC 17025: 2005 Standards ISO 9308-1: 2000 Standards ISO 7899-2: 2000

BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010