Development of a new device for the detection of gas production in coliform group bacteria determination

War. Res. Vol. 25, No. 9, pp. 1131-1135, 1991 Printed in Great Britain. All rights reserved

0043-1354/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc

DEVELOPMENT OF A NEW DEVICE FOR THE DETECTION OF GAS PRODUCTION IN COLIFORM GROUP BACTERIA DETERMINATION EIKO NAGAMACHII and YASUHIRO KANEMASA2 ~Department of Health and Welfare, Junsei Junior College, Takahashi, Okayama 716 and 2Department of Microbiology, Okayama University Medical School, Shikata-cho, Okayama 700, Japan (First received August 1990; accepted in revised form March 1991) Akstract--Pollution of public water is monitored mainly by chemical and physical assays. Moreover, it is evaluated by microbiological assay of coliform group bacteria. For a determination of the number of coliform group bacteria as the biological indicator, the most probable number (MPN) test is generally used. The MPN test is also used to monitor the bacterial contamination of foodstuff. As for the procedure, a test sample is added to Brilliant green-Lactose-Bile broth and the presence or absence of gas production is observed; it is judged by the presence of gas in an inverted vial (Durham fermentation tube). In routine work, numerous fermentation tubes are used for the multiple-tube fermentation test. Aiming at the development of a disposable and low cost substitute for the fermentation tube, we prepared and examined synthetic polymers for the detection of gas production. In conclusion, one type of the foamed synthetic polymers of polyurethane UFX-22 was found to be the most suitable and the results using this type were comparable to those obtained by the Durham fermentation tube method. Key words--water pollution, water examination, biological indicator, coliform group bacteria, multipletube fermentation test, MPN, gas production, new device, synthetic polymer, polyurethane

INTRODUCTION

The coliform group bacteria are indigenous to bacterial flora in human and animal intestines. Although the bacteria designated as the coliform group include not only human- and animal-derived bacteria but also a few bacterial species derived from plants, soil and water, the presence of such bacteria in public water or foodstuff means that the water or foodstuff is contaminated with human or animal feces (Wilson, 1983). In some cases, such water or foodstuff may be contaminated with various enteric pathogens. F o r detection of the number of coliform group bacteria, the multiple-tube fermentation method (Geldreich, 1985a), the pour plate method (Ginsburg, 1985), membrane filter method (Peterson, 1974; Geldreich, 1985b) or petrifilm method (Ginn et al., 1984) are used. Then, the multiple-tube fermentation method is carried out as the most standardized method for monitoring of a biological indicator of public water pollution, especially as the standard method of the Environment Agency of Japan. In the multiple-tube fermentation method, results of the examination of replicate tubes and dilutions are reported in terms of the most probable number (MPN). As for the procedure, a test sample is added to Brilliant green-Lactose-Bile (BGLB) broth and incubated at 37°C for 48 h and then the presence or absence of gas production is observed. The gas production is judged by the presence of gas in the

inverted vial (Durham fermentation tube) (Hoskins, 1934). In routine work, numerous fermentation tubes are to be used for the multiple-tube test. This creates various problems such as high cost and the problem of washing and breakage of the fermentation tubes. Nagamine et al. (1982) suggested the possibility that gas production by bacteria can be detected by using the foamed materials. Aiming at the development of a disposable and low cost substitute for the fermentation tubes, we prepared and examined various synthetic polymers for the detection of gas production. In conclusion, the foamed polyurethane was found most suitable for the detection of gas production in coliform group bacteria.

MATERIALS AND METHODS

To meet the objective described above, the newly developed foamed synthetic polymers should have specific gravity of more than 1.02 and bulk density of 0.25-0.04 g/ml. In addition, they should be porous but should hold the produced gas. We tried polyvinylchloride, polyester and polyurethane as raw materials for the preparation. Figure 1 shows three different types out of the many trial products that have been made and checked. These are 4--5 mm in size. As the polyurethane foams were more suitable than the other materials, several kinds of polyurethane foams are listed in Table 1, in which the physical characteristics of the foams are also shown. BGLB broth was prepared in the same manner as that for use with Durham fermentation tubes and 9 ml of the broth were dispensed in each morton tube. The foamed synthetic polymers were deposited in the bottoms of the morton

1131

1132

ELK() NAGAMACHI a n d YASUHIRO KAN[!MASA

foamed synthetic polymers was autoclaved at 121 C for 15 min. The water samples were collected from the river as usual and 1 ml of the 10 times stepwise dilution of them was inoculated into the sterilized media in the morton tubes. The inoculated tubes were incubated at 37C and examined at the end of 48 4- 3 h. Figure 2 shows the results of gas production using foamed synthetic polymer (in this case UFX-22). When there is gas production, UFX-22 came up to surface due to the trapping of gas in the foams. When there is no gas production, the foams remain at the bottom of the tubes. Therefore the reading is very easy. It is essential that the results obtained with the foamed synthetic polymers must be comparable to those obtained with the Durham fermentation tubes. Various types of foamed synthetic polymers were examined systematically for suitability by trial and error

R E S U L T S AND D I S C U S S I O N S

(I) Screening test of various types qfJoamed synthetic polymers .]'or the detection of gas production Various types o f foamed synthetic polymers were screened for the floating tube ratio (the n u m b e r o f tubes in which the foamed synthetic polymers come up to the surface o f the media) by using a type strain (Escherichict coli IID5208) and a heavily polluted water sample. Every inoculated specimen was prepared so as to give positive gas production in all the tubes with the D u r h a m fermentation tube method. We screened all the tbamed synthetic polymers and only the results o f several kinds of the polyurethane foams showing good results were listed in Table 2. UFX-12 and UFX-22 showed positive gas production in all the tubes with either of the specimens. As the flotation of these foams due to gas trapping was excellenL thesc were selected for further testing. Fig. 1. Physical configuration of the trial products. UFX-10: cubic bodies of polyurethane foam with filler. Bar = 1 cm. UFX-12: cubic bodies of polyurethane foam. Bar = 1 cm. UFX-22: columnar bodies of polyurethane foam which the filmy treatment was made on the lateral surface. Bar = 1 cm.

(2) Comparison of the results obtained with the foamed synthetic polymer and the Durham fermentation tube placed together in a morton tube UFX-12 and UFX-22 were selected for the combination tests with D u r h a m fermentation tubes. The results o f the combination tests were as shown in Table 3. In UFX-12, parallel results were obtained with normal gas production but some foams did not float with a small amount o f gas (pin-point). On the other hand, completely parallel results were obtained

tubes. When the foamed synthetic polymers were used together with Durham fermentation tubes for the preliminary test, the Durham fermentation tubes were placed in the medium first and then the foamed bodies were deposited. The medium with Durham fermentation tube and/or

Table 1. Types and characteristics o f polyurethane loams examined Polyurethane UFX-8 UFX-10 U F X - 12 UFX-15 U F X - 19 UFX-22

Bulk density and specific gravity 0.31 g/cm ~ 1,07 0.37 g/cm 3 1.20 0.25 g/cm 3 1.07 0.31 g/cm 3 1.07 0.25 g/cm ~ 1.07 0.25 g/cm ~ 1.07

Filler

Surface treatment

Form and size

-

4

~

+

....

+

Cubic, 4 nun Cubic, 4 mm Cubic. 4 mm Columnar. 4mm 0 x4mm Cnlumnar, 4ram 0 x 5 m m Columnar, 5 mm q5 ~ 5 mm

+ 4 +

l':ilnD Ireatment

Crushing

Detection of gas production by coliforms

Co~rol ~

Gaa(+)i

Gas(--)

Fig. 2. (A) Comparison between the tests using the Durham fermentation tube or polyurethane foam (UFX-22). (B) Confirmatory test of gas production using polyurethane foam (UFX-22). Left side: UFX-22 came up to the surface in all 5 tubes, i.e. positive gas production in 5 out of 5 tubes. Right side: UFX-22 came up to the surface in 3 tubes only, i.e. positive gas production in 3 out of 5 tubes. Table 2. Screening tests of several polyurethane foams (UFX) using E. coil and river water samples ~ p l e

1133

E. coli

Water

UFX-8 9/10" 8/10" UFX-12 10/10 10/10 UFX-15 5/10 7/10 UFX-19 9/10 10/10 UFX-22 10/10 10/10 *Denominator: number of samples tested, numerator: number of foams came up to the surface (gas production). with b o t h n o r m a l and a small a m o u n t s o f gas production in U F X - 2 2 . U F X - 2 2 is therefore considered to be the best a m o n g those tested. In addition, no flotation o f the synthetic polymers was observed with the samples which gave negative gas p r o d u c t i o n in the D u r h a m fermentation tubes.

Fig. 3. Sectioned fact profiles of polyurethane foams by SEM. (A) Control foam without bacterial growth. Bar = 100 pm. (B) Floating foam with the growth of coliform group bacteria. Bar = 100 #m. (C) High magnification of colonies of coliform group bacteria. Bar = 1 #m.

(3) Electron microscopic observations o f polyurethane foams floating due to trapped gas W h e n polyurethane foams float by trapping the gas produced, coliform group bacteria must be growing in the floating foams. To elucidate such a mechanism, the sectioned inqerfaces o f the foams were observed by scanning using electron microscope technique ( A m a k o et al., 1977) (Fig. 3). Figure 3 (A) shows the control profile o f U F X - 2 2 in which coliform group bacteria did not grow and (B) shows the colonies o f coliform group bacteria that grew in foam cavities and the gas spaces in upper area o f the individual cavity, (C) is a higher magnification o f a part o f (B). Moreover, only colonies o f Gram-negative bacteria were detected on Gram-staining s t a m p smears o f a section o f the foams (data not shown).

Table 3. Comparisonbetweenthe resultsby the Durham fermentationtube method and the polyurethanefoam method in the combinationtest Method

Water dilution series 10=~

1

10 2

Total

Gas production in Durham tube

+ (+) 40 0

0

+ (+) 21 8

11

+ 3

(+) l

36

+ (+) 64 9

47

UFX-12: flotation : no flotation

40 0

0 0

0 0

21 0

5 3

0 11

3 0

0 l

0 36

64 0

5 4

0 47

Gas production in D u r h a m tube

+ 40

(+) 0

0

+ 22

(+) 6

12

+ 4

(+) 3

-

+

(+)

33

66

9

45 0 45

UFX-22: flotation : no flotation

40

0

0

22

6

0

4

3

0

66

9

0

0

0

0

0

12

0

0

33

0

0

( + ) Small volume of gas production as pin-point. WR 2 5 / ~ H

-

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EIKO NAGAMACHIand YASUHIROKANEMASA

Table 4. Profile of positive tubes in the parallel study of the Durham tube method and polyurethane foam method with the same river water samples

A B C D E F G H 1 J

|o •

,d

Water dilution series

Water sample

10 5.

Method

1

Durham

5 5 5 5

UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22 Durham UFX-22

5 5 5 5 5 5

:q,

t0 -t 10 2 10-3 10-4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 4

4 3 5 5 5 5 3 2 5 5 5 3 2 1 3 1 3 2 0 1

0 1 1 1 2 3 0 0 2 2 1 l 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0

1.3 x 104 1.1 × 104 3.5 × 104 3.5 × 104 4.9 × 104 7.9 x 104 7.9 x 10~ 4.9 x 104 4.9 x 103 4.9 x 103 3.3 × 104 1.1 x 104 4.9 × l03 3.5 x 10 3 7.9 x 10 3 1.7 x 103 7.9 × 10 3 4.9 × 10 3 2.4 X 10 3 1.7 × 103

(4) The results of parallel study with the same river water samples M P N s o f v a r i o u s river w a t e r s a m p l e s were determ i n e d b o t h by t h e D u r h a m f e r m e n t a t i o n t u b e m e t h o d a n d by t h e U F X - 2 2 m e t h o d ( T a b l e 4). C o m parable results with the Durham fermentation tube m e t h o d were o b t a i n e d in t h e U F X - 2 2 m e t h o d , w i t h t h e s a m e river w a t e r s a m p l e s .

(5) Comparison of the MPNs between the tests with the Durham fermentation tube and the best one of /bamed polyurethanes (UFX-22) in field surveys T h e s a m e s a m p l e s f o r field s u r v e y were u s e d for testing the comparability between the UFX-22 method and the Durham fermentation tube method. T h e w a t e r s a m p l e s u s e d in t h e r o u t i n e t e s t i n g w i t h t h e D u r h a m f e r m e n t a t i o n t u b e s were tested w i t h t h e f o a m e d p o l y u r e t h a n e . T h e r e s u l t s o b t a i n e d by t h e D u r h a m f e r m e n t a t i o n t u b e m e t h o d were u s e d for c o m p a r i s o n . T a b l e 5 s h o w s a p a r t o f t h e m a n y results, of which both values had forgivable deviations from the concept of MPN. So f a r 100 river w a t e r s a m p l e s h a v e b e e n tested w i t h g o o d results. T h e c o r r e l a t i o n b e t w e e n t h e v a l u e s o b t a i n e d by t h e D u r h a m f e r m e n t a t i o n t u b e m e t h o d Table 5. Comparative study of both methods in the field surveys Water sample A B C D E F G H

•

MPN i

104.

E

~¢~ 103"

e •

e,

E •

102 102

•

•

•

{ ~ 1 4671 X ;''~45s°

103

104

105

MPN by Durham tube rnethod

Fig. 4. Correlation between MPNs obtained by the D u r h a m fermentation tube method and UFX-22 method.

a n d by t h e U F X - 2 2 m e t h o d is s h o w n in Fig. 4 (the d a t a in T a b l e s 4 a n d 5 were also statistically a n a l y z e d in Fig. 4 t o g e t h e r w i t h t h e o t h e r d a t a ) . T h e c o r r e lation coefficient w a s f o u n d to be very h i g h (0.930). In t u r n , s o m e w a t e r l a b o r a t o r i e s h a v e b e e n a s k e d to test t h e f o a m e d p o l y u r e t h a n e in their r o u t i n e w a t e r e x a m i n a t i o n s a n d a g a i n c o m p a r a b l e r e s u l t s were o b t a i n e d with t h e c o n v e n t i o n a l D u r h a m f e r m e n t a tion t u b e m e t h o d . T h e s t a n d a r d m e t h o d o f t h e M P N test for colif o r m g r o u p b a c t e r i a in w a t e r i n c l u d e t h e p r e s u m p t i v e test u s i n g lactose b r o t h a n d t h e c o n f i r m a t i v e test u s i n g B G L B b r o t h in t h e p a s t . In a l m o s t all o f t h e c o u n t r i e s as well as t h e E n v i r o n m e n t A g e n c y o f J a p a n , h o w e v e r , o n e step test u s i n g o n l y B G L B b r o t h h a s recently been a d o p t e d as r o u t i n e . T h e r e fore, we d e s c r i b e d o n l y t h e r e s u l t s o b t a i n e d by u s i n g t h e B G L B b r o t h . W e h a v e tried u s i n g U F X - 2 2 in l a c t o s e b r o t h a n d h a v e o b t a i n e d c o m p a r a b l e r e s u l t s with t h e D u r h a m f e r m e n t a t i o n t u b e m e t h o d . CONCLUSION In c o n c l u s i o n , o n e o f t h e f o a m e d s y n t h e t i c p o l y m e r s o f p o l y u r e t h a n e , U F X - 2 2 , w a s f o u n d to be t h e m o s t s u i t a b l e for t h e d e t e c t i o n o f g a s p r o d u c t i o n in coliform group bacteria determination and the results were c o m p a r a b l e to t h o s e o b t a i n e d b y t h e D u r h a m fermentation tube method.

MPN Durham tube 1.3 x 104 3.5 x 104 3.5 x 104 1.3 x 103 3.3 x 103 3.3 x 103 1.4 x 103 9.2 x 104

1

1.3 x 103

J K L

2.7 x 103 1.7 x 104 2.2 x 104

UFX-22 1.3 x 104 5.4 x 104 3.5 x 104 2.2 x 10~ 3.3 x 10~ 4.9 x 103 1.7 x 103 9.2 x 104 2.4 x 103 4.9 x 103 1.7 x 104 7.9 × 10 3

Acknowledgement--The authors wish to thank the Japan Exlan Co. Ltd for providing the newly developed foamed synthetic polymers. REFERENCES A m a k o K. and Umeda A. (1977) An improved method for observing bacterial growth by the Scanning Electron Microscope. J. Electron Microsc. 26, 155-159. Geldreich E. E. (1985a) Standard Methods for the Examination of Water and Wastewater, 16th edition, pp. 870-886. APHA, A W W A , WPCF, American Public Health Association, Washington, D.C.

Detection of gas production by coliforms Geldreich E. E. (1985b) Standard Methods for the Examination of Water and Wastewater, 16th edition, pp. 886-902. APHA, AWWA, WPCF, American Public Health Association, Washington, D.C. Ginn R. E., Packard V. S. and Fox T. L. (1984) Evaluation of the 3M dry medium culture plate (Petrifilm SM) method for determining numbers of bacteria in raw milk. J. Fd Protect. 47, 753-755. Ginsburg W. (1985) Standards Method for the Examination of Water and Wastewater, 16th edition, pp. 860-870 APHA, AWWA, WPCF, American Public Health Association, Washington, D.C.

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Hoskins J. K. (1934) Most Probable Numbers for evaluation of Coli-Aerogenes tests by fermentation tube method. Publ. Hlth Rep. 49, 393-405. Nagamine Y., Ikedo M. and Ishizuka I. (1982) Studies on the test of gas production by bacteria. Rinsho to Saikin (Clinics and Bacteria) 9, 75-78 (in Japanese). Peterson J. (1974) Comparison of MF technique and MPN technique for the estimation of coliforms in water. Publ. Hlth Lab. 32, 182-193. Wilson G. (1983) Principles of Bacteriology, Virology and Immunity (Edited by Wilson G. and Dick H. M.), 7th edition, Vol. 1, pp. 260-278. Arnold, London.