The Effect of the H Ion Concentration on Protozoa, as Demonstrated by the Rate of Food Vacuole Formation in Colpidium

THE EFFECT OF THE H-ION CONCENTRATION ON

PROTOZOA, AS DEMONSTRATED BY THE RATE OF

FOOD VACUOLE FORMATION IN COLPIDIUM

BY SYLVIA M. MILLS.

(Francis Maitland Balfour Student of Newnham College, Cambridge.)

(Received $th June, 1930.)

(With Six Text-figures.)

IN a short communication to the British Association at Leeds (1927) Saunders pointed out that the average number of vacuoles ingested by Colpidium in a given time, when fed with suspensions of Indian ink, varied with the H-ion concentration. The present series of experiments represent an attempt to analyse in greater detail the effect of the hydrogen-ion concentration on Colpidium, particularly in respect to its effect on the formation of food vacuoles. The work was carried out under the direction of Mr J. T. Saunders, to whom I owe my thanks for much help.

MATERIAL.

The Colpidium used for the experiments were obtained from hay infusion cultures. Solutions of known hydrogen-ion concentration were obtained by using the following buffer solutions:

1. Palitzsch's borax and boric acid mixtures diluted 1 in 10.

2. Sorensen's primary and secondary phosphate buffers diluted 1 in 10. 3. Prideaux's buffer solution (phosphoric, boric and acetic acids and sodium hydroxide) diluted 1 in 10.

4. Cambridge tap water and various solutions of carbonates of known alkali reserve adjusted to the required pH by blowing in regulated mixtures of air and CO2.

5. The water in which the Colpidium were living was obtained by filtering or centrifuging and adjusted as in 4.

The actual pH of the diluted 1/10 buffer solutions was measured by the glass electrode. Dilution affects the pH irregularly, and the very alkaline and very acid solutions are the most affected by dilution. In these solutions the pH on dilution 1/10 moves towards the neutral point to the extent of 0-5 pH. Buffer solutions near the neutral point appear to be little affected by the dilution, and the effects are here within the range of experimental error. After the effect of dilution had been measured by the glass electrode, comparison was subsequently made with standards by means of the ordinary sulphonaphthalein indicators.

i 8 SYLVIA M. M I L L S

The effect of different H-ion concentrations on the rate of food vacuole forma-tion was examined in the following way:

A series of buffer solutions giving the required range of pH was placed in small glass cells of i-|—-2 c.c. capacity, a few drops of hay infusion containing a thick culture of Colpidium were then put into each cell. The ciliates were allowed to remain in the solution for varying lengths of time, usually from 5 to 15 minutes, before being fed with Indian ink; this time allowed the protozoa to recover from any mechanical shock caused by transferring them to the cells. The animals were fed by adding a definite number of drops of the food suspension to each cell, the contents of which were then mixed to ensure an even dispersion of the particles. The Colpidia were left to feed for 5 or 10 minutes, after which they were fixed with a few drops of formalin. In every experiment the conditions in the cells were kept as constant as possible, the pH being the only variable factor. After fixation, the contents of the cells were placed on a slide and the percentage number of individuals containing o, 1, 2, 3, 4, etc., vacuoles coloured with Indian ink were counted. From this the average number of food vacuoles formed per organism within a given time at each pH was deter-mined. In every case, the averages were obtained by counting the vacuoles in 100 individuals. The actual number of food vacuoles formed at a given pH was found to vary in the different experiments, and only those performed simultaneously with individuals from one culture are quantitatively comparable. This is due to variations in the conditions of the hay infusion cultures which, though they affect the rate of feeding, do not alter the change in the rate of feeding with changes in the H-ion concentration of the medium. However, averages taken from the same culture at the same time and under exactly similar conditions were remarkably constant.

Colpidium can withstand a range of H-ion concentration from pH 4-5 to a point more alkaline than pH 10-5. Prideaux's (1916) buffer solutions (when diluted) appear to be completely non-injurious, but Sorensen's phosphates have a very slightly depressing effect. Borax and boric acid are more toxic and cause a general lowered rate of feeding, though Colpidium can exist in them for several days.

Table I shows clearly the general effect of pH on the rate of formation of food vacuoles.

Table I. The effect of H-ion concentration on the rate of vacuole

formation in Colpidium.

No. of vacuoles 0 1 2 3 4 5 Average

4 - 0

1 0 0 — — — — 0 4-S 6 2 1 4 6 2 3 4 1 98

Percentage of individuals containing

S-5 —

14 7 0 I S

3 ° 3

b-5

1 1 7 7 2 1 0

2 9 1

7 0

1 2 2 6 2 I S

2 9 1

7-5 — 3° °3 7 2-77 8 0 6 84 1 0 — 2-04 8-5 S 77 17 1 2-14 vacuoles (pH) 9 0 2 3 48 4 0 1

2 4 1

9/5 1 3 S i 45 — 2-40 io-o 1 4 7 i 2 4 — 2-18 10-5 1 0 75 1 5 — 2-05 II-O 5 2 3 75 2 — i-79

The most conspicuous feature is a marked depression in the region of pH 8-o. Typically the curve rises steadily from pH 4-5, the most acid point tolerated, to a maximum between pH 6 and 7. As the alkaline range is approached the curve falls, giving the characteristic depression point, which is followed by a slight rise in the region of pH 8-5-9-0. From this point the curve falls away gradually throughout

—-0—Prideaux's Buffer Solution

—A— Magnesium Carbonate O

—5?—Sodium Carbonate 0 0 4 N

—Q—Potassium Carbonate 0 0 4 N

65 70 75 80 85 90 95 100 105 110 pR of medium

FIG. I . Curves illustrating the effect of varied hydrogen-ion concentrations on the rate of vacuole formation in Colpidium. The results obtained from a series of different buffer solutions show the characteristic shape of the curves.

the alkaline solutions. With the diluted buffers employed, the ciliates were not killed during the experiment by the most alkaline point obtainable, though solutions of greater alkalinity than pK 9-5 were lethal after exposure for a day or two.

The pH of the hay infusion cultures from which the Colpidia are obtained vary considerably. By feeding individuals (from a number of such cultures selected to give a good range of pH), curves are obtained which conform to the type described above. Hence, under perfectly natural conditions, the rate of feeding is affected

20 S Y L V I A M . M I L L S

by the hydrogen-ion concentration of the medium in the same way as that of a simple culture exposed to an artificial buffer solution.

Variations from the typical interrupted curve occur most notably with borax and boric acid buffers, and to a lesser degree with the phosphates. These variations are chiefly connected with the position of maximum vacuole formation, and may perhaps be explained by the toxicity of the solutions.

In the experiments so far described, Indian ink was employed as the food material in every case. In order to ensure that the effects observed were in no way influenced by the ink, some of the experiments were repeated in which the ink was replaced by a variety of suspensions of finely granular coloured substances. Among those employed were carmine, iron oxide, carborundum particles and aquadag1. The curves ob-tained when the average number of vacuoles coloured with the particles was plotted against the pYL of the medium were of exactly the same shape as the controls fed with Indian ink. They differed only in that the organisms could not feed so readily on these substances, and consequently the average number of vacuoles was reduced. When, however, the suspension of aquadag was added to the solutions con-taining the Colpidia, an interesting phenomenon was observed. As the organisms swam through the fluid they appeared to drag behind them small masses of mucila-ginous material made visible by the graphite particles adhering to the surface. These collections of mucilage and graphite were towed about until they apparently got too heavy and dropped off. Other Colpidia seem to produce this mucus-like material rapidly and evenly all over their surface, and thus form round themselves a thick walled hyaline membrane. Such membranes have previously been described by Breslau (1921) and others. Paramecia under the same conditions leave behind them as they swim a trail of graphite particles adhering to mucilage. Here the trail of particles can be traced to the gullet, where the mucus appears to have its origin. The production of mucus for the entanglement of food particles is a well-recognised feature of ciliary feeding in metazoa. It is therefore only natural to find a similar mechanism in the ciliates, where it prevents the food, swept in by the inhalent currents, from being swept away by the exhalent currents. That aquadag is so effective in rendering this mucus production visible may, in part, be due to the fact that tannin is a precipitant of mucus.

That the viscous substance produced by Colpidium is actually a mucin is made probable by microchemical tests. Thus, when the Colpidia were treated intra vitam with a 1 per cent, solution of pyronine, the secretion acquired a pink colour in contrast to the protoplasm which remained unstained. With a o-i per cent, solution of thionin it stained a purplish pink, while the protoplasm stained blue. It is precipitated by acetic acid, and the precipitate is insoluble in excess of the acid, but can be dissolved in lime water. From this evidence it was concluded that the observed secretion is a mucin. That the mucus production was a pathological effect was shown to be improbable by its presence in quite considerable quantities in the culture medium in which the ciliates were growing.

1

THE RELATION BETWEEN THE RATES OF MOVEMENT AND FOOD INGESTION IN COLPIDIUM.

In the process of food vacuole formation the cilia, especially those lining the gullet, perform the important function of creating currents carrying a stream of water and suspended particles through the gullet. The rate of ciliary beat is known to be greatly affected by the H-ion concentration of their medium in marine organisms (Gray, 1922). It therefore seemed probable that changes observed in the rate of vacuole formation by Colpidium with changes in the H-ion concentration of their environment might largely be due to the influence of this factor on the rate of beat of the cilia. Evidence in favour of this view was obtained from the results of a number, of experiments carried out on the variations in the rate of movement of

Colpidium in the buffer solutions of different pUs. Changes in the actual work done

by the cilia cannot be obtained by this method since a large proportion of the available energy must be expended in overcoming the viscous resistance of the fluid through which they are moving. However, both the rate of movement and the strength of the feeding currents would be similarly affected by any change in the viscosity of the medium.

The following methods were employed to measure the rate of movement of both Colpidium and Paramecium.

1. The rate of movement over short distances was measured by means of an

eyepiece micrometer and stop watch. A small glass cell was filled with buffer solution at the required pH, and four drops of the culture containing the ciliates added. After 1 minute the buffer solution containing the ciliates was placed as a spread drop on a microscope slide. The time taken by the ciliates to cover a given number of divisions of the micrometer scale was found by readings taken at half-minute intervals for a period of 15 half-minutes. The rate of movement, as measured by the number of divisions of the micrometer scale (1 division = 0-04 mm.), travelled per second was plotted against the pH of the solution. Results were obtained by this method from both Colpidium and Paramecium.

2. The time taken by Paramecium to travel over greater distances than could be obtained by the first method was made possible by using a simple lens of low mag-nifying power. The distance travelled was measured by means of a ghost micrometer. This method was impracticable for Colpidia, which will not move in a straight line for greater distances than could be measured by the eyepiece micrometer (0-2 mm.).

3. When a suitable electric current is passed through the fluid containing

Colpidia, the organisms respond by travelling in a straight line towards the cathode.

2 2 _{SYLVIA M. M I L L S}

the mains. Details of the arrangement used can be seen in Fig. 2. A diagram of the rest of the circuit is given in Fig. 3. The experiments were carried out in the following way:

Four drops of the culture fluid were placed in a small cell containing the buffer solution at the required pH. After 2 minutes the buffer solution containing the ciliates was transferred to an elongated cell, 5 cm. long, 1 cm. broad and 0-4 cm. deep. This was connected at each end by a KC1 agar bridge to a non-polarisable electrode. The current was turned on and readings were taken, the ghost micrometer scale again being used. The current was reversed after every two or three readings, which were taken at intervals for a period of 10 minutes. The best results were obtained when a current of 1 milliamp was used. By a measurement of the specific

I—^ Osram rectifying

alve

FIG. 2. Diagram of the rectifying apparatus used to produce galvanotropism.

conductivities of the buffer solutions, the following values were obtained for the potential differences in the diluted buffers through the range of pH employed:

pH 4-5 = 3-69 volts per cm. 6-o = 2 7

7-0 = 2-61 80 = 2-399 10-0=2-15

4. Paramecium is negatively geotropic, and when placed at the bottom of a long tube containing water or dilute buffer solution, will swim vertically upwards. A piece of graduated tubing was therefore filled with a buffer solution at the required pH. Paramecium was introduced at the top and the tube was inverted at 5-minute intervals. A number of readings were obtained of the time taken to swim upwards through one division of the scale (1-2 cm.).

exactly comparable with those characteristic of the feeding curv,e. Thus from pH 7-0-8-0 the curve fell rapidly, rising slightly 2XpH 8-5 or 9-0, after which the rate

of movement steadily decreased with increasing alkalinity. Hence the type of curve representing the effect of pH on the rate of vacuole formation is also characteristic of that representing the effects of the same^Hs on the rate of movement. The only

Rectifier +

-Reversing" Switch

Milliammeter

Cell containing" Ciliates

FIG. 3. Diagram of the apparatus used to produce galvanotropism in ciliates to facilitate the measurement of their rate of movement.

Table II. The effect of H-ion concentration on the rate of movement of Colpidium.

pU of solution Seconds per cm. Average 4'5 IO-O 8-7 10-5 8-3 9 1 8-6 9-0 9 0 — — 9-15 5-5 9-S 8-4 9-0 8 9 io-o 9-1 8-7 9 3 8-2 8-7 8-98 6-5 8-7 8-8 8-7 9-0 8 9 8-6 8-6 8-3 — — 8-70 7-0 7-8 8-S

S"

9 ' 4

8-S 8 1 8 7 8-S 9 0 8-i 9 6 — 8-73 8 0 9-2 9 2 9-1 8-6 9-6 8 9 8-S 9-2 8-3 9 1 8-97 8-S 7-8 8-S 9-0 8-S 8-S 8-9 8-2 7-8 8-8 8-6 8-46 9-0 9-0 9-3 io-6 8-7 9-3 8-2 9 0 8-3 — — 9-os io-o IO-2 9 5 9 2 9 0 io-o 8-7 8-7 9-2 — — 9 3 2

distinguishable difference appears to be that whereas the pH giving a maximum vacuole formation usually fell slightly to the acid side of neutrality, the rate of movement through the buffer solutions was invariably greatest at/>H 70. There is, however, sufficient similarity between the two types of curve to show that a very definite relation must exist between the rates of movement and food ingestion.

SYLVIA M. M I L L S

are measured in solutions of different viscosity. The results given in Table III and Fig- 5 were obtained by using dilute neutral solutions of agar-agar whose viscosities relative to water were known.

The production of mucus by ciliates and its probable relation to the collection of food particles has already been discussed. Since the tests described show that

1 -16

1-14

1-12

1 1-10

6 1-08

1-06

1-04

1-02

1-0

-O- Rate of Movement

Rate of \acuole Formation

_L _L _L

1

i—U

2-6

2-4

2-2

§

2-0

1-8 XI

1-6

1-4

1-2

4-5 5-0 5-5 6-0 6-5 7"0 7-5 8-0 8-5 9-0 9-5 10-0

FIG. 4. Curves illustrating the relation between the rates of movement and vacuole formation of Colpidium at different hydrogen concentrations.

known about the physical properties of mucins, especially with regard to their relation to changes in pH. Owing to the minute quantities of mucus available from

Table I I I . The effect of H-ion concentration on the rates of movement and vacuole formation in Colpidium.

•a 8

pH of solution

4-₅

7-0

n

8-5

90

100

Rate of movement (mm. per sec.)

[•02 [•071 [•125 [-156 [•128 [•128 [•140 [-129 [•10

Average no. of vacuoles formed

in 5 min.

1-29 199 227 209 203 1 96 215 1-85 1-72

1-3

1-2

1-1

1-0

0-9

0-8

0-7

0-6

O

—

-I 1 -I -I

\ 0 ' _

1 1 • 1

1-0 1-6

- 3 - 0

- 2 - 9

2-8 §

2 - 7 |

2-6 2

- 2 - 5

- 2 - 4

M 1-2 1-3 1-4 1-5

Viscosity of agar solution (distilled water = i-o)

— • — Rate of movement. —©— Rate of vacuole formation.

26 SYLVIA M. M I L L S

Colpidium, mucus was collected from snails. Considerable quantities are readily obtained by inserting a glass rod into the mouth cavity of the common garden snail, Helix aspersa, and by twisting the rod the mucus can be brought out adhering to it (Levene, 1925). The mucus from 20 to 30 snails was collected in this way and allowed to stand for several hours. It was then filtered through glass wool in order to obtain

1-19

1!

1

'

•3

1 1-17

J 1-16

I 1-15

1-14-7-0 7-5 8-0 8-5

pH of mucus

9-0 9-5 10-0

FIG. 6. The effect of H-ion concentration on the viscosity of snail's mucus. The graph represents the average results of three experiments.

at pH 8-o. It fell again irregularly in the alkaline range, a small increase in the viscosity occurring at about pH 90.

Table IV. The effect of the viscosity of the medium on the rates of movement

and vacuole formation in Colpidium.

Solution

Distilled water ... Agar rel. viscosity = 1-047 Agar rel. viscosity = 1-107 Agar rel. viscosity = 1-219 Agar rel. viscosity = 1-570

Rate of movement (mm. per sec.)

1-29 1-17 1-06 090 o-66

Rate of vacuole formation, average no. formed

in s min.

3-°S 2-87 2'77 2-70 2-38

The swelling of dried mucin in buffer solutions again shows that the degree of hydration is also affected by the H-ion concentration. Dried flakes of mucin of known area were placed in a series of buffer solutions made up at suitable />Hs. They were left to soak for a few hours, after which the relative increase in size of the mucin flakes at each pH was measured. The proportional increase in size was found to get steadily greater from pH 5-O-8-O. There was then a marked drop at pH 8-5. This was followed by a slight rise at pH 9-0, but it decreased again with greater alkalinity.

These results show that changes in the physical properties of mucus, demon-strated especially by increased viscosity, do occur in the same range of H-ion concentration that is characterised by a depression in the curves representing changes in the rates of vacuole formation and of movement with pH. It is therefore suggested that through the range of pH occupied by this depression (i.e. pH 7-o-9-o) the viscosity of the fluid which immediately surrounds the Colpidium, and in which their cilia are beating, may be one of the limiting factors governing the rate of food ingestion. There is, however, a second way by which changes in the physical properties of the mucus with changes in pH may affect the rate of food ingestion. Fenn, working on the adhesive properties of mammalian leucocytes, has shown that measurable changes in the "stickiness" of leucocytes to different surfaces were obtained with variations in the H-ion concentration of the solution in which they were suspended. His results show that the adhesiveness of the leucocytes increased with the OH-ion concentration between pH 6-o and 8 0 (Fenn, 1922). Similarly, the stickiness of the mucus in the gullet of ciliates, and the ease with which food particles will adhere to the collecting surface, may vary with the H-ion concentration.

DISCUSSION.

28 SYLVIA M. M I L L S

concentration of the medium may show more exactly how these results have been arrived at.

1. The production by cilia of feeding currents bringing food to the base of the gullet is the first phase to be considered in the mechanism of food vacuole formation. Experiments on the rate of movement of Colpidium have shown that, except between pH 6 and 7, the rate of vacuole formation varies directly with the rate of movement when the H-ion concentration of the medium is altered. It therefore seems probable that changes in the rate of vacuole formation are largely caused by changes in the activity of the cilia. The exact way in which the ciliary activity affects the rate of food ingestion has not been determined. It is improbable that the number of particles reaching the base of the gullet in unit time should be sufficiently affected by the degree of change observed in the ciliary activity greatly to influence the rate of vacuole formation, and in these experiments it has not been possible to take the size of the vacuoles into account. Moreover, experiments on the density of the suspension of Indian ink in feeding experiments indicate that comparatively large differences in the amount of ink present give very small changes in the rate of food ingestion (e.g. halving the amount of ink present only made a difference of 0-05 in the average number of vacuoles formed).

2. A second phase in the formation of food vacuoles which may be affected by the pH of the medium is the accumulation of aggregated particles at the base of the gullet. This may depend firstly on the adhesive properties of the mucus present in the gullet, whose physical condition has been shown to be affected by their hydrogen-ion concentrathydrogen-ion. Secondly, it may depend on the rate at which particles are swept past the collecting area.

3. The actual envelopment of the collected food particles by the endoplasm and the formation of the vacuole itself is a process likely to be affected by changes in pH. These may be induced by alteration of the surface layers of protoplasm at the ingesting area and of the nature of the materials adsorbed on the surface of the food particles themselves.

Since changes in the rate of vacuole formation can largely be accounted for by changes in the rate of ciliary movement, the influence of pH on the accumulation and ingestion into the protoplasm of the collected particles is probably small, though it may perhaps account for the optimum reaction for vacuole formation being slightly more acid than that for ciliary movement.

SUMMARY.

1. The effect exerted by the/>H of their medium on Colpidium is determined quantitatively by counting the average number of food vacuoles formed in a given time when Colpidium is supplied with Indian ink.

3. Methods for measuring the rate of movement of ciliates are described, the most practicable being those in which their galvanotropic and geotropic reactions are used to control the direction of the movement. The effect of changes in the pH of the medium on the rate of movement of Colpidium was found to correspond very closely to the effect of similar pHs on the rate of food ingestion. It is, therefore, suggested that changes in the rate of ciliary movement are largely responsible for changes in the rates of food ingestion.

4. Mucus, produced for food collection, and probably also present in the fluid in which the cilia are working, is shown to have a maximum viscosity at pH. 8-0. It is suggested that the depression in the region of pH 8-o, seen in curves representing changes in the rates of feeding and movement with pH, indicate that the viscosity of the fluid in which the cilia are beating is one of the limiting factors in the rate of food ingestion through the range of pH occupied by the depression.

REFERENCES.

BRESLAU, E. (1921). Die Naturwissenschaften, 4, 57-62. FENN, W. O. (1922). Journ, Gen. Physiol. 61, 119-132. GRAY, J. (1922). Proc. Roy. Soc. B, 93, 104.

LEVENE, P. A. (1925). Hexosamines and Mucoproteins. PRIDEAUX, E. B. R. (1916). Proc. Roy. Soc. A, 92, 463-468.