6.1.01
0 g beef extract
AOAC Official Method 955.11
Testing Disinfectants
against Salmonella typhi
Phenol Coefficient Method
First Action 1955
Final Action 1964
(Applicable to testing disinfectants miscible with H2O or for standard
resistance of test bacteria. The 95% confidence limits are ±12%.)
A. Culture Media
(a) Nutrient broth.—Boil 5 g beef extract (Difco, No. 0126),
5 g NaCl, and 10 g peptone (Anatone, peptic hydrolysate of pork
tissues, manufactured by American Laboratories, Inc., 4410 S
102nd St, Omaha, NE 68127 USA) in 1 L H2O 20 min, and dilute
to volume with H2O; adjust to pH 6.8. (If colorimetric method is
used, adjust broth to give dark green with bromothymol blue.) Fil-
ter through paper, place 10 mL portions in 20 × 150 mm test tubes,
and autoclave 20 min at 121°C. Use this broth for daily transfers
of test cultures.
(b) Synthetic broth.—Solution A.—Dissolve 0.05 g L-cystine,
0.37 g DL-methionine, 0.4 g L-arginine⋅HCl, 0.3 g DL-histidine⋅HCl,
0.85 g L-lysine⋅HCl, 0.21 g L-tyrosine, 0.5 g DL-threonine, 1.0 g
DL-valine, 0.8 g L-leucine, 0.44 g DL-isoleucine, 0.06 g glycine,
0.61 g DL-serine, 0.43 g DL-alanine, 1.3 g L-glutamic acid⋅HCl,
0.45 g L -aspartic acid, 0.26 g DL -phenylalanine, 0.05 g
DL-tryptophan, and 0.05 g L-proline in 500 mL H2O containing
18 mL 1M NaOH. Solution B.—Dissolve 3.0 g NaCl, 0.2 g KCl,
0.1 g MgSO4⋅7H2O, 1.5 g KH2PO4, 4.0 g Na2HPO4, 0.01 g thia-
mine⋅HCl, and 0.01 g niacinamide in 500 mL H2O. Mix solutions A
and B, dispense in 10 mL portions in 20 × 150 mm tubes, and auto-
clave 20 min at 121°C. Before using for daily transfers of test cul-
tures, aseptically add 0.1 mL sterile 10% glucose solution per tube.
Grow cultures with tube slanted 8° from horizontal. Bactosynthetic
Broth AOAC (Difco) may be substituted.
(c) Nutrient agar.—Dissolve Bacto agar (Difco, No. 0140) to
1.5% in nutrient broth and adjust to pH 7.2–7.4 or in synthetic broth,
tube, autoclave, and slant. Use for maintenance of cultures.
(d) Subculture media.—Use (1), (2), or (3), whichever gives low-
est result (greatest resistance). (Commercial dehydrated brands
made to conform with preceding specifications may be used.) With
oxidizing products and products formulated with toxic compounds
containing certain heavy metals like Hg, (2) will usually give lowest
result. With products containing cationic surface active materials,
(3) will usually give lowest result. Other neutralizing chemicals can
be used appropriate to chemical under study and compatible to me-
dia used for growth of bacteria. (1) Nutrient broth.—Described in
(a). (2) Fluid thioglycolate medium USP XX.—Mix 0.5 g L-cystine,
0.75 g agar, 2.5 g NaCl, 5.5 g glucose⋅H2O, 5.0 g H2O-soluble yeast
extract, and 15.0 g pancreatic digest of casein with 1 L H2O. Heat on
H2O bath to dissolve, add 0.5 g sodium thioglycolate or 0.3 g
thioglycolic acid, and adjust with 1M NaOH to pH 7.1 ± 0.2. If filtra-
tion is necessary, reheat without boiling and filter hot through moist-
ened filter paper. Add 1.0 mL freshly prepared 0.1% sodium
resazurin solution, transfer 10 mL portions to 20 × 150 mm tubes,
and autoclave 20 min at 121°C. Cool at once to 25°C and store at
20–30°C protected from light. (3) “Letheen broth.”—Dissolve
0.7 g lecithin (Alcolec Granules, American Lecithin, PO Box 1908,
Danbury, CT 06813, USA, available in 25–50 kg quantities only)
and 5.0 g polysorbate 80 (Tween 80, or equivalent) in 400 mL hot
H2O and boil until clear. Add 600 mL solution of 5.
(Difco), 10.0 g peptone [Anatone, (a)], and 5 g NaCl in H2O, and boil
10 min. Adjust with 1M NaOH and/or 1M HCl to pH 7.0 ± 0.2 and
filter through coarse paper; transfer 10 mL portions to 20 × 150 mm
tubes, and autoclave 20 min at 121°C. (Can also be obtained from
Difco or BBL.) (4) Other subculture media.—Use (d)(2) with 0.7 g
lecithin (Alcolec Granules, American Lecithin), and 5.0 g polysorbate
80 (Tween 80, or equivalent) added; or suspend 29.8 g prepared fluid
thioglycolate medium (Difco, No. 0256), 0.7 g lecithin, and 5.0 g
polysorbate 80 in 1 L H2O, and boil until solution is clear. Cool, dis-
pense in 10 mL portions in 20 × 150 mm tubes, and autoclave 20 min at
121°C. Store at 20–30°C. Protect from light.
B. Apparatus and Reagents
(a) Glassware.—1, 5, and 10 mL volumetric pipets; 1, 5, and
10 mL Mohr pipets graduated to 0.1 mL or less (plastic pipets can be
substituted if sterility controls are included for pipets in the test pro-
cedure); 100 mL glass-stoppered cylinders graduated in 1 mL divi-
sions (plastic pipets can be substituted if sterility controls are
included for pipets in the test procedure); Pyrex lipped test tubes,
25 × 150 mm reusable or disposable borosilicate; bacteriological
culture tubes, 20 × 150 mm (test culture and subculture tubes). Cap
tubes with Morton enclosures. Sterilize all glassware 2 h in hot air
oven at 180°C. Loosely plug pipets with cotton at mouth and place in
closed metal containers before sterilizing.
(b) Water bath.—Constant temperature relatively deep H2O bath
capable of maintaining 20 ± 0.2°C, with cover having
≥10 well-spaced holes which admit test tubes but not their lips.
(c) Racks.—Any convenient style. Have holes well spaced to en-
sure quick manipulation of tubes. It is convenient to have them large
enough to admit tubes while dilutions are being made.
(d) Transfer loop.—Make 4 mm id single loop at end of 50–75 mm
(2–3 in.) Pt or Pt alloy wire No. 23 B&S gage or 4 mm loop fused on
75 mm (3 in.) shaft (available from Matthey-Bishop, Inc., 1401 King
Rd, West Chester, PA 19380, USA). Fit other end in suitable holder
(glass or Al rod). Bend loop at 30° angle with stem, Figure 955.11.
(e) Test organism.—Salmonella typhi, ATCC No. 6539. Maintain
stock culture on nutrient agar slants by monthly transfers. Incubate
new stock transfer 2 days at 37°C; then store at 2–5°C. From stock
culture inoculate tube of nutrient broth and make at least 4 consecu-
tive daily transfers (≤30) in nutrient broth, incubating at 37°C, be-
Figure 955.11—Transfer loop and manner of using in
phenol coefficient technique.
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Table 955.11A Methods for calculating phenol coefficient C. Operating Technique
number (example) Make 1% stock dilution of substance to be tested (or any other
convenient dilution, depending on anticipated concentration) in
Disinfectant (X)
glass-stoppered cylinder. Make final dilutions, from 1% stock dilu-
Dilution 5 min 10 min 15 min tion, directly into test tubes and remove all excess >5 mL. (Range of
1–300 0 0 0 dilutions should cover killing limits of disinfectant in 5–15 min and
should at same time be close enough for accuracy.) From 5% stock
1–325 + 0 0 phenol solution (1–20) dilute further to make 1–90 and 1–100 dilu-
1–350 + 0 0 tions, and place in medication tubes. Place these tubes, containing
5 mL each of final dilutions of disinfectant and of phenol, and tube
1–375 + + 0
containing test culture in H2O bath at 20°C and leave 5 min. Add
1–400 + + + 0.5 mL test culture to each of dilutions at time intervals corresponding
Phenol to intervals at which transfers are to be made. (Thus, by time 10 tubes
have been seeded at 30 s intervals, 4.5 min has elapsed, and 30 s interval
1– 90 + 0 0
intervenes before transference to subculture begins.) Add culture from
1–100 + + + graduated pipet large enough to seed all tubes in any one set.
350 In inoculating test tubes, hold them in slanting position after re-
Phenol coefficient would be = 3.89
90 moval from bath, insert pipet to just above surface of disinfectant,
and run in culture without letting tip touch disinfectant. After adding
culture, agitate tubes gently but thoroughly to ensure even distribu-
tion of bacteria, and replace in bath; 5 min after seeding first test
fore using culture for testing. (If only 1 daily transfer has been tube, transfer one loopful of mixture of culture and diluted disinfec-
missed, it is not necessary to repeat the 4 consecutive transfers.) Use tant from test tube to corresponding subculture tube. To facilitate
transfer of uniform drops of antibacterial mixture, hold tube at 60° an-
22–26 h culture of organism grown in nutrient broth at 37°C in test.
gle, and withdraw loop so that plane of loop is parallel with surface of
Shake, and let settle 15 min before using. liquid (Figure 955.11). After 30 s, transfer loopful from second test
(f) Phenol stock solution.—5% (w/v). Weigh 50 g USP phenol, tube to second subculture tube and continue process for each succes-
which congeals at ≥40°C, in beaker. Dissolve in H2O, rinse solution sive dilution; 5 min after making first transfer, begin second set of
into 1 L volumetric flask, and dilute to volume. Standardize with transfers for 10 min period, and finally repeat for 15 min period.
0.0167M KBr–KBrO3 solution, (g), as follows: Transfer 25 mL Gently agitate test tubes before taking each interval loop subsample
stock solution to 500 mL volumetric flask and dilute to volume with
for transfer to subculture medium. Before each transfer, heat loop to
H2O. Transfer 15 mL aliquot of diluted solution to 500 mL I2 flask
and add 30 mL standard KBr–KBrO3 solution. Add 5 mL HCl and redness in flame and flame mouth of every tube. Sterilize loop imme-
immediately insert stopper. Shake frequently during 30 min and let diately after each transfer (before replugging tubes) to allow time for
stand 15 min. Remove stopper just enough to quickly add 5 mL 20% cooling. Use care in transferring and seeding to prevent pipet or nee-
KI solution, taking care that no Br2 vapors escape, and immediately dle from touching sides or mouth of test tube, and see that no cotton
stopper flask. Shake thoroughly, remove stopper, and rinse it and
threads adhere to inner sides or mouths of tubes. Incubate subculture
neck of flask with little H2O so that washings flow into flask. Titrate
with 0.1M Na2S2O3, using starch indicator: Mix ca 2 g finely pow- 48 h at 37°C and read results. Thoroughly agitate individual subcul-
dered potato starch with cold H2O to thin paste; add ca 200 mL boil- ture tubes before incubation. Macroscopic examination is usually
ing H2O, stirring constantly, and immediately discontinue heating. sufficient. Occasionally 3-day incubation period, agar streak, micro-
Add ca 1 mL Hg, shake, and let stand over the Hg. 1 mL 0.0167M scopic examination, or agglutination with antityphoid serum may be
KBr–KBrO3 = 0.001569 g phenol. necessary to determine feeble growth or suspected contamination.

Phenol in stock solution, % = D. Calculation

(30 – mL 0.1M Na2S2O3 solution from titration) Express results in terms of phenol coefficient number, or highest
× 0.001569 × 1333 × 100/1000 dilution killing test organism in 10 min but not in 5 min, whichever
most accurately reflects germicidal value of disinfectant. Phenol co-
where 30 = mL 0.0167M KBr–KBrO3 solution added, 0.001569 = g
efficient is number obtained by dividing numerical value of greatest
phenol equivalent to 1 mL 0.0167M KBr–KBrO3 solution, 1333 = dilu-
tion factor, and 1000 = original volume phenol stock solution. dilution (denominator of fraction expressing dilution) of disinfec-
If necessary, adjust stock solution to 5.00 ± 0.05% phenol by add- tant capable of killing Salmonella typhi in 10 min but not in 5 min by
ing H2O or phenol. Keep in well-stoppered amber bottles in cool
place, protected from light.
(g) Potassium bromide–bromate solution.—0.0167M. Prepare as
in 947.13A (see A.1.08). Standardize as follows: Transfer 30 mL to I2 Table 955.11B Satisfactory readings for phenol control
flask, and add 25 mL H2O, 5 mL 20% KI solution, and 5 mL HCl. Shake
Phenol 5 min 10 min 15 min
thoroughly and titrate with 0.1M Na2S2O3, using starch indicator.
1–90 + or 0 + or 0 0
1–100 + + + or 0

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Table 955.11C Conditions needed to establish hypothetical If none of dilutions of disinfectant shows growth in 5 min and kill-
dilution
ing in 10 min, estimate hypothetical dilution only when any 3 con-
Disinfectant (X) secutive dilutions show following results: first, no growth in 5 min;
Dilution 5 min 10 min 15 min second, growth in 5 and 10 min but not in 15 min; and third, growth
1–300 0 0 0 in 5, 10, and 15 min.
1–350 + + 0 Example: See Table 955.11C.
1–400 + + +
To avoid giving impression of fictitious accuracy, calculate phenol
coefficient to nearest 0.1. Thus, in examples cited above, phenol coef-
Phenol
ficients would be reported as 3.9 and 3.4, instead of 3.89 and 3.42.
1–90 0 0 0
[Note: Although it is commonly accepted criterion that disinfectants
1–100 + + 0
be at dilution equivalent in germicidal efficiency to phenol against
325
Phenol coefficient would be
95
= 3.42 Salmonella typhi by calculating 20 × Salmonella typhi coefficient to
determine number of parts H2O in which 1 part disinfectant may be
mixed, this must be regarded as presumptive and is subject to confir-
mation by hard surface carrier test, 991.47 (see 6.2.02).]
greatest dilution of phenol showing same results. Table 955.11A il-
lustrates how to calculate phenol coefficient number. References: J. Roy. Sanit. Inst. 24, 424(1903).
Test is satisfactory only when phenol control gives one of follow- Am. J. Public Health 3, 575(1913).
ing readings: See Table 955.11B. U.S. Dept. Agric. Circ. 198 (1931).
JAOAC 32, 408(1949); 38, 465(1955).
Soap Chem. Spec. 34, No. 10, 79(1958);
47, 176(1964); 53, 860(1970); 56, 308(1973).

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