ONE OF THE MOST ACTIVE FIELDS of experimental biology deals with organisms that are too small to see. Yet microbiology attracts few amateur experimenters. The reasons are doubtless that experiments with microorganisms seem to require advanced techniques and elaborate apparatus, and even to involve the risk of dangerous infection. Actually the amateur can perform rewarding microbiological experiments with modest equipment and, given simple precautions, with a high degree of safety.

The cultivation of microorganisms resembles farming in miniature. The soil in which the organisms are grown consists of nutrient solutions or jellies; the climate is provided by artificial means. By manipulating this environment the characteristics of a microorganism can be sharply defined. The experiment described here demonstrates how certain bacteria are affected by bacteriostatic agents. It is presented by Robert Lawrence and Henry Soloway, students in the College of Medicine of the State University of New York.

"The experiment," they write, "is designed to demonstrate how certain drugs retard the growth of selected bacteria. Such drugs are called bacteriostatic.

"In testing such substances one first cultivates a selected bacterium in an environment which encourages its growth, subjects it to the bacteriostatic agent and then measures the result. Bacteria, like other forms of life, have preferences in foods, temperature, moisture and so on. Hence no universal culture medium, in which all organisms thrive equally, has been developed. Media must be compounded according to the preferences of the bacteria under study. However, one medium in which some thousands of organisms thrive consists of beef broth, the familiar consommé of the dinner table, which has been refined and specially treated. It is used both in liquid form and, when thickened by the addition of agar, as a stiff jelly.

"As in ordinary farming, 'weeds' must be kept down. One is often interested in the characteristics of a single species of bacterium, and since the size of the organism makes 'weeding' impractical, intruders must be prevented from gaining a foothold in the first place. This is accomplished by killing all microscopic life in the environment of the experiment except the desired organism. The culture medium and all equipment is sterilized, exposed only to sterilized air and otherwise kept scrupulously aseptic during the experiment.

"These conditions can be maintained if the amateur provides himself with an aseptic transfer chamber in which all critical operations are performed. This can be a simple wooden box two feet high, two feet wide and three feet long. It is fitted with a glass top, a small door and a pair of holes in one side large enough to admit the hands and forearms comfortably. The cracks should be caulked with cotton or sealing compound. A short pair of muslin sleeves may be tacked around the holes to serve as barriers against the outside air. Inside the box one should place, among other things, an alcohol lamp or Bunsen burner, and a small atomizer of the nosespray type containing Lysol or Clorox.

"Glassware should include two dozen Petri dishes, which have flat bottoms about four inches in diameter, sides about half an inch high, and are fitted with covers. The experimenter will also need three Erlenmeyer flasks of one-liter capacity and half a dozen of the quarter-liter size, a half-dozen test tubes of 20-milliliter capacity and a rack for supporting them, a dozen one-milliliter pipettes and a special rubber bulb or syringe for filling them, a 50-milliliter graduated cylinder, a wax pencil for ' marking the glassware, a dissecting needle fitted to a pencil-sized wooden holder, a small loop of thin wire fitted to a similar handle, a glass stirring rod about eight inches long, and a pair of tweezers. All these things should be assembled, together with the special wooden box or transfer chamber, on a substantial bench located where the materials will not be disturbed.

"All the materials are then sterilized. Petri dishes and pipettes are tightly wrapped in lots of six in brown paper. Larger items are wrapped individually. Test tubes and Erlenmeyer flasks may be plugged with wads of absorbent cotton instead of being wrapped in brown paper. The glassware is then placed in an oven and heated to 325 degrees Fahrenheit for at least two hours. None of the packages should be opened nor the cotton removed until the glassware is used.

"Dehydrated culture medium, both plain and in the form of an agar infusion, may be purchased from the Difco Laboratories, Detroit, Mich., or from the Baltimore Biological Laboratory, Baltimore, Md. A principal object of the experiment, however, is to provide the amateur with experience in the basic techniques of culturing bacteria. The beginner is therefore urged to prepare his own culture medium.

"Stir a pound of freshly ground hamburger into a liter of distilled water and put it in the icebox (at about 40 degrees F.) for 10 hours. Then skim off the fat which rises to the top and filter the remaining liquor through a single thickness of clean muslin. Add distilled water to bring the liquor back to a full liter, then add five grams of peptone and five grams of ordinary table salt and stir until the salt is dissolved. Pour 50 milliliters into a second flask and set it aside. Then add 15 grams of agar to the 950-milliliter portion.

"Bacteria, like other organisms, are sensitive to the acid-base balance of the medium in which they grow. Those grown in this experiment prefer a neutral medium. The two solutions just prepared will be slightly acid; they must accordingly be adjusted to neutrality (pH 7) by adding precisely enough sodium hydroxide to counteract the acid. Mix 10 grams of sodium hydroxide in a liter of distilled water. Test the beef broth with a piece of blue litmus paper. An acid broth will turn the blue paper red. The sodium-hydroxide solution will turn red litmus blue. Add a drop or two of sodium hydroxide to the liquor, stir, and with the glass rod put a drop of the solution on a piece of blue litmus. The paper in contact with the drop will probably turn pink. Add more sodium hydroxide to the liquor and test again. Continue this until the test drop causes no change in the color of either red or blue litmus.

"Each container of liquor is then heated almost to 212 degrees F. for half an hour. This will precipitate the proteins in the liquor. The proteins are removed by passing the hot liquor through coarse filter paper. Each filtrate is again brought up to volume by adding distilled water

"One hundred milliliters of the hot agar medium are poured into each of six Erlenmeyer flasks, which are then stoppered with wads of absorbent cotton. Five milliliters of the liquor containing no agar are poured into each of 10 test tubes, which are similarly stoppered.

"The media are now sterilized. The containers may be placed in boiling water for half an hour on each of three successive days. They may alternatively be sterilized in a pressure cooker. Put the containers inside the cooker, add two inches of water and pressure-cook for 20 minutes. Be sure to cool the cooker slowly. Rushing the job by quenching the cooker with cold water will cause the internal pressure to drop suddenly and the vessels of hot medium to boil over. Stoppered tubes of tap water and other solutions may be sterilized by either of these methods.

"Any nonpathogenic strain of bacteria may be employed for the demonstration of bacteriostasis. Micrococcus pyogenes var. albus, Proteus vulgaris or Alcaligenes faecalis can be used in the experiment and may be purchased at low cost from the American Type Culture Collection, 2112 M Street, N.W., Washington 6, D.C. Amateurs may wonder why one should go to the expense of buying bacteria if they are plentiful in the air. You can, of course, grow your own simply by exposing a quantity of the culture medium to the air and incubating it for 24 .hours. Let us emphasize that this is both pointless and extremely dangerous. It is pointless because the average amateur has no means of identifying the microbes he has caught. It is dangerous because he is likely to capture and cultivate deadly disease organisms. Incidentally, media that have been used should be sterilized and discarded immediately. Otherwise the experimenter runs a serious health risk. The strains recommended are inoffensive and have the further advantage of being accessible to all workers. Results of experiments may accordingly be compared. Purchased cultures can be perpetuated indefinitely by keeping them in beef broth at room temperature and inoculating a fresh tube of medium (by putting a drop of the old culture into it) every other day. If the culture can be stored at 40 degrees F., the growth of the bacteria is slowed and new media need be inoculated only once every six days.

"To perform the bacteriostasis experiment, first place a test tube of sterile beef broth, the tube containing the flourishing culture, and the wire loop inside the transfer chamber. The chamber is sprayed thoroughly with germicide and the droplets are allowed to settle for five minutes. The alcohol lamp or Bunsen burner is lit and the wire loop heated to redness as far as the handle. Hold both test tubes obliquely in the left hand and the sterile wire loop in the right. Remove both cotton plugs from the tubes with the last two fingers of the right hand. The mouths of both tubes are passed slowly through the flame. The wire loop is then dipped into the flourishing culture for about a second, withdrawn and inserted into the tube containing the sterile broth. Both cotton plugs are replaced and the wire loop is again sterilized by heating to redness. To avoid contaminating the pure culture the beginner should run through these operations with empty tubes a few times for practice.

"The freshly inoculated tube is permitted to incubate for two hours at room temperature and is then stored at 40 degrees F. At the end of 24 hours the tube is swirled in front of a light. The presence of sediment indicates that the inoculation has 'taken.' The purchased culture may then be sterilized and discarded. If at the end of 24 hours there is no sediment, the procedure should-be repeated. It is useless to wait another 24 hours.

"Antibiotics for diagnostic purposes may be procured in sterile containers with the trade name of Dia-Discs. They are manufactured by Reed and Carnrick of Jersey City, N.J. The assortment includes pellets of penicillin, bacitracin, streptomycin, Chloromycetin, Aureomycin and Terramycin in two strengths, the stronger having 10 times the potency of the weaker.

"The bacteriostasis test consists in exposing a series of increasingly concentrated cultures of bacteria growing on plates of agar medium to the action of the drugs. A duplicate set of plates is used as a control for detecting contamination. Begin the experiment by placing the following materials in the sterile transfer chamber: (1) a liter of sterilized tap water, (2) two packages of sterile Petri dishes, (3) a dozen sterilized one-milliliter pipettes and the sterilized rubber squeeze bulb, (4) a water bath heated to 112 degrees F. in which has been placed six Erlenmeyer flasks of agar medium, (5) a test-tube rack containing six test tubes, (6), the wax pencil and (7) an open bowl of germicide.

"The packaged glassware is opened and the cotton stoppers removed from the culture and test tubes. A small tuft of sterilized cotton is placed in the neck of each pipette. Nine milliliters of sterile tap water are poured into each of the six test tubes. The following operations are then carefully performed, each piece of glassware being labeled or coded as it is used. Fit the squeeze bulb to a pipette and with it transfer one milliliter of the culture to a test tube of tap water. This tube is Labeled 1:10, indicating that it contains one part of culture in 10 by volume. The tube is swirled for 30 seconds to assure thorough mixing. Remove the squeeze bulb from the pipette and drop the used pipette in the bowl of germicide. Select another sterile pipette and transfer one milliliter of the 1:10 mixture to a tube of tap water. Mark this tube 1:100. Again swirl the 1:100 mixture for 30 seconds, drop the used pipette into the germicide and with another sterile pipette transfer one milliliter of the 1:100 mixture to the third tube of tap water. Mark this tube 1:1,000 and proceed in the same way with the remaining tubes, labeling them 1:10,000, 1:100,000 and 1:1,000,000.

"A specimen of melted agar medium is now poured from each of the six Erlenmeyer flasks into six Petri dishes, each dish being labeled to correspond with the flask from which it is poured. The dishes are then covered with their glass tops and set aside to harden. After these control plates have been poured, each batch of melted medium remaining in the flasks is inoculated with one of the dilutions in the test tubes. Pipette one milliliter of the dilution into the appropriately labeled flask. Drop the used pipette into the bowl of germicide. The flasks are stoppered with cotton and swirled gently for 30 seconds to mix their contents. The water dilutions are sterilized and discarded.

"The control plates are incubated two days at 80 degrees F. The transfer box can be made to double as an incubator by fitting it with a 100-watt bulb controlled by a thermostat of the type used: in tropical-fish aquariums.

"Twelve Petri dishes, the Dia-Discs and a pair of forceps are next introduced into the transfer chamber. The chamber is sterilized as before. Two Petri dishes are then filled from each of the six inoculated flasks, each pair being labeled to show the culture dilution. The dishes are permitted to stand for about 20 minutes until the agar solidifies. The forceps are then passed through the flame, the box of Dia-Discs opened and the disc placed carefully on the agar medium means of the forceps. The weaker disc are placed on one plate of the pair an the stronger on the other. One way t keep track of the discs is to draw a radius on the back of each Petri dish with the wax pencil. The disc of Aureomycin is then placed on this line. All other discs are placed alphabetically, according to the name of the drug, in a clockwise circle. Crowding should be avoided; if space is limited, the last disc can be placed in the center of the plate. Covers are then placed on the dishes, and the culture is left to incubate for two days at 80 degrees F.

"The effects of the several drugs on the various concentrations of bacteria are then evaluated by observing the growth on the plates and the diameter of the rings around each drug where growth has been inhibited. The larger the ring, the greater the bacteriostasis. The results may be tabulated by using a minus sign to indicate no growth, a plus-and-minus sign for minimum inhibition, a plus sign for marked inhibition and two plus signs for extreme inhibition. Any growth on the control plates indicates contamination and invalidates the experiment. The test plates should be reread after four days of incubation, then sterilized and discarded.

"An interesting modification of the test permits the experimenter to chart the effect of the drugs with respect to time. Thus he can study the interval following inoculation at which each antibiotic exerts its greatest action, and the rate, if any, at which it loses its effect. This requires the construction of a relatively simple light-meter capable of reading the relative transmission of light through a test tube. Bacterial growth in beef broth increases the turbidity of the broth and reduces its transparency. When the broth is placed in a test tube its turbidity-and hence its population of bacteria-can be measured by the light-meter. The device consists of a lamp and lens for focusing a beam on the side of a test tube, a photocell on the other side of the tube for receiving the transmitted light, and a microammeter for reading the output of the cell. The light source, lens assembly, test tube and photocell are mounted in an appropriately compartmented and light-tight box [see illustration below].

"Here the operations are conducted in test tubes rather than agar. A tablet of antibiotic is dissolved in 10 milliliters of sterile tap water (in the aseptic transfer chamber). Dilutions of this solution are prepared as before, so that six dilutions span the range from 1:10 to 1:1,000,000. Observe that in this case it is the drugs, not the cultures, which are diluted. One milliliter of each of the dilutions is added to a sterile test tube which contains four milliliters of beef broth. The tubes are then inoculated with one loop of bacteria from a two-day-old beef-broth culture and left to incubate at 80 degrees F. At equal intervals during the incubation, say every three hours, the tubes are gently swirled and their turbidity is measured by means of the light-meter. Turbidity is then plotted against time The result is a set of graphs showing bacteriostatic activity. The test tubes should be inspected for optical uniformity by means of the light-meter before they are used. Professional light-measuring instruments used for this test are usually calibrated in accordance with Beer's law, which states that, for solutions of a given substance in a given solvent, light will be absorbed in proportion to the thickness of the solutions. Graphs made with instruments calibrated arbitrarily will show accurate rates of bacteriostatic effect, although the curves will not necessarily conform to those drawn with the aid of professional instruments. Tests which employ light-measuring devices can have great practical value because they show which antibiotic can be employed most effectively against a bacterium about which no data has been collected. They also disclose whether a known bacterial strain has mutated to become more resistant to a given drug.

"In the course of experiments employing plates of agar medium one may occasionally observe a small colony flourishing within the circle of inhibition. The chances are that this is a contaminant. There is always the possibility, however, that the organism is a mutant, a new strain which has been naturally selected over the original strain susceptible to the drug. All such unusual colonies should be isolated and cultured. (A portion of the colony is lifted from the plate with the tip of the dissection needle and transferred to fresh medium for incubation.) Tests can then be performed to learn if it is in fact a mutant or merely a contaminant.

Bibliography

MICROBES AND YOU. Stanley E. Wedberg. The Macmillan Company, 1957.

Suppliers and Organizations

The Society for Amateur Scientists
5600 Post Road, #114-341
East Greenwich, RI 02818
Phone: 1-401-823-7800

Internet: http://www.sas.org/