Improved method for phenotypical characterization of marine bacteria

Improved method for phenotypical characterization of marine bacteria

Journal of Microbiological Methods 13 (1991) 231 - 241 231 © 1991 Elsevier Science Publishers BN. 0167- 7012/91/$ 3.50 MIMET 00432 improved method ...

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Journal of Microbiological Methods 13 (1991) 231 - 241

231

© 1991 Elsevier Science Publishers BN. 0167- 7012/91/$ 3.50 MIMET 00432

improved method for phenotypical characterization of marine bacteria Geir H o v i k Hansen and Roald Sorheim Department of Microbiology and Plant Pkysiology, University of Bergen, Bergen, Norway (Received ?u November 1990; revision received 2 April !991; accepted 8 April 1991)

Summary A characterization system for marine bacteria using Difco Marine Broth 2216 and disposable 24-well tissue culture plates has been developed. Incubation was oerformed at desired temperature for up to 4 wk. Where anaerobic conditions were required, the plates were incubated under N 2 atmosphere. The nmjority of tests were based on colorimetric changes in the growth medium, the system allowing accurate reading of the tests, and thus minimizing the degree of subjectivity. This characterization system was compared with two commercial diagnostic kits, API 20E and API 20NE. The multidish system proved to be far mere suitable than the commercial kits in characterization of marine bacteria, both fish pathogenic and nonpathogen, and has applications both for numerical taxonomy and for diversity studies of bacterial communities.

Key words: API 20E; API 20NE; Characterization; Difco Marine Broth; Marine bacterium; Multidish method

Introduction In studies concerning ecological aspects of microbial activity; it is often necessary to do a phenotypic characterization of pure cultures of isolated bacteria. Such characterizations have mainly been performed by use of conventional testmedia and diagnostic kits [e.g., 1-4]. These methods are often unsuited for characterization of environmental isolates such as marine bacteria. The prevailing majority of testmedia [5- 7] and diagnostic kits, e.g., API, Minitek, Enterotube, OXI/FERM and MICRO-ID, are designed for use on common clinical bacterial specimens, often of human origin. As marine bacteria generally are more slowgrowing and biochemically versatile, many of them will not be able to grow, or will only show a faint growth, in these media. In addition many of them have demands for various salts and growth factors which are not satisfied by adding simple compounds (e.g., NaC1) to the testmedia. In many instances these demands can be satisfied by using seawater or artificial seawater in the Correspondence to: R. Sorheim, Center for Industrial Research, PO Box 124, Blindern, G314 Oslo 3, Norway.

232 media. Another factor of importance is the incubation temperature. Conventional tests are mostly designed for use at 37 °C, while the optimal temperature for most marine bacteria will be in the range of 12-20 °C. In addition, commercially available diagnostic kits are designed to be scored after a defined incubation time, usually 2 4 - 4 8 h. This short incubation time will often be insufficient for marine bacteria to express their phenotype to an observable degree. Various workers have developed miniaturized systems for characterization of bacterial isolates from different habitats, i.e., soil [8], milk 19, 101, ~reshwater [111, and clinical isolates [12, 13], but to the authors' knowledge no reports a:'e known concerning the use of a single medium as a base for a characterization system intended for use on marine bacteria. In this work we present a dynamic test system for marine bacteria, based on the use of multiwell trays, and Difco Marine Broth as a basal medium for 23 biochemical tests well suited for marine bacteria. Materials and Methods

General outline of ihe method Bacto Marine Broth (Difco) or agar was supplemented with various substrata and distributed into 24-well disposable polystyrene tissue culture plates (Gieiner, FRG, and Nunc, Denmark). The trays contained the same medium in a!! w'qls. Colonies of pure cultures were suspended in autoclaved 70% seawater. The method al '~-,wed a maximum of 23 strains to be inoculated at the same time onto different media by means of a multipoint inoculator. The trays were incubated at 15 °C for different time periods depending on the test assayed. Some of the tests were incubated anaerobically in a sealed glass container in which air was exchanged with N2 flushed (15 min) through a hypodermic needle that penetrated a rubber stopper in the wall of the container. Inoculation of multidishes Bacterial cultures grown for 5 - 7 days (15 °C) on Bacto Marine Broth 2216 supplemented with 1.5070 Bacto agar were suspended in 1 ml sterile 70% seawater in multidishes by means of sterile tooth picks. Bacterial suspensions used for inoculation contained 106-108 bacteria.m1-1. The multidishes were inoculated by depositing drops of the bacterial suspensions on ti~e agar surface or by stabbbag to the bottom of the wells by means of a multipoint inoculator. The tests were performed at least three times for each bacterial isolate. Washed agar Where solid media were used, 1.5°/0 Bacto Agar (Difco) was added. The agar was washed five times in distilled water, cen*.rifuged and frozen as a wet paste with a water content of = 90%. The agar was kept at - 2 0 °C until use. Biochemical tests Bacto Marine Broth 2216 (Difco) was used as basal medium for all tests exceot for the Simmon's citrate test. The concentrations given below are final concentrations in the media. A volume of 1 ml medium was used.well -l. Except for nitrate reductase, Voges-Proskauer, indole and methyl red tests, media were solidified with 1.5% washed

233 agar. Unless otherwise stated, the incubation temperature was 15 °C. APl 20E and API 20NE (Analytab Products, Moatalieu-Vercieu, France) were used for comparison. Autoclaved 700/o seawater was used as suspending medium for API 20E and for the first eight tests in AP! 20NE. API AUX medium used for inoculation of the remaining 20NE tests was supplemented with a 30°70 sterile NaCI solution to a final concentration of 1.5070. The chemicals used were of analytical grade, al~d unless otherwise stated, they were from Merck. Basal medium, Difco Marine Broth, was prepared according to the manufacturer's recommendation with the addition of filtering the medium through filterpaper (Whatman no. 1) after boiling, in order to remove possible precipitate. Removal of the precipitate had no effect on bacterial growth.

Metabolism of glucose. Oxidative/fermentative metabolism of glucose was assayed in basal medium supplemented with 0.001070 phenol red, 0.05°7o Tris (Sigma Chemical) and agar [141. After autoclaving, a sterile filtered (0.22/xm) 20070 glucose solution was added until a final concentration of 1070,and pH was adjusted to 7.6. Inoculated mu!tidishes for assaying oxidative metabolism were incubated in a temperature regulated incubator under air. Multidishes for assaying fermentative metabolism were incubated under N 2 atmosphere to ensure anaerobic conditions. None of the strains produced acid in the basal medium without extra C source. The fermentation tests were read after 7 days, the oxidative tests were inspected daily and read after 5 days. Aerobic production of acid from carbohydrates. Acid production from a diverse range of carbohydrates and carbohydrate derivatives was assayed in the same way as for the oxidative metabolism of glucose. The following substrata were used: fructose, sucrose, mannose, mannitol, glycerol, ribose and N-acetyl-glucosamine. The substrata were added to a final concentration of !°70as described under Metabolism ojglucose. pH was adjusted to 7.6. The multidishes were inspected daily for 14 days. Daily inspection of these tests are necessary because a reversible pH change was observed in some strains after 3 days of growth on some of the sugars. Such strains probably attack the peptone in the medium when the sugar content of the medium is exhausted leading to an increased pH. Amino acid decarboxylase and dihydrolase tests. Breakdown of L-arginine, L-!ysine and L-ornithine to alkaline amines was detected by using basal medium supplemented with 0.001070 phenol red, 0.000507o pyridoxal-HCl (Sigma Chemical) and agar [6]. After autoclaving, a sterile filtered 10070 solution of the appropriate amino acid was added to a final concentration of 1%; and glucose to a final cor~centration of 0.0107o. The pH of the medium was adjusted to 6.7 giving an orange/pale red medium. The wells were overlayed with 1 ml sterile liquid paraffin in order to inhibit diffusion of NH3 from one well to another, and were incubated under N2 atmosphere. The tests were read after 10 days and positive tests gave rise to a bright red colouration of the medium. Gelatinase. Gelatinase activity was assayed in basal medium supplemented with 0.5°/0 gelatin (Difco) [15] and agar. After 10 days the test was read, overlaying the wells with 0.5 ml of a 15°'/0 acidic HgCI 2 solution. Positive reaction was revealed by clearing of the medium after 45 min.

2a4

Amylase. 1070soluble starch and agar were added to the basal medium [6]. The medium was autoclaved for 10 rain at !15 °C. Higher temperature might hydrolyze the starch. The test was read after an incubation period of 10 days by flooding the wells with 0.5 ml kugol's iodine [5] di!uted 1:5. Positive reaction was demonstrated by a co]ourless zone around the colony in a dark blue background.

Chitinase. Colloidal chitin (Sigma Chem:cal) was prepared according to Herwig et al. [16]. Before autoclaving, the basal medium was supplemented with 50 g wet chitin paste.l-~ (10% wet wt) and agar [! 5]. The test dishes were inspected weekly and read after 4 wk. Strains exhibiting chitinase activity gave rise to a clearing of the medium. [3-Galactosidase. The method for assaying ~-galactosidase activity was modified from Sambrooke et al. [17]. Basal medium with agar was supplemented with 0.004% 5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside (x-ga',) ~Sigma Chemical), 0.18 mM isopropyl-/3-D-thiogalactopyranoside (IPTG) (S'.'gma Chemical) and 0.2070 glucose after autoclaving. Stock solutions of 40 mg x-gal in 2 ml dimethylfor~ amide and 0.1 M IPTG in distilled water were used. ~-galactosidase positive strains gave rise to bright blue bacterial growth. The test was read after 7 days. Urease. The urea medium of Christensen [6] was modified by using basal medium containing 0.01o70 phenol red and agar. pH was adjusted to 6.7. After autoclaving and cooling to 50 °C, sterile filtered 20°70 urea and 20°70 glucose were added to final concentrations of 2 and 0.02070, respectively. After 24 h growth, the wells were overlayed with 1 ml sterile liquid paraffin in order to avoid NH3 from escaping, and to hamper the diffusion of NH 3 into neighbouring wells. Urease positive strains gave rise to a bright red colouration of the medium due to NH3 production, and the test was read after an incubation period of 10 days.

(Dift.o) and 0.965 ~0 methy' greei~ [18]. Aiternatively medium without methyl green was used. The test was th,'n read after overlaying the wells with 1 N HCI [19]. The tests were read after an incubation period of 2 wk. DNase activity resulted in bleaching of the medium containing methyl green and in clearing of the medium without methyl green when .flooded with HC1.

Degradation of Tween 80. Test for lipolytic activity was assayed in basal medium containin~ agar and ~% Tween 80 [20]. Concentrated Tween 80 was au~oclz,-ed. separately and added aseptically to the basal medium after autoclaving. The test was read after an incubation period of 14 days. Positive strains gave rise t'~ an opaque precipitation in the medium due to Ca salts of oleic acid. Simmon's citrate. The Simmon's citrate medium [6] was modified by using artificial sea water (Ultramarine, Waterlife Research Middlesex, UK) as a base; omitting the dipotassium hydrogen phosphate and the magnesium sulphate in the original Simmon's citrate medium. Double-strength artificial seawater and a solution of trisodium citrate, bromthymo! blue and agar was autoclaved separately and mixed at 50 °C in order to

235 avoid precipitation. Positive reaction gave rise to a b:-~ghtblue colouration of the medium. The test was read after 10 days.

Nitrate reductase. The reduction of nitrate to nitrite was assayed in liquid basal medium containing 0.10/0 potassium nitrate [20]. After an incubation period of 7 days, the test was read overlaying the wells with a mixture of sulphanilic acid and ~naphthyiamine [5]. Positive strains develop a bright red colour within 2 - 3 rain. Powdered Zn was added to negative wells. This will eventually reduce remaining nitrate to nitrite. Development of a red co!our within 3 rain confirms a negative nitrate reductase reaction. Voges-Proskauerandmethylred. These tests were performed in liquid basal medium containing 1% glucose. Multidishes for the Voges-Proskauer test were overlayed with a mixture of u-naphthol and alkaline creatine, 0.2 and 0.1 ml.we!!-:, respectively [15]. Positive reaction was read as development of a cherry-red colour within 15 rain. The wells in the methyl red dishes were overlayed 0.2 ml of an ethanolic methyl red solution [5]. Methyl red-positive strains developea instantaneously a bright red colour. For both the Voges-Proskauer and the methyl red tests an incubation period of 7 days was used. Production of indole from tryptophane. Liquid basal medium with 0.04°70 Ltryptophane added was used. The test was read after 7 days overlaying the wells ~ t ~ 0.2 ml Kovac's reagent [5]. Positive strains developed a bright red colour within 20 rain. Bacterial reference strains The following reference strains were used. Vibrio anguillarum NCMB 6 and NCMB 2129; E ordalii NCMB 2167 and MSC2-75; E sc,lmonicida NCMB 2262 and NCMB 2245; E fischeri ATCC 7744; Aeromonas hyd:ophila NCMB 1105; A. ~aimonicida NCMB !!02; Yersinia ru~eri ATCC 29473; Edwardsiella tarda ATCC 15947; Proteus vuigaris IMP i i 6 - 1 ; Pseudomonasputida NCMB 1960; P..fluorescence NClVIB 1953; Cytophaga latercula ATCC 23177; Flexibacter iitoralis ATCC 23117; Flavobacterium balustinum ATCC 33487; Alteromonas denitrificans ATCC 43337 and Leucothrix mucor ATCC 25107. NCMB, National Collection of Marine Bacteria, Aberdeen, UK; ATCC, American Type Culture Collection, Maryland, USA; IMP, Culture Collection at Department of Microbiology and Plant Physiology, University of Bergen, Norway. Source of MSC2-75, U~iversity of Oregon, USA. Results

Results of the biochemical characterization of reference strains are presented in Table 1. There were no marked differences in test results Owingto mode of inoculation, but surface inoculation is recommended, minimizing the possibility of nutrien~ contamination from one medium to another when inoculating series of dishes. Because of the incubation temperature (15 °C) and time (5 days to 4 wk), the density of the inoculum was not critical. Variations in the order of 102-103 bacteria.ml -~ did not influence the results.

236 TABLE

I

RESULTS

OF BIOCHEMICAL

CHARACTERIZATION

OF REFERENCE

STRAINS

USING THE

MLILTIDISH TEST SYSTEM Test

1"

2

3

4

5

6

7

8

9

10

I1

12

13

0 gIc ~* Fglc

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+

+

NCglc

+

Acid from Fructose Sucrose Mannose

+

+

+

+

+

+

+

+

+

+

+ +

+

+

+

1-

Mannitol

+

+

+

+

Glycerol Ribose

q +

+ .4-

+

+

+ +

+ +

+ +

N-Ac-glucosam~ne

+

-,-

+

+

+

+

+

+

+

A D H *~-*

+

+

+

+

Amylase

+

Chitinase ¢3-Galactosidase Urease DNAse

+

+

Lipase(Tween80) Simmon's citrate Nitratereductase

+

+

+ +

+ +

-

Voges-Proskauer

+

+

+

lndole

+

+

Methyl red

-

+

~t,

+

+

+

-

+ +

_

4-

-

+

-

+

+

+

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

_

+ +

+

+

+

+

+

+

+

+

+

+

+

+ +

+

+

+

+ +

+

+

+

+

-

N C M B 6; 2, N C M B 2 1 2 9 ; 3, N C M B 2 i 6 7 ; 4, M S C 2 1105; 9, N C M B

+

+ +

+ +

+

+

+

+

+

+ +

+

+

-

+

-

+

+

+

-

+

+ +

+

+ +

-

+ +

+

19

+ + +

+

4

18

+

_

-

+

17

-

+

+

+

16

+

+ +

Gelatinase

15

~

+

LDC ODC

7 7 4 4 ; 8, N C M B

14

+

-

+

+

+

+

+

+ +

+

+

+

+

+

+

_

+

+

~_

+

+ +

+ +

+

+

+

+

75; 5, N C M B 2 2 6 2 ; 6, N C M B 2 2 4 5 ; 7, A T C C

1102; 10, A T C C 29~-73; l l0 A T C C

15947; 12, N C M B

1960; 13, N C M B

1953; 14, A T C C 2 3 1 7 7 ; ! 5, A T C C 2 3 1 1 7 ; 16, A T C C 3 3 4 8 7 ; 17, A T C C 2 5 1 0 7 ; 18, I M P 1 1 6 - I; t 9 , A T C C 43337.

oxidative metabolism o f in glucose medium. **O glc,

glucose; F glc,

fermentative metabolism of glucose; N C

glc, n o p H

change

* * * A D H , a r g i n i n e d i h y ~ r o ! a s ~ ; LDC~ l y s i n e d e c a r b o × y l a s e ; O D C , o r n i t h ! n e d e c ~ r b o ~ y ! ~ s e

~.',etabolism of carbohydrates. The multidish system gave results in accordance with the literaturc• in the test for fermentative metabolism of glucose. When using the same medium in tubes or multidishes with liquid paraffio, seal, as much as 2 0 - 30°'/0 of the isolates gave intermediate colour change in the medium. Hence, conventional use of liquid paraffin or mineral oil as a seal to obtain anaerobic conditions, does not provide sufficient reduction in the 02 level, leading to a variety of shades from red to yellow when reading the test. Use of N 2 atmosphere gave clearcut positive or negative reactions. With respect to fermentation of glucose, the API 20NE system scored < 21% positive, whereas the mu!tidish system scored 63% positive (Table 2). This is in agreement with the literature. The test for oxidative metabolism of glucose gave in the multidish syste~ a positive score of 73% (Table 2), which is ill accordance with the litera-

237 TABLE 2 P E R C E N T A G E P O S I T I V E R E S P O N S E S IN S O M E O F "~ ~ h~,--o . ... . ~. .'.~ T , ~ p ~ TESTS IN T H E "~ 'Mv, o .i . . ., .. ~ T H R E E T E S T SYSTEMS ( N = 19) Test /3-Galactosidase ADH LDC ODC S i m m o n ' s citrate Urease lndole Voges - P r o s k a u e r Gelatinase Glucose m e t a b o l i s m Fermentative Oxidative A e r o b i c acid p r o d u c t i o n Mannitol Mannose Sucrose ;'~" Ac-glu Nitrate reductase

API 20E

A P ! 20NE

Multidish

,~l . ~

gi .U

15.8 -

21.0 10.5 10.5 0 10.5 26.3 15.8 15.8

26.3 15.8 26.3 31.6

31.6 15.8 15.8 31.6 10.5 21.0 36.8 68.4

42.1

21.0 4 9 1*

63.0 73.7

52.6

15.8" 26.3* 47.4

31.6 42.! 21.0 57.9 52.6

-

-

2! .0 26.3

* M e a s u r e d as assimilation (growth).

ture. Using the API 20E and API 20NE, < 4 2 % of the isolates couid be scored as positive (Table 2). Comparing aerobic metabolism e f some of the carbohydrates meas~'.r~3 ~e ~ee;..r~. ,.'l.~t:,,', !,r.~,.,-rth~ ,.'~ __ADT

9N.N.T~

,=,;÷h " * r ~ h ; -

too;."] nrn~_~lot~nn ~n t h P

_I~_[ ! ] [ l d -

ish system, the multidish system gave a significant higher share of positive responses (T~bie 2). Metabolism of m a n n k o i is the on!y carl:3hydrate test for which the A P i 20E had a higher positive score (Table 2). The A D H , LDC and ODC tests gave a significanl higher positive score in the multio.ish system than in the AP~ <9~,,-.-o. ~. . . . . . . . . . . . nitrate reductase tests gave significantlv highe,: positive scores in the muitidish system than :,- the AP! systems. Other test,~ fo~"enzymatic degradation] of macromole~.~e~, " . . . . ~ L"C " ., g-~;~'; ~ , a t n ~ a o ~., . , .. a L i y l"a s e , chitinase, DNase and lipase gave resuRs corresponding to literature vzlues~ and wc~e all easily interpretable. The only exception was the geiatinase test which might r,e difficult to read, but that is also the case in other test systems using the HgCI2 method. The two ways of testing DNase activity g we correspogding results, bu* *he ~ ' ~ " " green was easier to interpret. In addition it was labour saving and allowed examination

of the multidishes during the whole incubation period. r~iscussion Many reports have questioned the use of commercially available diagnostic kits for characterization of bacteria isolated from environmental sources [8, 10, 21-26] and

OO ¢q

TABLE 3

AP1 20E

% Identification

API 20NE

[DENT!F~CATION OF T H E BACTERIA[, STRAINS RASED ON SCORES IN API 20E a~cl API 20NE (NI, strain not identified by the Analytical Profile Index of API 20E and API 20NE) Strain

Identified as

!

44.4/4 I. 1

75.2/22.7

98.

44/41 87.3

98.1

°70 Identification

Identified as 6 2129

-

-

-

-

NI NI Pest. haemol./Pseud, vesic. Moraxe!!a sop./ Pseud. diminutu i~i

97.5

NI Pasteurella multocida NI NI NI

99.9 98.9 99.9 86.5 67.2

2245 2262

NI NI NI Edwardsiella tarda Hafnia a!vei Proteus mirabilis Pseudomonas fluorescens Pseudomonas putida

97.8 38.0 38.i 39.2

anguillarum NCMB anguillarum NCMB ordalii NCMB 2167 ordali; M S C 2 - 75 salmo °uclda NCMB salmonicida NCMB

Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio

Flavobacterium spp. Achromogenes xyiosoxidans Achromogenes xylosoxidans Achromogenes xyiosoxidans

38.0

99.6 98.5

Vibrio fischeri ATCC 7744 Aeromonas hydrophila NCMB 1105 Aeromonas salmonicida NCMB 1102 Edwardsieiia tarda ATCC 15947 Yersinia ruckeri ATCC 29473 Proteus vulgaris IMP 116 - 1 P s e u d o m o n e s f h : o ~ s c e n x NCMB 1953 Pseudomonas putida NCMB 1960 Fla vo bacterium o"~u m '- a- :-, ,.um ATCC 33487 Fiexibacter litoralis ATCC 23117 Cytophaga tatercula ATCC 23177 Alteromonas denitrificans A T C C 43335

A chromogenes xylosoxltians

Vibrio vulnificus NI NI NI Moraxella spp./ Pseudomonas diminuta V. damsela NI Nl Pasteurella multocida NI N! P. fluorescens P. putida

Leucothrix mucor ATCC 25107

239 u n c o m m o n clinical isolates [27, 28]. At least for the API 20E system, there are inconsistencies i~; the results depending on the medium in which the bacteria are suspendea [29]. Many bioch,emical tests are based on eolorimetric changes in the media due to growth or to the presence oc different enzymes. ~nterpretation of colorimctric ~ests are often highly subjective; perception of shades of colour do differ from person to person. It is therefore essential ~ha* _~given test distinguish well between positive and negative results; avoiding intermediate shades of colour. This requirement is satisfactorily met for the colofimetric tests in our test system. The use of N2 atmosphere when a~avin~ for f e r r n e n t n t ~ w m o t ~ h m l i c r n o f r , ~ r h , - ~ h , , A r . ~ , ~ e h ...... ,1-,, ; . . . . . .a curacy of this test. In addition all the test dishes could be inoculated from the same inoculation dish, allowing inoculation of =-20 strains at a time in as many replicas as desired. The multidish method based on Marine Broth has proved itself superior to the cornpaired kits when characterizing bacteria of nonclinic~ origin. Working wire environmental, nonpathogenic marine isolates, the API 20E and API 20NE do not fulfii the requirements needed_ for identification purposes. Of the 19 bacterial reference strains used in this work, we were only able to identify Edwardsiella tarda, Pseudomonas fiuorescens and P. putida to the species level in the A P I 2OE system (Table 3); the two Pseudomonas spp. scoring only 86.5 and 67.2%, respectively, when measured as percentages of identification. Proteus vulgaris and Flavob~cterium balustinur~ were identified to the right genus, while none of the vibrios or aeromonads could be identified to this level. In the API 20NE system only P. fiuorescens and P. putida were identified to the species level (99.6 and 98.5%, respectively). In this system two of the Vibrio spp. could be identified to the genus level, namely E anguillarum NCMB 2129 and V. fiseheri which were identified as ,i. vulnificus (98.1%) and E damsela (87.3%), respectively. The rest of the reference strains could not be identified or were incorrectly idontifled by the API 20E ..,,.,-"'4APT. _'~.n~-l~ . . . . ~vstems.. Using the multidish system we were able to identify all the strains to genus level and the m~;,,,-;t,,...~...~~o. ~p,~eles_..level_ ,'ons~dting_ only Bergey's manual of systematic bacteriology [30]. The mulfidish test system constitutes a flexible system, new tests may easily bc added -=-, it • ..~1~,, to the test menu, ~,,u . . . . .on . . . ..... . o t~ ...v. . .,,.... . .,-,¢ . . .both . . . !iq,._,id~semi-solid and solid media. It is well suited for both numerical taxonomical purposes and for diversity studies of bacterial communities and has beer used in several Scandinavian microbiological laboratories over the last two years. In c ombination with microscopy, Gram-reaction, and some rapid colony tests (e.g. cytochrome oxidase and catalase) it has proven to be well suited for biochemical characterization of environmental isolates.

Acknowledgements The excellent technical assistance of Marl-Ann S~elc and Synneve Hansen is greatly appreciated. The Norwegian Fisheries Research Council (Grant V 101.017) and the _ :sc~emmc Royal Norwegian Council for -' " . . r and Industrial Research (Grant ~v .~..,,,~-., arc a,zk~ow!e.4ged for *.hci" ei,~neial supoort.

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