Enumeration Isolation and Characterization of Beggiatoa

The same enrichment medium, but without the acetate, was the best enrichment medium from which to obtain pure cultures becauseitsupported good growth ofthe beggiatoas withoutallowingthem

755-770 0099-2240/78/0036-0755$02.00/0

Copyright © 1978 American Society for Microbiology

36, No 5 Printed in U.S.A.

from Freshwater Sedimentst

WILLIAM R STROHL AND JOHN M LARKIN*

Departmentof Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803

Received for publication6September 1978

An accurate most-probable-number enumeration method was developed for

counting the number ofBeggiatoa trichomes from various freshwater sediments

The medium consisted of extracted hay, diluted soil extract, 0.05% acetate, and

15 to 35 U of catalase per ml. The same enrichment medium, but without the

acetate, was the best enrichment medium from which to obtain pure cultures

becauseitsupported good growth ofthe beggiatoas withoutallowingthem to be

overgrownby other bacteria.Atotal of 32strains of Beggiatoa were isolated from

sevendifferentfreshwater habitats and partially characterized The strains were

separated into five groups based on several preliminary characteristics Four of

the groups contained cells with trichomes ofapproximately the same diameter

(1.5 to 2.7,tm) andmay be Beggiatoa leptomitiformis or an unnamed species

The fifth group appeared to be Beggiatoa alba With the exception of three

strains, allof the strainsdeposited sulfur in the presence of hydrogen sulfide, and

all strains grew heterotrophically and deposited poly-fB-hydroxybutyrate and

volutin when grown on acetatesupplemented with low concentrations of other

organic nutrients Thin sections of sulfur-bearing trichomes indicated that the

sulfur granules were externalto thecytoplasmic membrane and thatthey were

surrounded byanadditional membrane.

Beggiatoa isafilamentous gliding bacterium

capable of oxidizing sulfide toelemental sulfur,

which it deposits in its cells (14, 36) When the

sulfide is depleted, the bacterium further

oxi-dizes the deposited sulfur to sulfate, which is

then releasedtothe environment (28) The

ecol-ogy, taxonomy, physiology, andmanyother

as-pects of Beggiatoa biology are poorly

under-stood.

Interest in the organism has been sporadic,

perhaps because of the difficulty of isolating and

maintaining cultures in the laboratory Recently,

interestin Beggiatoa hasbeen revivedbecause

of the fine work ofPitts etal (25) and ofJoshi

and Hollis (12), who suggested that Beggiatoa

and rice plantsmay occur togetherin a

mutu-alistic association in which the bacterium

oxi-dizes H2S in theroot zone, thus protecting the

plant from the toxic effects of H25, and the plant

roots excrete catalase, which decomposes the

toxicperoxides produced bythe bacterium

dur-ingitsmetabolism.

WehaveseenBeggiatoainclose association

withthe root zoneof themarsh grassSpartina

alterniflora (unpublished results), and J.

Charba hasseenit in close association with the

tDedicated to J L Stokes on the occasion of his

retire-ment.

rootsofwaterhyacinths (personal communica-tion) It is possible that Beggiatoa plays an important role in plant health in the entire flooded-soil/plantecosystem

Several techniques for theproduction of en-richment cultures of Beggiatoa from nature have appeared, the most recent by Joshi and Hollis (11) All are based on the techniques originally described by Cataldi (5), in which extracted hay (EH) is a prime ingredient Be-causeof the probable ecological significance of Beggiatoa in the flooded-soil habitat, it would

be valuable to know which enrichment tech-niques work the best, and ifanyof themcanbe adapted for the enumeration of Beggiatoa in its habitat.

In this paper we present our attempts to de-velop methods for the enrichment, enumeration, and isolation of Beggiatoa fromnature, aswell

as the preliminary results of our attempts to

characterize 32isolated strains insomeof their

morphologicalandphysiological features.

MATERIALS AND METHODS

Media.Allmedia for theenrichmentofBeggiatoa werebasedonthe method of Cataldi (5) andonthe various modifications of her technique as used by

others.Aningredientcommon toall of the enrichment mediawashay,orgrass,whichwasextractedatleast

755

756 STROHL AND LARKIN

five timesfor30min each inboilingwater, withtwo

rinses incold tapwaterbetween each extraction(EH)

When needed, a soil extract (SE) was prepared by

mixingapproximately 500g ofblack,

sulfide-contain-ing mud with1liter oftap water,allowingthecoarse

particlestosettleout, and thenfilteringthe

superna-tant fluid through Whatman no 2 filter paper

con-tained in a Buchner funnel For diluted soilextract

(DSE), the SEwasdiluted 1:2 with tapwater

Pringsheim (14) basal salt solution asmodifiedby

W Koch(personalcommunication) consistedof,per

liter, 5 ml ofa traceelement solution(14), 20mlofa

saturatedCaSO4solution,0.00045g ofNH4Cl,0.001 g

ofK2HPO4, and 0.0001 g ofMgSO4.7H20

BPmedium consisted of thefollowing ingredients:

basal saltsolution,0.05% sodium acetate, 0.05%

nutri-entbroth(DIFCO Laboratories,Detroit,Mich.),and

1.0% agar.Filter-sterilized catalasetogiveafinal

con-centration of 15 to 35 U/ml (3) was added before

pouring intoplates

MP medium consisted of thefollowing ingredients:

basal salt solution, 0.0001% sodium acetate, 0.03%

Na2S, and1.0% agar TheNa2Swasautoclaved

sepa-rately and added tothe medium before plates were

poured

Microcyclus-Spirosoma agar has been described

previously (19) Nutrient agar was obtained from

Difco

Evaluation of MPN techniques To determine

which mediumwouldyieldthe bestresults fora

most-probable-numberdetermination (MPN)ofBeggiatoa

in sediments, sediment samples were inoculated in

duplicate into five setsof tubes (three dilutions per

set)containingmedia that had been usedsuccessfully

forsimpleenrichmentbyothers(5,9, 11,35)ormedia

with variousmodifications that seemed appropriateto

us.The three media that gave thehighestcounts,plus

one newmediumsuggestedbytheresults,were

reex-amined inquadruplicate for theirabilitiestoprovide

suitable MPN results with additional sediment

sam-ples Each tube (25 by 250mm) in the MPN series

receivedapproximately0.5g ofEH,50ml oftheliquid

mediumtobetested, andasediment inoculum known

tocontainBeggiatoa.The tubeswereincubated for2

weeksat roomtemperature(approximately22°C) and

were then examinedmacroscopically for the presence

ofBeggiatoa by lookingfor the "fluff ball" tufts of

colonies, characteristic ofBeggiatoa (9) or for mat

formation supportedbyBeggiatoafilaments

Confir-mationofBeggiatoapresence, andanestimate of the

degreeofcontaminationbyotherbacteriain

presump-tively positivetubes,wasmadeby phase microscopy

The MPN obtained with each medium was

deter-minedfromastandard table(18)

To determine whether the MPN procedures that

resultedinthe highest counts would be accurate for

theenumeration ofBeggiatoa fromsediments,apure

cultureofBeggiatoawasgrown anddivided into four

aliquots.Onealiquotwasusedforadirect microscopic

countinahemacytometerto determine the number

oftrichomes present The viable population in the

secondaliquotwasdetermined with a plate count on

BP medium; another aliquat was divided, and the

beggiatoaswere enumerated by the MPNtechnique

with the mediachosen on the basis of theexperiments

described above The fourth aliquot was inoculated intoablacksulfide-emitting sediment which was then

stirred, divided, andassayed with the various MPN media to determine which one(s) gave the highest

counts and what percentage ofthe initial inoculum

wasrecoveredwith each technique Theseprocedures

werecarriedoutin triplicate using Beggiatoa isolate B14LD

Enrichments for the isolation ofBeggiatoa Thesamemediaasdescribed above were placed into 160-mlsterile prescriptionbottles and were inoculated with 1 to 2 g ofsulficontaining sediments as de-scribedby Joshi and Hollis (11) except that the bottles

wereautoclaved before use to hold down fungal

con-tamination Cycloheximide was added to a final

con-centration of 40 mg/ml to some cultures to reduce fungal and protozoan contamination After about 1 weekof incubationatroomtemperature, the enrich-ments were examined by phase microscopy for the presenceofBeggiatoa and for the degree of contami-nationby other microbes

For enrichment ofBeggiatoa strains from an S

alterniflora-containingsaltmarsh, a medium consist-ing of EH, DSE prepared from mud obtained at the collection site, and filter-sterilized catalase was used The salinity was adjusted to 0, 20, 30, 35, 40, 45, 50, 55,

60, or 65% ofsyntheticseawater(Seven Seas Marine Mix, Utility Chemical Co., Patterson, N.J.) because thesalinity of that site,nearLeesville, La., was about one-half that of sea water(W.Patrick, personal com-munication)

Isolation of Beggiatoa Beggiatoaswere isolated from enrichment media using a modification of Pringsheim'stechnique (27) for the isolation of fila-mentous gliding organisms Tufts of filaments from enrichment cultures were transferred with sharp-pointed forceps throughfourwashes in sterile basal salt solution made withtap water Afinal wash con-sisted ofa5-min soakin the samesolution with 100 U

of catalase addedper ml The washed tufts offilaments were driedby absorption of the excess water onto an agar plate Some of thepartially driedfilaments were then placed onto afreshly pouredplate of either BP

or MP medium

After2to4days of incubation at 28 to 35°C, the cultures wereobserved with a dissecting microscope, and isolatedfilaments were picked up by cutting out

ablock of agar beneath a trichome andtransferring it

to a fresh plate ofthe same medium Occasionally, isolation attempts could be made as early as 8 to 10 h after inoculation, butusually the filaments had not glided far enough away from the contaminants by that time, and attempts at 4 days proved best

Todetermine the optimum concentration of agar in the isolation medium, concentrations of 0.8, 1.0, 1.2, 1.4, 1.6, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5% were included A range oftemperatures, including 17, 23, 28, 35, and 45°C, wastested Concentrations of 0.001, 0.01, 0.05, 0.1, 0.5, and 1.0%sodium acetate and/or nutrient broth

wereadded todetermine the optimal concentration of eachnutrient The plateswereinoculated with washed filaments from enrichment cultures, and they were examined after2to4days with a dissecting microscope

to determine the level of contamination around the filaments and theability of thefilamentstoglide away

APPL ENVIRON MICROBIOL

BEGGIATOA FROM FRESHWATER SEDIMENTS 757 from thecontaminants This was repeated with several

purecultures after they were isolated

Use of inhibitors The antibiotic sensitivity of

Beggiatoa strainswasassayed to determine whether

theywould be useful as aids in the isolation of

Beg-giatoa Antibiotic diskswereplaced on the surface of

BPmedium in a petri dish, and then another layer of

BP medium was added to just cover the disks The

plateswere incubated for 6 to 8hto allow the

anti-bioticstodiffuse, and then a washed tuft of filaments

wasplaced on the agar above each disk They were

examinedperiodically withadissecting microscope to

determine the viability of the filaments, the degree of

contamination, and the ability of the filaments to glide

awayfrom the contaminants

Sodiumazidewasincorporated into the medium at

concentrations ranging from 0.001 to 0.5% todetermine

if it would beanaid in facilitating isolation of

Beggia-toabyreducing the level of contaminants

Physiological characterization of isolated

strains.Beggiatoa cultures were stab inoculated into

semisolid (0.2% agar) medium under three conditions:

MP medium, BP medium, and BP medium with a

sterile petrolatum overlayto provide anaerobic

con-ditions After 2, 4, 6, 8, and12daysof incubation at

28°C, the growth and position of the growth were

recorded

Gelatin and casein hydrolysis were assayed

accord-ingtoPringsheim's methods (27) Catalase production

was assayed by adding 3% hydrogen peroxide onto

actively metabolizing cultures of Beggiatoa and

ob-serving forbubble formation Cytochrome oxidase was

assayed by flooding plates of 48-h Beggiatoa cultures

witha1% aqueous solutionof

N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (Eastman

Ko-dakCo., Rochester, N.Y.) and observing for the rapid

formation ofapurplecolor The effect ofcyanide and

sodiumdodecyl sulfate (SDS) onBeggiatoa was

as-sayed on plates of BP medium (without catalase)

whichcontained 0.01or0.05%filter-sterilizedKCN or

SDS,respectively

Thedeposition of sulfur in trichomes growninthe

presenceofH2Swasdemonstratedbyamodification

of themethods of Skermanetal (38) and of Skerman

(37) Onedrop ofcellsuspension and1drop of

reagent-grade pyridine (Mallinckrodt Chemical Works, St

Louis, Mo.)weremixedonaslide,and thesuspension

wassealed withacoverslip andpetrolatum.Positive

resultswererecorded if thegranules disappeared from

the cells and if rhombicormonocliniccrystalsformed

extemal to thecellsasviewedby phase microscopy

Controls usingtrichomes grownonBPmedium, and

therefore without sulfurgranules,wereused

Poly-f-hydroxybutyrate (PHB) and volutin were

stained forlight microscopy usingSudanBlackBand

methyleneblue,respectively.Electronmicroscopywas

usedtoverifythe inclusionsinarepresentative strain,

B15LD

Electron microscopy Amodified

Ryter-Kellen-berger (33) technique wasusedforthe thin sections

Plates containing 96-h trichomes grown onBP

me-diumwerefloodedwith0.05%OS04in 0.1 MVeronal

acetate buffer at pH 6.0 for 20 min The trichomes

werethenscrapedoff theagarsurfaceandtransferred

to 0.1MVeronal acetate-buffered1%OS04for 16 hat

roomtemperature The trichomeswerethen rinsedin

Veronal acetate buffer, postfixed with 0.1 M Veronal acetate-buffered 0.5% uranyl acetate for2 h, and de-hydrated with 25, 50, 75, and 90% and two changes of 100%ethanol,followedbytwo washes in 100% propyl-ene oxide The fixed anddehydrated trichomeswere

embeddedinEpon812plastic (21) and then sectioned

on anLKBUltrotome (LKB Inc.,Stockholm, Sweden) usingadiamond knife The thinsectionswerepicked

upon300-mesh coppergrids and stained withuranyl

acetate (40)andthenleadcitrate(31) Allthin-section micrographs were obtained using an RCA EMU-2 electron microscopeat50kV.Cells containing sulfur granules were exposed toMPmediumfor4 hprior to

fixation

Both puffballsand surface colonies ofBeggiatoa were viewed by scanning electron microscopy The puffballswereprepared fromanaxenic liquid culture

of strainB15LD grown inastaticliquidBPmedium They were fixed for 2 h in 1 M Veronal acetate-buffered 3%glutaraldehyde The samples were then dehydrated with 25, 50, 75, and 90% and two changes

of 100% ethanol, and then they were critical-point dried with100%acetone asthetransitionsolvent The surface colonies of Beggiatoa were prepared by fixing strainB12LD, grown on MP medium, with 4% osmium vapors for 24hat roomtemperature Smallblocks of agar containing the trichomes were cut out of the plates, carefully rinsedwithdistilled water, andthen

dehydrated with acidified 2,2-dimethyoxypropane (23) They were then critical-point dried with 100% acetone as thetransition solvent Thescanning

elec-tronmicroscopesampleswerecoatedwith 15 to 20 nm

ofgold-palladium usingaHummerI Sputter Coater (Technics, Inc., Alexandria, Va.) andwereviewedon

aHitachi S-500scanningelectronmicroscope

Chemicals Cycloheximide and fungal catalase wereobtained from theSigma Chemical Co., St Louis,

Mo The latterwas always filtersterilizedand then

addedtothesterile mediain anamountsufficientto

give15 to35U/ml

Additional procedures Measurements of

fila-ment sizewere obtained with aFilar micrometer.A Gilletand Sibert microscope equippedwith aNikon

AFM camera attachment was used for phase and

bright-fieldobservations andphotomicrography

RESULTS Enumeration of Beggiatoa Usingthe

pres-ence ofpuffballs (Fig 1) ormats followed by microscopic confirmation (Fig.2),apreliminary screening of the various enrichment media as

possiblemedia for MPNtechniquesshowed that four media gave significantly higher MPN

re-sults (Table 1) The best resultswereobtained withamediumconsisting ofEH, DSE,and0.1%

acetate.However, this mediumwasbadly

over-grownbycontaminatingbacteria andso was not suitable as anenrichment medium from which

toattempt the isolation ofBeggiatoa. The ad-dition of catalase enhanced some media (cf.

stream water versus stream waterplus catalase,

or DSE versus DSE plus catalase) without ap-VOL 36,1978

758 STROHL AND LARKIN

TABLE 1 Preliminary comparison ofvarious

enrichmentmediafortheenumerationof Beggiatoa

Trichomes per g

Mediuma (reference) (wet wt) of

sedi-ment

Tapwater(5, 35)

Tapwater +catalase

Streamwater +catalase

Distilledwater,BSS,catalase Soilextract +catalase(11)

DSE

DSE+0.1%acetate.

DSE, 0.05% acetate, catalase

FIG 1. Typical appearance ofa Beggiatoa fluff

ballfromanenrichmentculture.Bar,100 pm.

FIG 2 Typicalappearanceof Beggiatoa froman

enrichment culture Thegranulesconsistof sulfur (S)

and PHB(P) Bar,10,um

pearing tostimulatethe growthof

contaminat-ing bacteria, so three of the above media were

reexamined; the fourth medium, consisting of

EH, DSE, 0.1%acetate,andcatalase,wasused

Using the highest MPN as a sole criterion, a

medium of EH,DSE, 0.05%acetate,and catalase

wasthebest (Table 2)

The three media thatappearedtobe the best

werethen examined for theirabilitytorecover

aknown number of trichomes from sediments

inoculated witha pureculture(Table 3).Aplate

countofa purecultureofthe inoculumyielded

3.4 x 104 trichomes per ml The same culture

yielded 1.6 x 104to3.7 x 104 trichomesperml

whencounted with the three MPN media After

inoculation of the culture intoanonsterile

sedi-mentandcorrectingfor the dilution and for the

6.4 3.2

l 3.5 13.2 35.5 13.0 9.5 25.0 73.0 45.0

aAll media contained EH and about 50 ml of the

appropriate liquidmedium.BSS, Basalsaltsolution

TABLE 2 Comparisonofthefourbest mediaforthe enrichment and enumerationof Beggiatoa

Trichomesb per g

sedi-ment

DSE, 0.1% acetate, catalase 120

DSE, 0.05% acetate, catalase 212

aAll media contained EH and about50ml of the appropriateliquidmedium

bAverageof fourreplicateseach

TABLE 3 Evaluationof three media for their abilitiesto recoverBeggiatoainoculated intoa

sediment

Counting procedure perTrichomes %Recovery

g(X104) ~Rcvr Viableplatecountof pure 3.4 100 culture

MPN of pureculture

MPN of sedimentafter

in-oculationb

aAllMPN mediacontainedEH, DSE, 15 to 35 U of catalase perml, and theamountof acetate indicated

bResultsshown areafteradjustment for dilution of the culture and for thebackground Beggiatoa

popu-lationin thesediment

background Beggiatoa population, recoveries rangedfrom 1.5x 104to3.2 x 104trichomes per

ml In each case the medium with no acetate

APPL ENVIRON MICROBIOL

BEGGIATOA FROM FRESHWATER SEDIMENTS 759

gavethe poGrestresults, with 44 to 47% recovery,

and the mediumcomposed of EH, DSE, 0.05%

acetate,and catalase (SACHmedium) gave the

highest recovery rates, with 94 to 109% of the

viable count being recovered Increasing the

acetate concentration to 0.1% resulted in a

de-crease intherecovery rate

The SACH medium was used to enumerate

the beggiatoas from a variety of flooded

sedi-ments in the BatonRouge area.Typical results

(Table 4) ranged from11 to 95 trichomes per g

ofwetsediment.

Attempts were made to adapt theSACH

me-diumtothe enumeration of Beggiatoa fromthe

flooded sediments associated with the marsh

grassS alterniflora in salt marshes SACH

me-diumwasused with salinities rangingfrom 0 to

65% that ofsea water The salinities that

sup-ported the best growth of those beggiatoaswere

40 to45% of that ofsea water.Salinity ofgreater

than 60% orlessthan30% of that of sea water

resultedinareductionorcomplete inhibition of

growth Trichomes ranging from3 to 35 ,im in

width were observed in the MPN tubes from the

TABLE 4 Population of Beggiatoatrichomes in

variousflooded sedimentsfromthe Baton Rouge

area

Samplesite

Elbow Bayou

Nicholson Road ditch

City Park bayou

Sorority Road bayou

Campus Lake

CapitolLake

Trichomes per g (wet wt) of sediment

13

11

95 39 39 17

salt-marsh sediments (Fig.3) Attempts to enu-merate these organismsby the MPN technique developed for the freshwater strains were not reproducible due tothe growth of a white floc-forming bacterium which mimicked Beggiatoa

and interferedwith itsgrowth The addition of Na2S, vitaminB12(27), orvariousconcentrations

of aoetatedidnothelp significantly.

Isolation of Beggiatoa. The best medium for isolationpurposes, becauseitcontained the lowest level of contaminating bacteria with a reasonably high recovery of Beggiatoa, was a medium consisting of EH, DSE, and catalase (EDC medium) Enrichments using nonex-tracted hay or undiluted SE resulted in high contamination levels and lowcountsof Beggia-toa(datanotshown).

From any Beggiatoa-containing enrichment from a freshwater sediment it was possible to isolate the organism, although it was easiest from EDC medium With some of the initial isolates, the utility of antibacterial agents as selectiveagents wasassessed Used singly, nitro-furantoin, sulfathiazole, penicillin G, and triple sulfa appeared to inhibit many contaminants while leaving Beggiatoa unharmed (Table 5)

Ampicillin, gentamicin, and polymyxinB either killed the beggiatoasor prevented their gliding

awayfrom the contaminants Tetracycline, kan-amycin, and streptomycin offered miniimal hope

as selective agents Those reagents that ap-pearedtobe useful when used singlywerethen tried in combination, with the combination of penicillin G plus nitrofurantoin and triple sulfa appearingtobe themostpromising Subsequent attempts toisolate Beggiatoa from enrichments were made on MP, BP,and BP-plus-antibiotic

FIG 3 Phasemicrographof Beggiatoa fromtheroot zoneofS.alterniflora fromasalt marsh Becauseof

thelarge diameter(15pm) ofthetrichome,thisorganismisprobablyB mirabilis Sulfur granulesmaybe

seenwithin the cells.Bar,10,um

VOL 36,1978

760 STROHL AND LARKIN

TABLE 5 Relativeefficacy ofvarious antibacterial

chemicalstoaidin the isolationof Beggiatoa

Antibacterial agent (concn)

Ampicillin (10,ug)

Gentamicin (10jLg)

Tetracycline (30mg)

Nitrofurantoin (300 tg)

Kanamycin (30 Mug)

Streptomycin (10 Mug)

Sulfathiazole (1 mg)

Penicillin G(10 U)

PolymyxinB (300 U)

Triple sulfa (1 mg)

Penicillin G (10 U) + streptomycin (10

,ug).

Neomycin (30 Mg) + streptomycin (10

Mug)

Penicillin G (10 U) + sulfathiazole (1

mg)

Penicillin G (10 U) + nitrofurantoin (300

Mg)

Streptomycin(10,g)+nitrofurantoin(300

Mg)

Penicillin G (10 U) + tetracycline (30,g)

Neomycin (30 Mg)+ sulfathiazole (1mg)

Triple sulfa (1 mg) + polymyxin B (300

U)

Penicillin G (10 U) +neomycin (30 Mg) +

nitrofurantoin(300,g)

Penicillin G (10 U) +sulfathiazole (1 mg)

+ tetracycline (30,g)

Penicillin G (10 U) + nitrofurantoin (300

Mug) +triplesulfa(1mg)

Relative effecta

+ +

++

+++

aResultsaregradedfrom+ to ++++ onthe

effec-tiveness of the agentsto preventgrowthof

contami-nantswhileallowingtheBeggiatoatoglideawayfrom

them Anegative sign indicates that Beggiatoawas

killed

media

Thirty-twostrains ofBeggiatoawereisolated

from seven different locations No two strains

from asingle enrichmentwere kept if they

ap-pearedtobe similaroninitial isolation Thebest

agarconcentration for their isolationwas1.0 to

1.2%, and the best nutrientconcentrationswere

0.0001%acetate (if0.03%Na2Swassupplied) or

0.05%acetate(if0.01to0.5% nutrientbrothwaw

added) The besttemperatureforisolationwa«

about33°C, but thecuturesdidnotsurvivepasi

3to4daysatthattemperature,andthetemper

aturewasdecreased to about25°Catthat time

Theuseof sodium azide in the medium atcon

centrationsranging from0.0001to0.05% didnot

facilitate isolation At concentrations of0.000]

to0.025% azide thecontaminantswere notsuf

ficiently inhibited Beggiatoawas notaffectec

by these low concentrations, but increasedcon

centrations first inhibited gliding and then in

hibited the growth of the trichomes

Although data with antibioticsindicated

that their incorporation into the media should

be ofhelp, it was found thatisolating the beg-giatoaswas relatively easy onthe other media and that the antibiotics offered no significant advantage

Characteristics of theBeggiatoa isolates Thirty-two isolateswereobtained, and the

mor-phological and physiological characteristics that

were shared by all ofour strainsare shown in Table 6 All strainsweremotileby gliding,were

abletogrow onboth MP and BPmedia, stored volutin and PHB as noted previously by Pringsheim and Weissner (30) whengrown het-erotrophically (Fig 4), andgrewinmedia made with freshwater but notwith salt water They

wereall oxidase and catalasenegative andwere

stimulated by the presence of catalase in the medium They failedtogrowinthepresenceof 0.05% KCN or 0.05% SDS All strains except

thosedesignatedas groupB(Table 7) deposited sulfur whengrownin thepresenceofNa2S The 32 strains tested were placed into five

groups (Strohland Larkin, Abstr Annu Meet

Am Soc Microbiol 1977, N84, p. 242) based

upon physiological and morphological charac-teristics (Table 7) Many isolates formed spiral

patterns when grown on an agar surface (Fig 6A), but only group A cells glided over them-selves to produce three-dimensional "super-coiled balls," which etched into the agar and rotated in place (Fig 5) The group A strains

were relatively fastidious and grew well with nutrient concentrations of less than 0.05% but

poorly onnutrientconcentrations above 0.05%

11

s

I

s

t

1) e r

s 'S

-t

1-t

1

r-d

1-i

TABLE 6 Characteristics sharedbyallof the

Beggiatoa isolates

GrowthonBP medium +

GrowthonMPmedium +

PHBdepositedonBPmedium +

VolutindepositedonBP medium +

SulfurdepositedonMPmediuma +

Production of catalase

Production ofcytochrome oxidase

-Growthon0.05%KCN

Growthon0.05%SDS Hydrolysis of gelatin.

SensitivitytopolymyxinB (300 U) + Sensitivityto neomycin (30jig) + Sensitivitytosulfathiazole(300jg)

Sensitivitytobacitracin(10tg)

Stimulation by catalase in the medium +

Growth in freshwatermedia +

Growth insalt-water media .-

Strictlyanaerobicgrowth

a Except for group B strains (Table 7), which grew wellon MPmedium butdidnotdeposit sulfur

APPL ENVIRON MICROBIOL

BEGGIATOA FROM FRESHWATER SEDIMENTS

FIG 4 Pure cultureof Beggiatoastainedtoshow the PHB(A)andvolutin (B) Bar,10pn.

GroupBstrainsweresimilar in sizeand in some

physiological characteristics to groupA strains,

although theydidnotdeposit sulfurinthe

pres-enceof H2S and they didnotnormally formthe

supercoiled balls Groups Cand D were similar

to each other but were differentiated on the

basis of temperature relationships, trichome

di-ameter,and theability to grow on nutrient agar

The group E strains were different from each of

the other strains.Theyhadwidertrichomes;the

average length of their trichomes was shorter;

they grew dispersed inliquid culture under

ap-propriate conditions; theyweresensitive to

pen-icillin and insensitive to 0.01% SDS, KCN, and

NaN3;they grew wellonnutrient agar; and they

all grew wellat00C.

Of 20 strains tested, 17 were viable after 6

weeks at 280C in a semisolid medium which

contained 0.03% Na2S as a hydrogen sulfide

source and 0.0001% acetate All of the strains

tested grew on MP medium plates, and they

were viable after 1 month when left at room

temperature.Althoughsomeautolysisoccurred

(especially with group E strains), the autolysis

wasless andwasslowerto occurthan whenthe

strainsweregrownonBPmedium.

When grownon amedium composed of basal

salt solution with acetate concentrations of

0.00001 to 0.05%, all of the strains except one

fromgroupCandonefromgroupD grewpoorly.

The same medium, but with 0.03% Na2S,

sup-ported good growth of all of the strains, including

those that didnot deposit sulfur (group B). In

liquid BP medium, group E strains grew best

when themediumwasshakenat200to300rpm

In semisolid (0.2% agar) stab tubes of BP

me-dium, growth beganatapproximately4 to5mm

belowthesurface,and in four of the five groups

thegrowthtook the form ofadenseringatthat

level. The fifth group (A) grew dispersed at a depth of 5 to 40 mm from the surface With Na2S (semisolidMP medium) the growth of the four ring-forminggroups occurred farther from thesurface,todepthsof 20 to 45 mm However,

none of them grew under the strictly anaerobic conditions atthe bottom of the tubes or under

apetrolatumseal

Cytology. The typical appearance ofa cell growing on MP medium is shown in Fig 6

Sulfurgranulesappearasdensely outlined gran-ules when viewedby phase microscopy (Fig 6A),

or asrefractile bodies when viewed by dark-field microscopy (Fig 6B) The granules were dem-onstrated to be sulfur by pyridine extraction (Fig 7).Inthinsections,the sulfurgranuleswere seen external to the cytoplasmic membrane in invaginated pocketsof the membrane, and they

wereenclosed within amembraneconsisting of three dark and twoelectron-translucent layers (Fig. 8) The sulfur granules consisted of a

washed-out space, as noted by Shively (36), which usually contained some electron-dense stringymaterial that may beproteinaceous.The cell wall appeared to be rather tight fitting around the cytoplasmic cylinder except in the

areaofasulfurgranule (compare Fig.8withFig. 9) At the point where the cell wall is pulled away itcan be seen that the wall is typical of gram-negative bacteria, but with an additional layer (Fig 10). When grown on BP medium, large lipid storage vacuoles (PHB) were ob-served in most cells (Fig 9), andin some cells the lipid occupied much of the cytoplasmic space

Whether thecells were grownin broth (Fig 11) or on anagarsurface(Fig 12),thecellswere connected by strands of extracellular slime Moreover,ontheagarsurface,thecellslefttrails

762 STROHL AND LARKIN

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where the trichomes had glided (Fig 12B) Cross walls are not seen in these scanning electron micrographs (Fig. 11, 12), indicating that the outer surface of the Beggiatoa trichomes was continuous and did not invaginate at the septa between individual cells of a trichome

DISCUSSION Beggiatoa cannotalways be enumerated di-rectly from its habitat, and especially from sed-iments, as some bacteria can (i.e., Escherichia coli), because ofthe lack ofa specificselective medium and because gliding cells may move about over the surface of an agar medium A reasonable approach tosolving this problem is

todevelopan MPN procedure usingan enrich-ment medium in which Beggiatoa is favored over competing microorganisms, so that high recoveriesmay beexpected.TheSACH medium providessuchanenrichment,andby confirming presumptively positive tubes by phase micros-copy an accurateenumeration of beggiatoa from freshwaterenvironmentsmay beobtained.From brackish andmarineenvironments, this medium does not always provide reproducible results eventhough beggiatoasmay grow init;we are trying various modifications to overcome this problem.

IntheSACHmedium the presence of catalase was important, presumably because it decom-posestheperoxidethatBeggiatoa produces (3). The concentration of acetate also appeared to

be critical, with 0.05% being the optimum con-centration

Our strains, with two exceptions, grew very poorlyon amedium composed of 0.0001% ace-tateplus catalase. TheadditionofNa2Stothis medium greatly stimulated growth, and thecells deposited sulfur We have notinvestigatedthe mechanism of H2S stimulation.

The relative abundance of Beggiatoa in the sediments of southern Louisiana lakes and streams(Table 4)wasnotsuprising Lackey(15), Lackeyetal (17), and Pringsheim(28) indicated thatBeggiatoa were presentin large numbers

in mostof the habitatsthat weresuited for them Suitable conditionsweregovernedby the avail-able nutrients, the proper salt balance, the proper 02-H2S balance, a supply of C02, and slightly alkaline conditions Pringsheim (28) notedthat standing water and black mud were typical environments that nearly always pro-duced thriving trichomesinenrichmentcultures, and ScottenandStokes(35)indicated that Beg-giatoa were common in most lake and river

sediments, sulfur springs, andmarine habitats Pitts etal (25) and Joshiand Hollis (12) have found Beggiatoa in association with rice roots

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APPL ENVIRON MICROBIOL

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FIG 5 Phase micrograph of strain B8GC (group A) growing on the surface of BP agar Two of the trichomes haveformed supercoiled balls, which rotate on the agar and etch the surface The large rotating (but flat) colony was associated with many strains Pringsheim (27) referred to the latter colony type as

circuitans,orC, typeofcolony.Bar,30p.m.

FIG 6 Appearanceof Beggiatoa growingonMP medium (A)Phasemicrograph (B)Dark-field micro-graph Bars, 10pm

under the annual flood-soil conditionsof

Louis-iana rice paddies Whereas Beggiatoa was not

found to be agood indicator of pollution (16),

someresearchers have reported thepresenceof

Beggiatoainpollutedlakes or streams (26) and

in activated sludge (8) The presence of the

larger forms of Beggiatoa in the Spartina salt

marsh (trichome widthup to 35Am) also

corre-spondstothereportsby Lackey (15) and Lackey

etal (17),whoreported that Beggiatoa

mirab-ilis and Beggiatoa gigantea, with arbitrary

tri-chome widths of15 to 21 pum and26 to 55,um,

respectively (20), were found onlyin marineor

brackish-water environments.

SACH medium,withtheacetatedeleted and withorwithout cycloheximide added,gave ad-equate recovery of beggiatoas without heavy contamination by other bacteria; itwastherefore used routinelyas an enrichment medium from whichtoobtainpurecultures.

Althoughourbasic isolation procedures differ little from some of the procedures reported in the literature (5, 9, 27, 35), the washincatalase and the blotting of the trichomeswere factors which greatly enhanced isolation By using those variations, along with optimal nutrient andagar

764 STROHL AND LARKIN

FIG 7 Extracionofsulfur fromBeggiatoaby pyridine (A) The trichomewaspreparedas a wetmount, pyridine wasadded, anda coverslipwassealedoverit.The empty cavities in the cellsareseen, andacrystal

hasformedexternally (B)The trichomeswereplacedon aslide anddrieduntilthey lysed.Adrop ofpyridine

wasadded, andthecrystalsformedwherethesulfur granuleshadbeen.Manynonextractedgranulesremain (arrow) Bars, 10pm

concentrations and optimal temperature,

iso-lates were routinely obtained from enrichment

cultures. Strainswerereadily isolatedby using

MPmediumwithorwithout antibioticdisks

The nature ofBeggiatoa nutritionhas been

the center ofcontroversy for several years

Al-though Keil(13) andBavendamm (2) reportedly

cultured autotrophic strains of Beggiatoawhich

oxidized H2S tosulfur for energy-yielding

pur-poses, there is somequestion asto thevalidity

oftheir results (39) Kowallik and Pringsheim

(14)andPringsheim and Kowallik (29) reported

thefacultatively autotrophicgrowth of 5 of their

14strains, and 2 strainswhich could be coaxed

to autotrophic growth if pregrown

mixotrophi-cally Pringsheim laterstated (28) thatnone of

the strains couldgrowautotrophically butthat

they deposited sulfur when in autotrophic,

non-growth conditions. Hefurtherstated thatall of

his strains required small amounts of organic

materialsfor growth, which demonstratedtheir

mixotrophy.Cataldi(5) and Faust andWolfe (9)

reportedtheisolationofBeggiatoa strains that

grewheterotrophicallyonmediacontaining low

amounts oforganic nutrients Faust and Wolfe

(9) showed that their strains could tolerate low

levels ofH2S, and they observed that the cells

deposited sulfur under those conditions; their

strains did not growautotrophically.Cataldi (5)

attempted to grow her strains autotrophically,

butshe neglected to include H2S as an energy

sourceand wasunsuccessful Scottenand Stokes

(35) isolated three strains which required H2S

and an organic substrate such as acetate for

growth,whichwasperhaps anindicationof

ob-ligate mixotrophy by those strains The other

two strains they studied, those supplied by Pringsheim (27), grew well in the presence of H2S,but like those of Faust and Wolfe (9)they didn'trequire H2Sforgrowth.Burton and Mor-ita (3) studiedastrain whichdepositedsulfur in the presence ofH2Sbutwhich also grew heter-otrophically on acetate or other organic acids and yeast extract We have not yet been ableto

grow anyof ourstrainsautotrophically

It does not seem likelythat our hay enrich-ments, orthose of Cataldi (5) or Faust and Wolfe (9),selected against autotrophic strains Thehay enrichments, with or without the addition of H2S-eminating mud, should have provided an environment similar to that in whichbeggiatoas are found, i.e., low levels of organic material, sulfide, and oxygen The presence ofcatalasein

our enrichments should be a stimulus to any autotrophic strains present Moreover, inthese enrichments, Pringsheim (14, 27) and we have notedthat the trichomescontained sulfur gran-ules, and upon isolation some of Pringsheim's strains weremixotrophic

It is possible that the isolation techniques, instead of the enrichmenttechniques,may have selected for the heterotrophic strains over au-totrophic strains, if the latter exist Pringsheim (27), Faust and Wolfe (9), Maier and Murray (22), Scotten and Stokes (35), and we have all usedheterotrophic media for the primary isola-tion ofBeggiatoa fromenrichmentcultures Ca-taldi (5) used a medium deficient in nutrients

APPL ENVIRON MICROBIOL