Methods have already been detailed (page 310) for the purpose of studying the vital resistance offered by micro-organisms to the lethal effect of germicides. But it frequently happens that the bacteriologist has to determine the relative efficiency of "disinfectants" from the standpoints of the sanitarian and commercial man rather than from the research worker's point of view. In pursuing this line of investigation, it is convenient to compare the efficiency, under laboratory conditions, of the proposed disinfectant with that of some standard germicide, such as pure phenol. In so doing, and in order that the work of different observers may be compared, conditions as nearly uniform as possible should be aimed at. The method described is one that has been in use by the writer for many years past, modified recently by the adoption of some of the recommendations of the Lancet Commission on the Standardisation of Disinfectants--particularly of the calculation for determining the phenol coefficient.

This method has many points in common with that modification of the "drop" method known as the Rideal-Walker test.

~General Considerations.~--

These may be grouped under three headings: Test Germ, Germicide, and Environment.

1. _Test Germ._--~B. coli.~

As disinfectants are tested for sanitary purposes, it is obvious that a member of the coli-typhoid group should be selected as the test germ. B.

coli is selected on account of its relative nonpathogenicity, the ease with which it can be isolated and identified by different observers in various parts of the world, the stability of its fundamental characters, and evenness of its resistance when utilised for these tests; finally since the colon bacillus is an organism which is slightly more resistant to the lethal action of germicides than the more pathogenic members of this group, a margin of safety is introduced into the test which certainly enhances its value.

B. coli should be recently isolated from a normal stool, and plated at least twice to ensure the purity of the strain; and a stock agar culture prepared which should be used throughout any particular test. For any particular experiment prepare a smear culture on agar and incubate at 37 C. for 24 hours anaerobically. Then emulsify the whole of the surface growth in 10 c.c. of sterile water. Transfer the emulsion to a sterile test-tube with some sterile gla.s.s beads and shake thoroughly to ensure h.o.m.ogenous emulsion. Transfer to a centrifuge tube and centrifugalise the emulsion to throw down any ma.s.ses of bacteria which may have escaped the disintegrating action of the beads. Pipette off the supernatant emulsion for use in the test.

_2. Germicide._--

_a. Disinfectant to be tested._--

The first essential point is to test the unknown disinfectant, which may be referred to as germicide-x, on the lines set out on page 311 to determine its inhibition coefficient.

This constant having been fixed, prepare various solutions of germicide-x with sterilised distilled water by accurate volumetric methods, commencing with a solution somewhat stronger than that representing the inhibition coefficient. The solutions must be prepared in fairly large bulk, not less than 5 c.c. of the disinfectant being utilised for the preparation of any given percentage solution.

_b. Standard Control._--~Phenol.~

The standard germicide used for comparison should be one which is not subject to variation in its chemical composition, and the one which has obtained almost universal use is Phenol.

The following table shows the effect of different percentages of carbolic acid upon B. coli for varying contact times, compiled from an experiment conducted under the standard conditions referred to under Environment. The results closely correspond to those recorded by the Lancet Commission on Disinfectants, 1909.

---------------------+----------------------------------- | Contact time in minutes.

Percentage of phenol +------+---+---+---+---+---+---+---- | 2-1/2| 5 |10 | 15| 20| 25| 30| 35 ---------------------+------+---+---+---+---+---+---+---- 1.20 | - | - | - | - | - | - | - | - 1.10 | - | - | - | - | - | - | - | - 1.0 | + | - | - | - | - | - | - | - 0.9 | + | - | - | - | - | - | - | - 0.85 | + | + | - | - | - | - | - | - 0.80 | + | + | + | - | - | - | - | - 0.75 | + | + | + | + | + | - | - | - 0.7 | + | + | + | + | + | + | - | - 0.65 | + | + | + | + | + | + | + | - ---------------------+------+---+---+---+---+---+---+----

- = No growth, i. e., bacteria killed.

+ = Growth, i. e., bacteria still living.

From this it will be seen that the following percentage solutions will need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per cent., 0.75 per cent., 0.7 per cent., as controls for each experiment.

Prepare solutions of varying percentages by weighing out the quant.i.ty of carbolic acid required for each and dissolving in 100 c.c. of pure distilled water in an accurately standardised measuring flask. The solutions must be prepared freshly as required each day.

~Environment.~--

_a. General._--

Close the windows and doors of the laboratory in which the investigation is carried out, to avoid draughts. Flush over the work bench and adjacent floor with 1:1000 solution of corrosive sublimate. Caution the a.s.sistant, if one is employed, to avoid unnecessary movement or speech.

_b. Contact Temperature_, ~15-18 C.~--

This is the temperature at which contact between the germicide and the test germ takes place, and is of importance, since some germicides (_e.

g._, Phenol) appear to be more powerful at high temperatures. 18 C.--practically the ordinary room temperature--is a temperature at which the multiplication of B. coli is a comparatively slow process, but variation of a degree above this temperature or of two or three degrees below is of no moment. If the room temperature is below 15 C. when the experiments are in progress, arrange a water-bath regulated at 18 C.

for the reception of the tubes containing the mixture of germ and germicide; if above 19 C. immerse the tubes in cold water, to which small pieces of ice are added from time to time to prevent the temperature rising above 18 C.

_c. Relative Proportional Bulk of Test Germ and Germicide_, ~50:1.~--

Five cubic centimetres is a convenient amount of germicidal solution to employ, and to this 0.1 c.c. of the emulsion of test germ should be added.

_d. Bulk of Sample Removed from Germ + Germicide Mixture at Each of the Time Periods_, ~0.1 c.c.~--

This is sufficient to afford a fair sample of the germ content of the mixture, and at the same time is insufficient to exert any inhibitory action when transferred to the subculture medium.

_e. Subculture Medium._ ~Bile Salt Broth.~--

A _fluid_ medium is essential in order to obtain immediate dilution of the germicide carried over; at the same time it is advantageous to employ a selective medium which favours the growth of the test germ to the exclusion of organisms likely to contaminate the preparation, and if possible one which affords characteristic cultural appearances.

Bile Salt Broth (page 180) combines these desiderata; it permits only the growth of intestinal bacteria, whilst the formation of an acid reaction and the production of gas in subcultures prepared from the germ-germicide mixture is fairly complete evidence of the presence of living B. coli.

The amount of medium present in each test-tube is a matter of importance, since the medium not only provides pabulum for the test germ, but also acts as a diluent to the germicide, to reduce its strength below its inhibition coefficient. For routine work each subculture tube contains 10 c.c. of medium, but it is obvious that if germicide-x possesses an inhibition coefficient of 0.1 per cent. the addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium would effectually prevent the subsequent growth of the test germ after a contact period insufficient to destroy its vitality. Hence the preliminary tests may in some instances indicate the necessity for the presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture tubes.

_f. Incubation Temperature_, ~37 C.~--

_g. Observation Period of the Subcultivations_, ~Seven Days.~--

In order to determine whether or no the test germs have been destroyed, observations must always be continued--when growth appears to be absent--up to the end of seven days before recording "no growth."

_h. Identification of the Organisms Developing in the Subcultivations after Contact in the Germ + Germicide Solution._--

This is based on the naked eye characters of the growth in the bile salt broth, supplemented where necessary by plating methods, further subcultivations upon carbohydrate media and agglutination experiments.

The sign (+) is used to indicate that growth of the test organism occurred in the subcultivations, and the sign (-) to indicate that the test germs have been destroyed and no subsequent growth has taken place.

METHOD.--

_Apparatus Required_:

Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).

Test-tube rack (Fig. 219).

Sterile graduated pipettes in case, 1 c.c. (in tenths).

Sterile graduated pipettes in case, 5 c.c. (in c.c.).

Circular rubber washers, 2.5 cm. diameter with central hole, sterilised by boiling immediately before use, then transferred to sterilised gla.s.s double dish.

Electric signal clock or stop watch.

Sterile forceps.

Sterilised gla.s.s beads.