CONTRATO DE INCREMENTO O INICIO DE ACTIVIDAD

57 .

SEC'fiON III

58 • .

IKI'RODUCTIOl'T .

The report o f Lillehoj and Ciegler (1 9 70 ) that bacteriophage · of

B . strain 899a could be induced by aflatoxin B1 was confirmed as reported in Sect ion

I .

T

h

e

s pecificity of phage induction makes it attractive as the basis of a bioassay system .

It se emed desirable that the s tudy of bacteriopl�ge induction should take place in a defined medium since unknown substance s pre sent in a com- plex medium may modify the effe ct of an inducing agent. Lilleho j

_{- -}

et al .

(1 967) have reported reversal of aflatoxin i��ibit ion of an

spe c ie s by yeast extract although yeast extract was included in the medium used in

the

experiments on induction of the B . bacteriophage by

aflatoxin. In a defined medium the effectivene s s of the aflatoxin might be incre a sed.

The

B . syste

m

was f irst isolated for study in 1 931 by

den

Dooren de Jong and was used by Lwoff and his c olleague s in their now cla s s ical stuU.ie s of lys ogeny in the early 1 950' s . Despite the attention that this system has re ceived t

he

re are inconsis tencies in the literature regarding the requirement s of the o rganism for growth and for the propagation of phage after induct ion.

Lwoff (1 950 ) used a complex mineral

salts

ba se c ontaining ammonium sulphate , po tass iili�

dihydrogen pho sphate ,

and

m

agn

es

ium sulphate as the main

ingredient s and

iron ,

manganese , cobalt , zinc , molybdate, bo rate and c opper as m inor

ingredient s .

This wa s

supplemented

with glucose , yeast extract and

furthe r

aDOU.Tlt s

o f

calcium and magne s ium . Try-�tone was sometimes added as

well. By contrast , Northrop (1 956 )

used a

medium c ontaining only ammonium sulphate, potassillin dihydrogen phospha te , magne sium sulphate , glucose and traces of i

ron.

The

requirements stated to be necc ss::try for induction and phage propagation are also confus ing. Gratia (1 936 ) claimed that calcium was required when plating phage on

the

B . 1 nJUtilat ' strain of

den Dooren de Jone; , a stra in which is very similar to the K11 strain used

59 .

and phos phat e ions for the

production

of ba cteriophage

in

a cultur

e

of the

lysoge

nic strain. Huybers in

1 953

reported that

ma

gnesium

ions were

nece ssary in nutrient b roth for induction to

occur after ultra-violet light

i

rradiat ion and Freidman and C owles

( 1 953 )

reported a

drastic reduct ion in

t he t itres o f phage whe n counted in at;ar

c

ontaining c

itr

ate. O n the other hand Gaal et al .

( 1 970 )

induced B.

899a b

acteriophage

with

mitomyc in C in a �edium c onta in ing only yeast extract, t ryptone, pota s s ium dih

yd

rogen phosphate

and sodium

chloride.

A s there wa

s

l

ittle cons i s tency in

the

previo

u

s report s on condit ions for groirth of B. and for propa

g

at ion o f its ba cter iophage , a

s

tudy

was made of

the strains that were t o be used in the induct ion assay.

Defined medium for B .

All

cultures for growth respons e experiment s were incuba ted in fla sks with s ide arms for opt ical density measurements. These flasks were

incubated sbaken in a water ba th at 3 7°C . In lat er experiment s

the

0 0 .

temperature was

lowere d t o 3 2 C a s 3 7 C

was

thousht t o be t oo close to t he maximum growt

h

t emperature of B. Growth

was followed

by mea suring t he opt i cal density

in

a Klett-Surr�erson colorimeter.

Attempts to grow B . stra in 899a �� basal medium

(BM)

supplemented with

0.1 �,;

gluco se

(EJ,:G)

wer e U:.'lsucce s sful and additional supp

le ment

s were incorporat ed. into the nedium. It was fo

u

nd that the

addit ion of

0.1

fl.s

/

_ml nicot inic a cid was nece s s_ary for the growth not only o f

strain 899a but

also o f strains 1

368 and KJ,r.

of the medium

Shaken culture s gre w more rapidly than unsh.:Jcen culture s .

This

wa s as sumed to b e

due

t o the incre a s e d availabilit

y of oxygen as

re ported

by

L':;off

( 1 950) .

To standardi se the

g

r

o

)rth c ondit ions culture s were grown in

BMG + nicotinic a c id at shake rutes varying betwe en

50 and 1 1 0

_{cycles/minute.}

Growth , a s mea sured by genera t io� t ime , wa s

unaffected between

70

and 1 1 0

cycle s/minute and all further experiments were performed at a rate o f 90

Gluco se reouirement s

of

B .

Strain o99a

was examined

for

the effect

of glucose concentration on

y

ield

. A culture wa s

grov.n in

BJ.: +

0 . 5 iJ.g/ml nicotinic acid and 0 .1 %

gluco

se

until it

was in

t he exponent ia l

phase of growth� The culture

was

centrifuged; the cells washed in buffer and resuspended in

BM.

This

suspension

was

used to inoculate

aliquot s of BM +

nicotinic acid containing

various

levels of gluco se .

Growth of these cultures at 3iC was followed

by taking optical density measurement s until no further increase occurred .

The max imum

growth at various levels of glucose for strains 899a ,

1 368 and

Kl.!

were determined and are presented in Figure 3 .1 . The regression line

wa s

calculated only on the re

sult s

from experiments using strain

899a ,

but

all three

strains

be

haved simila

rly

.

The

maximu.11

growth is reached at

approximately

0 . 3%

gluco se

beyond

v;hich

there

ma

y be

a

decline in yield.

During the series of

experiments de scribed above

it was

observed

that

the

re

l

a

tionship between cell

mass

and

optical density departed from

li..'l'learity above a Klett value of 1 1 0 .

Further investigation was

undertaken

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