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Flavobacterium Aquatile and Flavobacterium Meningosepticum: Glucose Nonfermenters with Similar Flagellar Morphologies

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Flavobacterium Aquatile and Flavobacterium Meningosepticum: Glucose Nonfermenters with Similar Flagellar Morphologies INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1979, p. 333-338 Vol. 29, No. 4 ~~0-7713/79/04-0333/05$O2.OO/O Flavobacterium aquatile and Flavobacterium meningosepticum: Glucose Nonfermenters with Similar Flagellar Morphologies JOHN A. WEBSTER AND RUDOLPH HUGH Department of Microbiology, School of Medicine and Health Sciences, George Washington Uniuersity, Washington, D.C.20037 Flavobacterium aquatile (Frankland and Frankland) Bergey et al. 1923, the type species of the genus Flavobacterium, was originally described as motile and later defined as a nonmotile glucose fennenter. The neotype strain of F. aquatile, ATCC 11947, is flagellated, spreads in semisolid agar, and produces oxidative acidity from glucose. Flavo bacterium meningosepticum King 1959 was reported to be nonmotile and both oxidative and fermentative. The type strain of F. meningosepticum, ATCC 13253, is flagellated, spreads in semisolid agar, and produces oxidative acidity from glucose. The flagellar morphologies of the two strains are similar. The genus Flavobacterium should not include species which ferment glucose. Flavo bacterium aquatile (Frankland and supplement. Casitone broth contains 2 g of Casitone, Frankland) Bergey et al. 1923, the type species 1 liter of distilled water, and thiamine supplement. A of the genus Flavobacterium, is motile according 5-ml amount of Seitz-filtered thiamine hydrochloride to Bergey et al. (2). However, the neotype strain (0.01 g/ml) was added aseptically to 1 liter of sterile, of aquatile (26) is nonmotile and nonflagel- molten, semisolid Casitone motility medium and to F. sterile Casitone broth. A l-ml amount of Formalin lated according to Weeks (29), and accordingly (38%formaldehyde) was added to each 4 ml of Casi- the species is described as nonmotile (30,31). tone broth culture. Formalinized cultures were centri- Flavobacteriurn meningosepticurn King 1959 fuged, washed in distilled water, and stained by the is reported to be nonmotile (17,28,31), but the method of Leifson (19). type strain appears to be motile. Flavobacte- OF media. Bromothymol blue OF (BTB-OF) me- rium Bergey et al. 1923 is suggested to be fer- dium contains 2 g of Casitone, 0.3 g of K2HPOs, 0.08 g mentative (28), and indeed the genus is included of bromothymol blue, 2 g of agar, and 1 liter of distilled among the fermentative bacteria in Bergey’s water. The composition of phenol red OF (PR-OF) Manual (31). King (17) reports that menin- medium is the same as that of BTB-OF medium, F. except that 0.03 g of phenol red replaced the bromo- gosepticum has “a predominantly oxidative type thymol blue. These media were modified by the asep- of utilization of glucose” in oxidative-fermenta- tic addition of 5 ml of Seitz-filtered thiamine hydro- tive (OF) medium. chloride (0.01 g/ml) to 1 liter of molten medium. Seitz- The neotype strain of F. aquatile, RH 3019, filtered 10% glucose was added aseptically to sterile and the type strain of F. meningosepticum, RH BTB-OF, PR-OF, their modifications, and OF basal 540, were studied with respect to spreading in medium (15) (catalog no. 0688;Difco Laboratories) to semisolid agar medium, flagellar morphology, a final concentration of 1%.The final pH of media and glucose metabolism in an attempt to resolve containing bromothymol blue was 6.8. The final pH of these conflicting observations and deductions. media containing phenol red was 7.3. Stiff petrolatum, sterilized at 170°C for 2 h, was used to seal media in Photomicrographs record the flagellar morphol- tubes. ogies. RESULTS MATERIALS AND METHODS The bacterial strains used in this study are Bacterial strains. The strains of F. aquatile and described in Tables 2 and 3. They were main- F. meningosepticurn used in this study and their cor- tained in a freeze-dried state. responding strain numbers are shown in Table 1. F. aquatile RH 3019 and F. meningosepticum Strain RH 3019 was received from the American Type RH 540 spread from the line of inoculation in Culture Collection, Rockville, Md. (ATCG) on 29 No- semisolid Casitone motility medium. Diffuse vember 1976. Strain RH 540 was received from E. 0. King on 28 March 1958. spreading growth of strain RH 3019 became Motility medium and flagellum stain. Semisolid apparent after 3 days of incubation at 22°C. Casitone motility medium contains 2 g of Casitone, 3 After 3 days of incubation at 35”C, the spreading g of agar, 1 liter of distilled water, and thiamine growth of strain RH 540 from the line of inoc- 333 334 WEBSTER AND HUGH INT. J. SYST.BACTERIOL. TABLE1. Corresponding strain numbers of the TABLE3. Characters of F. meningosepticum neotype strain of F. aquatile and the type strain of RH 540 ~~ F. meningosepticum Character Occurrence Corresponding strain no. of: Gram-negative rods ........... Yellow pigment ................ Collection" F. aquatile F* meningo- septicum Indophenol oxidasen ........... RH 3019 RH 540 Catalase ...................... Indoleh ..................... F36h Weeks Deoxyribonuclease ............. King 14 Oxidative acidity produced in:' 11947 13253 ATCC OF glucose .............. 8694 NCIB OF lactose ................. 9758' 10016 NCTC OF maltose ................ NCIB, National Collection of Industrial Bacteria, OF basal medium control ... Torry Research Station, Aberdeen, Scotland; NCTC, N,N-dimethyl-p-phenylenediamine monohydro- National Collection of Type Cultures, Central Public chloride. Health Laboratory, London, England. 'Casitone at 1% with 0.5% DL-tryptophan. Indole ' The following designations apparently are used for was detected with Kovacs reagent. descendants of the organism isolated by Taylor: F36 OF, OF basal medium (Difco). Carbohydrate con- (29), Taylor (291, Taylor 36 (31), Taylor F36 (see centrations were 1%. reference 1). "The numbers in parentheses indicate the number ' See reference 21. of days required for the detection of oxidative acidity. TABLE 2. Characters of F. aquatile RH 3019 Character Occurrence F. aquatile RH 3019 was incubated for 4 days at 22°C to obtain cells for flagellum staining. Gram-negative rods ............ + The flagellar morphology of strain RH 3019 is Yellow pigment ................ + shown in Fig. 1 and 2. F. meningosepticum RH Indophenol oxidasen ............ + Catalase ....................... + 540 was incubated for 4 days at 35°C to obtain Casitone broth with thiamine sup- cells for flagellum staining. The flagella of strain plement ..................... + (ah RH 540 are shown in Fig. 3 through 5. The Growth in brain heart infusion flagellar morphologies of the two strains were broth' ..................... similar. Most flagellated cells had a short, Oxidative acidity produced in:" straight flagellum on a pole or lateral surface PR-OF glucose ............. (Fig. 1 through 4). A few cells had a longer PR-OF lactose ............. flagellum at a pole (Fig. 5) or on a lateral surface. PR-OF maltose ............ The longer flagella sometimes had a greater PR-OF basal control ........ curvature and occurred less frequently than did N,N-dimethyl-p-phenylenediamine monohydro- the short, straight flagella. Cells with two or chloride. more flagella per cell were not encountered in The number in parentheses indicates the number either strairi. Flagella were seen on only a few of days of incubation required for the appearance of turbidity. cells of both strains. ' Difco catalog no. 0037. F. meningosepticum RH 540 grew in OF basal " PR-OF, basal medium with thiamine supplement. medium (Difco) at pH 6.8. The glucose medium Carbohydrate concentrations were 1%. near the top of the column in the open tube 'The numbers in parentheses indicate the number became distinctly yellow after 2 days of incuba- of days required for the detection of oxidative acidity. tion at 30"C, whereas the indicator in the me- dium under a petrolatum seal showed no signifi- ulation was less diffuse than that of strain RH cant change in color. After continued incubation, 3019. Strain RH 540 spread more rapidly at 35°C the medium under the seal became slightly yel- than at 22°C. The rate and degree of spreading low, but a distinct yellow color, as seen in the of both strains in semisolid Casitone motility open tube, did not develop. Inoculated basal medium was different from that of typical, mo- medium without glucose became deep blue near tile Pseudomonas aeruginosa strains which the top of the column in the open tube, whereas spread rapidly from the line of stab inoculation the pH indicator in the medium under the pe- within a few hours at 22°C. Although the Gard trolatum seal did not change color. The results plate technique (10, 14, 16) usually permits the of these experiments on strain RH 540 are shown selection of motile cells from a predominantly in color in reference 16. The type strain of Aci- nonmotile population, it did not promote spread- net0 bacter anitratus, ATCC 19606, oxidizes glu- ing or enhance the recovery of flagellated cells cose and produces the three control reactions in strains RH 3019 and RH 540. like strain RH 540. VOL. 29, 1979 F. AQUATILE AND F. MENINGOSEPTICUM 335 F. aquatile RH 3019 did not grow in OF basal medium (Difco), but it grew in PR-OF medium supplemented with thiamine. PR-OF glucose medium near the top of the column in an open tube became distinctly yellow after 2 days of incubation at 30°C, whereas the indicator in the medium under the petrolatum seal became slightly yellow. Inoculated basal medium with- out glucose became red near the top of the column in an open tube, whereas the pH indi- cator in the medium under the petrolatum seal did not change color. BTB-OF medium supple- mented with thiamine did not support the growth of strain RH 3019. F. meningosepticum RH 540 grew well in PR- OF and BTB-OF media. Although thiamine was not necessary for growth, it decreased the incu- bation time necessary for the indicators in these glucose-containing media to become yellow. After 1 day of incubation at 30°C, both glucose media supplemented with thiamine became yel- low at the surface.
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