an improved method for the selection of bacterial cells at division

6 0 8 SHORT COMMUNICATIONS

The exper iments here repor ted , toge ther wi th those repor ted by o ther workers 8,9, suppor t the thesis t ha t insulin can influence prote in synthesis in muscle at a site d is ta l to amino acid t ranspor t , p re sumab ly by an affect on some in t racel lu lar process. The resul ts do not exclude the poss ib i l i ty tha t insulin also s t imula tes amino acid t ranspor t , indeed, while there is no certain evidence tha t insulin promotes en t ry of na tu ra l amino acids into muscle, it is equal ly t rue tha t there is no exper iment tha t decis ively rules out the possibi l i ty. However , if insulin were to s t imula te the en t ry of amino acids into muscle then, ill the presence of [12C!amino acids, there should be grea ter di lut ion of the [14C~amino acids formed from [14C]pyruvate than occurs in t i le absence of the hormone ; insulin should, therefore, ac tua l ly decrease the incorpo- ra t ion of 14C into protein. The fact is, of course, tha t tile opposi te occurs and to t ha t ex ten t the results are agains t a t )romoting effect of insulin on th,,~ t r anspor t of amino acid into muscle.

We thank A. N. MOYER for vahlable assistance in carrying out the exper iments . The research was suppo r t ed by gran ts from the Nat ional Ins t i tu tes of Heal th

(A-4842), the John A. Ha r t fo rd Founda t ion , and The Abbo t t Memorial Fund. One of us (I.G.\V.) holds a Uni ted S ta tes Publ ic Hea l th Service Research Career l )evelop- ment Award.

Department of Plt3,siolog3,, The Univ~:rsitv of (?h/ca;,(), Chicago, Ill. (U .S .A . )

IR3. (i. \~'OOL M. E. KRAHL

1 K. L. MANCHESTER AND 1 ¢. (;. YOUNG, t3iochem. J., 7 ° {i(~58 ) 353. '-' I. O. WOOL AND M. E. I(RAHL, /t~1..]. Ph/siol., ~96 (1959) 901. ;/ 1). M. ](IPNIS AND 5I. \V. NOALL, Biochim. Biophys..~tcta, 2S (1958) 22o. .3 K. L. MANCHESTER AND I:. G. YOVNG. Biochem. J., 75 (190o) 487 . s H. AKEDO AND H. N. CHRISTENSEN, .[. Biol. Chem., 237 (I962) 118. 6 G. GUROFF AND S. UDENFRIENI), Biochim. I3iophVs. dora, 4 ° (196l) 38(,. 7 K. L. MANCHESTER AND I. G. WOOL, Biochem. J., in t h e press . s I. G. W o o l AND M. E. I(RAHL, Nature, 183 (I959) 1399.

I,Z. L. ~IANCIIESTER ANt) M. E. I£.RAttL, jr. Biol. Chem., 234 (I959) 2938. 10 ]( . L. MANCHESTER AND F. (;. YOUNG, t3zochem..[., 72 (1959) 13(). 11 C. A. VILLEE AND A. B. HASTINGS, .[. Biol. Chem., 181 ( I949) I31 . 121 R. GCHARFF AND I. (i. \VooL, u n p u b l i s h e d d a t a .

Received Sep tember qth, 1963 Biochim. Biophys. , i cla, S 2 (1964) 600 -0o~

sc 2394

An improved method for the selection of bacterial cells at division

A m e t h o d for ob ta in ing synchronous ly d iv id ing bac te r ia b y wi thdrawal of new daughte r cells from a growing cul ture has been descr ibed b y HFLMSTWrTER A~U) CUMMINGS 1. In tha t procedure a popula t ion of Escherichia coli B/r was first bound to an anion-exchange paper , and then growth med ium was passed th rough the paper . After elut ion had proceeded for 3 ° min, the vas t m a j o r i t y of ceils which appea red in the eluent were new daugh te r cells. I t was concluded tha t each bac t e r ium which e lu ted from the exchanger was t ha t sister of a new sister pair which was not involved

Biochim. Biophvs. dcta, ,'12 (i 904) 0o5 (~i o

SHORT COMMUNICATIONS 609

in the a t t a c h m e n t to the exchanger. While the selection of these cells was evident , cont inuous remova l of all the unbound sister cells was not achieved. This was p r o b a b l y due to the thickness of the ion-exchange paper and the consequent readsorpt ion of cells dur ing elution. A cons tant elut ion t i ter could be more near ly approached b y b inding the ceils to a single surface so tha t once a cell was avai lable for removal it could elute from the surface unobst ructed .

This repor t describes a simplified me thod which achieves this result. The surface to which the cells were bound was a membrane filter of pore size smaller than the cells. In this procedure a nea r ly cons tant yield of new daughte r cells was ob ta ined and these cells d i sp layed a much higher degree of synchronous growth than tha t ob ta ined with ion-exchange paper at equivalent bac te r ia l concentrat ions.

The organism used was E. coli B/r (ATCC I2 4o7). The min imal medium, growth condit ions and detai ls of the synchroniza t ion appa ra tus have been descr ibed 1. The bac te r ia were grown for 17 h at 37 ° to a concentra t ion of I . lO 8 cells/ml in 2.5 1 of minimal medium. I 1 of this cul ture was used for the b inding opera t ion and the re- mainder was fi l tered th rough a 0.65-/, pore, I52-mm d iamete r ( type DA) Millipore filter (Millipore F i l t e r Corp., Bedford, Mass.) and used as the eluent. The cells were bound to a o.22-/, pore, I52 -mm diamete r Millipore filter ( type GS) which was secured in a two-sect ion stainless-steel funnel. Tile b inding opera t ion was accompl ished b y passing the I-1 cul ture th rough the membrane filter under a pressure of approx. 5 lb/in2. This opera t ion was comple ted in 2. 5 min. The filter was then inver t ed and elution was begun with 1. 5 1 of med ium above the filter. The ini t ia l elut ion ra te was 50-60 ml/min. An ini t ial flow rate at least this high was necessary for efficient washout of unbound cells. In order to increase the concent ra t ion of selected cells, the flow rate was g radua l ly reduced b y allowing I 1 of eluent to pass th rough the filter before reeirculat ion of the e luent was begun. When the desired concent ra t ion was reached, the eluent was rec i rcula ted th rough a type DA Millipore filter wi th a S igmamotor 0V-22 pump. In this manner a cons tant l iquid level of about 500 ml was ma in t a ined above the filter and resul ted in a cons tant elut ion ra te of about 15 ml/min. The elut ion curve was ob ta ined b y collecting I -ml samples from the eluent a t the desired intervals . For the s?mehronous growth exper iment a sample of the e luent was collected for 2 min and incuba ted at 37 ° wi th vigorous shaking. Bacter ia l counts were de te rmined using a Coulter Counter Model B which was opera ted as descr ibed previously 1. The counts were de te rmined af ter dihl t ion in 0. 9 % NaC1.

Fig. I shows the elut ion of bac te r ia from the surface of a Millipore filter of o .22 / , pore size. The solid line indicates the results when the ini t ial elut ion ra te was 55 ml /min and then g radua l ly reduced to 15 ml /min during the first 4o min of elution. The dashed line gives the ini t ial shape of the elut ion curve when the elution was begun at a ra te of 15 ml/min. After the init ial wash-out of weakly bound cells, the bac te r ia in the eluent consisted p r imar i ly of new daughte r cells at near ly cons tant concen- t ra t ion. The observed per iodic i ty of cell number with the generat ion t ime (45 min) was most l ikely due to the age d is t r ibut ion of the cells bound to the membrane filter. In cont ras t to the results ob ta ined with anion-exchange paper , the age d is t r ibut ion of cells bound to the membrane filter was not uniform or the f luctuat ion in the elution curve would not have occurred. The age d is t r ibu t ion of the cells on the filter depended on the d is t r ibu t ion in the exponent ia l phase popula t ion and also on b inding proper t ies as a function of cell size and age.

Biochim. Biophys. Acta, 82 (1964) 6o8-61o

6 I O SHORT COMMUNICATIONS

20

l0 o x

50 c~

g

25

\

\ \

I I ] ~ - I .... !

\ \

J . . . . . I I _ _ I i _ _ l I

0 20 40 60 80 I00 120 140 ELUTION TIME Imin)

Iqg. I. E ]u t ion of bacter ia f rom a 0.22-ii M i ] l i - p o r e l i l ter . T h e c u r ~ e s a re d e s c r i b e d in d e t a i l

in t he t e x t .

Ic> x

<

m

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I I

I I 20 40 60 80

INCUBATION TIME (min) If'0 120 140

Fig. 2. S y n c h r o n o u s g r o w t h of bat t e r i a co l lec t - ed f rom the e l u e n t a f t e r 85 ra in of e lu t i on . T h e s a m p l e was co l l ec t ed for 2 ra in a n d t h e n

i n c u b a t e d a t 37 ~ w i t h v i g o r o u s s h a k i n g .

Fig. 2 shows the synchronous growth of bac ter ia in a sample of the eluent t aken af ter 85 min of elution. Al though the bac te r ia in the eluent after 35 min of elut ion were p r imar i ly new daugh te r cells, this selection was s l ight ly improved af ter elution had proceeded for 8o rain.

This procedure involves the in terac t ion between the bac te r ia and the membrane filter. JEXSEN AND HAAS 2 have recent ly presented a de ta i led analysis of the electro- s ta t ic in te rac t ion of Bacillus s,ubtilis with membrane filters. In our case, the cells were bound to a filter of pore size smaller than the bac te r ia so tha t the cells resided ()n the surface of the filter. When the filter was then inver ted, the med ium flowed th rough the pores and new daughters were e lu ted from the b o t t o m surface. The m a x i m u m t i le r of new daughte r cells e lu ted from a o.22-/~ Millipore filter var ied from 6. lO v to 8. lO 7 eells/min. Al though s l ight ly higher elution t i ters have been ob ta ined using Millipore filters of o.45 /~ pore size or larger, the highest qua l i ty of selection ot new daughters was achieved with the type (IS filter.

Laboratory of Molecular Biology, NINDB, National Institutes of Health,

Bethesda, Md. (U.S.A.)

CHARLES J~. HELMSTETTFA~*

D O N A L D .J. CUMMINGS

1 C. E . ]-fELMSTETTER AND D. J . CUMMINGS, Proc. Natl. Acad. Sci. U.5"., 5 ° (L~l~)3) 7o7 . 2 R . ,'\. JENSEN AND I ~'. [,. HAAS, J. Bacteriol., 86 ( [963) 73.

Received November 22nd, 1963

* P r e s e n t a d d r e s s : U n i v e r s i t y I n s t i t u t e of Mic rob io logy , (:5ster F a r i m a g s g a ( t e 2A, C o p e n h a g e n 1,7, D e n m a r k .

Bzochim. Bioph3,.,. A cta, 82 (E 904) 608 01 o