The study of bacteria may be taken up for the disciplinary and pedagogic value of the study of a science; with the idea of extending the limits of knowledge; or for the purpose of learning their beneficial or injurious actions with the object of taking advantage of the former and combating or preventing the latter.
Since bacteria are classed as plants, their successful study implies their cultivation on a suitable soil. A growth of bacteria is called a “culture” and the “soil” or material on which they are grown is called a “culture medium.” In so far as the culture medium is made up in the laboratory it is an “artificial culture medium” as distinguished from a natural medium. A culture consisting of one kind of bacteria only is spoken of as a “pure culture,” and accurate knowledge of bacteria depends on obtaining them in “pure culture.” After getting a “pure culture” the special characteristics of the organism must be ascertained in order to distinguish it from others. The discussion of the morphology of bacteria in Chapters II, III, and IV shows that the morphological structures are too few to separate individual kinds. They serve at best to enable groups of similarly appearing forms to be arranged. Hence any further differentiation must be based on a study of the physiology of the organism as discussed in the chapters on Physiological Activities of Bacteria.
The thorough study of a bacterium involves, therefore:
1. Its isolation in pure culture.
2. Its study with the microscope to determine morphological features and staining reactions.
3. Growth on culture media for determining its physiological activities as well as morphological characteristics of the growths themselves.
4. Animal inoculations in certain instances.
5. Special serum reactions in some cases.
Since isolation in pure culture requires material for growing the organism, the first subject to be considered is culture media.
A culture medium for a given bacterium should show the following essentials:
1. It must contain all the elements necessary for the growth of the organism except those that may be obtained from the surrounding atmosphere.
2. These elements must be in a form available to the organism.
3. The medium must not be too dry, in order to furnish sufficient moisture for growth and to prevent too great a concentration of the different ingredients.
4. The reaction must be adjusted to suit the particular organism dealt with.
5. There must be no injurious substances present in concentration sufficient to inhibit the growth of the organism or to kill it.
Ordinarily, more attention must be paid to the sources of the two elements N and C than to the others, for in general the substances used to furnish these two and the water contain the other elements in sufficient amount. For very exact work on the products of bacteria, synthetic media containing definite amounts of chemicals of known composition have been prepared, but for most of the work with bacteria pathogenic to animals such media are not needed.
Culture media may be either liquid or solid, or for certain purposes may be liquid at higher temperatures and solid at lower, as indicated later. Liquid media are of value for obtaining bacteria for the study of morphology and cell groupings and for ascertaining many of the physiological activities of the organisms. Solid media are useful for studying some few of the physiological activities and especially for determining characteristic appearances of the isolated growths of bacteria. These isolated growths of bacteria on solid media are technically spoken of as “colonies,” whether they are microscopic in size or visible to the unaided eye.
It is clear that the kinds of culture media used for the study of bacteria may be unlimited but the undergraduate student will need to use a relatively small number, which will be discussed in this section.
Meat Broth (Bouillon).20—This itself is used as a medium and as the basis for the preparation of other solid and liquid media.
Finely ground lean beef is selected because it contains the necessary food materials. Fat is not desired since it is a poor food for most bacteria and in the further processes of preparation would be melted and form an undesirable film on the surface of the medium. The meat is placed in a suitable container and mixed with about twice its weight of cold water (not distilled) and allowed to soak overnight or longer. The cold water extracts from the meat water-soluble proteins, blood, carbohydrates in the form of dextrose (occasionally some glycogen), nitrogenous extractives and some of the mineral salts. The fluid is strained or pressed free from the meat. This “meat juice” should now be thoroughly boiled, which results in a coagulation of a large part of the proteins and a precipitation of some of the mineral salts, particularly phosphates of calcium and magnesium, both of which must be filtered off and the water loss restored by adding the proper amount of distilled water. The boiling is done at this point because the medium must later be heated to sterilize it and it is best to get rid of the coagulable proteins at once. The proteins thus thrown out deprive the medium of valuable nitrogenous food material which is replaced by adding about 1 per cent. by weight of commercial peptone. It is usual also (though not always necessary) to add about 0.5 per cent. by weight of common salt which helps to restore the proper concentration of mineral ingredients lost by the boiling. The chlorine is also an essential element. The reaction is now determined and adjusted to the desired end point, “standardized,” as it is called. The medium is again thoroughly boiled and filtered boiling hot. The adjusting of the reaction and the boiling ordinarily cause a precipitate to form which is largely phosphates of the alkaline earths with some protein. The filtered medium is collected in suitable containers, flasks or tubes, which are plugged with well-fitting non-absorbent cotton plugs and sterilized, best in the autoclave for twenty minutes at 15 pounds pressure, or discontinuously in streaming steam at 100°. If careful attention is paid to titration and to sufficient boiling where indicated, the meat broth prepared as above should be clear, only faintly yellowish in color and show no precipitate on cooling.
The conventional method for standardizing an acid medium is as follows: Take 5 cc of the medium, add 45 cc of distilled water and 1 cc of phenolphthalein as indicator. Boil the solution and while still hot run in from a burette N/20 NaOH solution until a faint pink color appears. From the number of cc of N/20 NaOH used to “neutralize” the 5 cc of medium it is calculated how many cc of N/1 NaOH are necessary to give the desired end reaction to the volume of medium which is to be standardized. The resulting reaction is expressed as % acid or alkaline to phenolphthalein. If it is necessary to add to each 100 cc of the medium 1 cc of N/1 NaOH to make it neutral to phenolphthalein, the reaction is called 1% acid: if to each 100 cc of medium there is added 1 cc of N/1 alkali in addition to the quantity necessary to neutralize, the reaction is called 1% alkaline.
In order to obtain a pink color when titrating with this indicator not only must the “free acid” be neutralized by the alkali but also loosely combined acid and any other substances present which will combine with the alkali rather than with the indicator so that in many media more alkali is added than is necessary to neutralize the “free acid,” i.e., the free H ions present.
It is well established that the controlling factor in the growth of bacteria in so far as “reaction” is concerned is not the titratable substances present but only the “free acid,” i.e., the number of free H ions, consequently it is better to determine the concentration of H ions and to standardize to a definite H ion concentration. Phenolphthalein as shown above is not a good indicator for this purpose.
The H ions present can be determined accurately in all cases only by electrolytic methods. The apparatus necessary is usually relatively expensive and scarcely adapted to the use of large classes of students. There are a number of indicators each of which will show color changes within rather narrow ranges of H ion concentration. Standardization by the use of these indicators, the “colorimetric method,” is recommended by the Society of American Bacteriologists and is coming into general use.
The H ion concentration is ordinarily indicated by the conventional symbol PH, e.g., the concentration in pure water which is regarded as neutral is expressed as PH 7; of normal HCl, PH 0; of normal NaOH, PH 14. The figure after PH does not in reality represent the concentration of H ions in the solution. This, like the concentration of acids, is expressed on the basis of normality, i.e., as compared with the concentration of a normal solution (1 g. equivalent) of H ions. Concentration of H ions in pure water is N/10,000,000, i.e., is 1/10,000,000 of the concentration in a normal solution of H ions. Expressed in other words, it is the concentration in a normal solution of H ions diluted ten million times. 10,000,000 = 10 to the 7th power = 107. Hence the figure after the PH indicates the logarithm of the number of times the solution is diluted. Therefore this number increases with the dilution, and the larger the figure after the PH, the less acid the solution is.
Most saprophytic organisms and many parasitic ones grow within a wide range of H ion concentration so that titration with phenolphthalein gives sufficient accuracy for media for such organisms. On the other hand, many organisms grow within a very narrow range of H ion concentration, hence accurate standardization to a definite H ion concentration is necessary. It is also evident that for comparative work, such standardization is essential because this reaction can be reproduced in other media and by other workers.21
Broth may be prepared from Liebig’s or Armour’s meat extract by adding 5 grams of either, 10 grams peptone and 5 grams NaCl to 1000 cc of water, boiling to dissolve, then titrating and filtering as above.
The author after much experience finds meat juice preferable to meat extract for broth and other media for pathogenic bacteria, and has abandoned the use of meat extracts for these organisms.
Glycerin Broth.—Glycerin broth is made by adding 4 to 6 per cent. of glycerin to the broth just previous to the sterilization. The glycerin serves as a source of carbon to certain bacteria which will not grow on the ordinary broth—as Mycobacterium tuberculosis.
Sugar Broths.—Sugar broths are used for determining the action of bacteria on these carbohydrates, since this is a valuable means of differentiating certain forms, especially those from the intestinal tract. Broth free from sugar must first be made. This is done by adding to broth prepared as already described, just previous to final filtering and sterilization, a culture of some sugar-destroying organism (Bacterium coli is ordinarily used), and then allowing the organism to grow in the raw broth at body temperature for twenty-four hours. Any carbohydrate in the broth is destroyed by the Bacterium coli. This mixture is then boiled to kill the Bacterium coli, restandardized and then 1 per cent. by weight of required sugar is added. Dextrose, saccharose and lactose are the most used, though many others are used for special purposes. After the sugar is added the medium must be sterilized by discontinuous heating at 100° for three or four successive days, because long boiling or heating in the autoclave splits up the di- and polysaccharids into simpler sugars and may even convert the simple sugars (dextrose) into acid.
Various other modified broths are frequently used for special purposes but need not be discussed here.
Dunham’s peptone solution, frequently used to determine indol production, is a solution of 1 per cent. of peptone and 0.5 per cent. of salt in tap water. It does not need to be titrated, but should be boiled and filtered into tubes or flasks and sterilized.
Nitrate Broth.—Nitrate broth for determining nitrate reduction is 1 per cent. of peptone, 0.2 per cent. of C. P. potassium nitrate dissolved in distilled water and sterilized.
Milk.—Milk is a natural culture medium much used. It should be fresh and thoroughly skimmed, best by a separator or centrifuge to get rid of the fat. If the milk is not fresh, it should be titrated as for broth and the reaction adjusted. The milk should be sterilized discontinuously to avoid splitting up the lactose as well as action on the casein and calcium phosphate.
Litmus Milk.—Litmus milk is milk as above to which litmus has been added as an acid production indicator. The milk should show blue when the litmus is added or be made to by the addition of normal NaOH solution. It should be sterilized discontinuously. Frequently on heating litmus milk the blue color disappears due to a reduction of the litmus. This blue color will reappear on shaking with air or on standing several days, due to absorption of O and oxidation of the reduced litmus, provided the heating has produced no other change in the milk, as proper heating will not.
Gelatin Culture Medium.—Gelatin to the extent of 10 to 15 per cent. is frequently added to broth and gives a culture medium of many advantages. It is solid at temperatures up to about 25° and fluid above this temperature, a property which is of great advantage in the isolation of bacteria. (See Chapter XVIII.) Further gelatin is liquefied (that is digested, converted into gelatin proteose and gelatin peptone, which are soluble in water and do not gelatinize) by many bacteria and not by others, a valuable diagnostic feature. The gelatin colonies of many bacteria are very characteristic in appearance, as is the growth of many on gelatin in culture tubes.
Gelatin medium may be prepared by adding the proper amount of gelatin (10 to 15 per cent. by weight) broken into small pieces (powdered gelatin in the same proportion may be used) to broth, gently warming until the gelatin is dissolved, standardizing as for broth, filtering and sterilizing. It is usually cleared before filtering by stirring into the gelatin solution, cooled to below 60°, the white of an egg for each 1000 cc., and then thoroughly boiling before filtering. The coagulation of the egg albumen entangles the suspended matter so that the gelatin filters perfectly clear, though with a slight yellowish color. The filtering may be done through filter paper if the gelatin is well boiled and filtered boiling hot, but is more conveniently done through absorbent cotton, wet with boiling water.
Or, the gelatin may be added to meat juice before it is boiled, then this is heated to about body temperature (not too hot, or the proteins will be coagulated too soon) until the gelatin is dissolved. Then the material is standardized and thoroughly boiled and filtered. The proteins of the meat juice coagulate and thus clear the medium without the addition of egg white. Commercial gelatin is markedly acid from the method of manufacture, hence the medium requires careful titration, even when made from a standardized broth.
Gelatin should be sterilized by discontinuous heating at 100° on three successive days, because long boiling or heating above 100° tends to hydrolyze the gelatin into gelatin proteose and peptone and it will not gelatinize on cooling. It may be heated in the autoclave for ten to fifteen minutes at 10 pounds’ pressure and sometimes not be hydrolyzed, but the procedure is uncertain and very resistant spores may not be killed. The medium should be put into the culture tubes in which it is to be used as soon as filtered, and sterilized in these, since, if put into flasks these must be sterilized, and then when transferred to tubes for use, it must be again sterilized unless great care is taken to have the tubes plugged and sterilized first, and in transferring aseptically to these tubes. These repeated heatings are very apt to decompose the gelatin, so it will not “set” on cooling. The prepared and sterilized tubes of gelatin should be kept in an ice-box or cool room, as they will melt in overheated laboratories in summer or winter.
Agar Medium.—Agar agar, usually called agar, is a complex carbohydrate substance of unknown composition obtained from certain seaweeds along the coast of Japan and Southeastern Asia. It occurs in commerce as thin translucent strips or as a powder. It resembles gelatin only in the property its solutions have of gelatinizing when cooled. Gelatin is an albuminoid closely related to the proteins, agar a carbohydrate. Agar is much less soluble in water, 1 or 1.5 per cent. of agar giving a jelly as dense as 10 to 15 per cent. of gelatin. It dissolves only in water heated to near the boiling-point (98° to 99°) and only after much longer heating. This hot solution “jells,” “sets” or gelatinizes at about 38° and remains solid until again heated to near boiling. Hence bacteria may be grown on agar at the body temperature (37°) and above, and the agar will remain solid, while gelatin media are fluid above about 25°. No pathogenic bacteria and none of the saprophytes liable to be met with in the laboratory are able to “liquefy” agar.
An agar medium is conveniently prepared from broth by adding 1 or 1.5 per cent. of the finely divided agar to the broth and boiling until dissolved, standardizing, clearing, filtering, and sterilizing. The agar must be thoroughly boiled, usually for ten to fifteen minutes, and the water loss made up by the addition of distilled water before titration. Agar is practically neutral so that there is little difference between the titration of the dissolved agar and the original broth. The agar solution should be kept hot from the beginning to the end except the cooling down to below 60°, when the egg white for clearing is added. Though filtration through paper is possible as with gelatin, if the agar solution is thoroughly boiled and filtered boiling hot, it is more satisfactory for beginners to use absorbent cotton wet with boiling water and to pour the hot agar through the same filter if not clear the first time. The solidified agar medium is never perfectly clear, but always more or less opalescent. The agar medium may be sterilized in the autoclave for fifteen minutes at 15 pounds pressure as the high temperature does not injure the agar.
Potato Media.—Potatoes furnish a natural culture medium which is very useful for the study of many bacteria. The simplest, and for most purposes the best, way to use potatoes is in culture tubes as “potato tube cultures” (No. 8, Fig. 119). These are prepared as follows: Large tubes are used. Large healthy potatoes are selected. Each end of the potato is sliced off so as to have parallel surfaces. With a cork-borer of a size to fit the tubes used, cylinders about one and one-half inches long are made. Each cylinder is cut diagonally from base to base. This furnishes two pieces each with a circular base and an oval, sloping surface. The pieces are then washed clean and dropped for a minute into boiling water to destroy the oxidizing enzyme on the surface which would otherwise cause a darkening of the potato. (The darkening may also be prevented by keeping the freshly cut potatoes covered with clean water until ready to sterilize.) A bit of cotton one-fourth to one-half inch in depth is put into each of the test-tubes to retain moisture and a piece of potato dropped in, circular base down. The tubes are then plugged with cotton and sterilized in the autoclave at 15 pounds pressure for not less than twenty-five minutes, since potatoes usually harbor very resistant spores, and it is not unusual for a few tubes to spoil even after this thorough heating.
Potatoes are sometimes used in “potato plate cultures.” The term “plate culture” is a relic of the time when flat glass plates were used for this and other “plate cultures.” Now glass dishes of the general form shown in Fig. 115, called “Petri dishes,” or plates are used for practically all plate culture work. For “potato plates” slices from potatoes are cut as large and as thick as the relative sizes of potato and dish permit (Fig. 116). The slices should be thin enough not to touch the lid and thick enough to be firm.
It is a good plan to wrap each dish separately in paper to retain the lid securely, then sterilize as for potato tubes, and leave plates wrapped until wanted.
It sometimes happens that the natural acidity of potatoes is too great for the growth of many organisms. The acidity is sufficiently corrected by soaking the pieces of potato in a 1 per cent. solution of sodium carbonate for an hour before they are put into the tubes or plates.
Glycerinized potato tubes are conveniently prepared by covering the potato in the tube with glycerin broth, sterilizing and pouring off the excess broth immediately after sterilizing, taking care that the tubes do not become contaminated which is not very probable if the work is quickly done while the tubes are still hot.
Blood Serum Media.—Blood serum, usually from the larger, domestic animals on account of convenience in securing it in quantity, is used in the study of the bacteria causing disease in man and animals. Most commonly the serum is collected from the clotted blood after it has well separated (usually about forty-eight hours is required for this). It is then run into tubes which are plugged with cotton and placed in an apparatus for coagulating the serum by heat. A copper water bath with a tightly closed air compartment or the horizontal autoclave (Fig. 81) is sufficient for this purpose, though special forms of apparatus are to be had. It is important that the temperature be raised slowly so that the blood gases escape gradually. Three to five hours or longer should be allowed for the temperature to reach the boiling-point. If the tubes are heated too rapidly, the serum is filled with bubbles and badly torn since the gases are driven off suddenly. Löffler’s serum is made by adding one part of dextrose broth to three parts of serum and then coagulating as above. The solidified serum in either case is best sterilized discontinuously, though with care the autoclave at 15 pounds pressure may be used for a single sterilization. This is very apt to cause a greater darkening of the serum and frequently also a laceration of the solid mass by escaping gases.
Blood serum is also used in the liquid state. For this purpose it is best to collect it aseptically; or it may be sterilized discontinuously at a temperature of 55° or 56° on seven to ten consecutive days. Novy has recently suggested dialyzing the serum to free it from salts and thus prevent its coagulation when heated. Whether the removal of the various “extractives” which diffuse out with the salts deprives the serum of any of its advantageous properties remains to be ascertained.
From the discussion of the physiological activities of bacteria in Chapters IX–XII it is apparent that a very great variety of culture media other than those described is necessary for the study of special types of bacteria, but such media are beyond the scope of the present work.
The ideal culture media are without a doubt the synthetic media, that is media of definite known chemical composition, so that the various changes due to the growth of bacteria can be accurately determined and thus a means of sharply differentiating closely related organisms be secured. Such media have been prepared and every bacteriologist believes strongly in their future usefulness when media of wider application shall have been devised. An example of this type of culture media is Uschinsky’s synthetic medium, of which the following is one of the modifications:
| Distilled-water | 1000 parts |
| Asparagin | 4 parts |
| Ammonium lactate | 6 parts |
| Disodium phosphate | 2 parts |
| Sodium chloride | 5 parts |
A criticism of this medium is that the elements K, Ca, Mg, Fe, Mn, and S which have been shown to be essential are not present if chemically pure salts are used in the preparation.