WO1985005691A1

WO1985005691A1 - Apparatus for microscopic examination

Info

Publication number: WO1985005691A1
Application number: PCT/GB1985/000227
Authority: WO
Inventors: John Scholefield
Original assignee: University Of Strathclyde
Priority date: 1984-05-25
Filing date: 1985-05-28
Publication date: 1985-12-19
Also published as: FI860358A; CA1264117A; EP0185698A1; FI860358A0; AU4407085A; NO860266L; JPS61502279A; DK31886A; DK31886D0; GB8413425D0

Abstract

Apparatus for examining microscopic bodies, more especially for enumerating micro-organisms which have been visualised by the direct fluorescent staining technique which is the subject of British Patent No. 1,503,828, uses a travelling flexible tape of plastics material, such as polyester, to which smears of a sample are applied. The sample is fixed to the tape which is then passed through a sequence of treatment, normally staining, and washing steps, finally dried and presented to a microscope for continuous examination under U.V. excitation.

Description

APPARATUS FOR MICROSCOPIC EXAMINATION

This invention relates to apparatus for the examination of microscopic bodies using a microscope. Particularly, but not exclusively, this invention relates to apparatus for the examination of micro- organisms in samples of, for example, food and body fluids.

Our British Patent No. 1,503,828 describes and claims a method of enumerating micro-organisms by staining same with a fluorescent stain after treatment with a phosphate in order to enhance the fluorescence. Numerous examples of chemical treatment techniques for enhancement of the fluorescence of the stained micro¬ organisms are given. The present invention may utilise the staining techniques described in Patent No. 1503828 but is not restricted thereto. Reference is directed to Patent No. 1,503,828 for details of staining tech¬ niques suitable for use in this invention.

An object of this invention is to provide apparatus for use in the automated examination and, optionally, enumeration of microscopic bodies such as, but not exclusively, micro-organisms.

According to the present invention there is provided apparatus for the microscopic examination of microscopic bodies, comprising an elongate flexible tape, means for moving the tape through the said apparatus, means for applying to a surface of the tape a quantity of liquid sample containing microscopic bodies to be examined, means for fixing the sample to the tape, a microscope stage, means permitting travel of the sample-bearing tape over the stage, a microscope focussable on the tape and means for holding the tape on the stage and in the focal plane of the microscope.

Preferably, the apparatus of this invention includes means for treating the fixed sample on the tape with treatment liquid before microscopic examination.

The means for treating the sample with treatment liquid may be a liquid-holding bath having therein a plurality of tape-guide rollers permitting immersion of the travelling sample-carrying tape in the liquid. In a more preferred embodiment, the means may have a form substantially the same as the wash/dry unit described hereinafter.

The means for fixing the sample to the tape may be a heating block over the surface of which the tape passes. The apparatus may also include means for examining the sample as it passes under the microscope. Such means may include a photomultiplier tube for detecting brightly illuminated fluorescing microscopic bodies. The elongate flexible tape is conveniently of plastics material such as a polyester.

The tape used in this invention may be provided with a generally centrally disposed dark, preferably black, stripe to provide a dark background, the tape being other- wise transparent. It may be desirable to apply the said stripe in situ by means of an ink-applying pad, brush, roller or the like.

The means for applying the sample to the tape may be a motor-driven syringe or micropipette having a chisel point so as to smear the sample as a band on to the moving tape. The apparatus may also include means for sampling sequentially a plurality of discrete specimens, for example, a turntable or travelling band arrangement which presents a plurality of specimen tubes sequentially to an automatically operating sampling device which withdraws a sample of each specimen from the specimen tube and delivers it to the syringe or micropipette sample applicator and hence to the moving tape.

Once the smear of sample has been applied the sample is fixed to the tape preferably by the application of heat, In this respect a heating block having a shallow channel thereon for the tape may be provided. The heating block is preferably heated electrically. The sample is fixed to the tape by the heat with or without the addition to the sample of a quantity of fixing, agent, such as alcohol, to the sample before it is applied to the tape.

After fixation the sample may be subjected to one or more treatment and wash/dry steps. The apparatus of this invention may therefore include one or more units for effecting these steps. Each such unit may be a bath having therein a series of rollers enabling the tape to travel through the bath in serpentine fashion and be immersed in and the sample treated by liquid in the bath. The sample may be washed and dried in a wash/dry unit, which may also, preferably, be used for the treatment steps, which includes a block having a channel with a perforate floor and a drainage channel below. The tape travels atop the perforate floor and jets of wash liquid or treatment liquid are delivered on to the sample. Excess liquid runs off the tape and through the perforate floor into the drainage channel whence it may be drawn away by suction to drain or recirculated. It may be advantageous to provide a multiple unit incor¬ porating a plurality of treatment and wash/dry stages. The final drying of the sample may be effected by delivery on to the tape of a stream of warm air or in a second heating block of the type used for fixing the sample.

The fixed, treated dry sample-bearing tape passes across a specially adapted microscope stage which includes means for holding the sample in the plane of focus of the microscope. Preferably the microscopes incorporate- an automatic focussing facility. the microscope stage has a shallow tape-carrying channel and a pair of rows, transverse to the tape, of apertures. suction being applied to the apertures to draw the tape into tight contact with the floor of the channel on to which the microscope is focussed.

The invention will now be described by way of example, with reference to the accompanying drawings, of which:

Fig. 1 is a schematic representation of apparatus of this invention;

Fig. 2A shows the wash/dry or treatment unit shown in Fig. 1 and Fig. 2B is a cross-section thereof;

Fig. 3 is a detail of the adapted microscope stage indicated in Fig. 1;

Fig. 4 shows a cassette arrangement containing various parts of the apparatus and will be described later;

Referring now to Fig. 1, apparatus for examining microscopic bodies 1, has a tape dispenser 2 having a dispensing reel 3, a take-up spool 4 and means, not shown, for winding a tape 5 from reel 3 to spool 4 through various intermediate stages of the apparatus 1. On leaving reel 3, the tape 5 passes below a sample applicator 6 which is a motor or otherwise driven syringe 7 which dispenses liquid sample on to the tape 5 via a chisel-tipped needle 8 positioned so as to deliver a band or smear of sample on to the tape 5.

Between the dispensing reel 3 and the applicator 6 there may be provided means (not shown) for applying a dark ink stripe on to the tape. The sample is then applied on to the stripe. The tape 5 then passes over a heating block 9 where the sample is heat-fixed to the tape 5.

The tape 5, carrying the fixed sample, then passes through one or more treatment stages: one is shown at 10 in Fig. 1. Treatment stage 10 is an immersion bath 11 for holding a liquid such as a biological staining medium, having a series of rollers 12, 12' (etc.) around which the tape 5 passes in serpentine fashion while immersed in the treatment liquid. The residence time of the tape in the bath 11 is determined not only by its speed but also by the number of times it folds back and forth around the rollers.

Following the treatment bath 11, or more than one of such baths, the tape 5 is washed and dried in wash/ dry unit 13 (shown in detail in Fig. 2). The tape may also be washed and/or dried between baths when more than one is used.

The treatment stage 10 described above may be re¬ placed by a treatment unit of generally similar construc¬ tion to the wash/dry unit 13 which will be described in detail later.

After washing and drying, the tape passes below the microscope 14 for examination and hence on to take-up spool 4. As the tape passes below the microscope it lies in a shallow channel on the microscope stage 15 and is pulled into tight contact with the stage by means of a pair of transverse rows of apertures through which suction is applied to the tape to draw it on to the floor of the channel. A sub-stage 16 allows fine adjustment of the position of the tape below the lens of the microscope to centre the sample in the field of view. Fig. 2 shows the wash/dry unit 13, which may also be used as a treatment stage, in more detail. A block of plastics material 131 has a channel 132 with shoulder 133. A first perforate plate 134 and second perforate plate 135 locate upon the shoulder 133. A drainage channel 136 is formed below the perforate plates 134 and 135 and the floor of channel 132. A top closure device 137 of generally similar construction fits on to the block 131. Perforate plate 138 within the top closure 137 has a pair of side by side longitudinal protrusions 139 which, in use come into contact with the edges of the tape, leaving a central channel 140 through whichthe sample-bearing portion passes. Wash liquid, normally water or treatment liquid such as stain or buffer solution is pumped under pressure on to the fixed sample. The used liquid is drained away via channel 136 in the base unit.

Fig. 3 shows the microscope stage 15. It is a block of material 151 having a channel 152 for guiding the tape. A pair of rows of apertures 153 extend trans¬ versely across the direction of travel of the tape. Suction is applied to these two rows of apertures 153 to draw the tape flat on to the floor of channel 152. The stage 15 is located such that the area between the rows ot apertures 153 is in the field of view of the micro¬ scope which is then focussed on to the sample.

Fig. 4 shows a cassette unit which is a flat plas¬ tics container 20 having a recess 21 for locating the sample applicator and heating block (6,9). The con- tainer also contains a reel of tape 22. The lower end of container 20 provides a treatment bath 11 with its associated rollers 12, 12' etc., the tape being threaded around these rollers. A wash/dry unit 13 may be provided within the cassette or an aperture in each wall may permit such unit to be inserted.

The apparatus of this invention is intended for counting micro-organisms which have been visualised using the fluorescent staining technique which is the subject of British Patent No. 1,503,828 in which numerous examples of fluorescent staining techniques for various micro-organisms are disclosed and to which reference is directed.

However, this invention is of much wider application than the counting of fluorescing micro-organisms. For example, by u__ing available techniques for size, shape and colour discrimination the system may be used to identify and enumerate specific micro-organisms in the presence of others, conduct particle size analysis and similar measurements. The bodies being examined need not be fluorescent.

The following Examples illustrate various chemical staining procedures but many systems are available for use in respect of different microscopic bodies. Many suitable procedures are given in our British Patent No. 1,503,828. Example 1

To a sample of urine for bacteriological examination a small amount of surfactant was added. A portion of the sample was then applied to a travelling tape which was then passed, as described hereinbefore through the following steps.

(a) Fixation on the heating unit at 20°C

(b) Staining with 0.1% aqueous acridine orange (AO) buffered to pH 3.0, for 2 to 5 seconds. (c) Washed for 1 to 5 seconds in the wash/dry unit

(d) Drying on a second heating unit.

Example 2

As Example 1 with an addition of an extra wash with

0.005 to 1.0M aqueous calcium chloride for 0.5 to 5 seconds prior to the washing step (b) of Example 1.

Example 3

Sample was stained with 0.1% W/V of AO in 0.05M diethanolamine/hydrochloric acid pH 9.0 buffer for about

2 to 5 seconds followed by calcium chloride and water washes as described in Examples 2 and 3 above.

Claims

1. Apparatus for the microscopic examination of microscopic bodies, comprising an elongate flexible tape, means for moving the tape through the said appa¬ ratus, means for applying to a surface of the tape a quantity of liquid sample containing microscopic bodies to be examined, means for fixing the sample to the tape, a microscope stage, means permitting travel of the sample-bearing tape over the stage, a microscope focus- sable on the tape and means for holding the tape on the stage and in the focal plane of the microscope.

2. Apparatus as claimed in claim 1, including also means for treating the fixed sample on the tape with treatment liquid before microscopic examination.

3. Apparatus as claimed in claim 1 or claim 2, in which the means for fixing the sample to the tape is a heating block over the surface of which the tape passes.

4. Apparatus as claimed in claim 1 or 2 or 3, in which the means for treating the sample with treatment liquid is a liquid-holding bath having therein a plurality of tape-guide rollers permitting immersion of the travelling sample-carrying tape in the liquid.

5. Apparatus as claimed in any preceding claim, in which the tape is of synthetic plastics material.

6. Apparatus as claimed in claim 5, in which the said material is polyester.

7. Apparatus as claimed in any preceding claim in which said tape has a generally centrally disposed dark stripe.

8. Apparatus as claimed in claim 7 including means for applying a dark stripe generally centrally on to said tape.

9. Apparatus for the microscopic examination of micro¬ scopic bodies comprising, sequentially a dispensing reel of tape, means for applying to said tape a dark stripe, an applicator for applying a liquid sample to said tape on the dark stripe, a heating block for heating the said tape to fix and dry said sample, one or more units for applying treatment liquid to said fixed sample and one or more units for washing excess treatment liquid from the sample, means for drying said sample, a micro¬ scope for examining the sample and a tape take-up spool.