6.3.01 S. aureus, also follow procedures under 991.48A(b) (see 6.2.03).

AOAC Official Method 955.16 Prepare, in sterile glass-stoppered cylinders, NaOCl solutions
Chlorine (Available) in Disinfectants
Germicidal Equivalent Concentration
First Action 1955
Final Action
(Applicable to H2O-miscible disinfectants for determining
available Cl germicidal equivalent concentrations with products
offered for use as san i tiz ing rinses for pre vi ously cleaned
nonporous surfaces, especially where speed of action and capacity
are essential considerations.)
A. Reagents
Use reagents specified in 955.11A, B(e), and (f) (see 6.1.01), and
in addition:
(a) Sterile distilled water.—See 991.47A(f) (see 6.2.02).
(b) Sterile phosphate buffer solution.—pH 8.0. Add 97.5 mL
solution containing 11.61 g anhydrous K2HPO4 in 1 L H2O to 2.5 mL
solution containing 9.08 g anhydrous KH2PO4 in 1 L H2O and
autoclave 20 min at 121°C in cotton-plugged Erlenmeyer.
(c) NaOCl standard stock solution.—Approximately 5%. Store
NaOCl stock solution in tightly closed bottle in refrigerator, and
determine exact available Cl concentration at frequent intervals by
As2O3 titration. Transfer 20 mL sample to 1 L volumetric flask and
dilute to volume. Pipet 50 mL aliquot of mixture into 200 mL
Erlenmeyer. Add excess As2O3 solution and then decided excess
NaHCO3. Titrate excess As2O3 with standard I2 solution, using
starch solution (mix ca 2 g finely powdered potato starch with cold
water to thin paste; add ca 200 mL boiling water, stirring constantly,
and immediately discontinue heating; add ca 1 mL Hg, shake, and let
solution stand over Hg), or use the I2 as its own indicator. Subtract
volume I2 solution, corrected to 0.1M, from volume As2O3 solution
used, and from this value and specific gravity of solution, calculate
percent NaOCl.
1 mL 0.025M As2O3 = 0.003722 g NaOCl
(d) Test organisms.—Use Salmonella typhi ATCC No. 6539 or
Staphylococcus aureus ATCC No. 6538 or both.
B. Apparatus
See 955.11B (see 6.1.01).
C. Operating Technique
Determine resistance of test culture to phenol as in 955.11
(see 6.1.01), and use cultures with resistance spec ified. For
containing 200, 100, and 50 ppm (µg/mL) available Cl in sterile
buffer solution, A(b). Transfer 10 mL of each solution to 25 × 150 mm
medication tubes, place tubes in 20°C water bath, and let come to
temperature.
Starting with tube containing 200 ppm (µg/mL) available Cl, add
0.05 mL test culture prepared as in 955.11B(e) (see 6.1.01), shake,
and return to water bath. After 1 min, make transfer to tube of
appropriate subculture medium, 955.11A(d) (see 6.1.01), using
flamed 4 mm loop. At 1.5 min, add another 0.05 mL culture to the
200 ppm (µg/mL) Cl solution, shake, and return to bath. After
additional 1 min interval (2.5 min in test), make second subculture in
same manner, and in 30 s, or at 3 min time in test, add another
0.05 mL culture, shaking and returning to water bath. After another
1 min interval (4 min in test), make another transfer to tube of
subculture medium.
Repeat operation to give total of 10 added increments. This
requires total time of 14.5 min for each solution and addition of
0.5 mL total culture with subculture at standard 1 min intervals after
addition of culture aliquots. At conclusion of test shake all
subculture tubes and incubate 48 h at 37°C.
Repeat operation with solutions containing 100 and 50 ppm
(µg/mL) available Cl. Prepare solution of germicide to be tested at
concentration recommended or selected for study in sterile water in
glass-stoppered grad uate. Transfer 10 mL to 25 × 150 mm
medication tubes, place in water bath, and let come to temperature.
Repeat operation with this solution.
To be considered equivalent in disinfecting activity to 200 ppm
(µg/mL) available Cl, unknown germicide must show absence of
growth in as many consecutive tubes of subculture tube series as
200 ppm (µg/mL) available Cl standard. Determine activity
equivalent to 100 and 50 ppm (µg/mL) available Cl in same manner.
See example, Table 955.16.
In this example, 25 ppm (µg/mL) solution of germicide X could
be considered equivalent to 200 ppm (µg/mL) solution of available
Cl, and 20 ppm (µg/mL) solution equivalent to 100 ppm (µg/mL) of
available Cl, but 10 ppm (µg/mL) solution of germicide X would not
be considered equivalent in germicidal activity to 50 ppm (µg/mL)
of available Cl.
Draw con clu sions rel a ti ve to ger mi cidal equiv a le nt
concentrations only when resistance of test culture to NaOCl control
is such that ≥1 negative increment is obtained at 50 ppm (µg/mL)

a
Table 955.16. Example for determination of chlorine germicidal equivalent concentration

Concentration, ppm Subculture series

Germicide (µg/mL) available Cl 1 2 3 4 5 6 7 8 9 10
NaOCl control 200 – – – – – + + + + +
100 – – – + + + + + + +
50 – – + + + + + + + +
Unknown (X) 25 – – – – – + + + + +
20 – – – – + + + + + +
10 – + + + + + + + + +
a
– = No growth; + = growth.

 2005 AOAC INTERNATIONAL

concentration and 1 positive increment is obtained at 200 ppm
(µg/mL) level.
References: Soap Sanit. Chem. 27, No. 2, 133(1951).
JAOAC 38, 274(1955); 40, 755(1957).

 2005 AOAC INTERNATIONAL