The method generally employed for the evaluation of germicides is to rate them according to their phenol coefficients. In addition to this, a number of newer methods have been proposed, which are designed to measure the toxicity of germicides for tissue as well as for the test bacteria.
1. Phenol-Coefficient Method:
The phenol-coefficient test was first proposed by Rideal and Walker (1903) for comparing and rating substances intended for the destruction of bacteria. Since that time, many modifications of the original method have been recommended.
At the present time, the standard procedure in this country is that proposed by Ruehle and Brewer (1931) of the Food and Drug Administration, U.S. Department of Agriculture. The test, in one form or another, is universally employed for examining and rating disinfectants.
Definition:
The phenol coefficient may be defined as the killing power of a germicide toward a test organism as compared with that of phenol under identical conditions.
The conditions that must be specified include:
(1) Time and Temperature of germicide,
(2) Presence and amount of organic matter,
(3) Organism used in the test,
(4) Age of the culture,
(5) Composition and reaction of the culture medium.
(6) Proportion of disinfectant to culture, and
Variations in one or more of the conditions will affect the final result. It is, therefore, of utmost importance to specify the conditions of the test; otherwise the final result will be worthless.
1. Time and Temperature:
In general, germicidal action is increased with time. This means that a higher dilution may be employed with an increase in the period of action. This applies also to temperature. An increase in temperature increases the effectiveness of a germicide, making higher dilutions possible. Germicides are not affected to the same degree by an increase in time and temperature, and for this reason, no general rule can be made.
An important exception to the rule that germicidal action is increased with time is iodine. This germicide is a vigorous oxidizing agent and acts almost immediately when placed in contact with bacteria.
2. Organic Matter:
Probably all germicides are largely reduced in activity in the presence of organic matter, although some are affected more than others. This is especially true in the presence of proteins, amino acids, and compounds of a similar nature. Results of the evaluation of germicides in aqueous solutions are quite different from those obtained when organic matter is added. The kind and amount of organic matter must be mentioned in reporting the efficiencies of germicidal substances.
3. Organism:
Germicides vary considerably in their action on different bacterial species. Some are more effective against Gram-positive than against Gram-negative bacteria, and vice versa. Still others display approximately the same degree of toxicity toward both groups of organisms. The name of the organism used in the test must be mentioned. The organisms generally used are Staphylococcus aureus (Gram +) and Salmonella typhosa (Gram -).
4. Age of the Culture:
In general, old organisms are more resistant to adverse environmental conditions than young ones. In practically all procedures for evaluating germicides, 24-hr. cultures are specified. This precaution must be observed in order that constant and comparable results be obtained.
5. Composition and Reaction of Medium:
Variations in composition and pH of culture media also affect the final results. Goetchius (1950) employed beef extract from three different sources and obtained wide variations in the phenol coefficients. Klarmann and Wright (1945) obtained similar results, which led them to propose the use of semisynthetic media for more constant results in phenol-coefficient determinations.
In general, an organism is more resistant to adverse conditions at its optimum pH. A change in the reaction of the medium on either side of the optimum pH increases the susceptibility of an organism to a germicide.
6. Proportion of Disinfectant to Culture:
A parallelism exists between the number of organisms employed in the test and the smallest amount of germicide required to destroy them. If the number of organisms is increased or decreased, the concentration of germicide required to destroy them is likewise increased or decreased.
2. Food and Drug Administration Method:
The Food and Drug Administration (FDA) method of Ruehle and Brewer (1931) is as follows: A series of dilutions of phenol and germicide to be compared are prepared in sterile distilled water contained in test tubes measuring 25 mm. in diameter and 150 mm. in length. Each tube must contain not more than 5 ml. of germicidal dilution.
The tubes are placed in a rack in a water bath, previously adjusted to a temperature of 20°C., and allowed to remain for at least 5 min. in order to bring the temperature of the germicidal dilutions to that of the water bath.
The test organism should be transferred daily for five successive days previous to use. A 24-hr. culture must be employed in the test. The culture is shaken vigorously to break up small clumps of bacteria and is then placed in the water bath for 15 min. to permit large suspended particles to settle out. One-half milliliter of culture is pipetted into each dilution of the germicide.
At intervals of 5, 10, and 15 min. a 4-mm. loopful of material is removed from each tube and transferred to a corresponding tube containing 10 ml. of broth. The subculture tubes are incubated at 37°C. for 48 hr. If the germicide is suspected of being bacteriostatic, the subculture tubes should be incubated for a longer period of time.
If mercurials, silver preparations, dyes, or other compounds exhibiting strong bacteriostatic properties are tested, it is necessary:
(i) To make secondary subcultures from the first subculture tubes immediately after the test has been completed, or
(ii) To make the first transfers to 100 ml. amounts of broth contained in flasks, or
(iii) To make transfers to broth containing substances that combine with or destroy the germicidal agent.
For example, bacteria treated with mercuric chloride contain Hg+ ions adsorbed to their cell walls. In this condition, the bacteria are not necessarily killed but merely prevented from multiplying. The numbers of Hg+ ions may be insufficient to produce death but sufficient to produce a bacteriostatic effect.
Sodium thioglycollate contains a sulfhydryl group that is capable of reacting with Hg+ ions. If mercury-treated organisms are transferred to a broth medium containing sodium thioglycollate, the germicide is removed from the bacteria by the sulfhydryl groups. This destroys the bacteriostatic effect of the mercury and permits growth of the organisms.
The phenol coefficient is calculated by dividing the highest dilution of germicide killing the test organism in 10 min. but not in 5 min. by the corresponding dilution of phenol. For example, the phenol coefficient would be 350/90 = 3.89. This means that germicide A is 3.89 times more effective than phenol.
Since Salmonella typhosa was used as the test organism, the value is referred to as the S. typhosa phenol coefficient.
Limitations of the Test:
The phenol-coefficient test was originally designed to be used for comparing the toxicity of phenol with phenol-like compounds. However, the method has been used to test compounds which are totally unlike phenol in composition and mode of action, as for example, chlorine and its compounds, mercury compounds, iodine, and quaternary ammonium germicides, leading to considerable variation in results.
Another cause of variation has been the liberties which certain investigators have taken with the test itself, modifying it to favour the compounds being tested. For example, water is employed as the diluting agent in the official test procedure; yet alcohol has been substituted for the water to improve the germicidal powers of the compound under examination.
Another departure has been the use of water containing alkali for certain germicides which are insoluble in water but soluble in alkaline solutions. Phenol coefficients so obtained do not represent true comparisons of the germicides with phenol.
Still another limitation of the test is that it is of no value in determining the efficiency of a germicide intended for clinical application. A phenol coefficient attempts to compare the toxicity of a germicide for a given organism with that of phenol but gives no information as to its effect on living tissue.
It can be seen that a germicide having a high phenol coefficient and a proportionately high toxicity to tissue would have no advantage over one having a low phenol coefficient and a proportionately low toxicity to living tissue.
3. Tissue-Toxicity Method:
A number of methods have been proposed for determining the effects of germicides on living tissue cells as well as for their ability to kill bacteria.
Several workers have tested germicides by using the inhibition of phagocytosis as a criterion of tissue toxicity. Witlin (1942), Green and Birkeland (1942, 1944), and Gershenfeld and Witlin (1947) tested the effect of germicides on the infected chorioallantoic membrane of the developing chick embryo. Nungester and Kempf (1942) swabbed the tails of anesthetized mice with a broth suspension of the test organism and allowed the culture to dry. The tails were next dipped into a solution of the germicide.
After a period of drying, the tip of the tail was cut off and inserted into the peritoneal cavity through a small incision. Survival of the animals indicated that the germicide killed the test organism.
Salle and Lazarus (1935), Salle, McOmie, and Shechmeister (1937), Salle et al. (1938, 1939), Shechmeister and Salle (1938), Foord, McOmie, and Salle (1938), and Salle and Catlin (1947) tested germicides for their effect on the viability of chick heart-tissue fragments as well as for their ability to kill bacteria. Paff, Lehman, and Halperin (1945) employed a modification of the method.
A number known as the toxicity index may be calculated from the results, which is defined as the ratio of the highest dilution of germicide required to kill the tissue cells in 10 min. to the highest dilution required to kill the test organism in the same time and under similar conditions.
Theoretically, an index less than 1 means that the germicide is more toxic to the bacteria than to the tissue; an index greater than 1 means that the germicide is more toxic to the tissue than to the bacteria. The smaller the toxicity index, the more nearly perfect the germicide.
Several germicides and their corresponding toxicity indexes. The highest degree of efficiency of the agents tested by this technique, combining low tissue toxicity with high germicidal potency against Staphylococcus aureus.