bacteriology laboratories, university of notre dame, notre ...single cell isolatilon single cell...

STUDIES IN MICRURGICAL TECHNIQUE

J. ARTHUR REYNIERS

Bacteriology Laboratories, University of Notre Dame, Notre Dame, Indiana

Received for publication, November, 1, 1932

I. THE ADAPTATION OF SINGLE CELL TECHNIQUE TO ROUTINE USE

Single cell methods of isolating bacterial cells as they exist atpresent are neither as exact as they might be nor are they suffi-ciently mechanized for adaptation to routine. By way of exam-ple, when hundreds of such isolations are necessary, in problemsconcerned with heredity, etc., or when a single cell isolation isdesirable in the course of a more general problem, the presentmethods are inadequate. They are inadequate because theydepend too much on individual skill and because the necessaryapparatus requires too much time and attention to set up. Thetechnique at present is too detailed in its application and for thatreason presents a mental hazard which has resulted in its notbeing as widely used as it might be. If, however, the apparatuscould be combined into one mechanism specialized for the pur-pose of single cell isolation, if the accessory details such as makingmicro-pipettes and isolation droplets could be done by machinemethods, if in other words the isolation of bacteria could beaccomplished by machine-if these things could be done, thenthere is no good reason why such a technique should not be moregenerally used. Certainly the accumulated mass of evidencefor variation and dissociation, as well as the many basic problemsin heredity, yet to be properly investigated warrant the carefulstudy of single cell methods with a view toward routine adopta-tion. Whether or not a technique so highly specialized as thiscan be adapted to general laboratory procedures will depend onthe completeness with which it can be mechanized and in the final

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analysis, upon the trend that bacteriology will follow and thenecessity this trend will create for cell selection.

Because of the difficulty of presenting a problem such as this,I wish first to discuss the problem of mechaniizing single cellisolation and adapting it to routine as well as to present a surveyof the available literature on the subject for comnparison with mymethods. The second section of this paper will describe the iso-lation apparatus; the third, a mechanical pipette puller; and thefourth paper will discuss the use of all the apparatus and will a1lsoinclude a presentation of an automatic isolation droplet former.

In the prosecution of the work I have been assisted by Dr.Francis J. Wenninger and Dr. Theodore Just, both of the Uni-versity of Notre Dame, to whom I hereby make grateful acknowl-edgement. Without the help of my father, 'Mr. Leo A. Reyniers,the development of the apparatus would have been impossible.

Single cell isolatilonSingle cell isolation, be it mechanical or semi-mechanical,

involves three factors: (1) the necessary apparatus, (2) the tech-nique used to isolate the cell, and (3) the means taken for remov-ing the selected cell to culture. As used in this paper the term"semi-mechanical" refers to technique similar to plating or thehanging block type of isolation; while "mechanical isolation"implies methods in which a micromanipulator is employed tomove a minute pipette with which the selected cell is handled.The semi-mechanical methods of isolating bacteria in pure cul-

ture such as were first practiced by Koch involved the use ofliquefiable solid media to separate the cells; and the cells wereeither trapped in an isolated state as the medium solidified orwere spread on its surface after solidificationi. B3urri, Hansenand others, altered the original technique slightly in that theisolated cell was now "spotted" under the microscope, and wasallowed to grow into a colony while under direct observation.The macroscopically v-isible colony while under direct observationlwas then removed as a pure culture. Plating based on the trap-ping effect of solidifying media which holds the scattered cellsapart cannot, however, be as surely controlled. The value of

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the original method consists in its ready adaptability to routineand mass work, which is, of course, not possible with thesemodifications.

While the early modifications of Hensen (1883), H. W. Hill(1902) and Burri (1909) were efforts to improve the original tech-nique, the later attempts of Hewlette (1918), Holker (1919),Hort (1920), Tropley, Barnard and Wilson (1921), Qrskov (1922),Paine (1927), Schoutens (1921), have been outgrowths for themost part, of attempts to improve mechanical single cell isolationand at the same time to supply a method easily adapted to routineprocedures.Most semi-mechanical modifications are based on the following

procedures: (1) The use of a solid medium, (2) the smearing ofthe organisms on the surface of the medium, (3) the "spotting"of an organism sufficiently separated from its neighbors, (4) themarking of the position of the selected organism, (5) the incuba-tion of the organism until a colony is formed, (6) the inoculationof a culture tube from the colony.

Procedures, semi-mechanical in nature, involve the use ofverniers or markings on the isolation surface for the accuratelocation and relocation of the isolated cell. In this technique thecells are separated from one another by smearing a diluted cultureover an agar surface, or, as in the methods of Burri and Paine, asteel mapping pen is filled with a diluted culture and small drop-lets are made with it on the agar surface. Once the cell has beenisolated and its position spotted it may be allowed to multiplyinto a colony or it may be removed directly by taking up thecell together with a larger portion of the agar by means of a hol-low needle.

Distinct modifications of the procedures just outlined are thoseof Holker (1919) who uses a flattened glass capillary and of Trop-ley, Barnard and Wilson (1921) who kill off all but a shieldedand selected cell by means of ultra violet rays.

Mechanical single cell isolationThis type of isolation involves a direct handling of the indi-

vidual cell to effect its isolation and culture. It is essentially a

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mechanical method and as used today is virtually the same aswhen introduced by Barber (1904), or as described in the paperof Gee and Hunt (1928).

In considering a technique of this kind for the purpose of mech-anization these factors must be considered: (1) the isolator, (2)the moist chamber, (3) the isolation instruments, i.e., micro-pipettes, (4) the removal of the isolated cell to culture.

Concerning the isolator. Many instruments have been designedfor micro-manipulation, but few of them have been designedespecially for bacteriological work. In some cases of specializa-tion, efforts have been made to simplify the isolator as a unitbut no one has gone so far as to incorporate the accessories as apart of the manipulator. As a consequence of not carrying thespecialization far enough the routine requirements have not beenfulfilled. Again, isolators have been designed which are deli-cately adjustable but here also little attention has been paid tothe fundamental demands of the problem at hand. If the basicidea of single cell work as originated by Barber be called to mindit will be seen that it is not the single cell which is handled but adroplet of fluid containing the cell and, of course, many timeslarger than the cell. Furthermore, it is unnecessary to makemore than one isolation droplet in a single field but the fieldscan be changed and the isolation droplets scattered over theentire surface of a cover glass which is moved between the pipettetip and the objective by a mechanical stage. If these ideas bekept in mind it will be obvious that undue attention need not bepaid to the refining of movement distances but that care shouldbe directed to bringing the pipette tip up to an exact point in asingle checked movement. Once this is obtained in an instru-ment, attention may then be directed toward changing thepipettes, and refining the control of environmental conditionsunder which the cell is isolated.

Micro-manipulators have been designed by Schmidt (1859),Chabry (1887), Doty (1900), Schoutens (1904), McClendon(1907), Tschaoutine (1912), Hecker (1916), Malone (1918), Bishopand Tharldison (1921), Chambers (1922), Kahn (1916), Peterfi(1923), Taylor (1923), Johnson (1924), Briedigan and Chang

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(1924), Dunn (1927), Fitz (1931), Emerson (1932), and Reyniers(1932). Most of these instruments move the pipette or tool inthree planes. The exceptions to this, move the tool in one plane,

1.

FIG. 1. (A) DIAGRAMMATIC SIDE VIEW OF ISOLATOR; (B) ToP VIEW OF ISOLATORDiagram A. 1, pipette holder tubing; 2, side-to-side adjustment of pipette tip;

3, holding post; 4, guide-pin which fits into a slot cut into the pipette holder; 5,plunger rod for lowering the pipette tip; 6, adjusting screw for raising or loweringthe pipette tip; 7, hollow screw in end wall of cradle housing; 8, hinged door overchanging compartment; 9, moist box; 10, pipette changing compartment; 11,extensions which fit into the arms of a mechanical stage; 12, microscope stage; 1$,set screw for holding the clamping post.

Diagram B. a, thermostat; b, set screw for holding pipette tubing in place;c, movement lever for raising the pipette tip into contact with the cover glass;d, screw head by which the moist chamber cradle is moved from front-to-back ofthe microscope stage; e, screw head for moving the cradle housing from side-to-side on the microscope stage;f, arms of mechanical stage; g, screw head for adjust-ing pipette tip in the microscopic field; h, tongued guide bar to keep the cradlehousing in line; i, guides for keeping the clamping post in line when pipette tipis being adjusted by screw head g.

up-and-down as illustrated by the instruments of Malone, Tscha-outine, Bridganin and Chang, Doty, Malone and Johnson. Theseinstruments achieve movements in other planes by the use of amechanical stage.

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J. ARTHUR REYNIERS

Strictly speaking, one movement only of the pipette is essentialto bacterial isolation and that is a movement in an up-and-dowindirection. If this motion is supplied to the isolator independentlyof the microscope stage, a further advantage is gained in that thetip of the pipette is at all times centered in the microscope field,and the droplets that are formed are always, roughly speakinig,in focus. Two other movements in the same plane but at rightangles to each other are necessary to scatter the droplets but thesemay be supplied to the moist chamber by a mechanical stage.

If the pipettes are mechanically made to the same length andto a desired diameter, then the one movement can be derivedfrom a checked lever and a single push of the finger. Whatevercentering of the pipette is necessary can be supplied by move-ments whose total sweep is never wider than a microscope fieldand which are never used in the isolation except to center thepipette when it is changed.The isolation of a single bacterial cell may be effected mechain-i-

cally in four different ways: (1) in hanging droplets, i.e., themethod of Blarber, (2) by separation of the cells as they lie scat-tered on an agar surface, which is accomplished by forming acone of moisture around them (method of Dickinson, 1926), (3:)by touching the isolated organism with a dry needle, (4) by acombination of semi-mechanical principles.

NTow, in any mechanical single cell isolation a moist chamberis necessary to prevent drying of the isolation droplets. Thesuccess of the method also depends upon the clearness with whichthe isolated bacterium can be defined in the isolation dropletand finally upon the ease and certainty with which the isolationdroplet and cell can be removed to culture. Certain physio-logical considerations pertaining to the bacterium must be keptin mind and such environmental effects as oxygen tension, mois-ture and temperature should be controllable. These factors canbe more or less completely conitrolled with apparatus of the properdesign.The methods I offer for mechanizing single cell culture are

essentially those of Barber but specialized in every detail, so thattheir application is purely mechanical and nmore exact. B3efore

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mechanical single cell isolation methods could be adapted toroutine, many considerations, both mechanical and physiologicalhad to be taken into account. To effect this purpose the follow-ing steps were taken:

(1) Micro-pipettes. These are made mechanically on a pipettepuller and are drawn to a uniform size and shape.

(2) Isolation droplets. These are made automatically, are per-fectly flat with well defined edges and are mechanicallyspaced. An isolation surface of this type requires no skillto use.

(3) Micro-manipulator. This is controlled by checked leverswhich are operated by a single push.

(4) Moist chamber. This is incorporated as a part of the manipu-lator and is interchangeable so as to permit many experi-ments to be carried out on the same machine. It iselectrically heated and automatically controlled at a settemperature. It is never exposed to the atmosphere evenwhen the pipettes are changed which permits an accuratecontrol over environmental conditions such as moisture,temperature, and oxygen tension.

(5) Pipette change. This is simplified by a new type of adaptorwhich allows the pipette to be accurately set in place so thatit is always in line with a predetermined position.

(6) Isolation surface and droplet formation. Surfaces other thanthe automatic type may also be prepared by machine.

In the specialized technique the pipette is moved in a singledirection (up-and-down) and is controllable by an adjustable andaccurately checked lever. Since the pipettes are made by ma-chine the tip is accurately in line with the shaft and when suchpipettes are mounted in their adaptors very little centering inthe microscope field has to be made. By controlling the pipettewith a lever and moving it in one plane only the tip is alwaysreturned to the same point and is, at this point, always in focus.The two movements necessary to expose the different areas ofthe isolation surface are accomplished by a mechanical stagewhich may also be moved by levers. Such problems as constanttemperature, oxygen tension, etc. were solved by devising a

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moist chamber which is enclosed at all times and is electricallyheated. Better optical conditions for defining the isolated cellsare obtained by flat droplets. Pipette replacement is simple,sure, and does not involve sealing in with special wax or cement.

II. A SPECIALIZED SINGLE CELL ISOLATOR FOR USE IN

BACTERIOLOGY

Repeated attempts to overcome the difficulties attendant onmechanical isolation of single bacterial cells have shown thatthese can be successfully conquered only when the entire processis made automatic. Any apparatus designed for such isolationsmust take into account the use of limited checked movement dis-tances as well as all accessory details. For instance, my appa-ratus of 1932, while undoubtedly simplifying the procedure involved in single cell isolation, lacks refinement for specializedwork. It wants certain accessories necessary for routine work,and for unifying the entire technique. So essential an accessoryas, for example, an enclosed moist chamber is lacking. As aconsequence, environmental factors cannot be controlled.The machine here described is designed for handling small par-

ticles, and incorporates all the essential features demanded byroutine work. It is so constructed that, once it is adjusted, theaction is initiated by the mere push of a button. The entireaction is automatic.A moist chamber, enclosed at all times even during the changing

of a pipette, and warmed by electricity, effects the exact controlof moisture and temperature. It is so constructed that it maybe removed and its contents incubated while another moistchamber is substituted in its place. This construction allowsthe closest possible control of environmental conditions and per-mits a number of separate experiments to be carried on and ob-served at the same time. Other details, such as the making andmounting of pipettes, have been considered with an eye to facili-tating the technique.The manipulation of this compact apparatus requires no par-

ticular skill on the part of the operator, other than the ability torecognize an isolated cell. The simplicity of the apparatus as well

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s'I'UDIES IN MICRURGICAL 'I'TECHNI('E

as the rapidity and ease of its operation should recommeind itsuse to technicians in genleral.

In the construction of an inst-rument for specialized single cellisolation, the following details demand careful consider-ation: (1)The change of pipettes, (2) the attachment of the pipette to theadaptoi, (3) the making of uniform pipettes, (4) the changing ofcover slips, (5) the enclosure and heating of the moist chamber,(6) the removal of the cell to culture, (7) the preparation of isola-tion surfaces, (S) the lighting of the isolation droplet, (9) theadjusting devices which center the tip of the pipette in the fieldunder the high powers of the microscope, (10) the r-aising of the tipof the pipette into contact with the isolation surface, (11) theformation of micro-droplets in which the cells are isolated.

Descriptlololn the apparatutsl'or1 convenience in desciiption the apparatus is divided into

these parts: (1) The housing which caiiies the moist chambercradle, (2) the moist chamber cradle, (3) the moist chamber, (4)the pipette system, ('5) the stage upon which the isolator is moved,(6) the pipette adapt,ors, (7) heating the moist chamber, (S)mechanical action of the isolator.

1. The cradle housing. This housing (fig. 2, diagram 1B) isbuilt in the form of a box with sides one-half as high as the endsand without roof or floor. A partition is built 2 inch in from eachend and acts as a support to the side walls as well as a guide for amoving block. This block is 2 inch thick and is as high as thepartitions but {- inch less in width. It is made to cairry the pipettesystem and to act as one of three centering devices for adjustingthe pipette tip. rMovement is imparted to the block by a leadscrew anchored to the side wall of the cradle. To insure accu-racy of movement the block is guided in a straight line by foulpins, two on each side of the block.

2. T'he mtioist chamlber cradle. The cradle (fig. 2, diagiam B3) isbuilt to move from side-to-side, inside the housing. It is coIn-structed in the form of a box and incorporates a replaceable Imioistchamber as well as two pipette-changing chambers. T'he moistchamber fits into the center of the cradle which is cut out to

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J( . ART'H It ItEYN IERIS

adn111iit it. he Ithetiioist (haiIlr)ei isin place t\\() cIlIrtmiienit s

a11e formued oi either side of it. these comnpartments are roofedwsith small, closely fitting, trap doors wN-hich openi upward. rheen'(1s (f the ca(llade are. cutt (m)i1t to adhlmit I he pipette holder. The

F I G. 2. (A) ENSEIBLE \VIEW (F SPCI('ALLZED) If.sLA.roi; (11) DET.\i, 0' .\loinv,LD)iagram A. Shows a lahb(ratOry Imo(del fr(I i which the insullatim inhas heen

removed t O shO(oloStruost iIl.D)iagram 1B. 1, riolist chainer taken aplart to show ininer and outer shell; 2

moi.st chainher (ra(dle; 3, mloist ('eh.am1Iher housingr and pipette system; 4, stage ofisolator.

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flBoor of the cradle extenlds out oIn either eni(l an11(l wheni the cradleis in place pass under the end walls of the housing. This cradlemust be accurately machined to fit the inside of the housing sothe sliding fit is aiIr tight but smooth in action.

3. The itioist chamtiber. The moist chambers may be built intwo fornms, i.e., square oIr round. The box type of chamber fitssmnoothly into the ways cut for it in the inside of the moist chan-ber cradle. It is as high as the criadle but one-third as long. Theends are cut out to adnmit the pipette holders. 'T'hese openingsmay be closed off l)y sliding doors which fit into grooves cut inthe sides of the inoist chamber. In an earlier model of the iso-lator these doois were mande to operate from the top instead offronm the sides of the chamber but proved to be unsatisfactoryw-hen operated in this manner. The floor of the chambei iscovered with mica which is fastened into place by a frame screwedtight to the bottom of the chamber. 'T'he roof of the chamber,which is in most instances also the isolation surface, is also heldin place by a frame. T'wo pipes which screw into the side wallsof the chamber can be used as gas inlet, anid outlet pipes.

T'he cylinder type of moist chamber consists of two pieces ofbrass tubing, one fitting snugly inside the other. Slots are cutthiough both cylinders. The inner cylinder is a, bit loniger thanthe outei cylinder and the shoulder formed by this constiuctionj)erinits the moist chambei to be fitted into the bottom of thecradle. In oIdei to close the moist chamber it is necessary torevolve the outer cylinder until the slots are covered. The roofand flooi of the chamber are hel(d in place by a cap which fitssnugly over the shouldeIrs formed oIn the top and bottomn of thec.ylinder. Pipes for gas control may also be built inlto thechamber.

4'. PIiette system. The pipette system consissts of a centeringinechanism anid a, lever for moving the pipette tip to a point in asingle checke(d motion. Two types of pipette systems are shownin the model pictured in figure 3. One is based on a ball-anid-socket principle and moves the pipette tip in anl arc. The oppo-site movement works in straight lines. The ball-and-socket typeof movement has proved to be unsatisfactoiry because of the

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21J. A1RTHURt1{EYNIERS

complexity of its mechanmism wN-heII it i.s prop)e(rly Correcte(t. r)rthis reasoni it will Iot be (describ)ed in this paper.The straight liiie type of movement system (fig. 1) is used oIn

b)oth sides of the isolator. Trhis sv,stem is built inlto the enid( ofthe housing by drilling aI ciinder hole in its middle. Theupper end(I of the hole is threcaded to take a hollow screw. Theluiimen1 of the screw is threaded ml aidmnits -Ia second, smaller a11nd

Fi. 3. ENiSEEINI L, LEW ()1' 1,Ami3t.vIr()n-lIlY\ ,i ,( -i'llESIEI AS IZIIXAED SI NGILE CEL.LI .sO 1,ATOIt

loilgerI, hollow screw wiwhich is htte(l a plunger mod. The seeommc (lhollow screw presses against at cylinder beat'ring wN-hich in tUrInI is

)ressed atgainst a sprinlg. A piece of tub)ing is plassed( throulgth theceniter- of this be.-tring. In order to cover the elong'ated slot wN-hichmust be cUt through the wN-atlls of the cylinder hole to peimit miove-mnent of the pipette sleeve, a flat plalte is fasteniedl to this sleeve.(Guides .are falstened to the outside of the end wall to keep this

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plate close to its surface and to form an air-tight joint. Thecover plate and the guides are made of steel so that the closestpossible lapped fit may be made. If the guides are shimmed upto compensate for any uneveness in the sliding fit by tightening orloosening the screws which hold them in place. This arrange-ment also allows the guides to act as gibs to the cover plate.The up-and-down movement is initiated by a lever which is

fastened to the plunger rod and permits this rod to be easily andquickly worked. A clamping post, fastened to the stage of theisolator, holds the pipette holder in place. This post is a small,hollow, cylinder, open at the top and having three elongated slotscut in its walls. Its base is square and fits between two guidebars so that the clamping post can be moved from front-to-backon the stage. It can be clamped to the stage by means of a setscrew. A ball is fitted into the cylinder and acts as a bearing.This ball is bored out to permit the pipette holder to be passedthrough it and is also fitted with a set screw for clamping thepipette holder in place. A specially made nut is screwed over theend of the holder tubing and presses against the clamping post.The action of this system takes place in straight lines. The

adjusting screw for the up-and-down movement is in the end wallof the housing. The front-to-back movement is made with thelead screw in the side wall of the housing. The side-to-side move-ment is accomplished by moving the nut on the pipette holdertubing. These three adjustments supply the necessary centeringmotions. The mechanism for raising the pipette tip into contactwith the cover glass is a plunger rod which is worked by a lever andfits through the up-and-down adjusting screw.

5. The stage. The stage (fig. 2, diagram B) is made of twobrass plates which are bolted together. The upper plate acts asthe surface upon which the isolator is moved and must be per-fectly smooth. A hole is cut in the center of the stage to admitlight. The moist chamber passes directly over this hole. Sincethe housing must be moved in one direction only, side-to-side,two-tongued, guide bars are placed parallel to each other andthe exact width of the housing, apart. The tongued part of theguides fit into slots milled into the side walls of the housing.

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The stage should be made interchangeable with the one usuallyfound on a microscope.

6. Pipette nounting.-The pipettes used with this isolator haveshort tapered poinits. Trhev are nmade mechanically on an elec-trically heated pipette puller to a uniform size. Because suchexact mechainical requirements aIe necessary to this apparatusthe usual manner of mountiing the pipettes cannot be satisfac-torily used.The adaptor on which the pipette is mounted is made from a

piece of 16-gauge hypodeermic wire beent to a modified V shiape

511P34

FIG. 4. (A) PIPETTE ADAPTO1; (B) DETAIL OF ADAPTORt SHONVING I'IPETTESEALED IN PLACE

Diagiram 13. 1, pipette wall; 2, 27 gauge hypodermic wire; 3, vallseline or paraf-fin seal; 4, wvall of 18 gauge hyp)odermic w-ire; 5, solder.

(fig. 4, diagram A). One arm of the 11 is bent to fit into a threadedcap which screws on the tube-like pipette holder. The otherarm of the U is cut off short and a piece of 27-gauge hypodermicis soldered into it so that -8 inch protrudes from the larger tubing.The hypodermic wire is easily bent to shape over a Bunsen flame.A number of these adaptors may be made and sterilized in apetri dish.

Specially constructed forceps may be used for handling thepipettes more conveniently. These forceps are curved at thetips and have two pivoted, grooved, jaws fastened between them.A clamping device is incorporated in the forceps which when setwill hold the pipette in place without pressure from the fingers.

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A pipette may be mounted in the following manner: It is cutto length (- inch) and placed between the grooved and pivotedjaws in the forceps. The clamp on the forceps is set to hold thepipette firmly. A bit of vaseline or melted paraffin is touchedto the base of the fine needle in the U-shaped adaptor. Thepipette based is warmed slightly and placed over this fine needle.The sealing substance at its base runs up a short way into thepipette, sealing it very effectually without danger of contamiiia-tion either by bacteria or by the substances in the seal used.

7. Heating the moist chamber. Heating is accomplished elec-trically by means of coils placed between the two brass plateswhich make up the stage. Since the correct winding of thesecoils, together with a certain amount of difficulty in adjusting thecoils to the apparatus, is a matter for one skilled in such work,it is best to have the apparatus wired at some electrical supplyhouse.

Coils, used for this purpose, are wound on sheets of mica orother insulator. Temperature is controlled by a small thermo-stat such as is used on microscopic warming chambers and whichis mounted on the upper surface of the stage. The exposed sur-faces of the stage and moist chamber are insulated with thinstrips of cork. With this arrangement the temperature canl becontrolled to within 0.50 to 0.75°C. It has been suggested to methat a more accurate control of temperature could be obtained ifthe heating units were wound directly around the moist chamberor its housing. The method for heating the moist chamber asused in my present apparatus dissipates too much heat by unevenheating of the brass parts and, since it is practically impossibleto make many of the moving parts from copper, the temperaturecannot be controlled accurately enough for the closest work.

8. Mechanical action of the isolator. Movement, other thanthat in a vertical plane, is supplied to the isolator by a mechanicalstage. The arms of such a stage are fitted closely to the exten-sions which form a part of the base or floor of the moist chambercradle. Any movement of the stage from side-to-side moves thehousing and the moist chamber in a straight line. A movementfrom front-to-back moves only the moist chamber cradle and

JOURNAL, OF BACTERIOLOGY, VOL. XXVI, NO. 3

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allows the housing to remain stationary. The action is basicallythat of a crOss slide principle. Since the pipette holders are heldstationary 1y the clamping posts n0o movement results in the pi-pette tip when the housing is moved. Because the pipette h1old(eris fixed in the housing which does not move when the cradle isworked, the pipette tip is again held immobile. The mnechanicalstage which imparts suclh movements may be worked by a rachetand pawl if accurate spacing of the moist chamber is desirable.Otlher motionis imparted to the pipette tip by the centerinig aindraising devices have already been described elsewhere in thispaper. It should be needless t,o say tlat the satisfact,ion obtainiedfroimi anl instrument of this type is directly proportionial to thecare with which it is made.

III. AN APPARATUS FOR MECHANICALLY CONSTRUCTING GLASS

MICRO-PIPETTES AND CAPILLARY GLASS TUBINXG

Glass micro-pipettes with openings of five micra or less havebeen used successfully for the isolation of single bacterial cells.They have also proved to be satisfactory instrument.s for handlingsmall droplets of culture containing the bacterial cell, when em-ployed in a specialized isolator such as that described above.These pipettes are generally made by hand over a micro-flame aprocedure that requires a certain degree of skill ancd which is,therefore, an objection to mechaniical single cell isolation. Anyone can, with some practice, turn out pipettes by hand. Butthese products will always v-ary in size at the tip so that only alimited number of pipettes are usable at all. Hand-made pipettesfor this reason are useless in specialized isolation where mechanicalconditions are constant. A pipette for use in a specialized iso-lator must be straight, the tip must be in line with the shaft, thesize of the lumen must to some extent be predetermined, and thetip opening must be smooth. Furthermore, if mechanical siiglecell isolation is to become a routine procedure the making of isola-tion pipettes or needles must become mechanical so that theirmanufacture will not demand too much skill or attention on thepart of the operator.

In this paper is presented a simple and inexpensive apparatus

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for mechanically makinig micro-pipettes, needles, an(d capillarytubing of glass. The apparatus is easily controlled and, if pIrop-erly regulated, will turn out pipettes of any desired diameter andshape. While the apparatus is intended for specialized isolationwith the instrument described in this paper, it can be successfullyused in other micrurgical procedures where fiine points -are desiired.If it is built largei and with specially constructed heating unlitsit can be used for imaking capillary tubing.The literature on miciurgy, so far as I know, records but three

published attempts for dlrawing isolation pipettes mechanically.One method is described by C(habry (1887), another by S. L.Schoutens (1907), and a thiid by DuBois (1931). But, whilethese instruments seem to be satisfactory for certain purposes theyare either too difficult to manipulate, or too complex of construc-tion, or not accurately controllable.The manufacture of micro-pipettes mechanically involves a

considertation of these factors: (a) The source of heat, (b) thearea of glass wall heated, (c) the diamneter of the glass tubing fromwhich the pipette is to be made, (d) the distance through whichthe pulling force acts, (e) the strength of the pull exerted, and (f')the length of time for which the healt is allowed to act before thepull is started.

In the apparatus described in this paper the source of heat is asmall platinum oIr nichIome wire coil. The area of glass wallheated depends on the temperatuie of the coil and the strength ofthe pull. The temperature of the coil is controlled by a rheostatand the timing of the pull by the amount of heat and weight ap-plied. An apparatus such as is here described is designed to pullcapillary tubing not greateI than 10 mm. in outside diameter.By using a different set of clamping jaws and making the appa-ratus larger it can be used for drawing out any size glass tubingor rod.

Description of a mnechanmical p)ipette pullerThe apparatus described in this paper (figs. 6 and 7) consists of

a frame made from a siingle length of cold-rolled steel on whichtwo arms of brass are fixed with a heating element between them.

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The upper armiii, designated as A (fig.support rod WNhile thef lower ar.BB.the rod.

7 ), is fixed to one end of the(aII1 b)e moved1Up-l-(lddoII

A B C D E

FIG. 5. I)VVEiRENT lTYPEs o 100 tETTF:-10IF(PI1TT( I)P()N \IE(HANLIOAL P10TLiJtloow heat, light 1)u111, (ItO11 (,il ; B, slow heat, li'ghit p)uill, t \v() tll'II 'c()'il

C, Show\ hea t IIghIIt puIIi, ooie1 toII 1. ('()il, pil)pet tt' IIIaa(le iII t \w( p0 t11Is; I), hIoI}h t'.lt,hea-v weig(hlt oneic turnl 0(cil; E. s -owheat ioe(oliouo weI'ight one tuirn c( oil.

F1(G. Gi. SHiO WINOIAGiLAORATORII NIM)ELni `01 1WtI'WllIATiCNT11i 1,i I'lA-kMENT11 SE1) '1'() tI1)1'i()tI 'l1T Ii I IEATING'O CO II,

The suppmoting ro(d has .t groove milled aloig its enlltire lengthand is fitteI MltO a hrass p)late which is ulse(l to Illounlt the instrI-

ment to the desk to)p. The fixed aIrm, A, hals aI jVeweler' S Pi1-NiSefitte(l inito otIe (11(1 of it. These piil-Vis(es are little thiee-ijawe(d

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chucks that jewelers or machinists use to hold small brooches,files, or fine wires. The jaws are opened or closed by screwing a

5

.I

8

FIG. 7. DIAGRAMMATIC ENSEMBLE VIEW OF MIECHANICAL PIPETTE PULLER1, stationery pin-vise; 2, capillary glass tubing; 3, heating coil; 4, pin-vise; 5,

check collar; 6, pulling arm; 7, adjustable collar to regulate the pull distance; 8,weights; 9, stationery arm A; 10, support rod; 11, adjusting screw for heating coil;12, adjustable arm B; 13, set screw; 14, set screw for coil centering mechanism;15, base attached to table top.

tapered collar over them. Usually a vise of this nature has tohave a hole drilled through it in order that a long length of glass

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capillary may be fed into it from the back end, and clamped atdifferent lengths.The movable arm, B, is a bar of brass the same size as A, but

it can be moved up and down the supporting rod and clampedat different distances on it. One end of the bar is drilled to fitthe supporting rod and a small stud of steel, the exact thickness ofthe groove in the supporting rod and as wide as the groove isdeep, is fastened to the inside. This stud keeps the moving armin line with the stationary arm. A set screw is fixed on one sideof the moving arm to hold it in place by pressing against the sup-port rod. The pulling arm, which slides through one end of arm,B, is made from a piece of steel rod and has a hanger plate screwedto its end. This arm should slide through a polished steel orbrass bushing fixed in arm, B.One end of the pulling arm is drilled out and a pin-vise fixed

in place with a set screw. To check the movement of the arm amovable collar is placed on the rod between the pin vise and theupper surface of arm B. Another wider set collar is placed on thepull rod between the weights and the under surface of the sta-tionary arm. The pulling arm may be graduated. When thelarger collar is fixed a certain distance below the arm B, thepulling distance is regulated and the collar allows it to be accu-rately checked.The weights used on the pulling arm are made in the form of

disks. When several weights are hung on the arm there is a tend-ency towards jarring when the arm is checked suddenly. Toprevent this jarring from breaking the capillary tip a set collarmay be made to press against the disks.

It is important that the centers of the two pin-vises be exactlyin line with each other and that the pulling arm move in a straightline. For timing the pull it is likewise important to have thepulling arm slide smoothly and evenly through the bushing.Pin-vises have been found the only satisfactory means of holdingthe capillary tubing from slipping. If these vises are accuratelymade they will hold, safely and securely, glass tubing as fine asa hair.

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Heating element

Gas micro-flames are useless in this work because they cannotbe accurately regulated. Since heat must be applied equally allaround the glass capillary tubing in order that the point be inline with the shaft, a small platinum or nichrome wire coil wassubstituted for the gas flame. The coil can be made from anygauge wire but I have found an 18-gauge most satisfactory.These coils are made by winding the wire around an 8 inch rod.Coils of a smaller diameter are unsatisfactory because the capil-lary tubing tends to touch the sides of the coil and fuse with it.For all practical purposes a coil of more than one turn is seldomnecessary.Two types of mounting are shown in this paper. The one made

from an electric light filament (fig. 6) can be easily made in thelaboratory but is not quite so accurately mounted as that shownin figure 7.

If an experimenter desires to construct his own elements hemay do so by winding the coils on a rod and mounting them inthe elements of an electric light bulb of the long necked type.The glass is carefully cracked away and the filaments as well

as the glass rod supporting the filaments are removed. Thisleaves two supporting wires which are fused into a glass tube andonto a brass thread base with which the bulb is screwed into alight socket. The coil is mounted across the supporting wiresby bending the wire and pinching it tight. Several such basesmay be prepared with coils varying in diameter and in numberof turns.When used as a heating element the bulb is screwed into an

electric light socket supported by an iron stand (fig. 6). Thebase of this stand is fastened to the table top near the pullingapparatus and the coil centered between arms A and B and inline with the center of the pulling vises. A rheostat is shuntedinto the electric line between the coil and the current outlet, and aspring switch is used to make or break the circuit. This arrange-ment permits control of the temperature as well as the timethrough which the heat is allowed to act. Since the tiny coil

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cools quickly once the current is switched off the pull may heaccurately controlled.The coil mounting shown in figure 7 is more accurately made

and more easily adjusted. It has the further advantage of allow-ing the coil to be changed quickly. The wire coil is sealed intopieces of glass tubing and fastened into a fiber base which hastwo brass pronges in it. This fiber base is plugged into a socketwhich fits on the support rod and which is adjustable by a screwin one direction and by swinging it from right to left in the othernecessary directions.

Shapes of isolation pipettesThe shape of an isolation pipette is determined to some extent

by the purpose for which it is intended. Figure 5 shows a seriesof pipettes designed for micrurgical work. For single cell isola-tion and particularly with specialized apparatus, it is necessaryto hav\e the point of the pipette taper to its smallest diameter ina very short distance from the capillary tubing in order to conservespace and to give ridigity to the pipette. Generally speaking,these short pipettes are also satisfactory for other micrurgicalwork unless the mass to be penetrated is relatively large andrequires a longer shank. The opening or mouth of the pipettemust be smooth and not jagged, in order that the forces of capil-larity may be utilized to the best advantage in forming the iso-lation droplets.

This apparatus can be regulated so that it will turn out pipettesof any desired shape, i.e., with a longer or shorter shank and apoint which may taper out or be abrupt. The apparatus isusually set to make a pipette with a single pull. A pipette madein a single pull and with a "slow temperature" will take the shiapeshown as E in figure 5. The end will be fire polished and squaredwith the shaft, or it will be capped with a minute cone of fusedglass, dependinig entirely upon the choice of the operator. Theopening can, of course, be regulated by adjusting the pull distanceand weight. A pipette answering this description meets all therequirements of single cell isolation and most of the requiremenltsof cell injection. Pipettes of a very large diameter when made

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on this machine will not, of course, pait in the center. Such pi-pettes can be broken satisfactorily by nickiing the sides of thecapillary tubing with a very fine oil stone. One investigator,A. Belkin (1928), has devised a micro-guillotine to cut capillariessquarely. This could possibly be used to advantage for thispurpose.

U18e o/J t/le appa ratts1/811 itiali-i isolat/on pipettesCapillary tubing is made by dIrawing out pieces of laiger glass

tubing over a wing-top Bunsen flame. Since the lumens of thesecapillaries run in ratio to their outside diameters and the outsidediameters of the larger tubing it is easy to select tubing of a cer-tain size by passing it though a wire gauge plate. Graded capil-lary tubing may be kept on hand by storing it in longer pieces ofglass tubing well stoppered. Capillary tubing made by handover a B1unsen flame has one disadvantage in that it tapers andis not of a constant diameter for any length. A more satisfactorytubing can be made with a large model pipette puller of the samedesign as the smaller model described in this article. However,by the use of a gaIuge plate it is fairly easy to select pieces of almostthe same diameter. In making the pipette the following stepsare necessary:

(1) h'lie set scriew in arIn B is loosenedl an(d the arm plishedl up thesupporting rod to within 2 inches of armi A.

(2) The heating coil is lined up with the centers of both pin visesand at an equal distance between theim.

(3) A piece of capillary tubing is selectedI an(d is pushed throughthe fixed pin-vise in arm A and into the pin-vise on the pul-ling arIn. T'he pin-vise in the pulling armi is tightened.

(4) T'he pull distance is pre-determined and the large collar under-neath arm B is moved down the pulling rod until it is atthe required distance when it is fixed in place.

(5) The set screw in arm B is loosened and the arm moved againstthe previously set collar.

(6) Current is turned into the coil land is shut oft as soon as thepulling arm starts to move freely. If the pipette is made inone pull and the factors of weight and distance have beenaccurately calculated the result will be two pipettes. The

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pipette in the pullingyIarni xNiii have aI lon(gel.shank thanlI theone in the fixe(I a1l111.

( B)B)t h I)ii)pettes a.11e remolve(ld (nd)brokell (o to the proper lethls'isb)V t()llChillr the si(le of the basew'-itlh a verv finle (grade ()iISt (on1e'.

IIG. X. (A) MIC1R()-P1IOT()GRIA 111 (OF IIPIETTE .\I A O)E( (N TlE, MIE(CHANIAAL.PIPETTE PULiiEiiR, X 50(); (B) Tiie ()I PIPETTE SHOWN IN (A), X 120()

I)iagran A. INot,eettetip filled by eapillalitV shon as a light area at tip. N(tsvn.Iniel(t 1rv.

Diagram 1B. 1)Dve fle(l ti1) of plipette shownIts daIrk area.

Ict(lor.s to be CoW/(!rer(I i'/i m/(I/iQ capllar/)Ill' I)iIWt1C.s

Figure (S showN-s the tVpe of pipette male wNith thi.s mIachillne.i\laking these pipettes is largely a inatter of exl)erience in knowingt,he results thait miaty be expected fron a1 number ()f coitributing

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factors. The following short discussion may be of assistance tothe operator.The lumen diameter at the tip of an isolation pipette is depend-

enit on these factors: (a) The weight or force of the pull, (b) thelength of the pull distance, (c) the degree of heat supplied fromthe coil.These factors vary with the size of the capillary tubing. An

increased wall thickness, for instance, will require an increasein weight of pull and the time through which it acts if the heat iskept constant. Timing is an important factor in determining thesize and shape of the capillary. With this instrument the timingis automatically regulated by the force of the pull which actsconstantly for the same degree of heat and the same size tubing.If the heat is kept constant, the timing of the pull will vary withdifferent sizes of capillary tubing. If the weight is increased inproportion to the diameter of the capillary tubing the heat mayremain the same. The more weight added for the same heat, thequicker will the pull act and the more needle like and pointedwill be the resulting capillary. If the weight is increased toomuch the pipette breaks before it forms into a point.

It is best for practical purposes to keep the heat "slow" forany weight. If the heat is "high" and the weight light the resultis a pipette which does not point but tends to draw out to a hairwith very fine walls. The same is true if the heat is high and theweight heavy since the tendency is to draw out the glass into ahair-like process. This tendency is readily explained when oneconsiders that the capillary tubing starts to soften from the out-side toward the lumen in the form of a cone with the apex towardthe lumen and that the acting weight tends to taper the capillarytubing from the outside inward giving a decreasing wall thicknessand shape as shown in figure 8. If the heat is high the apex ofthe cone is wider and the tendency is a more gradual decrease inlumen diameter and outside diameter. The result is a hair-likeprocess.The pull distance is a factor in drawing capillary tubing. By

setting the distance so that the capillary does not have a chanceto point, any size capillary tubing can be obtained. Thus in

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making a pipette with a long shank and a short point the pulldistanice is set short, the fiiner part of the capillary is formed,the coil moved to the ceniter of the finer capillary and the poinltdrawn on. B3y propeirly regulating pull distance and keepinig thefactors of heat, weiglht, aind capillary tubing constant, pipetteswith a conlstant, size opening may be made anid the result predicted.A glance at the table of actual settings and results may giV(e

the reader a better understaniding of the problem.

TABLE 1Mlechanically alade micro-pipettes

LENGTH OF P'IPETTE DIAM1ETER CAPILLARY TUBINGWEIGHT OF DISTANCE RHEOSTAT PIIPETTE ___

PULL OF PULL SETTING SHANK Inner Outer IIloier Outer

gratms 0 1. 011. 71 M1. 711101. /1 ti.

226.1 7 mm. 5. 5 4 0.012 0.023 0.710 1.077226.1 8 mm. 5.5 3.5 * 0.0007 0.703 1.074226.1 5 mm. 5.5 30 0.0958 0.071 0.617 0.915226.1 1 cm. 5.5 3a0 * 0.0007 0.718 1.(8226.1 5 mm. 5.5 3.0 0.062 0.085 0.711 1.09246.5 1 cm. 5.5 2 5 * * 0.585 0.762246.5 1 cm. 4.2 Did not part to a point 0.607 0.904178.9 1 cm. 5.5 2.0 * 0.0007 0.722 1. 1113.5 7 mm. 5.5 4.0 0.021 0. 035 0.681 1. 0(52.5 1 cm. 4.0 0.1 0.0004 0.001 0.426 0.639

* Indicate pipette openings too small to bie measureed. All pipettes wsere o)enat the end and could l)e filled l)y capillarity. The measuremenits givein in table 1were selected at random from experimental data obtained from many trials. Thecoil used throughout these experiments was made from 20 gauge nichrome wirewound once around a rod -8 inch in diameter. The lowN-er number rheostat readingsindicate a higher degree of heat.

IV. THE TECIINIQUE OF ROUTINE SINGLE CELL ISOLATION

AND CULTURE

Specialized mechanical single cell isolation and culture can beadopted to many problems in bacteriology where a pure strain ofbacteria is desirable. The various steps for accurately carryingout such isolations are not the same in all problems. They varyslightly with the material from whicll the isolation is made as wellas with the organism which is to be isolated. In order to appre-ciate the fullest possibilities of this technique, its basic factors

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must be considered. Accordingly, then, it is the purpose of thispaper briefly to consider some of these factors as well as todescribe the use of the specialized apparatus.These factors are concerned with single cell isolation and cul-

ture: (1) The moisture conditions within the moist chamber, (2)droplet formation and preparation of the isolation surface, (3)the lighting and definition of the isolated bacterial cell, (4) thepreparation of the isolation medium.

The noist chamberThe purpose of a moist chamber is to prevent evaporation

changes from taking place in the isolation droplet. To effect thispurpose the interior of the chamber must be saturated withmoisture. Saturation depends on the temperature of the chamberand upon its being entirely enclosed. Any opening in the cham-ber admits the possibilities of changes in the interior. Successfulisolation depends, in many instances, on the prevention of anychange in the isolation droplet no matter how small. There are,however, several objections to enclosing a moist chamber entirelyfor any length of time. For instance, when a grease film is usedto form the isolation droplets, as well as to collect the moistureof the chamber as a dew, there is a tendency toward the dilutionof the isolation droplet followed by a certain amount of spreadingand loss of outline. This condition is rectified by the use of a newtype of isolation surface and with it a construction permittingenclosure and a regulated temperature, can be successfully in-cluded in the design of the specialized isolator.

Saturation may be supplied to the chamber from pads of porousmaterial previously saturated, from agar barriers or droplets(Barber, 1914), or from pads of unglazed porous porcelain(Peterfi, 1927). The material used makes little difference in theresults obtained although the porcelain plates are more satisfac-tory because they can be mounted and handled more easily.

Isolation surfacesThe type of isolation surface used, varies with the source of

material and the purpose of the isolation. Generally speaking

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it must be: (1) Perfectly flat, (2) free from debris, (3) thin andtransparent, (4) easily prepared in a routine fashion, (5) able tomaintain the isolation droplet without permiitting it to spread,(6) able to support, the isolation droplet in a flattened state.These requiremenits can be more or less satisfactorily met by

spreading a thin film of oil on a cover glass. A new type of isola-tion surface which I have invented, forms an exception to thisin that it is opaque and utilizes no surface film. The successfulroutine use of the "film" surfaces depends to a great extent on aproper attention to details of depositing the film. Both the filmsurfaces and the newer "cell" surfaces have their uses and neithermethod can replace the other for certain work. Furthermore,several types of isolation surfaces may be advantageously com-bined to serve a special need. The failure in the past to specializethe surface to the work and to combine advantages of a non-mechanical with those of a more mechanical nature accounts to acertain extent for the failure to use single cell methods morewidely.

Isolation films may be prepared from grease, oil, agar, collodion,or some substance non-miscible N-ith water. A film may be putdown by rubbing, by flowing the material over the surface of acover glass, or by depositing it from a solution. The details ofcleaning cover glasses and of applying a grease film are wl-elldescribed by Gee and Hunt (1928). It should be rememberedthat throughout such a preparation the materials used must befree from dust or dirt. If v-aseline is used for the purpose it isbest to buy it in metal tubes because such a receptacle preventsthe contents from becoming soiled.The usual grease film is put on a cover glass by polishing off

the excess grease. To polish and distribute the film I use a motordriven polisher made from a small toy motor. The polishing diskis mounted on the motor shaft so that it revolves parallel to thetable top. A base of washed chamois skin is drawn tightly overthe disk and a wiping surface of two or three thicknesses ofwashed ciepe silk is stretched over this base. AMetal bands orstring may be used to bind the wipers in place. The wipingsurface must be free from w-rinkles and tightly stretched. It

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must be changed frequently and when not in use can be coveredwith a petri dish to protect it from dust.A grease film is put on a cleaned cover slip as follows: The

cover glass is removed from the storage jar and is wiped drybetween several layers of grease-free, washed, silk. The coverslip is laid flat on the silk and a bit of vaseline is touched to itssurface with a dauber, made by winding several layers of silkaround the end of a swab stick. If there is an excess of grease it isremoved with a clean dauber. The cover slip is polished by hold-ing it film side down on the polishing disk. It is held in placewith the fingers which are covered with several layers of silk.After it is polished the cover glass is flamed, film side down, overa Bunsen flame. Flaming must be done quickly so that thegrease has no time to be burnt or crystallized. Isolation surfacesprepared in this manner are quite uniform.Agar films. The chief advantages of an agar film are the pro-

tection it affords an isolated cell, the arresting of motility, theopportunity it offers to cultivate a cell in situ, and the ease withwhich the material containing the cells can be spread over itssurface. The disadvantages are the difficulty of obtaining a per-fectly flat surface, removal of the isolated cell to culture apartfrom the isolation surface, the preparation of an agar which isgranule free, and the difficulty of obtaining films thin enough tobe practical. Furthermore it is difficult not to mar the surfaceof the film wlhen the culture is spread upon it.The agar used for this purpose may be made as a saline agar

or it may contain a nutrient. In either case the agar which is tobe used for making isolation films should contain 40 grams of dryagar shreds per liter. The film is made by allowing agar to flowevenly over the surface of a clean cover glass from a pipette heldperpendicular to the glass surface. The thickness of the film isregulated by drawing the excess agar back into the pipette.

Isolation cell surfaces. For want of a better name the term"isolation cell surface" is used tentatively to designate a newtype of metal isolation surface in which a series of very fine holesis drilled. These surfaces were devised so that the size of theisolation droplet could be limited and better optical conditions

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might be obtained. Experience has taught that a mere film orflattened droplet provides better optical conditions for observinga single isolated bacterial cell, than a deep, hemispherical droplet.These flattened droplets are hard to maintain on the usual type

of isolation surface. Furthermore, in making an isolation drop-let of this nature the cell is often exposed and in many instancesharmed. Trhe problem has been satisfactorily solved by the useof a new type of isolation surface which differs in principles fromany heretofore used and which by nature of its automatic isola-tion droplet formation is well adapted to routine.

Such a surface may be made from a very fine metal gauze (200or more meshes to an inch) or from a platinum plate perforatedwith holes 0.002 of an inch or less in diameter and spaced as theoperator wishes.

If wire gauze is used a small piece must be run between steelrollers to flatten the wire and partially close up the openings.The gause is then drawn tight over a frame and is soldered in place.A more satisfactory type of "cell isolation surface" may be

made from platinum or gold or some metal which will cause aminimum of harm to the cells and be stiff enough to maintain itsshape. The thickness of the plate may range from 0.01 to 0.005inch or thinner. Its size is determined by the opening in themoist chamber roof. A series of holes is drilled in this plate withMarple wire drills. These holes may vary in diameter from0.002 to 0.005 of an inch and must be free from burr. The sur-face of the plate is buffered to a high polish. Unless the operatoris familiar with fine tools, time and expense is saved by having theplates drilled by a manufacturer of platinum articles or at thenearest competent jewelers. After the plate has been made itmay be rolled again between steel rollers to flatten it further andto partially close up the holes. For practical purposes holes lessthan 0.002 inch in diameter are seldom necessary, although bydrilling holes of this size and by carefully rolling the plate, theholes may be reduced to less than 0.0005 inch in diameter.

In order that the exposed surface of the plate be protected it iscovered with a cover glass. The glass must not touch the platebut should rest on a ledge made of a lacquer or gold size. This

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ledge must be just thick enough to support the cover slip so itcannot touch the surface of the plate. Several larger holes aredrilled just inside the ledge so that when the cover glass is inplace gas and moisture from the chamber can circulate beneath it.

Definition of the isolated cellThe source of light used in most of these experiments is a 100

watt nitrogen filled bulb behind a layer of milk glass which iscovered in turn with frosted glass. Because of the height of themoist chamber a special series of condensing lenses are used.These are mounted in a frame under the stage of the microscopeso that they can be swung into place separately. The light ispassed through a heat-absorbing glass and a red or yellow filter.The colored glass can be quickly swung aside and a more criticalwhite light substituted for the short time that it takes to examinea droplet.

Micro-droplets are slightly spherical on greased surfaces andwhile isolated bacteria may be easily defined in them there is aslight aberration and the edges of the droplet cannot be so criti-cally lighted. If the droplet is flattened slightly, as often happenswhen it merges with the collected dew, the lighting is better.The difficulty with a flattened droplet is that the edges of theoriginal droplet are soon lost or hard to make out. Peterfi(1926), in adapting single cell isolation to dark field illuminationhas taken advantage of the flattening effect by dropping piecesof glass wool on the isolation surface to flatten the droplet andprovide a satisfactory film. Other investigators advise the useof a greaseless cover glass or the use of agar films which likewisepermit a flat field to be obtained.The spherical droplet, because of its small size, is satisfactory

for isolation purposes with larger organisms but a more criticalillumination is necessary for work with smaller organisms orwith the reproductive products of certain cells. For this purposethe isolation droplet should be flat. If it can be flattened andthe cell protected from injury which would otherwise result fromexposure the desideratum is attained.When an agar surface is used the isolation droplet at the in-

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stant it is made is absorbed inlto the film of moisture found on theagar surface, the cell is deposited on the agar anid the field hasthe necessary flatness. An isolated bacterium is easily seen ona film of agar if the film is thin eniough an-d the agar free fromgiranules.The most satisfactory type of isolation droplet as far as visi-

bility is concerned is that formed in an isolation cell. Here tlhedroplet is stretched to a film the thickiness of the isolation plate,and the surface tension exerted is great enough to prevent assump-tion of a spherical surface. The edge of the droplet is clearlydefined against the opaque edges of the pore. This allows adefinite limited area to be searched.

Manner of isolating a bacteriutit withi specialized apparatutsa. Adjusting the a,ppparatus. (1) The moist pads are saturated with

distilled water delivered from an eye-dropper. (2) A temporary coverglass is fastened into place over the moist chamber. (3) Current isturned into the heating coils and the temperature of the chamber re(ru-lated by means of the thermostat. Temperature can be read directlyfrom a clinical thermometer fastened into the side wall of the moistchamber. (4) The pipette holders are pulled back into the changingchambers and the moist chamber is closed off. The trap doors closingthe changing chambers are opened and the pipette holder exposed. (5)The guide pin in the pipette hiolder sleeve is lifted and a sterilizedpipette adaptor screwed into place. (6) Pipettes are prepared on thepipette puller and are cut off into lengths of 2 inch. These pipettes areclamped into a curved pair of forceps. A daub of paraffin, vaseline orsome sealing substance is touched to the base of the fine needle in theadaptor tip. The base of the pipette is heated over a flame and ispushed over the fine needle. The wax at its base, melts and runs upinto the pipette base to furnish a seal. (7) The pipette holder on theopposite side of the isolator is treated as in steps (4), (5), (6). (8) Botlhpipettes are lowered so that the changing chamber doors can be closed.(9) The moist chamber is opened, the pipette adaptor turned sideways,a.nd the pipette is pushed into the chaiimber where it is once more re-turned to an upright position. (10) The pipette tips are centered inthe low poweer field. Front-to-back adjustment is made with the leadscrew in the side of the rnoist chamber housing. Side-to-side adjust-ment is made with the screw on the pipette holder tubing. Up-and-

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down adjustment is made with the screw which is a part of the pipetteraising system. Once the pipette tip is accurately centered with lowpower it can be further adjusted to a higher power. One side of thepipette systemn is centered at a time. When one side has been satis-factorily centered the pipette tip can be removed slightly out of fieldby loosening the set screw in the base of the clamping cylinder. Theopposite side can then be centered. Since the pipette tip is removedonly a short, set distance out of field, a quick push will return it toview. (11) With the pipette tips centered in the microscopic field theapparatus is ready for isolating the bacterial cells. Any future pipetteadjustments will be quite limited.

b. Isolating bacterial cells. Assuming that the new type of isolationsurface is to be used and the cells are in proper dilution the proceduretakes the following steps: (1) The temporary cover glass, which hasbeen placed over the moist chamber, is removed and a "cell isolationsurface," fastened in its place. (2) A loopful of diluted culture isspread over the isolation surface so that the isolation pores are filled.The excess culture is removed with the inoculating loop. (3) A cleanedcover slip may now be pressed down over the top of the isolation surfaceto protect the isolation films or the manipulations may be made withoutthe protecting cover. (4) Isolating the cells consists in bringing theisolation pores into the center of the microscopic field and examiningthe flat film for isolated bacteria. (5) When a pore is found in which asingle cell has been isolated the pipette tip is raised into contact with theisolation film and the cell collected by capillarity. It might be well tomention at this point that a micro-tip drawn from glass rod may besubstituted for the micro-pipette.

c. Removing the isolated cell to culture. When the isolated bacteriumhas been taken up in a micro-pipette the next step is to culture it. Thismay be done on another part of the isolation surface or in a test tubeapart from the isolation chamber. Assuming that the latter is the caseand the cell is to be cultured in a test tube, the procedure is as follows:(1) The pipette tip is dropped away from the isolation surface and ispulled back into the changing chamber. (2) The moist chamber isclosed off and the changing chamber exposed. (3) The pipette is re-moved from its adaptor with the curved forceps. (4) Since the isolatedcell, together with a small amount of broth, is in the extreme tip of thepipette it is only necessary to break off this tip by rubbing it against theside of a tube of broth. The broth may be washed over the spot wherethe pipette tip has been broken. (5) A new pipette is pushed over the

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J. ARTHUR REYNIERS

adaptor and is returned to the field of operation. Since the pipettesare uniformly made and mounted only very limited adjustments willhave to be made. ]klore cells can then be isolated.

The use of the single cell technique in various problemsGenerally speaking, single cell methods might be used to obtain

a pure strain from a broth or agar culture, in selecting certaincells or their products, in separating a pure strain from a mixedculture, in watching the development of an isolated bacteriummicroscopically, in microbiological tests, and finally, in the selec-tion of certain protozoon forms.

Pure strains of bacteria obtained from single cells isolated outof a broth or agar culture are useful in problems of heredity, inthe preparation of antigens, the study of dissociation or in anyproblem where the pedigree of an organism is desirable. Withthese methods it is possible to select bacteria from tissues orexudates and to cultivate them as pure cultures with a consequentsaving in time in many cases. It is also possible to use the tech-nique in diagnostic bacteriology, a field not yet fully open tosingle cell methods. All in all, single cell methods have manyuses of a very practical nature which time and space will not per-mit outlining but with which an operator using the technique soonbecomes familiar.

SUMMARY

In this paper is presented a consideration of certain factorsconcerned with mechanizing single cell methods as well as a newtype of automatic isolation surface. This paper is an attempt tomechanize the technique of pure culture, for the purpose ofmaking it more exact, more direct, automatic in nature, andcapable of closer control. The present technique is not intendedas a substitute for plate culture methods any more than any exactand highly specialized procedure can be substituted for a moregeneral method. In certain instances, particularly in researchproblems of the nature I have already mentioned, single cellmethods are essential. The proper investigation of these prob-lems involves series of many hundreds of single cell isolations.

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Thus the special problem becomes one of routine. It is towardthis end that I have offered my methods in an attempt to systema-tize and unify mechanical single cell technique. If the newmethods are adapted to a more general use it is because the gen-eral use has become partly research in nature.The first section of this paper considers (1) the methods used

by other investigators to isolate single bacterial cells, (2) thenecessary principles to completely mechanize such a technique,(3) a review of the methods used by the writer to accomplishthis mechanization.A new micro-manipulator for mechanical isolation of single

bacterial cells is described in detail in the second section. Theinstrument embodies the following advantages: It is specializedfor work in bacteriology, and is easy to manipulate. The moistchamber is never exposed to the air, permitting an accurate con-trol over environmental conditions and, depending on the careused in the construction of the manipulator, it affords a more orless complete control over anaerobic conditions to be maintained.The moist chamber or isolation box is replaceable, permittingmany experiments to be carried on simultaneously. Pipettemounting and changing are simplified.

Specialized mechanical isolation of single bacteria demands auniform pipette made by a mechanical device. The instrumentpresented in this paper fills the requirements by automaticallyregulating the factors entering into pipette pulling and furnishesan instrument which is (1) simple in construction, (2) fool proof,(3) accurate, (4) simple to manipulate, (5) the heating elementsmay be constructed quite easily in the laboratory.

If capillary tubing of uniform diameter is used pipettes with auniform opening may be made. The apparatus will make pi-pettes with openings varying between those too small to be meas-ured with the microscope to any size chosen by the operator.

REFERENCES

AVERY, R., AND LELAND, S. 1927 Jour. Exper. Med., 45, 1003.BARBER, M. A. 1904 Jour. Kansas University Medical Society, 4, 489.BARBER, M. A. 1907 Kansas University Science Bulletin, 4, 3.BARBER, M. A. 1908 Jour. Infec. Dis., 5, 379.

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BARBER, M. A. 1911 Jour. Infec. Dis., 8, 348.BARBER, M. A. 1914 Phil. Jour. Sci.; Trop. AMied., Sec. 3, 9, 307.BARBER, M. A. 1920 Jour. Exper. Med., 32, 295.BARNARD, J. E. 1925 Brit. Jour. Exp. Path., 6, 39.BELKIN, M. 1928 Science, N. S., 68, 138.BisHop, G., AND THARLDISON, C. 1921 Amer. Naturalist, 55, 381.BRIEDIGAN, F. T., AND CHIANG, T. M. 1924 Jour. Lab. and Clin. Med., 9, 572.BROWN, W. 1914 Phytopathology, 4, 115.BURRI, R. 1907 Centr. f. Bakt., Abt. II, 20, 95.BURRI, I. 1909 Das Tuscheverfahren. Jena. Gustav Fischer.CHABIRY, H. 1887 Jour. de l'anatomie et de physiologie., 23, 167.CHAMBERS, R. 1918 Biol. Bull. Marine Biol. Lab., 34, 121.CHA'MBERS, R. 1922 Jour. Infect. Dis., 31, 334.CUNNINGHAM, B. 1922 Amer. Microseop. Soc., 45, 55.DICKINSON, S. 1926 Ann. Bot., 40, 273.DICKINSON, S. 1926 Proe. Roy. Soc. Med., 19, 1.DOTY, H. A. 1900 Jour. Applied Microscopy, 3, 991.DuBoIs, D. 1931 Science, N. S., 73, 345.DUFF, D. C. B. 1929-30 Jour. Lab. Clin. MIed., 15, 167.DUNN, F. L. 1927 Jour. Infee. Dis., 40, 383.EDGERTON, C. W. 1914 Phytopathology, 3, 115.FITZ, G. W. 1931 Science, N. S., 73, 72.GEE, A. H., AND HUNT, G. A. 1928 Jour. Bact., 16, 327.HAHN,)M., SCHUTZ, F., AND WAMOSCHER, L. 1926 Z. Ilyg. Infectionskranikh.,

106, 191.HANSEN, E. 1884 Medd. Fra. Carlsburg Lab., 2, 152.HILL, 11. W. 1902 Jour. MIed. Res., 7, N.S., 202.HECKER, F. J. 1916 Jour. Infee. Dis., 19, 306.HEWLETTE, R. W. 1918 Manual of Bacteriology, 6th edition, page 535.HOLKER, J. 1918-1919 Jour. Path. and Bact., 22, 28.HORT, E. 1919-20 Jour. Hyg., 18, 361.JOHNSON, H. 1923 Jour. Bact., 8, 573.KAHIN, M\. C. 1922 Jour. Infee. Dis., 31, 344.LEVINTLIRAL, W. 1927 Z. Hyg. Infectionskrankh., 107, 380.MALONE, R. H. 1918 Jour. Path. and Bact., 22, 222.MCCLENDON, J. F. 1907 Biol. Bull., 12, 241.MUTCH, N. 1919 Jour. Roy. M\icro. Soc., 1, 221.QRSKOV, J. 1922a Compt. Rend. Soc. Biol., 86, 221.ERIKOV, J. 1922b Jour. Bact., 7, 537.

QRSKOV, J. 1924 Centr. Bakt. Parasitenk., I A)t., Orig., 92, 312.PAINE, S. 1927 Jour. Bact., 14, 441.PETERFI, T. 1923 Die Naturwissenschaften., 6, 81.PETERFI, T. 1923 Aberhalden, E., Handbuch der hiologischen Arbeitsme-

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Technik. von Kraus und Uhlenhut, page 2471. Published by Urbanund Schwarzenberg, Berlin.

PETERFI, T. 1926 Z. Wiss. Mikroskop., 43, 186.


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PETERFI, T. 1924 Z. fur. Wissen. Mikro., 41,263.PETERFI, T. 1927 Z. fur. Wissen. Mikroskop., 44, 296.PETERFI, T., AND WAMOSCHER, L. Z. Hyg. Infectionskrankh., 106, 191.PETERFI, T. Die Technik der Zelloperationen (Mikrurgie) Method der wissen-

schaftlich. Biologie von Peterfi, Bd. 1, page 559. Published bySpringer, Berlin.

REYNIERS, J. A. 1932 Jour. Bact., 23, 183.SCHMIDT, H. D. 1859 Amer. Jour. Med. Science, N.S., 37, 2.SCIIOUTEN, S. L. 1905 Z. Wiss. Mikroskop., 22, 10.SCHOUTEN, S. L. 1907 Zeit. fur Wissen. Mikrop., 24, 258.STARIN, W. A. 1924 Jour. Infec. Dis., 34, 148.STEARN, E., AND STEARN, A. 1927 Jour. Lab. and Clin. Med., 13, 276.STIURGIS, W. 1928 Jour. Bact., 15, 20.TAYLOR, C. V. 1923-25 Univ. Calif. Pub. Zool., 26, 443.TROPLEY, W. W. C., BARNARD, J. E., AND WILSON, G. S. 1921 Jour. Hyg., 20,

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bacteriology laboratories, university of notre dame, notre ...single cell isolatilon single cell...

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