6 1 2 . 112. 3 . 0 8 3

A METHOD E'OR OBTAINING ACCURATE AND REPRODUCIBLE RESULTS IN EXPERIMENTS ON PHAGOCYTOSIS

G. E. DAVIES Prom the Biological Laboratories of Imperial Chemical Induatriee Limited,

Hexagon House, Blackley, Manchester

THE opsonic technique has been largely abandoned in recent years, mainly because of the difficulty of obtaining reproducible quantitative results. Most authors have mentioned this dif5culty : thus, Gordon and Atkin (1938) wrote (p. 206) " . . . phagocytosis experiments do not lend themselves to definite quantitative measurements . . .". In consequence, Gordon and Atkin contented themselves with demon- strating gross effects. For the work we had in mind, namely a study of the action of chemical compounds on phagocytosis of staphylococci by human leucocytes, the mere demonstration of gross effects was useless. By the technique described in this paper, however, w0 have been able to obtain reproducible quantitative results. Since by slight modifications it can be adapted to the study of many other problems connected with phagocytosh, we feel that it may be of some interest.

MATERIALS AND METHODS

Bacterial suspension

The aim was to obtain a suspension of bacteria which would, as far as possible, consist of individual cells in constant numbers from day to day. This was achieved as follows :-

The afternoon before a test, 0.5 ml. of a 24-hour broth-culture of the organism is pipetted on to the surface of 5 ml. of sterile nutrient agar contained in a 50 ml. conical Pyrex flask. After tilting to allow the liquid to spread evenly over the surface of the agar, the flask is incubated a t 37' C . On the following morning, 10 ml. of sterile saline and about 200 glass beads 2 mm. in diameter are added and the growth suspended and homogenised by rotating the flask by hand for 6 minutes. With any particular strain of organism the results of viable counts on suspensions prepared in this manner are fairly constant from day to day. The viable counts obtained with & strain of Staphylowccwr aurew on six occasions are shown in table I. Table I1 shows that 85-97 per cent. of the cells occur singly or in pairs as shown by stained films. Some experiments carried out using autoclaved suspensions of bacteria were unsatisfactory, in that autoclaving caused the cells to clump and to stain less readily.

Preparation of leucocytes

After a seriea of preliminary experiments, using animal leucocytes obtained in various ways, it was found that human leucocytes were preferable since

J. PATE. BICT.-VOL. LXUI 149 9 2

150 G. E. DAVIES

41.5 55.5

1.5 1.0

0.5 ... ... ... ... ... ... ... ... ...

they are bigger and stain better, and are more easily recognisable than those of the mouse, guinea-pig or rabbit. To avoid agglomeration of leucocytes caused by the clotting of plasma in the presence of coagulase-producing staphylo-

TABLE I

Reproducibility of viable counts of Staph. “ Dodd ” from day to day

- _ _ - - ~ ~

45.5 43.5 44.5 46.5 46.5 50.5 50.5 49.5 47.5 47.0 2.0 4.0 4.0 5.5 5.5 1.5 1.0 1.5 0.5 1.0 0.5 0.5 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 0.5 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 0.5 ... ... ... ... ... ... ... ... ... ... ... ...

Date of expt

4.5.49 5.5.49 6.5.49

27.5.49 1.6.49 4.6.49

Xo, of viable bacteria per nil. 1

3,510,000 2,595,000 3,460,000 3,460,000 4,590,000 2,500,000

I

TABLE I1

Size of bacterial aggregates after suspensions had been rotated with glass beads (approximately 200 groups of cells counted)

1 2 3 4 5 6 7 8 9

10 11 12 13 14

Percentage distribution of groups in successive esperimrnts

Staph. (Dodd)

1

36.0 57.5 2.5 2.5 0.5 0.5 ... ... ... 0.5 ... ... ... ...

__ 2

~

30.5 62-0 2.5 3.5 1 .o ... ... ... ... ... ... ... 0.5 ...

__

Staph. I

3

34.5 65.0

5.0 4.5 0.5 ... ... ... 0.5 ... ... ... ... ...

32.0 53.0 6 6 5.0

1 .o 0.5 0-5 0.5

...

...

...

...

... 1 .o

5 -

39.0 48.0 4.5 6.5 1 .o 0.5 0.5 0.5 ... ... 0.5 ... ... ...

cocci, we used defibrinated blood collected in the apparatus shown in fig. 1 . This consists of a small glass tube fitted with a siphon and U-shaped glass stirring-rod, one arm of which protrudes well above the top of the tube.

Blood is obtained by means of Wright’s “ sliver-pricker ” (Wright, 1941-42). The drop of blood having been obtained, the siphon is applied to it and sufficient blood allowed to flow into the tube. The siphon is then withdrawn and the stirrer twirled between the finger and thumb until defibrination is complete ; not less than 2 minutes should be allowed for this to happen. The start of defibrination is shown by an increased frothing of the blood.

Preparation of mixture of leucocytes and bacteria

Capillary tubes as recommended by Wright and Colebrook (1921) were chosen in preference t o other types of tubes because they require only small amounts of blood. Tubes internally coated with paraffin-wax are used to avoict loss of leucocytes caused by their adherence to the walls of the tube.

T E C H N I Q U E OF PHAGOCYTOSIS E X P E R I M E N T S 151

To prepare the mixture of blood and bacteria a rubber teat is fitted to the end of a Pasteur pipette internally coated with paraffin wax, and a mark made with grease pencil a t a point about in. from the distal end of the capillary. Defibrinated blood is drawn up to this mark and then a bubble of air allowed t o enter. A second volume of blood and one volume of bacteria, suspended either in saline or in a solution in saline of the compound being tested, are also introduced, each portion being separated from the next by a bubble of air. The contents of the pipette are then expelled on to a microscope-slide coated with paraffi-wax and mixed by drawing them into and expelling them from the pipette five times. Finally the liquid is drawn well up into the capillary and the tip of the latter sealed in the pilot-flame of a Bunsen. Control experiments showed that no measurable phagocytosis happened during preparation of the tubes.

Incubation of the mixtures

The temperature of incubation (37" C.) being an obvious choice, it remained for us to consider whether the tubes should remain stationary or be rotated. Rotation results in a greater amount of phagocytosis but with staphylococci under the conditions described, adequate phagocytosis occurs if the tubes are left untouched for 30 minutes.

Preparution of films and staining

The sealed end of the capillary is broken off, a rubber teat fitted to the barrel and the contents mixed on a paraffined slide as before. Drops of the mixture are then placed on clean microscope slides and films spread with the aid of the concave-edged spreader described by Wright and Colebrook. A rapid pushing action is used when spreading in order to obtain a film of suitable thickness. The films are rapidly dried in air, fixed in methanol for 2 minutes and stained for 45 minutes in 1 in 50 Giemsa stain (Gurr's Improved Giemsa Stain R. 66) in pH 7 buffer. They are then washed in buffer solution and allowed to dry without blotting. This

DEFl0RlNATOR

SIPHON.

FIG. 1.--Apparatus for collecting and defibrinating small quan- tities of blood. X 2.

~

method of spreading and staining results in large, well-stained leucocytes. Bacteria are easily differentiated from leucocytic granules by their deeper blue stJain. As many as 80 cocci have been counted in one leucocyte.

Estimation of t l ~ e amount of phngocytosk

The slides are examined with a 1/12 in. oil-immersion lens. Fifty neutrophilic polymorphs are observed and the number of bacteria present in each is recorded. Although neutrophils were the only cells used in the investigation. eosinophils were frequently seen to contain bacteria.

152 G . E . DAVIES

1.04 1.19 1.23 1.13

I

TABLE 111

The influence of 4-methyl-5-o-carboxy-n-valeryZthiazoZ-2-one (1 : 60,000) on the phagocytosis of coagulase-negative staphylococci by humn Zeucocytes

Distribution of bacteria in leucocytes

Bacteria suspended in saline 3acteria suspended in solutiou of drug No. of bacteria per

leucocyte Tube 1 Tube 2 Tube 1 Tube 2 - Slide 2

- Slide 2 Slide 2

- Slide 2 Slide 1

-

4

4 1 3 5 7 6 8 2 1 1 1 3 1

...

...

...

...

...

... 1 2 ... ... ... ... ... ... ... ... ... ...

Slide 1 -

2 1 6 2 2 2 5 3 5 2 6 3 1 1 2 1 1 1

1 ... ... ... ... 1 ... ... 1 ... ... ... ... 1

Slide 1 Slide 1

2 2 2 1 6 6 3 1 4 5 2 2 2 1 2 1 2 2 1 1 ... ... ... 2 ... ... ... ... ... ... ... ...

2 1 4 6 4 1 2 3 4 2 3 1 4 2 1

3 4 1 1

1

...

...

...

... 1 ... ... . . . ... ... ... ...

1 4 5 2 3 5 7 4 4 3 2

3 1 2 1 1

...

...

... 1 ... ... ... ... ... ... 1 ... ... ... ... ...

5 1 2 4 2 6 6 3 1 2 3 2 4 2 1 ... ... ... ... ... 2 ... ... 2 1 1 ... ... ... ... ... . I .

2 2 3 5 4 4 6 4 3 2 5 1 4 1 1 ... ... ... 1

1 1

...

...

...

... ...

...

...

...

...

...

...

4

3 6 4 3 5 4 1 6 2 1 1 1 1 2 1 2 1 1 1

...

...

...

...

...

...

... 2 ... ... 1 ... _-

8.84 -

0 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 40

Means 7.24 8.72 ~

8.96 8.98 7.18 __

8.26 7.20 -

7.87 8.48

8.48 7.87

Phagocy t io coefficient = ~ = 1.08

TABLE IV

The effect of various substances on the phagocytosis of coaguhse-negative staphylococci by human Zeucocytes

Substance Mean ingest (test)

Mean ingest (control)

4.7 4.7 4-7 4.7

Phapocytic coeffirient

Arginine hydrochloride 1 : 60,000 Aneurin hydroch lo r ide 1 : 60,000 Histidine hydroch lo r ide 1 : 60,000 Nicotinamide 1 : 60,000 .

4.9 5.6 5.8 5.3

TECHNIQ WE OF PHAGOCYTOSIS EXPERIMENTS 153

Expression of results

The effect of the compound on phagocytosis is expressed in terms of its phagocytic coefficient.

Phagocytic coefficient =

Average no. of bacteria ingested by one polymorph in test solution Average no. of bacteria ingested by one polymorph in saline ~-

TYPICAL EXPERIMENTS

Table I11 shows the results of a typical experiment in which the effect of 4-methyl-5-w-carboxy-n-valerylthiazol-2-one (Swain, 1949) was investigated. Table IV shows the effect of arginine, aneurin, histidine and nicotinamide. The compounds tested in this work were chosen partly at random and partly because of their known biological activity or their relation to such compounds. None showed any obvious stimulation of phagocytosis. Table V shows that the

TABLE V

Application of the method to the measurenzent of the rate of phagocytosb of staphylococci by human leucocytea

Period of incubation of blood and bacteria

Hean no. of bacteria per P h w o m e

0.12 3.05 5.46 5.77 7.85 7.94

10.68

Percentage of phagocyte containing bacteria

5 77 92 88 95 96

100

method may be applied to measure the rate of phagocytosis. The percentage of phagocytes which contain bacteria quickly rises to a maximum but the average ingest per phagocyte increases much more slowly. This shows that the ingestion of a second bacterial cell is not prevented by the previous ingestion of cocci. It also shows that, as a measure of phagocytosis, the average ingest per phagocyte is a more sensitive figure than the percentage of phagocytes containing bacteria.

STATISTICAL ASSESSMENT OF THE RESULTS

There is considerable variation in the number of bacteria observed per leucocyte on each slide and the distribution of bacteria per leucocyte is very skew. Typical values, taken from many similar experiments, are shown in table VI. The ratio of the variance to the mean in all cases is greater than unity, indicating that phagocytosis is not a perfectly random procedure. Some leucocytes engulf B

relatively larger number of bacteria and others a relatively smaller

154 G . E. DAVIES

number than would be expected from purely chance causes. This indicates either that leucocytes vary considerably in their ability to

TABLE VI Means and variances of the results of typical experiments

with the bacteria suspended in saline

Blood

GED BYB GED GED BYB GED BYB

Btcteria

Staph. I

Sta;h. B

St&. E

,,

,

No. of slides

2 3 3 3 3 4 4

Mean no. of bacteria per

lenc,ocyte

13.3 20.6 18.8 14.0 9 . 5

14.2 10.3

Variance

64.1 70.4

204.9 44.9 55.9 86.7 70.6

Coefficient of variation

per leucocyte (per cent.)

60 41 76 48 79 66 82

of mean per slide

(per cent.)

8.5 5 .8

10.7 6.8

11.2 9.3

11-6

engulf bacteria, that the bacteria tend to agglutinate, or that when a leucocyte has engulfed a bacterium its ability to engulf other bacteria is increased thereby, or to some combination of these causes.

Large variations in both the mean and the variance occur from one control experiment to another. When the standard deviation is plotted against the mean for each slide in the whole of the data, it is seen that the standard deviation tends to increase linearly with increases in the mean (fig. 2 ) . This indicates that the coefficient of variation (that is, the standard deviation expressed as a percentage of the mean) is a more appropriate measure of the variation than the standard deviation. Selected values of the coefficient of variation are included in table VI together with the coefficient of variation of the mean per slide, which is the former value divided by d/50, 50 being the number of leucocytes counted per slide.

Examination of a large number of tests in this laboratory shows that there is no evidence of real variability between slides from the same capillary tube and between tubes treated alike in the same experiment. It does not necessarily follow that, this is universally applicable and it is recommended that two tubes and two slides per tube be taken for each drug and each control: this represents the normal experiment in this laboratory. Such data are readily amenable to the “ Analysis of Variance.” The coefficient of variation of the mean per slide varies from about 5 to 12 per cent. but is usually between 7 and 9 per cent. in the experiments carried out in this laboratory. This is used as a guide to the order of accuracy that can be achieved. Other laboratories may obtain different values for the coefficiont of variation, but if the experiment is carried out as recommended above, a valid estimate of the experimental error may be obtained from each experiment.

If v per cent. is the coefficient of variation of the mean per slide,

TECHNIQUE OF PHAGOCYTOSIS EXPERIMENTS 156

the coefficient of variation of the mean over the two tubes, with two slides per tube is vl2 per cent. since in our experiments there was no evidence of variation caused by the preparation of the tubes or the

1.10 0.91

1.07 0.93

2oj '

I

1.16 1.24 0.86 0.81

1.11 1.17 0.90 0.85

..

One tube ; 2 slides per tube

2 tubs; 2 SLdes per tube

tubes; slides per tube

. . . -

Upper limit ( 1 +2w/100) . Lower limit 1 / ( 1 + 2 ~ / 1 0 0 ) .

Upper limit (l+wdq100) . Lower limit I / ( I + w v ' ~ / ~ o o ) . .

Upper limit 1+2wd/z/100dnZn . Lower limit 1/(1 +~wd/z/100dntn .

..

. .

MEAN

FIG. 2.

slides. From this we may readily derive the probable limits of error of the phagocytic coefficient, which is defined as :- Average no. of bacteria ingested by one polymorph in test solution.

Average no. of bacteria ingested by one polymorph in saline. TABLE VII

95 p e r cent. l i m i t s of error of the phagocyt ic c o e f i c k n t

Set-up

I

Coefficient of variation of mean per slide = 2' per cent

5 per 8 per 12 per cent. I cent. I cent .

________

... ... ...

. . . / . . . / . . .

156 G. E . DAVIES

The coefficient of variation of this ratio is approximately 2/%/2 = w/.\/Tper cent. and the 95 per cent. limits of error are twice this, that is v2/2per cent. For v = 8 per cent. this amounts to 11.3 per cent. Deviations from unity of the phagocytic coefficient greater than 11.3 per cent. can then be regarded as significant at the 5 per cent. level ; that is, values outside the range 1.113 to 0.898 differ significantly from unity. In practice, a phagocytic coefficient less than 0-8 or greater than 1.2 has been considered of sufficient significance to warrant further investigation. Any smaller deviation from unity was not considered of practical interest and indeed, a coefficient of 1.2 would only serve as a guide in the choice of more active substances.

DISCUSSION The technique described above commends itself in several ways.

It requires simple apparatus, and while a certain amount of skill is required, this is readily attainable by practice. A complete experiment can be performed in half a day, and, above all, the limits of error of the method can be readily determined. The technique has a wide application in problems connected with phagocytosis. By simple modifications it can be adapted to such problems as the comparison of two or more sera; the comparison of leucocytes from different sources ; the influence on phagocytosis of immunisation, change in diet, administration of drugs or hormones, etc.

The germ of the idea behind this method was derived from the writings of Sir Almroth Wright. His aversion to statistical treatment is to be regretted, since it renders many of his conclusions of doubtful value. For instance, his practice of counting as 20 any “large” number of bacteria in a phagocyte, can lead to serious errors.

SUMMARY By modifications of Wright’s capillary tube method for the

measurement of phagocytosis a technique has been evolved of which the limits of error can be readily determined. Errors due to the presence of bacterial clumps, faulty staining, loss of leucocytes that adhere to glass and the method of preparing the films have been either eliminated or reduced.

The method can be adapted to problems which involved the comparison of various influences on phagocytosis.

I wish to record my gratitude to Dr 0. L. Davies who was responsible for the statistical aspect of this work.

REFERENCES GORDON, J., AND ATEIN, W. R. . 1938. Brit. J . Exp. Path., xix, 204. SWAIN, G. . . . . . . . 1949. J . Chem. Soc., p- 2898. WRIGHT, A. E. . . . . . . 1941-42. Proc. Roy. SOC. Med., xxxv, 161. WRIGHT, A. E., AND COLEBROOK, 1921. Technique of the teat and capillary

L. glass tube, 2nd ed., London.