ON THE FARM where the editor of this department grew up we tried to purify drinking water by pumping it through several layers of cheesecloth. We changed the cloth when it became clogged with water bugs that lived in our well. Our neighbors used similar filters.

After some years we learned that smaller bugs came through the cheesecloth: organisms that spread dysentery and typhoid fever through town and country alike in the days of the open well, the privy, the open sewer and the manure pile. Subsequent events have proved that the idea of purifying water with a filter is perfectly sound. All we needed on the farm was a sieve with smaller holes, such as those in the filters used by the Apollo astronauts for screening bacteria out of their drinking water. This novel filtering material consists of a plastic membrane that contains millions of holes per square inch, all exactly the same size. The development of the membrane filter has given rise to a dozen new fields of experimentation that should appeal to amateurs, particularly those who have an interest in pollution control.

The new filtering material is made by mixing two kinds of plastic and forming the mixture into a membrane. The molecules of one plastic are stable. The molecules of the other plastic are relatively volatile, but they can be stabilized. To perforate the sheet the manufacturer allows molecules of the volatile plastic to evaporate until holes of the desired size form in the membrane; volatilization is then halted.

The filters are available commercially with uniform holes in a range of sizes down to a diameter of a millionth of an inch. Pores that small, which are beyond the resolving power of optical microscopes, will hold back particles as small as the virus of poliomyelitis. Although the membrane is actually clear, it resembles white paper because the perforations scatter light. Twenty percent of the membrane is solid; the rest is empty space. In contrast, the porosity of ordinary window screening is less than 55 percent.

Water passes through the membrane some 40 times faster than it does through filter paper that would retain particles of the same size. When the membrane is saturated with clear oil with a matching index of refraction, it becomes almost as transparent as glass. Indeed, a filter on which organisms have collected can be converted into the equivalent of a glass microscope slide with a drop of immersion oil.

One might assume that a plastic membrane containing 80 percent holes would be easily crushed. In actuality membranes that are properly supported withstand pressure differentials of up to five tons per square inch. Most filter papers become unreliable at pressures above 20 pounds per square inch. Incidentally, all filters can be put in two basic categories, depth filters and screen filters, according to the filtering mechanism. Depth filters consist of a thick mass of fibers, grains or fragmented substances that create a maze of flow passages where particles become lodged. Screen filters, of which the plastic membrane is an example, consist of a perforated sheet-a sieve-on which the particles collect in an accessible layer. Particles are retained on a screen filter rather than in it, with at least one important consequence: all the collected particles are exposed to view.

The fact that filtered particles collect as a uniformly distributed layer on a relatively inert sheet of porous plastic makes the membrane a convenient surface for culturing microorganisms. It takes only seconds to saturate a pad with nutrient solution and put a filter of organisms on it for growth. Nutrient reaches the organisms by diffusing through the porous membrane. When growth is complete, colonies of organisms can be examined with a microscope. They can also be converted into a permanent record of the experiment by fixing the colony with a solution of phenol glycerin.

Membrane filters are available commercially from the Millipore Corporation, Bedford, Mass. 01730. They are available in a range of sizes from about half an inch to 12 inches in diameter (and also in tubular form) and are .006 inch thick. Normally the fluid to be filtered is passed through the membrane under pressure.

Most specimen fluids can be filtered with greatest convenience by the use of a vacuum apparatus, which typically consists of a funnel provided with an outlet that supports the filter. The outlet, in turn, must make an airtight fit with a flask that receives the filtrate [see illustration at right]. Atmospheric pressure is exerted on the specimen fluid when air is pumped from the receiver flask.

It is possible to build the vacuum filtering apparatus at home. Alternatively, an apparatus specially designed for membrane filtration is available inexpensively from the Millipore Corporation, which also sells a kit that includes the vacuum filtering apparatus, a plastic syringe that functions as a vacuum pump, an alcohol lamp, an apparatus for sterilizing water, a variety of culture mediums, membrane filters, pipettes, Petri dishes and all other essentials for performing a broad range of experiments. The kit, which is called the Experiments in Environmental Microbiology Kit, includes a carrying case for convenience in making pollution tests in the field. The experiments to be described were done with the kit.

An introductory experiment that demonstrates something of the power and versatility of the filters involves the analysis of water for pollution by organisms that cause disease. Pathogenic organisms, such as Salmonella typhosa (the cause of typhoid fever), can be extremely difficult to identify. For this reason specialists routinely check water for the presence of indicator organisms that are easy to detect. Reliable indicators are the coliform bacteria that live in the intestines of animals. Their presence indicates pollution by sewage. Forty-two of the 50 states employ this test as a standard method of analyzing water pollution.

Coliform bacteria are easy to identify because they have the unique ability to transform the complex sugar known as lactose into a known sequence of simpler compounds, some of which are aldehydes. The presence of aldehydes in a growing culture positively confirms the existence in the culture of coliform bacteria. The test for aldehydes involves the use of an ingeniously contrived culture fluid known as MF-Endo medium, which contains lactose, other nutrients and basic fuchsin, a stain that is normally red but is bleached in the medium to a pale pink by the addition of sodium sulfite.

The first action of bacteria that are grown in MF-Endo medium is to partly reverse the effect of the sulfite. Some of the fuchsin and sulfite react, and the fluid turns a medium red. Most colonies of multiplying bacteria take on the red stain, but no species is distinguished by color until aldehydes form. When the aldehydes appear, some of the unreacted fuchsin-sulfite complex, the portion that did not react to change the color of the medium from red to pink, attaches itself to the aldehyde molecules and forms a shiny green coating. Only coliform bacteria convert lactose into aldehydes. Therefore colonies in the growing culture that change from red to green are coliform bacteria and provide proof that the specimen is polluted by sewage.

The analysis of water for sewage pollution proceeds in a series of steps. It begins with the selection of a specimen that is large enough to genuinely represent the water source. The specimen is filtered. Organisms that collect on the membrane filter are cultured in MF-Endo medium, and the culture is examined for the presence of coliform colonies.

How many milliliters of specimen water should be passed through the filter? The answer depends on the nature of the water. The specimen of polluted water should be sufficiently large to yield (after culturing) not more than 200 colonies, of which from 20 to 80 colonies are coliform bacteria. In the case of potable drinking water a specimen of from 100 to 200 milliliters is usually adequate. It is likely that from 50 to 100 milliliters of untreated potable water, such as water drawn from a well or a spring, would yield an equivalent count.

When testing raw water, as from rivers or lakes, the experimenter might try three volumes serially: .1 milliliter, 1 milliliter and 10 milliliters. To ensure an even distribution of the organisms across the surface of the porous membrane such small volumes of specimen water would be mixed with at least 10 milliliters of sterile water before they were filtered. In the extreme case of raw sewage the dilution would be made serially to perhaps or to provide a significant coliform count.

The concentration of organisms in water from various sources varies so widely that no hard and fast rule can be established for determining the optimum volume of a specimen to be filtered. The experimenter must exercise common sense, based on experience, and rely on trial and error when that procedure is necessary. Collect a bulk sample of water to be analyzed in a sterile container with a tight lid. Sterilize all apparatus that will come in contact with the specimen by boiling the parts for three minutes in a container of water. Sterilization should include the vacuum filtering apparatus.

The vacuum filtering apparatus that comes with the Millipore kit consists of the funnel, the filter holder, a receiving vessel, a plastic syringe, flexible tubing, a plastic valve mechanism and a funnel lid that has four tubular ports. Three of the ports are closed with plastic caps. The fourth is fitted with a filter holder, known as a "Swinnex," which is similar to a pipe union. Its two halves screw together. A filter clamped between the halves admits air to the funnel but catches contaminating particles and airborne organisms.

The syringe functions as a vacuum pump. A tapered hole in one end of the valve mechanism makes an airtight fit with the inlet of the syringe. A sidearm on the valve mechanism mates with the flexible tubing. The opposite end of the tubing fits an inlet port in the receiving vessel of the filtering apparatus. Air is pumped from the receiving vessel by operating the plunger of the syringe. A dozen strokes usually will reduce the pressure sufficiently for an adequate rate of filtering.

The Millipore kit includes a supply of Type HAWG filters and Type GS filters. The Type HAWG membrane disks are 47 millimeters in diameter and have uniform pores .45 micron (about 18 millionths of an inch) in diameter. A single 47-millimeter membrane filter is therefore perforated with some 960 million uniform pores. These membranes fit the filter holder of the vacuum filtering apparatus. The Type GS filters are 25 millimeters in diameter and have .22-micron pores. They fit a special Swinnex filter holder in the kit that mates with a companion plastic syringe. The syringe is used to force tap water through the GS filter, thus sterilizing it for experiments.

Sterilize all parts of the filtering apparatus, including the Swinnex fitting that admits air to the funnel but excluding the receiver flask. The receiver does not require sterilization because the experimenter is concerned only with the specimen organisms that remain on top of the filter membrane and anything that touches them. After boiling lift the funnel and filter base from the water with tongs. Put them on a clean sheet of wrapping paper that has been sterilized by heating to 250 degrees Fahrenheit in an oven. The funnel should rest upside down, on its larger rim, and the filter base rests with the filter-support area up. Do not wipe these units or touch the inside of the funnel or the top of the filter support.

After the pieces have drained for a short time, press the filter base down firmly over the top of the receiver flask. If the red silicone rubber O ring should fall out of its groove during the boiling, put it back with sterile forceps. Screw the blue filter base into the funnel until the O ring seats firmly. Sterilize the forceps by dipping the tips in alcohol and passing them immediately through the flame of an alcohol burner. The ignited alcohol will sterilize the tips. When the tips have cooled, use them to lift a single HAWG filter from its packet, close the packet immediately and with the forceps center the filter carefully on the holder.

Incidentally, the filters are white but are inscribed with a grid of fine dark lines that are convenient for counting colonies. The packets contain, in addition to filters, a supply of relatively thick pads in the form of disks that match the diameter of the filters. They are used for supplying nutrient to the cultures.

A pad is put in a Petri dish and saturated with nutrient solution. A filter with its collection of organisms is placed on top of the pad. Nutrient from the pad diffuses through the filter to the organisms. Remove one of the pads with the sterilized forceps and store it in a sterilized Petri dish. The packet also contains disks of blue waxed paper. They are separators and can be discarded.

Most experiments require a supply of sterile water for diluting the specimens. The sterile water can be prepared from ordinary tap water. Usually tap water has been treated with chlorine, which must be removed or it will retard the growth of the cultures. Boil the water for a minute or two to remove the chlorine. Water thus dechlorinated can be stored in a stoppered glass bottle. Sterilize the dechlorinated water immediately before use by passing it through a Type GS filter.

Put a filter in the 25-millimeter Swinnex. Observe sterile procedure. Draw a quantity of dechlorinated water into the mating syringe. Insert the tip of the syringe in the Swinnex. By operating the plunger of the syringe, force the water through the filter and into a sterilized container. Remove the syringe from the Swinnex before withdrawing the plunger. If the plunger is withdrawn while the Swinnex is joined to the syringe, air pressure will act against the porous membrane from its supported side, causing it to bend toward the unsupported side and rupture.

Assume that the initial experiment involves the analysis of water from a reasonably clean pond or stream. With the 25-millimeter Swinnex, filter 10 milliliters of dechlorinated water into the funnel of the vacuum filtering apparatus. Add about one milliliter of specimen water to the funnel and mix the solution thoroughly by swirling the funnel. Cap the funnel with its cover. It is assumed that the Swinnex admitting air to the funnel has been equipped with a filter and that the three remaining ports of the cover are closed by removable plastic caps. Connect the vacuum pump to the receiver flask with the flexible tube. Operate the plunger of the syringe to induce a brisk flow of filtrate.

Break the top from an ampule of pink MF-Endo medium and with the contents saturate the absorbent pad that was formerly stored in the Petri dish. After admitting air to the receiving flask uncouple the funnel. With sterilized forceps lift the cover from the Petri dish and transfer the membrane holding organisms from the filtering apparatus to the absorbent pad. Replace the cover with the forceps and let the culture incubate for 48 hours at room temperature (or for 24 hours at 37 degrees Celsius if you have access to an incubator). After incubation remove the membrane with flamed forceps and put it on a clear blotter for 30 minutes. With a hand magnifier count the number of colonies that have a greenish sheen. Each of the colonies will have started as a single bacterium. Multiply the number of colonies by 3,785 (the number of milliliters in a gallon) to find the number of coliform bacteria that would be present in a gallon of water.

Cultures, particularly those that disclose the presence of coliform bacteria are potentially hazardous. They should be handled with the utmost care. If they are to be retained as records, they should be treated with phenol glycerin and sealed between sheets of glass. Otherwise they should be promptly destroyed. Using forceps, remove the covers from the Petri dishes, put the dishes and their covers and contents in a large beaker or pan and cover them with liquid household bleach, straight from the bottle. After 10 minutes, and while wearing rubber gloves, remove all the parts and rinse them thoroughly under running tap water. The wet pads and filters can be put in a plastic bag for disposal.

Immerse the Petri dishes and their covers in a 70 percent (by volume) solution of alcohol diluted with tap water for 10 minutes. Rubbing alcohol of the kind that is available in drugstores is adequate. Remove the dishes and stack them upside down with the edge of each dish resting on top of its neighbor so that air circulates freely on the under surface. When the dishes dry, replace the covers and store the dishes for use.

Culture mediums have been compounded to help the experimenter in gathering specific information of various kinds. MF-Endo medium is an example of what is termed a "tagging" medium. In effect it tags coliform bacteria by imparting a distinctive color to colonies of this organism. It is also classed as a selective medium, meaning that it favors the growth of coliform bacteria and discourages the growth of other microorganisms.

Culture mediums that are similarly selective and to which chemicals or dyes have been added for imparting a distinctive color to a specific kind of organism or to its surroundings have been compounded for identifying a number of bacteria. The use of these preparations enables the experimenter to make a census of a mixed population without resorting to more laborious techniques, such as microscopic examination. Information concerning the techniques of using these and other mediums is available, particularly in publications such as the Difco Manual, published by Difco Laboratories, Detroit, Mich. 48201.

At the other extreme are mediums that impartially encourage the growth of most organisms. These compounds are useful when the experimenter wants to make a total count of all organisms in a specimen. For example, the analysis of a milliliter of pond water may turn out to contain 26 colonies of coliform bacteria. Are other bacteria present? If so, how many In order to make a complete census o the microscopic population follow the procedure of the introductory experiment but substitute Total Count medium for MF-Endo medium. A supply of Total Count medium is included in the Millipore kit.

In general, cultures that are grown for making a total count of organisms require smaller specimens than those grown on selective mediums. For ample, a specimen that yielded 200 colonies when cultured with MF-Endo medium might display several times that number when grown on Total Count medium. Most colonies, including those of coliform bacteria, are colored red by Total Count medium.

Membrane filters can of course be used for separating particles from fluid other than water. Aerosols such as pollen, particles of smoke, spherules of tar minute crystals of chemical compound and numerous similar suspensions can b collected from the air, particularly in metropolitan areas and in the vicinity of industrial establishments. The collected particles can be examined microscopically by applying a drop of immersion oil to a sheet of glass of the kind used for protecting color slides, and placing the filter membrane on top of the oil. Th membrane will appear to vanish as oil saturates the pores. The particles can then be viewed as if they were mounted on a conventional glass slide.

Readers who are not disposed to undertake experiments of this kind should not overlook a practical application o the membrane filter: its use as a mechanism for the cold sterilization of water. Campers, hunters and people on vacation in underdeveloped countries may have occasion in emergencies to drink raw water. A Swinnex equipped with .22-micron filter of the GS type will separate the smallest-known pathogenic bacterium from the filtrate. With this device and a 22-cubic-centimeter syringe one can remove the bacteria from a quart of water in a matter of minutes. (There is slight risk that polluted water will contain viruses, such as the one that cause hepatitis. They will pass through a pore structure of .22 micron.) Slime, algae and similar gross suspensions may quickly clog a membrane filter. Raw water of this kind should be cleared by passing it through a few layers of cloth or conventional filter paper before cold sterilization.

Bibliography

MICROBIOLOGICAL ANALYSIS OF WATER. Application Report AR-81. Millipore Corporation, Bedford, Mass.

ANALYSIS OF MICRON-SIZED PARTICLES. J. P. Lodge in Analytical Chemistry, Vol. 28, No. 3, pages 423-424; March, 1956.

NEW TECHNIQUES IN COLD STERILIZATION. Winston Brown in Annals of Allergy, Vol. 25, No. 5, pages 282-283; May, 1967.

The Society for Amateur Scientists (SAS) is a nonprofit research and educational organization dedicated to helping people enrich their lives by following their passion to take part in scientific adventures of all kinds.