Up to the present we possess no material which we can cali. hand, and of the r6le of colloids in the processes of life, on the

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1 PART I IGOR ASHESHOV State Bacteriological Laboratory, Dubrovnik, Yugo-Slavia, and Bacteriophage Inquiry, I.R.F.A., Patna, British India Received for publication, June 22, 1932 PREPARATION OF GRADUATED MEMBRANES The ever-increasing interest in, and comprehension of, the importance of the new field in microbiology, which relates to the so-called ultraviruses and filtrable forms of bacteria, on the one hand, and of the r6le of colloids in the processes of life, on the other, urgently call for further developments in the technique necessary for their study. More adequate methods of filtration, and particularly ultra-filtration (in the widest sense), are especially required. The object of this study is to record certain facts and observations made on the technical side of the preparation of collodion membranes developed during my studies on bacteriophage. I do not intend to attempt any theoretical discussion or explanation of the facts observed either by others or myself. Want of access at the time to the extensive literature on the subject prevents my entering into such a discussion. Up to the present we possess no material which we can cali ideal for filtering purposes. An ideal filter can be defined as one which will act as a simple sieve alone, i.e., will mechanically retain or let pass particles suspended in a liquid, without changing their physical or chemical nature. In such a case the results of filtration would depend solely on the relative dinensions of the pores of the filter and of the particles suspended. With the filters at present in use, however, it is very seldom indeed that the finest suspensions can be mechanically separated. In the great majority of cases such a separation is produced by a 323 JOURNAL OF BACTERIOLOGY, VOL. XXV, NO. 4

2 324 IGOR ASHESHOV much more complicated process in which incompletely studied phenomena such as adsorption, coagulation, etc., intervene. Filters made out of inorganic materials-porcelain, silica, gypsum etc.-are comparatively coarse. Their power of adsorption is great and their porosity can neither be exactly determined nor varied outside certain limits. The use of animal membranes (amnion, alantois, bladder, etc.) or specially prepared paper membranes is restricted. These can only be used for special purposes, e.g., dialysis. Collodion membranes, on the other hand, offer much wider possibilities. In the literature on the subject a "collodion membrane" has generally been taken to mean a "collodion sac." In this study the term "membrane" is used to describe any membrane forming a film of more or less permeable material. The term "membrane filter" is reserved for a disc of collodion film used in a special funnel for fitration purposes. A considerable amount of work on the different uses of collodion sacs and membranes has been published and different methods of preparation have been advised. Some workers have recognised the variability of the membranes obtained and the difficulties encountered in producing membranes of a desired permeability. Many studies in different fields of biology have, however, appeared in which membranes have been used without paying the necessary attention to their properties. A very superficial description of the technique employed for the preparation has sometimes preceded, indicating the solvents and strength of collodion solution alone. Very few authors actually have introduced necessary controls and proved that all the membranes which they have used in their study were of comparable properties. COLLODION SACS During my researches on the properties of collodion sacs I came to the conclusion that it was almost impossible to prepare two sacs of equal permeability. Malfitano's method of dipping a test tube in a solution of collodion, taking it out and rotating it until the collodion sets, in particular, produced sacs which could not

3 bear comparison with each other. The technique of Gates' proved to be better in that one could obtain from 4 to 6 sacs of almost the same permeability at one time. But a second series of sacs of a permeability approximate to that of the first could only be obtained by chance. The cause of such divergent results lies in the extreme sensitiveness of the membrane to the different factors governing the formation of its final structure during the period of drying. In 1IValfitano's method the resulting permeability of the sac depends upon the following factors: 1. The composition of the solution used, nature of the solvents, percentage of celloidin, brand of celloidin, etc. 2. The number of times the solution was used (e.g., the amount of evaporation of the solvents which has occurred during previous manipulations). 3. The rapidity with which the tube is withdrawn from the solution, and its rotation until the collodion sets, factors which are responsible for differences in the uniformity of the sac wall. 4. Atmospheric conditions (temperature and humidity) prevailing at the time. 5. The duration of the period of the drying of the sac. In Gates' method, besides the points already mentioned, the following factors are of importance: 1. The diameter of the tube used. 2. The time the tubes are left above the collodion solution before the cork is removed. 3. The position in which the tubes are left to dry, i.e., whether with the mouth of the tube upwards or downwards. I have made a very complete study of collodion sacs made by these two methods, but chiefly by that of Gates', and as a result of my experiences have found the following technique to be the best for measuring the comparative permeability of different types of the sacs. To begin with, the sacs are cut to the length of 6 cm. and mounted on tubes exactly 20 cm. long, in such a manner as to Jour. Exp. Med., 1922, 35, 635.

4 326 IGOR ASHESHOV leave free 5 cm. of the length of the sac. They are then filled to the top of the tube with distilled water and their intactness controlled by observing the surface of the sac after carefully drying it with filter paper; should a drop of water appear continually at any particular point on the surface, the sac is rejected. Downloaded from FIG. 1. PERMEOMETER FOR COLLODION SACS The sacs are then immersed in distilled water to a depth of exactly 6 cm. By so doing and by keeping the sac and tube filled with water a constant pressure equal to the height of the column of the water in the tube was assured in every case. After about half an hour the permeability of the sac becomes constant. The permeability of the sacs was then measured by a very simple device (fig. 1). A 0.1 cc. pipette about 25 cm. long is mounted in on October 10, 2018 by guest

5 an India rubber stopper in such a way that its proximal end just passes through the stopper. A mark is then made on the pipette about 5 cm. from the distal end and a second one 10 cm. further down. Before fitting the cork into the tube it is essential that the latter should be completely filled with water so that as the cork is inserted the water rises and completely fills the pipette also. The level of the water in the pipette immediately starts to fall depending on the permeability of the sac. As soon as the water-level reaches the upper mark a stop-watch is put in motion and the time necessary for the water to reach the lower mark is noted. This expresses the permeability of the sac in seconds. It is important that this test be carried out with the sac immersed in water, otherwise the flow through the membrane will be irregular, especially with sacs of low permeability. By means of this test I have found it possible by Gates' technique to obtain a series of from 4 to 6 sacs of similar permeability. The preparation of a new series of sacs of the same permeability as the first series was, however, purely a matter of chance, and that chance never came when it was most wanted. Certain experiments which gave valuable results completely lost their significance for want of confirmation, as it was found impossible to prepare sacs identical with those used in the first experiment. As the result of my experience with these types of collodion sacs, I have been forced to the conclusion that the uses of such sacs are very limited and that they should not be employed for researches in which the permeability of the membranes is an important factor. MEMBRANE FILTERS 327 On the other hand, it is much easier to prepare membrane filters of comparable permeability. It is true, one cannot be certain of preparing an exact replica of a large membrane filter (although several small ones, which will then have equal properties, may be cut out of one large sheet), but by observing the same technique, membrane filters of practically equal properties can be obtained, and their preparation involves much less of the personal element. The permeability of a membrane depends on the following three

6 328 IGOR ASHESHOV factors: (a) the composition of the collodion solution; (b) atmospheric conditions (temperature and humidity); (c) the length of time the membrane takes to dry before being immersed in water. These three factors are easier to control than all the factors involved in the preparation of sacs. MATERIAL FOR USE Collodion is a low nitrated cellulose. Its nitrogen content is not constant and varies with different samples. A highly nitrated cellulose such as pyroxylin or smokeless powder, for instance, cannot be used for the purpose, as it is not completely soluble in alcohol-ether. I advise the use of a nitrocellulose of known make, which is guaranteed to be of more or less constant composition, so that the results will not vary from batch to batch. Different brands of nitrated cellulose vary considerably in adsorptive power. By filtering a 1 per cent dilution of serum in physiological salt solution through a membrane prepared from the collodion under examination and determining the quantity of albumen in the first 10 cc. of ifitrate by the addition of 20 per cent sulfosalicylic acid, sufficient information on this point can be obtained. If the reaction is frankly positive, the adsorptive power of the celloidin is low enough;if not, another brand must be obtained. Some of the brands are not sufficiently responsive to the action of the chemicals used to obtain membranes of different permeabilities, and should not be used. STOCK SOLUTION OF COLLODION In the present study I have used Schaering's celloidin. This celloidin is commonly employed for histological work, and was the only one obtainable. It is pure nitrocellulose of comparatively constant composition sold in air-tight tins in tablet form. Each tablet contains about 40 grams of nitrocellulose saturated with alcohol so that it weighs more than 100 grams. This brand is not an ideal product, as it still contains a considerable amount of oxycellulose which is not soluble in alcohol-ether. I was not able to get another brand and so my experiments have been confined to this one alone. The stock solution, which in my hands

7 has proved to be the most convenient for the purpose, has the following composition: 329 Pure nitrocellulose grams Alcohol, 96 per cent cc. Ether cc. The solution can be prepared directly from the "alcohol" tablets mentioned above or else the tablet can be dried and weighed before dissolving. Experience shows that the two methods give different results; a solution prepared from dry celloidin always gives less permeable membranes. In the first case the whole tablet is cut into thin long slices; small pieces should be avoided, as they get stuck together in a mass and are then difficult to dissolve. The slices when cut are weighed and should weigh 121 grams: 40 grams of cellulose plus 81 grams (the weight of 100 cc. of 96 per cent alcohol). If their weight is more than this they must be quickly dried by fanning. If the total weight is less than 121 grams the slices are put into a tared bottle of 500 cc. capacity, antd alcohol (96 per cent) added until the proper weight is obtained. Three hundied cubic centimeters of ether are then added and the bottle shaken, preferably in a shaker. A bottle with a good cork stopper must be used. Rubber stoppers are soluble in ether, and glass ones either do not fit close or stick to such an extent that they cannot be taken out. If dry celloidin is used the tablet is grated through an ordinary kitchen grater oir passed repeatedly through a meat grinder. With the latter method it is necessary to keep the celloidin moistened with alcohol to prevent its particles sticking together. The celloidin is then left in a dish, covered with filter paper, until a constant weight is established. The weight is then noted and the necessary amount of alcohol added. After the celloidin is thoroughly soaked in alcohol for a few hours or overnight the ether is added and the bottle shaken in a shaker until solution is complete. It is better to keep nitrocellulose in solution. If exposed to the air in the dry condition, it slowly oxidises to oxycellulose, which is insoluble in the alcohol or ether. This applies also to the tablets of celloidin sold in air-tight tins. When

8 330 IGOR ASHESHOV the tin is opened the whole tablet must be dissolved at once, otherwise it will not give a transparent solution. PREPARATION OF MEMBRANES Apparatus necessary 1. A small levelling table. This consists of a board of 50 by 50 cm. with four levelling screws at the corners. The board is painted black to make the air bubbles in the membrane visible, FIG. 2. TANK WITH HOLDER FOR WASHING MEMBRANES and is covered with a glass plate to insure the flatness of the whole surface. 2. A good spirit level, large and very sensitive. 3. Six flat cast glass plates 20 cm. square and 5 or 6 mm. thick. These must be plane-parallel and must be free from scratches. The best variety is made of the so-called crystal or mirror glass. 4. A copper tank with a holder like those used in photography into which the plates can be put vertically, some 4 or 5 cm. apart, when the membranes on them are ready to put into water (fig. 2). 5. An aluminium stick about 3 mm. thick and 15 cm. long. 6. Thick-walled test tube with 1 cc. graduations up to 15 cc.

9 7. Cork stoppers of the best quality (velvety), so that no cork dust may pass into the solutions, to fit the test tubes. These must be about 4 cm. long and not too conical. 8. A Petri dish cover of about 90 mm. outside diameter. 9. A small sharp scalpel, or better a safety razor blade in a suitable holder. 10. A few 1 cc. pipettes graduated to 1/100. SOLUTIONS 1. Ten per cent solution of celloidin. 2. Mixture of 25 parts of 96 per cent alcohol and 125 parts of ether to which about 0.1 per cent of concentrated HCl is added. 3. Acetic ether and formic ether. TECHNIQUE OF PREPARATIONS OF MEMBRANE FILTERS The principle of the technique consists in pouring a properly diluted stock celloidin on the glass plate and leaving it for a definite time until the ether evaporates and the celloidin sets. The plate is then put into water to wash out the solvents. A disc can then be cut out of the film, which may be used as a membrane filter in Zsigmondy's funnel. To change the permeability of the membrane, measured amounts of acetic or formic ether are added to the diluted celloidin solution. PREPARATION OF THE GLASS PLATES Before use, the glass plates should be carefully cleaned with acetone and polished with a cloth. However, it was observed that in spite of every care in cleaning the glass, the membranes would sometimes stick to the glass, or sometimes become detached from it too early and shrink without the necessary support. After numerous trials it was found that this was not due to the condition of the glass surface alone, but mainly to the reaction of the celloidin solution; a slightly alkaline film sticks to the glass, while a slightly acid one detaches itself very early. The addition of a very minute quantity of the hydrochloric acid to the alcohol-ether diluting mixture always produced an easily detachable membrane. The maximal amount necessary for this purpose is 0.1 per cent 331

10 332 IGOR ASHESHOV HCl, and very often considerably less. On the other hand, the premature detachment of the film before the solvents are all washed out introduces another difficulty in the form of ultimate shrinkage and a change in the quality of the membrane, usually in the production of a membrane which is difficult to use owing to its uneven surface. To overcome this latter difficulty the following method was used: Any strong cylindrical object such as a strong tin or porcelain or flat glass jar 11 cm. in diameter is taken and placed in the center of the glass plate. A moistened disc of filter paper under the jar will help to hold it in position. By means of the aluminum stick a circle is drawn on the glass plate around the jar. This procedure is not easy, for it is necessary to apply pressure and to traverse the same area with the pencil repeatedly. The surface of the glass must be damp, not wet; the best way of keeping it so is to breathe on it constantly. If the surface of the glass has been previously treated with a little sodium silicate, the procedure becomes easier. When properly done, the circle should give a silvery reflection, the line itself being almost 1 mm. wide. This line can be removed only with a strong NaOH solution. If an acidulated solution of celloidin is now poured in the center of the circle and spread so as to overlap this line, the collodion will stick to it at the edges. At the same time, due to the presence of acid in solution, it will not adhere to the remaining surface of the glass, thus remaining stretched. Once the membrane is washed, a disc which can be removed from the glass without difficulty can be cut from the center. The aluminium line wears out by use and must be renewed from time to time. If the film still detaches from it, it means that too much acid has been added to the diluting mixture. Sometimes difficulties are experienced in getting the celloidin to spread evenly when poured within the circle. This means that the glass is not clean. If for any reason the addition of hydrochloric acid is not desirable, the best method of preventing the membrane from sticking to the glass is to cover the surface of the glass with a very thin film of gelatin. It is sufficient to smear the glass with cotton

11 333 wool soaked in 2 per cent gelatin and leave it to dry. The aluminium line must, however, be drawn afresh every time the glass has been coated with gelatin. PREPARATION OF COLLODION MIXTURE The ingredients are mixed fresh in the graduated test tubes in quantities sufficient for one disc. The ingredients which are intended to change the permeability of the membrane are first added, i.e., the acetic or formic ether, as the case may be. As very small quantities of these substances produce considerable changes in the membrane, they must be accurately measured by means of a 1 cc. pipette having 0.01 cc. dinvsions. The acidulated alcohol-ether mixture is then added up to the 9 cc. mark and well mixed. The stock solution is added last by pouring it in a fine stream into the middle of the liquid up to the 15 cc. mark. The tube is then corked with the cork stopper and vigorously shaken so as to produce a thorough mixture of the contents. A good deal of froth and numerous bubbles are produced. If now the cork is taken out very abruptly, with a jerk, an instantaneous vacuum is created above the collodion, and the bubbles rise quickly to the surface. That is why a sufficiently long and alnost cylindrical cork must be used which acts as a piston. When all the bubbles have come to the surface and burst, the solution is ready to be poured on the glass plate. MAEING THE FILM Before mixing the collodion solution, the glass plate must be placed on the levelling table and levelled carefully. In order to mark each film for record purposes the number of the film is now written in reverse characters (as in "mirror writing") with an ordinary laboratory wax pencil on the surface of the glass. When the film becomes detached the number will adhere to the membrane. The celloidin mixture is now quickly poured into the middle of the circle. If the glass is clean and properly levelled the liquid distributes itself equally in all directions and is assisted to do so by means of the mouth of the test tube so that its edges finally overlap the aluminium circle to the extent of about 5

12 334 IGOR ASHESHOV mm. If air bubbles appear, they can be easily removed by touching them with a metallic or glass rod. This must be done within the first minute of the liquid being poured on, for if done later it will alter the structure of the film. A wooden object or a piece of paper is not suitable for this purpose, as they only increase the number of bubbles. Small bubbles which may appear later are not of importance and the membrane should not be rejected if there are a few of these left. It is natural to think that a membrane containing bubbles wrn be more permeable than one without them, but experience shows the contrary to be the case as the following experiment proves. Two membrane filters are mounted in a Zsigmondy funnel. The first of these, which has some bubbles in it, is of sufficient permeability to permit the partial passage of India ink particles. The second, which is placed below the first, can be of any permeability. Diluted India ink is now filtered through the membranes. The smallest particles of ink pass through the upper membrane and stain the lower one when the particles have passed through. Now, if the bubbles in the first membrane were more permeable than the other parts, we should expect to find black spots on the second membrane corresponding to the places occupied by the air bubbles in the first membrane. No such condition, however, is ever found for the following reasons: The air bubbles do not make a hole in the membrane. They are really small chambers covered on both sides with a very thin film. This film dries more quickly than the rest, and by so doing produces a less permeable membrane. Thus, at the site of a bubble the liquid has to pass through two membranes instead of one. The removal of bubbles is recommended only because they are unsightly. At the moment the mixture of collodion is poured on the glass plate the stop-watch is started. A photographic darkroom timer is most convenient for this purpose, as it will signal the time when the membrane must be put into water. The time taken in drying is most important. The permeability of the membrane depends upon the quantity of alcohol and ether present in the film at the time when the membrane is put into

13 water, and also upon the humidity of the air. This last factor is one which is not under our control in the ordinary laboratory. Ceteris paribus, however, the more humid the air is the more permeable is the membrane obtained. In my experience a membrane prepared when the humidity of the air is nearly 90 per cent is about twice as permeable as one prepared in dry weather when the humidity is 40 per cent. These variations in humidity are used by Zsigmondy and Bachmann in their patent method of preparing graduated membrane. In order to reduce discrepancies from this source to a minimum, the period of drying should be cut down as much as possible. The correct period is the time required for the collodion to set on the glass, i.e., the time necessary for the ether and other solvents to evaporate to a certain extent, but not the alcohol. The humidity of the air has practically no influence on the rapidity of evaporation of the solvents. The time required for the collodion to set is influenced mainly by the temperature of the air and by the amount of solvent used. The following equation, which has been evolved after numerous observations, probably gives the most accurate estimate of the time required: T = K(30-t) n 335 where T is the optimum time required to dry the membrane in minutes, t = the temperature (centigrade) of the air, n = the number of cubic centimeters of solvents other than ether-alcohol added, and K = a constant found experimentally for every batch of collodion. Usually K lies somewhere between 0.25 and 0.3. Thus, if the temperature of the air is 210C., 0.5 cc. of acetic ether is added to the mixture, and K is 0.28, the optimum time of drying will be ten minutes forty-five seconds. For practical purposes the equation can be plotted on a squared paper as a straight line. The constant K is easily found experimentally. Celloidin of proper strength prepared from a new batch is poured as already indicated, and the stop-watch set in motion at the time the solution reaches the glass. The correct time to stop evaporation by immersion in water is reached when the sensation of a liquid

14 336 IGOR ASHESHOV under the superficial film disappears and is determined by feeling the membrane lightly with a finger.2 Another formula which also gives satisfactory results, particularly for the higher temperatures (28 C. and upwards) is T - T + 2.5n where 210 is a constant found experimentally for the majority of batches. This method gives uncertain results with temperatures lower than 20 C. When the membrane has set sufficiently, the glass plate is put into running water, in which it must remain until all the solvents are washed out. This process is complete when the water over the surface of the membrane spreads evenly and usually takes about fifteen to thirty minutes. To prepare a filter for use, a Petri dish cover 91 to 93 mm. in diameter, is put over the middle of the film and a round disc is cut out of it with a knife. The knife must be very sharp, otherwise the edges of the disc will be ragged. The disc can be preserved either in 0.5 per cent formalin or in chloroform water. I prefer the former, as it is less volatile, but still disappears completely during sterilization. The discs are kept in round covered glass jars 14 cm. in diameter. In addition to the single discs described, it is also possible to prepare a membrane from which several discs can be cut. Such a procedure has the great advantage of considerably shortening the time of preparation required for a number of membranes, andwhat is more important-of obtaining several filters of the same permeability. For this purpose a square must be drawn on a large glass plate with the aluminium pencil. The larger the surface the wider must be the line in order to withstand the greater tension of the film. Alternately, a circle of some 30 cm. can be drawn making a celloidin disc 31 cm. in diameter. Seven membrane filters can be cut from a circle of this size. To get results with larger membranes comparable to those with single mem- 9 Pierce in a recent paper advocates a better method; the film is sufficiently dry if its edge leaves a clean surface when lifted with a knife blade.

15 337 branes, the quantity of celloidin solution per unit of surface must naturally be the same. The quantity is easily calculated, knowing that, for the surface of a single disc of 113 sq. cm. area (12 cm. in diameter), 15 cc. of freshly made celloidin solution are used. As will be shown in my next paper, the freshly made mixture can be diluted with any convenient amount of ether without appreciably changing the permeability of the membrane. This dilution makes it easier to pour the celloidin solution over a large area. Even if all the conditions of preparation are apparently identical, however, a large membrane is eventually more permeable than a single membrane if prepared in a wet atmosphere, and less permeable if prepared in a dry one. This is due to the longer time required to dry larger membranes. The correct time of drying for large membranes is determined in the same way as that for the single standard membrane, K being found experimentally for each size of the covered surface. Once the filter has been cut the remnants can be dried and used again in the same way as dry celloidin, as the solvents do not change its chemical composition. They must not be kept too long in the dry state however, as nitrocellulose changes to oxycellulose in the presence of air. The amount of dexterity required in making large membranes is much greater than that required in the production of small ones. I, therefore, advise that their preparation should not be attempted until the technique required to produce the smaller filters has been thoroughly mastered.