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]