AU511384B2 - Washing apparatus - Google Patents

Description

V]7

Specitlcati!on

2 COMMONWEALW OF AUSTRALIA Form 1

COMPLETE AFTER PROVISIONAL SPECIFICATION Ilk. PatentsAct 19521973 0 7 95 7 APPLICATION F OR A ATEINT REGINALD HENRY BUTCHER

of Sum.t... DxA..v.e..Bualei.,..Victori4a.,...3.10.5 ust.ra.M.a

hereby apply for the grant of a Patent for an invention

WASHING. APPARATUS -APPLICA-T'1ON q ACCEPTED AND AMFNDi o N I.2O 9

ALLQWFD-

which is described in the accornpanyin rvsoa specification.

MY address for service is SALE-RC2O.CK HAW-THO.RN,

OZJK Victoria,'3122, Australia.

Dated this .1th. day of ember.

REGINALD HENRY BUTCHER,

by his Pa te Attorneys,

THE COMMISSIONER OF PATENTS L

(Signature)

SANDERCOCK CO.

This form must be accompanied by either a provisional specification (Form 9 and true copy) or by a

complete specification (Form 10 and true copy).

11805/75-L 1 180/75-LF. D. ATKINSON, Government Printer, Canberra

AXN4 11 Wk

.CoMMON~WEALTH OF AUSTRALIA Form 7 PatentsAct 1952-19609

DECLARATION INSUPPORT OF AN APPLICATION FOR A PATENT OR PATENT OF ADDITION In support of the Application made by qin a ~d ~yButcher

[Henryi

fora atntfor an invention entitleci WAS HING APP ARAT US

0*

of .Summit Drive, BULLEEN, Victoria, 3105 *.00 do soleminly and sincerely declare as follows:-

S S r the applicant forthPae. 1.J

(or, in the case of an application by a body corporate) f. f ail UntlhU liL~d by

thu-P~ if j~t~flte. to nrztke. tbis deeia±rtt err-

2. 1 am the actual inventor of the invention. (or, where a person other than the inventor is the applicant) 2 o f

is fte'nettl i-retm of ffi. Xn 1 6~

-aii~--he act ~i~n hkhthe applieeatiect are as fdllew-.

Declared at this, day of

THE COMlNUSSIONER OF PATENTS.

of Declarant)

(IMPORTANT-Cross out inapplicable words in the above Form.)

10217J73--L 102173-L.ArxKn430N, -Government Printer, Canberra

S(11) AU-B1-19,508/76

(12) PATENT SPECIFICATION ABRIDGEMENT (19) AU

(21) 19,508/76 511,384 (22) 12.11.75 (23) 10.11.76 (24) 12.11.75 (43) 18.5.78 (44) 14.8.80 (51) G01N 23/00 GO1N 31/14 GO1N 33/16 A47L 15/06 B67C 1/06 (54) Washing apparatus Butcher, R. H. (74) SA (57) Claim 1. Washing apparatus comprising an outer casing for containing washing liquid having a central vertical axis, an annular receptacle centred about said axis for receiving and retaining articles to be washed, said receptacle comprising a plurality of circmfferentially positioned cages for said articles surrounding a space which is in the centre of said casing and said receptacle. an overflow weir over which used washing liquid may flow, washing liquid inlet means located in said space and adapted to direct washing liquj-' generally radially of said axis at a- depth below said weir, and stirrer means located in said space and adapted to rotate about said axis and constructed and arranged to produce a washing liquid motion having radially outward and radially inward components located one about the other and a compound around said axis.

30.5

&U KO

Form kPATENTS ACT 1952-1 973

COMPLETE SPECIFICATION (OR IGINAL) FOR OFFICE USE Class: T.44 d0unlxentcntj Int. Cl: ane~~nna ~ie 1lav&

~ectio~for Application Number: Lodged:

Complete Specification-Lodged: @0 Accepted:

0* 0 Published:

Priority: 0O 0

0

0*0@

Releted Art:

TO BE COMPLETED BY APPLICANT Name of Applicant.: REGINALD HENRY BUJTCHER

Address of Applicant: 4 Summit Drive, Bulleen, Victoria, Austral ia.

*::Actual Inventor: REGINALD HENRY BUTCHER moos0 too0 oNQ Address for Servce: SANDERCOCK SMITH 432-434 Burwood Road, Box 410) HAWTHORN 3122 VIC Complete Specification for the invention entitled: "WASHING APPARATUS

The following statement is a full description of this invention, including the best mnet!,cd of performing it known to me:- Ito: The description is to be typed in double spacing, pica type face, in an area not exceeding 250 mrm in depth and 160 mm in width, ILI on tougt, white paper of good qualty and it 's to be inserted inside this form.

Pb1111 /76-L F. 0. Atkinson, Govetinent Pr n ot. Conberra ii r ~iii

This invention relates to washing apparatus.

In a particular aspect, this invention relates

to a was'hing apparatus suitable for washing paper discs of

the type used in certain laboratories for assay purposes.

Applicant has recently developed a useful procedure

for serum gentamicin assay by enzymatic adenylation and in the

course of that development has found the need of, and has ej developed, apparatus for washing paper discs. However, that 4,9v ~4 apparatus may be put to a number of other uses.

The present invention provides washing apparatus *00 0 0 comprising an outer casing for containing washing liquid

having a central vertical axis, an annular receptacle centred

006 about said axis for receiving and retaining articles to be *0*

0 00 20 washed, said receptacle comprising a plurality of circum- S

ferentially positioned cages for said articles surrounding a

space which is in the centre of said casing and said receptacle,

an overflow weir over which used washing liquid may flow,

washing liquid inlet means located in said space and adapted

to direct wshing liquid generally radially oi said axis at a 00

depth below said weir, and stirrer means located in said space

and adapted to rotate about said axis and constructed and

arranged to produce a washing liquid motion having radially

out,;ard and radially inward components located one above the

other and a component around said axis.

PREFERRED ASPECTS OF THE INVENTION

The effect of radially outward and inward com-

ponents of mo'ion can be enhanced if the apparatus has a

bottom 'ich slopes downwardly and inwardly as the slope will

have a directing effect on liquid flow.

2.

i 7 L II

-P-RFRRED ASPECTS OF Tl I.VENTIO

In practice, applicant has f nd that the

washing liquid motion u.sually has mponents radially

outward and inward of said axi and if the stirrer means.is

arranged to stir through ly a part of the depth of liquid

the outward and inwar components will be located one

above the other ad when the apparatus is used to wash

circular pap discs then those discs usually rotate during

washing. This effect can be enhanced if the apparatus

has bottom which slopes downwardly and inwardly as the

pewill have a directing effect on liquid flos

*c S If, as is preferred, the stirrer means inc.cdes

S* a number of impeller blades then a circular wave motion

around the space seems to be set up which has a generally

sinusoidal form and in practice this seem,s to cause paper

*r 0* discs to bob up and down and, dependant to some extent on i the depth of washing liquid, angularly reciprocate back

and forth in their cages. I The impeller blades are preferably carried by 0

a body adapted to rotate and that that body has a cavity S

therein to which wasning liquid is delivered and an

aperture extending outwardly from that cavity through 5 which washing liquid may be projected radially outwardly. s Preferably, only one such aperture is provided as assymetry 1

of flow appears desirable. Further, the generally sinu-

soidal form referred to above seems to have greater wave

length and amplitude than if more than one aperture is used. i 11i 309D 3.

t (

In this respect, the greater wave length and the greater

amplitude appear to produce more positive movement of

the paper discs.

The cages are preferably open topped to allow

5 loading of articles thereinto. 0*S*

It is preferred that thc weir is located

relatively inward of the cages so that, in use, the

discs are located outwardly of and around the weir.

This arrangement of the weir has 'the advantage that

washing liquid can flow outwardly upwardly and then

inwardly and over the weir so that used washing

liquid tends to be passed over thF weir and mixing of

fresh and used liquid is minimized. An outwardly

located annular weir milht be used but is not

15 considered as desirable as a less circular flow of

washing liquid is obtained.

An outlet fro. the outer casing may be

provided through the bottom thereof.

The stirrer means preferably comprises a

number of magnets and is used ii conjunction with a

magnetic stirrer outside the outer casing. The stirrer

means could, however, be driven positively such as

by a shaft.

157H

The preferred form of the apparatus of the

present invention establishes different flow patterns at

different points within the outer casing but as those flow

patterns will cause the discs to move up and down, angularly reciprocate, and :'otate, each of the discs and substantially

all areas of the discs will be subjected to substantially

the same washing conditions.

The apparatus may also be used for washing

slides.

The preferred washing apparatus of this invention

is useful in gentamicin assay as it is capable of producing

consistent and reproducable washing conditions.

The apparatus is particularly useful in a s

method of gentamicin assay which comprises forming an

15 adenylation mixture containing labelled adenosine triphosphate and an enzyme obtained from E. coli,

incubating that mixture and a. serum sample, depositing a

sample of the incubated mixture on an absorbent disc,

washing the disc, drying the disc and determining a measure

of the labelled atoms on the disc so obtained to obtain a

measure of gentamicin.

A specific construction of washing apparatus in

accordance with this invention will now be described with

the aid of the accompanying drawings.

DESCRIPTION OF THE VIF1f OF THE DRAWINGS

Figure 1 is a plan view of the apparatus,

Figure 2 is a cross-section on line 2 2 in

Figure 1

157H

i, i

Figure 3 is a cross-section on line 3 3 in Figure 2, and Figure 4 is a detail of part of the apparatus. DETAILED DESCRIPTION

The apparatus shown in the drawings comprises an

outer casing 1 which has a cylindrical side wall 2 and a

bottom 3. The outer casing is open at the top.

Within the outer casing 1 is an article holding

rack 5 made up of cages 4. The rack 5 includes outer,

inner and bottom rails 6, radial barriers 8 and a lifting

frame 7 provided with a lifting member 7a. The barriers may be made of nylon mesh, stainless steel, plastics coated metal or any other suitable material. The cages 4 are

closed at their bottoms and radial inner and outer ends

15 by the rails 6 but are open at their tops.

A: will be seen from Figure 1, the cages 4 are

arranged as an annulus about a space 9.

Within that space 9 is a boss 10 around which a

S.. stirrer 16 is free to rotate. The boss 10 has a threaded

20 projection on which an open ended cylindrical body 14 is

screwed. The body 14 is fitted with a tube 11 having an outlet 12 directed downwardly and inwardly.

The stirrer 16 has two horizontally directed

magnets 17, a central cavity 18 which registers with the

outlet 12 and a single egress aperture 19.

The upper end 20 of the body .14 serves as an

overflow weir 20 and an outlet hole 21 in the projection 12 communicates with an outlet tube 22.

In use, the apparatus shown !l Figures 1 4

is located above a magnetic stirrer (not shown), the tube

11 is connected to a source of washing liquid; for instance,

a tap or a flask containing a predetermined volume of

washing liquid and provided with a siphon tube connected

to tube 11.

A tap operated suction device is connected

to the tube 22 or an ouclet hose extends to a drain.

Fow from the flask is initiated, the suction

o device is operated to take off any flow and the magnetic

*o'oo stirrer is operated.

Washing liquid enters via the tube 11, passes

into the cavity 18, the stirrer 16 rotates, the magnets 17

15 stir, washing liquid passes out of the egress aperture 19,

passes over the weir 20 and out the outlet tube 22.

In consequence it has been found that the washing

liquid will flow as shown by arrows 33 in Figure 2 and will

cause paper discs in the cages to rotate, as shown by the

20 arrows 34 in Figure 2 and also has a circular (arrow 32)

wave motion which causes paper discs to bob up and down

as shown by arrows 35 in Figure 2 and angularly reciprocate

as shown by arrows 36 in Figure 1.

The above described washing apparatus has been

found to give gentle but thorough washing action and has

enabled good and quick assays to be :,roduced.

A preferred assay procedure will now be

309D

I

described with the aid of the following Example.

EXAMPLE

The following preparations were made in the

manner stated

Adenosine 5'-triphosphate (ATP) mixture

ATP (Unlabelled) Solution

ATP, disodium salt (Sigma A3127, Sigma Chemical

Company, Saint Louis, Missouri.) was dissolved in cold distilled water to give a 30 mM solution.

ATP lLabelled) Solution

Adenosine 5'triphosphate, tetra sodium

salt (C- 14 C) 40-60 mCi/m mole. S NEC 541 (obtained from

u New England Nuclear, Boston, Massachusetts.). 56

5 0 The labelled and unlabelled ATP solutions were

compounded as follows

S 30 mM A.T.P. Solution in distilled water. 0.5 ml. 14C. A.T.P. 54 uCi/ml. (Amersham) 4.7 ml.

Ethanol (AR) absolute 4.8 ml. Mix and store in the deep freeze. It is liquid at -18 C). S..

to form the ATP mixture.

Broth No. 1 0

Oxoid brain heart infusion broth (Cm 225) 185 gm. 1M Phosphate buffer (Table 7) 100 ~il., 6 Distilled water 4900 ml.

Sterilised in B.S.L. media sterilizer (or iarge

flask) and cooled to 370C.

Broth No. 2

Broth No. 1 plus gentamicin to a concentration

of 10 microg/ml.

309D

i-C~ ICs~m.l~b*m7c

1M Phosphate Buffer. KH2 P04 (AR) 27.2 g. NaHPO 4 (AR) 113.6 g. Distilled water to 1000 ml. The pH of a 1/20 dilution =7.4 at 37 0C

TRIS Buffer No. I TRIS Hydrochloride (Sigma T3253) 13,0 g. (tris (hydroxymethyl) aminomethane hydro chl1 onr de TRIS Base (Sigma T1503) 2.30 g. Distilled water to 100 M1l. The pH of a 1,120 dilution at 37 0 C 7.26 at 37 0 C. 'C S S

es TRIS Buffer No. 2 V C C 9*OS TRIS (Sigmd T3253) 8.04 g. C SO C S C C TRIS (Sigma T1503) 5.94 9. C

S. S C Distilled water to 100 Ml.

0 The pH of a 1/20 c~lution at 370 C 7.8

TRIS Buffer No. 3

*5 TRIS buffer No. 1 10 ml. C.

S NaCl (AR) 1.75 g.

Distilled water to 1000 ml.

pH =7.26 at 37 0 C. C

Buffered Sucro-se -Solutilon 'S

S TRIS buffer No. 1 30 ml Tetra sv)eium EDTA 1.25. g Sucrose (AR) 200 g. Distilled Wiater to 1 1i tre.

309D

1M Magnesium chloride solution

Ng.C1 2 6H2 0 (AR) 20.3 g.

Distilled water to 100 ml.

0.2M Dithiothreitol solution

Dithiothreitoi (Sigma D0632) 0.309 g.

Distilled water to 10 ml.

Store in the deep freeze.

mM Magnesium Chloride solution

1M Magnesium chloride 0.5 ml.

Distilled water 1000 ml.

C. Enzyme preparation *r 4 0 0* Organism. The organism E coli K12 W667/HJR66 is S C stored in the refrigerator as a stab culture in a 5 C Co nutrient agar deep containing lOug/ml gentamicin. 0o 4o 0* o Innoculum. Subculture the organism to 50 ml of 0r

eog... broth No. 2 and incubate 16 18 hours at 37 0 C.

Aseptically add the innoculum to 5000 ml of broth

No. 1 at 37°C in the BSL media sterilizer, which *s C geoe

*0 is used as a stirred incubator. Monitor the o0

growth by measuring the optical density at 515nm

against the sterile broth No. 1 as a blank. When 0 the growth measures an optical density of 0.7-0.75 c (approx 5k hours) stop the growth by adding a

bucket of crushed ice to the water surrounding the

pot containing the culture. Alter the thermostat

to cut out.the heater.

Centrifuge (approx. 5000g for 20 min. i.e. 6000 rpm

using angle head of MSE multiplex centrifuge) and

309D

~lla r--I

Y

S 1

carefully decant the supernant (SNF) down the siJe (prevent splashing) and into a bu.cket. Resuspend the deposits (use a magnetic stirrer

and stir bar) in a total of approx. 1000 ml TRIS buffer No. 3.

6. Repeat step 4.

7. Resuspend the deposits in 500 ml of 20% sucrose solution and stand 15-30 minutes at room

temperature. 8. Repeat step except centrifuge for 30 minutes

and carefully drain and wipe out the shoulders

of the centrifuge bottles.

9. Cool the deposits in an ice bath and shock by

adding a total of 150 ml of ice cold 0.5 mM MgC1 22

15 disperse the deposits quickly with a magnetic

stirrer and stir bar.

Deep freeze over night with the magnetic stir bars. 11. Thaw in luke warm water (approx. 350C) and use the

magnetic stirrer to agitate the mixtures, hence

20 speeding the thawing process.

12. Centrifuge, step 4 for a time of 1 hour.

13. Decant the SNF. through an 0.8 micron membrane filter. Several membrane changes may be necessary.

The filtrate is further filtered through an 0.45 micron membrane.

14. Store the filtrate (Freeze. shock enzyme (FSE) in suitable volumes in the deep freeze.

3090 11.

Enzyme Mixture

Enzyme (Freeze shock prepared) 25 ml

1M TR.IS Buffer pH 7.8 37°C 3 ml

IM Mg.C1 2 25 ml

0.2M Dithiothreitol 25 ml

Mix and store in 0.8 Ml volumes in

the deep freeze. (-180°C).

Paper Disc wash liquid concentrate

KH2PO 4 27.2 g.

NaHPO 4 113.6 g.

NaCI 1461 g. o

Distilled water to 5000 ml.

Warm to effect solution and filter through a

coarse paper. To prepare the wash liquid for *o assay, dilute 100 ml. of concentrate to 5000 ml

cf distilled water. 0o

Gentamicin assay standards.

Gentamicin su" hate reference powder (Roussel

Pharmaceuticals, Sydney, Australia) was used to

prepare the routine assay standards 1, 6, and

12 ug/ml for the adenylation method. Antibiotic,

free, pooled human serum w's used as diluent.

O**O Composition of adenylaticn mixture.

Concentrated enzyme 10.0 ml.

1M TRIS buffer (pH 7.8, 37°C) 0.5 ml

1M Magnesium chloride 0.1 ml.

0.2M Dithiothreitol 0.5 ml.

mM ATP 0.1 ml. 14 250 uCi/ml ATP C 0.1 ml.

3090 12.

Gentamicin adenylation assay procedure

The adenylation mixture was stored frozen RoC)

in two parts: the A.T.P. mixture and the Enzyme mixture.

For assay, the adenylation mixtyre is prepared by thawing

the required amount of enzyme mixture and adding 10% A.T.P.

mixture. for 0.8 ml of enzyme mixttre, add 0.08 ml of

A.T.P. mixture). Store the adenylation mixture in an ice

bath until used.

ul of cold adenylation mixture is added with

mixing to 20 ul of patient's or standard serum in 'V'

bottomed plastic tubes held in an ice bath. All tubes are 60 S: incubated at 37 0 C for .15 minutes and then returned to the

ice bath. From each tube, a 50 ul subsample is pipetted

5 onto a 2.5 cm phospho-cellulose paper disc (Whatman P81) 15 Each paper disc is allowed to dry at room temperature until

o 2 other tubes have been subsampled. The disc is then

transferred to the paper disc washer containing wash liquid;

subsequent discs are transferred in sequence to the washer

after the next tube has been subsampled. This procedure is o

20 followed to ensure that all subsamples on the paper have

similar adsor-tion and air drying times. 6 When all the discs have been transferred, the 0

washer is placed on a magnetic stirrer; the wash liquid 0

o. outlet and wash liquid inlet tubes connected and washing c*

commenced. The wash liquid consist of 5 litres of a 4 mM

phosphate buffer containing 0.1M NaCI and flows into the

washer over an approximate period of 6 minutes.

309D 13.

-rnr xlU ~m i n )~-rrrrmsr~rrii

On completion of washing, the paper discs are

lid out on clean filter paper to absorb excess liquid

before drying in a hot air oven. Each disc is then

counted in 10 ml of toluene scintillant for one minute

after an equilibration time of 5 minutes in an automatic

liquid scintillation counter (Unilux 1, Nuclear-Chicago,

Des Plaines, Illinois). As the labelled adenylated

gentamicin is firmly bound to P81 paper, the scintil'ant

is harvested, filtered and re-used.

Typical assay times are set out below.

TIME V0 a *s e 1. PIPETTING

0.02 ml standard or patient *a serum C0

0 COe C 0.04 ml adenylation mixture

ma *ice bath*

2. INCUBATION 37 0 C. C

4J C 3. RETURN TO ICE BATH AND SUBSAMPLE

0.05 ;nl to pap-

[a~ 0 aa

4. WASH 8

DRY 110 0 C m 6. COUNT 0

m 0 Total time (minutes) C These times are for a total of 9 tubes, i.e.

S1 patient's serum and three standard sera all tested in

duplicate, plus a pooled human serum control.

The whole procedure takes 1 hour.

For each additional serum tested a further 4

minutes is required. Thus an assay of 8 test sera plus

standards can be completed in 1 hours.

3090

Liir~i'"' ~i~iiii -Irll ~n~y~ll

The ratio gentarnicin/counts per minute showed. a substantially linear relation in the range 0 16 qglml and resul ts were reproducable to within 6%. ftod H at oms aid adaptios ma be me'ad to above described without departing from the rit and scope of this invention which include ery novel featu.re and combination of feat isciosed herein. c f form parttolaims of the disclosure of this

9 00 a 0a 0

fAle

a 3 0

Claims (1)

1. The claims defining the invention are as follows:

   1. Washing apparatus comprising an outer casing for

   containing washing liquid having a central vertical axis, an

   annular receptacle centred about said axis for receiving and

   retaining articles to be washed, said receptacle comprising a

   plurality of circumferentially positioned cages for said

   articles surrounding a space which is in the centre of said

   casing and said receptacle, an overflow weir over which used g

   washing liquid may flow, washing liquid inlet means located

   in said space and adapted to direct washing liquid generally

   i radially of said axis at a depth below said weir, and stirrer

   ?means located in said space and adapted to rotate about

   said axis and. constructed and arranged to produce a washing

   liquid motion having radially outward and radially inward

   components located one above the other and a c mponent around

   said axis.

   2. Washing apparatus as claimed in claim 1, wherein

   the stirrer means includes a body mounted to rotate about said

   axis and said washing liquid inlet means includes a cavity

   within that body to which washing liquid is fed in use and a

   single aperture through said body extending outwardly from

   the cavity.

   3. Washing apparatus as claimed in claim or claim 2,

   wherein the stirrer means includes a number of impeller blades.

   4. Washing apparatus as claimed in any preceding claim,

   wherein the inside bottom of the casing slopes downwardly

   inwardly towards said axis.

   Washing apparatus as claimed i, any preceding

   claim, wherein said weir is located radially inward of said *01 receptacle.

   6. Washing apparatus substantially as hereinbefore

   described with reference to the accompanying drawings. 0e

   @0 0 *0 0

   DATED this 5th day of JUNE, 1980. 0 0

   0

   REGINALD HENRY BUTCHER S

   By his Patent Attorneys:

   S. SANDERCOCK, SMITH BEADLE S S

   00 S Fellows Institute of Patent @0* Attorneys of Australia. 0 0

   0 0 00* ***If

   157H

   1~

   19,50 8176