CONTINUAR LABORANDO DESPUÉS DEL VENCIMIENTO DEL CONTRATO MODAL.

tre2.tecl '!J ith r. fJ utoxin

',','hen lysogenic cu lt�;re s o f are

treated

with

a flatoxin

there i s a sudden i nc rease in t r�t� numbers of free phage o ccurring 80-90 !",inut e s after trcc.tnent. Tl':is i s fo ll o·::ed by an almos

t

equally rapid

d.e c:n�a in ;,unbers. The level t o vthich the

phage numbers

fall i s still

grea t e r than _thc:.t in

untreated

cult u re s _and continue s to rise sl

o

VIly

a

fter

a

r::in:i.!''UI.il r,s.s bee n

r<:cached.

'.i.'ne st,;.G.den :i ncrease in phe.ge nurr

,

b

e

rs c�n reasonab

l

y be

interpreted

as the lysis and phae;e releas caused by tbe induct i or1 of the

prophage

by

aflatoxin . The decrease i.rnnc diately foll

o

wi

n

g

the

incre a s e i s

less

ea sily e):plained.

I n

_Sect

i

on I I J the adsorption o f mcgaphage 8

9

9a to

1. 23 .

B . stra in K}.� i s de scribed. l!nfo rtunc,tely no i nve st igat ions

of the

o.O.sorp t icn of :2he.ge to strc.in

29So.

\7ere me.de but it i s 2. s sumed in

the follovring ciiscuss ion tllLt the kine t i c s o f adsorpt ion of

ph2.e;e

t o

stra in 899a are sic i la r to those t o strain XJ,: . It wa s n o t p o s s ible

t

o

deri ve o.n adsorption constant u s i11g cc 2.l num

b

er_s

since the

nw:Jbers could

not a c c u r�tely be a s se ssed. A constnnt us ing optical de n sity in

Klett

unit s as a measure of c e l l mass ;va s derived

instead

ttus : -

K =

1

2 . 3 log

Po

ODt p

whe re OD = optical density

t = t ime in minut e s

Po = initit::.l plmee count

p phage count. at t irr•e t

Data from a

m:;;Jber

o f adsorptio

n

eY.!Jeriments at optical

densitie s ranging

fro� Klett

26

to Klett 400 Gave a nean value of

9X1 0-4•

U sing thi s value

one finds t hat <lt an opt i cn.l dens ity of

50

Kle tt u

n

its,

9q:;

of

the phage

would be adsorbed in 1 30 sinutes. I n the

experioents where enough data

is available to co.ke an e ::;t imate , the peak of free phage occurred

between

80 and 9 0 minute s o.nd he.d been reduced t o a 1 0th of the p e a k value by

betwe en 1 20 .:1nd

1 50

minu te s . 'l'hus the time in \'ihich

the phage nurnbers

dec l i ne d by sq. cou l d

be

ns s hort a s

30

o r as long as

70

minutes.

It

seems

t

wre f ore tbu t a d s orp tion is not of the sar::e order as the d

e

crea s e

of ph:: §:;e in a�'lc. to}:in-trec.ted culture .:; n f t e r t l:e init i a l pe ak o f

_phage

prc::luction C:l1d c:c:.nr..o t a c c ount for al1 o f that decre as e. It he.s

a

lr

e

ady

been coc.:1ented that the trnnsient and varinble nature · o f

the

peak

made

i

t

unso.tisfn.ct ory c• .:> ::1 mea sure o f o. flatoxin c oncentration. Part of

the

variEtion betr;een experim e n t s could be due to 'Wriation s i n adsorpt ion

rate in cultt<TC S o f diffe rent optic:::. l den sity.

2c .

Be co.use cf tha difficult i e s a ssoc:i.ded w it h _{gett ing} a reproducible

of induced c e l l s. _{The main} problem was to distingui

s

h c e l l s induced at

a

par

ticule.r time from lat e r , spon tane ous induct ion. :::t did. not se em

likely

th:.:.t

chem ically in0.uc ed cells c ould be dist insu::. s�.e d. fror;1

spontaneou::>ly induced eel]

s

but i t did pro

v

e p o s s ible t 9 de•:elo

p

a system

where the nun:ber of c e l l s induced by af

l

at

ox

in t re at:::el'lt cculd be coll!

p

ared

with the spontane ously ind

u

ced cells in an untrea t e d c

o

ntr

o

l. ':'hi s wa s

achi evE:d by allo

w

i

n

� sufficient time for the tre atsent t o b e e ffe c t ive

a nd then rer1oving t h e c ells int o an agar medium. Sufficient t ime wa s

then allo,;:ed fo r the l,:yt ic cycle to be complet ed and � r:y rer::e. i

n

ing

unlysed cell s were kill.eu by t reatment viith stre ptorr.ycin. The pb.age-

sensitive organi sms , being s tre pt omycin-re- i stant, �ere st i _l able to grcv1 a nd be i

n

fected by tr.e phage released from induc e C. and lysed cells.

ThJ s

s

ys

t

em appeare d t o g ive a suffic iently good correlat ion betwee n

aflat oxin · :.mcentrat ion and re sponse to provide a quc.n-: itat ive bioassay.

The rcproC..u c i b i1 ity of the do::;e

/

re sponse re lat ionship is no t as good a s that at

t

aina_ble by the inhibition sy

s

tem but i s . pot e ntially I:lore spec ific. S ensit ivity i s al s o le s s than that of the i

n

hibit io

n

s�· s t e� by a factor

o f twenty . Improvemen t s in s en s it ivity Dight be obtain�ble using the

potcntiating system o f Ame s

�

a l .

( 1 9 73 )

usi

ng live r extr.2.cts

to convert the t oxins into their act ive form.

The a ssay o f o ther knO\'m c cmpounC.s by the inC.uct ion s

ys

tem supported

t he suggest ions of Lecator

(1 966)

and others t hat c

o

=

po�.:

nC.s known or

suspe cted. to be carc inogeni c are a l.r-o capable o f ce.us :..r,g i

n

ci

u

ctio

n

.

Aflatoxin B1 , st cri��,t o cyst in a nd ochrat oxin A have all b e e n re

p

orted t o

be carc inoccnic

(i?et.1Cl1 1 969 )

aml a ll were a c t ive ' n the i

n

G.

u

ction bioa ssay. The usefu lne s s of the as say sy stc:n for toxin s in ckiry proC.ucts i s limited at present. by the uifficulty in obtainj ng sufficie.::tly pure extract s of

toxin from these produ cts. Fu rther inv e s t igat ion of cethoC.s of extractio n

and purificat ion should provide the solution to thi s diffi c ulty.

Corrma ri. scn \'iith

Frequently the a s so.ys sucge stecl for a flatoxins i

n

volve ext

r

action,

1 25 .

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