CALIFORNIA STATE UNIVERSITY, NORTHRIDGE

PATTERNS OF GROWTH AND GLIDING ~10TILITY !t OF SIMONSIELLA AND ALYSIELLA

A thesis submitted in partial satisfaction of the requirements for the degree of Master of Science in Biology by

Gordon Emery ____Buchanan, ,/ Jr.

January, 1976 The thesis of Gordon Emery Buchanan, Jr. is approved:

California State University, Northridge December, 1975

ii ACKNOWLEDGt4ENTS

I am especially indebted to the members of my graduate committee, :or. Daisy A. Kuhn, Dr. Marvin H. Cantor and Dr. Charles R. Spotts, who :helped me in manY ways during my years as a graduate student at California State University, Northridge. I am thankful for the continual assistance and encouragement from my colleagues, David Gregory, Roland Gunther and Christie Jenkins, who also participated in the research on the taxonomy of Simonsiella and ~siella. I also acknowledge gratefully the help and advice given to me by Carla Bowman, Ruth Jung, David Oakland, Flora Price, Bernardine Pregerson, and Edward Rau.

iii TABLE OF CONTENTS

Page

List of Tables...... v List of Figures. vi Abstract. .vii

Introduction ...... • • • • • • • • • • • • • • • • • • • • • • • • • • • • ot ••••••••••••••• 1 Methods and Materials. 7

Results and Discussion. 17 Conclusions ...... 76 References ...... - ...... 80

iv LIST OF TABLES Page Table 1. Colony characteristics of Simonsiellaceae ...... l8 Table 2. Types of patterns of growth and gliding motility.; ...... 29 Table 3. Rates of gliding motility ...... •...... 36 Table 4. Effect of temperature on growth ...... 41 Table 5. Effect of temperature on patterns of growth and gliding motility ...... •...... 43

.Table6. Effect of pH on growth ...... •.... ~ ...... 46 Table 7. Effect of pH on patterns of growth and gliding motility ...... •...... •...... 47 Table 8. Effect of NaCl on growth ...... 48 Table 9. Effect of NaCl on patterns of growth and gliding mot i 1i ty ...... ·...... 51 Table 10. Effect of 11 Flexibacteria Medium 111 on growth ...... •...... 53

, Table 11. Effect of 11 Flexibacteria Medium 111 on patterns of growth and gl i d1 ng moti1 i ty ...... 55 Table 12. Effect of further modifications of 11 Flexibacteria Medi urn 1 11 on growth ...... 56 :Table 13. Effect of further modifications of 11 Flexibacteria Medium 111 on patterns of growth and gliding motility ...... 58 Table 14. Effect of omission of salts from BSTSY on growth ...... 60 'Table 15. Effect of omission of salts from BSTSY on patterns of growth and gliding moti 1i ty ...... 61

Table 16. Effect of humidity on grm.,rth and gliding motility. I. .... 63

Table 17. Effect of humidity on growth and gliding motility. !! .... 64 Table 18. Effect of agar concentration on growth ...... 65 Table 19. Effect of agar concentration on patterns of growth and gliding motility ...... 66 Table 20. Effect of used BSTSY broth on growth. 1 ....•...... 69

v Page Table 21. Effect of used BSTSY broth on patterns of growth and gliding motility. I...... 70

Table 22. Effect of used BSTSY broth on growth. !! ...... 72 Table 23. Effect of used BSTSY broth on patterns of growth and g1 i d i ng mot i1 i ty . I I...... 73

LIST OF FIGURES

Figure 1. Colony morphology types of dog Simonsiella strains ...... 22 Figure 2. Colony morphology types of human Simonsiella strains ...... 24 ,Figure 3. Colony morphology types of sheep Simonsiella strains ...... •...... 26 Figure 4. Colony morphology types of cat Simonsiella strains ...... 28

vi ABSTRACT

PATTERNS OF GROWTH AND GLIDING MOTILITY OF SIMONSIELLA AND ALYSIELLA by Gordon Emery Buchanan, Jr. Master of Science in Biology January, 1976 Forty six strains of Simonsiella and two strains of Alysiella, large filamentous, gliding bacteria from the oral cavities of warm- blooded animals, were grown under various environmental conditions in .order to study aspects of the taxonomy of these organisms. Differences in growth with changes in temperature, pH, and NaCl and synthetic sea water salts concentrations segregated the strains into several :individual groups which correlated with the isolation of the strains from dogs, sheep or humans. These findings add to the information derived from studies on the cellular morphology and on the biochemistry

:and physiology (Kuhn et ~·, 1974; Pangborn et al_., 1973 and 1974; . Nyby, 1974; Gregory, 1975), on the fatty acid profiles (Jenkins, 1976), and on the mole percent G+C content (Kuhn et .!!_., 1974). Microscopic features of gliding motility and growth patterns of Simonsiellaceae are recorded for the first time. Growth on agar viewed microscopically revealed that a small fraction of Simonsiellaceae . strains glided, whereas other strains displayed types of colony

vii morphology that ranged from entire-edged to pronounced filamentous growth. Gliding motility rates were found to range from 5 llm/min ; to 23.8 llm/min. Gliding motility was manifested by individual, frequently well-separated fi 1aments rather than by 11 armi es 11 of closely-associated cells as described by Stanier (1942a) for the cytophagas. Gliding motility appeared to occur as _necessary to ·reach areas on the agar surface supplying fresh nutrients, and ,when these areas were reached gliding motility was arrested and :secondary colony formation ensued. Gliding motility was frequently most pronounced in regions of heavy growth bordering unoccupied agar ·surfaces, suggesting that filaments glide in response to gradients of either decreasing waste metabolites or increasing fresh nutrients. The influence of other factors such as light, agar concentration and 'humid incubation are considered.

viii ~~- --- ·-· -·· - - -·· ·· · · -~ NTROilucnoN ·· ···· ····- -- ·-· · - ~~ -- - - - ···· '

1.' ! 1 i / Observations of gliding motility among procaryotic organisms were

1reported as early as the 18th century by botanists studying the morph- 1 !ology and taxonomy of the cyanobacteria (Burkholder, 1934). Experi­ i ! :mental investigations of gliding motility began in the late 19th : :century. Unusual aspects of gliding motility such as the swarming of i i"armies 11 of cells (Stanier, 1942a) in the cytophagas and in the

! :myxobacteria, and mucilaginous sheath production and axial rotation of ; i ~gliding filaments in Oscillatoria (Burkholder, 1934; Halfen and :castenholz, 1970 and 1971) have been described. Theories for the 'mechanism of gliding motility have been proposed based on (1) osmotic or surface tension gradients along gliding filaments (Burkholder, ;1934); (2) slime production (Burkholder, 1934; Dodd, 1960), and (3) :rhythmic contractile waves (Jarosch, 1962; Doetsch and Hageage, 1968). -- i ' ';Energy requirements for gliding motility have been calculated (Ha1fen .and Castenholz, 1971). Researchers have observed with transmission electron microscopy and freeze-fracture techniques cell wall features possibly related to the structural apparatus responsible for gliding :motility (Pate and Ordal, 1967; Halfen and Castenholz, 1970 and 19-71; 1Burchard and Brown, 1973). However, in spite of experimental work on 'gliding motility, the basic cellular mechanisms involved remain 'largely a mystery (Doetsch and Hageage, 1968). Vets certain observed !features of gliding motility can be summarized. Gliding motility is found among some species of both the bacteria land the cyanobacteria, the two groups of the Kingdom Procaryotae, as I joutlined in Bergey•s Manual of Determinative Bacter-iology, Eighth L_~ 1 2

:Edition (Buchanan and Gibbons, 1974). These procaryotic gliding 'organisms may be divided into the following groups: (1) the unicellular and multicellular, filamentous cyanobacteria, represented by the orders Chroococcales and Hormogonales, respectively (Desikachary, 1973); (2) the fruiting myxobacteria of the order Myxobacterales; and (3) all other non-fruiting unicellular and multicellular, filamentous bacteria composing the order Cytophagales which includes the families Cyto­ phagaceae, Beggiatoaceae, Simonsiellaceae, Leucotrichaceae and, possibly, Pelonemataceae and Achromatiaceae (Soriano and Lewin, 1965; Buchanan and Gibbons, 1974). Gliding motility may occur among some or all members of each group, or, occasionally, may be limited to certain juvenile or reproductive forms such as the hormogonia of some cyanobacteria, or the conidia or gonidia of Thiothrix and Leucothrix, respectively (Doetsch and Hageage, 1968). Gliding motility is defined as an active movement of an organism in contact with a solid substratum where there is neither a visible organ responsible for the movement nor a distinct change in the shape of the organism (Jarosch, 1962). Gliding motility among some filamentous cyanobacteria may occur even when a filament makes contact with a substrate along only a fraction of its length (Stanier, l942b), or may occur within medium solidified with 0.5% to greater than 3.5% agar (Castenholz, 1973). Gliding organisms placed in a liquid medium have occasionally shown darting, jerking or rolling motions, particularly after making contact with the side of the vessel (Doetsch and Hageage, 1968). Other workers have observed a bending or flexing of the apical portions of the filaments (Burkholder, 1934; Doetsch and Hageage, 1968). All these phenomena point to an inherent 3

.. -···-- --- .. --- --· .. -~------·· .. . ---··-----·- ·-- ... ------... ------···------1 flexibility of either the individual cells or intercellular 1 - I connections within a multicellular filament (Costerton, Murray and Robinow, 1961; Doetsch and Hageage, 1968), and suggest an inherent irritability and responsiveness for movement. Gliding motility is generally manifested as a slow, persistent, non-jerking movement resembling somewhat the progress of a snail (Castenholz, 1973). It may continue in one direction for many minutes or several hours, followed by frequent or regular reversals of direction presumably due to environmental factors (Castenholz, 1973). In spite of the slowness of gliding motility, such locomotion may convey distinct survival value over the course of time (Doetsch and 'Hageage, 1968; Castenholz, 1973). Gliding motility, which requires :direct contact with the substratum, might be necessary for effective .enzymatic activity in obtaining essential nutrients within the ;substrate (Doetsch and Hageage, 1968; Castenholz, 1973). Or such :locomot·ion possibly represents the means by which the organism ·physically secures itself within its habitat. The fastest gliders, the multicellular, filamentous cyanobacteria,

display rates of 2 ~m to 11 ~m per second (Castenholz, 1973). Hetero­ cystous and uni ce 11 ul ar cyanobacteria, myxobacteri a and cytophagas have

gliding motility rates of less than 1.0 ~m per second (Stanier, 1942a; ,Doetsch and Hageage, 1968). In contrast, flagellated eubacteria

~commonly attain speeds of 20-300 ~m per second, and vibrios apparently ;reach speeds as high as 50-200 vm per second (Castenholz, 1973). A rich production of slime is frequently associated with gliding i ,motility and, inevitably, has been suggested as the mechanism for :gliding motility (Burkholder, 1934; Doetsch and Hageage, 1968). 4

------~-----·---- .. --. -----· --· -·------·------rCyanobacteri_a_ are--particularTynoteworthy for slime secretion which i ~esults in the formation of mucilaginous sheaths, frequently completely· I ' ' ' !investing the gliding filaments (Burkholder, 1934; Doetsch and Hageage,; ! I jl968). Such sheaths can be demarcated with India ink particles (Dodd, ' :1960;' Costerton, Murray and Robinow, 1961; Walsby, 1968), and Hosoi I '(1951) reported observing the flow of slime around a cyanobacterial filament, using clarkfield microscopy. Likewise, during the formation pf fruiting bodies, myxobacterial cells glide into the developing I fruiting body structure, raising themselves up on stalks of polysac- ! charide material given off by the 11 armies 11 of such gliding organisms !(Dworkin, 1973). Other investigators, however, deny that significant, i i :if any, slime production occurs among some gliding organisms (Doetsch and Hageage, 1968). Thus, slime production varies greatly among species of gliding organisms. Where it does occur, slime may facilitate gliding by enhancing adherence to and maximizing surface contact with

~he substratum (Doetsch and Hageage, 1968).

The presence of etched 11 trails 11 or 11 tracks 11 left behind by 6rganisms gliding on agar surfaces is graphically as intriguing as mucilaginous sheath formation, and they serve as an unmistakable guide to the presence and extent of gliding motility. Whether such etched trails are the product of enzymatic activity on the agar as the

~rganism glides over the nutrient-containing agar surface, or whether they arise from a physical wearing away of the surface is an unanswered question. A number of investigators have reported on the influence of en vi ronmenta 1 factors on gliding motility, e.g. the effect of 1i ght

~ ntens i ty and wave 1ength, exposure to a 1tet~na ti ng periods of l______5

G------.-· -----·------·------,------·--·-·-----·····------·· ---- ··------· ------·--··------·· ---· ·---- .. -· ··------· - ---·--·------, !illumination and darkness, temperature, pH, oxygen ava·ilability,

I I lnedium viscosity, age of cultures, and avaifability of moisture ! I !(Burkholder, 1933 and 1934; Stanier, 1942a; Pringhseim, 1951; Doetsch I ~nd Hageage, 1968; Halfen and Castenholz, 1970 and 1971; Castenholz, ! h973). Little attention has been given to the influence of nutrients, ! ~ons, agar concentration, and other factors such as responses to 'chemical gradients. Detailed studies of the influence of environmental factors on gliding motility per se, comparable to such work on eubacterial flagellated motility (Adler and Templeton, 1967), are ! ~acking, although review articles, such as that of Burkholder (1934), ! have occasionally devoted considerable attention to the question. The present work looks in detail at the gliding motility of Sin1onsiella and Alysiella. They are aerobic, multicellular, filamentous bacteria, composing the family Simonsiellaceae of the order Cytophagales (Buchanan and Gibbons, 1974). Their habitat is the oral i . cavity of humans and other warm-blooded animals. They offer distinc- tive investigative value in regard to gliding motility because of their extremely large size. A filament of cells can measure 3-10

~m in width and hundreds of micrometers in length. Hence, aspects of gliding motility are more readily discernible with Simonsiella than with other species of bacteria. Additionally, Simonsiella possesses a unique dorso-ventral flattening of its filaments (Pangborn, Kuhn and Woods, 1973 and 1974), and the possibility of an associated differentiation of the cellular structures of the two surfaces might readily assist in the search for the structural basis for gliding motility.

i i [______6

~-----ft1e-1Tfera til-re·e:-on-1:ai_n_s--onf.y --5-can_t_reie-rence- ta9-l icii-n9~~oti1-ii::Y___ " I lin Simonsiella and Alysiella. Fellinger (19~4) was the first person Ito report gliding motility in Simonsiella, viewed in a hanging-drop I !preparation of saliva. Steed (1962) observed gliding motility in I !jYoung serum broth cultures of Simonsiella and Alysiella placed on

I !slide-mounted agar medium. ' . ' The present investigation examined the phenomenon of gliding lmotility and the interrelated patterns of growth under various environ­ ! i !mental conditions. Forty-six strains of Simonsiella and two strains i )of Alysiella were subjected to numerous variations in environmental ! !conditions in order to determine whether growth and gliding motility I !patterns have value in the taxonomy of the family Simonsiellaceae. i 1 such patterns might offer one more tool, in addition to cellular 1 i ,morphology and biochemical and physiological information reported by iKuhn et al. (1974), Pangborn et al. (1973 and 1974), Nyby (1974), _.t -- -- I ;Gregory (1975) and Jenkins (1976), and to mole percent G+C content

!information (Kuhn et ~., 1974) in the classification of the collection i 'of 48 strains of Simonsiellaceae isolated from sheep, cats, humans and dogs. Pringsheim (1951) attempted to group mixed cultures of gliding :organism, then tentatively identified as Vitreoscilla and related ;organisms, according to patterns of growth and gliding motility. To my knowledge, this is the only taxonomic study of gliding organisms ·undertaken with microscopic patterns of gliding motility and growth :as a prime determinative consideration. ! I I I l______- -- ~------·----~------METHODS AND- -MATER-IALs------:·------~ I - I l I !Strains of Simonsiellaceae. Three strains of Simonsiella and one 1 !strain of Alysiella were isolated from the oral cavity of sheep at Los l ~ngeles Pierce College, and one Simonsiella strain each was isolated i ' ifrom the oral cavity of a cat and a dog by the technique developed by I ' !Nyby (1974). These strains are part of the collection of Simonsiel- ilaceae strains deposited with the International Collection of Phyto­ l ! :pathogenic Bacteria (ICPB), University of California, Davis, that was i examined in this study. The labels Sim 1 through Sim 47 correspond t 'to strains ICPB 3601 through ICPB 3647, and Alysiella strain Aly 1 ~orresponds to strain ICPB 3652. Simonsiella strain S6 and Alysiell~ ! :strain Al, the respective neotypes of Simonsiella crassa and Alysiella filiformis (Steed, 1962), were obtained from the National Collection of j ~ype Cultures (NCTC), London, England, and have the labels ICPB 3651 :(NCTC 10283) and ICPB 3653 (NCTC 10282), respectively.

l Maintenance of strains. The strains were maintained on BSTSY agar {Nyby, 1974) of the following composition: Tryptic Soy Broth w/o Dextrose (Difco) ....•...... •.•• 27.5 g Bacto-Yeast Extract (Difco) .•...... •...... •. 4.0 g

Bovine serum (North American Biologicals, Inc.) ..• ~ ... lOO ml Bacto-Agar {Difco) ..•.•....•....••...•...•.••...... •... l5.0 g Distilled water ...... •.•.•...... lOOO.O ml The bovine serum was added aseptically to the other ingredients l ~fter they had been autoclaved at 121 C for 15 minutes and cooled to i i 45-50 C in a water bath. ! 7 8 r------·-·-·------·-----·--··--·-----···------· ------·· -·------··------··-· ----·-·--··------·------, 1 The strains were cultivated on BSTSY agar plates, incubated at 1 f7 C, and transferred every 48 or 72 hours, depending on the charac- !

~eristic viability of individual strains. A duplicate set of the I ! strains was maintained at 30 C and transferred every 6 or 7 days. I I iI Culture inocula. Cultures grown on BSTSY agar at 37 C for 24 ' ~ours or less served as the inocula unless specified otherwise. ! Observations of gliding motility in BSTSY broth medium were made on 6- I to 24-hour old BSTSY broth cultures which had been transferred i i previously in BSTSY broth every 24 hours for 2 or 3 days. l i Microsco~. A Wild phase contrast microscope was used for most obser- ; i vations of growth patterns and gliding motility. Organisms on solid media in petri plates v1ere surveyed with a lOX Fluotar achromat objective lens [numerical aperture (n.a.) 0.22], a 40X Fluotar phase Fontrast objective lens (n.a. 0.75) or a HI SOX Fluotar phase contrast objective lens (n.a. i.O), and a condenser (n.a. 0.9). The lOX objective lens was adequate for most routine observations and permitted viewing the organisms on ~he agar surface with the petri plate inverted and its cover in place, thus minimizing the chance of culture contam- ination. Observations of either BSTSY broth wet mounts or slide-mounted BSTSY agar cultures were made with either a Wild phase contrast micro­ scope with the above-mentioned objective lenses or with a HI lOOX 'Fluotar phase contrast objective lens (n.a. 1.30) or with a Zeiss GFL "t ' microscope, using the 40X Neofluar (n.a. 0.75) or the lOOX Neofluar 9 frrl:·-a~·-·r:3-6f-phase ·-contrast-ob}ecti ve-Te·n-ses' and--a--I IZ-cc)nde-ns--er______l l(n.a. 0.9). I I !Colony morphology. Simonsiellaceae strains were transferred three l !times at approximately 12-hour intervals on BSTSY agar. After 12 l I :hours of incubation, cells from the third set of plates were added to I !5 ml BSTSY broth in culture tubes, shaken with a Vortex Jr. mixer, and I i !streaked on BSTSY agar plates. Morphology of individual colonies was i !observed after 36 hours of incubation at 37 C and the characteristics ! ;were recorded using the terminology of Rogosa et ~- (1971). i I jColony photomicrography. The inocula were prepared by transferring the i I ~strains twice in succession on BSTSY agar at 12-hour intervals, and :then suspending 12-hour old organisms in BSTSY broth and streaking them I :on BSTSY agar plates. After 12 hours of incubation at 37 C the !colonies were photographed with a 16X Neofluar objective lens (n.a. :0.40) mounted on a Zeiss photomicroscope with the Optovar set at 1.25X i iand the projective lens set at 3.2X. The colonies were illuminated :without the use of a condenser by a 12 v, 50 w tungsten filament lamp !operated with a regulating transformer at 4 amps. Photomicrographs were recorded on 35 mm Kodak Plus-X panchromatic ifilm printed on Kodabromide F·2 paper, and processed in Kodak DK-60 i and Kodak Dektol developers, respectively. [

!Examination of cultures. In all the following experiments cultures )growing on agar media were examined macroscopically for growth and !microscopically (as described previously) for patterns of growth and L __ ., __ 10

f9iidin9-llloiiTft:Y-:-- ·-c·uftures ___were incubated at 37 c·unie-ss inCiTca-teX ---, otherwise.

!Temperature tolerance. BSTSY agar plates were divided into quadrants I !with a marking pen and one strain per quadrant was inoculated by first, I !making a point inoculation in an outermost corner of the quadrant and, ! !then, streaking to cover the agar surface of the quadrant. Plates ! :were incubated at 27, 30, 33-34, 37, 40, 43, and 45 C. After 24, 48 i i land 120-144 hours of incubation they were examined. I I ;pH tolerance. BSTSY agar plates of varying pH were prepared by i I iadjusting tryptic soy-yeast extract (TSY) broth to pH 5, 6, 7, 8, 9, :or 10 with 1 N HCl or 1 N NaOH before adding the agar, autoclaving I :and ·supplementing the media with bovine serum. The pH values were idetermined with a Beckman Zeromatic pH meter with the electrodes -· i I !applied directly to the surface of the solidified BSTSY agar. Two :sets of plates were made, and several pH readings were recorded for

i :representative plates of each set. The pH values for the plates of both sets had the following ranges: 5.4-5.5; 6.15-6.25; 7.2-7.25; I !7.85-8.0; 8.45-9.45. Plates were streaked in quadrants and examined iafter 24, 48 and 96 hours of incubation.

;salinity toierance. BSTSY agar contains 0.5 percent NaCl, contributed ·by the Tryptic Soy Broth without Dextrose (Difco). It was supplemented jwith NaCl to achieve final NaCl concentrations of 1.0, 1.5, and 2.0%. ! i !The plates were streaked in quadrants and examined after 24, 48 and 196 hours of incubation. I_____ - - .. -······· 11

Sea v.,rater tolerance. Modifications of "Flexibacteria ~1edium l" (Lewin and Lounsbery, 1969) were prepared with varying quantities of the following synthetic sea water mineral salts solution:

NaCl ...... 25. g

MgS04-7H20 ...•.... :··································· 5 g CaCl 2·2H20 ...... •...... •...... l g KC 1 • • • . . • • . . • . . . . • • • • • • . . • • • . • . . • ...... • ...... • . . . 1 g Distilled water ...... 1000 ml :Three media with either 100%, 50% or no sea water mineral salts solution were prepared in liter quantities containing the following common ingredients:

KNO ...... ~ ...... 0 . 5 g 3 Na glycerophosphate ..•...... •...... O.l g Tris [(2-amino-2-) hydroxymethyl]- 1,3 propanediol (Matheson, Coleman and Bell) .... l.O g Trace element solution* ...... l ml Bacto-Tryptone (Difco) ...... 1.0 g Bacto-Yeast Extract (Difco) ...... l.O g Bacto-Agar (Difco) ... ·...... l5.0 g

*The trace element solution contained the following components: H3B03 ...... •.•...... •...... 3.0 g Feso4.7H20 ...... 2.5 g MnSO 4 · H20 ...... 1 . 5 g CoS04 · 7H 20 ....••...... •...... •...... 50.0 mg CuC1 2·7H20 ...... •...... 30.0 mg NaMoO 4 · 2H 2o ...... ~ ...... •...... 25. 0 mg Znso4.7H20 .....•.•...••. ,-...... 40.0 mg Distilled water ...•...... lOOO.O ml 12

r------· ---' -~------··-· --·---····------, tEach medium was adjusted to pH 7.2 with l N HCl, autoclaved, and I /supplemented with 10% bovine serum. I i Plates were streaked in quadrants and examined after 24, 48-53 I land 114-125 hours of incubation.

I !Modified sea water agar. 11 Flexibacteria Medium 111 plates were prepared

/as described above, except that either NaCl, Mgso4.7H2o, CaC1 2·2H20, I I lor KCl was eliminated. In a second series of experiments, all plates I !additionally lacked KN0 and sodium glycerophosphate. Control plates I 3 I !contained all four sea water salt compounds. i I Selected Simonsiella strains were streaked on sets of test I /plates which were examined after 24, 48 and 96-120 hours of incubation. !

!Gliding motility rates. Gliding motility was observed with a Wild ;microscope, housed in a 37 C incubator equipped with a modified i iplexiglass door, enabling an observer to monitor gliding motility at jthe optimum growth temperature of 37 C of Simonsiellaceae. The I !incubator door contained a recessed plexiglass box with two openings, i ,allowing the microscope ocular lenses to protrude. Two additional !circular openings in the plexiglass door were fitted with heavy duty i I irubber gloves to reach the microscope stage and focus adjustment knobs. I i ! Four- to 12-hour old cultures, transferred previously every 24 !hours for 2 or 3 days, were streaked onto thin layers of BSTSY medium i !containing 0.5% agar. Agar blocks, approximately 1.5 em x 1.5 em, I I ! !were cut and removed vdth alcohol-rinsed cover slips to alcohol­ ! /name-sterilized microscope slides. Alcohol-rinsed cover slips, I ~=-=--~-~~-~~-~-~e~~- placed over the agar blocks and seale~ -~~-~~-~aspar 13 i(an equal mixture by weight of vaseline and paraffin). These cover l :slips provided air chambers on each end of the agar blocks, permitting ,temporary aerobic growth and gliding motility of the organisms, at least at the extreme edges of the agar blocks. Gliding motility rates were measured with a stop watch and a 'calibrated micrometer in a Zeiss 12.5X ocular lens. The rates were recorded as the distance travelled in micrometers per minute by the leading end of a filament. Occasi ana lly, gliding motility rates were determined for BSTSY ,broth cultures mounted on microscope slides without the agar block and covered with 22 x 22 mm cover slips and sealed with vaspar.

Influence of humidity. Selected known gliding strains of Simonsiella :were streaked in duplicate onto BSTSY agar plates. One set of plates was placed in a 37 C incubator as usual. The duplicate set of plates was placed in a covered plastic box containing a bowl of water with a half-immersed sponge. The box was sealed with tape and placed in a 37 C incubator. Plates were examined after 24, 48 and 96-120 hours of incubation.

The effects of omission of salts found in BSTSY. Tryptic Soy Broth without Dextrose (Difco) consists of 3 g Soytone, 17 g Tryptose, 5 g NaCl, and 2.5 g dipotassium phosphate per liter. Modified BSTSY agar was made with Soytone (Difco) and Tryptose (Difco) but without NaCl and dipotassium phosphate. Additional modified BSTSY agar was made with Soytone (Difco) and Tryptose (Difco) to which NaCl and dipotassium phosphate were, again, added. Control plates of BSTSY 14

c-·------~ ------·------·------~------·-·------·------.------··· ·- rgar were made with commercial Tryptic Soy Broth without Dextrose J(Di fco). I Plates were inoculated with selected Simonsiella strains and bbserved after 24 and 48 hours of incubation. I ~he effects of agar concentration. BSTSY medium was prepared with agar I \ koncentrations of 0.8, 1.5, 3.0 ana 4.0%. Plates were dried prior to I i use at room temperature for one to several days, or at 37 C overnight. i ,Plates were then either used immediately or refrigerated in a closed i I Plastic box for future use. I The plates were streaked with selected Simonsiella strains, except. ! :1n! • the case of the soft BSTSY agar the inoculum was applied to the i I medium' surface with a dabbing motion of the loop. The plates were j ' 'examined at several of the following incubation times: 6-8 hours, I I no-12 hours, 24-29 hours, 48-58 hours and 3-6 days. i ! I The effects of serum concentration. BSTSY agar plates were prepared ' ~ith bovine serum concentrations of either 5, 10, 20 or 40% (v/v).

~he plates were inoculated with selected Simonsiella strains and

~xamined after 24-31, 48-56 and 96-120 hours of incubation.

I ~he influence of used BSTSY broth incorporated in BSTSY agar. BSTSY agar plates were prepared containing various proportions of fresh ;BSTSY and Seitz- or Millipore-filtered BSTSY broth in which selected l :Simonsiella strains had been incubated for various durations. Two I • 1ser1es of media were prepared. l - 15

··- -- -· --~------. -.------· ~----···-----: ~---·- ·-(1 )---Fl asks--~o~ta-i-~i-ng--50 ml fresh BSTSY broth were inoculated \in duplicate with either Simonsiella strain Sim 39 or strain Sim 40, land incubated with shaking at 37 C. After 4 hours and 8 hours of I !incubation the broths were filter-sterilized and added aseptically to !150 ml of sterile, fresh BSTSY medium containing sufficient agar so i lthat the resultant medium contained 1.5% agar. I I (2) Simonsiella strain Sim 39 was cultured in several flasks f !of BSTSY broth incubated at 37 C with shaking. After approximately i h5 hours of incubation the broth cultures were Millipore-filtered and I !dispensed into a series of flasks. Each flask contained an equal i i !volume of fresh, sterile BSTSY with doubled agar concentration to I I :produce BSTSY agar plates composed of 50% used BSTSY broth and either ! !o.5, 1.0, 1.5 or 2.0% agar. Control plates of fresh BSTSY medium with

:agar concentrations of 0.5, 1.0, 1.5 and 2~0% were used. All plates were inoculated with selected Simonsiella strains, and examined at several of the following incubation times: 6 hours, 12 j !hours, 19-24 hours, 27 hours, and 40-48 hours.

The effect of silica gel. BSTSY medium plates were prepared with ;silica gel substituted for agar. The silica gel was prepared !according to the method of Funk and Krulwich (1964) and adapted to i I ;the use with BSTSY broth in the following manner: i 1. A 20 percent orthophosphoric acid solution was prepared. 2. A fresh solution of silicic acid (500 mesh) dissolved in 7 percent KOH was prepared and dispensed in 20 ml quantities into 125 ml flasks. 3. Double strength TSY broth was prepared. 16

----···--·------·------·------·------·-----. «< ------·------·-· ------·------..------1 I 4. All the above ingredients were autoclaved at 121 C for 15 minutes. 5. Twenty percent bovine serum (v/v) was added aseptically to I I the double strength TSY broth. Twenty ml quantities of this double strength BSTSY broth were added aseptically to individual

I! j flasks of potassium silicate. 6. Three ml sterile phosphoric acid were added as rapidly as possible to the BSTSY-potassium silicate solution. The acid was added from a pipet while vigorously swirling the medium­ containing flask and, then, the medium was poured immediately into a glass petri plate. Gelation was extremely rapid, there­

fore, the technique required skill and had to be performed by bJO people. The pH of the BSTSY-silica gel plates was measured with the electrodes of a Beckman Zeromatic pH meter placed directly on the medium surface, and ranged from pH 7.3 to 8.0. The plates were incubated with selected Simonsiella strains and examined after 24 hours. ~------~------~:----rffsuLrs ANn orscussroN ------:------1

I This investigation began as a study of the environmental ! jconditions under which gliding motility occurs in Simonsiella and I jA 1ys i ell a . These organisms, discovered by MUller (1911) and studied I \by Simons (1922), Langeron (1923) and Fellinger (1924), have received : I trecent attention by Steed (1962) fn a taxonomic study which provides ! 1much of our current knowledge about these unusual bacteria. Steed ! I !{1962) made brief comments about observing gliding motility. ! The present study revealed that gliding motility was inter- i !related with various patterns of growth. The investigation, accord­ ! :ingly, was enlarged to consider aspects of the patterns of both growth I ! .and gliding motility in 46 Simonsiella and 2 Alysiella strains under 'numerous environmental conditions. tolony morphology Table l lists the colony morphology characteristics of the Simonsiella and Alysiella strains. All strains display comparable macroscopic colony morphology, although slight differences were noted ,in colony size and in the extent of yellow pigmentation. General 'colony morphology characteristics for Simonsiellaceae strains can be

1 :summarized as follows: opaque, ivory to very faint yellow color; smooth, glistening, butyrous texture; and entire, low convex form, with maximum colony diameters ranging from less than l mm to somewhat more i [than 2 mm. I ! Closer examination of colony morphology, using a hand lens and iholding the plates against a light source, revealed that colonies of t_ .. ,.... _ 17 18

--- ··----~---~~-----·· ---~---- ··-----·-·------~------~------, ,.---·-····------. ---·--···-···--· --- ··-----·· --- . -·--· --- --· ··-·- . - ·-·- ··------· -- ITABLE 1. Colonial characteristics of Simonsiella and ·Al,isiella strains - after 36 hours of incubation at 37 c on BSTSY agar. I I I ! CHARACTER .f-) Diameter s:: X Ol !' Cl.l Cl.l s:: u > Cl.l Ill •r- :l s:: .f-) :l s:: I E r- Cl.l Cl.l 0 res 0 Cl.l ..s:: E Ill :l s... u s:: s... .f-) .f-) ..s:: E s:: 0'" ·.- 0 >. Ill 0 Ol E ~ 1.0 res res .f-) 3: ..0 .f-) •.- 0 :l I I s... 0. s:: 0 E :l r- E 0 I STRAIN r- N N .f-) 0 Cl.l r- :l ..0 Ol Ill s... i v v

!CAT I I I 1 ( +) + + + + + + + I ill (+) + + + + + + + ! !12 ( +) + + + + + + + h8 (+) + + + + + + + l i19 i

!DOG I I 2 + + + + + + + i 3 4 + + + + + + + 5 (+) + + + + + + + 6 + + + + + + + 7 + + + + + + + 8 + + + + + + + 9 + + + + + + + 10 + + + + + + + .17 + + + + + + + '22 + + + + ( +) + + + l23 + + + + + + + 26 + + + + (+) + + + i27 + + + + ( +) + + + :28 (+) + + + + ( +) + + + ·32 + + + + + + + i38 (+) + + + + + + + ! !39 + + + + + + + !40 + + + + + I + + i I L 19

-~---- _ _. ------~------~ . ·~·-··~-----·- ~·- --··------·------·'------______·------· ------;TABLE 1 (c~on ti nuedT~-~-~ -- I I I ! ! :HUMAN

I!13 + + + + + + + h4 ! I jl5 + + + + + + + :16 + + + + + + + 24 ( +) ( +) I + + + + + + + '25 + + + + + + + + 33 + + + + + + + + :34 (+) :35i + + + + + + + i36 37 + + + + + + + Al + + + + ( +) + + + ( +) :42 + + + + ( +) + + + (+) 43 + + + + ( +) + + + (+) 44 + + + + + + 45 + + + + + + 46 + + + + (+) + + + (.f.) ,47 + + + + + + +

SHEEP 20 + + + + + + + 29 + + + + + + + 30 + + + + + + + 31 + + + + (+) + + + (+) 56 + + + + + + + Al + + + + + + + Aly l + + + + (+) + + + 20 some strains possessed a slightly rough texture (Simonsiella strains :Sim 35, 41, 42, 43, 46), or a slightly umbonate form (Simonsiella strains Sim 20, 22, 24, 26, 28, 31, 40, 41, 42, 43, 46, and Alysiella strain Aly 1), or a distinctive opalescence (Simonsiella strains Sim 31, 35, 41, 42, 43,· and 46).

[Note: Routine transfer of cultures, employing a direct loop .. transfer of inoculum without first suspending in BSTSY broth, results :in marked differences in growth among strains. The extent of growth t'anges from faint to heavy and confluent; pigmentation ranges from off-white to light yellow.]

Patterns of growth and gliding motility The course of growth among the Simonsiella and Alysiella strains varied significantly and resulted in an array of microscopic patterns of both growth and gliding motility (Figures l-4). Colony morphology ranged from entire-edged colonies, where all filaments remained closely adherent to the other filaments composing the colony (Table 2, pattern ·1), to colonies displaying advanced gliding motility, where countless filaments glided away from the colony edge for great distances (Table 2, pattern 8). Between these two extremes there occurred either gliding mot·ility of lesser degrees, or filamentous growth characterized by multicellular filaments, sometimes of very long lengths, protruding from the colony edge at various angles onto the surrounding agar surface. While many Simonsiella strains display filamentous growth to moderate degrees, several strains show significant filamentous growth. Following approximately 24 hours of incubation, such growing Simon- 21

r·------. ------··············- -··········-··-········-·- ... ······-··-··· ····-··-- ·······--·-··-·--·------·- ·-·-·· ··-···- ··------····-·------~.

I I

i ',1 I

1 I I I ' '

FIGURE 1. The four types of colony morphology of dog Simonsiella trains as represented by strains Sim 4, 5, 27 and 28, observed after 2 hours of incubation on BSTSY agar. Magnification: 320X.

23 r------...... - . . . -1 I

I I

FIGURE 2. The four types of colony morphology of human Simonsiella strains as represented by strains Si1n 14, 36, 43, and 46, observed

~fter 12 hours of incubation on BSTSY agar. Magnification: 320X. 14 25

~------.. ------· ----··------. ------1 i I

I I I ! I i I l' ! !

FIGURE 3. The three types of colony morphology of sheep Simonsiella strains as represented by Sim S6, 20 and 20, observed after 12 hours of incubation on BSTSY agar. Magnification: 320X.

27

~~---~-~~------····----· ------· ------~~-- .. ·- -·-·-···-··-·--·· ---············---·····~---··-····-~------··-·····------····------~---l

I

FIGURE 4. Three types of colony morphology of cat Simonsiella strains as displayed by strains Sim 1, 11 and 12, observed after 12 hours of incubation on BSTSY agar. Magnification: 320 X.

29 r------:------, \TABLE 2. Patterns of growth and gliding motility of Simonsiellaceae !strains on BSTSY agar after approximately 24 hours of incubation. !Magnification: lOOX. i i ' !GROWTH/GLIDING MOTILITY PATTERN SYMBOL PATTERN OBSERVED I AT COLONY EDGE ! '------iNo growth !

!Neither gliding motility nor filamentous :growth at colony edge. Individual_ filaments :are parallel to the colony edge and are 11 11 jeither on their sides ( rose petal pattern ) :or flat. I l !Individual filaments occasionally extend :from the colony edge. i ' Filaments frequently extend from the colony ,edge. 2

' 1Groups of closely associated filaments extend from the colony edge .

.Numerous fi 1aments extend from the colony !edge, giving a fringe-like appearance. 3

Numerous, extremely long filaments extend ifrom the colony edge, producing straight or curled patterns. Filaments may be 4 ,flat or on theit· sides.

Prominent groups of filaments extend from the colony edge, forming "peninsulae-" or 5 11 : fl ame-li ke" outgrowths. 30

,-··--·-·-······· ·------········ .. - ..... ··- --·--·-·--·----··-·· .. ... -----·-·· ··-·· ...... ---- ...... ·-·····. ----·------·-··· -· ·····-···-······-·-----·------·-l ~TABLE 2 (continued) 1 I I !Slight gliding motility. A few to many '~~ l /filaments leave the colony edge and disperse \\ .' ~~&./). ifor short distances, forming refractile 6 ,, !tracks which mark their path. i ~-~~ 1------[Protiounced, well-established gliding moti1 i ty ,~ I 't •producing a vast network of refractile -~ ·tracks. Some filaments may have glided for 11 ;long distances. New colonies, referred to :as secondary growth, develop within the 1 netw01~k of tracks at distances from the edge ~d;-\) ( ,s] 11 'of ori gina l co 1ony. '-.': c ,_-,_, :?"'.,!! ! _.... e::-r ~ ~ __,. ~ .

!Long distance gliding motility. Large popu­ ilations of individual filaments glide 'randomly for long distances onto the 8 :surrounding agar surface. Secondary colony formation may be absent.

IL __ 31

;siella filaments might protrude from the colony edge for hundreds of 'micrometers, extending straight along a groove formed by the loop while streaking the culture (Table 2, pattern 4). More commonly, complex patterns of filaments develop, manifested as wavy extensions or whorls (Figure 3, Sim 20) as seen in Vitreoscilla (Pringsheim, 1951) and in Oscillatoria (Castenholz, 1973). Simonsiella strains isolated from sheep, such as strain Sim 20, frequently displayed this pattern. One other distinctive pattern of filamentous growth observed among Simonsiellaceae consists of irregular-shaped co-lonies composed of flat sheets of cells with 11 peninsula-like 11 or "flame-like 11 extensions. Fot' example, the colonies of Simonsiella strain Sim 46 give the impression that constituent cells divide and spread in sheets, perhaps by gliding or 11 Sliding 11 (Henrichsen, 1972) in mass, and that such spreading occurs only to a limited extent, presumably sufficient to maintain a monolayer of closely-adherent cells. Table 2 describes and illustrates the range of observed patterns of growth. Figures 1 through 4 are photomicrographs of the represen- tative colony morphologies of Simonsiella strains incubated at 37 C on BSTSY agar for 12 hours. Each figure groups strains from a particular animal source as follows: Figure 1, dogs; Figure 2, humans; Figure 3, sheep; Figure 4, cats. The figures reveal that a particular pattern of growth may be found in Simonsiella strains isolated from several or all four animal types. Strains isolated from the same animal type may exhibit three or more of the growth and gliding motility patterns presented in Table 2. Each strain of Simonsiella and Alysiella exhibits one of these patterns of growth and gliding motility and, presumably, a particular 32

.. -----~-----·· ------··· -·-·- •t 'pattern is a constant feature for a strain grown on BSTSY agar at 37

C. Fo~~ example, pronounced gliding motility (Table 2, pattern 7) was displayed consistently by strain Sim 28, as photographed in Figure 1, whereas 11 peninsula-like 11 colonial growth (Table 2, pattern 5) was 1 :typical of strain Sim 46, as photographed in Figure 2. Numerous ;attempts to detect gliding motility among strains not known to iglide failed. BSTSY agar cultures of a representative sampling of 1 Simonsiella strains were examined repeatedly, including during initial 'phases of incubation, with higher than usual magnification {500X) ;but gl-iding motility was observed only among those strains of ' Simonsiella known to be consistent gliding strains.

'A~ects of gliding motility Only a small number of the Simonsiella strains exhibited gliding motility and neither Alysiella strain was observed to glide. Simon­ lsiella strains Sim 10, 17, 26, 28, 39, and 40, all isolated from dogs, ! :became known as the consistent gliding Simonsiella strains. Several iother Simonsiella strains (Sim 2, 3, 12, 13, 14, 19, 20, 22, 27, 34, i 37, 44, 46, and possibly 25 and 35) showed occasional instances of

gliding motility. t~ost of the gliding strains glided consistently 'test after test under standard and experimental conditions. The ability to glide tended to diminish in some strains after several months of maintenance on BSTSY agar. For example, gliding motility disappeared from strain Sim 17 within a year of culturing on BSTSY agar. Sim 9, previously described as a gliding strain

(Nyby, 1974), lost its gliding ability prior to the start of this present work. Furthermore, Sim 28, having remarkably extensive 33

- .. - .. --- ... -----·- .. ·-······------. ;gl·iding motfHtY at the time of isolation, showed significantly reduced· i gliding motility within a year after isolation. Gliding motility could be detected most readily after several l ~ours of incubation following transfer to fresh BSTSY agar. Gliding ' motility in progress could not be witnessed on standard BSTSY agar

' . due to the very slow rates of progression. For example, filaments might i ·advance one micrometer in 30 minutes. One could most easily detect ,that gliding moti 1 i ty had occurred by the presence of refracti 1e tracks .. ! Patterns of these tracks were easily seen at the periphery of colonies I where individual multicellular filaments depart from the edge of the colony and move out onto the surrounding agar surface. In discussing gliding motility the term 11 SWarming 11 is given to the· large scale creeping locomotion found among myxobacteria and cytophagas, resulting in a continuous extension from the colony periphery of long, often pointed, cell masses which creep out across the substrate like miniature armies (Stanier, l942a). Likewise, the term 11 pseudoplasmo­ dium11 was used in the early literature (although subsequently abandoned) to describe such an active increase in colony size among myxobacteria

(Stanier, l942a). Howeve~, such swarming was not observed as a characteristic feature among gliding strains of Simonsiella. Only under specially altered media conditions, e.g. media containing low agar concentr·ations, was swarm-like gliding motility observed. Such

,swal~m-like or long distance gliding motility will be examined later.

Typical gliding motility among Simonsiella strains gl~owing on

~STSY agar appeared as a conservative, conditional phenomenon. ' ~liding motility was manifested as locomotion exercised by individual, l frequently well-separated filaments. Gliding motility occurred L_ .. 34

'principally in heavy-growth areas amongclosely-·associated colonies or ' i' ;masses of growth. It appeared to be spurred by congested growth and, i ~dditionally, required that filaments have access to fresh, previously i junoccupi ed agar surfaces. ' Gliding motility appeared as a transitory event among Simonsiella j :strains and consistently gave rise to the formation of secondary . ' 'colony centers once new areas of previously unoccupied agar surface had been reached. Further gliding motility occurred at the periphery of secondat·y co 1oni es upon continued i ncuba ti on, presumably among terminal segments of growing Simonsiella filaments having access to the

~urrounding agar surface. Consider·ing this observed cycle of gliding motility followed by non-gliding secondary colony grO\"'th, exhibited consistently in Simon­ siella strains Sim 26 and Sim 28, it is attractive to propose that gliding motility occurs to enable filaments to reach fresh nutrients for subsequent periods of growth. The distinctive dorso-ventral flattening or ribbon-shaped morphology of Simonsiellaceae (Pangborn et ~., 1974) may help explain the cyclic phenomenon of gliding motility and colony formation just discussed. Growth, as clearly observed in young colonies, occurs with the filaments lying on their edges and frequently curled around each other like rose petals (Steed, 1962). Gliding motility occurs only when the undersurface of the filament makes contact with the substratum, and filaments are immobilized while on their sides (Steed, 1962). Thus, sustained growth and cell division forces filaments to turn on ! 'their sides with a resultant loss of ability to glide. Gliding I 'motility may be resumed at the periphery of colonies by the terminal 1 1--·- 35

- -~ ------~------.. - -- ~- ___ , ____ -~------G~gments of filaments whose ventral surfaces either cdntinue to ! ' ~aintain contact with the substratum or are able somehow to I Ire-establish contact. i !

:Gliding motility on glass in BSTSY broth and on soft BSTSY agar Steed (1962) reported observing gliding motility among Simonsiella and ~lysiella isolates with the use of sealed microscope slide ,Preparations of young broth cultures. This technique provided a good .means for observing maximum rates of gliding motility among Simons·iella

·strains in the present investigation. r~aximum gliding motility rates were also achieved with Simonsiella cultures placed on BSTSY medium

11 'so 1i di fi ed with 0. 5% agar, termed .. soft BSTSY agar • Motility rates varied from trial to trial for a particular stl'ain and among individual filaments of the same preparation. Filaments glided at rates between 2.1 ~m/min and 37.5 ~m/min (Table 3). Average 'rates for individual Simonsiella strains were as follows: Sim 19,

~ ~m/min; Sim 20, 10 ~m/min, Sim 26, 23.8 ~m/min; Sim 28, 13.8 ~m/min;

Sim 46, 22.3 ~m/min. Steed (1962) provided sequential photomicrographs displaying several gliding filaments of a sheep Simonsiella strain, from which a rate of 5-10 ~m/min could be approximated. Thus, the range of 5

~m/min to 40 ~m/min appears to be a reliable estimate of gliding motility rates of Simons·iella. Simonsiella gliding motility rates are extremely slow. They are

~omparable to those found among myxobacteria and unicellular cyano- 1 ~acteria (Castenholz, 1973; Doetsch and Hageage, 1968). To the 36

--·· ---- -··---··---- . ---·- -·------!TABLE 3. Rates -~f gliding -;;t-ility of some Simonsiella strains on 'eithet~ glass in BSTSY (or HSTSY*) broth or BSTSY agar under the )individual culture conditions noted.

;sTRAIN RATES CULTURAL CONDITIONS I. Broth Wetmount Preparations 19 5 pm/min (approx.) 41 hr BSTSY broth, observed after one hour on illuminated scope, at ------~r~o~o=m~temperature 20 5 pm/min 18 hr BSTSY broth, observed immedi­ ately at room temperature ,------~------'~--'----'--'--"-=----C--'----~-'-- 20 4.1-13 pm/min 48 hr HSTSY broth, observed immedi------· ______i _a t-'-e'-l~y_a_t_r_o_o_m_te_m__,_p_e_r_a--'-tu_r_e:.._,:;___.__p_H_=_8_ i 20 5-27.5 pm/min 23 hr HSTSY broth, observed immedi­ ; ately at room temperature 20 7.5-12.5 pm/min 24 hr HSTSY broth, observed after one hour, at room temperature 28 10.0-10.75 pm/min 6 hr BSTSY broth, observed immedi­ ately, at 37 C 28 7.5-17.5 pm/min 8 hr BSTSY broth, observed after one hour at 37 C II. Slide-mounted Agar Preparations 26 15.75-31.75 pm/min 0.5% agar-BSTSY, observed immedi­ ately, at 37 C

28 2.1-25 11 m/rrri n 0.5% agar-BSTSY, observed immedi­ ately, at 37 C. From 24 hr-old ------"a_,ga r culture 28 7.5-12.5 pm/min 4 hr-old 0.5% agar-BSTSY, observed at 37 C 28 15-30 pm/min 12 hr-old 0.5% agar-BSTSY, observed at 37 C ; 46 12.5-20 pm/min 22 hr-old 1.5% agar-BSTSY, observed '------·--- at 37 C 46 19.25-37.5 pm/min 1 hr old 0.5% agar-BSTSY at 37 C. From 7 hr-old agar culture

*TSY broth supplemented with 10% horse serum (North American Biologicals, Inc.). 37

~-·-- -· ---·~-----~---· ·- -. ·-· ···------··--- . , ______---··-·--···· ------· ----·- !'obser·ver, gliding . motility in Simonsiella at the slowe-r rates is barely discernible, even at 1250X magnificaiion. 1 Gliding motility in broth cultures seemed to be activated and ·enhanced by either illumination or heat. In young, healthy cultures i' :filaments brought into the illuminated microscope field sometimes :began distinctive swaying and flexing motions, and gliding motility sometimes ensued. In one slide preparation of a fresh human saliva sample, Simonsiella filaments clustered on an epithelial cell quickly

~ispersed when the epithelial cell was brought into the microscope i :fie 1d. A BSTSY broth culture of Simonsiella strain Sim 19, viewed for several hours with the microscope field diaphragm closed to transmit only a small circle of light through the microscope field, revealed several filaments gliding back and forth between the illuminated and ·darkened regions until finally coming to rest just outside of and

parallel to the illuminated circle. This behavior sugg~sts a negative phototaxis and a positive thermotaxis. The latter taxis would have been promoted under the experimental conditions since the organisms were observed at room temperature and, yet, have been shown to grow optimally at 37 C (as discussed later). Observations of slide preparations of BSTSY broth or soft BSTSY 'agar cultures after 24 hours of incubation at 37 C showed a great proliferation of filaments. Such growth led to accumulations of

11 11 granular or fibrillar or cellular debris • Simonsiella filaments had

laid down unmistakable brush stroke-like 11 trails 11 of this debris on the soft BSTSY agar substratum. Points of origin of such trails were marked frequently by distinct, large piles of material that are 38

1presumed to be remnants of cells. Occasionally, se~eral filament i !segments were linked together vJith 11 rope-like 11 fibrillar stl~ands, I I !presumably similar in makeup to the other fibrillar debris. i Filaments would sometimes depart the soft BSTSY agar and glide ! 'onto the glass slide in a liquid channel that extended from the filament' back to the edge of the agar. When the liquid dried, trails of

~ranular or fibrillar debris remained, sometimes concentrated at the ~ides of the trails, rendering a rail-like appearance. Since uninoculated BSTSY broth sometimes accumulates stringy or fibrillar material with long term storage, it is possible that the i fibrillar accumulations described above are contributed by the BSTSY medium. Fibrillar material found attached to filaments might arise through interaction of macromolecular materials of the serum­ supp1emented medium with surface material of the filaments. If accumulations of debris in growing, gliding Simonsiella

~ultures are the macromolecular products of cellular metabolism, then such materials might be satisfactorily classified as 11 Slime 11 production, as observed in other gliding organisms (Burkholder, 1934; Doetsch and Hageage, 1968). Sheath-ltke slime structures, characteristic of some cyanobacteria (Burkholder, 1934; Dodd, 1960) were not observed for Simonsiella. Accumulations of slime-like debris might be classed as i quite meager in Simonsiellaceae, in accord with the observations of Vahle and Meyer-Pietschmann (cf. Doetsch and Hageage, 1969; Weibull, 1960) for some myxobacteria. i Gliding motility is observed in BSTSY broth cultures only when ! transfers are made successively at short intervals for several days prior to observations, thus insuring a population of young, growing 39

J·r-liamet1ts-with peak gl·iding ability. Gliding filaments are consistently' I :phase-dense, smooth and sleek, and lack protruding blebs so charac- !teristic of involution forms. i Gliding motility occasionally does not occur for no apparent :reason, even after successive transfers of cultures. It is conceivable: that particular batches of media differ in composition, possibly due to the quality of bovine serum.

Agar concentration and rates of gliding motility Gliding motility was slower on BSTSY solidified with 1.5% or

~reater agar concentrations than on BSTSY of lower agar concentrations ..

~he question arises: What differences exist between growth and gliding motility in broth and on soft BSTSY agar as compared with such behavior on 1.5% agar-solidified medium? The higher concentration of agar must be imposing significant physical resistance to motility as available water decreases. Gliding on glass in broth or over soft BSTSY agar may provide the necessary solid substrate, yet allow filaments to straddle ,the substratum because they are surrounded by liquid. The liquid milieu of broth and of soft BSTSY agar presumably allows a more efficient exchange of nutrients and waste metabolites. Possibly growth without gliding motility may be favored on BSTSY of high agar concentrations due to a savings of cellular energy. Several Simonsiella strains which exhibited gliding motility in broth did not display motility on BSTSY agar. Sim 20, known to glide in broth, displayed pronounced filamentous growth on agar. long filaments extended· over the agar surface for hundreds of micro­ l ~eters in straight, undeviating projections or as complex patterns of L______, ,I' 40

------·- . ---- ·-- ;\~avy tangies of filaments (Figure 3). It appears that growth of Sim 20 l i ton agar medium is committed to the extension of long filaments over I l jagar without significant fragmentation into short segments. Perhaps lsim 20 filaments growing on agar-solidified medium do not have the \ ;capabi 1i ty to segment readily or, perhaps,· growth on agar pro vi des the ! static, stable environment required for such pronounced filamentous . ' growth that is not provided by the broth medium. It can be proposed 'that the extensions of long filaments over the agar surface allows the cells of such filaments to reach areas of the agar supplying fresh nutrients and to escape regions of accumulated waste products. This

'Simonsiella stl~ain m·iyht achieve by pronounced filamentous growth what other strains attain by gliding motility.

Effect of temperatul~e Table 4 records the effect of temperature on the extent of growth of Simonsiellaceae, and Table 5 records the effect of temperature on microscopically-viewed patterns of growth and gliding motility.

Browth occ~rred, with few exceptions, between 30 C and 40 C, with optimum growth at 37 C. Growth at 27-30 C and at 40-43 C was frequently poor or questionable (it was possible to mistake a heavy inoculum for growth) and restricted to the areas of heaviest inoculation. Thus, it was difficult to determine precisely at what temperature extremes growth still occurred. No growth, however, occurred at 45 C. The effect of temperature on the extent of growth varied among 1the strains, and was related to the source of the strains. Sheep :Simonsiella strains grew very well at 27 to 43 C. Human Simonsiella L 41

---·- ---1 ;TABLE 4. The effect of temperature on the growth of Simonsiellaceae 'strains after 48 hours of incubation on BSTSY agar. l -.--I I I TEMPERATURE (oc) $TRAIN ! I 27 30 33-34 37 40 43 CAT I i 1 + + + + ll + + + + 12 + + + + + 18 + + + + ,19 + + + +

DOG •2 + + + + .3 + + + + !4 + + + + 5 + + + + :6 + + + + '7 + + + + 8 + + + + i9 + + + + + 10 + + + + 17 + + + + 22 + + + + 23 + + + + 26 + + + + 27 + + + + 28 + + + + 32 + + + + 38 + + + + 39 + + + + 40 + + + + 42

(continued)

+ + + + + + + + + + + + + + + 16 + + + + + 24 + + + + + 25 + + + + '33 + + + + 34 + + + + 35 + + + + + '36 + + + + 37 + + + + + Al + + + + :42 + + + + + '43 + + + + + 44 + + + + + 45 + + + + 46 + + + + + :47 + + + + +

SHEEP S6 + + + + + + 20 + + + + + + 29 + + + + + + 30 + + + + + ;31 + + + + + Al + + + + ;Aly l + + + + + +

44

.. --·------~------·-.- .. ------·-·------~·--·------··· ------··· ------TABLE 5 (continued)

.HU~1AN I !13 2 2,6 2,3,6 2,3,6 3 :14 2,3,6 2,3 3,4 3,4 2,3,4 ;15 1 l l l l 16 2,3 2,4 2,3 3 3,4 24 2,3 3,4 2 2 2 25 2 2,3 2,4,(6) 3,4 33 3,6 2 2 2 ;34 2,6 2,6 2,3,4 2,3 (6) 35 2 2,3~4 2,3 3,4,(6) 3 36 2,3 2,3 2,3,4 2,3,4 37 2 2,3,4 2,3 2,3 2,3 41 2 1 2 2 42 3 3,4 3,4 3 3 43 2 3,4 2,3 3 2,3 44 3 3,4,{6) 3,4 3,4 2,3,4 45 3,4 3,4 2,3,4 3 46 3' ( 6) 3,6 3,6 3,6 3,6 47 1 2 2 2 2,3

SHEEP S6 2 2,3,4 2,3 2,3,4 2,3 2 20 2 2 2,3 3,4 2,3 2,3 , 29 1 2,3 2,3 2 3 I 30 2 2,3,4 3,4 3,4 3,4 31 2,3 3,4 3,4 3,4 1 A1 1 1 1 1 A1y l 1 l 1 1 1 2,3 45

·- -· - ---. -·--·--·--·-. strains-showed gene1hally good gl~owth at 27 C to 37 or 40 C. In contrast, dog Simonsiella strains showed poor growth at 27 C and 43 C

and, sometimes~ also at 30 and 40 C.

P1~ominent gliding motility occurred almost exclusively among dog Simonsiella strains and at temperatures which supported good growth. iAt 27 and 43 C, where growth was poor or questionable, gliding :motility was much reduced or non-existent. Among sheep and some human Simons}ella strains, pronounced lfi l amentous growth occun'ed most commonly in the temperature range of 'good growth, that is from 30 C to 40 C.

Effect of pH Optimum growth of Simonsiellaceae occurred on BSTSY agar of pH 7.2, and no growth occurred among any strains with pH values deviating by more than one pH unit to either side of pH 7.2 (Table 6). Growth among SimonsieJla strains was correlated strongly with the source of isolation. As revealed in Table 6, sheep strains displayed the greatest pH tolerance, growing in the pH range of 6.1 to 7.8-8.0.

Human Simonsiella strains grew well at pH 6~1 to 7.2, whereas dog Simonsiella strains grew satisfactorily at pH 7.2 to pH 7.8-8.0. Gliding motility (Table 7), observed among dog Simonsiella strains

Sim 2, 10, 17, 26, 28, 39, and 40, was restricted generally to the pH range of good growth (Table 6) i.e., pH 7.2 to 8.0.

:Effect of NaCl concentration Table 8 shows the effect of adding 0.5 or 1.0% sodium chloride to BSTSY agar on growth of Simonsiellaceae strains. These quantities 46

--·--··-1 -- -- ~ -- ~ABLE 6. The effect of pH on the growth of Simonsiella and Alysiella ;strains on BSTSY agar observed after 43-48 hours of incubation. I i I ! I pH pH 'STRAIN STRAIN 6.1-6.2 7.2 7.8-8.0 6.1-6.2 7.2 7.8-8.0

,CAT HUMAN : 1 + + 13 + + i 11 + + 14 + + ,12 + + 15 + + :18 + + 16 + + :19 + + 24 + + 25 + + DOG 33 + + + ~2 + + 34 + + i :3 + + 35 + + '4 + + 36 + + '5 + + 37 + + .6 + + 41 + + + 7 + + 42 + + 8 + + 43 + + 9 + + 44 + + 10 + + 45 + + )7 + + 46 + + 22 + + 47 + + 23 + + 26 + + SHEEP 27 + + S6 + + + 28 + + 20 + + + 32 + + 29 + + + 38 + + 30 + + + 39 + + 31 + + + 40 + + A1 + + A1y 1 + + + 47 h~ABLE-7-.- The effect of pH on the pattet'ns of grovrth and gliding !motility of Simonsiellaceae strains on BSTSY agar. Entries present ithe most distinctive patterns observed at 24 and 48 hours. Symbols in !Table 2. I i pH pH !sTRAIN STRAIN :I 6.1-6s2 7.2 7.8-8.0 6. l-6.2 7.2 7.8-8.0

'CAT HUMAN I ! l 2 2,3 13 2 2,3 ll 2 l 14 2 3,4 12 2,3 2 15 1 2 l l 16 2 2,3 2 2 24 l 2 25 2,3 4 DOG 33 2 2 1 2 2,4,6 2,4 34 1 2,4 3 2 3 35 3 2,4 4 2 2,3 36 2,3 2 5 1 2 37 2 2

I 6 1 2 41 2 2 2,3 7 2 2 42 2 3,4 8 2,3 2 43 2 2 9 2 2 44 2 2,3,4 ilO 2,6 2,6 45 2,3 2,4 17 2,6 2,6 46 2 2,3 22 2,3 2,3 47 2,4 2 23 2 2,3 :26 6 6 SHEEP ,27 1 1 S6 2 2,3 2 28 6,7 6,7 20 3 2,4 4· 32 2 1 29 3 3,4 2,3,4 :38 2 2,3 30 3 2,4 3 ;39 6 6 31 3 2,4 3,4 l40 4,6 3,6 A1 1 l Aly 1 1 l 1 48 lTABLE 8. The effect of NaCl concentration on the growth of Simons i e 11 a· iand Al_,ts_i_~_Ll~ strains on BSTSY agar after 48 hours of incubation. )

0.5% NaC1 :sTRAIN (standard BSTSY) 1% NaCl 1. 5% NaC1 2% NaC1 t i ;CAT

1 i' • + + + ' 11 + + + :12 + + + 18 + + + :i 9 + + +

.DOG 2 + + + 3 + + + 4 + + + 5 + + + 6 + + + 7 + + + 8 + + + 9 + + + i ·10 + + + 17 + + + :22 + + 23 + + + 26 + + + ·27 + + + ;28 + + + '32 + + 38 + + + )39 + + + 40 + + + 49

jiABLE--8-Tconti nuecf)~------­ !HuMAN 113 + + 114 + + 115 + + + + + + I1~: 125 + + i !33 + + ! !34 I + + \35 + +

I136 + + 137 + + + !41 + + 42 + + 43 + + 44 + + ,45 + + I :46 + + + i + + iSHEEP :s6 + + + ;20 + + + 29 + + + 30 + + + :31 + + + ! !Al + + iAly 1 + + +

i i I I i L______-- -- 50

--·------.------·------~- ---- .. ---····· ---~-- -·------~f sodium chloride supplement the 0.5% sodium chlorid~ already in I \unsupplemented BSTSY (contributed by the Tryptic Soy Broth without i !Dextrose (Difco).

1 Human Simonsiella strains grew in the presence of 1.0% NaCl but :did not generally grow on 1.5% NaCl. All other strains showed a gradua i ~iminution of growth on BSTSY agar of 1.0 and 1.5% NaCl. No Simon-

I :siellaceae strains grew on BSTSY a~ar of 2.0% NaCl. Microscopic obser-. ' :vati' ons revea 1ed an increasing 11 bl eached-out 11 appearance, as NaCl .concentrations were increased. Gliding motility and pronounced filamentous growth were greatly ! .reduced with the addition of NaCl (Table 9), in gliding Simonsiella strains Sim 2, 10, 17, 22, 26, 28, 39, 40 and in filamentous Simon- siella strains Sim 8, 11, 14, 16, 20, 29, 31, 37, 44 and S6, respectively.

Effect of 11 Flexibacteria Medium 111 Table 10 records the growth of Simonsiellaceae strains on the three modified 11 Flexibacteria Medium 1•• (Lewin and Lounsbery, 1969). One hundred percent sea water eliminated growth of all strains. Growth on the other two media was correlated with the source of the strains. Human Simonsiella strains grew on the medium prepared with distilled water and generally did not grow on the medium prepared with 50% sea water. Most other strains grew on both these media. Sheep Simonsiella and Alysiella strains grew better on the medium prepared with distilled water whereas dog Simonsiella strains generally grew slightly better on the medium with 50% sea water salts. 51

. ~· .. -·-···------·· ·----· ------, -··· ·-----~-... ------· ·--- -- TABLE 9. The effect of NaCl concentration on the patterns I of growth and gliding motility of Simonsiella and Alysiella strains on BSTSY agar. The most distinctive features observed at 48 hours of incubation are recorded. Symbols in Table 2.

0.5% NaCl STRAIN (standard BSTSY) l% NaCl 1.5% NaC1

CAT 1 2,3 2,3 1 11 4 3 1 12 1 2 1 18 1 1 1 19 2 2,3 2

DOG 2 2,3 3,6 slight 6 3 5 2 1 4 3,5 2 1 5 1 3 1 6 1 2 1 7 2 1 1 8 2,4 1 1 9 2,3 3 1 10 2,3,6,7 6,7 slight 6 17 2 2,6 1 22 1 3,6 23 1 2 1 26 7 7 slight 6 27 1 1 1 28 4,6,7 3,6 slight 4,6 32 2 2 38 1 2 1 39 3,6,7 3,6 slight 6 40 3,6 6 slight 6 52

- ···-·---·.. --·--- -· -- ·------·---~---- TABLE ~f(conti nued) HUMAN 13 1 2 14 4 4 15 2 2,3 16 3,4,5 2,3 24 3,5 2,3 25 3,5 3 33 2,5 3 34 3,5 3 35 5 2,3 36 5 2,3 37 4 4,6 2 41 2 2,3 42 3,5 2 43 3,5 2,3 44 4 2,3 45 2 2,3,4 46 5 5 2 47 1 2

SHEEP S6 2,3,4 2 2 20 4 2,4 1 29 3,4 1 1 30 1 2 1 31 4 1 1 A1 1 1 A1y 1 1 1 1 53

------·~--··--·- ·---·-·· ·-- ~--- !TABLE 10. The growth of Simonsiellaceae strains on modififed Flexi- ,ibacteri_____ a Medium 1 • observed after 48 hours incubation.

:I Salts + Salts + Salts+ Salts + Salts + Salts+ !STRAIN seawater l/2 seaw DisH 0 STRAIN sea~tJater 1/2 seaw Di sH 0 :---- 2 2 !cAT HUMAN

I 1 + -/+ 13 + : ,11 + + 14 + :12 + 15 + '18 + + 16 + i :19 + + 24 -/+ ---- 25 + DOG 33 + :2 + + 34 + 3 + + 35 + + 4 + + 36 + ,5 -/+ -/+ 37 + + •6 -/+ 41 -/+ + ' ..., i I + -/+ 42 -/+ '8 + + 43 -/+ + .9 + + 44 -/+ 10 + + 45 -/+ 17 + + 46 + 22 + + 47 + 23 + -/+ 26 + + SHEEP 27 + -/+ S6 + + 28 + + 20 + + 32 + -/+ 29 + + 38 + 30 + + 39 + + 31 -/+ + 40 + + A1 + Aly 1 + +

j I L._. 54

·- ·- ----~~-. --~-· ----- .. ·····-- -··--·-; . ~------Th~-~-ffe~t--~-f these modified special salts media on the patterns !Of growth and gliding motility of Simonsiellaceae strains (Table 11) i !suggests that distinctive filamentous growth and gliding motility I ! toccur under optimum growth conditions. For example, modest or ;pronounced filamentous growth occurred among human strains on medium prepared with distilled water but not on medium containing 50% sea :water where these strains grow poorly or not at all. These special salts media were employed to determine if particular 1concentrations or combinations of ions might spur gliding motility in stra·lns not known to glide on BSTSY agar, but they did not. However, gliding motility among Simonsiella strains known to glide consistently :on BSTSY agar occurred significantly only on the medium containing 50% 'sea water salts and to a diminished extent or not at all on the medium pre~ared with distilled water. This suggests that salt concentration ;or the presence of particular ions is indispensable for gliding motility to occur in Simonsiellaceae.

Further modification to Flexibacteria Medium 1 The effect of salts on gliding motility, demonstrated pr-eviously by dog _Simonsiella strains Sim 2, 10, 17, 26, 28, 39 and 40 on the .medium containing 50% sea water salts, was further tested by omitting

.either NaCl, MgS04, CaCl 2 or KCl from this medium. Growth and ,gliding motility on these modified media were compared with growth and gliding motility on the medium containing 50% sea water salts and on BSTSY agar. Most strains grew satisfactorily on all media except the medium lacking NaCl (Table 12). 55

··- ---- TABLE ll • The effect of modified Flexibacteria Medium 1 on the patterns 'of grovrth and gliding motility of Simonsiellaceae strains. Entries ~resent most pronounced patterns observed after 48 hours incubation. ,symbols in Table 2.

Salts + Salts + Salts+ Salts + Salts + Salts+ STRAIN seawater l/2 seaw DisH 0 STRAIN seawater 1/2 seaw Dis H 0 ---- 2 2 ,CAT HUMAN ' 1 2,3 l 13 l ll l 3 14 4 12 2,(6) 15 4 18 1 l 16 3 :19 1 3 24 1 25 2,3 DOG 33 1 2 2,6 2 34 2 3 2 l 35 1 2 4 1 2 36 1 5 2 1 37 1 2 ·6 1 41 1 3,4 7 2 1 42 3 ·8 1 '2 4 43 1 2 9 1 2 44 2 10 6 3 45 1 17 3,6 2,3 46 3 22 2,3 3 47 2,3 23 2 2 26 7 6 SHEEP 27 2 1 S6 1 1 28 7 (6) 20 1 1 32 3 2 29 1 1 38 1 30 1 1 :39 7 3 31 1 1 40 7 1 A1 1 A1y 1 1 2 56

TABLE. 12·.···. The growth of selected Simonsiella and Alysiella strains on modified 11 Flexibacteria t~edium 111 prepared with 50% sea water 11 11 C'medium 5 ) or with either NaCl, MgS04, CaCl2 or KCl ( media l thru 11 4 , respectively) omitted, and compared with growth on BSTSY after 48 hours of incubation

STRAIN Medium Medium Medium Medium t~edi urn BSTSY 1 2 3 4 5

CAT :n + + + + + 12 +

DOG 10 + + + + +++ 17 + + + + +++ 22 + + + + + +++ 26 + + + + +++ !28 + + + + +++ 39 + + + + +++ 40 + + + + + +++

HUMAN 13 +++ 46 + + +++ ------· SHEEP 20 + + + + + +++ 30 + + + + + +++ 31 + + + + +++ A1 +++ Aly 1 + + + + + +++ 57

------· -----·--. ---- . -~---··-·--·------All of the media failed to spur gliding motility among Simonsiel- laceae ~trains not known to glide on BSTSY agar. However, all media except that lacking NaCl supported good gliding motility among the usual gliding Simonsiella strains Sim 10, 26, 28, 30 and 40, and in Sim 22, known to glide occasionally. (Sim 17 had lost it gliding

.ability by the time of 'this investigation.) In fact, a 11 long distance" type gliding motility, not generally observed on BSTSY agar, occurred ,on these media (Table 2, pattern 8). Advanced, long distance gliding motility on these media,· ;summarized in Table 13, was characterized by populations of individual filaments gliding from the borders of heavy growth areas for long

1 distances onto the surrounding previously unoccupied agar surfaces. These observations suggest that salt concentration plays an important role in the culturing of Simonsiellaceae, particularly since removal of NaCl eliminates growth among most Simonsiella strains tested and eliminates all gliding motility.

The sodium chlo~ide concentration of the Flexibacteria Medium l is 1.25% whereas the other salt concentrations range from 0.05 to 0.25%. It is not known whether sodium chloride owes its growth and gliding motility stimulatory effect to the presence of sodium and chloride ions, or to its major contribution in a non-specific manner to total ion concentration.

Omission of salts found in BSTSY agar The previous experiments suggest an important, if not indis­ pensable, role for salts in the growth and gliding motility of Simonsiellaceae. Salts in BSTSY agar are contributed by all 58

-. TABLE 13. The patterns of growth and gliding motility of selected Simonsiella and Alysie11a strains on media modified from 11 Flexibacteria Medium 111 {compositions described in Table 12). The most prominent features observed after 48 hours of incubation are recorded. Symbols in Table 2. 11 At edges 11 denotes that the observed pattern was restt·icted to the periphery of colonies and heavy growth areas.

STRAIN Medium Medium Medium Medium Medium BSTSY 1 2 3 4 5

CAT ll l l 2 2 3 12 1

DOG 10 6,8 @ 6,7-8@ 6-7,8@ 8 @ 1 edges edges edges edges 17 2 1 2 l 2 22 l 3,6,7 @ 3,6 2,6;8, 6-7,8@ l edges @ edges edges 26 7,8@ 7,8@ 6-7,8@ 7,8@ 7 edges edges edges edges 28 7,8@ 7,8@ 6-7 7,8@ 7 edges edges edges 39 6-7 6-7,8@ 6-7 6-7~8@ 6-7 edges edges 40 1 6,8 @ 6-7,8@ 6-7,8@ 3,6-7,8 6-7,8@ edges edges edges @ edges edges

HUMAN 13 1 46 3,4,5 1 5

SHEEP 20 3,4 4 3,4 4 3,4 4 30 1 l l 1 1 l 31 2,3,4 2 l l 2,4 Al l Aly 1 1 2 l 2 1 1 59

[ingr~-edi enls-~ i ncludi ng~Trypfic Soy. Brotn wi thouf Dextrose -(D1 fco), - ----~-~ I I I I iknown to contain 0. 5% NaCl and 0. 25% K2HP04. To test further the . ' fffect of salts on growth and gliding motility, the NaCl and K2HP0 4 of ~ryptic Soy Broth without Dextrose (Difco) were omitted. Salts of ' unknovm amounts contributed by other ingredients remained.

· The omission of NaCl and K2HP04 from BSTSY agar had no detectable ~ffect on the extent of growth of Simonsiella (Table 14). Gliding

I motility, however, among the consistent gliding Simonsiella strains i(Sim 10, 26, 28, 39 and 40) was eliminated or reduced on the modified 'asTSY medium (Table 15). When gliding motility did occur its

I appearar.ce was delayed for many hours. Simonsiella strains Sim 26 and 28, important gliding strains on BSTSY agar, displayed pronounced filamentous growth on the modified medium. Gliding motility in these strains was delayed for possibly 24 hours, and pronounced filamentous growth may have continued to be the dominant pattern with subsequent incubation. When salts were added again to modified BSTSY medium, micro­ scopically-viewed growth and gliding motility did not seem to equal that on regular BSTSY agar. It appears that the Tryptose (Difco) and Soytone (Difco) components of Tryptic Soy Broth without Dextrose (Difco), which were added individually in salt-deficient BSTSY, were ,inferior in quality to those of commercial Tryptic Soy Broth without Dextrose (Difco). Thus, it is likely that this affected the observed patterns of grm'lth and gliding motility. It is thought, however, that the experiment revealed to some extent the expected patterns of reduced ' ' gliding motility with reductions in salt concentrations. 60

~------fABLE 14:---the effeCt-a{ omi-ssion- o{ NaCl-and--KzHPo4 from ______! BSTSY on the growth of some Simonsiella strains. "BS/SOY" contained Tryptose + Soytone + Yeast Extract + 10% bovine serum. "BS/Soy + sa 1ts" contained the ingredients of · 11 BS/Soy" plus the restored salt concentration of BSTSY. BSTSY was prepared in the usual manner with Tryptic Soy Broth without Dextrose (Difco) (a commercial preparation containing Tryptose, Soytone, NaCl and K2HP04), Yeast Extract and 10% bovine serum. Observations were made after 48 hours of incubation.

. - STRAIN BS/Soy BS/Soy + salts BSTSY

DOG 10 + + +

22 + + +

26 + + + 28 + + +

39 + + + 40 + + +

HUMAN

46 + + +

SHEEP 20 + + + 61

,------·------·------; iTABLE 15. The patterns of grm

HUMAN

,46 5, slight 6 pronounced 5 pronounced 5

SHEEP 20 2 3-4 pronounced 4 62

,.------~------~------~------·------, !The influence of humid atmosphere I I Tables 16 and 17 record the results of two trials attempting to : Jshow an influence of humidity in the incubation chamber on the extent I . !of growth and on the patterns of growth and gliding motility of known i igliding Simonsiella strains Sim 10, 26, 28 and 40. These strains grew ! i well on BSTSY agar at 37 C, regardless of regular or humid incubation.

~umid incubation, however, significantly enhanced and prolonged gliding I motility of all four strains, which could be viewed both macroscopically ' . ~nd microscopically (Table 17). That gliding motility may be prolonged ' With humid incubation indicates that viability can be sustained for ' ~onger periods of time. i I '

~ffect of agar concentration Previous evidence showed that maximum gliding rates occurred in

~STSY broth, and that humid incubation enhanced gliding motility on agar· medium. To study in more detail the effect of the availability of water and of the fluidity of the environment, BSTSY medium was prepared with agar concentrations ranging from 0.8 to 4 percent. Table 18 records the effect of agar. concentration on the extent of growth of selected Simonsiella strains after 48 to 56 hours of incubation at 37 C. Growth is most abundant on BSTSY containing 0.8% agar and least ! abundant on BSTSY containing 4% agar, indicating a primary effect of availability of water on growth of Simonsiella. Gliding motility among some strains was most pronounced on BSTSY

~ontaining 0.8% agar and noticeably reduced or arrested on BSTSY ' ~ontaining 4% agar (Table 19). Simonsiella strains Sim 10, 26, 28 and ! ~9 displayed extensive gliding motility in the presence of 0.8% agar L. .. --. 63

r.;:lTA.BLE"f6~--T-heeffe~t-o.fhum-idi-ty.on" g~-~~th···~~d--~iiici"i"~9-~~-1:i"1"i1:y" of selected gliding strains of Simonsiella after four days incubation on ·1 BSTSY agar. I jsTRAIN Regular Incubation Humid Incubation l ! 110 Growth: good Growth: good l Motility: pronounced Motility: Much more pro­ nounced than regular incuba­ tion. At the outermost edges of growth areas there is a broad band of gliding motility.

26 Growth: good Growth: good Motility: pronounced Motility: significantly more pronounced than regular incubation for all growth areas.

28 Growth: good Growth: good Motility: pronounced; very f4otility: very pronounced, pronounced at the outermost particularly at the outermost edges of growth areas. edges of growth areas.

40 Growth: good Growth: good Motility: secondary growth Motility: very pronounced has obscured most of the particularly at the outer­ gliding motility. most edges of growth. 64

~------. --- . ------1 1TABLE 17. The effect of humidity on the extent of growth and on the ! 1\patterns of grov1th and gliding motility of selected gliding strains '

10f Simonsiella on BSTSY agar after 66 hours-of incubation. 1------~ !sTRAIN Regular Incubation Humid Incubation 1------llo Growth: good Growth: good Growth and motility patterns: Growth and motility patterns: Macroscopic view: Macroscopic view: Slight "halo" around Extensive "halo" around colonies due to gliding. colonies due to gliding. Microscopic view: Microscopic view: Pronounced gliding motility Very pronounced gliding and secondary growth motility and secondary I growth. ~-2-6 ______G_r_o_w-th_: __ g-oo_d ______Gr_o_w_t_h_:_g_o_o_d ______

Growth and motility patterns: Growth and motility patterns: Macroscopic view: Macroscopic view: No distinctive features. Extensive 11 halo" around Microscopic view: colonies due to gliding. Moderate-pronounced gliding Microscopic view: and secondary colony Very pronounced gliding and formation. secondary colony growth.

Growth: good Growth: good Growth and motility patterns: Growth and motility patterns: Macroscopic view: Macroscopic view: Slight "halo" around Extensive "halo" around colonies due to gliding. colonies due to gliding. Microscopic view: Microscopic view: Pronounced gliding and Very pronounced gliding secondary growth motility and secondary colony growth.

40 Growth: good Growth: good Growth and motility patterns: Growth and motility patterns: Macroscopic view: Macroscopic view: No distinctive features. Slight "halo" around Microscopic view: colonies due to gliding. Occasional slight to moder­ Microscopic view: ate gliding. Pronounced Pronounced gliding motility. filamentous growth. 65

:TABLE 18. The effect of agar concentration on the growth of some :Simonsiella strains on BSTSY medium, observed after 48-56 hours of ,incubation.

11 11 11 11 :Symbol explanation: - = no growth; ± = faint growth (consisting of microco1onies); "+ 11 =fair growth (consisting of small, widely­ spaced colonies); 11 ++ 11 = moderate growth; 11 +++ 11 = excellent, generally confluent growth; 11 ++++ 11 = extremely wet, confluent growth. An asterisk indicates that.growth is confined to heavily inoculated area.

STRAIN 0.8% agar 1.5% agar 3% agar 4% agar

10 ++++ +++ ++ ++

'17 ++++ +++ +++ Individual colonies start to predominate

22 ++++ ++ ++ ++

26 ++++ ++ to +++ ++ +to++

28 ++++ +++ +++ ++ to +++ Individual colonies start to predominate

39 ++++ . +++ +++ ++ Individual Mostly colonies individual start to colonies predominate

40 ++++ +++ ++ ++ 66

... .. -·· ' - -. -~· ·- .. !TABLE 19. ~-The effect of agar concentration on the patterns of growth 'and gliding motility of selected Simonsiella strains on BSTSY agar, :observed after 48-56 hours of incubation. Addition, significant observations made after 4 days of incubation are bracketed. Symbols ,in Table 2.

STRAIN 0.8% agar 1.5% agar 3% agar 4% agar

10 7,8 7 7 7

17 3,6? 7 4 [7] [7,8] [4,6]

22 1 2 4 3

26 8 7 4 3-4

28 8 8 7 4,6 [4,6-7] [6 reduced]

39 6-7 6-7 4,6 3 [7-8] [7-8] [6-7] [4]

40 1 7 4 4 67

bharacterized by spreading and long distance gliding among many

~ndividual filaments. Sim 22 and Sim 40, however, manifested entire­ edged colonies on BSTSY containing 0.8% agar, and gliding motility or filamentous growth on BSTSY containing 1.5% agar. This suggests that good growth on 0.8% agar can be achieved among some strains without filamentous growth or gliding motility, or that filamentous growth and gliding motility among some strains requires a less fluid substrate thari that provided by medium containing 0.8% agar. With gliding Simonsiella strains Sim 17, 26, 28, 39 and 40 pronounced filamentous growth occurred on BSTSY containing 3% agar, del~ying or replacing completely gliding motility. The situation was essentially the same on BSTSY containing 4% agar except that Sim 22, 26 and 39 showed instances that filamentous growth was also being impeded at this agar concentration. The marked diminution of gliding motility and, apparently, filamentous growth on BSTSY of high agar concentrations suggests that lack of water imposes significant resistance to gliding motility and to the extension of growing filaments. Or it is conceivable that growth on such media with a less fluid substratum causes metabolites to build up while reducing the availability of fresh nutrients, thus inhibiting filaments from actively increasing the colony size through gliding motility or filamentous growth.

Effect of serum concentration Bovine serum concentration of BSTSY was adjusted to 5, 10, 20 or 40% to see if serum concentrations other than the standard 10% bovine serum concentration might enhance gliding motility in gliding strains 68 iand promote gliding motility in non-gliding Simonsiellaceae strains. ! ,Several trials performed over a period of a year produced conflicting :results. High bovine concentrations displayed either growth inhibitory or growth enhancement effects.

BSTSY medium incorporating used BSTSY broth Repeated observations of gliding motility of Simonsiella on BSTSY agar revealed that motility was restricted to or, at least, more pronounced in regions of heavy growth or among closely-associated 'colonies. This recurring pattern suggested that gliding motility might be promoted by metabolites produced by growing Simonsiella cultures. To test for the influence of growth metabolites and to attempt to spur motility among non-gliding Simonsiella strains, BSTSY agar was prepared which incorporated portions of filter-sterilized BSTSY broth used previously to grow selected Simonsiella strains. Tables 20 and 21 record the effect of used BSTSY broth in which either Simonsiella strain Sim 39 and 40 had been incubated for four ot· eight hours on the extent of growth and on the patterns of growth and gliding motility, respectively, of selected Simonsiella strains. The overall results indicate that growth on BSTSY medium incorporating used BSTSY broth is less than that on regular BSTSY agar. Only on BSTSY agar incorporating used BSTSY broth preincubated for four hours with Sim 39 was the extent of growth among most strains comparable to that on regular BSTSY agar. BSTSY medium incorporating used BSTSY broth preincubated with Sim 40, also led to reduced gliding motility. In contrast, BSTSY medium incorporating used BSTSY broth preincubated with Sim 39 enhanced 69

TABLE 20. The effect of used BSTSY broth incorporated into fresh BSTSY ~gar on the growth of selected Simonsiella strains, observed after 48 hours of incubation. Symbols in Table 18. ! BSTSY agar containing 25% BSTSY broth preincu- bated with the Simonsiella strain and for the STRAIN BSTSY duration as follovJs: Sim 39 Sim 39 Sim 40 Sim 40 4 hours 8 hours 4 hours 8 hours

10 +++ ++ ++ ++ +

,17 ++ + + ±

20 +++ +++ +++ +++ ++

26 ++* + ± + ±

28 ++ +++ + +

39 +++ +++ ++ +

40 +++ +++ ++ ++

46 +++ +++ ++ ++ ++ 70

TABLE 21. The effect of used BSTSY broth incorporated into fresh BSTSY agar on the patterns of growth and gliding motility of selected Simon- siella strains, observed after 48 hours of incubation. Symbols in Table 2. 11 At edges 11 indicates pattern is restricted to the edges of heavy growth areas.

BSTSY agar containing 25% BSTSY broth preincu- bated with the Simonsiella strain and for the STRAIN Regular quration as follows: BSTSY Sim 39 Sim 39 Sim 40 Sim 40 4 hours 8 hours 4 hours 8 hours

10 6-7 6 6-7 6 6-7

17 1 1 1 1 1

20 3-4 4 3-4 3 3-4

26 7 6-7 6-7 6 6

28 7 6,8 @ 6-7 6-7 l edges

39 6-7 6-7,8@ 6-7,8@ 1 1 · edges edges

40 6 6-7,8 @ 6,8 @ 6-7 1 edges edges

46 1 7,8@ 7,8 @ 3,6? 3,6? edges edges 71

------··· -· ~liding motility of known gliding strains Sim 28~ 39 and 40, and ;spurred gliding motility in Sim 46, a strain exhibiting gliding

~otility only on soft BSTSY agar. Gliding motility on this medium was exceptionally pronounced and characterized by extensive long­ distance gliding, particularly at the borders of heavy growth areas and previously unoccupied agar. Pronounced, long-distance gliding motility could not be repeated with the same batch of plates approximately a week later, however, suggesting that the wetness of the agar surface may have an important role in long distance gliding motility. Therefore, BSTSY agar medium incorporating used BSTSY broth preincubated with Sim 39 was prepared with agar concentrations of 0.5, 1.0, 1.5% and 2.0%. Standard BSTSY medium prepared with these agar concentrations was used as a control. The extent of growth (Table 22) on regular BSTSY agar and on BSTSY agar incorporating used BSTSY broth was essentially comparable, regardless of the agar concentration, although growth was slightly better on media containing 0.5% agar. The results presented in Table 23 indicate that used BSTSY broth frequently enhances gliding motility in comparison to that observed on regular BSTSY agar of equivalent agar concentration. This was true for all strains tested, that is Sim 10, 26, 28, 39, 40 and 46. Used BSTSY broth containing 2% agar sometimes appeared to enhance filamentous growth, as seen for Sim 26, 28 and 46. The overall results suggest that gliding motility is enhanced, if not initiated and promoted by the products of metabolism, and that this effect is made apparent only on media containing agar concentra- tions less than 1.5 or 2.0%. It can be proposed that gliding - TABLE 22. The effect of both agar concentration and used BSTSY broth on the growth of selected Simonsiella strains, observed after 20 and 48 hours of incubation. The used BSTSY medium was prepared by incubating with shaking for 15 hours BSTSY broth inoculated with Simonsiella Sim 39, then filter-sterilized and added to an equal volume of fresh, sterile BSTSY agar. Symbols in Table 18.

STRAIN 0.5% agar 0.5% agar 1% agar 1% agar 1.5% agar 1.5% agar 2% agar 2% agar Fresh BSTSY Used BSTSY Fresh BSTSY Used BSTSY Fresh BSTSY Used BSTSY Fresh BSTSY Used BSTSY • 10 20 hours: +++ ++ to +++ +++ ++ ++ to +++ ++ to +++ ++ to +++ ++ to +++ 48 hours: +++ +++ +++ ++ to +++ ++ to +++ ++ to +++ +++ +++ 26 20 hours: ++ to +++ ++ to +++ + ++ to +++ + ++ + ++ 48 hours: +++ +++ ++ to +++ +++ +++ ++ to +++ ++ to +++ ++ to +++ 28 20 hours: ++ ++ to +++ ++ ++. +++ ++ ++ +++ 48 hours: +++ +++ ++ to +++ ++ to +++ +++ ++ to +++ ++ to +++ ++ to +++ 39 20 hours: +++ +++ ++ + ± to + + ++ ± to + 48 hours: +++ +++ +++ ++ ++ to +++ ++ to +++ +++ ++ to +++ 40 20 hours: +++ +++ ++ +++ ++ to +++ ++ to +++ ± to + ++ to +++ 48 hours: +++ +++ +++ +++ +++ +++ + ++ to +++ 46 20 hours: ++ ++ +++ ++ to +++ ++ to +++ +++ ++ to +++ ++ to +++ 48 hours: t+ to +++ ++ to +++ +++ ++ ++ to +++ ++ to +++ ++ to +++ ++ to +++ ...... N TABLE 23. ·The effect of both. agar concentration and used BSTSY broth incorporated into fresh BSTSY agar· "on····: the patterns of growth and gliding motility of selected Simonsiella strains, observed after 12, 20 and 48 ·. hours of incubation. See Table 22 for preparation of used BSTSY medium. Symbols in Table 2. . ' . . . . '

STRAIN 0.5% agar 0.5% agar 1% agar 1% agar 1. 5% agar 1.5% agar 2% agar 2% agar . Fresh BSTSY Used BSTSY Fresh BSTSY Used BSTSY Fresh BSTSY Used BSTSY Fresh BSTSY Used BSTSY i

10 12 hours: 6-7 6-7 6 7 6-7 7 6-7 6-7 20 hours: . 7 7 6 7 7 7 7 7 48 hours: 6 8 7 8 8 8 8 8

26 12 hours: 6 8 6 7 6 8 6 7 20 hours: 6 8 6 6-7 6 7 6 7 48 hours: 6 7 7 8 6-7 8 4,6 4,7

28 12 hours: 4,7 4,7-8 6 7-8 4,7 4,7 4 4 20 hours: 6-7 7 2,6 4,7-8 4,7-8 4,7 3-4,6-7 4,7-8 48 hours: 3 6-7 6-7 7-8 3-4,6-7 8 4,7-8 4,7-8

...... , w . ··---···-·· TABLE 23 (continued)

39 12 hours: 6-7 6-7 6 2-3,6-7 2 3,6 2 6 20 hours: 6,8 @ edges 6-7 1 2-3,6-7 2-3 6 2 6 48 hours: 4,8 @ edges 4,8 @ edges 3-4 4?6 4,6 4,6 2,6 2,3

40 12 hours: 6 6 1 8 6 8 1 1 20 hours: 3,8 @ edges 3,8 @ edges 1 8 4 8 1 3 48 hours: 3,6-7 3,6-7 1 6-7 1 6-7 3 1

46 12 hours: 6-7 6 5 8 5 5,8 5 5 20 hours: 7-8 7-8 5,6? 8 5 8 5 5 48 hours: 7-8 6-7 5 8 @ edges 5 8 5 5

"""'..j::o 75

,motility is prompted by the buildup of metabolites and continues in ,response to a gradient of decreasing metabolites and increasing fresh nutrients. Gliding motility presumably would cease when the gradient no longer exists and where fresh nutrients can support renewed growth.

The effect of silica gel Silica gel was substituted for agar as a solidifying agent for BSTSY medium. It was hoped that silica gel might have some influence on the patterns of growth and gliding motility. For example, the texture of silica gel might alter the nature of the 11 etched tracks 11 which are formed by filaments gliding on agar. Any observed differences in the nature of the tracks might assist in determining if the tracks result from a wearing away of the agar or from enzymatic activity on agar. Two attempts to grow Simonsiella strains Sim 10, 26, 28, 39 and 40 on BSTSY solidified with silica gel were unsuccessful. It is possible that the high levels of phosphate in silica gel were growth inhibitory, since phosphate appeared to be growth inhibitory in preliminary experi­ ments with Simonsiellaceae. Roslycky (1972) has pointed out that the :silica gel medium of Funk and Krulwich (1964) used in the present work has the disadvantage of high phosphate levels. CONCLUSIONS

The extent of growth of Simonsiella strains in response to ranges of temperature, pH, NaCl concentration and synthetic se,a water salts concentration (Lewin and Lounsbery, 1969) varied according to the source of isolation of these strains. The influence of these environ- mental factors on growth suggested that human, dog, and sheep Simonsiella strains belong to individual groups of organisms. The cat Simonsiella strains did not present uniform results in response to environmental factors, and no conclusions regarding their classification can be presented. The range of pH supporting growth was particularly fixed for a group of Simonsiella strains isolated from the same type of animal, and it seemed that this index alone invariably correlates with the source of isolation of the strains. Thus, growth responses of Simonsiella to a range of environmental conditions appears to offer additional information for the classification of Simonsiel1aceae, in addition to information derived from cellular morphology and

biochemical and physiological tests (Pangborn et ~., 1973 and 1974; Nyby, 1974; Gregory, 1975), fatty acid profiles (Jenkins, 1976), and mole percent G+C content (Kuhn et ~., 1974). Gliding motility on BSTSY agar was observed among only a fraction of the Simonsie11a strains and was not observed in either Alysiella strain. Non-gliding Simonsiellaceae strains display patterns of growth ranging from colonies with entire edges to colonies with varying degrees of filamentous growth. Gliding motility on standard BSTSY agar could not be observed directly due to extreme slowness. Instead, the etched tracks formed 76 77

'on agar surfaces at the periphery of colonies by gliding Simonsiella filaments after many minutes or hours of incubation was an indicator ,of the presence and extent of gliding motility. Simonsiella strains cultured on slide-mounted agar medium demon­ strated that filaments could grow and glide simultaneously. However, the overall growth and gliding motility pattern produced with long term incubation showed that gliding motility was generally transitory for particular filaments and was followed by secondary colony formation in regions of fresh agar. Swarm-like gliding motility (Stanier, 1942a), seen, for example, in myxobacteria, where 11 armies 11 of closely­ associated cells actively glide over the entire agar surface, was not seen in Simonsiella. A type of long distance gliding could be promoted on medium containing used BSTSY broth or synthetic sea water salts ( 11 Flexibacteria Medium 111 of Le\'iin and Lounsbery, 1969), particularly when agar concentration was 1.0% or less. Gliding motility on BSTSY agar occurred to the greatest extent in areas of heavy growth and among closely-apposed colonies if access to fresh agar existed. Long distance gliding was also promoted in these regions. Gliding motility could be observed directly in BSTSY broth or on soft BSTSY agar. Average gliding motility rates of young, growing cultures of selected Simonsiella strains were as follows: Sim 19, 5 vm/min; Sim 20, 10 vm/min; Sim 26, 23.8 vm/min; Sim 28, 13.8 vm/min; Sim 46, 22.3 vm/min. Overall observations of growth patterns and gliding motility in Simonsiella prompt the following general conclusions: 78

- - . r· ------L' Gliding moti 1 i ty and filamentous growth vary among the Simon- ! :siella strains, and presumably, are a constant feature for each strain grown aerobically at 37 C on BSTSY agar.

2. These patterns vary ev~n among the group of strains isolated from the same type of animal, but several subgroups can be established :to accommodate the diverse strains of a group. 3. Substantial gliding motility and filamentous growth generally occur only where good growth occurs. 4. Low agar concentrations and humid incubation enhance gliding 'motility among known gliding Simonsiella strains and, occasionally, will promote gliding motility in strains not known to glide on BSTSY agar. 5. Reductions in NaCl concentration in BSTSY agar and in modified 11 Flexibacteria Medium 111 (Lewin and Lounsbery, 1969) inhibited or reduced gliding motility. It is also attractive to present the following proposals to attempt to interpret some of the observed manifestations of gliding motility and filamentous growth: 1. Gliding motility·may be promoted by accumulations of waste metabolites. 2. Gliding organisms may glide in response to gradients of increasing nutrients and decreasing metabolites, and may cease to glide (and begin the formation of secondary colonies) where such gradients no longer exist, i.e. in regions of agar surface supplying ·fresh nutients. 3. Gliding motility may occur only as necessary to reach fresh nutrients. 79

: .. ~ 4.- Growing fi 1 aments become immobilized when they turn on their

sides (Steed, 1962). Gliding motility may resume at the periphery of secondary colonies among the terminal segments of filaments whose ventral surfaces either do not lose contact with the substrate or somehow re-establish contact as conditions favoring gliding motility reappear .. 5. Pronounced filamentous growth may be an alternative to gliding motility to reach fresh nutrients on BSTSY agar. One Simonsiella strain, Sim 20, which glided in BSTSY broth, displayed pronounced filamentous growth on BSTSY agar. Its failure to glide on agar suggests either an inability to move across the agar surface, an inability of the filaments to fragment into motile segments, or a responsiveness for filamentous growth which allows access to fresh nutr1ents. REFERENCES

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