OPERATED FOR THE UNITED STATES ATOMIC ENERGY COMMISSION 8Y SANDiA CORPORATION I ALBUQUERQUE NEW MEXICO LIVERMORE CALIFORNIA

Issued by Sandia Corporation, a pr ime contractor t o the

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Planetary Quarantine Applied Science Division 1742

Sandia Laboratories, A l b u ~ ~ ~ ~ ~ u ~ 87115

ABSTRACT

A method ies described fo of T4 bacteriophage. This de n i s of sufficient deta i l t o allow those unfamil t o successfully u t i l i z e and s Key Worda: eparation - Ass

eparation and assay

1-2

provided by more than one

i s t i c s of l i v i

y have also provided investigate s with eas i ly comprehensible

models for life processes which are cha a c t e r i s t i c of more complex forms.

these reasons ~ i r ~ s e $ 8. e eminently su i tab le i n ~ n ~ e $ t i ~ a t i o n ~ on the molecular

basis of l i f e ,

For

~ a ~ t ~ ~ i o ~ h a g ~ s ~ viruses tha t employ baete i a BEJ hosts fo r

3

bac te r i a l host c e l l l y s i s serve as the bas i s for the assay procedure t o be

described i n t h i s document. Further studies, particzalarly over the past

nty years, have delineated i n grea te r d e t a i l t h e p a r a s i t i c l i f e cycle of

bacterio-ghage and, i n addition, have elucidated the s t ructure and genetic

map of T4 phage.

Such research e f f o r t s have resulted i n the publication of numerous papers

dealing with bacteriophages, and much of t h i s information has been coiqiled

i n t o several excellent t e x t s (2, 3) on bac te r i a l viruseso

apparently i s no detai led description of how one prepares and actual ly caryies

out an assay on v i r a l ac t iv i ty .

i n suff ic ient d e t a i l , how such an assay may be accomplished.

However, there

The purpose of t h i s document is t o out l ine,

atfon of E. col i and T4 gh

u l t w e d from the

2.

Dspta, 12301 9

5

3 e

4,

S B one l i t e r beaker covered with aluminum f o i l .

6 , centrifuge tubes,

two Erlenmeyer flasks (1 l i t e r ) with rubber. stopperse

one l i t e r Milbigore suction f l a s k sealed with aluminum f o i l ,

Firs t , the E, c o l i B working stock is prepared by inoculating the 3% T,S,B*

solution with the i n i t i a l bac t e r i a l cul ture , The f l a s k is resealed with the

s t e r i l e aluminum f o i l and shaken gently a t room temperature u n t i l the solution

i s ju s t v i s ib ly turbid (10

T,S.B, solut ion with 5 aril. of a bac te r i a l culture containing 10

inoculum was v is ib ly turbid after shaking f o r 5 hours,

7 cells/ml), I n o w laboratory we inoculated the

cells/ml, 8 This

When turb id i ty i s reached, pour approximately 500 m l of the E. co l i B

suspension in to a s t e r i l e one l i t e r Erlenmeyer f lask , s ea l w i t h a rubber

stopper, and s tore under re f r igera t ion (4"C,).

the EO c o l i B working stock and should be so labeled,

This mixture w i l l serve as

The remaining 500 ml of the suspension is inoculated wi th T4 phage 8 (approximately LO

and allowed t o shake overnight ( -18 hrs.) e

bacter fa l suspension i s routinely accomplished i n t h i s laboratory by first

adding the phage t o 100 m l of 3% T,S,B, and then slowly adding t h i s phage

mixture t o the bac ter ia l suspension while gently swirling,

too low a r a t i o of bactekia t o phage because of the poss ib i l i t y t ha t the

phage could lyse the bacter ia without f irst undergoing phage multiplication

(the so-called " l y s i s fYom without")

bacteria per phage p a r t i c l e is desired a t t h i s phage inocdat ion step.

phage pa r t i c l e s ) , re-sealed with the s t e r i l e f o i l cap,

Addition of the phage t o the

One should avoid

Hence, a r a t i o of a t l ea s t ten

6

for the turbid nsfon i

C d a t 3000 r p

is stored i n a steri

r is a ~ h i e ~ e d b

a 0,45 i d is used t o

plrocess. A s i n t s

ed because o t h a t it might entrap o damage the Qhage

p a r t i c l e s dwf l t r a t e , which contains the T4 ghage, is

n ch i l led t o 4 " - 5 " C a

A , the c lear f i l t a t e i s t ~ a n ~ f @ ~ r e d t o a s t e r i l e one l i t e r

and the solut ion is ac id i f ied t o pH 3.9-4,O with 0.1 N HCL (approx,

25 id), The solut ion is swirled during the addition of the acid, and the

resu l t ing cloudy suspension is allowed t o stand overnight a t 4°C. The

i s i so la ted by centrifugation a t 3000 rpm f o r 10 minutes.

resuspended in a minimal volume of 15 T.S,B. to

ion which i a stored a t 4 " C e Thi

ion is the T4 ph e w ~ r k i ~ stock and should be so labeled. Heriott an

c ip i t a t ion t echla

TABLE P

i f i ca t fon of T4

Treatment Volume ( m l )

Lysate 1000

(Supernat ant ) 1000

1,o x 10 lo 1 x ro13

8,o x i o 8 x

Millipore F i l t r a t i o n ( F i l t r a t e ) loo0 6,2 x l o 6.2 x 10 12

4 ( Prec ip i ta te ) a 1 2 3.3 x 10 11 4,12 x 10

100

62

33

C) Assay of T4 bacteriophage,

This phage assay is based on the l y s i s of a sui table bac ter ia l host

after phage infect ion and multiplication. For c l a r i t y , a b r i e f summary

of the assay protocol i s presented.

An al iquot of the E. c o l i B working stock i s gently shaken a t room

temperature two hours p r io r t o the actual phage assay. This yields a

suspension which contains approximately 10 c e l l s per ml. For e f f i c i en t

phage multiplication, the bac te r i a l host should be undergoing logarithmic

growths The two hour incubation period f u l f i l l s t h i s requirement, Once

the E. c o l i la have reached t h i s l eve l of gro h, a cer ta in amount of the

E , c o l i B suspension i s added t o a special agar-nutrient mixture referred

to a s soft agarD

is pipetted i n t o v ia l s .

a re mafntained a t 42"C0

and is not deletepious t o the bacter ia ,

a

After mixing, 9.5 m l o f t h e bacteria-soft agar suspension

These Vials, containing the bacter ia and s o f t ag

This ternperatme keeps the agar i n a liquic! s t a t e

Concurrent w i t h preparation of the v i a l s containing bacter ia and so f t

A small agar, the phage samples are di luted t o a countable concentration,

amount of (0,5 m l ) cf the phage, appropriately d i lu ted , i s added t o each

8

e infection and ImultB-

The amount of material needed fo

the scope of the experiment, The f o l

needed for the deter nation of a ~4

e bacteriophage assay depends on

ng sample defines the materials

one hour. Ab1 materials must be steri l i

-containiPlg t e s t tube

control - 2 (~uglicate! samples ar F data p o i n t # )

60 miw. - 2 -

9

6 ,

y e 1 m 1 2 5 nil, and 10 m l pipe t tes ,

E. eohf I3 i n logarithmic phase of glp --

I n summary, the following sterfPe materials are needed t o conduct

t h i s assay: 6 test tubes with l i d s , 24 d i lu t ion containers, 24 d i lu t ion

v i a l s with screw caps ( s ize 25m, I D , 95m, h t ) , 230 m l of so f t agar, 46

p e t r i dishes containin

E, c o l i B and p ipe t tes ,

agar ( s ize 20mm x lOOmm, Falcon p l a s t i c disposable )

Prepare the d i lu t ion bo t t l e s with 1% TeSIB., autoclaving the bo t t l e s

and stoppers separately, After cooling, adjust the volume i n each b o t t l e

t o the 99 m l mark and add the stopper. The 50 m l d i lu t ion beakers are a l s o

s t e r i l i zed but contain no T,S.B. medium, Nine m l of s t e r i l e 1% T.S.B. w i l l

be added ju s t before the assay t o each beaker needed,

The amount of soft agar needed depends on the number of virus v i a l s

Therefore, 24 via ls

The so f t agar is prepared i n

used, since each v i a l contains 9.5 ml of t h i s agar.

require approximately 230 m l of sof t agar.

the following manner:

add 250 ml dionized water,

0.8% (wt , /vo l , ) i n agar.

weigh out 7e5 gm of T.S.B. and 2.0 gm agar-agar and

This solut ion is 3 (wt,/vol.) i n T,S.Be and

It i s autoclaved, cooled, and maintained a t 42'Ce

regare p e t r i dishes by adding 10 ml of 476 TeSeAa to each dish and

allowing t o cool.

24 hours previously, since drying of the agar must be avoided i f good

plaque s ize is t o be obtained, I n t h i s laboratory, the p e t r i dishes are

prepared on the day of the v i r a l assay.

am t G be prevented, heavy condensation of water vapor on the top of the

o p e t r i d i s h i s used i f it has been prepared more than

If s t reaks i n the bac ter ia l lawn

the glates and

stock is placed on a sha

amount of

e Consider th

of E. c o l i B

Hence approxi

8 ml of Ea coli B are necessary for the 230 lnil of soft agar

8 approximately 10 cells/

in which the small

i B suspension from

ren t ly with the

stribution o

i s di%ution

schedule produeas the indie ted phage ~ ~ ~ e e ~ t ~ a ~ ~ o ~ per di lu t ion con-

at ion in the control assay t e s t tube of

PP

Dilution D i lut ion C ont a i ner Container

Control w d91, bo t t l e I mP assay + 99 mL 1

B beaker 1 ml d i p , A -I- 9 Kl ld/ml C d i l , b o t t l e 1 mP d i l , A + 99 tnl 1% T,S.B. 10 /ml

B beaker 1 ml d i l , C + 9 ml l 0 3 / ~ l

E d i l , bo t t l e 1 mf. d i l , C + 99 pnl 1O2/miL

F beaker 1 m l d i l , E -I- 9 ml 1% TeSoBD l O / m l

4

The phage assay i s carr ied out by first deciding which d i lu t ions w i l l

insure a countable number of phages per p e t r i dish, I n the above example,

d i lu t ions C through E may be used, Remove 0.5 m l from the selected dilu-

t i o n containers and add t o the appropriately labeled virus d i lu t ion v i a l s

which contain 9.5 m l of the E, c o l i B - so f t agar mix, This r e su l t s i n a

twenty-fold d i lu t ion of the phage concentration f o r a given d i lu t ion con-

t a ine r , The v i a l is capped, inverted t e n times t o mix i n the viruss and

placed i n a 42OC, water bath u n t i l a l l pipet t ings and mixings have been

accomplishedl A 5 m l blow-out p ipe t te i s then used t o del iver 2 m l

(blow out ) of the contents of each v i a l i n to each two appropriately

labeled p e t r i dishes, The d i sh is quickly rotated t o spread the agar

over the surface before it hardens. Since the experiment is performed

with duplicate samples, the second virus v i a l holding the duplicate phage

sample is pipetted according t o the same .schemee A f resh s t e r i l e 5 m l

p ipe t te i s used t o remove the two 2 m l portions from t h i s second v ia l ,

The p e t r i dishes a re placed i n a 37"C, incubation chamber overnight

(-18 hcurs) and the plaques are counted the following day.

d ica tes the type of data obtained from such a protoeol, This data i s

Table 2 in-

presented i n Figure 1 as par t of the survival curve data foir the syner-

g i s t i c inact ivat ion of T4 phage by thennoradiation,

Treatment

Control (No Heat)

30 mine heat

60 min. heat

Survival Data for T4 Phage at 66°C.

Dilution

E

D

E

D

C

D

C

B

142,155 153,160

68 ,71 60,68

1 6 4.9 x 10

1.53 x lo5

6.7 x lo3

3.13 x lom2

1.37 x lom3

* I n t h e course of t h e assay 0.5 m l of phage, d i lu t ed appropriately, was added t o 9.5 m l a f t e r bacter ia-sof t agar suspensions One must correct

f o r t h i s d i lu t ion i n order t o obtain a corrected phage concentration.

For example, t h e cont ro l sample y i e lds 4.9 x 10 6 phage/ml

1

P

a

14 I

e author would l i k e t o express h i s s incere appreciation t o

amy J. Laible for her highly competent technical

ass is tance

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