J O U R N A L OF U L T R A S T R U C T U R E R E S E A R C H 64,261-269 (1978)

A Centrifuge Head for Back-and-Forth Centrifugation to Improve Specimen Infiltration by Viscous Plastics

FRITIOF S. SJ()STRAND AND HOLMES HALMA

Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024

Received June 7, 1978

For embedding of small pieces of tissue a special centrifuge head was designed for back-and- forth centrifugation of the tissue to speed up infiltration of the tissue by a viscous plastic which is poorly miscible with the solvent used for dehydration. The centrifuge head was designed in such a way that the tube holders flip between two positions with a difference in orientation of close to 180 ° by varying the speed. This way the tissue moves back and forth in the centrifuge tube with the surfaces of the pieces of tissue being exposed continuously to virtually fresh plastic. This way a steep concentration gradient solvent-plastic is constantly maintained at the surface of the tissue. This centrifugation-infiltration procedure is also useful whenever plastics of high viscosity are used even when the plastic and the solvent used for dehydration are easily miscible.

The high viscosity of many plastics used for embedding tissues for thin sectioning imposes problems due to the slow rate of exchange between the solvent used for de- hydration and the plastic. It limits the size of the pieces of tissue that can be infiltrated in a reasonable time and makes the embed- ding procedure time-consuming and cum- bersome.

This problem becomes much worse when a solvent is used for dehydration that is very poorly miscible with the plastic.This applies to the use of ethylene glycol for dehydration combined with embedding in Vestopal. This combination was found to be particularly favorable when trying to limit the denaturation of proteins by embedding the tissues according to one of the low denaturation techniques developed by SjSstrand and Barajas (1968).

The high viscosity of certain plastics that are particularly suitable for embedding tis- sues for electron microscopy is due to the fact that the manufacturers have partially polymerized or cross-linked the plastics be- fore marketing them. When applying the low denaturation technique of SjSstrand and Barajas it is best not to use a mono- meric plastic for embedding since even wa- ter-miscible monomeric plastics are strong

261

denaturing agents. Partial polymerization or cross-linking of the components in the plastic reduces the concentration of plastic in a monomeric form and therefore is likely to reduce the denaturing action of the plas- tic. It is also desirable to remove the eth- ylene glycol as quickly as possible by speed- ing up the infiltration procedure.

The infiltration time is reduced by embedding small tissue fragments obtained by gentle homogenization of briefly cross- linked tissues and by keeping these frag- ments moving through the embedding me- dium by means of centrifugation. To allow sufficient time for exchange using this method the material must be mixed with a relatively large volume of the plastic in sealed centrifuge tubes that make it possi- ble to centrifuge the material back and forth in the tubes by frequently turning the tubes 180 °. Still the infiltration time is sev- eral hours with only a few minutes of cen- trifugation between turnings of the tubes. This makes this procedure impractical if the turning has to be done by hand.

To overcome this problem a special cen- trifuge head was constructed that made the tissue fragments move back and forth in the centrifuge tube by automatically rotat- ing the tubes 180 ° at regular intervals. This

0022-5320/78/0643-0261502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

262 SJOSTRAND

special head was designed for the Sorvall GLC centrifuge.

Although this centrifuge head was devel- oped for the embedding of material accord- ing to one of the low denaturation tech- niques used by SjSstrand and Barajas (1968) it also was found very useful in con- nection with conventional preparatory pro- cedures when a viscous plastic such as Ves- topal or Araldite is used. This seems valu- able since these plastics are those with par- ticularly excellent properties for cutting thin sections and long ribbons of serial sec- tions and the disadvantage of their high viscosity has limited their use.

RATIONALE FOR THE METHOD

The rate at which the solvent used for dehydration is replaced by the embedding medium depends upon the steepness of the concentration gradient of the plastic at the surface of the pieces of tissue to be embed- ded. It is furthermore dependent upon the mean distance the solvent molecules and the molecules of the plastic must diffuse to establish this exchange of plastic for sol- vent.

It was therefore important to find that tissues that had been cross-linked even very briefly through 3 min of perfusion with 1% glutaraldehyde tolerated gentle homogeni- zation without damaging the structure of subcellular components. Thus pieces of tis- sue consisting of single cells or a few cells, as well as fragments of cells, can be ob- tained.

Such small particles sediment extremely slowly in a viscous plastic, and as a conse- quence the plastic next to the surface of these pieces becomes saturated with dehy- dration solvent, thus preventing the estab- lishment of a steep concentration gradient of the plastic at the surface. Moving the pieces of tissue through the plastic by cen- trifugation corrects this situation by mak- ing the pieces of tissue move fast enough that they are continually exposed to pure plastic.

The method was first tried by pelleting the pieces of tissue and then transferring

AND HALMA

the pellet to a new centrifuge tube filled with plastic. This was repeated several times using a Beckman microfuge and the small (0.4 m]) tubes designed for that cen- trifuge. The pellets were transferred by cut- ting off the end of the tubes and placing the cut-off end in a new centrifuge tube filled with Vestopal with the cut surface dipping into the Vestopal.

This technique improved the infiltration, but it required such a large number of cen- trifugations that it became too time-con- suming. The situation was improved by suspending the material in a large volume of plastic in a 5-ml plastic tube with a cap that was sealed to allow centrifugation with the tube in either the upright or turned- over position.

A few minutes of centrifugation at low speed makes the tissue move to one end of the tube. After turning the tube 180 ° the centrifugation was repeated. Repeated cen- trifugation with turning the tube makes the tissue pieces move back and forth through the tube at a sufficiently high speed that the surface is continuously exposed to pure plastic. The volume of the tissue is so small compared to the volume of the plastic that contamination of the plastic by ethylene glycol is negligible.

Even under these conditions the infiltra- tion requires a long time, and it became obvious that the centrifugation procedure should be made automatic.

PRINCIPLE OF DESIGN

The centrifuge head utilizes spring force to hold the tube carriers in one position. Excentric location of the center of gravity of the tube carriers makes it possible to turn and keep the tubes in a second position by applying a centrifugal force larger than the return force of the springs. The differ- ence in the two positions is about 180 ° . The schematic drawing in Fig. 1 and the pho- tographs of the head in Fig. 2 illustrate the design and operation. Initially, the rotor is run at low speed (800 rpm), and the spring force is stronger than the centrifugal force, so the carriers remain in position (a). At

CENTRIFUGATION-INFILTRATION

a. Low speed

centrifugal force

center of mass of carrier / ,o ,se.io . \

/l S weight ~ ' ~ /

return spring

centrifugal force

axis of centrifuge

263

b. High speed

FIG. 1. Principle of design.

the end of an adjustable time (1 to 10 min) the centrifuge speed is increased automati- cally to approximately 1200 rpm. During this speed increase the centrifugal force on the carriers becomes larger than the spring force and the carriers suddenly flip to po- sition (b), where they remain for the second adjustable time period. Then, the centri- fuge motor is turned off, and as the rotor almost comes to a halt, the spring force takes over again, and the carriers flop back to position (a), thus completing the cycle. The control unit, described below, allows the complete cycle to be repeated as long as desired. To ensure that all four carriers move in unison, and thus avoid rotor im- balance problems, all four carrier axes are interconnected with bevel gears.

DESCRIPTION OF THE CENTRIFUGE HEAD AND OF THE CONTROL UNIT

(a) Centrifuge Head

The specific design is for a Sorvall GLC- 1 centrifuge which was available in our

laboratory, but it could be adapted to al- most any similar small centrifuge.

An unfinished HL-4 rotor was obtained from Sorvall. It was balanced, but without the usual slots for hooking in the swing carriers. The pads at the extremities of the arms were just large enough to have preci- sion 0.5-in.-diameter holes bored through their centers and needle bearings pressed in. The carrier shafts fitted inside these bearings and could rotate freely without friction problems.

Consideration was given to fabricating the carriers out of sheet stainless steel. However, it was finally decided to machine them out of solid blocks of high-strength aluminum. We felt it would be easier to match the units and thus avoid imbalance problems. Each carrier is bored out with six sockets to accommodate plastic tubes (No. N-1086, made by Cole Scientific).

Instrument-type flexible steel cable was used to connect the springs to the shafts.

Part of the off-axis weight which causes

FIG, 2. Rotor in top view (upper picture) and bottom view (lower picture).

264

.3~ TYP CARRIER - 4 REQ

M'T'L: 7075-T6Aluminum Weight: 206 gms each

YP

3t ̧

' S H A F T - 4 REQ I / 4 - 4 0 Two holes CIL --~ ~"--.,87TYP 05

U I ~ i l Iii - - ' i~l I - - - F : ~

! I - 6.600 % ClL /, 3/8

.25 T ~ - -

I_.

Clear for 10-32 Two holes

PLATE - 4 REQ I n

M'T'L: :/t6 Hard Aluminum

Cleor for 6-32

Cleor for 8-32 % .t5

4 - .050 holes I'PTI~ ~ on 0.60 circle

~ - ~ DISK -t REQ j ~ _ r - M'T'L: Aluminum

Fro. 3. Mechanical parts.

265

"-f0-32 Two holes - ~-.312 x .875

~. .3t0 TYP

\ ~ - - _ .3i0 ._. L,4_ t~-i,-. .5oo ~ e- i I t.ioo + ~ 500

I .350 _

-- 2.250. ,-- 3.600 Deep

M'T'L: Steel

®

WEIGHTS - 4 REQ

M'T'L: Brass- .093 thick (see text)

/ - C l e a r for 4-40 t I , / / " Four holes

e - - e"--q-- I : : d ; ~ , ~ _ _ , ~ .5o l / T l - - I

SPACER - 8 REQ M'T'L: Aluminum

266 SJOSTRAND AND HALMA

flipping is integral to each carrier in the form of a top panel (see Figs. 2 and 3). Additional weights are then added to these panels as required to adjust the empty rotor flip speed to 1000 rpm. These weights are made of brass, 1.50 in. long, 0.50 in. wide, and 0.093 in. thick, and of weight approxi- mately 10g each. However, they probably will have to be modified slightly when the unit is placed in service (see "Initial Oper- ation").

The specifics of the mechanical fabrica- tion are shown in Fig. 3. The key mechan- ical components are: 1. Needle bearings: 1/2-in. o.d., 5/1e-in. i.d.,

Torrington No. B59; 2. Plastic bevel gears: Stock Drive Prod-

ucts, New Hyde Park, N. Y., No. 1M4- Y24036;

3. Springs: Century Spring Corporation, Los Angeles, Calif., No. 183B (No. C- 69, same spring sold in hardware stores); specifications: 0.035 diameter music wire, 45 turns, 0.25 o.d., 1% in. between inside of hooks.

(b) Control Unit

An automatic control unit was designed using two "delay on make" solid-state time delay relays. The schematic is shown in Fig. 4.

The speed control circuit inside the GLC- 1 was modified as shown on the schematic. A three-wire shielded cable was connected which exits through a slot cut in the bottom plate and terminates in a three-pin (plus shield) female connector which plugs into

I Control Switch (SWt)

. . . . ~ , , & Automat c - - Input' II ~ Off , I

Cord I I A Manual I I J _ m _ I I

I x First Second I ' Cycle Cycle . ,

AC I Ti~mer N.O. Timer ~ xJ.C. N

Neufrol ' i q[--"(,Z_) L~ (" i

- t ~ ' \ .. . I Relay 2 I ~ • 'JtJ'5V Outlet . I (time

Ground, ~ " - - f - / for GLC . , .~. I Delay)

",, - . Second Cycle -*1 F~rst Cycle t Speed Control', RI S p e e d C ° n r ° l , ~ ~

I, ,I, , +¢ 1 ~ _ ~ ~ 3 - p i n Connector ( Shield Connection) R2 Relay t

~ Shielded 3-wire Cable

=-E- I - X ~ Speed Control Potentiometer I I q on GLC Front Panel open this connection

FI~. 4. Control unit, schematic.

CENTRIFUGATION-INFILTRATION 267

the control unit. The centrifuge power plug also plugs into the control unit.

Operation is initiated by moving switch SWl to "Automatic." One hundred-fifteen volts is supplied to the GLC through the "Second Cycle Timer" (terminals 8 to 5). One hundred-fifteen volts is also supplied to the "First Cycle Timer" through Relay 2, thus initiating the timing period set by the front knob control (1 to 10 min).

For this type of timer the output relays are not activated until the end of the timing cycle. Thus, contacts 1 and 3 (First Cycle Timer) remain open, Relay 1 remains deac- tivated, and R1, the first cycle speed con- trol, is connected into the centrifuge and is used to set its speed (approximately 800 rpm).

At the end of the timed period contacts 1 and 3 close which activates Relay 1 and the Second Cycle Timer. Speed control of the GLC thus switches to R2 which is ad- justed to about 1200 rpm. Timing in the Second Cycle Timer continues to the end of the indicated period, when contacts 5 and 8 open, removing power from the GLC; it then slows down almost to a stop. Simul- taneously, contacts 1 and 3 close, applying power to Relay 2, a thermal time delay relay. Its contacts continue closed for about another 90 sec, then open for a few seconds. This interruption of power resets both cycle timers and also applies power to the centri- fuge again. A few seconds later Relay 2 closes again, applying input power to the First Cycle Timer, and the whole cycle begins anew; it continues until the unit is switched off.

By moving SWl to the "Manual" posi- tion the speed control on the centrifuge front panel is reconnected; thus the unit may easily be used for other work. Key control unit components are: 1. Timers: Master Electronic Controls,

P.O. Box 25662, Los Angeles, Calif. 90025, Model DMPOKll5A600;

2. Relay 2: Amperite No. 115C90T delay relay;

3. R1 and R2: Linear potentiometer, 50,-

000 ohms (for GLC-1). The GLC-2B model is believed to require a poten- tiometer of 500,000 ohms. We were unable to obtain the value for the GLC-3 model;

4. Cabinet: LMB No. 685 (8 in. wide, 5 in. high, 6 in. deep).

Initial Operation

This rotor is potentially dangerous, es- pecially because of its moving parts. Pre- cautions should be taken that no screws come loose during operation because pieces could then fly off and/or the rotor could become badly unbalanced.

Also, if the rotor is allowed to run too fast the springs themselves will be perma- nently stretched and thus mined. We cau- tion and recommend that the maximum speed be kept below 1500 rpm.

It is likely that the offsetting weights will have to be modified slightly to adjust the flip speed to 1000 rpm (empty rotor). The heavier (thicker) the weights, the lower the flip speed (and vice versa). If lighter weights are required, it is simple to machine the 0.093-in.-thick weights down a few thou- sandths of an inch at a time. For heavier weights we suggest using 0.010-in.-thick brass shim stock laid out to the same pat- tern. These should then be clamped under the thicker weights.

Loading the Rotor

After adjusting the empty rotor to flip at 1000 rpm we found that symmetrical load- ing of the carriers with a few tubes did not affect the flip speed. However, as more tubes were added the flip speed increased to an unacceptable level. The solution we worked out was to load the carriers asym- metrically, such that the off-axis weight was increased; thus it was possible to maintain the flip speed acceptably close to the de- sired 1000 rpm. Figure 5 summarizes this rotor loading information.

Another possibility (although we did not try it) would be to fully load the rotor with water filled tubes, then add to the weights

268

One pair of opposite

carriers

SJOSTRAND AND HALMA

Other pair Total Flip of opposite number speed

carriers of tubes RPM

0 @ 0 O@O

0 @ 0 0 @ 0

@0@ @0@

@0@ @0@

O 0 0 0 0 0

O®O 0 0 0

0 @ 0 0 0 0

@@@ 0 @ 0

4 970

6 700

t0 860

t6 740

@@@ ®@@

@0@ ®0@ @ loaded

FIG. 5. Rotor loading chart.

20 4040

until the flip speed was adjusted down to 1000 rpm. The rotor would then always be kept fully loaded (24 tubes).

To use the rotor, embedding-material- filled tubes would then be substituted for water-filled tubes as required. This is pos- sible because the weight of the embedding- filled tubes is sufficiently close to the water- filled tubes that there is no major change in the flip speed.

PRACTICAL APPLICATION OF THE FLIP-FLOP CENTRIFUGE HEAD

This centrifuge head has been used when embedding material prepared according to one of the low denaturation techniques de- veloped by SjSstrand and Barajas (1968) which involves rapid and incomplete de-

hydration in 90% ethylene glycol and embedding in Vestopal W. Due to the low miscibility of ethylene glycol and Vestopal the infiltration of even small specimens such as isolated mitochondria takes 1 week or more when the material is tumbled by slow-speed rotation. With the flip-flop rotor the infiltration time is 24 to 48 hr for small tissue fragments at room temperature.

The cycle that has been used involves centrifugation at 800 rpm and 1200 rpm for 3 min at each speed. The speed and the times are adjusted to allow most of the material to move the whole length of the tube back and forth each cycle.

To prevent extensive aggregation of the material when starting the infiltration the centrifugation times are made very short

CENTRIFUGATION-INFILTRATION 269

during the f~rst 10-25 min of infiltration. The centrifuge is then controlled manually.

DISCUSSION

The infiltration of tissues by highly vis- cous plastics used for embedding is speeded up considerably by back-and-forth centrif- ugation. This reduces the disadvantages of the high viscosity of the plastics and the low miscibility of a dehydrating agent and a plastic. In the case of the low denaturation technique mentioned here this is of consid- erable importance because the infiltration times are exceedingly long without centrif- ugation-inffitration and perfect embed- dings are hard to obtain even after long- time infiltration using other techniques to speed up infiltration.

Centrifugation-infiltration will be an im-

portant technique to apply when dehydrat- ing and embedding at low temperatures where even monomeric plastics become highly viscous.

Due to the low speed used for centrifu- gation-inffltration there is little risk that the material will be damaged by the forces applied during centrifugation.

This work was supported by NSF Grant BMS 74- 20390. We would like to thank the Sorvall Company for furnishing us with the unfinished rotor mentioned in the text. We would also like to thank the Life Sciences Design and Development Facility, University of California, Los Angeles, for their careful and accu- rate mechanical work which certainly contributed to the success of this development.

REFERENCE

SJSSTRAND, F. S., AND BARAJAS, L. (1968) J. Ultra- struct. Res. 25, 121-155.