A ITU>Y OP 1111 IF'FICT OJ' DJLVl'IOlf IATI 81' IMCT.DJAL POPVLATI019

., •••• ... ••• 11 •••••••

Tll•••• •••• , •••• , •••• o ••••••• '•••ltJ ••••• Vtratata Pol1t••••t• l••tit•t• ,. • ••• , ••• , ••• ••• •••••• •f

llAITD OJ' ICJDCI

ill ... ,, .. , ... , ...... .

••••••• 1•••

1.

ll.

Ill. u.

'· n.

Vil,

nn. u. x.

XI.

Ul.

·2·

•••• IftlODUCTIOH··-···-·················--·-··-· 3 llYIIW or LlftMTUIB············-··········• S

OBJICT-·----------------------------------·- 14 HITHODI AID MlTllJALI····----···-·-········-15

lllULTS•···-·--·--·-······--·--·-·-··-··•••• 29 DlSCUSSlOll•••··--·······-··--·------·----··· 38

lllftlt.USlORS··--·-···--·-·-······----·--·-·•• 42 SUllfll.Y··························------··••• 43 A~•·•••••••••••••••••••••••••• 45

UTA···--··-········-····-···-······-·--···· 46 IDLlOG-IAftY••····--··-····-··-·-··--·-····• 47

APRllD11BS··---------·--------·-------------49 Appa4:1.x A • Theory of the Coulter

Couater·······-·------·-··•· 49

Appeadix I • Cleaalag of Clae1Var .. -·-··· 53

ImROOUCTION

y ~roblems in sanitary en~1nooring require

tau•l•d1e of the growth dyna•lcs of microbial ftF•t•~$.

for ez••Pl~, ourrently, •est reaearcb on blcl•glcal

wa~t• t~•ata•nt is b&$ed on ht empirical approache~.

Eluc1d~tien ~t tb• t~ndamental biol~~Y is neCG$~a~y

before a meanS••fal approaeb fa~ i•praw1n1 o••plex

biological proo••••• can be tor•ulated. A knowledge

culture

ot populat ton dyruu111hu11 in such iu1vircu11\l$t1iiil a~ streams,

sewage treatment facllttl••• and water tr•at•ent

t~cili U.t11s can b$ i<•ached. .Mixed cultures studitu! by

batcb t~chalquea tend to give re$ult• whlcb are

dlfflcult to tnte•p•et beoau5e •f almulta~eoue ehan1••

, nutrient

oonce~tr~tion, waste p~oduot accumulation, m~tabolie

int~rmediat~ pool•, and the lik•. Contin~ou~ flow

praeedure• t•nd to ••tabll•b steady-state• wblcb bold

constant sue~ envi~onmental faot~r$ a~ ~ut~ient

ccncentratien, averag• physiological ace, and phys!o-

cb•mical condition•.

At fi~•d nutrient co~centration and dilution

~ate not •xoeeding the maxi~um growth r•te, a pure

culture will In theory •~aob a •t•ady•8tate population.

Wiuim the iii ion rat• h~ si;reater than th• grow th rate

of tb• organlsa. wa6h•aat should eccar. In a rand••

atsed cultura, dilution rate sbaul• ha•• the eam~

lnteractin1. ••1antsas with slower 1rawth rat•&

•hould be waab~d t•om the srowth wea••l befo•• tboae

t AU~b•r of sp•c1eG of or~ani$m$ ~hould th••• d•Or@a&~ with only-• more hardy and faster •rowln1

orgaaS~M• ••~alnlng.

thl~ ••~earcb tbe Gffect• of dilution rate

on population we~e studiee for a pu~~ culture of ~.1..c.J..llu~

·H~~e:gs~, a mix•d cultur• of Bscillus e.it,JNtU. •UUl 11\•roba,ete,r

~·¥-:~g1ntt§, and 11 complet•lY ra.a:uhu1 mi~~Hi cn1li.:.t.t1<•. The

object$ of thl~ atud' •~••: 1) to det•rmln~ whether

e·t~uuty-stattt popi1lat:lcru1 a1'• ?'eaohed in gnn·• o•nt:hlueiusi

flo• cultures of •noap•alated organism• saoh as Baolllas

Qt•111, 2) to compare tbe•e reGult$ to tha centlnaeus

flow theory, 3) ta deter•lne tbe effect or di tion rate

upen population Of the defined aixed CUltur• 1 1 a•••¥! and A1 •!••«•nasA and, 4) te deter•lne th• eff•ct• of

dilution •at• upon a randoa ~ixed eu1ture.

ba«te•la la eoatiaueas ••ltu••· The ••$eat

thl~ ol•••I06l aaalJ•I• ol tb~ •••ti••••• l• that aic•••l&l a•••th I• thl$ s1•••• aoble••• a

•

lat• a oompletely •ia•d 1rowth •••••I •••'al•l•1 a

p~u·• eult1:n••• Bue~ •u:•vit-n~••nt~l fact~Ya; •uo.h -.s

coruh\Htt•atto~ of nutf'tent• 1 Mt&bollc pl'odtu~'h~ -.~ul

Cans ith~r b&Ot•rlA 1.-•wlng itt 11n1eb a c~ttt

oultur•. the laflQWiftt: mfluUa @ent.ai:n.i ~ $:ht

l:h1!ttn1 «n•1a.~ie llntbetrate at • CUUlOflUttl.-atton s1 • It

a.~d •~Yl•Hl flupply is alw.,y~ ~.uSeqvatu~ i Uu; $~f>p1:;

*t o~gani .. e l!uhstra'fa» t~ 'h•r•:foa-• tile l!!lOl• ~:a"OW"tb•

1 i h~r fact°'.-. Tae Y4Ut1a..,le• of iat!ll'thtt iu•e 1uut

s~b!\ltratfi 0~11cu1:n.tl"&thu1 and th$ di1utio~ ~ei.te. A

of tb.fHH' eat'h"OlUHnttal faot(lra. •.aa affect 1rowtb rat•

an4 cencentration ~f o~ganisn$.

... t;r:.=Dx dt

( l)

D • th• dllutlo• •ate (11•• •ate eaterlna t•e

t)Ht:l!'efC'l'e if k "7 l) the YalUfb U Wi dt

(3)

D? k. t1i. i«t rutg&thre and the o:rg&l.'li«tu'I eea.etu.it:t~tion will dt

•••r•ase, ev•ntuallf to ••••• tber•t•re the cultu•e will

p,~ = 0 •nd :.: l~ OO!Uita:nt; •t

•t•t~ t•• effect 01 dilution •ate

awtn1 oat at • aonae ••tl•n a.

y = ""''i-·---s1 ... s

•

(5)

to cell

co~ .. D S (6) j{

(1)

bour~ k.,. O.

_,_

ean. t••refel'e •• •Xpl'e•••• &8 11. • • + k yl

••••trat• ••••vat •r k • yl uc. - •)

(8)

(t)

la t•• ••••l•p•it •f •••ti••••• f1•• ••••tl••• t•• c••-'• ••t• l• ueua11J ••••-• t• II• a ••••taat

l•&etl•• of tile ••ll•t•at• r•••••l rate1 (10)

ct••• .,,, U. • • J.L. • ks/Y cat Ydt

(11)

"•• '' .. ,, ••••••• , ......... (10) .... , •••

••••1•• ••••••• of a ••all •••••• ef ••ll•t••t• •••--pttea

•Ill•• i• ••••••• •••• if •• crewtla •••••• •• tlaat

... •••

•••

.U. • • C ax + kx/Y1"> .,

.ti. • k •• -<• + k1/Y1) .. , ...... , .. (13) ••k•• t.t

rt•J.• Y at Y&l'teu J.eyeJ.s

y. k -"• ...... , .... y. k

n + (k/YI)

(18)

P•••t.111• to •••P•t• , .. Of ke Br d•flntttoa

(14)

(11)

• A

.!!, =:: D S1 - D S .,.,. x <n + k/Y1 > (16) tit

ste•dY ~tate •ondi t1ons wht1u1 4.x = o w• dt

D • k~ (1 • e·• r,

fl lB ( ka ) k 11 .... D

1 . l ... -=Yd'- -1s1 'D -

( 17)

( l.10

(20)

valua

u @I ( s:; ) c sl k 'f

OU)

s

nutrit to

eul ture.

b' ••an• •f the aAe•••tat.

aueb $ft~iehmentg a~• t•s

rl•a• war• of dlreettn1

lealar •••Pl• of water ••• dl~eu•••d.

.tUHi QWStU'ibtHi

I.on o:f :intet'•

aetlon~ ••tween twe or more •P••I•• af ba•ter

11~1

at aar point on th•

•Ith t•• s•••••tlaa tl••• of eaok or t

th~ er1aai•~~ •••• 1•••n wltb ••••

• Shlndala. et al ( ) •h•••d tbe lnteraetloa ot

has ne ef feet an the other.

The b~eterial 1rowt• was shown to fol

a relatl•D•hlp that ••• nat llnea~.

culture •h•• o•clllatlea1 In s•••th rat•

of dllutl•• rat•

ebae1•. latien a•a•t• were done by tke •l•ctrenlo

Coult•r Counte~ ••tbod.

The initial object of this reeearch was to tna,uly a

raudo• mixed culture in a continuous flcnr 1yatem. The initial

plan was to detemiae the amowt of variation of oraaniem in

the mixed oulture and by foretna environmental eon41tious to

be able to select .simple: species from the m11'ture.

tube.

not <Umtttttuen\s &f tlu1 $lf.m.r.) .tc ·the ~ample l;)y ~bet

P•tt<ott•fhu:uuuu· aethod.

Whcui sllae 'luuua.c1iu11 't•0 h4UiVJ t~e tf<J•••l walls

we~~ f.IC th • t~J!llt tublt • i!lli \HU!! UIJ 11

t O~ CHI!· .a

i'U$1l

ealaYed at 15 p.•.i• fOI' 15 .$ • b pro.:uHss wae

.t. 1 'l'.O•~.

•U•

1-

AIR INTM<E

(~ A

B

I.

tb• •••ptla aeries of tbe

PY•Vl•••lr •eatlan•• except •• eo•1taRt wa•

••quired, ••r• e n•ot•d to a 11-11ter pyrex re•ervoir

~ ••• •applied to tb1• r•••

CC Fss. ZJ and tb••

i' i~oi:t the

tub•· xqow

1rawtb •••••l• were •lllllt•r

fla$k• with ~G$ ~ a~~ •ffl~•~t tub~•

•••s • t• p•••lt ove•flow at aoo •lltllt•r•.

WI$ :re at

Fi~. 2}.

•o•• •h•k•r ••••S•ted •f a pl board

by fo•r ooll epr ab••• a b•••·

s•• pl ••• eenneot•d a •teel aanaectln1

FOtil to a at•$1 tlywatu!l ttt attain 4 thJ<.ee b 8t •

f 1p•h•el wa• driven br a

E tri~ ~~t$r, Th•

-19·

the entire apparatua wa• co1111ected and auto-

c lavecl for 15 lliaut•• at lS , ••• 1. the arowth proce••

took place in a 2o•c± 1 c• con.teat temperature room.

After tbe arowth v••••l• were partially filled

with llediua, uoh ••• 1d.entically inoculated with the

rudom culture grown in the prwiou• experiment.

------------·-------------..

B

PUMP

Figure Z

D

:;: 0 ..J u.

3. I• th• 4•f1Md caltv• •terf.meata tile

ceetl .... flew a,,...atu MU1ete4 of a 12•U.ta

r .. enolr f ... l .. an lrl...,._ f1-ak ooataild.ag 116

allllltaa with afflut111t throuah a &laaa tube a7atem

(l'f.1. 3). Aeratioa vu ac...,Uahed b7 1tlowlq air from

the 18oratory CGlllpr .. ae4 &11' •1•t• (Ftg. 3) into tile

culture ...... 1 ~ • cottoa fltlaaed al••• t .....

The culture ...... , vu lae14 1• a 30•c water ltath, aacl

aecli• •• fe4. witlt a perlataU.o ,_, (8f.p&110tor

eo.pay, Middleport, lew York). MapetS.e llb1 .. of

tile .cmlt•r• ,, .. ,.1 .., t11111ploJed. The •Y•t• la ahowa

ln figure 3.

a_,1 .. WR• takea froa tbe af flaent tube fer

retroff•lla\le•• ... Coult• countlq. One m11111t•r

of tbe ._,1. •• aMecl to 99 milUf.tera of a Hlvtioa

tbat ceataiaed 0,9 ar-. of lael per 100 miUUtar•

of 411tl1le4 wtar. rua ._,1. •• thea couatecl 1»1

tile elfftl'oale Coulter Couater with a 50 alcron apertva.

Tbe Coulter Coater had ••ttlnp of the follow1 .. :

1) thrulaolcl re.Mlq of 4 1 pln aelectioa of 3 1

aad aa .,_.t•I'• ovrr•t ••tt1ag of 7.

After aMl .. 10 U.tara of Mcl•(Table Z)

to tbe ruenolr the .,,.ratu •• conaecte4 aacl ••to·

clavecl at 15 p.a.1. for 15 al1111tu. rollowiq coollq

tbe vlt•i•• Wft& a44e4 to the aeclim aacl opel'&tioa of tbe

ter th~ grewth wes••l content~ had reaehed

111 •lllllt•r•, pamplftS ••• •topped ~n tneculu~ could

be 8dded ta th~ grewth vea•el•. The air Intake w•• al•• diaeonneeted lrom the labarat•rJ eoapres•ed air

av•••• to pr&went eonta•inatlon of the media r••ervolr,

anti t& prayf!ln t 11u1tedla t@ wtushout of the !tuH:n1ltu11,

Culture!!!! ot 'B.d. c~l'•-q!lt and .l!u • .. ~ui:rcs~~.e,, whS.ch

Wl't'«J ctown in t•J!lt tuh•illl f'.!f ltlfHHa (Table 2) at S7°C

w•r• usu!td 1u11 ino«11ultun. ln 1 t: tally ~P.!~.!.! waui.1 added

ta the 1row~b •easels of the continuous flow •r•tea. The or1a•l•ms were g•own in bateh oultu•e to• 11 hour~

bet••• stQrtlns continuous flow. Aft&r the population

of 81 •~E•s• al•ne waa det•••lned at ••••~•l flow rate~.

4. urogen~,u; wes added to the iay9tem without interupttns

tbe flow.

B

AtR

fi9ure 3.

B. 9MlturM el tHIE !tbtw•• Cult•i9 ••ed ,. cm f1r1t tw ..,..laeatl

were eellected by 1 ... 1a1 tlae arowtb vee1el1 of tM

Wttal aperlaeat opea to tM atm.,Mre for ... ..._

expe1ure to ra.._ Mcterla. The t7P8• of l:tacterla

prueat in the culture,wres ... u l'oQ witla a •t-ter

of rovply OM aicroa, 1cae eacapaulated and ._. not;

tarp ro41 wttll .U.-ter• •• Iara• •• 5.0 atcrou,

.... euapulat .. alld a.e aot; 1Mll an4 Iara• cocei

.... free and otllc;• tr..,.. la •U•. There wu aln

a arut IUllter of 1U.me flocnlu Mtla lara• a1ld IMll.

Bulllg HUii (ltr• l) an4 AtroMISS

'V'MM' (ltTaln 1) uaed la tM .. ff.Md culture apeYlaeat•

were •talaed from die nltul'• eellectlon of tlae Mcterf.oloa

..,.rtaeat, Vlrpala hl)'tedmlc lutltute. 11 nrnt

11 a ro4 alaaped erpal•, l.O to 1.2 by 3.0 to s.o microu,

with •caur• -.. aerol:ttc, fanltatlft ••••le, .... 117

ocnrtaa la ellort to toa1 taaaled .Uiaa. The epecl••

•ult wltk wu fl1-tou, eacapeulated aad ar• vartule.

Opt.._ arowth occur• at 30•c. A. IH!UMI 11 • rot 1baped

orpal•, 0.5 to O.I by 1.0 to 2.1 llteron. oenrtaa

•l-larly, ._,..lated, ar• aegatlft, urol:tlc. aa• pn'vMr. 8pt..._ anwtll occur• at 30•c. Belk culture• ._..

iaitlally arowa •• Difee CJ•tt .. tl')'ptlc aaar tlaea transferred

by uae of a loop ..-1. to tut tubes of medt• (table 2).

C. 9.P•i<ifLtjgfl. an(\ f£8R•.~~tjpn g:t Med~!

.~...,, ............ of tbe e~dUon e~n l~ l

w~re u1:utd i'a

~itm of c~sition. in

uu1d <r;U. cultu.t'ea.

in

wll!ra diuolved

a m1Hpotte rn .. b:rane:

ddin9d

led wat~n·

lter to

used, atwaya morll:li th.al\ 5

sto,ck so wtitre added to tb~

of media.

ware

of t:he vitaod.n

li tet" rGaervoir

l

~i~ ! ~nens 9JM!nt1tx

fotanium pbospMte. monobaetc (KH2ro4) 6 g

Po~asdum phosphate, diabad•~ o:.2HP04) g

• 1

(MH4)2 S04

D~trose (c6u'.! 2 06 • e2o

Distil b1d Water

0.2 g

O.O(HO i

3.0 g

2.0 g

•II•

Table 2

§i!swcm•t

Dextroee (C6H12o6 • a2o

<•4>2•04 K B2104

~lll'04

Socllum Citrate 2 a2o Ha so4 • 1 a2o Diat1lled Water

Viteip

for lxperimllat C and D

Biotin

Calcium fantothenate

1 • inoaitol

Thiamine

Pyrid oxine

Qyytitx

4 I

2 I

6 I

14 I

1 I

0.2 I

10 liter•

Quantity/100 al of a2o

10.0 ..

1.0 ..

s.o .. 1.0 ..

200 ..

Iu the &.'!:peri.metlts with random mi~ed cultures the

•l di 1u.tion rate.; werEi kept coru1tant at D • 0. 6 hr for the

Population as a fu~ction of time ia given in fig. 4. The

popul•tton suuwa to oac:U late randomly. This great oscillation

h in U.ne with tne results of C.uell 6't al ( Ht64).

Since random oscillattc:ms of population leve1s occurred

production waa great as in the open mixed culture experiment~.

There was no way to do quantitative work because of clump.a

growtb floccules similar to activated a 1.udge floe.cutes were

noted. Abo, frO&'l1 visual observations sHme formed in the

vesse a at different rates and variations were evident from

CIO l 0 ....

12.0 j I ! l 10.0 1 I

I I l .,, 8.0 ~

~ l

..... G>

(.)

I l

ti(! p """ 6.0 -i ~·

.,..j ;t Vl

Iii ... ,.. """ 4.0

2.0

0 1.0 2 .o

-1 .D .. 0.6 Hr

-1 '"' 0.2 Hr

--.------·------.----·----·r-·-·----·--~--··---,.-·-··----·-·t··---·-·----

3. 0 4.0 5.0 6.0 7.0 8.0 Hours After Run Began

Figure 4 Population Changes in a Random Mixed Culture

the start. After continuous flow operacion was stopped,

the a lime cm th!!! $id~$ of" the vesa• l& took from one to four

w~ekllJ of batch culture to slough off. A gre;ater time was

required for the morQ dense dime to slough th6 container

walls.

Since resu from tluui~ random mbed culture growth

syatems ware hard to interpret, it va• decided to de pofulation

studies on pure. cult~.u:H of l:uu::taria at var1011s dilut::lon rates.

A different medium {Table 2) • 1ncn:-e suited to the selected

cultures. liiB& used. Sterile tttst tubes of thb medium were

1.ooculated \1ith the various orpnbms. Thi.I batch method of

growth provided an iudex of the ability of the organifn1~• to

grm;~ ia tl1i.1J m~ia. &esulu from thb ttiHlt are •1u:r;.rn i.n Table 3.

Table 3

Growth of different cest culturtuJ in the chemically

defined medium (Table 2)

Organism

S~a2nxio~9ccus ~R&~e~m!d~a

la~111us c•[;eus

f\erQba~ter ~er21en$Jt

l,rotau! wlar&s

Ser:c:atia r&n."c.rusearuJ

~s.cherr,iel\4. co.Ji

Siu.:ch!rP?!lc~~ eet:!Ji.tti.\'\fl

Growth

0

·tt

+H

0

0

++++

-++++

:rate. !at~r be

W8$ i,: a cui cure

!b.....!~F.fJl~;tte,J. d o\rerstoH B. cereu1.

or floes

to tbe bi1hest dilution rate cei

reduction in population did not follow

in culture. It

the two apeci~s

Popuhu:::ion le;;els of

and e()ntinued up -1 0.72 h:t • The

flow theory

-co I 0 --

s.o~ I l 7 .o -1 l i

I 6.01

I i

5.0 ~ I

l I :l i {

4.0 -j

J ~ ' J I !

3.0 i ., I l !

I 2.0 1

l l ~

l 1.0 J

j l·

! I

0 1 0

-33-

Petroff-Hausser Counts

8.0

7 .o

6.0

5.0

~4.0 I l 3.0 ! ( f

! j

I )... 2.0 ( ;.

~ '

-, ------T-----·----.---··----.-·------r----r--·----i-0.2 0.4 0. 6 0.8 1.0

Figure 5 Population of B. cereus as a function of Dilution Rate

-'° I 0 --Ul --~ u

6.

Aft•t, the

count ~-;,; to

1<r.a•

corud.a Ot

D

wa1

det.e~ined that 1C1t

and

~ o. , 1t

• i

t:o

di 1:'$.telil,

froo1

•on

it

l

t1j

populatio11 •l hr

f

rate tUHMl

!nte~:tate

not

~ult.uro in

..... a

Oo • 0.8 0 ..... ~ u ~

~ u ~ 0 ~ 0.6 ~

~

0 ~ u ~

u ~ u M 0.4 ~ ~

0 s 10 IS 20 2S 30 Hours after lnnoculation of A. aerogenes

Figure 6 Trends of Total Counts of.Baclilus cereus and Aerobacter aerogenes vs Time after lnnoculation of A. aerogenea

• (.,J r

\

I"-• 0 ~

>< .,, u c: ;:I

8 M ~ .., ..... ;::I

8

.,37 ..

6.0

.5 .o

4.0

-l 3.0 ~ I> - 0.370 hr

•4\ A £... ~ I & -/ir

2~0 ~ I

1.0 J

0 -+---------...... ----------..---~-----,..-----------r----------0 o.s . l.O 1.5

Time (lirs.) 2.0

Figure 7 Population Counts of a Culture of Bacillus cereus and Aerobacter aprogenes . by the Coulter Counter Method

e~~i~re ~o rafutcm

population •e•~d to

swi~1· popuJation at·lil

ScrM~ tActcr«t

J latiotu~

1n.terf$& i.: and

eatuie f fit"et the varittb '·• iuoculaticn

interaction

t:bi! t:x.·e• awblmet' ~v• oseH illted

1 it~creaeed unt1 v•ll'tU~ t be

flow c•.ltur• syst.-

s and &ict

.iuu;;,m Si t.e«ui y• 1;H:.a t *-

became attached tu

rat• of die 1

th~ $lim4t eont:f.muil

I {;If f

gr~th pattern~.

that

~II

IS

l'.·and"°1~

l({

OOL

., ...

level• clecrea•ed •• clUutioa ratu 1acrea•ed. fopulation

lft818 d14 aot follft coatlJmoua c.ltuee tbeo17 in that

population level• decr .. •ed 1reduall7 a.er a •eri•• of

di batloa ratu inatud of decr••iaa •barply at tbe

critical clUution rate. Since I• WN fol'Mll ea,.ulea

aad soaa It-.- attadled to tbe arewtb .uael walh, it

•• tmpeaaU.te to provide the coaatent control over arewtb

factor• that ...... 111 would be espected in a coatinuoue

flw •7•t•. IU.11• vlliah eatrapped th•• orpni ... could

poeaibl7 lane clecr•aed ... , ... detention ancl incr••ed

os1aen •"PPlY per uait volume by clur ... iaa tbe volume

of coatat.Mr. Also tbe ti• intenat required for wa•bout

of bacteria aM aetahU.c wute pl'Ntacta *>ulcl U.. been

iacruaed fr• theae iaillu belaa lwlcl firm in tbe •lime

couected to tbe veaael wall.

Wban A1 IHllWI •• added to the •J'•t• already

contaialaa I, u.rw, it,••• beU.eved that tbe •re rapU

anvlaa A• MU• .. would .-pletel7 take over tbe •Y•t•• Total pepulation le.el• deer••_. after tbe acldltloa of

6, ..,..,., htnff•Bauaer 11DUDta •bowed after 1..eral

daya tbat tlae population vaa oompoa_. of 78' of As nrpam•

encl lOl of Ir um1. It u beU.....a tut t1ae only reaaoa

tbat 11 •w wu .. t wuW ...,1etel7 from tlw •Jet•

waa ........ of tile COlltilBIOWI ret.culation of tllia

orpnl• fr. the alime on the container •lla.

in

quit"

l. of

-41-

By finding the condition of minimum slime production

a greater number of mixed cultures can then be studied

in the future.

Other research suggestions are:

1. The affect of sinusoidal application of flow

to a defined mixed culture of B. cereus and

A. aerogenes to determine whether there is an

immediate response in population levels.

2. The use of sinusoidal application of medium

to a defined mixed culture of B. cereus and

A. aerogenes to determine the growth response

of these organisms to food supply.

3. The use of sinusoidal pH variance on mixed

cultures in general to determine the growth

responses of these organisms to pH.

These are only a few suggestions for future research projects

on mixed cultures, but much research is still needed.

2. s i~ produe;;.ic:m incrl'ilaa11:1d s the

mi:hed cu t:ure l'UUS and 'l>l'lil3 control

as

a .., Population eve I of ., .

a 1harp red.~H".,t:ion in

ly aver a ••ri•• of

free iquid.

An astlpt1e ccn:u:tnuou.s flow syster.i fen: the study of a

random mi:u~d Cl.\ tura was deecribed. The quick production of

great advantagtt ever the syfltem with co1·uuant e:1tposure to

the atmospher~. Activated sh.Jdgfa ike t !o,~cules and 1

were noted to grew at tlu;~e different rate8 in the 1..1.u:ee

idendc.a irowth veueh.

~act l luti cex·et.ls population liltve ls were noted at various

flow rates in a continuous

formed and some: 31 im$ was ootad cor:uiected to the growth vessel

walls. Population lwcda taken by Coulter Counter method did

not confor!lft to the contiru.tous f a pure culture in

that there was not an i~edhte ~uction in popuhition level

at the ct"it:lcal dilution r~te but: a series of reductions

over & range of several dilution rat:.u.

.<;.~;eus and Ae,t:~bacter ae:rogEui~" were s A uduetion

in population of t:he culture from~ ch.at of: a

pure culture of i •. cer,e.!lJ. was noted.

shooed seven l ~ya

mixed culture w.iu appro:dm.aeely

Petrcff•IiautlH~r counting

population of the

of tile ttr·i.gina • &,

a11ir1tos,e~e1 population eounu vere taktm iat.: S&\!'(:ral di h.ttion

:rates. Thefle lev~ls did iwt follow contiooou!S :flow theory for

was ooted that) the count at a lm1t:r d:Uution rate. It wa3

decided that sli~e on tb8 w.dh; of the gro~t.h vcuuutl ccrntiAu41 ly

to control

fl ow prcc~1H11 •

Also thh n~thod 'tt.lllS too laborious and it \<J'!Ut almost

aad advice !•om hi• tbe•i• adwi••r, • H, R. sa11 te

Dr. w. A. Pa••••• to• bl~ 1••••••~ ••eo••~s~ment •ft~•••

darlns this eear~• •f studJ; to • B. G. Willard fer

bi• ti~• •••Ins techaSea1 •••lstance; to my oell••I•••

who f~und tS•e amld~I their awn ••s•arob l••••t Ion

io lend a belpint; ~uuu~ and •UtCJ)~t"a1'•1Uuu1t, te the N•tional

stltate of B•alth I•~ financial help and to th• Ylr•l•I•

T•ob At•letle soelatloa I•• time and P•••l••lan te

•••• sraduate •t~dy while •mployed by tbea.

A •e•t er sratltude I• •l•• extended to •Y

The vita has been removed from the scanned document

, N. Yi.•

llt.

1964.

J ey) J. ~; • '

4. liar~ert. ll. > E th J Tt1tl • a. c. t

i.tt~• Cu.H:ura o.t' J ~ 1 a.rid

G58,

•

7. Olmcla, A., ad lobaJ'&IAl, II., "Productioa of I UM

•••tw• lD KbM Calblr.. .. ....... Jf 2 ! W!!llsp

aa4 Aet.WslR Y'M1t"'&•" llhll• 191, 1201-1, 1961.

a. ••rar, 1.. 1 .... a.,.an, M. 1., "lntnaetS... of the

Ol:al Hicni»S..ta, • a,.t .. for the Defl.Md ltudJ of

IUxecl c.aicua," butt4'111 ti 'M llCUD' w

t. ldml&e, c. i.., "The A.ctlwtecl 11-.. ln-• u a

Ce•ttawu flew Calture," !IS• •9' IM't !RU• 526-

ssa, .,.., ••. 1H4.

11. lkhctala1 A., -._,, II. l. 111, lrlea1 I. I..; a4

CallMart, &., ....._..a.ttue lateraecteu," al!R'Ml d. IMSMMltV• It, Ml•6t6, Mna, 1965.

11. lllWMlala, A., "G'N'Wth lB Mbeil Calttr• of MlCl'ON'palw,"

Dl1aertatioa &tr IHcer of rtdl ..... J lB Cf.vtl laaf.aeertaa

fna firalala lolytHbaio lutlblte, 1•114 1 .._.. .. , 196'.

11. 1'u Itel, c. 1., "llatual lelHtioe la the Mlereltlal

W.l'ld," Janll ,, e=eu1 RllWtitlt&t• tJ, zo1.111,

195J.

In th.~ Cou ! ter Celntt.er a guspftsion is Nd• to

tlu:ol.lgh a aJMH ap~rture in a &laH 'Wall separa.t41s the

su~pension :bu:.o tl>to aleeti·ical isolated po:t·tions. lHectrodes

placed in the ~uspe.nt.rion on ~ithe.r side th• wal are

oot•mectfid tc 4 direct eun·e-nt SQpply. the total curl'e'!lt

passi~ through tlli\ S~8pt!UiOtl. uet~een WUU::

p.ua

pordoti vari•s its area,

oi :ta thfl aperture w:iU be very much bigb.er

than th• ~•8t the susp&~$1on. a~d tn~ eu~r~nt be•

tween thf! &leotrode~ will depend t on

t'e"iatanca this t~shta:nce l ba con.atatu: •

when a parti.ele ei:en the ~u.une:l a reduction i!!l. the of

th~ fluid bt th~ ap•rt:urti oectn:-e 1 tbe raei:iu;al1.~e riiu-1111 •

currant bet:wee.n al•c~rod$• A• a particle p:1U1tJe$

apertur• the cunent will

to its or:igioal levelt magnitud.•

11 the 'VOl~e of partit;:le.

conversion factor

diamete:r of a ephare

l and th.en riHt again

cu:r:rent:

use a 8uitable

tbrC11osbold

d~te~nation

above whi~h puls1H s.1."e counted euablt>.s the

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s~ le i• ¢~illlt:ei:A

ap•rture. _,,.u<•·A~lB!lo 1$

the mill1i'P'"1'1Y"il1

oo~tacts

ccat~lcts

JU!!CO~d

alway• coiuur.iid

f ir&t elecctrode11

f laid a. A ~d;lll timt" ell

atopP*'f

pardcl«u!l

flowius

pert(ld §tboot s~11uzoud• watt U$ed tbroua~ut this res~H•rch.

'fhi~ as1i1ted a great d~l in tbe tr1!1l~lt.tentt

pltiqin1 the 5:0 •tcron orifi<:e b;y reducing tia n•c~1utsry

for u11i£om wlwne:B ff>l' couatitta+ Umel' f.bo tU.:f.mi:uted

the t:Lme coosaini teduaiq;u~

unoutrer af tei- cout. ea U.tiratton at~

tn thti

CU!fl:.~~~~lll& Q f this

Moi11 ----iompllfier

..._.. ld.•t• , .. titlu of vttat.u er lald.•iter•

Id.pt enae an•• en:or• l• tke ruulta of tke acrtttoaal part of dd• wrk, aad larp _._.. of dut parclelu Id.Pt ef feet tke Coulter Glnmter Cftlltl or aue tile SO 111.•n•

ortftce to plq, lt •• ....,..,. to take ar•t ear• ta ti.

cl••laa of au aJ.unare u8'. '111.9 11a11War• •• ff.rat

Hake4 o 11100 Ml•tUla 71. 4eter,_t. (Llllltro auild.oal

Cellpa1lJ, llev Hafta, c:. ... ctnt) for Mffl'al hou• aatl tha

1cn&Mt8' ud rtue4 10 tlau la tap •t•r aafl I ti.au o

4f.ati118' •t•. '111.9 11&•-r• •• tkea allakea lt7 haa4 aa4

tavert8'.,..,.,.. towel• ud left o the air to •rJ• AU

.. ..._. _.. tllat coataOed ...,1.. i.r tu C..lt• Glnmt•

ODt1Bt1 were rtu8' 5 t._.. vltb 411ttll .. •t•r ltefere

atelaa ued. tut •• u.t o the pr.,_atioa of cu -.ie

for tu fetroff•Baua• Mttlod of COUDttaa aa4 al• .... to

ceUect ...,1 .. .,.. ••Md ud riu8' •• p1nioul7

Mlltioaecl aeept tU -.. .,... ctrtad o aa .... for over I b.oura

at 200•c for aterllt .. tl••· nae pf.pate• ......... la 11100 71. •l•tloa for •r• than

34 heva anti tUa rtuad wttb aa auc-tlc oeau ...... 4ftlce for

at luat oae -.. o aold tap vat•. !bale plpett• ••• tUa

pla_. ta al•a.- coatalw• aad placed ta •• ova at 200•c for

atarlltut&oa.

OSTIACT

tepulatloD levela of a aixed culture that waa .-1ently

apo•ed to bacteria in the at:ID•phere wa• atucH.ed at varioua

conataat diluttn ratu. &aadola fluctuatiou iD the free n1-er

population were noted. Aa aaeptic ceatimaoua flow •1•t• proved

of DO p-.. t 8'vantage over that which bacl .-1eat iDDOculatlon,

however, aliM productioa did not foll• identical FOWth patteru

la tu .. iclential arowth ..... i..

Coulter eeunta were made on a pure culture of I• ur1111,

arowa in a ceatlnuou• flow •J•t• at yarioua clilutioa rat••·

Thea• eounta clicl aot foll• ~ flow theory for a pure

culture l• that there •• DO •herp clrop f.D count at the critical

dllutloD rate but arac&ual recluctlou over a rana• of aeyeral

clUutioa ratu.

Total populatioD count• by the Coulter Counter were made

oa a cleftmcl aixed culture of a, cuw allCI A, pm11Mf. After

a •neral day• retroff•llau••er ceuata •bew.S that 70'& of the

,.,.1atloD ••A, '"PllP''• Total population waa reduced from

that of a pure culture of ! 1 urwt. st .. d7•atate populatlou were

noted at YArioua cU.lutloD ratu. Thea• at ... J••tatu • bowner • ell.cl

not foll• eontlnuou• flow theory for a pure culture la that

larger cowau were aot.S at a hiah•r clllutioa rate than .... cowat•

at a lower dilution rate. lt • ....,... that alime on ltae arovth YU••l•'

wall• continuall7 cbaq.S the .... s.romeat had a , .... t•r effe.:t and ucle

it .... ••I.bl• to control all the ...,,lromaautal factor• av• in a

contf.mulue fl• procua.