specifications for identity and purity of some … · 2021. 3. 22. · of official agricultural...

SPECIFICATIONS FOR IDENTITY AND PURITY

OF SOME ANTIBIOTICS

• Food and Agriculture Organization of the United Notions

World Health Organization

1969

IN.., Y.f.D

Specifications for Identity and Purity of Some Antibiotics

Corrigenda

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0

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Kl/90326 /1

110364

FAQ Nutrition Meetings Report Series No. 45 A WHO/Food Add./69.34

Last but one line. Read 11 6 mm(+ 0.1 mm)" for the words 11 6 mm (:t, 1 mm)". -

Fourth line from bottom. Replace "7.81" by the figure "7.8o".

Para 3.5. Replace figures "1.2.3" wherever they occur by the figures "3.2.3".

Fourth line from top. Delete the figures "1.2.3" after "saline T.S.".

18th line. Change"!. 0.2" to"+ 0.2".

Para 1.2.3. Second line. Change "2.5 ml" to "25 ml".

Para 1.3. Fifth line. Delete "0.4 ml". Ninth line. Add "4.4 ml" in between 4.5 ml" and "4.2 ml".

l6th line. Change "!. 0.2" to "+ 0.2".

Second line from bottom. for "FeNHiso

4)".

Read "FeNH (SO)" 4 4 2

Put an asterisk mark on "0.3 unit/g'' and a foot note explaining "Basis: l million units .. 1 g of nisin".

Para 1.1 Change "quantity" to "quality" after the words "Agar according to".

Para 1.2.1. Read the figures "6947" as "6.941"·

Para 1.2.3. Read the figures "57539" and "1407" as "57.539" and "1.407" respectively.

Seventh line. Change the figures "(l.1.2)" to "(1.2.2)".

Page 100

Page 101

\

-2-

Fourth line foot note. Delete the words "0.05 u/ml standard" appearing second time and substitute them by the words "untreated sample".

Second paragraph, fourth line. Add the words "aliquot is 10. 5 ml. After addition of the 25 ml of aqueous pyridine the total sample dilution is 15 g" between the words "15 gm" and the words "to 35.5 ml".

Para 2.3.2. Fourth line. Read "lON'' as "10 N''.

FAO Nutrition Meetings Report Series, No.45A WHO/Food Add./69.34

SPECIFICATIONS FOR IDENTITY

ANO PURITY

OF SOME ANTIBIOTICS

The contents •of this document are the result of the deliberations of the Joint FAQ/WHO Expert Committee on Food Additives, which met in Geneva, 1·8 July 1968 (Twelfth Report of the Joint FAO/WHO Expert Committee on Food Additives, FAQ Nutrition Meetings Report Series No.45, and World Health Organization Report Series No.430).

Wortd Hulth Organization Food and Agriculture O'l•niz•tion of the United Nnion1

1969

CONTENTS

lntrod.1.1e:&n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Am.inoglycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Streptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Dihydristreptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Neomycin................................. 6

~crolides . . . . . . • . . . . . . . . . . . . . . • . . . . . . . • . . . . . . 9

Erythromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Oleandomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Spiramycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Tylosin ..... · . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Penicillins • · • • • • • . . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 9

Penicillins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9

Polyenes . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Nystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Pinnaricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Polypeptides . . . . . . . . . . . . . . . • . . . . . . . . . . . . • . . . . . . 5 2

Bacitracin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . · 52 Nisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Polymyxin 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Tetracyclines •....•................. ~ ·. . . • • . . . . . . 69

Tetracycline . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 69 Chlortetracycline . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Oxytetracycline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Annex • • • • • • • • • • . • • . . • • • • • • . . • • • . . . • • • • • • . . . • 86

INTRODUCTION

The monographs on specifications of various antibiotics contained in this publication are the result of the deliberations of the Joint FAO/WHO ·Ex.pert Committee on Food Additives, which met in Geneva from 1-8 July, 1968. The monographs should therefore be consulted together with the report of this Twelfth Session.1

The Committee decided to classify various antibiotics on a chemical basis, i.e. aminoglycosides, macrolides, penicillins, polyenes, polypeptides, tetracycline and others. Wherever the specifications for an antibiotic are available in the International Pharmacopoeia, it was agreed to make a reference to the same, rather than rewriting the specifications. Similarly, in case of accepted assay procedures for antibiotics in animal feeds appearing in the AOAC (Methods of Analysis of the Association of Official Agricultural Chemists, U.S.A.) a reference to the relevant method was considered adequate. For an individual estimation, where procedural details are not given in this publication the corresponding method in the International Pharmacopoeia should be used.

The Committee has further stated that:

"(a) Analytical methods are continually being developed to improve sensitivity, specificity, accuracy and reproducibility. The methods cited in this publication were considered acceptable according to their present state of knowledge. Alternate methods or refinements of those prescribed may be available now or in the near future.

(b) For the assay of antibiotics in the tissue, to determine possible antibiotic residues, discrimination as to the actual type of residue present is necessary. This can be based upon qualitive tests, which may include direct simple zone inhibition methods with tissues or urine specimens, separation by electrophoresis, chromatography, etc. An alternate approach woµld be to submit the sample to each of the quantitative assays for different groups of antibiotics. However, quantitative measurement of any specific antibiotic requires a knowledge of the antibiotic involved, as different response curves may exist for different antibiotics in the same group.

1Twelfth Report of the Joint F AO/WHO Expert Committee on Food Additives. F A.O Nutrition Meetintt Reoort Series. No. 45; World Hulth Organizotion Technical Report Series No. 410.

( c) In practice, more than one antibiotic may be present in a material. The method\ described herein are for each individual substance. In cases where interference may occur, appropriate analytical procedures should be used.

( d) The specifications contained in this publication are considered in temtS of the pure antibiotic and pure forms thereof. Similarly, tissue residue methods and safety were studied in terms of the active antibiotic moiety involved rather than on the basis of the form in which the antibiotic was administered.

(e) A certain amount of degradation of antibiotics may occur during the processing and storage of tissues that may contain residues of antibiotics. Antibiotics, like any other biologically active substance may undergo metabolic modification in the animal body. Ideally, analytical procedures. would delineate and identify the degradation products; however, for those products which the Committee considered, the assay methods in general were not capable of such exact discrimination and differential quantification, and only measure total antimicrobial activity. The Committee therefore recommended that future information on existing and new products should cover this aspect of the problem so that a more complete analytical review of the substances and their main degradation products can be made."

Any new information and comments relating to the specifications of these antibiotics, the accepted methods of analysis of the pure material and of the material in feeds and foods should be addressed to: Food Standards, Additives and Regulation Section, Nutrition Division, FAO, Rome, Italy.

Comments and additional information, if any, relating to biological data and their evaluation should be addressed to: Food Additives Unit, World Health Organization, Avenue Appia, Geneva, Switzerland.

.MUNOGLYCOSIDES

STREPTOMYCIN

Specification.,

Streptomycin sulphate. Specifications for the Quality Control of Phannaceutical Preparations. International Pharmacopoeia WHO, Geneva, 2nd Edit., Page 535

Amys

1. In animal feed: Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) 1965, lOth Edit., Page 660; 33.lSS-33.158

2. In tissue, milk and eggs: As given in the Annex

Sensitivity of assay

Sensitivity of assay reported to be as under:

In animal tissue 1.0 ppm (as streptomycin base)1

In milk

In eggs (whole)

0.2 ppm (as streptomycin base)1

0.5 ppm (as streptomycin base)1

1 Basis ·1 lU = 1 microgram of streptomycin base.

3

DIHYDROSTREPTOMYCIN

Chemical Name: Dihydrostreptomycin sulphate

Chemical fonnula: (C21 RuN,012h, 3H1SO ..

Structural formula:

NH ,, HO NH-C

' NH NH2 ~ C-NH O CH

HO OH I 2 CH O HC-OH I I I CH HO-~H

3 CH

I

CH20H

Molecular weight: 1461.5

2

Deflllition: Dihydrostreptomycin sulphate is a salt of the antibiotic derived from controlled hydrogenation of the antibiotic substance streptomycin produced from microbial ·sources Streptomyces griseus; s. bilcinieusis; or s.mashuensis. Dihydrostreptomycin sulphate contains not less than 700 international unit~ per mg.

Description: Dihydrostreptomycin sulphate is a white or almost white powder.

Solubility: Very soluble in water; very slightly soluble in alcohol, in · ether and in chlorofonn.

Identification tests

A. Dissol~e 0.05 gm in 2 ml of N/ 1 sodium hydroxide, acidify with dilute hydrochloric acid (about 10 percent w/w of HCl), and add 0.05 ml of ferric chloride solution (5 percent w/v in water); not more than .a faint violet colour is produced, ( distinction from streptomycin sulphate).

B. It complies with the tests A and C given in the International Pharmacopeia, 2nd Edit., for streptomycin sulphate.

4

Purity tests

Streptomycin. Dissolve a quantity of the powder equivalent to 200 OOO units in 10 ml of water, add 2 ml of N/1 sodium hydroxide, heat on a water-bath for 10 min., cool to room temperature, add 2.4 ml of N/ l hydrochloric acid and 5 ml of 0.25 percent w/v solution of ferric chloride in N/100 hydrochloric acid, and dilute to 25 ml with water; the extinction of a 1 cm layer of this solution at t:1e maximun at about 550 nm is not greater than that of a solution prepared by treating 6 OOO units of streptomycin sulphate in a similar manner.

Loss on drying. When dried at 60° in vacuo for 3 hrs. loses not more than 5.0 percent of its weight.

Undue toxicity. Complies with the tests given for streptomycin sulphate in the International Pharmacopeia, 2nd Edit.

Assays

l. Assay of the material

Proceed by the method for the biological assay of antibiotics ( streptomycin) as given in the International Pharmacopeia, second edition, page 742, using the standard preparation of Dihydrostreptomycin. For purpose of the assay and calculations, the stated potency is taken to be 800 international units per mg. The estimated potency is not less than 87.5 percent of the stated potency. The fiducial limits of error of the estimated potency (P = 0.95) are not less than 80 percent and not more than 125 percent of the stated potency.

2. Assay in msues, milk and egs - As given in the AMex

Sen.1itmty of aaay


In animal tissues - 1.0 ppms(as Dihydrostreptomycin base)1

In milk - 0.2 ppm (as Dihydrostreptomycin base)1

In eggs - 0.5 ppm (as Dihydrostreptomycin base)1

1 Basis 1 IU = 1 microgram of Dihydrostreptomycin base.

s

NEOMYCIN

Specifications

Neomycin sulphate. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia WHO, Geneva, 2nd Edit., Page 379

Assays

t. Assay in animal feed

1.1 Test organism: Staphylococcus epidermidis {ATCC 12228)

Transfer the growth from an agar slant culture to a Roux bottle containing 300 ml of culture medium and incubate for 16 hours at 37°. Harvest growth with 100 ml of sterile physiological saline.

1.2 Culture media

1.2.1 Culture medium:

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . . 6.0 gm T9"ptic digest of casein . . . . . . . . . . . . . . . . . . . . 4.0 gm Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Yeast extract ....... ·. . . . . . . . . . . . . . . . . . . . 3.0 gm Glucose . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . 1.0 gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gm Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . 1 OOO ml

pH = 6.55 ± 0.05

1.2.2 Test medium

Same as Culture medium, but adjust so that ph is 8.0 ± 0.1

'· .3 Reagents

Phosphate buffer ph 8.0. Dissolve 16.73 gm K2 HP04 and 0.523 gm KH2 P04 in distilled H2 0 and dilute to 1 liter.

Phosphate-bicarbonate buffer - pH 8.0. Dissolve 16.73 gm K2 HPO•, 0.523 gm KH2 P04 , and 20 gm NaHC03 in distilled H2 0 and dilute to 1 lit er.

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1.4 Standard preparation

Dissolve the working standard of Neomycin in phosphate buffer pH 8.0 to give a concentration of 100 IU ml.

l.S Preparation of sample

Grind coarse or pelleted samples to "eO mesh" and mix thoroughly. Weigh 10 gm of feed sample into a 250 ml centrifuge tube, add 100 ml of phosphate-bicarbonate buffer, stopper, and shake for 15 minutes. Centrifuge 10 minutes at 2000 rpm. Remove 20 ml of supernatant, adjust to pH 2.0 with IN HCl, let stand 5 minutes, and adjust to pH 8.0 with N NaOH. Dilute the adjusted solution with enough phosphate buffer pH 8.0 so that 1.0 ml of the final dilution should contain 1.0 IU of neomycin.

1.6 Test procedure

1.6.1 Preparation of plates

Base layer - Pipiette 12 met of melted test medium into each plate.

Seed layer - Add 10 ml of the inoculum suspension to 1 OOO ml of test medium. Pipette 6 ml of this inoculated test medium over the base layer. Tilt the plate back and forth to spread agar evenly over the surf ace. Refrigerate plates at least one hour.

1.6.l Standard curve

Dilute the working standard to 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 3.0, and 5.0 IU/ml with phosphate buffer pH 8.0. Dilute an independerit 1.0 IU/ ml solution for the ref ere nee print.

1.6.3 Determination

Use 3 plates for each assay solution. Place 6 cylinders equidistant on each plate. Fill 3 cylinders with reference standard and 3 cylinders with sample. Incubate 16 hours at 37°. Alfter incubation, measure the diameter of the zones of inhibition, average the standard readings and sample re~dings, plot corrected standard values and calculate potency.

7

2. Assay in ~ue, milk and eggs - As given in the Annex.

Sensitivity of ~y


In animal tissues 0.5 ppm (as neomycin base)1

In milk

In eggs (whole)

0.15 ppm (as neomycin base)1

0.20 ppm (as neomycin base)1

1Basis 1 IU = 1 microgram of neomycin base.

8

MACROLIDES

ERYTIIROMYCIN

Specifications

Erythromycin. Specifications.for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia WHO, Geneva, 2nd Edit., Page 199

Assays

1. Assay in animal feed

1.1. Culture media

1.1.1 Agar medium A. Dissolve 6.0 g peptone, 4.0 g pancreatic digest of casein, 3.0 g yeast ext., 1.5 g beef ext., 1.0 g anhyd. dextrose, and 15 g agar in water, and dil. to 1 liter. Adjust with 1 N NaOH or HCl ( 1 + 9) so that after sterilization for 30 min. at 121° pH is 6.5-6.6.

1.1.2 Agar medium E. Use·agar medium A adjusted with 1 N NaOH or HCI (1 + 9) sc;, that final pH is 7.95-8.05.

1.1.l Broth medium. Dissolve 5.0 g peptone, 1.5 g yeast extract, 1.S g beef ext., 3.5 g NaCl, 1.0 g. anhyd. dextrose, 3.68 g anhydrous. K2 HP04, and 1.82 g anhyd. KH 2 P04, in H2 0,, and dil. to 1 liter. Adjust with 1 N NaOH or HCl ( 1 + 9) so that after sterilization for 30 min. at 121° pH is 6.95-7.05.

1.2 Reagenu

Phosphate buffer - pH 8 - Dissolve 0.523 g anhydrous KH2P04 and 16.73 g anhydrous K2 HP04 in water and dil. to 1 liter.

1.3 Apparatus

1.3.1 Cylinders ( cups). Polished stainless steel qlinders, 8 ± 0.1 mm outer diameter, 6 ± 0.1 mm inner diameter, and 10 ± 0.2 mm high.

9

1.3.2 Petri dishes (plates). 20 X:. 100 mm with porcelain covers gla1An on outside

1.4 Stock cultures of test organisms

Sarcina lutea - ATCC 9341. Incubate stock culture at 26° fol" 18 h.

1.S Plates

Roux bottle culture - Wash growth from 24 h. slant culture with ea 3 ml broth medium, and transfer liquid to surf ace of 300 ml agar medium A in Roux bottle. Spread suspension evenly over entire surface with aid of sterile glass bea~. Incubate 24 h at 26° and wash growth from agar surface with ea 15 ml broth medium. Using photoelectric colorimeter and 18 mm diam. test tube as absorption cell, det. transmittance of 1 : 10 diln. fo this bulk suspension at 650 nm, and, if necessary, adjust by diln. so that 1 : 10 diln. gives 10 percent transmittance. Use adjusted bulk suspension (not 1 : 10 diln.) in preparing seed layer. (Bulk suspension may be stored at ea 10° several months.) Before actual assay, det. by prepn. of trial plates the optimum concn. (usually 0.3-0.5 percent) of inoculum to be added to agar medium E to obtain zones of inhibition of adequate size and sharpness. Add appropriate amount of organism suspension to agar medium E which has been melted and cooled to 48°. Mix thoroughly and add 10 ml to each plate. Distribute agar evenly by tilting plates from side to side with circular motion and allow to harden. Use plates prepared the same day.

1.6 Standard solutions

1.6.l Stock solution. Weigh accurately about 50 mg of Erythromycin Reference Standard. Dissolve in enough methanol to give a concentration of exactly 1 OOO mcg per ml. Store in the dark at about l 0° not longer than 7 days.

1.6.2 Standard solutions. Dilute appropriate aliquots of stock solution 1.6. l with enough pH 8 buffer to obtain concentrations (a,b,c,d, and e) of 0.05, 0.1, 0.2, 0.4, 0.8 per ml. Reference concentration is 0.2 mcg per ml (c).

10

2. Preparation of standard curve

Place 6 cylinders on each plate at ea 60° intervals on 2.8 cm radius. Fill 3 alternate cylinders with reference concentration and other 3 cylinders with one of other concentrations. of standard. Use 3 plates for each concentration required for standard curve, except reference concentration ( total of 12 plates). Incubate plates 16-18 h at 30° and read diameters of zones of inhibition by means of mm ruler, calipers, or calibrated projection device. In each set of 3 plates average the 9 readings of the reference concentration and the 9 readings of concentration being tested. Average of all 36 readings of reference concentration from 12 plates is correction point for curve. Correct aver value obtained for each concentration to the figure it would be if reference concentration reading on that set of 3 plates were same as correction point. For example, if in correcting second concentration of standard curve, aver. of 36 readings of reference concentration is 20.0 mm., and aver. of 9 readings of reference concentration of this set of 3 plates is 19.8 mm., correction is + 0.2 mm. If the aver. reading of second concentration on same 3 plates is 17.0 mm. corrected value is 17 .2 mm. Using the ff. equations, calculate the corr. zone diameter for the lowest and highest concentration of the standard curve (L and H respectively).

3a+2b+c-e 3e+2d+c-a L = and ff=

s s

Plot the values obtained for L and H and connect with a straight tine.

3. Preparation of asay solution

3.1 Transfer 10~0 g, accurately weighed, of feed containing approximately SO mcg erythromycin activity to, a sintered-glass funnel of coarse porosity (30 x 60 mm), fitted to a 250 ml suction flask.

3.2 Add lS ml of chloroform and thoroughly blend with the feed, allowing the solvent to filter through by gravity (S minutes).

11

3.3 Complete the filtration by applying vacuum for about l minute.

3.4 Repeat steps 3.2 and 3.3 using, in sequence, 15 ml of chloroform, 10 ml of methanol, 15 ml of chloroform, 15 ml of chlorpform, and 15 ml of n-hexane.

3.S Place the suction flask in a water bath, heated to between 40° and 50° and evaporate the solvent just to dryness by means of a stream of air.

3.6 Dissolve the residue from step 3.5 in 50 ml of n-hexane and transfer to a 125 ml separating funnel.

3.7 Rinse the suction flask with 50 ml of 1 percent pH 8 phosphate buffer and transfer to the separatory funnel containing the n-hexane.

3.8 Extract the erythromycin activity from the n-hexane by gentle mixing for about 5 minutes.

3.9 Separate the aqueous layer, filtering through Whatman N° 1 paper into a 250 ml volumetric flask.

3.10 Repeat steps 3.7 through 3.9 using, in sequence, a mixture of 10 ml of methanol and 40 ml of 1 percent pH 8 phosphate buffer, and 50 ml of 1 percent pH 8 phosphate buffer.

3.11 Wash the filter paper with sufficient pH 8 buffer to bring the combined extract,; to volume. Each ml of this solution theoretically contains 0.2 mcg of erythromycin activity.

4. As&1y procedure

Use 3 plates for each assay solution. On each plate fill 3 alternate cylinders with reference concentration and dilute other 3 cylinders with assay solution. Incubate plates 16-18 h at 30°, and read diameters of zones of inhibition. Average the 9 readings of ref ere nee concentration and the 9 readings of assay solution. If assay solution gives larger average than reference

12

concentration, add difference between them to reference point on standard curve. If assay solution gives smaller value than reference concentration subtract difference between them from reference point. on standard curve. Using corrected value of assay solution det. quantity of antibiotic from standard curve.

5. Asay in ti~e. milk and eggs - As given in the Annex.

Sensitivity of asay


In animal tissue 0.3 ppm (as erythromycin base)

In milk

In egg (whole)

0.04 ppm (as erythromycin base)

0.3 ppm (as erythromycin base)

13

OLEANDOMYCIN

Chemical name: Oleandomycin Phosphate

Chemical formula: C37 H67 N0 13 (approx.)

Definition: Oleandomycin Phosphate is a phosphate salt of a kind of Oleandomycin, a basic antibiotic produced by a strain of Streptomyces antibioticus, or a mixture of two or more such salts. It contains not less than 775 micrograms of Oleandomycin per mg.

Description: Oleandomycin Phosphate is a white crystalline powder.


A. Dissolve about 10 mg of Oleandomycin Phosphate in 5 ml of hydrochloric acid and heat the solution in a water bath; greenish yellow colour is produced.

B. The pH in a solution containing 100 mg per ml is not less than 3 .0 and not more than 6.0

Purity tests

Water. Not more than 5.0 percent (Karl Fischer method)

Residue on ignition. Not more than 1 percent, when tested by the procedure described in the International Pharmacopoeia.

Heavy metals. Not more than 100 parts per million, when tested by the procedure described in the International Pharmacopoeia.

Undue toxicity. Complies with the test given in the International Pharmacopoeia, when using a test dose of 0.5 milliliter of solution in sterile saline T.S. containing 8.0 milligrams of oleandomycin base equivalent per milliliter.

Assay

1. Assay of the material

1.1 Cylinders

Stainless steel cylinders with an outside diameter of 8 mm (± 0.1 mm), an inside diameter of 6 mm (± 1 mm) and length of 10 mm(± 0.1)

14

1.2 Culture media

1.2.l Make nutrient agar for the seed and base layer as follows:

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . . 6.0 gm Pancreatic digest of casein . . . . . . . . . . . . . . . . . . 4.0 gm Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 gm Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 gm Agar ........... : . . . . . . . . . . . . . . . . . . . . 15.0 gm Distilled water, q.s. . . . . . . . . . . . . . . . . . . . . . . 1 000 ml

pH 7 .8 to 8.0 after sterilization

1.2.2 Use the nutrient agar described above for maintaining the test organism, except that its pH after sterilization is 6.5 to 6.6. In lieu of preparing media from the individual ingredients specified here, they may be made from a dehydrated mixture which, when reconstituted. with distilled water, has the same composition as such media.

1.3 Working standard

Dissolve a suitable weighed quantity (usually 25 milligrams or less) of the working standard in 2 milliliters of ethyl alcohol, then add sufficient 0.1 M potassium phosphate buffer, pH 8.0, to give a concentration of 1 OOO micrograms of oleandomycin base per milliliter . This stock solution may be kept in the refrigerator for 3 days.

1.4 Preparation of sample

Dissolve the sample in sufficient 0.1 M potassium phosphate buffer, pH 8.0 to give a convenient stock solution. Further dilute in O.lM potassium phosphate buffer, pH 8.0, to give a final concentration of 5 .0 micrograms of oleandomycin per milliliter ( estimated).

1.S Preparation oi test organism

The test organism is Staphylococcus epidermidis (ATCC 12228), which is maintained on slants of agar described in 1.2.1. Wash the organism from the agar slant with 3 milliliters of sterile saline T .S. onto a large agar surface such as that provided by a Roux bottle containing 300 milliliters of the agar

15

1.2.1. Spread the suspension of organisms over the entire agar surface with the aid of sterile glass beads. Incubate for 24 hours at 32° to 35° and then wash the resulting growth from the agar surface with about 50 milliliters of sterile International Pharmacopoeia saline T.S. Standardize the suspension so that a 1 + 24 dilution of it with sterile saline will give 25 percent light transmission, using a suitable photoelectric colorimeter with a 580-millimicron filter and a 13-millimeter diameter test tube as an absorption cell. Run test plates to determine the quantity of the bulk suspension ( usually 0.16 milliliter) that should be added to each 100 milliliters of agar to give clear, sharp zones of inhibition of appropriate size.

1.6 Preparation of plates

Add 21 milliliters of the agar prepared as described in 1.2.1 to each Petri dish (20 millimeters x 100 millimeters). Distribute the agar evenly in the plates and allow it to harden. Use the plates the same day they are prepared. Melt a sufficient amount of the agar 1.2.1, cool to 48°, add the proper amount of the test organism as described in 1.5 and mix thoroughly. Add 4 milliliters of this inoculated agar to each Petri dish. Distribute the agar evenly in the plates, cover with porcelain covers glazed on the outside, and allow to harden. After the agar has hardened, place 6 cylinders on the agar surf ace so that they are at approximately 60° intervals on a 2.8-centimeter radius.

1. 7 Standard response line

Prepare the daily standard response line by further diluting the 1 OOO micrograms per milliliter stock solution in 0.1 M potassium phosphate buffer, pH 8.0, to obtain concentrations of 3.2, 4.0, 5.0, 6.25 and 7.80 micrograms of oleandomycin per milliliter. Use three plates for the determination of each point on the response line, except the 5.0 micrograms per milhliter concentration, a total of 12 plates. On each of three plates, fill three cylinders with the 5.0 micrograms per milliliter standard and the other three cylinders with the concentration under test. Thus, there will be 36 5-microgram determinations .:tnd 9 determinations for each of the other points on the response line. After incubation, read the diameters of the circles of inhibition in the plates Average the readings of the 5 .0 micrograms per milliliter concentration and the readings of the

16

point tested for each set of 3 plates and average also all 36 readings of the S .0 microgram per milliliter concentration. The average of the 36 readings of the S .0 micrograms per milliliter concentration is the correction point for the response line. Correct the average value obtained for each point to the figure it ~ould be if the S .0 micrograms per milliliter reading for that set of 3 plates were the same as the correction point. Thus, if in correcting the 4.0 microgram concentration, the average of the 36 readings of the S .0 microgram concentration were 20 millimeters and the average of the 5.0 microgram concentration of this set of 3 plates were 19.8 millimeters, the correction would be + 0.2 millimeter. If the average reading of the 4.0 microgram concentration of these same 3 plates were 19 .0 millimeters, the corrected value would be 19.2 millimeters. Plot these corrected values, including the average of the S .0 micrograms per milliliter concentration, on a 2-cycle semilog paper, using the concentrations' in micrograms per milliliter as the ordinate (the logarithmic scale) and the diameter of the zone of inhibition as the abscissa. Draw the standard response line through these points, either by inspection or by means of the following equations:

where:

L =

H =

c =

a, b, d, e =

L

H

=

=

3 a + 2 b + c -e

s 3e+2d+c-a

s

Calculated zone diameter for the lowest concentration of the standard response line;

Calculated zone diameter for the highest concentration of the standard response line;

Average zone diameter of 36 readings of the S .0 micrograms per milliliter standard;

Corrected average values for 3.2, 4.0, 6.25 and 7.81 micrograms per milliliter standard solutions, respectively.

Plot the values obtained for L and H and co_Qnect with a straight line.

17

1.8 Assay procedure

Use 3 plates for each sample. Fill 3 cylinders on each plate with the standard 5.0 micrograms per milliliter solution and 3 cylinders with the 5 .0 micrograms per milliliter ( estimated) sample, alternating standard and sample. Incubate all plates, including those containing the standard curve at 32° -35° overnight, and measure the diameter of each circle of inhibition. To estimate the potency of the sample, average the . . zone readings of the standard and the zone readings of the sample on the 3 plates used. If the sample gives a larger zone size than the average of the standard, add the difference between them to the 5 .0 micrograms per milliliter zone on the standard response line. If the average sample value is lower than the standard value, subtract the difference between them from the 5.0 micrograms per milliliter value on the response line, and read the potencies corresponding to these corrected values of zone sizes.

2. Assay in animal feeds: Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) 1965, lOth Edit., Page 659; 33.148-33.151

3. Assay in tissues

3.1 Cylinders

Use the same cylinders as in 1.1.

3.2 Culture media

3.2.1 Use the medium described in 1.2.1 for carrying the test organism and for the base layer.

3.2.2 Use this medium for the seed layer:

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . . 6.0 gm Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 gm Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gm Distilled water, q.s. . ..................... 1 OOO ml

pH 6.5-6.6 after sterilization

18

3.2.3 Broth medium. Use this medium for preparing an inoculum of the test organism:

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . 5 .0 gm Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 gm Sodium chloride . . . . . . . . . . . . . . . . . . . . . . . 3.5 gm Dipotassium phosphate . . . . . . . . . . . . . . . . . . . 3.68 gm Monopotassium phosphate . . . . . . . . . . . . . . . . . 1.32 gm Distilled water, q.s. . .................... 1 OOO ml

pH 6.95-7.05 after sterilization


Use the standard solution described in 1.3

3.4 Preparation of sample

Weigh 1 gram of tissue, add 9 ml 0.1 M phosphate buffer, pH 8.0 and thoroughly homogenize the sample.

3.S Preparation of test organism

Maintain the test organism Sarcina lutea (ATCC 9341) on agar slants of agar described in 1.2.1 and transfer to a fresh agar slant about once every two weeks, incubating overnight at 32° to 35°. Prepare an inoculum for the plates by washing the culture from the agar slant into 100 ml of broth medium described in 1.2.3 with 2 to 3 ml of distilled water. Incubate this broth for 48 hours by continuous shaking on a shaking machine at room temperature. This culture when refrigerated may be used for at least two weeks. Determine per cent of inoculum to be used by determination of the best zones of inhibition obtained on test plates. Generally, this will be obtained within a range of 2 to 5 per cent inoculum. An alternate method and the one usually used in preparing the test suspension is as follows: Maintain the test organism on slants of agar described in 1.2. l and transfer to a fresh agar slant once a week. Streak an agar slant heavily with the test organism and incubate for 24 hours at 26°. Wash the growth off with 3 ml of broth medium 1.2.3. Use the suspension so obtained to inoculate the surface of a Roux bottle containing

19

300 ml of agar medium 1.2.1. Spread the suspension over the entire surface with the aid of sterile glass beads. Incubate for 24 hours at 26°. Wash growth from the agar surface with 50 ml of sterile saline T .S. l .2.3. If an aliquot of this bulk suspension. when diluted 1 + 39 Sterile Saline T.S., gives 25° lo light transmission in a suitable photoelectric colorimeter at a wavelength of 580 millimicrons with a 13-millimeter diameter test tube as an absorption cell, the bulk suspension is satisfactory for use. It may be necessary to adjust the bulk suspension by dilution so that an aliquot of the adjusted s1:1spension diluted l + 39 gives 25° lo light transmission. (The adjusted bulk suspension only and not the 1 + 24 dilution of it is used in preparing the seed layer.). The. bulk suspension may be used for several rnonths or' longer. The adjusted suspension is usually used in the proportion of 0.3 to 0.5 ml of suspension for every 100 ml of seed layer agar.


Add 10 ml of agar medium 1.2.1 to each Petri dish (20 X l 00 mm). Distribute the agar evenly in the plates, over with porcelain covers glazed only on the outside or other suitable covers, and allow it to harden. Use the plates the same day they are prepared. Add the appropriate amount (as determined in 3.5) of the broth culture or organism suspension to each 100 ml of seed agar medium 1.2.2, which has been melted and cooled to 48°. Mix the culture and agar thoroughly and add 4 ml to each of the plates contain~ the 10 ml of the uninoculated base agar. Tilt the plates back and forth to spread the inoculated agar evenly over the surf ace.

3.7 Standard response line and assay procedure

Standard response line. Place 6 cylinders on the inoculated agar surface so that they are equidistant. Use three plates for each sample. Fill three cylinders on each plate with the 0.8 microgram/ml standard and 3 cylinders with sample, alternating standard and sample. At the same time, prepare a standard response line using concentrations of the standard of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 microgram/ml in pH 8.0 buffer. The 0.8 microgram/ml concentration is the reference point. Use 3 plates for the determination of each concentration on the curve except the 0.8 microgram/ml concentration, a total of 18 plates. On each of 3 plates, fill 3 cylinders with the

20

concentration under test and 3 with the 0.8 concentration. Thus, there will be 54 of the 0.8 detenninations and '9 detenninations for each of the other concentrations on the curve. Incubate the plates for 16 to 18 hours at 26° and measure the diameter of each circle of inhibition. Average the readings of 0.8 microgram/ml concentration and the readings of the point tested for each set of 3 plates and average also all 54 readings of the 0.8 microgram/ml concentration. The average of the 54 readings of the 0.8 microgram/ml concentration is the correction point for the curve. Correct the average value obtained for each concentration to the figure it would be if the 0.8 microgram/ml reading for that set of 3 plates were the same as the correction point. Thus, if in correcting the 0.4 microgram concentration, the average of the 36 readings of the 0.8 microgram concentration is 20.0 mm, and the average of the 0.8 microgram concentration of this set of 3 plates is 19.8, the correction is ± 0.2 mm. If the average reading of the 0.4 microgram concentration of these same 3 plates is 17.0 mm, the corrected value is then 1 7. 2 mm. Plot these corrected values, including the average of the 0.8 microgram/ml concentration, on 2-cycle semi-logarithmic paper, using the concentration in microgram/ml as the ordinate (the logarithmic scale) and the diaqleter of the zone of inluoition as the abscissa. Draw the standard response line through these points. To estimate the concentration of the sample, average the zone readings of the standard and the zone readings of the sample on the plates used. If the sample gives a larger average zone size than the average of the standard, add the difference between them to the 0.8 microgram zone on the standard curve. If the average sample value is lower than the standard value, subtract the difference between them from the 0.8 microgram value on the response line. From the response line, read the concentrations corresponding to these corrected values of zone sizes.

Seositmty of assay

Sensitivity of amy reported to be as under:

In animal tissues 0.3 1,:pm (as oleandomycin base)

In milk

In eggs (whole)

0.1 S ppm ( as oleandomycin base)

0.1 ppm ( as oleandomycin base)

21

SPIRAMYCIN

Chemical name: Spirarnycin

Chemical formulas: Spiramycin I: Spiramycin II: Spiramycin III:

C4 s H, a01 s N2 C41Hao016N2 C43Ha2016N2

Structural formulas: The chemical structures of these spiramycins are not yet fully known. It is however known that spiramycins II and III are the acetic and propionic mono-esters of spiramycin I.

Molecular weights: Spiramycin I 887.1 Spiramycin II 929 .1 Spiramycin III 943.2

Definition: Spiramycin is the term used to describe a mixture of antibiotics (spiramycins I, II and III) having similar chemical and biological properties, usually produced by Streptomyces ambofaciens. Spiramycin consists of a mixture of three Spiramycins in the following proportions:

Spiramycin I Spiramycin II Spiramycin III

The biological activity of each is:

Spiramycin I Spiramycin II Spiramycin III

63 ± 10 percent 24 ± 5 percent 13 ± 5 percent

3 500 u/mg 2 590 u/mg 3 100 u/mg

Spiramycin base contains not less than 2 700 IU per mg.

Description: Spiramycin is a white or slightly yellowish, amorphous non-hygroscopic powder. It is bitter in taste and has a slight odour. Spiramycin is used in the form of spiramycin embonate in animal feeds.


A. Solubility

Ethyl alcohol, benzene, chloroform water petroleum ether

22

- highly soluble - fairly soluble - practically insoluble

B. Melting Points Spiramycin I 134° to 137° Spiramycin II 130° to 133° Spiramycin III 128° to 131°

c. Ultra violet absorption 1%

Ecm at 232 nm=

Spiramycin I 322 Spiramycin II 307 Spiramycin III 327

D. Specific Rotation (2 percent solution in acetic acid, dilute, R)

Spiramycin I 80° [ a ]2J Spiramycin II 86°

Spiramydn HI 84°

E. Dissolve 0.5 g of spiramycin in 25 ml of water and 11 ml of a 0.1 N surphuric acid; bring the pH to 8 by the addition of 0.1 N sodium hydroxide and fill to 50 ml with water. This solution gives a cream yellow precipitate with potassium mercuric iodide neutral solution TS, a brown precipitate with iodine solution TS, and a bright yellow precipitate with picric acid TS.

F. To 5 ml of the above solution, add 2 ml of an aqueous solution of sulphuric acid obtained by mixing two volumes of concentrated sulphuric acid TS and one volume of water: the resultant solution has a rose colour.

Purity tests

Loss on drying sulphated ash Heavy metals Nitrogen*{on dry wt.basis)

not more than 2 percent - not more than 0.1 percent

not more than 20 ppm - 2.95 percent to 3.25 percent

* Dissolve about 0.5 g (accurately weighed) sample in 10 ml of acetic acid R; add 75 ml of dichloroethane R and two drops of a solution of a naphtholbenzein R in acetic acid. Titrate with O.IN acetic acid solution of perchloric acid R until the solution turns green. Carry out a blank titration. Each ml of O.IN perchloric acid corresponds to 0.0014 g g of nitrogen.

23

percent nitrogen = 0.14 (n - n')

p

When n and n' are the ml of O.IN perchloric acid used for the titration of the sample and the blank, respectively, and p is the weight of the sample.

Undue toxicity: Complies with the Test for Under Toxicity described in Appendix 42 of the International Pharmacopoeia, the test dose being 0.5 ml of a solution in saline TS containing 0.004 g in each ml.

1. ~y of spiramycin base

1.1 Diffusion method

l.1.1 Preparation of standard solutions and samples

Dissolve aseptically, in a 100 ml calibrated flask, an accurately weighed assay sample of about 0.03 g of standard spiramycin base in l ml of. methanol and fill to 100 ml with a potassium phosphate buffer solution of pH 8 in order to obtain a stock solution containing approximately 1,000 units per ml. This stock solution may be used for l week when stored in a stoppered flask at temperatures of between 0° and 5°. Prepare from this stock solution, in a buff er solution of pH 8, solution a, b, c, d and e of the following concentrations:

200 - 100 - 50 - 25 - 125 IU/ml

Proceed in the same fashion with the sample to be assayed to obtain a solution presumed to contain 50 IU/ml. If a 3 x 3 design is used, prepare three standard solutions E,, E2, E.J of concentration 100-50-25 IU/ml respectively and, with the sample to be assayed, three solutions X1 , X2 , X3 of a titer presumed equal to the first three.

l.1.2 Procedure

Follow the procedure described in Appendix 41, Biologial Assay of Antibiotics, given in International Pharmacopoeia for streptomycin.

24

2. Amy in animal feeds

2.1 Extraction solution

Dissolve 3.92 g of phosphoric acid, 2.4 g of acetic acid and 2.48 g of boric acid in distilled water and make to 100 ml. Take 40 ml of the solution and add 28 ml of a solution of 0.2 N sodium hydroxide to make the pH of the solution 9.2. Take 30 ml of this solution and add 70 ml of methanol to make the extracting solution.

l.l Extraction

l.l.l Samples containing over 11,250 IU of spiramycin per Kg: Accurately weigh about S g of material and place in a 300 ml Erlenmeyer flask.. Add 40 ml of extraction solution (2.1 ). Stopper carefully; agitate for 20 minutes on a vibrating table; centrifuge. Collect the supernatant (solution A). On the material sample of the same composition not containing spiramycin make a similar extraction of the same proportion (solution 8). After having checked on an assay plate or dish that this extract has no antibiotic effect capable of interfering with the assay response, use solution 8 to prepare standard solutions and to dilute solution .• A and bring it to an estimated titer of close to 2 IU/ml (solution A1 )

1

2.2.2 Samples containing less than 11,250 IU of spiramycin per Kg

Weigh accurately about 10 g of the material and place in a 300 ml Erlenmeyer flask. Add 100 ml of extraction solution (2.1). Stopper carefully; agitate for 20 minutes on a vibrating table; centrifuge. Take SO ml of supernatant (solution A'); concentrate under vacuum to a volume of l S ml. Add 20 ml of dichlorethane and agitate in a separatory funnel for 2 minutes. Separate the dichlorethane phase. Again add 1 S ml of d.ichlorethane and repeat the operation. Make a third extraction with 1 S ml of dichlorethane under the same conditions as the first two. Combine the three dichlorethane phases and bring to SO ml by adding dichlorethane. Concentrat~ the extract obtained to a volume permitting a final concentration of about 2 IU/ml (solution A' 1 ).

10.1 M phosphoric acid buffer solution, pH 8.0, may be used for dil , tion, provided the same amount of buffer is used for diluting the sample and the standard.

25

Make a similar extraction with the same proportions of a sample of the material of the same composition not containing spiramycin (solution B'). After having checked to make sure that it has no antibiotic effect, use it to prepare standard solutions.

2.3 Culture media

2.3.1 Culture mediwn for assay (D'):

Peptone ...................... ·. · · · · , · · · · Meat extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. NaCl ........................... · · ·. · · · · Agar ..................... , , , , · · · · · · · · · ·

5 g 1.5 g 1.5 g 3.5 g

17.5 g

Distilled water in sufficient quantity to make up to 1 OOO ml of medium of pH 8.3 prior to sterilization. Sterilize in the autoclave for 20 minutes at 120°.

2.3.2 Culture medium for the inoculum (E')

The assay organism is Sarcina lutea ATCC 9341 stored on inclined agar tubes of the following composition:

Peptone ....... , .......................... 10 g Meat extract . ·. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Agar •........•.•.....•............•..... 25 g

Distilled water in sufficient quantity to make up to 1 OOO ml of medium of pH 7 .1 prior to sterilization. Sterilize the inclined tubes of agar in the autoclave for 20 minutes at 120°.

2.4 Preparation of the inoculum

Cultures of Sarcina lutea on inclined agar of medium (E') must be renewed weekly after incubation for two days in the incubator at 30°. Prepare the inoculum by placing in suspension in 10 ml of sterile, distilled water the culture obtained on an inclined agar tube. This suspension diluted 1/20 has an

26

absorptivity of about 1 at 630 nm Inoculate the assay medium (D'), that has previously been melted and cooled to 49° with I percent of the inoculum (Medium F'). ·

2.5 Preparation of assay dishes

In each of the IO·cm diameter Petri dishes place 13.5 ml of medium (D') and, after it has solidified, 4 ml of medium F'. Store these dishes in the refrigerator up to the time of their use.


Use a reference standard of spiramycin. Prepare a stock solution containing 2000 IU of spiramycin per ml by dissolving 50 mg (weighed to an accuracy of 0.1 mg) in methanol. Then dilute this solution with either solution B ( see para 2.2.1.) or solution B' (see para 2.2.2.) to obtain solutions of the following concentrations:

8 - 4 - 2 - 1 - 0.5 IU/ml.

The concentration of 2IU/ml is the reference solution. Keep all these solutions in carefully stoppered flasks. Dip 12-mm diameter discs of filter paper in the various solutions, then allow them to dry on glass plates at room temperature for two hours. After this drying time, place the standard discs on the inoculated agar surface of 12 assay dishes in order to obtain the standard response line.

2. 7 Preparation of sample

Solution A1 (or A'r) is used to prepare sample discs in the same manner as the standard discs on each of three inoculated assay dishes, place three sample discs and three discs of the reference standard (21U/ml) alternately. Place these dishes in the incubator at 30° for 20 hours.

2.8 Readings and calculations

After having measured the diameters of the inhibition zones, <;onstruct the standard response line on semi-logarithmic paper, plotting the concentrations on the logarithmic scale and the measured diameters on the arithmetic scale.

To determine the concentration of each sample solution, average the diameters of the inhibition zones for the standard reference concentration on the three dishes under consideration

and calculate the differenc.e between value obtained and that read on the standard response line for the concentration of 2 IU/ml. Add, ~gebraica1ly, the difference to the averageffigure of diameters of the inhibition zones of the sample on the same three dishes under consideration. Plot this figure, corrected in this way, on the standard response line and determine the corresponding concentration of spiramycin. If a is the concentration (in IU/ml); p is the weight in grarrunes of the amount assayed; v the final volume in ml of the extract A1 ( or A' 1 ); the titer t (in IU/g) of the material is calculated from the following equation:

a x v t (IU/g) =

p

3. Assay in tis.mes

3.1 Extraction solution. As given in para 2.1.

3.2 Extraction

Grind about- 10 g (accurately weighed) of the tissue with sand in mortar. Place in a 300 ml. Erlenmeyer flask. Add 100 ml of extraction solution (2.1). Stopper carefully; agitate for 20 minutes on a vibrating table; centrifuge. Take 50 ml of supernatant (solution· A'); concentrate under vacuum to a volume of 15 ml. Add 20 ml of dichlorethane, agitate in a separatory funnel for 2 minutes. Separate the dichlorethane phase. Again add 1 S ml of dichlorethane and repeat the operation. Make a third extraction with 15 ml of dichlorethane under the same conditions as the first two. Combine the three dichlorethane phases and bring to SO ml by adding dichloretjlane. Concentrate the extract obtained to 2.5 ml (solution A'1 ).

Make a similar extraction with the same proportions of a tissue sample of the same composition not containing spiramycin (solution 8'). After having checked to make sure that it has no antibiotic effect, use it to prepare the standard solutions.

28

3.3 Procedure

Proceed exactly as under para 2 for sample of feed containing less than 11 250 IU of spirarnycin per Kg.


In Animal Tissues 0.025 ppm (0.08 IU/g)1

1 Basis 3,200 IU = 1 mg spiramycin base.

29

lYLOSIN LACTATE

Chemical name: Jylosin lactate

Chemical formula: C4 8 H81 020N

Structural formula:


(;H3 -..·- OH ,,...,,,... '--,yot-t • I

---~o--)_CH3

Definition: Tylosin lactate is a salt of tylosin, an antibiotic usually produced by Streptomyces fradiae. 1 Tylosin lactate contains not less than 850 IU of tylosin per mg.

Description: Tylosin lactate is a white to light yellow crystalline powder. It is odourless and has a bitter taste.


A. Solubility. Soluble in water. Very slightly soluble in methanol and acetone. Insoluble in cyclohexane and benzene.

B. Melting point. Approximately 146°.

C Ultra-violet absorption. Maximum absorption wavelength is 1%

is 290 ± 2 mu and Elem = 210 ± 2.

1 Tylosin base, Tylosin tartrate and Tylosin phosphate are also available.

30

Purity tests

Water: Not more than 5.0 percent (Karl Fischer method)

Sulphated Ash: Not more than 5.0 percent

Asay

l. A.my of the material

1.1 Media

1.1.1 Stock medium

Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 g Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . 4.0 g Peptic Digest of Meat . . . . . . . . . . . . . . . . . . . . . 6.0 g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 g Agar ................................ 17.5g Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . 1 OOO ml

Dissolve the ingredients in a quantity of hot distilled water and adjust the pH to 6. 7 before the solution is diluted to its final volume. Divide the hot solution into approximately 7.0 ml aliquots and transfer into culture tubes. Plug the tubes and sterilize by autoclaving for 30 minutes at 121 ° and cool on a slant. When cooled, store in a refrigerator.

1.1.2 Culture medium

Mix equal amounts of Basal Medium (1.1.3) and distilled water. A 200 ml aliquot of this solution is placed into each of several Erlenmeyer flasks, which are plugged and sterilized by autoclaving for 15 minutes at 121°C. When cool, store in a refrigerator.

1.1.3 Basal medium

Peptic Digest of Meat ................... . Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium chloride . . . . . . . . . . . . . . . ....... . Glucose ............................ .

31

10.00 g 3.00 g 3.00 g 7.00 g 2.00 g

Pota~ium phosphate, dibasic . . . . . . . . . . . . . . . 6.68 g Potassium phosphate, monobasic . . . . . . . . . . . . . 2.60 g Distilled water q.s. to .................... 1 OOO ml

Dissolve the ingredients in a quantity of distilled water and adjust the pH o 7.0 before the solution is diluted to its final volume. Place the solution in several Erlenmeyer flasks, plug and sterilize by autoclaving for 15 minutes at 121°. Store this solution in a refrigerator.

1.2 Standard solution

1.2.1 Tylosin standard

Use a specific lot of tylosin calibrated in terms of International Units of Tylosin.

l.2.2 Phosphate buff er - pH 7 .0

Pota~um phosphate, dibasic . . . . . . . . . . . . . . . . 13.6 g Potassium phosphate, monobasic . . . . . . . . . . . . . 4.0 g Distilled water q.s. to ..................... 1 OOO ml

Place the solution in a bottle, plug and sterilize by autoclaving for 30 minutes at 121 °

1.2.3 Stock Standard solution

An accurately weighed quantity, 25 mg of tylosin standard 1.2.1 is rinsed into a 2.5 ml volumetric flask with 2.5 ml methanol. Bring the flask exactly to volume using phosphate buffer pH 7 .0 (1.2.2). This solution may be used to prepare the daily standard solution for a period not to exceed 14 days if kept under constant refrigeration.

1.2.4 Daily standard solution

On the day of the test further dilute a portion of the Stock Standard Solution using phosphate buffer pH 7.0 so that 1 ml of the final dilution contains exactly 2.0 lU of tylosin.

1.2.S Sample solution

Dilute an accurately weighed quantity, 30 mg of tylosin lactate to be tested according to the same procedure as that used in

32

the preparation of the Daily Standard Solution, so that 1 ml of the final dilution contains 2.0 lU of tylosin.

1.2.6 Test organism suspension

Staphylococcus aureus (ATCC 6538P) is transferred to fresh slants of Stock Medium ( 1.1. l) for subculturing each day. Incubate the freshly transferred cultures for 16 to 20 h. at 37.5° and store in a refrigerator. Wash the fresh culture slant with 5.0 ml of Culture Medium ( 1.1.2). Transfer this cell suspension into a flask containing 200 ml of Culture ,Hedium Incubate this broth culture for 16 to 20 h. at 37.5°. Just prior to use, inoculate the Basal Medium ( 1.1.3) with this culture to fonn a l O I 0 · suspension. A fresh suspension is prepared for each day's assay.

1.3 Assay procedure

Use nine rows of 3 culture tubes, 18 x 150 mm in size, for the standard solutions as follows: A 5.0 ml, 4.8 ml, 4.7 ml, 4.5 ml, 4.4 ml, 4.3 ml, 4.2 ml. 4.1 ml, and 4.0 ml, add aliquot of distilled water to each set of 3 tubes, respectively. Then add a 0.0 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, and 1.0 ml aliquot of the Daily Standard Solution to each set of 3 tubes, respectively. Prepare five rows of 2 tubes for Sample Solutions of the materials to be assayed as follows: Add a 4.7 ml, 4.5 ml, 4.2 ml and 4.0 ml aliquot of distilled water to each set of 2 tubes, respectively. Then add a 0.3 ml, 0.5 ml, 0.6 ml, 0.8 ml and 1,0 ml aliquot of Sample Solution to each set of 2 tubes, respectively. Just prior to incubation, add a 5.0 ml aliquot of the chilled Test Organism Suspension to each tube of the standard solution or sample solution. Cover each tube to prevent contamination during the incubation period and place in a 37.5° constant temperature water bath for approximately 4 h. 1 Then place the tubes in a steam sterilizer for 5 to l O mins. When cool, the turbidity produced in each tube is measured using a suitable photoelectric colorimeter.

1The optimum incubation period is determined by reading the turbidity produced in the tube containing no standard solution. When the turbidity comes between 20 and 30 percent transmission in comparison to LOO percent transmission for water, all the tubes should he removed from the water bath and sterilized.

33

Obtain the average of the turbidmetric readings for each set of 3 tubes. Draw the standard response curve by plotting these averages against the concentrations of the standards. Calculate the tylosin content in each Sample Solution from this standard response -curve, by interpolation, and compute the potency of the sample of tylosin lactate.

2. A~y in feed: Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC), 1965, lOth Edit., Page 660; 33.159-33.162.

3. ~y in Twues

3.1 Cylinders

Stainless steel cylinders with an outside diameter of 8 mm. (± 0.1 mm.), an inside diameter of 6 mm.(± 0.1 mm.), and a length of 10 mm.(± 0.1 mm.).

3.2 Culture media

3.2.1 Agar for carrying the test organism and for the single seed layer.

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . ·Pancreatic digest of casein ................ . Yeast extract ..... : ................... . Beef extract .......................... . Dextrose ............................ . Agar .............................. . Distilled water, q.s. . ................... .

pH 8.45 - 8.55 after sterilization

6.0gm 4.0 gm 3.0 gm 1.5 gm 1.0 gm

15.0 gm 1 OOO ml

3.2.2 Nutrient broth for stock cell suspension and inoculum

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . 5.0 gm Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Dextrose . . . . . . • . . . . . . . . . . . . . . . . . . . . . . 1.0 gm Sodium chloride . . . . . . . . . . . . . . . . . . . . . . . 3.5 gm Dipotassium phosphate . . . . . . . . . . . . . . . . . . . 3.68 gm Monopotassium phosphate . . . . . . . . . . . . . . . . . 1.32 gm Distilled water q.s. . . . . . . . . . . . . . . . . . . . . . . 1 OOO ml

pH 6.95 - 7.05 after sterilization

34

In lieu of preparing media from the individual ingredients specified here, they may be made from a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such media.


Dissolve a suitable weighed quantity (usually 25 milligrams or less) in 2 millilitres of ethyl alcohol, then add sufficient 0.1 M potassium phosphate buffer, pH 8.0 to give a concentration of l,000 micrograms of tylosin base per milliliter. This stock solution may be kept in the refrigerator for three days.

3.4 Preparation of samples

3.4.1 Muscle, liver and kidney

Grind the tissue in a food grinder. To 30 gm of ground tissue add 30 ml. of water. Blend in a homogenizer and adjust the pH of the slurry to 4.5 with hydrochloric acid. Centrifuge the suspension for I hour. Decant and collect the supernatant. Discard the sediment. Adjust the pH of the supernatant to 8.0 with sodium hydroxide and use as the assay solution.

3.4.2 Fat. Proceed as in 3.4. l except add 45 milliliters of water to 30 grams of sample.

3.S Preparation of test organism

3.S.I Preparation of stock suspension

The test organism, Sarcina lutea (ATCC 9341), is maintained on agar slants containing 10 millilitres of agar medium 3.2. l. The slants are inoculated and incubated at 32° - 35° for 24 hours. Three milillitres of sterile saline T.S. is used to wash the growth from the agar slant onto a large agar surface, such as a Roux bottle containing 250 millilitres of agar medium 3.2.1. The suspension of organisms is spread over the entire surface of the agar in the Roux bottle with the aid of sterile glass beads. The Roux bottle is incubated for 24 hours at 32° - 35°. The resulting growth is washed from the agar surface with 50 millilitres of sterile saline T .S. to give a stock suspension.

35

3.S.2 Standardization of the organism suspension

A 0.5-1.0 millilitre portion of the stock suspension is diluted with sterile saline T .S. in a proportion of 1 + 39 and the light transmiSsion detennined at a wavelength of 580 millimicrons using a suitable photoelectric colorimeter and a 13-millimeter diameter test tube as an absorption cell. If the light transmiSsion of the diluted suspension is about 25 percent, the stoc_k suspension is ready for use. If it is less than 25 percent, the stock suspension should be diluted with sterile saline T.S. so that when it is diluted 1 + 39, a 25 percent light transmission value will be obtained. The diluted suspensions used to determine light transmiSsions are discarded. The optimum amount of stock suspension to be added to each 100 millilitres of agar medium 3.2.1. is determined by running test assay plates using 0.5 millilitre per 100 millilitres as a starting point.


Add the appropriate amount of standardized organism suspension (as determined in 3.5) to each 100 millilitres of agar medium 3.2.1 which has been melted and cooled to 48°. Mix the culture and agar thoroughly and add 5 ml to each Petri dish (20 x 100 mm.) .. Tilt each dish back and forth as the agar is added to spread the seeded agar evenly over the surface, and then cover each dish. After the agar has hardened, place 6 cylinders on the surface so that they are at approximately 60° intervals on a 2.8-centirneter radius.

3. 7 Standard Response Line

Prepare the daily standard response line for each type of tissue by further diluting the 1,000 micrograms per millilitre stock solution with an extract of control anbiotic-free tisstie prepared as in 3.4 to obtain concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 micrograms per millilitre - the 0.4 microgram per millilitre concentratiqn as the reference point. Use three plates for the determination of each point on the response list, except the reference point, a total of 12 plates. On each of three plates fill three cyclinders with the 0.4 microgram per millilitre concentration (reference concentration) and the other three with the concentration under test. Thus, there will be 36 reference concentration zones of inhibition and nine zones of

36

inh.t'bition for each of the four other concentrations of the response line. After incubation, read the diameters of the circles of inhibition in the plates. Average the readings of the 0.4 microgram per millilitre concentration and the readings of the point tested for each set of 3 plates and average also all 36 readings of the 0.4 microgram per millilitre concentration. The average of the 36 readings of the 0.4 microgram per millilitre concentration is the correction point for the response line. Correct the average value obtained for each point to the fJgure it would be if the 0.4 microgram per millilitre reading for that set of 3 plates were the same as the correction point. Thus, if in correcting the 0.2 microgram concentration, the average of the 36 readings of the 0.4 microgram concentrations were 17.0 millimeters and the average of the 0.4 microgram concentration of this set of 3 plates were 16.8 rnillimeters, the correction would be ± 0.2 millimeter. If the average readings of the 0.2 microgram concentration of these same 3 plates were 14.0 millimeters, the corrected value would be 14.2 millimeters. Plot these corrected values including the average of the 0.4 microgram per millilitre concentration, on 2-cycle semilog paper, using the concentrations in micrograms per millilitre as the ordinate (the logarithmic scale) and the diameter of the zone of inhibition as the abscissa. Draw the standard response line through these points, either by inspection or by means of the following equations:

where:

L =

H =

c =

a, b, d, e =

L 3a + 2b + c - e =

5

H 3e + 2d + c - a = 5

Calculated zone diameter for the lowest concentration of the standard response line;

Calculated zone diameter for the highest concentration of the standard response line;

Average zone diameter of 36 readings of the 0.4 microgram per millilitre standard;

Corrected average values for 0.1, 0.2. 0.8 and 1.6 micrograms per millilitre standard solutions, respectively.

37

3.8 Assay Procedure

Use 3 plates for each sample. Fill 3 cylinders on each plate w!th the standard 0.4 microgram per milWiter solution and 3 cylinders with the 0.4 microgram per milliliter (estimated) sample, alternating standard and sample. Incubate all plates, including those containing the standard curve at 30° overnight, and measure the diameter of each circle of inhibition. To estimate the potency of the sample, average the zone readings ·of the standard and the zone readings of the sample on the 3 plates used. If the sample gives a larger zone size than the average of the standard, add the difference between them to the 0.4 microgram per milWiter zone on the standard response line. If the average sample value is lower than the standard value, subtract the difference between them from the 0.4 microgram per milliliter value on the response line, and read the potencies corresponding to these corrected values of zone sizes.

Sensibility of assay


in animal tissues 0.2 ppm 1

1·Basis 1 I.U. = 1 mcg. of Tylosin base.

38

PENICILLINS

PENICILLINS

Specifications

Benzylpenicillin potassium. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia WHO, Geneva, 2nd Eq.it., Page 77.

Benzylpenicillin sodium. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia WHO, Geneva. 2nd Edit., Page 79.

Insoluble forms often employed are Procaine benzylpenicillin and Ben.zathine benzylpenicillin.

Assays

l. In animal feed. Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC), 1965, lOth Edit., Page 654, 33.118-33.121

2. In tissue, milk and eggs - As given in the Annex.

Sensitivity of assay 1


In animal tissues 0.10 IU/gram (0.06 ppm)1

In milk 0.01 [U/ml (0.006ppm)1

In eggs (whole) 0.03 IU/gram (0.018 ppm)1

1 Further work is required to determine the exact type of penicillin, e.g. phenoxymethyl penicillin, nafoillin, oxacillin, cloxacillin, ampicillin, etc., which may be present in the tissue. This is important, as quantification of possible penicillin residues requires a knowledge of the form of penicillin present.

1 Basis 1667 units = one mg of benzylpenicillin sodium.

39

POLYENES

NYSTATIN

Specifications

NystatinSpecifications for the Quality Control of Pharmaceutical Preparations. [nternational Pharmacopoeia WHO, Ge'neva, 2nd Edit., Page 394

Assays

l. In animal feeds. Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) l 965, IOth Edit., 33.144-33.147, Page 658

2. In tissue

2.1 Media

2.1.1 Inoculum broth: Penassay Broth, dehydrated (Difeo), l 7.5 g/l; glucose, 10 g/1; yeast extract (Difeo), 5 g/l; tryptone, 10 g/1; and distilled water. Inoculum agar for weekly transfers of the test organism consists of inoculum broth with LS percent agar added.

2.1.2 Assay broth: Casitone (Difeo), 9 g/1; glucose, 70 g/1; yeast extract (Difeo), 5 g/1; sodium citrate, 10 g/1; K2 HP0 4 , l g/1; KH 2 P04 I g/1; and distilled water. Just before use add to each litre of sterile assay broth 0.13 ml of a dimethyl sulfoxide (DMS) solution containing 4 OOO units of nystatin per ml (final concentration equals 0.5 units/ml) 1 and to control bacterial contamination add l gm each of penicillin and streptomycin.

2.1.3 All media are sterilized for 15 min. at 120°.

1With some samples, levels of nystatin less than 0.5 unit/ml enhances gas production. The addition of 0.5 unit/ml of ny,tatin causes partial inhibition of respiration, therefore any further addition of nystatin cannot stunulate gas production, but results in inereased inhibii:on.

2.2 Inoculurn

Test Organism: Saccharomyces mellis (Squibb No. 1647). Prepare a fresh stock slant each week on inoculum agar from the slant of the previous week and incubate for 24 hours at 37°. From this slant, inoculate a 500 ml Erlenmeyer flask containing l 00 ml of inoculum broth and incubate for 17 hrs. at 37°, on a reciprocal shaker (120-l-li2 in.strokes per min.) Transfer a 25 ml aliquot of this inoculum to a second 500 ml Erlenmeyer containing l 00 ml of inoculum broth and incubate for 3 hrs. at 37°, on a reciprocal shaker. After 3 hrs. remove the f1ask and store at 5°, until needed, but for not longer than one week.

2.2.2 Just prior to use, blend a 100 ml aliquot of the inoculum for 1-1-1/2 minutes in a blender to break up aggregates formed during incubation. The blend is centrifuged to concentrate the cells. The yield of cells should be approx. 2 percent (V/V). The supernatant is discarded and the cells resuspended in approx. 10 ml of assay broth to produce a 20 percent cell suspension.

2.3 Preparation of tissues for standard curve

2.3.1 Nystatin solutions - Prepare a 30 OOO unit/ml 1 solution of nystatin in dimethyl sulfoxide (OMS). For use with no fatty tissues dilute this solution in OMS to each of the following concentrations: 16 OOO; 4 OOO; l OOO: 250 units/ml. For use with fatty tissues dilute the 30 OOO units/ml solution in methanol to the following concentrations: 3 200; 800; 200: 50 units/ml.

2.3.2 Standards for non fatty tissues - Grind the tissues in a meat grinder before processing, rinsing the grinder with methanol between samples. Place a 10 gm portion of nystatin-free tissue in a graduated cylinder and dilute to 95 ml with assay broth containing 0.5 unit/ml of nystatin. Blend in a blender for 1 mins, and then pour it into a flask. To dissipate the foam add two drops of mineral oil and place the flasks at 5° for about l hr. Add 5 ml of the 20 percent S. me/lis suspension and mix thoroughly. Pipette 10 ml aliquots of the inoculated blend into each of the

10ne mg equals about 3 OOO units of pharmaceutical gr:ide nystatin.

41

5 test tubes (1" x 4") and number the tubes 1 through 5. Dose the tubes with 0.01 ml of nystatin standard solution as follows:

Tube Nystatin standard Final concentration No. concentration units/ml units/g of tissue

1 16 OOO 160 2 4000 40 3 1 OOO 10 4 250 2.5 5 (0.01 ml of DMS) 0

2.3.3 Standards for fatty tissue - Grind the tissues in a meat grinder, rinsing the grinder with methanol between samples. Place five 2 g portions of the ground, nystatin-free fatty tissue in separate mortars and dose with 0.1 ml of nystatin solution as follows:

Mortar No.

1 2 3 4 5

Methanolic nystatin standard sc;>lution, units/ml

3 200 800 200 so

(0.1 ml methanol)

Final concentration units/g

160 40 10 2.5 0

Dry each portion under vacuum (about 3 mm of Hg.) to remove the methanol (20 to 30 min.). Wash each 'portion with hexane three times using 20 ml for the first wash and 10 ml each for the second and third washes. It is essential to mash the fatty tissue with a pestle during the first wash to effect intimate contact between the solvent and the fat. Centrifuge each wash to precipiate suspended nystatin. Discard the clear hexane supernatants. Air dry the contents of

• the centrifuge tubes and mortars to remove residual hexane. Moisten the tissue residue in each mortar with dwo drops of assay broth, add 10 to 15 mg of sand and grind the mixture to a paste with a pestle. Suspend the paste in 18 ml of assay broth

42

containing 0.5 unit nystatin per ml. Pool this suspension with its respective three precipitates from the hexane washes. To this, add 2 ml of 20 percent cell suspension.

2.4 Preparation of samples containing an unknown quantity of nystatin

Treat unknowns the same as the zero concentration of nystatin (Tube No.5), shown in the appropriate procedure above 2.3.2 or 2.3.3.

2.S Syringe assay

Lubricate the plungers and barrels of 5 ml syringes with low viscosity mineral oil to prevent gas leaks and to lessen friction. Draw 2 ml of inoculated tissue suspension from each tube, as described in section 2.3, into a syringe and cap with undrilled needle nub. Incubate the syringes in a 30° water bath. During incubation, remove the syringes from the bath at 15 minute intervals and invert several times to aid the release of dissolved C02 • Incubate until the syringe containing the O units/ml of standard produces approx. 2 ml of gas. This requires 1 to 4 hours depending on the nature of the tissue. Read the volume of gas produced directly from the calibrations on the syringe barrel. Construct the standard curve by plotting C02 volume vs. log concentrations of nystatin and read, from the curve, the concentration of nystatin in an unknown.

3. In milk and eggs - As given in the Annex

Sensitivity of a~y


In animal tissue 20 IU/gm (7.1 ppm)1

In milk

In eggs (whole)

3 IU/gm (1.1 ppm)1

12 IU/gm (4.3 ppm)1

1 Basis 2 800 OOO units = l gm of Nystatin.

43

PIMARICIN

Chemical name: Pimaricin

Chemical fonnula: C33H4,.N013 Structural fonnula:

HO


Definition: Pimaricin is an antifungal antibiotic of the polyene rnacrolide group usually produced by Streptomyces natalensis. It contains not less than 98.0 percent of C33 H41NOu calculated with reference to the substance dried at 60° for 4 hours at a pressure not greater than 5 mm of mercury.

Description: Pimaricin is a white, tasteless, odourless powder.


A. S<'. ubility

Soluble in glacial acetic acid and dimethylformamide. Slightly soluble in methanol. Insoluble in water.

44

B. Ultra-violet absorption

The solution prepared for spectrophotometer assay has absorption maximum wave-length of 290 run, 303 run, and 318 run.

C. Add a few crystals of Pimaricin to a drop of hydrochloric acid in a spot plate. A blue colour develops. Repeat using concentrated phosphoric acid. A pale red colour develops. Repeat using sulphuric acid. A green colour develops.

Purity Tests

Loss on drying: When dried at 60° at a pressure not greater than 5 mm of mercury for four hours, loses not more than 7.0 percent of its weight.

Sulphate ash: Not more than 0.5 percent

Heavy metals: Not more than 30 ppm

~ys

1. Microbiological assay

1.1 Materials and apparatus

1.1.1 Gla3Sware

Petri dishes: diameter 10-11 cm, flat bottom

Agar pipette: wide tip; 25 ml graduated in 5 ml wide tip; 10 ml graduated in 2.5 ml

Stainle~ steel cups: cylinders, outer diameter 8 mm; inner diameter, 6 mm; height 4 mm

Blow-out pipette: see figure

(a 1 ml graduated pipette may be used, if necessary)

Water bath: 45°

(ncubator: 30°

Ruler: white, graduated in mm.

45

1.1.2 Test organism

Sacclzarom,vces cerei·,szas. ATCC 9763. Maintain the yeast on malt-agar. To prepare the inoculum transfer a loopful to liquid malt extract medium and incubate for not less than 38 hours at 26°. This culture may be stored in the refrigerator. Prepare fresh inoculum weekly.

l.1.3 Nutrient media

Wiffen-agar (buffered): Dissolve 2.6 g yeast extract (Difeo) and 15 g agar in 600 distilled water. Dissolution is effected by heating for 30 min. at 110°. Filter the solution hot, mix with 400 ml of a wann buffer solution and sterilize for 30 min. at 110°; pH after cooling 6.4

Composition of buffer: A = 34 g KH 2P04/ l

B = 10 g NaOH/1

Mix 250 ml A + I 50 ml B

Before use add 2 ml sterile 50 percent glucose solution to 100 ml cooled ( 45°) Whiffen:agar (the glucose solution is sterilized by Seitz-filtration).

Malt-agar (for maintenance of the test organism): Dilute malt-extract with water to 10° Balling (this corresponds with 100 g Difeo malt extract 'per litre), and add 2 percent agar. Dissolve by heating over a flame or preferably by keeping the mixture at 100-110° for 30 min. Filter and sterilize for 20 min at l 00° Final pH, after cooling: 5.4 The test-organism is transferred monthly; it is incubated for 24 hrs at 26°-30° and stored at 5°-15°

Malt-extract medium (for preparation of inocµlum): Sterilize malt exw-act of 12° Balling (this corresponds with 125 g Difeo malt extract per litre) for 1 hr at 110°, filter, sterilize again for 20 rnin at 120° and filter while still hot. Fill small flasks with 30 ml of this medium, plug and sterilize for 20 min. at 110°.

46

1.1.4 Antibiotic: standard solutions

A preparation of pure pimaricin is required of which the potency has been determined and is given in "'(/mg. The preparation is stored in the dark in a cool, dry place. Prepare a stock solution containing 1000 "'(/ml by dissolving the appropriate amount of pimaricin standard in 100 percent methanol. Make working standards by first diluting the stock solution l: l with distilled water. Use solution obtained (which must be made fresh every 2 days) containing 500 "'(/ml in 50 percent methanol to prepare a series of dilutions with 100, 75, 50, 30, 20 and 15 -y/ml ( diluent SO percent methanol).

1.2 Method

1.2.1 Pcuting of the test-plates; setting-up of the cups

Place 25 ml hot Whiff en-agar in a sterile Petri dish. After hardening, pour a second layer on top of the first. The second layer consists of 6 ml of a mixture of 100 ml Whiffen-agar cookd to 45° and 1 ml inoculum. Place 6 cups on each plate in a circle, at regular intervals, 20--25 mm from the outside of the plate

1.2.2 Filling of the cups

Use small 50 mm3 blow-out pipettes for this purpose. Fill the capillary carefully with the sample or with a standard solution, then blow out on a piece of filter paper. Repeat until the pipette is well washed (at least 3 times). Use the pipette to transfer the measured amount of sample or standard to a cup on the test-plate. (Graduated pipettes of 1 ml may be used, but they are considerably less convenient). With each set of unknowns, use 15 standard plates: a series of. 5 standards, carried out in triplicate. Arrange the standard solutions on these plates in the following manner:

Plate 1: Plate 2: Plate 3: Plate 4: Plate 5:

3 cups l 00 -y/ml each + 3 cups 50 "'(/ml each 3 cups 75 'Y/ml each + 3 cups 50 -y/ml each 3 cups 30 'Ylm1 each + 3 cups SO -y/ml each 3 cups 20 -y/ml each + 3 cup~ 50 'Ylml each 3 cups 15 'Y/ml each + 3 cups 50 "'(/ml each

47

For the samples, which must be diluted to contain 25-75 -ylml, the tests are also carried out in triplicate: each of 3 plates carrying 3 cups with sample solution and 3 cups with the 50 'YI ml standard.

1.2.3 Incubation: taking the readings; calculation of the results

After the standards and samples have been pipetted, leave the plates uncovered. for 2 hrs at 15° -18°. Close the top and incubate for 18 hrs at 30°. After incubation, measure the diameters of the zones of inhibition to the nearest 0.5 mm with a ruler. Calculate the average diameters for the samples and the standards of each set of triplicates, as well as the grand-average for the 50 -ylml standard. Correct the average diameter of the standards and the sample. For each set of triplicates a correction value is calculated as the difference between the grand-average for the 50 'YI ml standard ~d the set-average for that same solution. Each set-average is now corrected by adding (algebraically) its correction value. Plot the corrected values thus obtained for the standard solutions on semi-logarithmic graph paper: concentration along the loga~thmic abscissa and corrected diameters along the ordinate. Draw a straight line from which unknown concentrations may be read with the help of the corrected diameters of their inhibition zones.

2. Spectrophotometric ~y

Introduce approx. 100 mg Pirnaricin, accurately weighed into a 200 ml volumetric flask. Add 10 ml of buffer solution pH 10, made by dissolving 54 mg of arrunonium chloride and 350 ml concentrated arrunonia solution (25 percent) in distilled water anc! diluting to 1000 ml; shake well and allow to stand for about 5 minutes in the dark. Now add 100 ml of methanol and shake mechanically in the dark for 30 minutes. Fill up to mark with methanol and rt#· Pipet 10 ml of this solution into a 100 ml volumetric flask, fill up to mark with methanol and mix. Of this solution pipet I O ml into a 100 volumetric flask and fill to mark with a 1 percent solution of acetic acid in methanol. Prepare the last solution in subdued light. Determine the transmittance at the maximum absorbing wave lengths (about 303 and 318 nm) in a 1 cm cell against a blank of the 1 percent

48

solution of acetic acid in methanol. The concentration of pimaricin is read from a calibration curve obtained with pimaricin solutions. containing 1 to 8 micrograms per ml, determined as described above. Calculation:

micrograms pimaricin per ml in sample (from curve) X 200

wt. of the sample in mg. = % pimaricin

3. Assay in cheese, cheese rind and plastic coating

3.1 Qualitatii•e

3.1.l From rind and plastic coating

Cut off a slice of rind approx. l mm thick, or peel off the plastic coating. Cut out discs with a borer ( 8 mm diameter) from the rind or plastic coating. Place the discs outer side down on the agar of the test plates (see under Microbiological Assay).

3.1.2 From cheese

Cut slices about 1 mm thick parallel to the cheese surface. From these slices cut out 8 mm diameter round discs. Place the discs 0uter side (nearest side to the rind) down on the agar of the test plates.

Examination of the test plates

Zones of inhibition develop around the discs if the discs contain pimaricin. In case of doubt as to whether the activity found is caused by pimaricin or not, further examination must be carried out by taking the extract and examining it spectrophotometrically, (For extraction proceed as under 3.2)

3.2 Quantitative

3.2.l From rind and plastic coating

Cut off a slice of rind approx. 1 mm thick, or peel off the plastic coating. Weigh approx. 20 cm2 of this, and then cut it into little pieces. Extract the pieces with 10 ml of l 00 percent

49

methanol by shaking at room temperature in the dark for half an hour. Centrifuge and repeat the extraction on the residue with 5 ml methanol. This procedure is performed a third time. The extraction is complete if the last extract contains no measurable quantity of pimaricin. The extracts are diluted l: 1 with water. Each of the 50 percent-methanol extracts is assayed microbiologically in 3 dilutions (undiluted, 1/2 and l/4 dilution with 50 percent-methanol). Calculate the total pimaricin content from the sum of the assay values of the extracts.

3.2.2 From cheese

Cut slices about 1 mm thick parallel to the cheese surf ace. Weigh approx. 20 cm2 of this and extract by shaking for half an hour with 5 ml of 100 percent-methanol at room temperature in the dark. Centrifuge and repeat the extraction on the residue with 5 ml 100 percent-methanol. This procedure is repeated a third time. Dilute and assay the extracts in 3 dilutions as given under 3.2. l above.

Sensitivity of a~y in cheese

The method is estimated to be capable of detecting 25 ppm.

so

cork tube oppro11 70 cm moolh piece

'-~~~~~--~~~~~-_________________ a~- o

V')

cop i II or y tube volume 50mm3 50mm3btow pipette

POLYPEPTIDE~

BACITRACIN

Specifications

Bacitracin. Specifications for the Quality Control of Phannaceutical Preparations. International Phannacopoeia, WHO, Geneva, 2nd Edit., Page 58.

Zino bacitracin, Manganese bacitracin and Bacitracin methylene disalicylate are also used.

Assays

1. In animal feed. Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) 1965, l Oth Edit., page 655 33.122 - 33.130

2. In tissue, milk and eggs. As given in the Annex

Sensitivity of as.uy


In animal tissue 0.03 IU/gram (0.7 ppm)1

In milk 0.05 IU/ml (l.2 ppm) 1

In eggs (whole) 0.20 IU/gram (4.8 ppm)1

1 For the purpose of this document 4200 lU have been taken to be equal to l gm of bacitracin.

52

NISIN

Chemical name: None

Chemical formula: The structural formula is not known. Nisin contains the following amino acids:

Lysine, histidine, aspartic acid, serine, lathionine, B-methyllanthionine, praline, glycine, alanine, valine, methionine, insoleucine, leucine (Cheeseman, G.C. and Berridge, N.J., Biochem. J. 71, 185, 1959.) lt has been claimed (Berridge, Newton and Abraham. Biochem. J. 52, 529, 1952, that there are 4 or possibly S closely related polypeptides having similar properties.

Molecular weight: The molecular weight was reported by Cheeseman and Berridge (loc.cit) as approximately 7000. Cross, E. and Morell, J.L. (J.Amer.Chem.Soc., 89, 2791, 1967) however calculated a value of 3510.

Definition: Nisin consists of several closely related polypeptide antibiotics produced by strains of Streptococcus lactis, Lancefield group N. Nisin concentrate contains not less than 900 units per mg. in a mixture of non-fat milk solids and a minimum sodium chloride content of SO percent. The most potent preparation of nisin yet obtained is 40,000 units/mg. The Unit has been redefined by Trarner and Fowler, J.Sci.Fd.Agric., 15, 522 (1964) in terms of a standard preparation. This approximates to the activity unit described by Berridge (Biochem.J. 45, 436, 1949.

Description: Nisin concentrate is a white, micronised, spray-dried powder.

Stability: Nisin concentrate is stable at ambient temperatures. Both purified nisin and nisin concentrate are stable to heating under acid conditions, and will withstand 121° for 30 minutes _at pH 2.0 and 15 minutes at pH 3.0. Nisin is less stable at higher pH values; heating at 121 ° in buff er for 15 mitt. results in the following percentage decreases in activity

pH ~.0-29%, pH S.0-69%, pH 6-86%, pH 7.0.99.7%


A. Melting point

None. Chars on heating

53

B. Solubility

(a) Purified preparation of nisin containing 40,000 units/mg.

Solvent

Water, pH 2.5 Water, pH 5.0 Methanol ·acidified with

HCl to pH 2.8

Insoluble in non-polar solvents

(b) Nisin concentrate

Units/ml

4.8 x 106

1.6 x 106

3.2 x 106

Forms a cloudy suspension in water due to the presence of denatured protein, but the nisin component behaves similarly to the purified material.

C. Differentiation from other antibiotics - See Appendix

Loss on drying:

Heavy metals:

Purity tests

Not more than 3.0 percent when dried to constant weight at 102-103° (British Standard Method of the Chemical Analysis of Dried Mille B.S.1743: 1951).

Lead (Pb) Arsenic (As)

Assays

Not more than 2.0 ppm Not more than 1.0 ppm

l. Determination of sodium chloride content of nisin concentrate

Weigh ea. 5 g material, transfer with 80 percent alcohol to 100 ml volumetric flask and add enough 80 percent alcohol to give volume of ea 50 ml. Shake well to suspend all insoluble material. Add 1 ml HN03 and with pipet add excess of 0.1 N AgN03 soln. Dilute to 100 ml with alcohol. Transfer mixture to centrifuge bottle and centrifuge 5 min. at ea 1800 rpm. Pipet 50 ml supernatant into 300 ml Erlenmeyer flask, add 2 ml saturated FeNH4 (S04 ) solution and 2 ml HNO 3 and titrate to permanent light brown with 0,1 N NH4 CNS. Divide ml

54

0.1 N AgN03 used by 2 and subtract ml NH4 CNS solution used. Multiply difference by 0.005844 to obtain g NaCl present.

2. A.ay of nisin concentrate

2.1 Preparation of test organism

Streptococcus cremoris, lPS (NCOO 495 1) is subcultured daily

in sterile separated milk by transferring (Based on the method of Friedman and Epstein, J. Gen.Microbiol. 5: 830, 1951) one loopf ul to a McCartney bottle of litmus milk and incubating at 30°. Inoculated milk for the assay is prepared by inoculating a suitable quantity of sterile separated milk with 2 percent of a 24-hour culture, and placing it in a water-bath at 30° for 1-1/2 hours. It is then used inunediately.

2.2· Preparation of standard solution

The Standard Stock Solution is prepared by dissolving an accurately weighed quantity of standard nisin in N/50 hydrochloric acid to give a solution containing 5 OOO. units/ml. The stock solution is diluted further with N/50 HCl immediately before use to give 50 units of n.isin per ml.

2.3 Preparation of sample solution

The weight of sample taken for preparing the stock solution of the sample is such that corresponding tubes of the sample and standard series match, i.e. within close Limits the sample and standard are of the same concentration with respect to nisin content. The sample stock solution is diluted in N/50 hydrochloric acid. to give an estimated concentration of SO units of nisin per ml.

2.4 Preparation of reSllZurin solution

A 0.0125 percent solution of resazurin in distilled water is prepared immediately before use.

1 National Collection of Dairy Organisms, National Institute for Research in Dayring, Shinfield, Berksh.ue, England.

55

2.S Ass~v procedure

Graded volume (0.60, 0.55, 0.50, 0.45, 0.41, 0.38, 0.34, 0.31, 0.28, 0.26 ml} of the 50 unit per ml sample and standard solutions are pipetted into rows of 10 dry 6

11X 5/8"

bact<uiological test-tubes, and 4.6 ml of the inoculated milk is · added to each by means of an automatic pipetting device. The addition of inoculated milk is made in tum aero~ each row of tubes containing the same nominal concentration not along each row of ten tubes. The tubes are placed in a water-bath at 30~ for 15 minutes, then cooled in an ice water bath while 1 ml resazurin solution is added to each. The addition is made with an automatic pipetting device, in the same order used for the addition of inoculated milk. The contents of the tubes are thoroughly mixed by shaking, and incubation at_ 30° is continued in a water-bath for a further 3-5 minutes. The tubes are examined under 2 X 20 watt Osram "Natural" fluorescent light in a black matt-finish cabinet. The sample tube of the highest concentration which shows the first clear difference in colour (i.e. has changed from blue to mauve) is compared with tubes of the standard row to find the nearest in colour. Further matches are made at the next two lower concentrations of the sample and standard. Interpolation of matches may be made at half dilution steps. As the standard tubes contain known amounts of nisin, the concentration of nisin in the sample solution may be calculated. The three results are averaged.

3. ~ay in foods

3.1 Assay by plate diffusion technique

3. t .1 Assay medium

Bacteriological peptone . . . . . . . . . . . . . . . . . . . . . . . 10 g Bacteriological beef extract . . . . . . . . . . . . . . . . . . . . 3 g Sodium chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Autolysed yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Brown sug!1r (unrefined).... . . . . . . . . . . . . . . . . . . . 1 g Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Distilled water to ............................ l OOO ml

3.1.2 Tween 20 - (Polyoxyethylene sorbitan monolaurate)

56

3.1.3 Assay organism - Micrococcus flavus (NC 18 8166)

The organism is maintained by subculturing on slopes of the assay medium and incubating at 30° for 48 hr. Prepared slopes may be stored at 4° until required up to a maximum of 14 days. When required for use the growth on the slope culture is suspended with 7 ml of nonnal saline solution.

3.1.4 Nisin standard - 0.1 g of standard nisin (1000 units/mg) is weighed accurately and suspended in about 80 ml of 0.02N-hydrochloric acid and set aside at room temperature for 2 hrs. The suspension is then made up to 100 ml with 0.02N-hydrochloric acid. It contains l OOO units of nisin/ml and is further diluted with 0.02N-hydrochloric acid to give the required concentration. This can be satisfactorily stored for 7 days at 4°. Otherwise a fresh nisin standard should be prepared each day.

3.1.S Preparation of standard curve

The prepared assay medium is melted and cooled to approx. 50°. Two percent of a l: l mixture of Tween 20 and sterile distilled water, previously held for 20-30 min. at 48°, is added and thoroughly mixed with the medium. The suspension of the test organism is diluted l in l O with nonnal saline solution and 2 ml of this dilution are added to each 100 ml of melted medium at 48° with thorough mixing. The inoculated medium is poured to a depth of 34 mm into previously levelled assay plates or flat-bottomed sterile Petri dishes and allowed to solidify. The plates are then inverted and stored at 4° for 1 hr to facilitate the boring of holes. With the aid of a sterile hollow steel borer, 7-9 mn, diameter the required number of holes are cut into the solidified medium, the discs so produced being removed and discarded. With a standard dropper or Pasteur pipette, the known and unknown nisin solutions are delivered, randomised, in unifonn quantities into the holes, which are almost filled. The plates are then covered and carefully transferred, without displacing the liquid in the holes, to a 30° incubator and left overnight. The next day the diameters of the zones of inhibition are measured to the nearest 0.1 mm by means of callipers or suitable projection devices. A straight line response should be produced when the log nisin concentrations of a critical range are plotted against linear zone diameters. It is advisable to work with four points in quadruplicate and to

57 •

•

calculate the concentration of the unknown solutions from points falling on the straight line. Between 0.5 and 10 units of nisin per ml are nonnally expected to give a straight-line response.

3.1.6 Assay" Procedure

When estimating residual nisin in foodstuffs one has to consider the absorption of nisin on proteins, the heat stability of the antibiotic at a given pH value and various substances present in the_ food which may interfere with the test. lt has been found that nisin can best be freed from proteins as follows: A 20 percent W/v suspension of nisin containing macerated food is prepared in 0.02 N HC l and the pH value lowered to 2.0 with strong HC l, followed by boiling for S min. and centrifuging. The supernatant is divided into two fractions, Fraction l and ll. To account for the interfering substances in the food extract, the nisin present in a portion of the food extract (Fraction l) is inactivated by adjusting the pH value to at least 11.0 by addition of 5 N NaOH keeping the solution at 65° for 30 min. or 44° for 2 hrs., cooled and then re-acidifying to pH 2.0 with S N HC 1. While under these ~onditions nisin is, for all practical purposes, inactivated, the interfering substances remain unimpaired. This procedure makes it possible to use food extracts thus freed from nisin as controls, by simply incorporating into them certain known concentrations of the antibiotic. Nisin, · from a stock suspension of 1 OOO units/ ml in 0.02N-hydrochloric acid, is diluted with Fraction I, which has received alkali treatment, to give 10, 5, 2 and 1 units/ml, these being the reference concentrations. Fraction II is diluted 1: 1 and 1: 3 with Fraction I, which has received the alkali treatment, these being the unknowns. The reference concentrations and unknowns are delivered, randomised, in quadruplicate into the holes on an assay plate. After overnight incubation at 30° measure the zone diameters and determine the average zone diameter for each of the nisin concentrations. The average zone diameters for the reference concentrations are plotted against the log nisin concentration, and the nisin content of the unknows read off the straight line so produced.

Sensitivity of assay - reported to be 0.4 unit/ g.

3.2 Assay by the reverse-phase disc technique

58

3.2.1 Agar - 1,5 percent Ionager No. 2, containing 0.9 percent sodium chloride.

3.2.2 Nutrient disc

13 mm. Whatman AA discs, dipped for 5 sec. into sterile 20 percent Evans peptone solution and dried overnight over phosphorus pentoxide under vacuum.

3.2.3 Spores of B. stearothennophilus (NC[B 82241) prepared by

inoculating an active culture into glucose nutrient broth and incubating for 16 hrs. at 55°; then harvesting the cells by centrifugation; washing twice with sterile distilled water to remove any nutrients and resuspending the cells in a further volume of distilled water. The suspension is spread on the surface of a series of agar plates containing no fermentable carbohydrate and incubated for 1-2 days at 55° or until the production of large numbers of spores occurs. The spores are removed from the agar surface by washing with sterile distilled water, the washings being transferred to a sterile bottle and stored at 4° until required for use. The spores are heat-shocked by boiling for 10 min. and incorporated into the saline non-nutrient agar to give a concentration of approximately 4 X 106 per plate. Ten ml quantities are poured into levelled, sterile, flat-bottomed Petri dishes and allowed to solidify. Suitably packed, the plates can be kept satisfactorily for some time at 4°. The plates should not be stored at room temperature since a rapid drop in the nuinber of viable spores may occur.

3.2.4 Food extract - A 20 percent w/v or other suitable strength acid extract is prepared as described under 3.1.6 and pH adjusted to 1.5 before testing.

3.2.S Procedure

The nutrient disc is immersed in the pH-adjusted test solution, touched against a filter paper to remove excess solution and placed on the agar plate. The plates are incubated for 18-24 hrs. at 55° and the zones of inhibition produced are measured

1 NCIB: National Collection of lndustrila.l. Bacteria, Torry Research Station, Aberdeen, Scotland, U.K.

59

by some suitable means, bearing in mind that this method is only· semi-quantitative. Appropriate amounts of nisin are incorporated into the nisin-free extracts, or into alkali-treated extracts prepared as described under 3.1.6 (Fractions I and II) for use as controls. This method of preparation of controls has been found necessary because interfering substances present in the food also affect this type of assay. The reverse-phase method could be very useful in the assessment of residual nisin concentrations in foods, particularly where only limited laboratory facilities are· available. By testing a range of dilutions of the neutralised acid extract, a fair estimate of the concentration of nisin present can be obtained if one accepts that between 0.5 and 1 units is required to produce a definite zone of inhibition. The test may also be valuable as a sorting test, prior to quantitadve assay by the plate diffusion technique.

Sensitivity of as.say - r~ported to be 1 unit/ g.

3.3 · Assay by using heat-damaged spores

3.3.1 ~tedium for spore production.

Basamin-Busch (Obtainable from Anheuser-Busch, Inc., St. Louis, 18 Missouri) . . . . . . . . . . . . . . . . . . 3.0 g

Trypticase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g K2 HP04 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 g KH 2 P04 • • • • • • • • • • • • • • . . . • • . • • • • • • • • • • • • • • 1.0 g \tnS04 • • • • • • • • • • • • • . • • . • • • • • • . . . • • • • • • • • • • 10.0 n1g Bacto agar .................. : . . . . . . . . . . . . . . 20.0 g Distilled H'2 •••••••••••••••••••••••••••••••• l OOO ml

The medium is stirred until the soluble ingredients are in solution, autoclav.ed 15 min. at 5 lb. and distributed in appropriate containers for sterilization. (Prior to distribution, the pH is adjusted, if necessary, to 6.8-7 .0). The medium is sterilized by autoclaving 20 min. at 15 lb.

60

3.3.2 Test organism

Highly heat-resistant strain of Bacillus stearothermophilus (A TCC 7935).

3.3.3 Production of spores

Slants of Basamin agar are inoculated with the B. stearothermophilus and incubated 48 hrs. at 54°. Three ml of sterile distilled water are added to each 48 hrs. slant culture. With an inoculating loop, the growth is acraped from the slant and suspended in the water. Suspensions from the slant tubes are collected in a sterile screw-cap dilution bottle containing a few glass beads. The bottle of suspension is then autoclayed 5 min. at 10 lb pressure. This heated suspension is used to inoculate bottle slants of Basamin agar for mass spore production. Screw-cap dilution bottles are convenient for making bottle slants. Fifty ml of Basamin agar are placed in each ISO-ml dilution bottle and sterilized by autoclaving 20 min. at 15 lb. After sterilization, the bottles are slanted so as to obtain maximum agar surf ace. Each bottle slant is inoculated with 2 ml of the heated spore suspension, prepared as described above. Each bottle is then tilted back and forth until the inoculum covers the entire surface. The inoculated bottles are incubated 3 days at 54°. (During incubation, the bottles are slanted at about a 45° angle so that the excess inoculum and condensation collects toward the bottom of the bottles). After incubation, 2 ml of sterile distilled water are added to each bottle, aJ!d the growth is scraped from the agar surface with an inoculating loop. Suspension from all bottles are then combined in an appropriate sterile container. Two ml of sterile distilled water are again added to each bottle, and the remaining growth is washed from the surfaces and combined with the first washings. The suspension thus prepared is well mixed and distributed in sterile centrifuge tubes. After 5 min. of centrifugation at 2500 rpm, the supernatant is discarded and the spores are resuspended in sterile distilled water and again centrifuged. This washing procedure is repeated twice more (3 washings in all). After the fmal washing, th~ spores are resuspended in sterile distilled water and all suspensions are combined and mixed well. This suspension is then filtered through sterile glass wool

61

into a convenient container for storage. The suspension thus prepared is stored at 2° -4° for use as needed.

3.3.4 Estimation of spores in suspension

K small portion of the above suspension is heated 5 min. at 100° to destroy the vegetative cells and activate the spores for germination. This heated suspension is then serially diluted so that about one-third of 0.1 ml portions contain spores and about two-thirds contain none. (The correct dilution is arrived

. at by limited sampling of a number of dilutions and estimating spore counts is done as described below). After arriving at the correct dilution, 0.1 ml of the heated suspension (immediately after cooling) is transferred to each of at least 30 tubes of Basamin agar. The inoculum and agar are mixed well and the agar allowed to solidify. All inoculated tubes are then incubated 72 hr. at 54°. After incubation, the colonies may be counted directly or the number of tubes positive for growth may be counted. If counted directly, the average per tube is multiplied by the dilution factor to obtain the spore count. If the number of positive tubes is det~muned, the most probable number of spores per ml may be calculated by the following equation.

x = 2.303 n log-

0.1 q

Where: x = most probable number per ml; n = total number of tubes; q = number of tubes negative for growth.

The x value is then multiplied by the ·appropriate dilution factor to obtain the total count.

3.3.S Preparation of standard curve

3.3.S. l Preparation of heat-damaged spores. Dilute the stock spore suspension, prepared as above, so that each 0.1 ml of diluted suspension contains approximately 100 spores. Twenty ml of the diluted suspension is transferred to a sterile dilution bottle containing a few glass beads. This, in tum, is placed in a preheated autoclave, heated 35 min. at 110°, and cooled immediately. This heated suspension is stored at 2-4° until used which should never be later than 2 h{s._ aftet heating.

62

3.3.S.2 Nisiri solutions for assay. Nisin concentrate is diluted with sterile skim milk so as to give dilutions containing 0, 1, 2, 3 and 4 unit per ml of skim milk. {The skim milk is sterilized. in 100-ml quantities in screw-cap dilution bottles by autoclaving 15 min. at 10 lb pressure).

3.3.S.3 Procedure. To each of at least 12 tubes of sterile melted Basamin agar (exactly 20 ml agar per 22 x 175-mm tube) is aseptically added 0.1 ml of the 0-unit skim-milk-nisin solution. To each of an equal number tubes is added 0.1 ml of the I-unit nisin solution. This procedure is repeated with 2-, 3- and 4-unit nisin solutions. This will give five lots of tubes containing 0.005, 0.01, 0.015 and 0.02 unit of nisin per ml of medium. To each of these 60 or more tubes is now added 0.1 ml of the spore suspension, prepared as described above, containing heat-damaged spores. The contents of each tube are then thoroughly mixed with a vortex mixer. All tubes are incubated 24 hr. at 54° and the bacterial colonies developed in each tube are then counted. The counts are averaged for each nisin level. The logarithm of colony count per 6 or 12 tubes is then plotted against nisin concentration.

3.3.6 Assay procedure

To assay a food of unknown nisin concentratiO!l the food. sample is diluted with sterile distilled water so a.s to obtain at least 2 nisin levels that fall within the range of those on the standard curve, when an appropriate quantity of food sample is added to 20 ml of medium. Using food samples greater than 1 ml should be avoided where possible. Used for foods suspected of containing less than 1.0 unit per ml., are l ml of the diluted food {where possible), 1 ml of a 1 :2 dilution, and of a 1 :4 dilution. For foods suspected of containing higher levels of nisin, dilutions are made to approximate these levels or proportionately less food sample is used. Each diluted food sample to be assayed is distributed in Basamin agar tubes, and heat-damaged spores are added exactly as is done in establishing the standard curve. Colonies developed in 24 hr. at 54° in the presence of each diluted food sample are counted. Always included as a control is one set of tubes with no food sample added. Colonies developed in the presence of each diluted food sample are averaged, and the average count is referred to the standard curve to obtain nisin level in the

63

medium contammg the food sample. This value is then multiplied by the appropriate dilution factor .

. 015 X 5 X 21 = 1.575 unit per gram

Sensitivity of assay - reported to be 0.3 unit/g.

3.4 Nisin in processed cheese - British standard 4020: 1966

Assay organism -Microccocus jlavus NCIB 8166 1

Sensitivity of assay - reported to be 0.4 unit/g.

1National Collection of Industrial Bacteria. Torry Research Station Aberdeen, Scotland

64

APPENDIX

DIFFERENTIATION OF NISIN AND OTHER COMMON ANTIBIOTICS IN FOOD EXTRACTS

The microbiological assay of nisin in foods is not specific and other antibiotics present in food could interfere. [n preparation for nisin assay, samples of food are acidified and boiled in order to bring nisin into the aqueous phase. As a second stage the nisin-containing extracts are made alkaline and heated, causing rapid inactivation of nisin, in order to provide a suitable nisin-free diluent for the nisin standard. The fate of other antibiotics when subjected to heat under acid and alkaline conditions is an important factor when considering the possibility of interference during rJsin assay.

(a) Suspension of processed cheese to which various antibiotics had been added were adjusted to pH 2.0 by the addition of cone. HCl and boiled for 5 minutes. Antibiotic activity in the acidified suspensions was measured by diffusion assay against M. flavus before and after boiling. Table I gives the loss in activity, expressed as a percentage, attributable to boiling at pH 2.0.

Table I

Antibiotic,

Nisin ................................. . Tylosin ................................ . Polymyxin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tetracycline ............................ . Cloxacillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Grarnicidin ............................. . Bacitracin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ampicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenoxyethylpenicillin ..................... . Benzylpenicillin .......................... . Neomycin .............................. . Novebiocin ............................. . Streptomycin ........................... . Erythromycin ........................... . Chlorarnphenicol ......................... . Phenoxybenzylpenicillin .................... . Phenoxymetyl penicillin .................... . Phenoxypropyl penicillin .................... .

65

0 / 0 loss of activity

0 0 0

68 32 61 50 72 94 98 87 93 96 99 99 97 97 74

(b) Cheese suspensions as in (b) were adjusted to pH 11.0 by the addition of SN NaOH and heated at 63° for 30 minutes. The suspensions were cooled and the pH adjusted back to 2.0 with cone. HCl. Antibiotic activity was measured before and after treii.tment. Percentage lo$ses are listed in Table II.

Table II

Antibiotic

Nisin ................................. . Tylosin ................................ . Polymyxin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tetracycline ............................ . Ooxacillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gramicidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacitracin . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . Ampicillin .............................. . Benzylpenicillin .......................... . Neomycin .............................. . Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Streptomycin ........................... . Erythromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenoxybenzylpenicil4n .................... . Phenoxymethylpenicillin .................... . Phenoxyethylpenicillin ..................... . Phenoxypropylpenicillin .................... .

0 / 0 loss of activity

100 100 100 64 58

100 100 87 64 78

100 99

100 99 24 93 66 75

Tylosin, polymyxin, gramicidin, bacitracin, novobiocin and erythromycin behaved in the same way as nisin.

From the results obtained in (a) and {b) above it appears that two antibiotics in particular, namely tylosin and polymyxin B, might be confused with nisin when measuring antibiotic activity in foods.

It is known that the Streptococcus lactis strains which produce nisin will grow in reasonably high concentrations of the antibiotic. The same strains· may, however, be sensitive to a wide range of other antibiotics, thus providing a fairly simple means of distinction.

Sensitivity tests were set up in which sterile litmus milk containing aerial dilution of antibiotics was inoculated with 0.1 percent of an

66

overnight milk culture of Streptococcus lactis NCIB 8586. After 18 hours incubation at 30° the sensitivity of the organism to a particular antibiotic was taken as the lowest concentration in which no growth occurred (MIC}. The results are given in Table Ill.

Table Ill. - Sensitivity of Streptococcus lactis to antibiotics

Antibiotic

Nisin ........................... . Polymyxin B . . . . . . . . . . . . . . . . . . . . . . . Gramicidin . . . . . . . . . . . . . . . . . . . . . . . ·. Benzylpenicillin . . . . . . . . . . . . . . . . . . . . . Bacitracin . . . . . . . . . . . . . . . . . . . . . . . . . Tylosin .......................... . Chloramphenicol . . . . . . . . . . . . . . . . . .. . Tetracycline ...................... . Streptomycin ..................... . Neomycin ..............•.......... Novobiocin . . . . . . . . . . . . . . . . . . . . . ... Erythromycin . . . . . . . . . . . . . . . . . . . . . .

67

MIC

· >5 OOO > 100 > 100 L

1.5 12

1.0 3 1.5 0.75 2.5 1.5 0.3

units/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml

POLYMYXJNB

SpecificatioRs

Polymyxin B. Sulph:ite. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia WHO, Geneva; 2nd Edit., P:ige 464

Assays

1. In animal feed. Not so used

2. In tissue, milk and eggs. As given in the Annex.



In animal tissue

In milk

In eggs (whole)

SIU/gm

2 IU/ml

5 IU/gm

68

TETRACYCLINES

TETRACYCLINE

Specifications

Tetracycline Hydrochloride. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia, WHO, Geneva, 2nd Edit., Page 572

Amys

1. Amy in animal feed

1.1 Culture medium

Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 g Tryptic peptone . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g Meat extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 g Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 ml Phosphate buffer pH 5.5(1.2) . . . . . . . . . . . . . . . 10.0 ml 5 percent boric acid solution . . . . . . . . . . . . . . . . 15.0 ml Solution of 0.5 percent methylene blue in ethanol . 4.0 ml Agar according to quantity . . . . . . . . . . . . . . 10.0-20.0 g Distilled water, q.s. . ..................... 1 OOO ml After sterilisation, the pH should be 5.8

1.2 Reagents

1.2.l Phosphate buffer pH 5.5

KH2P04 . . . . . . . . . . . . . . . . . . . . . . . . . . . 130.86 g Na2HP042H20....................... 6 947 g Distilled water, q.s. . . . . . . . . . . . . . . . . . . . . 1 OOO ml

l.2.2. Buffer pH S.5 diluted to 1 : 10 ·

69

1.2.3 Phosphate buff er pH 8

Na2 1iP042H2 0 ...................... . K.H 2P04 •••••••••••••••••••••••••• • •

Distilled water, q.s .•....................

1.3 Stock culture of test organism

57 539 g t 407 g 1 OOO ml

Inoculate the test organism B. cereus (ATCC 11778) on agar slants consisting of the culture medium ( l. l) but without qi.ethylene blue and boric acid. Incubate overnight at 35° Preserve in refrigerator at 4° to 10°. Maintain this culture on agar slants by transfers every fourteen days.

1.4 Preparation of inoculum

Wash the bacterial growth from agar slant ( 1. 3) with 2 to 3 ml sterile distilled water and transfer liquid to surface of 300 ml of culture medium ( l.1) in a Roux bottle; this medium must be free of methylene blue and boric acid, and contain 3 to 4 percent agar. Spread suspension evenly over entire surface. After incubation for 3 to S days at 28~30° wash the growth with 1 S ml of 20 percent ethanol. Homogenise. The suspension of spores keeps under refrigeration ~or at least S months. It is used to inoculate the culture medium ( 1.1)

l.S Standard solution

Prepare ·a stock solution containing SOO Jtg tetracycline-HCl/ml in OJN HCl. This solution will keep for one week under refrigeration. From this stock solution prepare a working standard solution S 8 of 1.0 µg tetracycline-HCl/ml, and therefrom make the following dilutions:

S 4 ........................•....... 0.5 µ.g/ml S 2 .. , •.• , , , .. , .•..... , . : .•.•.•..•. Q.25 /Lg/ml Sl ................................ 0.12Sµg/rnl

1.6 Extraction

In case s,f feed samples having an assay under SO ppm, the extraction is done with freshly prepared dilute formamide (70 percent v/v) adjusted to pH 4.5 with the aid of H2 S04 • For samples with assay above 50 ppm use methanol-HC 1 mixture (98 parts of methanol and two parts of hydrochloric acid, sp. gr. 1.19)

70

.Shake for 30 minutes, extract and proceed inmediateJy on the basis of its presumed antibiotic content to make the dilutions U8 (1.0 Jtg/ml) U4 (O.S 11.g/ml) U2 (0.25 /Lg/ml) and Ul (0.125 g/ml) in the following manner:

Extracts obtained with the aid of formamide

Make all dilutions directly with the aid of phosphate buffer pH 5 .S diluted to 1: 10 ( 1.1.2). The fonnamide concentration in dilution U8 must not exceed 40 percent.

Extracts obtained with the aid of methanol-BC 1

Adjust the pH of an aliquot of the extract to S .S using phosphate buffer of pH 8.0 ( 1.2.3) in the presence of bromocresol green (turning from yellow to blue). Bring to a known volume with phosphate buffer pH S.S diluted to 1 : 10 {1.1.2) and make necessary dilutions with this buffer.

1.7 [)ifjusion test

1.7.1 Inoculation of the culture medium

Inoculate the culture medium (I.I) with the inoculum (1.4). Preliminary tests are made to determine the quantity of inoculum required to obtain sharp zones of inhibition for the different concentratiops in the standard range. Generally, a volume of 0.2 to 0.3 ml/I OOO ml of culture medium is used. Inoculation temperature must not exceed 60°


Preferably use flat glass plates supporting a perfectly flat aluminum or plexiglas ririg of 200 mm diameter, 20 mm deep. Into the vessel thus formed pour about SO ml of inoculated culture medium to form a layer about 2 mm deep. Cool. Make 16 holes of 10 to 13 nun diameter in the agar. Into each hole pipet an accurately measured quantity of antibiotic solution 0.10 to 0.15 ml depending on the diameter of the hole. It is essential that each plate be provided with all 4 standard solutions (S 8, S 4, S 2, and S 1) and the 4 solutions of the extract to be tested (U 8, U 4, U 2 and U 1). Prepare at least two plates per sample.

1.7.3 Incubation

Incubate the plates for 18 hours at about 28-30°

71

1.8 £i,a/uation of results

Read the diameters of the zones of inhibition by projection. Plot the readings on semilong graph paper using the logarithmic scale .for the concentrations and the arithmetic scale for the diameters of the zones of inhibition. Draw the standard and extract curves; check the two straight lines for parallelism. The logarithm of relative activity is given by the ratio

The margin of error is ± 20 percent.

2. Assay in tissue, milk and eggs - As given in the Annex



In animal tissues In milk In eggs (whole)

0.5 ppm 0.1 ppm 0.3 ppm

72

CHLORTETRACYCLINE

Specifications

Chlortetracycline Hydrochloride. Specifications for the Quality Control of Phannaceurical Preparations. International Pharmacopoeia, WHO, Geneva, 2nd Edit., page 123.

Assays

I. In animal feed. Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) 1965, 1 Oth Edit., Page 656, 33.131 • 33.134 amended as under.

33.131 Reagents

(a) Stock soln. - as in lOth ed.

(b) Standard solns. and curve for samples containing > 50 ppm chlortetracycline. HC 1 - As in 33.131 (b).

(c) Standard Solns and curve for samples containing not > 50 ppm chlortetracycline. HC l - Prep. as in (b) but dil. with inactivated diluent, (d) instead of buffer soln. and include concns. of 0. 0.005 and 0.32 ,,g CTC. HCl/ml. Draw best line of fit by inspection.

(d) Inactivated diluent. To 10 ml acid-acetone feed ext. (Prepd. from feed under test as in Preparation of Assay Solution, 33.133 (b) in 600 ml beaker, add 90 ml pH4.5 phosphate buff er, 33.133 (g) and adjust to pH 4.5-4.7 with IN NaOH. Add 1.0 ml fresh 5.25 percent naOC 1 soln. (corn.household bleach is satisfactory) and stir .1 • 2 min. rinsing sides of beaker. Heat, stirring thoroughly at 10 min.intervals. in uncovered beaker in boiling H2 0 bath 30 min. Cool to room temp. under tap H~O stream and quant. transfer to 100 ml vol. flask. Rinse beaker with acerone, adds rinsings to vol. flask and dil. to vol. with acetone. Quant. transfer to another flask and dil. with enough pH 4.5 buffer so that final concn. of feed ext. is same as that in assay sol.

73

33.132 Plates

(a) Base layer - as in lOth ed.

(b) Seed layer - as in lOth ed. but in line 15 after "HCL/ml" insert "for assaying samples contg > SO ppm and 0.005 µ /ml for samples contg. not > SO ppm ~TC. HCl."

33.133 Preparation of Assay Solution

(a) Samples containing > SO ppm chlortetracycline. HCl - Place, 2, 10 or 20 g sample, resp., contg CTC.HCl not < 10 g/lb (> 2 percent) > 400 ppm to 2 percent, or 50-400 ppm in 1 SO ml beaker and pipet in 40 ml acid-acetone soln. 33.113 G). Stir ea 2 min. with glass rod, let stand 2 min. and stir. Adjust pH to 1.0 - 1.2 with HC 1, if necessary and note vol. HC 1 added. Transfer to 1 qt. high speed blender jar using addnl 20 ml (minus vol. equiv. to HCl added in adjusting pH) acid-acetone· to rinse beaker and pH meter electrodes.· Cover jar and blend 3 min. at high speed. Transfer mixt. to 100 ml centrifuge tubes. Wash blender jar with 40 ml acid-acetone and combine washings with ext. in centrifuge tubes. Shake well 5 min. Centrifuge ea 15 min. at 2000 rpm. Combine and mix clarified exts. Adjust 10 ml aliquot to pH 4.5 with IN NaOH. Oil. adjusted soln. with enough pH 4.5 buffer, 33.113 (g), to obtain estimated concn. of 0.04 1-'g/ml. Designate soln as assay solo.

(b) Samples containing not > SO ppm chlortetracycline. HC 1 - Place SO g sample in 250 ml beaker and pipet in 100 ml acid-acetone soln. 33.133 G). Stir, adjust pH, and blend as in (a), using SO ml (less vol. equiv.to HC 1 added in adjusting pH) acid-acetone to transfer to blender jar. After blending, transfer quant. to 250 ml. centrifuge bottle, rinsing jar with SO ml acid acetone solo. Shake bottle thoroughly and centrifuge ea 15 min at 2000 rpm. Pi pet s. ml clear supernatant into SO ml beaker, add ea 40 ml pH 4.5 buffer, 33.133 (g), mix, and adjust

74

4.5-4.7 with IN NaOH. Quant. transfer to flask, rinse beaker and pH meter electrodes with pH 4.5 buff er and add rinsings to flask. Add enough pH 4.5 buffer to obtain estd concn. of 0:04 µg/ml. Designate as assay soln.

33.134 Assay

As in lOth ed.

2. In tissue, milk and eggs - As given in the AMex


Sensitivity of assay reported to be as under

In animal tissues 0.05 ppm 1

In milk 0.02 ppm 1

In eggs (whole) 0.05 ppm 1

Use in foods

Limited application has been made of chlortetracycline hydrochloride as an aid in the preservation of certain foods.

1 Basis I I.U. = 1 microgram of chlortetracycline hydrocloride.

75

OXYTETRACYCUNE

Specifications

Oxytetracyclin~ Hydrocloride. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmapocopoeia, WHO, Geneva, 2nd Edit., Page 419.

Asmys

1. Assay in animal feed. Official Methods of Analysis of the Association of Official Agricultural Chemists (AOAC) 1965, IOth Edit., Page 660, 33.152 - 33.154 amended 1 as under for low levels (not more than 220 ppm) of oxytetracycline in final feeds

33.168 Standard solutions

(a) Stock solution: Accurately weigh suitable quantity exytetracycline NF Reference Standard and dissolve in enough O.lN HCL to give concentration of exactly 250 µ. g oxytetracycline HC 1/ml {l µ.g oxytetracycline base is equivalent to 1.08 µ g oxytetracycline HC 1 ). Store in dark at ea 1 o:. not > 5 days.

(b) Standard curve: Dilute appropriate aliquots of stock solution, (a), with enough pH 4.5 buffer, 33.113 (g), to obtain concentrations of 0.05, 0.10, 0.20, 0.40, and 0.80 µ g oxytetracycline HC 1/ml. Reference concentration is 0.20 µ. g/ml.

33169 Plates

Use single inoculated agar layer prepared as follows: Transfer growth from stock culture of B. cereus var. mycoides, 33.115 ( d) to 100 ml broth medium A, 33.112 (j). Incubate overnight at 30° with continuous mechanical agitation. Prepare slant culture on agar metlium A, 33.112 (a), and incubate overnight at 30°. Wash growth from slant culture with ea 3 ml sterile

1 J. Assoc Official Agricultural Chemists 51, 500 ·(1968)

76

H10 and transfer to surface of 250 ml agar medium A in Roux bottle. Spread suspension evenly over entire surf ace and incubate 6 days at 30°. Wash growth from agar surface with ea 20 ml sterile H1 0, dilute to 50 ml. and heat suspension 30 min. at 65°. Centrifuge and decant. Wash residual spores 3 times with sterile H 2 0, centrifuging and decanting each time. Discard H2 0 washings. Transfer washed spores to sterile high speed blender with ea 50 ml sterile H20 and blend throughly. Heat suspended spores 30 min. at 65° and store at ea 10°. Before actual assay, determine by preparation of trial plates optimum concentration (usually 0.03-0.10 percent) of inoculum to be added to agar medium D, 33.112 ( d). to obtain zones of inhibition with as little as 0.05 µ g oxytetracycline HC 1/ml. For actual assay add appropriate amount of inoculum to agar medium D previously melted and cooled to 48°. Mix thoroughly, and add 9.0 ml to each plate. Distribute agar evenly, and let harden on perfectly level surface. Refrigerate plates not < 1 hr. before use, and use same day prepared.

33.170 Assay Solution

Weigh accurately 20 g ground finished feed into 250 ml extn. flask, add 100 ml acid-MeOH. 33.113 (k), stopper and shake mechanically S min. Centrifuge ea 5 min at 2000 rpm. Remove 20 ml supernatant and adjust to pH 4.S with IN NaOH. Dilute adjusted solution with enough pH 4.S buffer, 33.113 (g), to obtain estimated concentration of 0.20 g/ml and filter though Whatman No 2V paper, or equivalent. Designate as assay solution.

33 .171 Assay

Using oxytetracycline HC 1 standard curve, assay solution and plates, proceed as in 33.116-33.117, incubating at 28-30°.

2. Assay in tissue, milk and eggs - As given in, the Annex.

77

Sensitivity of a~y

Sensitivity of assay reported to be as under

In animal tissue In milk In eggs (whole)

0.25 ppm (as oxytetracycline base)1

0 .1 ppm ( as oxytetracycline base) 1

0.3 ppm (as oxytetracycline base) 1

Use in foods

Limited application has been made of oxytetracycline as an aid in the preservation of certain foods.

1 Basis 1 IU = 1 mcg. of oxytetracycline base

78

OTHER

NOVOBIOCIN

Specifications

Novobiocin Calcium. Specifications for the Quality Control of Phannaceutical Preparations. International Pharmacopoeia WHO, Geneva, 2nd Edit., Page 392

Novobiocin Sodium. Specifications for the Quality Control of Pharmaceutical Preparations. International Pharmacopoeia, WHO, Geneva, 2nd Edit., Page 393

Nov~biocin acid is used in feeds

Assays

l. In animal feed. Official Methods of Analysis of the Association of Official Agricultural Chemists {AOAC) 1965, lOth Edit., p.657, 33.140-33.143

2. In tissue, milk and eggs. As given in the Annex.



In animal tissue 0.5 ppm (as novobiocin acid)1

In milk 0.15 ppm (as novobiocin acid) 1

In eggs (whole) 0.1 ppm (as novobiocin acid) 1

1 Basis l 1.U. = 1 microgram of novobiocin acid.

79

ANNEX

~y and Reporting of Data Pertaining to Antibiotic Residues in Milk, Dairy Products and Animal Ti§ues

Section I - MILK

l. l. l General instructions

All glassware and equipment must be chemically clean. Control milk used to prepare the standard curve or dilute the unknowns, should be first tested to ensure that it exhibits no activity on the appropriate assay plates. The control milk should be the same type of milk as the milk being tested. In all cases, the milk should be fresh. For standardization, use an instrument such as a Bausch and Lomb Spectronk 20 with a round cell 1.2 cm i.d. or a Beckman Model B spectrophotometer with 1.0 cm square cell, both set at a wavelength of 580 nm. The light transmission reading should be used as a guide only and not considered absolute. The amount pf inoculum recommended should also be considered as a guide rather than as absolute. For each assay, prepare test plates with varying concentrations of the standardized suspension to determine the amount of inoculum that allows the most desirable zones of inhibition.

l.1.2 Media

Medium A

Peptic Digest of Meat . . . . . . . . . . . . . . . . . .. . . . 6.0 gm Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . 4.0 gm Yeast Extntct ........... ·. . . . . . . . . . . . . . . 3.0 gm Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Dextrose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l.O gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gm Distilled water q.s ........................ l OOO ml

(pH of 6.55 ± 0.05 after sterilization)

80

Medium B

Same as Medium A, except that it also contains 300 mg of Mn S04 H20 per litre.

Medium C

Peptic Digest of Meat .................... , 6.0 grn Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 gm Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gm Distilled water q.s .......................• l OOO ml

(pH 6.55 ± 0.05 after sterilization)

Medium D

Peptic Digest of Meat . . . . . . . . . . . . . . . . . . . . . 6.0 gm Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 gm Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 gm Agar ............................ - . . . . 15.0 gm Distilled water, q.s. . ..................... l OOO ml


Medium E

Same as Medium D, except pH is adjusted so that it is 5.8 ± 0.1 after sterilization.

Medium F

Same as Medium A, except pH is adjusted so that it is 8.0 ± 0.1 after sterilization

Medium G

Pancreatic digest of casein ................. . Papaic digest of soybean meal .............. . Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . •

17.0 gm 3.0 $ffi 5.0 gm

Dextrose . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 gm Dipotassium phosphate . . . . . . . . . . . . . . . . . . . . 2.5 gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 gm Distilled water, q.s. . ..................... l OOO ml

(pH 7 .3 ± 0.05 after sterilization)

81

~tedium H

Pancreatic digest of casein . . . . . . . . . . . . . . . . . . 17 .0 gm Papaic digest of soybean meal . . . . . . . . . . . . . . . 3.0 gm Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 gm Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . 5 .0 gm Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . 2.5 gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.0 gm Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 gm Distilled water, q.s. . ..................... l OOO ml

(pH 7 .3 ± 0.05 after sterilization)

Medium J

Same as Medium D, except in lieu of Agar use Special Agar (Noble)1 and adjust pH of the medium so that it is 8.0 ± 0.1 after sterilization.

Medium K

Same as Medium A, except in lieu of Agar use Special Agar (Noble) and adjust pH of the medium so that it is 8.0 ± 0.1 after sterilization.

Medium L

Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gm Distilled water, q.s. . ..................... l OOO ml

MediumM2

Peptic digest of meat . . . . . . . . . . . . . . . . . . . . . . 5 .0 gm Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 gm Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 gm· Distilled water, q.s. . ..................... 1 OOO ml


1.1.3 Buffers

(a) Percent phosphate buffer, pH 6.0 ± 0.05. Dissolve 8.0 gm monobasic potassium phosphate and 2.0 gm dibasic potassium phosphate in distilled water and dilute to l litre with distilled water.

1Noble and Tonney. J. Amer. Water Works Assoc., 27: 108-120 (1935) 2 All o,. the above media may be obtained from commercial sources in

dehydrated form.

82

(b) 0.lM phosphate buffer, pH 4.5 ± 0.05. Dissolve 13.6 gm monobasic potassium phosphate in distilled water a·nd dilute to l litre with distilled water.

(c) O.lM phosphate buffer, pH 8.0 ± 0.1. Dissolve 16.73 gm dibasic potassium phosphate with 0.523 gm of monobasic potassium phosphate in distilled water and dilute to l litre with distilled water.

( d) 0.2M phosphate buffer, pH 8.0 ± 0.1. Dissolve 33.46 gm dibasic potassium phosphate and 1 046 gm of monobasic potassium phosphate in distilled water and dilute to 1 litre with distilled water.

(e) 20 percent phosphate buffer, pH 6.0 ± 0.05. Dissolve 160 gm monobasic l?otassium phosphate and 20 gm dibasic potassium phosphate in distilled water and dilute to l litre with distilled water.

(f) 10 Percent phosphate buffer, pH 6.0 ± 0.05. Dissolve 80 gm monobasic potassium phosphate and 20 gm dibasic potassium phosphate in distilled water and dilute to l litre with distilled water.

(g) Phosphate-sulphate buffer, pH 1.5. Adjust buffer (c) to pH 1.5 with concentrated surphuric acid.

1.1.4 Cylinders

Use stainless steel cylinders with an outside diameter of 8 mm ( ± 0.1 mm). and an inside diameter of 6 mm ( ± 0.1 mm) with a length of I O mm ( ± 0.1 mm).

1.1.S Petri Dishes

Use P~tri dishes that are 20 x 100 mm in size, equipped with porcelain covers, glazed on the outside, or cover lids with filter pads capable of absorbing the water of syneresis. Comparable plastic Petri dishes may be used if desired.

1.1.6 Working Standards

Authentic antibiotic reference standards may be obtained from U. S. P. Reference Standards, 46 Park Ave., New York, N. Y., 10016, and the Committee on National Formulary, American Pharmaceutical Association,, 2215 Constitution Ave., N.W., Washington, D.C. 20037. Follow the· label directions for

83

preparJtion and storage of the standards. Prepare stock solutions by weighing carefully. in an atmosphere of 50 percent humidity or less, a small amount of the standard and diluting the weighed powder in the appropriate diluent to obtain a solution of a convenient concentration.

1.1. 7 Orga,,11 isms

Cultures of the test organisms may be obtai~ed from American Type Culture Collection, 12301 Parklawn Drive, Rockville, l\faryland 20852. Preparation of suspension is described under specific antibiotic assays.

1.1.8 Standard Curve

A standard curve should be prepared as described in the following example:

Prepare a stock solution of penicillin by dissolving an accurately weighed portion of the penicillin standard in sufficient l percent phosphate buffer. pH 6 to obtain a solution containing 1000 units/ml. The stock solution may be used for two days. Further dilute this stock solution in antibiotic-free milk to obtain concentrations of 0.0063, 0.0125, 0.025, 0.05, 0.1 and 0.2 unit/ml. lt will be noted that the concentrations are related geometrically by a factor of 2.

In Petri dishes previously prepared, place six cylinders equally spaced on the inoculated agar surface and fill three alternate cyli:lders with the 0.05 unit/ml concentration and the other three cylinders with one of the other concentrations of the standard. Use three plates for each point in the curve for a total of 15 plates. The three plates containing the lowest concentration of standard are for determining the final sensitivity of the method. The other 12 plates are used for constructing the standard response line. This, then, will give (36) 0.05 unit/ml determination and nine determinations for each of the other points on the line. After the plates have been incubated, read the diameters of the circles of inhibition as accurately as possible, at least to the nearest 0.5 mm, by means of a millimeter ruler, vernier calipers, or by projecting the inhibition zones on a calibrated screen. Other measuring devices of equal accuracy may be used. Average the readings of the 0.05 unit/ml concentration and the

84

readings of the point tested for each set of three plates and average also all 36 readings of the 0.05 unit/ml concentration. The average of the 36 readings of the 0.05 unit/ml concentration is the correction point for the curve. Correct the average value obtained for each point to the figure it would be if the 0.05 unit/ml reading for that set of three plates were the same as the correction point. Thus, if in correcting the 0.025 unit concentration, the average of the 36 readings of the 0.05 unit concentration is 20 mm, and the average of the 0.05 unit concentration of this set of three plates is 19 .8 mm, the correction = 0.2 mm. If the average reading of the 0.025 unit concentration of these same three plates is 17.0 mm, the corrected value is then 17.2 mm. Plot these corrected values inclduing the average of the 0.05 unit/ml concentration on two cycle, semi-log paper, placing the concentration in units/ml on the logarithmic scale and the diameter of the zone of inhibition •on the arithemtic scale. Construct the best straight line through these points, either by inspection or by means of the follozing equations:

L = 3a + 2b + c - e

5 H= 3e + 2d + c - a

5

where L · and H = calculated zone diameters for the lowest and highest (0.0125 and 0.2 u/ml) concentrations of the standard response line. a, b, c, d, e = average zone diameters for each of the solutions used for the standard response line.

1.1.9 Calculation of potency

To calculate the antibiotic content of a sample, average the zone readings of . the standard and the zone readings of the sample on the three plates. If the sample gives a larger average zone size than the average of the standard,' add the difference between them to the zone size of the reference standard on the curve. If the average sample value is lower than the standard value, subtract the difference between them from the zone size of the reference standard on the curve. From the curve, read the concentration corresponding to this adjusted sample zone size. If the sample was diluted, multiply by the dilution fact<>r to obtain its final concentration.' Some antibiotics, such as

85

chlorietracycline are finally diluted 1 : 3. These final dilutions must be taken into consideration in calculating the final potency of the sample.

1.2 Details for assay of specific antibiotics in milk

l.2.1 Penicillin

(i) Preparation of bacterial suspension

The test organism is Sarcina lutea (ATCC 9341) which is maintained as a stock culture on agar slants of Medium A and transferred to a fresh slant approximately once every two weeks. This culture is incubated overnight at 30°. Prepare an inoculum for the plates as follows:

Streak an agar slant heavily with the test· organism and incubate for 18-24 hours at 32-35°. Wash the growth from the slant with 1-2 ml of sterile' physiological saline and transfer to the dry surface of a Roux bottle containing 300 ml of Medium A. Spread the suspension evenly over the entire surf ace with the aid of sterile glass beads. Incubate for 18-24 hours at 32-35°. Wash the growth from the agar surface with 50 ml of saline. If an aliquot of this bulk suspension when diluted 1 : 35 gives a 25 percent light transmission, the bulk suspension is satisfactory for use. It may be necessary to adjust the bulk · suspension by dilution so that an aliquot of the adjusted suspension when diluted 1 : 35 will give the desired 25 percent light transmission. (Note: The adjusted bulk suspension only and not the 1 : 35 dilution of it is used in preparing the seed layer). Before the actual assay, determine by trial plates the optimum amount of the bulk suspension prepared i.r). this manner to be added to the seed agar to obtain the best zones of inhibition. Generally, this will be obtained within the range of only 0.1·0.5. ml of inoculum per 100 ml of inoculum per 100 ml of Medium C. Store this stock suspension in the refrigerator no longer than two weeks.

(ii) Preparation of plates

Add 10 ml of Medium A to the Petri dishes. Distribute the medium evenly and allow to harden on a flat, level surface. Add the appropriate amount of the culture to

86

each 100 ml of Medium C which has been melted and cooled to 48°. Mix thoroughly and add 4.0 ml of this inoculated agar to each of the plates which have previously received 10 ml of the base layer. Distribute the agar evenly by tilting the plates from side to side in a circular motion. Allow to harden and use the plates the same day they are prepared. Place six cylinders, evenly spaced, on the inoculated plates, using three plates for each sample to be tested. Fill three alternate cylinders on each plate with an 0.05 u/ml standard of penicillin, and the remaining three cylinders with the sample under test. Place covers back on the plates and incubate at 30° for 16-18 hours. After incubation, invert the plates to remove the cylinders and measure the diameter of each circle of inhibition.

(iii) Preparation of Sampl«= 1

Test the sample undiluted. If a concentration is expected or found to be greater than 0.2 u/ml, dilute the sample to an estimated 0.05 u/ml with antibiotic·free milk.

(iv) Standard Curve and Calculation of Potency

Proceed as described in paras 1.1.8 and 1.1.9.

1.2.2 Bacitracin


The test organism is Sarcina subflava (ATCC 7468) or Micrococcus flavus (ATCC 10240) either of which is maintained at refrigerator temperature on slants of Medium A. Inoculate a Roux bottle containing 300 ml of Medium A with a fresh culture slant of the test organism. Incubate at .32-35° for 18-24 hours. Harvest the growth from the surface of the agar with 50 ml of sterile physiological saline. Adjust the bulk suspension so that a 1 : 25 dilution gives a 25 percent light transmission.

~ To provide for identification of any activity as due to penicillin take a port10n of the samp!e, add penic~ase concent,rate at the rate of 0.5 mi per 10

. ml. of sam~le, and mcubate 30 minutes at 3 7 . On an additional plate, fill two cylinders with the 0.05 u/ml standard, two cylinders with the 0.05 u/ml standard two cylinders with ~h.e pe_n~cillin~se-treated sample. Incubate the plate overnight at ~~ ~· A zone ot inh1b1t1on with the untreated sample j111d no zone with the pemciltinase-treated sample is a positive tes~ for penicillin.

87

Bdore the actual assay, determine by trial plates the optimum amount ( usually 0.1-0.5 ml) of the adjusted bulk suspension to be added to 100 ml. of Medium A, which has been melted and cooled to 48°. Keep this stock suspension in the refrigerator no longer than two weeks.

I ii) Preparation of plates

Add 10 ml of Medium D to each Petri dish, distribute evenly and allow to harden. Then, add 4.0 ml of Medium A, which has been seeded with a suspension of iH. flavus or S. subflava. Proceed as described for preparation of plates under penicillin, except use the bacitracin standard. Incubate at 30° for 16-18 hours.

(iii) Preparation of sample

Test the sample undiluted. If a concentration is expected or found to be greater than 0.8 u/ml, dilute the sample to an estimated 0.2 u/ml with antibiotic-free milk.

(iv) Standard curve

Dissolve an accurately weighed sample of 30-50 mg of plain bacitracin in sufficient 1 percent phosphate buffer, pH 6, to give a stock solution of a convenient concentration (for example, 100 u/ml). If zinc bacitracin is used as the working standard, suspend an accurately weighed sample equivalent to about 10 OOO units in 5 ml of distilled water. Add 0.5 ml of 3M HCl, stir until the sample is dissolved, transfer to a 100 ml volumetric flask and make to volume with distilled water. The stock solution may be used for seven days. Dilute the stock solution in antibiotic-free milk to give concentrations of 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 u/ml. The reference concentration is 0.2 u/ml. Proceed as described in para. 1.1.8.

(v) Calculation of potency

Proceed as described in para. l.1.9

88

1.2.3 Chlortetracycline


The test organism is Bacillus cereus var. mycoides (ATCC 11778), which is grown at 30° and maintained at refrigerator temperature on Medium A. Transfer the growth from a fresh slant with 2.0-3.0 ml of sterile distilled water and heat for 30 minutes at 70°. Wash the culture three times with sterile distilled water ( centrifuging and decanting). Heat for 30 minutes at 65° and resuspend in 30 ml of sterile distilled water. Keep this stock in the refrigerator. Determine by trial plates the percent of spore suspension necessary to give a sensitivity by plate test to 0.005 ug/ml of chlortetracycline in buffer. Generally, this will be obtained with a 0.03-0.10 percent inoculum. The spore suspension may be used indefinitely if protected from evaportation and contamination.


Add 6 ml of Medium E to each Petri dish, distribute evenly and allow to harden. Then add 4.0 ml of Medium E, which has been seeded with a suspension of B. cereus var. mycoides. Proceed as described for preparation of plates under penicillin, except use the chlortetracycline standard. Incubate at 30° for 16-18 hours.


Dilute one part of the sample with two parts of O. lM phosphate buffer, pH 4.5. If a concentration is expected or found to be greater than 0.48 ug/ml, dilute the sample to an estimated 0.12 ug/ml with antibiotic-free milk and then further dilute one part plus two parts of buff er. Multiply by the appropriate dilution factor after the assay has been completed, in order to obtain the final potency.

(iv) Standard curve

Dissolve about 30-50 mg of the standard in sufficient 0.0lN HCl to give a stc,::k solution of 1 OOO ug/ml. The stock solution may be used for seven days. Dilute the stock solution in antibiotic-free milk to give

89

concentrations of 0.015, 0.03, 0.06, 0.12, 0.24 and 0.48 ug/ml. Further dilute one part of each of these solutions with two parts of pH 4.5 buffer to give final concentrations of 0.005, 0.01, 0.02, 0.04, 0.08, and 0.16 ug/ml. The reference concentration is 0.04 ug/ml. Proceed as described in para.1.1.8.

(v) .Calculation of potency

Proceed as described in para. 1.1. 9

1.2.4 Oxytetracycline


Proceed as directed under chlortetracycline.


Proceed as directed under chlortetracycline.


Dilute one part of the sample witt:. two parts of 0. lM phosphate buffer, pH 4.5. If a concentration is expected or found to be greater than 2.4 ug/ml, dilute the sample to an est.4nated 0.60 ug/ml with antibiotic-free milk and then further dilute one part plus two parts of buffer.

(iv) Standard curve

The standard curve is prepared in the same manner as described for chlortetracycline, except the standard is dissolved in O.lN HCl to give a stock solution of 1 OOO ug/ml. The stock solution may be used for seven days. Dilute the stock solution in antibiotic-free milk to give concentrations of 0.075, 0.15, 0.3, 0.6, 1.2 and 2.4 ug/ml. Then dilute each of these solu.tions with buffer to give.final concentrations of 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 ug/ml. The reference concentration is 0.2 ug/ml. Proceed as described in para. l.1.8.


Proceed as described in para. 1.1. 9.

90

1.2.S Tetracycline

Proceed as in the case of oxytetracycline, except that, use the tetracycline standard as a basis of comparison.

1.2.6 Streptomycin


The test brganism is Bacillus subtilis (ATCC 6633}. Grow the organism five days at 37° in a Roux bottle containing 300 ml of Medium B. Suspend the growth in 50 ml of sterile physiological saline, centrifuge and decant the supernatant liquid. Reconstitute the sediment with 70 ml of sterile physiological saline and heat-shock the suspension by heating for 30 minutes at 70°. Maintain the spore suspension in the refrigerator. Determine by appropriate tests the quantity of spore suspension to be added to each 100 ml of agar for the secondary layer that will give sharp, clear zones of inhibition. The spore suspehsion may be used indefinitely if protected from evaporation and contamination.

(ii} Preparation of plates

Add 10 ml of Medium J to each Petri dish, distribute evenly and allow to harden. Then add 4.0 ml of Medium K which has been seeded with a suspension of B. subtilis. Proceed as described for preparation of plates under penicillin, except use the streptomycin standard. Incubate at 37° for 16-18 hours.


Using IN sodium hydroxide, adjust the pH of the sample to 8.0 and shake thoroughly. If a concentration is expected or found to be greater than 2.0 ug/n.l, dilute the sample to an estimated 0."5 ug/ml with antibiotic-free milk and then adjust the pH of the sample to 8.0 and shake thoroughly. The pH adjustment of the sample with sodium hydroxide is not considered a dilution if the change in volume is not significant.

91

(iv) Standard curve

Dissolve an accurately weighed sample of 30-50 mg ofstreptomycin standard in sufficient distilled water to give a stock solution of convenient concentration. The stock solution is kept under refrigeration and may be used for 14 days. Dilute the stock solution in antibiotic-free milk previously adjusted to pH 8.0 with 1 N sodium hydroxide to give concentrations of 0.0625, 0.125, 0.5, l.0 and 2.0 ug/ml. The reference concentration is 0.5 ug/ml. Proceed as described in para. 1.1.8.


Proceed as described in para. 1.1.9.

1.2.7 Dihydrostreptomycin

Proceed as outlined above for streptomycin, except use the dihydrostreptomycin standard as the basis of comparison.

1.2.8 Neomycin


The test organism is Staphylococcus epidermidis (ATCC 12228) which is maintained as · stock culture on agar slants of Medium A. Transfer the· growth from a fresh slant with 2.0 ml of sterile physiological saline to a Roux bottle containing 300 ml of Medium A and incubate for 18-24 hours at 32°- 35°. Harvest the growth from the surface of the agar with 50 ml of physiological saline. Determine the dilution of the suspension to be used (usually 1 : 25) that will give 25 percent light transmission. Before the actual assay, determine by trial plates, the optimum amount (usually l.0-2.0ml) of the dilution of the suspension to be added to l 00 ml of Medium F which has been melted and cooled to 48° Store the suspension in the refrigerator no longer than one week.


Add 10 ml of Medium K to each Petri dish, distribute evenly, and allow to harden. Then· add 4 ml of Medium K

92

which has been seeded with a suspension of Staph. epidermidis. Proceed as described for preparation of plates under penicillin except use the neomycin standard. Incubate at 32° - 35° for 16-18 hours.

{iii) Preparation of sample

Using lN sodium hydroxide, adjust the pH of the sample to 8.0 and shake thoroughly. If a concentration is expected or found to be greater than 4.0 ug/ml, dilute the sample to an estimated 1.0 ug/ml with antibiotic-free milk and then adjust the pH of the sample to 8.0 and shake thoroughly. The pH adjustment of the sample with sodium hydroxide is not considered a dilution if the change in volume is not significant.

(iv) Standard curve

Dissolve the weight of working standard in O. lM potassium phosphate buffer, pH 8.0, to make an appropriate solution (for example, l OOO ug/ml). The ~tock solution may be used for 14 days. Further dilute the neomycin stock solution in antibiotic-free milk previously adjusted to pH 8.0 with IN sodium hydroxide, to give final concentrations of 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 ug/ml. The 1.0 ug/rnl concentration is the reference concentration. Plae all fmal dilutions of standard and sample in an 82° water bath for five minutes, remove and cool radidly. Proceed as described in para. 1.1.8.


Proceed as described in para. l.1.9.

l.2.9 Polymyxin


The test organism is Bordete/la bro11chiseptica (ATCC 4617) which is maintained as a stock culture on agar slants of Medium A. Transfer the growth from a fresh slant with 2.0 ml of sterile physiologcial saline to a Roux bottle containing 300 ml of Medium A and incubate for 18-24 hours at 32° - 35°. Harvest the growth from the

93

surface of the agar with 50 ml of sterile saline. Determine the dilution of the suspension to be used (usually l : 25) that will give 25 percent light transmission. Before the actual assay, determine by trial plates the optimum amount (usually 0.1-0.2 ml) of the diluted suspension to be added to 100 ml of Medium H which has been melted and cooled to 48°. Store this stock suspension in the refrigerator no longer than two weeks.

(ii} . Preparation of plates

Add 10 ml of Medium G to each Petri dish, distribute evenly, and allow to harden. Then, add 4 ml of Medium H which has been seeded with a suspension of Bordetella bronchiseptica. Proceed as described for preparation of plates under penicillin, except use the polymyxin standard. Incubate at 37° for 16-18 hours.

(ill} Preparation of sample

Dilute one part of the sample with one part of 20 percent potassium phosphate buffer, pH 6.0. If a concentration is expected or found to be greater than 40 u/ml, dilute the sample to an estimated 16 u/ml with antibiotic-free milk and then further dilute one part with one part of 20 percent potassium pho

0

sphate buffer, pH 6.0.

(iv) Standard curve

Dissolve the weight of working standard in sufficient sterile distilled water to make an approi;riate stock solution (for example, 1 OOO u/ml). The stock solution may be used for 14 days. From this stock solution, prepare the standard curve by diluting in antibiotic-free milk to concentrations of 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 u/ml. Immediately before filling, further dilute with equal parts of 20 percent potassium phosphate buffer, pH·o.0, to final concentrations of 1.0, 2.0,4.0, 8.0, 16.0 and 32.0 u/ml with the '.0 u/rnl serving as the reference point and its. average value as the correction factor. Add the solution to the cylinders as rapidly as possible. Proceed as described in para. 1.1.8. ·


Proceed as described in para. l.l.9.

94

1.2.10 Erythromycin


The test organism is Sarcina lutea (ATCC 9341). Prepare a suspension of the organism in the manner described under penicillin. Before the actual assay, determine by trial plates the optimum amount (usually 0.1-0.5 ml) of the bulk suspension to be added to 100 ml of Medium F which has been melted and cooled to 48°. Store the suspension in the refrigerator no longer than two weeks.


Add 10 ml of Medium F to each Petri dish, distribute evenly, and allow to harden. Then add 4 ml of Medium F which has been seeded with a suspension of Sarcina lutea. Proceed as described for preparation of plates under penicillin except use the erythromycin standard. Incubate at 30° for 16-18 hours.


Test the sample undiluted. If a concentration is expected or found to be greater than 0.8 ug/ml, dilute the sample to an estimated 0.2 ug/ml with antibiotic-free milk.

(iv) Standard curve

Prepare a stock standard solution by dissolving 30-50 mg of erythromycin standard in 2 ml of methanol and then adding sufficient O.lM pH 8.0 phosphate buffer to give a concentration of 1 OOO ug/ml. The stock solution may be used for 14 days. Dilate the stock solution in antibiotic-free milk to give concentrations of 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 ug/ml. The reference concentration is 0.2 mcg/ml. Proceed as described in para 1.1.8.


Proceed as described in para. 't. t. 9.

95

1.2.11 Novobiocin


The test organism is Staphylococcus epidermidis {ATCC 12228). Prepare a suspension of the organism in the manner described under neomycin. The adjusted bulk suspension only and not the dilution of it is used in preparing the seed layer. Before, the actual assay, determine by trial plates the optimum amount ( usually 1.0-2.0 ml) of the bulj suspension to be added to l 00 ml of Medium A which has been melted and cooled to 48°. Store the suspension in the refrigerator no longer than one week.


Add 10 ml of Medium D to each Petri dish, distribute evenly, and allow to harden. Then add 4 ml of Medium A which has been seeded with a suspension of Staph. epidermidis. Proceed as described for preparation of plates under penicillin, except use the novobiocin standard. Incubate at 32-35° for 16-18 hours.


Test the sample undiluted. If a concentration is expected or found to be greater than 2.0 ug/ml, dilute the sample to an estimated 0.5 ug/ml with antibiotic-free milk.

(iv) Standard curve

Prepare a stock solution by dissolving 30-50 mg of a standard novobiocin acid in. 2 ml of absolute ethyl alcohol and then adding sufficient O.lM pH 8.0 phosphate buffer to give a concentration of 1 OOO ug/ml. The stock solution may be used for 14 days.· Dilute the stock solution in antibiotic-free milk to give concentrations of 0.06.25, 0.125, 0.25, 0.5, 1.0 and 2.0 ug/ml. The reference concentration is 0.5 ug/ml. Proceed as described in para. 1.1.8.


Proceed as described in para 1.1.9.

96

Section II - DAIRY PRODUCTS, ANIMAL ~UES AND EGGS

Methods are herein described for assaying antibiotics in cheese, butter, buttennilk, ice cream, dry powdered milk, evaporated milk, animal tissues and eggs. The basic assay method for each antibiotic is the same as that described in Section I. However, the preparation of the sample and the standard curve are different. To assay a sample of cheese, an antibiotic-free cheese should be diluted as recommended in Table I and tested to detennine that it does not contain inherent inhibitory substances. If it does, it will be necessary to prepare greater dilutions with buffer and to modify the standard curve.

2.1.1 General instructions

An antibiotic-free product comparable to that being tested should be used for preparing the standud curve. Dilute the control product as described for the puticular method and test this dilution on the appropriate plates to ensure that no zone of inhibition occurs. Use a high speed blender where blending of the product is prescribed. Blenders must be cleaned thoroughly before use. At the time of the assay, the buffer used for the particulu assay should fust be blended and placed on test as a control on the blender itself. It is essential to use cylinders on the plates for the assay of buttermilk. If paper discs are used, false positives will be obtained with antibiotic-free buttermilk'.

2.1.2 Standard curve

(a) Cheese, butter, ice cream, animal tissue and eggs: Prepare working solution by diluting the stock solution of standard in the appropriate buff er to give solutions whose concentrations equal 10 times the dilution factor times the final standard curve concentrations. Then blend 10 gm of the control product and I ml of each of the working solutions with enough buff er to give the prescribed dilution. Blend each point of the standard curve separately. For example: to prepare the standard curve for penicillin in cheese, proceed as follows:

Prepare dilution of penicillin standud in 1 percent phosphate buffer, pH 6.0, to give concentrations of 0.315, 0.625, 1.25, 2.5, 5.0 and 10.0 u/ml. Then blend 10 gm of control cheese plus 39 ml of buffer

97

plus 1 ml of each of these working solutions separately to get final concentrations of 0.0063, 0.0125, 0.025, 0.05, 0.1, and 0.2 u/ml. These are the final concentrations for the standard curve.

(b) Buttermilk and evaporated milk: Dilutions of the standard are first made in an antibiotic-free product and then final dilutions are made with the appropriate diluent in

· the ratio shown in Table I in the column headed "Dilution" to give the final standard curve concentrations shown. For example, the dilution shown for penicillin in evaporated milk is 1 + 1 and the diluent is distilled water. Therefore, to prepare the standard curve for assaying penicillin in evaporated milk, dilute the penicillin stock solution in an antibiotic-free evaporated milk to 0.0125, 0.025, 0.05, 0.10, 0.20 and 0.40 u/ml, and then further dilute each of these solutions one part plus one part of distilled water to give final concentrations of 0.006, 0.0125, 0.025, 0.05, 0.10 and 0.20 u/ml.

2.1.3 Preparation of sample

To assay cheese, butter, ice cream, or animal tissues, weigh accurately 10 gm of the sample and blend with the appropriate amount of buffer as indicated by the dilution factor in Table I. For example, .to assay penicillin in cheese, Table I shows a 1 + 4 dilution. Therefore, blend 10 gm of the sample with 40 ml of buffer. To assay dry powdered milk, weigh accurately 10 gm of the sample and dissolve the weight in the appropriate amount of buff er as indicated by the dilution factor in Table I. To assay buttermilk or evaporated milk, dilute one part of the sample with the appropriate number of parts of diluent shown in Table I. For example, to assay penicillin in evaporated milk, dilute one part of the sample with one part of distilled water. To assay eggs, mix the shelled contents of one or more eggs in a beaker, using a stirring rod. Weigh a portion of the mixed egg sample and "to it add the amount of appropriate diluent indicate in Table I. Mix thoroughly by shaking and use this mixture as the sample solution. If water: methanol is used as the diluent, centrifuge the mixture for about 30 minutes, decant off the clear supernatant fluid. Recentrifuge the clear supernatant fluid for about 5 minutes. The final clear solution is the sample solution.

98

If a concentration of antibiotic greater than the highest concentration of the standard is expected or found in a sample of butter, cheese, ice cream, animal tissue, or dried milk, then the sample should be mixed with sufficient diluent to give an estimated concentration equivalent to the reference. concentration of the standard. In this case, it would be necessary to prepare a standard curve with a control product diluted in the same ratio as the sample. It is not necessary to prepare a separate standard curve for buttermilk or evaporated milk since they can be diluted with a comparable control product and then further diluted with diluent in the same ratio as used for the standard curve.

2.1.4 Perfonnance of assay

Prepare the sample and standard curve as shown in Table I. Then proceed as described in Section I for the performance of the specific assay. In calculating final potency of the sample, remember to multiply by the appropriate dilution factor.

2.2 Bacitracin in animal tissues

Proceed as described for bacitracin in milk (1.2.2) except


Use a single inoculated layer of l O ml of Medium A. This is seeded with 11. flavus (O. l percent inoculum, or such concentration that a 0.05 u/ml bacitracin standard in 5 percent pH 6.5 buffer ·will produce zones 15 to 17 mm). Incubate the plates for 18 hours at 3 7°.

2.2.2 Standard curve

Dilute the stock solution of the standard in 5 percent pH 6.5 buffer to give concentrations of 0.0125, 0.025, 0.05, 0.10 and 0.20 u/m1. The reference concentration is 0.05 u/ml. The 5 percent pH 6.5 buffer is prepared by dissolving 27.85 gm monobasic potassium phosphate and 22.15 gm dibasic potassium phosphate in distilled water and diluting to l litre with distilled water.


Thoroughly mince the tissur by passing it twice through a meat grinder using a fine cutter blade. Weigh a 15 gm aliquot of the

99

minced tissue in a beaker. Add 25 ml of 33 percent aqueous pyridint:! and stir thoroughly for five minutes. Transfer to a round bottom centrifuge tube and centrifuge at I 200 rpm for five minutes. Transfer 10 ml of supernatant to a 100 ml Erlenme¥er (hsk. Add 30 ml of methanol, shaking the flask while adding the solvent. Transfer flask contents to a round bottom centrifuge tube and centrifuge at I 200 rpm for a few minutes. Transfer half of supemate to a Petri dish bottom and aerate to dryness. When almost dry, add the remaining supernate and continue aerating until the entire sample is dry. Add 3 ml of pH 6.5 buffer (5 percent) and adjust the pH to 6.5 with lN NaoH (the amount needed varies between 0.2 and 0.3 ml depending on the tissue). Add more pH 6.5 buffer so that the final volume of the aliquot will be equivalent to the original volume of the tissue. This final volume of the aliquot is detennined as follows:

Prior to running the bacitracin assays, determine the moisture content of the minced tissue. As an example, assume that the tissue contains 70 percent water. Then the water content of a 15 gm to 35.5 ml, and the supernate of the first extraction represents a dilution of l to 2.37. The 10 ml aliquot of the supernate, which is treated with 30 ml of methanol and then later _dried, should be dissolved in a total volume of 4.22 ml (10/2.37), to restore this aliquot of the sample to its original volume.

2.2.4 Calculation of potency

Since the extraction procedure enables one to bring the extracts of the tissue back to a volume equivalent to the original sample, no dilution factor is necessary and u/ml is also u/gm.

2.2.5 Rccorery experiments

Using a control sample of minced tissue, prepare six 15 gm aliquots to contain 0.0 l, 0.02, 0.03, 0.04, 0.05, and 0.10 u/gm by adding (use a 1/10 ml KaJrn serological pipette) to each 15 gm sample 0.015, 0.030, 0.045, 0.060, 0.075 and 0.150 ml of a I O u/rnl standard solution of bacitracin. Stir minced samples thoroughly to distribute the antibiotic, and carry out the extraction procedure described above for the unknown samples. Prepare also sufficient blank extract of a control sample that

100

has no added bacitracin. Determine the mean recovery of added bacitracin and use this correction factor to detennine the potency of bacitracin. As an example, assume the mean recovery is 75 percent. If bacitracin is found in any unknown sample, a correction factor of 100/75 should be applied to the assay.

2.3 Streptomycin in animal tissues

Proceed as described for streptomycin in milk (1.2.6) except:


Use 9 ml of Medium I for the base layer and 4 ml of Medium M for the seed layers.


Blend one part of tissue with four parts of phosphate-sulphate buffer, pH 1.5. Centrifuge the homogenate, and by decanting collect and measure the volume of the clear solution. Adjust the pH to pH 8.5 with ION sodium hydroxide, added drop-wise with stirring, centrifuge again, and use the clear solution for assay.

2.3.3 Preparation of standard curve

Proceed as in para. 2.3.2 above and prepare a tissue extract of antibiotic-free tissue. Use this extract as a diluent to prepare final concentrations.

2.3.4 Dilution factor =

Vol. of clear portion of blank extract after adjustment to pH 8

------------------ x 5 Vol. of clear portion of sample extract before

adjustment to pH 8

2.4 Novobiocin in animal tis.mes

Proceed as described for novobiocin in milk (1.2.11) except:

2.4. t Standard curve

Dilute the stock solution of the standard in 1 ° /0 pH 6.0 buffer to give concentrations of 0.05, 0.1, 0.2, 0.4 and 0.8 mcg/ml. The reference concentration is 0.2 mcgimL

101

2.4.2 Preparation of samples

Grind the tissue in a meat grinder. Place a 10 gm weight of the ground tissue .in a 50 ml Lourdes Tissue Homogenizer and add 30 to 35 ml of ethyl acetate. Immerse the homogenizer in an ice bath and extract the tissue for two minutes. Filter the extract through filter paper in a Buchner Funnel. Repeat the extraction of the tissue two more times with ethyl acetate, adding the tissue and the filter paper to the homogenizer each time; Combine the filtered extracts and evaporate at 50° to 60°

to about 20 ml. Transfer the extract to a separatory funnel and add 0.4 ml of 0.5N KOH. Extract as rapidly as possible six times with 5 ml of cold 4 percent pH 10.0 potassium phosphate buffer. Drain the buffer extracts into a graduated cylinder containing 2 ml of I : 10 phosphoric acid. Adjust the pH of the combined buffer extracts to 6.0 with 1 : 10 phosphoric acid and bring the final volume to 40 ml with 1 percent pH 6.0 phosphate buffer. The dilution factor for the calculation of potency is four.

2.4.3 Recovery experiments

L'sing a control sample of ground tissue, prepare, in triplicate, aliquots of control tissue containing 0. 2 and 1.0 ppm of novobiocin. Add the novobiocin at the time of homogenization. Carry out the extraction procedure described above for the unknown samples and determine the mean recovery of added novobiocin. Use a recovery correction factor to determine the potency of novobiocin. As an example, assume the mean recovery is 75 percent . If novobiocin is found in any unknown sample, a correction factor of 100/75 should be applied to the assay.

102

---

TAliLE I - DETAILS FOR ASSAY OF ANTIBIOTICS IN DAIRY PRODUCTS AND ANIMAL TISSUES

Dilution 1-'inal Concentrations Antibiotic Product Dilution Diluent Factor for Standard Curvel

Penicillin cheese, butter, 0.0063, 0.0125, 0.025, ice cream I +4 buffer (a) 5 0.05, 0.10 and 0.20 U/ml

animal tissues I +4 buffer (a) 5 Same as above

dry powdered milk 1+3 buff r (a) 4 Same as above

buttermilk none negative buttermilk I Same as above

evaporated milk I+ 1 distilled water 2 0.006, 0.0125, 0.025, U.05,

§ 0.10 and 0.20 11/1111

eggs 1 +2 buffer (a) 3 Same as above

Bacitracin cheese, b~tter, 0.025, 0.05, 0.10, 0.10, ice cram I -1-4 buffer (a) 5 0.4, 0.8 u/ml

animal tissues !il!e 5%pll 6.5 0.0125, 0.025, 0.05, 0.10, para 2.2 buffer 1 0.20 11/ml

dry powdered milk 1+3 buffer (a) 4 0.125, 0.25, 0.5, I.U, 2.0, 4.0 u/1111

evaporated milk I + I distilled water 2 0.075, 0.15, 0.3, 0.h, 1.2, 2.4 11/ ml

eggs 1, t I water: rni:thanol ( I + 2) 2 Same as above

I ll1lul ion I· i11al ( 'onn:11lral ions

\1111hi111il' 1'1,,dud llil11lio11 l>ilunll l':1d1n for Sl:111dan.l ( 'urvcl

Strq11t1111yd11 ( 'hlT"'· hUlll:I, 0.5, ! .0, 2 .0, ··1.U, l<.O,

... il'C l'l"l'.1111 I l 4 l>uffr1 hi) s I h.ll ug/1111 . I >ihy,lrosl .-c1,1umy dn animal tissues sc,· para 2.3 0.05, IJ. I, 0.2, U.·I, IJ.8,

I.(, ug/ ml

dry po\\Lkn:,l milk I ·t .l buffer (d) 4 1.25, 2.5, 5.0, /I).I), 20.IJ, 40.0 ug/1111

h11lkr111ilk I + I butler (ii) 2 1.5, J.O, C..O, 12.U, 24.0, 48.0 ugiml

cvapmall'd 111ill,, t + I buffer (ii) 2 0.5, 1.0, 2.0, 4.U, 8.0, 16.0 ug/ml

l'g~s I+ I water: 111clh,111ol ( I + 2) 2 0.05, 0.1, 0.2, IJ . ./, IJ.8,

52 1.6 ug/ml

Nl'omydn chcl:Sl', ict· cream I +4 buffer ld) 5 0.075, 0.15, O.J, OJ,, 1.2, 2.4 ug/ml

a11i111al tissues t t 4 buffer (d) 5 0. t 25, 0.25, 0.5, 1.0, 2.0, 4.0 ug/1111

butter 1+4 buffer (d) 5 0.012, 0.025, 0.05, U. I, 0. 2,

0.4 ug/ml

dry powdcrl'O milk 1+3 buffer (d) 4 0.7.b, 1.5, 3.0, 6.0, 12.0, 24.0 ug/1111

buttermilk t + 1 buffer (d) 2 1.5, 3.0, 6.0, 12.U, 24.0, 48.0 U!!/ 1111

i:vaporatcd milk 1 + 1 buffer (d) 2 0.5, 1.0, 2.0, 4.0, 8.0, t 6.0 ug/1111

t:ggs 1 + 1 O.OZN NaOH 2 0.075, 0.15, 0.3, U.<>, 1.2, (final pll-8) 2.4 11~/1111

·-.

Dilution Final Concentrations Antibiotic Product Dilution Diluent Factor for Standard Curvel

~ystatin milk milk 1 + 1 buffer (e) 2 1.5, 3.0, 6.0, J 2.0, 24.0, 48.0 u/ml

eggs 1 + 1 buffer (e) 2 1.5, 3.0, 6.0, 12.0, 24.0, 48.0 u/ml

(Use .\.O.A.C. Nystatin Feed Assay Plates)

Chlortctracyclinc chcc~. butter, 0.025, 0.05, 0.1, 0.2, 0.4, ice L"ream 1 +4 buffer (b) 5 0.8 ug/ml - anima

0 v. I animal tissues 1 +4 buffer (b) 5 0.005, 0.01, 0.02, 0.04, 0.08,

0.16 ug/ml

dry powdered milk 1 +3 buffer (b) 4 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 urJml

buttermilk 1 + 2 buffer (b) 3 0.05, O. IO, 0.20, 0.4, 0.8, 1.6 UtVIIII

evaporated 111ilk I +2 buffer (b) 3 0.05, 0.1, 0.2, U.4, 0.8, 1.6 ug/1111

c~gs 1 + 2 buffer (b) 3 0.005, 0.01, 0.02, 0.04, 0.08, II. I 6 ug/ 1111

l>ilu tion Final ( 'om:cn !rations .\ntihoti, l'r11d11d l>ilution l>ilul'nl Fai:tor for Slandanl <. 'urvl' 1

Tt."lracyclinc d1eese, butler, 0.125, 0.25, 0.5, I ./J, 2.0,

01' il'l' i:1ea III 1 +4 buffer (b) 5 4.0 ug/1111

(hy tetracycline animal tissues I t 4 buffer (b) 5 0.025, 0.05, 0. I, 0. 2, 0.4,

0.8 ug/ ml

dry powdered milk I + 3 buffer (h) 4 0.5, 1.0, 2.0, 4.0, 8.0, 1 b.O ug/ ml

buUcr111ilk 1+2 buffer (b) 3 0.25, 0.5, 1.0, 2.0, 4.0, -~ 8.0 ug/1111

evaporated milk I +2 buffer (b) 3 0.25, 0.5, 1.0, :!. 0, 4.0, 8.0 ug/ml

c~gs 1+2 buffer (b) 3 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 ug/ml

,

hythromycin animal tissues 1+4 buffer (b) 5 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 ug/ ml

t:ggs 1 + 1 water: methanol ( 1 + 2) 2 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4 ug/ml

·- ----

------

0 -...J

!\ ntibiotic Product Dilution

Polymyx.in cheese, ice ere am 1 +4

animal tissues 1 +4

bulter 1+4

dry powdered milk 1+2 then 1 + l

bulter111ilk 1 + 1

evaporated milk I +I

eggs 1+2

Novobiodn animal tissues sec para 2.4

C!!l(S I -i I

I The figures in italks arc the n:fcn,ucc co11ccnlratio11.

Dilution Final Concentrations Diluent Factor for Standard Curve I

buffer (t) 5 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 u/1111

buffer (t) 5 Same as above

buffer (I) 5 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 u/1111

distilled water 3) 1.0, 2.0, 4.0, 8.0, 16.0, 6 32.0 u/ml

buffer (t) 2)

buffer (I) 2 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 u/1111

buffer (I) 2 1.25, 2.5, 5.0, /0.0, 20.0, 40.0 11/1111

buffer (I) 3 0.25, 0.5, 1.0, ].0, 4.0, 8.0 u/1111

0.05, 0.1, 0.1, 0.4. buffer (2) 4 0.8 u!?J ml

buffer (;i) 2 Sarne as ahovL,

specifications for identity and purity of some … · 2021. 3. 22. · of official agricultural...

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