5.3.01 pancreatic digest of gelatin, 1.5 g yeast extract, 1.

5 g beef extract,
AOAC Official Method 957.23 3.5 g NaCl, 1.0 g anhydrous glucose, 3.68 g anhydrous K2HPO4, and
Antibiotics in Feeds 1.32 g anhydrous KH2PO4 in H2O, and dilute to 1 L. Adjust with 1N
Microbiological Methods NaOH or HCl (1 + 9) so that after sterilization pH is 6.95–7.05.
(Difco Penassay Broth [antibiotic medium 3] and BBL Antibiotic
A. Culture Media Assay Broth have been found satisfactory.)
(Deionized H2O may be used for preparation of media.) (o) Broth medium B.—Dissolve 5.0 g pancreatic digest of casein,
5.0 g pancreatic digest of animal tissues, and 20 g anhydrous glucose
(a) Agar medium A.—(Antibiotic Medium 1.) Dissolve 6.0 g in H2O, and dilute to 1 L. Adjust with 1N NaOH or with HCl (1 +
pancreatic digest of gelatin, 4.0 g pancreatic digest of casein, 3.0 g 11) so that after sterilization pH is 5.6–5.7. (Difco Fluid Sabouraud
yeast extract, 1.5 g beef extract, 1.0 g anhydrous glucose, and 15 g Medium and BBL Sabouraud Liquid Broth Modified have been
agar in H2O, and dilute to 1 L. Adjust with 1N NaOH or HCl (1 + 9) found satisfactory.)
so that after sterilization pH is 6.5–6.6. (Difco Penassay Seed Agar
[antibiotic medium 1] and BBL Seed Agar have been found satis- B. Reagents
factory.) (a) Phosphate-bicarbonate buffer.—pH 8. Dissolve 16.73 g an-
(b) Agar medium B.—(Antibiotic Medium 4.) Dissolve 6.0 g hydrous K2HPO4, 0.523 g anhydrous KH2PO4, and 20 g NaHCO3 in
pancreatic digest of gelatin, 3.0 g yeast extract, 1.5 g beef extract, H2O and dilute to 1 L.
1.0 g anhydrous glucose, and 15 g agar in H2O, and dilute to 1 L. (b) Phosphate buffer.—pH 8; 0.1M. Dissolve 16.73 g anhydrous
Adjust with 1N NaOH or HCl (1 + 9) so that after sterilization pH is K2HPO4 and 0.523 g anhydrous KH2PO4 in H2O and dilute to 1 L.
6.5–6.6. (Difco and BBL Yeast Beef Agar have been found satisfac- (c) Phosphate buffer.—pH 7.0; 0.1M. Dissolve 13.6 g anhydrous
tory.) K2HPO4 and 4.0 g anhydrous KH2PO4 in H2O and dilute to 1 L.
(c) Agar medium C.—(Antibiotic Medium 2.) Dissolve 6.0 g (d) 5% Phosphate buffer.—pH 6.5. Dissolve 22.15 g anhydrous
pancreatic digest of gelatin, 3.0 g yeast extract, 1.5 g beef extract, K2HPO4 and 27.85 g anhydrous KH2PO4 in H2O and dilute to 1 L.
and 15 g agar in H2O, and dilute to 1 L. Adjust with 1N NaOH or (e) 10% Phosphate buffer.—pH 6. Dissolve 80 g anhydrous
HCl (1 + 9) so that after sterilization pH is 6.5–6.6. (Difco Penassay KH2PO4 and 20 g anhydrous K2HPO4 in H2O and dilute to 1 L.
Base Agar and BBL Base Agar have been found satisfactory.) (f) 1% Phosphate buffer.—pH 6. Dissolve 8.0 g anhydrous
(d) Agar medium D.—(Antibiotic Medium 8.) Use agar medium KH2PO4 and 2.0 g anhydrous K2HPO4 in H2O and dilute to 1 L.
C adjusted with 1N NaOH or HCl (1 + 9) so that final pH is 5.7–5.9. (g) Phosphate buffer.—pH 4.5; 0.1M. Dissolve 13.6 g anhydrous
(Difco Antibiotic Medium 8 and BBL Base Agar with low pH have KH2PO4 in H2O and dilute to 1 L.
been found satisfactory.) (h) Acid-acetone.—Mix 1 volume 4N HCl, 13 volumes acetone
(e) Agar medium E.—(Antibiotic Medium 5.) Use agar medium and 6 volumes H2O.
C adjusted with 1N NaOH so that final pH is 7.8–8.0. (Difco (i) Acid-methanol.—Mix 1 volume HCl and 50 volumes metha-
Streptomycin Assay Agar [antibiotic medium 5] and BBL Strepto- nol.
mycin Assay Agar with Yeast Extract have been found satisfactory.) (j) Ethyl acetate.—99% undenatured grade.
(f) Agar medium F.—Adjust agar medium A with 3.5N NaOH (k) Buffer-acetone extractant.—Mix equal volumes pH 6 buffer,
(2.8–3.8 mL/L) so that after sterilization pH is 8.9–9.1. (f), and acetone.
(g) Agar medium G.—(Antibiotic Medium 32.) Use agar medium (l) Tris buffer.—pH 8.0, 0.05M. Dissolve 6.05 g tris(hy-
A to which is added 300 mg MnSO4⋅H2O or 0.4 mL 1% MnCl2 droxymethyl)aminomethane (THAM, primary standard, available
solution/L. from Fisher Scientific Co.) in 900 mL H2O, adjust pH to 8.0 with
(h) Agar medium H.—Dilute 1 L agar medium A to 1.2 L and HCl, and dilute to 1 L.
adjust to pH 8.1. (m) Calcium chloride solution.—2M. Dissolve 294.04 g
(i) Agar medium I.—Dissolve 9.4 g pancreatic digest of gelatin, CaCl2⋅2H2O in H2O and dilute to 1 L.
4.7 g yeast extract, 2.4 g beef extract, 10.0 g NaCl, 10.0 g anhydrous (n) Sodium chloride-calcium chloride solution.—Dissolve 200 g
glucose, 13.0 g anhydrous KH2PO4, 1 g Na2HPO4⋅7H2O, and 23.5 NaCl in H2O, add 10 mL 2M CaCl2, and dilute to 1 L.
g agar in H2O, and dilute to 1 L. After sterilization pH is 5.3–5.5. (o) Sodium hypochlorite solution.—5.25%. Use freshly opened
(BBL Nystatin Assay Agar supplemented with 13.0 g anhydrous bottle commercial solution. (Clorox has been found satisfactory.)
KH2PO4 and 1 g Na2HPO4⋅7H2O has been found satisfactory.) Store in dark at 2–10°.
(j) Agar medium J.—(Antibiotic Medium 11.) Use agar medium (p) Sterile isotonic saline solution.—Dissolve 9.0 g NaCl in H2O
A adjusted with 1N NaOH so that final pH is 7.9–8.0. (Difco and and dilute to 1 L. Autoclave 20 min at 121°.
BBL Neomycin Assay Agar have been found satisfactory.) ( q ) L e a d a c e t a t e s o l u t i o n .— D i s s o l v e 3 0 3 m g
(k) Agar medium K.—To each L agar medium J add 12.5 mL 2M Pb(CH3COO)2⋅3H2O in H2O and dilute to 1 L with H2O.
CaCl2 after autoclaving and just before pouring plates.
C. Apparatus
(l) Agar medium L.—Dissolve 0.69 g K2HPO4, 0.45 g KH2PO4,
2.5 g yeast extract, 10.0 g anhydrous glucose, and 15.0 g Difco Noble (High-speed blender jars, after disassembling, must be cleaned with
agar in H2O and dilute to 1 L. Adjust to pH 6.0 with HCl before use. great care to eliminate all traces of antibiotics. All apparatus which
(m) Agar medium M.—Dissolve 2.5 g yeast extract, 10.0 g glu- contacts sample and solutions must be thoroughly cleaned and be
cose, 0.69 g K2HPO4, 0.45 g KH2PO4, and 20.0 g agar in H2O and detergent-free.)
dilute to 1 L. Before adding inoculum, adjust liquified medium to
pH 6.0 with 1N HCl (ca 2 mL/L). (a) Cylinders.—Polished open stainless steel cylinders, 8 ± 0.1
(n) Broth medium A.—(Antibiotic Medium 3.) Dissolve 5.0 g mm od, 6 ± 0.1 mm id, and 10 ± 0.1 mm high (obtainable from S &
L Metal Products Corp., 58–29 57th Drive, Maspeth, NY 11378).

© 1998 AOAC INTERNATIONAL

Alternatively, wells cut in agar may be used in assay. (1) Roux bottle culture.—Wash growth from 24 h slant culture
(b) Agar cutter.—Capable of cutting precise circular wells that with ca 3 mL broth medium A, and transfer liquid to surface of 300
are vertical to and reach bottom of 100 × 15 mm Petri plate. Diameter mL agar medium A in Roux bottle. Spread suspension evenly over
of cutter may be from 8 to 12 mm (±0.1mm) (available from Purdue entire surface, using sterile glass beads, and incubate 24 h at 26–32°.
University, Dept. of Biochemistry, West Lafayette, IN 47907). Wash growth from agar surface with ca 15 mL sterile isotonic saline
Equivalent system may be used. Use only 1 size cutter in assay. solution. Store bulk suspension ≤2 weeks at 2–10°.
Remove agar plugs manually or preferably by using agar cutter (2) Broth culture.—Wash growth from stock culture with ca 3 mL
connected to vacuum strong enough to remove plugs so that neither broth medium A, and transfer liquid to 100 mL broth medium A.
cracks nor rough edges occur. Volume of assay agar should be Incubate 48 h at 26–32° with continuous mechanical agitation. This
enough to form cup that can be dosed with at least 100 µL sample 48 h culture is inoculum. Store ≤2 weeks at 2–10°.
(between 15 and 20 mL medium/plate). Add same volume of stand- Use for erythromycin, lincomycin, novobiocin feed supplement,
ards and samples to wells. oleandomycin, penicillin, and tylosin assays.
(c) Petri dishes (plates).—Glass or plastic; 100 mm wide × 20 mm (f) Staphylococcus epidermidis.—ATCC No. 12228. Incubate
deep. Porcelain covers glazed on outside or cover lids with filter pad stock culture at 32°. Inoculate 30 mL broth medium A in 300 mL
inserts are satisfactory for absorbing H2O of syneresis. Glass or flask with 1 loop from stock culture, and incubate overnight at
plastic covers may be used if they are raised slightly to allow escape 26–32°. Prepare daily. Use for neomycin and for novobiocin final
of H2O. feed assays.
(d) Cylinder dispenser.—May be used to place cylinders on (g) Saccharomyces cerevisiae.—ATCC No. 9763. Incubate stock
plates. Shaw Dispenser, available from Arthur E. Farmer, 47 Frazier culture on agar medium I at 37°. Prepare inoculum by one of
St, PO Box 1785, Trenton, NJ 08618. following methods:
(1) Broth culture.—Inoculate 100 mL broth medium B with 1 loop
D. Stock Cultures and Preparation of Test Organism from stock culture and incubate overnight at 37°. This culture is
Suspensions
inoculum. Store ≤2 weeks at 2–10°.
For appropriate test organism designated below, prepare slant
(2) Roux bottle culture.—Wash growth from stock culture with
culture on ≥1 tube of agar medium A. Incubate overnight at indicated
ca 3 mL sterile isotonic saline solution, and transfer liquid to surface
temperature held constant to ±0.5°, and then store in dark at 2–10°.
of 300 mL agar medium I in Roux bottle. Spread suspension evenly
Do not use if >2 weeks old.
over entire surface, using sterile glass beads, and incubate 24 h at
Prepare suspensions of test organisms as follows:
37°. Wash growth from agar surface with ca 15 mL sterile isotonic
(a) Micrococcus luteus.—ATCC No. 10240. Incubate stock cul-
saline solution. Store ≤2 weeks at 2–10°.
ture at 32–35°. Wash growth from stock culture with ca 3 mL broth
Use for nystatin assay.
medium A and transfer liquid to surface of 300 mL agar medium A
(h) Escherichia coli.—UC 527 (available from The Upjohn Co.).
in Roux bottle. Spread suspension evenly over entire surface, using
Incubate at 36°. Inoculate 30 mL broth medium A in 250 mL flask
sterile glass beads, and incubate overnight at 32–35°. Wash growth
from stock culture of E. coli and grow 18–24 h at 36°. Prepare daily.
from agar surface with ca 25 mL sterile isotonic saline solution. Store
Use for spectinomycin assay.
bulk suspension at 2–10°. Use for bacitracin assay.
(b) Sarcina subflava.—ATCC No. 7468. Incubate stock culture E. Preparation Standards
at 32–35°. Prepare suspension as in (a) and use as alternative Except bacitracin and hygromycin, a smaller amount of standard
organism for bacitracin assay. material, 10–15 mg, may be weighed. If desired precision is ± 0.01
(c) Bacillus cereus.—ATCC No. 11778. Incubate stock culture at mg, then 5-place electronic balance should be used; if desired
30°. Wash growth from stock culture with ca 3 mL sterile H2O, precision is ± 0.1 mg, then 4-place electronic balance should be
transfer to surface of 300 mL agar medium A, and incubate 7 days used.
at 30°. Wash growth from agar surface with ca 25 mL sterile H2O
and heat suspension 30 min at 65°. Centrifuge and decant. Wash F. Design and Plotting of Standard Response Line
residual spores 3 times with sterile H2O, centrifuging and decanting Prepare concentrations of reference standard as described for
each time. Discard wash H2O. Heat residual spores 30 min at 65° each antibiotic. In general, it is preferable to use shorter 4-fold
and resuspend in sterile H2O. Store this stock suspension at 2–10°. range between lowest and highest doses of standard line. Use
Use for chlortetracycline and oxytetracycline assays. indicated concentration as reference concentration. (Values of
(d) Bacillus subtilis.—ATCC No. 6633. Incubate stock culture at standard or reference concentration could slightly vary from
37°. Wash growth from stock culture with ca 3 mL sterile isotonic those indicated for each antibiotic without affecting validity of
saline solution, transfer to surface of 300 mL agar medium G in Roux assay.)
bottle, and incubate 7 days at 37°. Wash growth from agar surface Prepare plates with appropriate base agar layer and/or appropriate
with ca 50 mL sterile isotonic saline solution into centrifuge bottle. seed agar layer; one layer of media can be substituted for 2 layers of
Heat suspension 30 min in 65° H2O bath to destroy vegetative cells. media if reference concentration gives adequate zone size as de-
Centrifuge, decant, and resuspend cells in ca 50 mL sterile isotonic scribed for each antibiotic. Distribute agar evenly by tilting plates
saline solution. Repeat heating, centrifugation, and suspending from side to side with circular motion, and let harden. Use plates
twice, or until supernate is clear. Final suspension is stock spore same day prepared.
suspension. Store at 2–10°. Use for hygromycin B, monensin, and Place 6 cylinders on each plate at ca 60° intervals on 2.8 cm radius.
streptomycin assays. Fill 3 alternate cylinders with reference concentration and other 3
(e) M. luteus.—ATCC No. 9341. Incubate stock culture at cylinders with one of other concentrations of standard. Use 3 plates
26–30°. Prepare organism suspension by one of following meth- for each concentration required for standard response line, except
ods: reference concentration. Incubate plates overnight at appropriate

© 1998 AOAC INTERNATIONAL

temperature, and measure diameters of zones of inhibition as accu- Plot values for L and H and connect with straight line. Reference
rately as possible. (In most cases, it is possible to estimate zone point is zone size intercept on arithmetic scale. This corrected reference
diameters to nearest 0.1 mm.) Values given in each method for zones point is to be used for sample calculations (if corrected reference point
of inhibition to be obtained with reference concentrations of antibi- diameter varies significantly from average reference diameter, error in
otics are for guidance only, but it is important that lowest concen- preparation of standard solutions is indicated and validity of assay is in
trations on standard response line give measurable zone and that question.) For more accuracy in calculation, determine slope of standard
slope of response line be adequate. In each set of 3 plates average response line B = (H – L)/(log h – log l), where h and l are high and low
the 9 readings of reference concentration and the 9 readings of standard concentrations, respectively, and B is the increase in zone size
concentration being tested. Average of all 36 readings of reference for each 10× increase in drug concentration.
concentration from 12 plates is correction point for response line. Computer or calculator can be used to calculate standard lines
Correct average value obtained for each concentration to appropriate whether standard concentrations are equally spaced or not. Least
figure if reference concentration reading on that set of 3 plates was square fitting using linear or polynomial equations may be per-
same as correction point. formed based on best fit (polynominal fitting is most appropriate,
For example, if in correcting second concentration of standard especially for long range 8× or 16× range).
response line, average of 36 readings of reference concentration is
20.0 mm, and average of 9 readings of reference concentration of G. Determination of Potency
this set of 3 plates is 19.8 mm, correction is +0.2 mm. If average Use 3 plates for each assay solution. On each plate fill 3 alternate
reading of second concentration on same 3 plates is 17.0 mm, cylinders with reference concentration and fill other 3 cylinders with
corrected value is 17.2 mm. Plot corrected values, including correc- assay solution. Incubate plates overnight at appropriate temperature
tion point, on semilog graph paper, using logarithmic scale for and measure diameter of zones of inhibition. Average the 9 readings
concentration and arithmetic scale for average zone diameters. Man- of reference concentration and the 9 readings of assay solution. If
ual plotting of standard lines is possible but could be subject to large assay solution gives larger average than reference concentration, add
variation. Response lines would be more accurate if calculated. difference between them to reference point on standard response
When standard doses are equally spaced, i.e., interval between line. If assay solution gives smaller value than reference concentra-
successive doses is the same, calculate L and H (calculated zone tion, subtract difference between them from reference point on
diameters for low and high concentrations, respectively, of standard standard response line. Using corrected value of assay solution,
response line) as follows: determine amount of antibiotic by reading concentration from stand-
For method specifying 5 doses of standard, ard response line.
Alternatively, determine log relative potency M′ = (Yu – Ys)/B,
L = (3a + 2b + c – e)/5 where Yu and Ys are average of 9 readings of assay solution and
reference concentration, respectively, and B is slope of standard
H = (3e + 2d + c – a)/5 response line. Antilog M′ = potency of assay solution relative to
standard; and (antilog M′) × 100 = potency of assay solution as %
where a, b, c, d, and e = corrected average zone diameters for each
of standard reference concentration.
concentration of standard.
For calculation of sample potency by computer or calculator, enter
For methods specifying 4 doses of standard,
sample data and calculate antibiotic potency based on least square
L = (7a + 4b + c – 2d )/10 linear or polynomial lines.
For calculations, 1 ton = 908,000 g.
H = (7d + 4c + b – 2a)/10
References: JAOAC 40, 857(1957); 72, 105(1989).
For methods specifying 3 doses of standard, Revised: March 1998
L = (5a + 2b – c)/6

H = (5c + 2b – a)/6

© 1998 AOAC INTERNATIONAL