Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 1 of 17

UNCONTROLLED COPY

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 514

Supplemental Assay Method for the Determination of Hydrogen Ion 

Concentration, Total Nitrogen, TCA Nitrogen, Phenol and Clarity in 

Intradermic (Filtrate Produced from Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

Date: June 17, 2022

Number: SAM 514.09

Supersedes: SAM 514.08, February 8, 2016

Standard Requirement: 9 CFR 113.406

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 2 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 3 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

UNCONTROLLED COPY

1. Introduction

This Supplemental Assay Method (SAM) describes the procedures for determination of 

hydrogen ion concentration, total nitrogen, trichloroacetic acid precipitable (TCA-ppt) nitrogen, 

phenol content and clarity in intradermic (filtrate produced from cultures of Pn, C, and Dt strains 

of Mycobacterium tuberculosis) tuberculin; as prescribed in title 9,Code of Federal Regulations

(9 CFR), part 113.406. 

The hydrogen ion concentration shall be determined with a pH meter which has been 

standardized with a pH 7.0 buffer just prior to use. The total nitrogen content shall be determined 

by the Kjeldahl method on duplicate 15 mL samples, consisting of 5 mL from each of three vials.

The determination of precipitable nitrogen by a final concentration of 4 percent trichloroacetic 

acid shall be made by the Kjeldahl method on duplicate 15 mL samples, consisting of 5 mL from 

each of three vials. The phenol content shall be determined by direct titration with a standardized 

bromide-bromate solution. (A correction factor of 0.04 should be subtracted from the final value 

in the determination of phenol in tuberculin.) The product shall be optically clear and free from 

any extraneous particles.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 pH meter, with combination pH electrode (i.e., Orion® ROSS 8103)

capable of measuring from pH 0.0 to 14.0

2.1.2 Disposable beaker, 5-mL

2.1.3 Balance, top loading, capable of measuring 0.001 g 

2.1.4 Digestion unit (Büchi)

2.1.5 Distillation unit (Büchi)

2.1.6 Volumetric pipettes, Class A, 5-, 10-, and 25-mL

2.1.7 Volumetric flasks with barrel head glass stopper, Class A, 500-mL and 

1-L

2.1.8 Erlenmeyer flasks, 125-mL
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Testing Protocol Page 4 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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2.1.9 Burets with PTFE stopcocks, precision bore, Class A, 10-, 25-, and 50-mL

2.1.10 Graduated cylinders, Class A, 50-, 100-, 250-, 500-mL, and 1 L

2.1.11 Glass-stoppered Erlenmeyer flasks, 250-mL

2.1.12 Heating/stirring plate with stirring bars

2.1.13 Filter paper, 11μm particle retention (Whatman No. 1)

2.1.14 Timers, 30 seconds to 1 minute

2.1.15 Dropper, i.e., transfer pipette, Pasteur pipette, dropper bottle

2.1.16 Pipettor and tips to accurately dispense 100- to 1000-μL

2.1.17 Small spot light lamp

2.1.18 Vortex mixer

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

chemicals are reagent grade, unless specified. 

2.2.1 Total and TCA-ppt nitrogen

1. Sulfuric acid (H2SO4), CAS# 7664-93-9, Purity: Minimum 95.0%, 

Maximum 98.0%

2. Kjeldahl Catalyst Tablets, 1.5 g K2SO4 + 0.075 g HgO

3. Sodium hydroxide (NaOH), CAS# 1310-73-2, Purity: 98.5%

4. Boric acid (H3BO3), CAS# 10043-35-3, Purity: 99.9%

5. Methyl red, CAS# 493-52-7, Purity: 98.0%

6. Hydrochloric acid (HCl), CAS# 7647-01-0, Assay: 36.5-38.0%

7. Sodium carbonate (Na2CO3), CAS# 497-19-8, Purity: 99.9%

8. Bromo phenol blue, CAS# 115-39-9, Purity: 98.0%
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Testing Protocol Page 5 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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9. Trichloroacetic acid (TCA), CAS# 76-03-9, Purity: 98.0%

10. Control Sample – Either a pool of PPD tuberculin products with 

established protein and phenol values as tested by PL-CAS; or a product 

produced for use as a control sample and tested by PL-CAS.

11. Protein Standard Reference Material, Bovine Serum Albumin (current 

lot of SRM 927 from National Institute of Standards and Technology)

2.2.2 Phenol (some reagents same as for protein)

1. Hydrochloric acid (HCl), CAS# 7647-01-0, Assay: 36.5-38.0% 

2. Water (H2O), Purity: distilled, demineralized, reverse osmosis or 

equivalent.

3. Methyl orange, CAS# 547-58-0, Purity: 98.0%

4. Silicotungstic acid hydrate (H4[Si(W3O10)4]*26H2O), CAS# 12027-43-

9, Purity: 99.0%. Store at 4°C.

5. Sulfuric acid (H2SO4), CAS# 7664-93-9, Purity: Minimum 95.0%, 

Maximum 98.0%

6. Arsenic trioxide, anhydrous (As2O3), CAS# 1327-53-3, Purity: 99.9%

7. Sodium hydroxide (NaOH), CAS# 1310-73-2, Purity: 98.5%

8. Phenol (C6H6O), CAS# 108-95-2, Purity: ≥ 99.0%

This can be a purchased NIST standard and diluted, if necessary, to the 

appropriate level.

9. Sodium bicarbonate (NaHCO3), CAS# 144-55-8, Purity: 99.9%

10. Potassium bromate (KBrO3), CAS# 7758-01-2, Purity: 98.5%

11. Potassium bromide (KBr), CAS# 7758-02-3, Purity: 99.0%

2.2.3 Hydrogen ion concentration

1. Commercial buffers, certified pH 7.00 and pH 4.00
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Testing Protocol Page 6 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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2. pH electrode storage solution

3. Reference electrode filling solution 

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have experience or training in this protocol. This includes 

working knowledge of the use of general laboratory equipment, glassware and chemical 

safety; and specific training in the operation of the laboratory equipment and reagents

listed in Section 2. 

Analysts performing this procedure should first conduct at least 2 trial runs using controls 

and standards and obtain results within acceptable limits.

3.2 Preparation of equipment/instrumentation

All equipment must be operated and maintained according to manufacturers’ 

recommendations and monitored in compliance with applicable standard operating 

procedures.

3.2.1 Hydrogen ion concentration

Calibrate the pH meter with appropriate electrode according to manufacturer’s 

instructions using either one buffer, pH 7.0 or two buffers, pH 4.0 and pH 7.0.

3.2.2 Total and TCA-ppt Nitrogen Tests 

1. Prepare digestion and distillation units according to manufacturer’s

recommendations.

2. Check levels of water and sodium hydroxide tanks on the distillation 

unit, fill if necessary.

3. Prime the buret by rinsing with standardized HCl.

3.2.3 Phenol Test

Prime the buret by rinsing with test fluid. 
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 7 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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3.3 Preparation of reagents/control procedures

Reagents are stable for 6 months from date of preparation and stored at room 

temperature, unless otherwise noted. Prepare reagents in volumes appropriate to demand 

to minimize waste due to expiration. 

Glassware used for preparation of reagents must meet ASTM requirements; 

measurements are based on the measurements of uncertainty outlined in those 

requirements.

All references to “water” indicate distilled, demineralized, reduced oxygen or water of 

equivalent purity (Section 2.2.2(2)).

In the following steps the acronym QS is used. It is defined as quantity sufficient; as 

much as is sufficient.

3.3.1 Total and TCA-ppt Nitrogen Tests

1. Standard, 1.0 ± 0.1 mg/ml Protein (0.016% nitrogen): Dilute protein 

standard reference material (Section 2.2.1(11)) to the range of 1.0 ± 0.1 

mg/mL protein with water. Prepare sufficient dilution to provide several 

10 mL portions. Store at 4°C. 

2. Control Sample: Either (1) a pool of tuberculin products with 

established nitrogen and phenol values as tested by PL-CAS; or (2) a 

product produced for use as a control sample and tested by PL-CAS.

(1) Combine any sample volumes remaining after test completion in a 

pool. Record all identifying information, CAS protein result and 

expiration date for each sample. Control sample protein concentration is 

the mean of CAS results for all samples included in the pool. 

(2) Obtain a product produced for use as a control sample. Analyze the 

product a total of three times, mean of these trials must be 1.0 ± 0.1 

mg/mL protein. Expiration date is as indicated on product. Store at 

4°± 10° C.

3. 32% Sodium Hydroxide (NaOH): Caution!! NaOH is caustic--

Avoid contact with skin. Dissolve 640 g ± 1 g sodium hydroxide in 

approximately 1.4 L water in a 2-L volumetric flask on a stir plate. 

Solution will be HOT!! Cool to room temperature. QS with water. Store at 

room temperature.

4. Saturated Boric Acid (H3BO3): Use a container with at least twice as 

much volumetric capacity as your final volume. Add 15.0 ± 0.1 g boric 
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Testing Protocol Page 8 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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acid to 100 mL water. Stir, with heat, until all boric acid dissolves. Some 

boric acid recrystallizes when cool. Store at room temperature.

5. 0.1% bromo phenol blue: Dissolve 0.1 ± 0.1 g in 100 mL water. Store 

at room temperature.

6. 0.5% methyl red: Dissolve 0.5 ± 0.1 g in 100 mL ethanol. Store at 

4°C.

7. 4.0% Trichloroacetic acid (TCA): Dissolve 4.0 ± 0.1 g TCA in 

75 mL water in a 100-mL volumetric flask. QS with water. Store at room 

temperature.

8. Standardized 0.01 - 0.02N Hydrochloric acid (HCl): Caution!! 

Concentrated HCl is corrosive – Handle in fume hood. Avoid contact 

with skin.

Preparation: Add 1.72 mL hydrochloric acid to approximately 900 mL

water in a 1-L volumetric flask. QS with water. Store at room temperature. 

Standardization: Weigh approximately 0.010 g dried sodium carbonate. 

Record weight. Dissolve in 25 mL water. Add three drops 0.1% bromo 

phenol blue (indicator). Titrate with prepared 0.01 – 0.02N hydrochloric 

acid to an endpoint color of green, not bluish green nor yellowish green. 

Calculate the normality of the hydrochloric acid solution as below. 

Perform three trials and use the calculated mean as the normality of the 

hydrochloric acid solution. 

Calculation:

NN HHHHHH = [(gg NNNN2 CC3)(1000)]

[(mmmm HHHHHH)(52.994)]

3.3.2 Phenol test

1. Standard, 0.50% phenol: Dissolve 5.0 ± 0.01 g phenol in 

approximately 500 mL water in a 1-L volumetric flask; QS to 1 L with 

water. 

2. Control, Pool of Previously Analyzed Samples: 

Either (1) a pool of PPD tuberculin products with established protein and 

phenol values as tested by PL-CAS; or (2) a product produced for use as a 

control sample and tested by PL-CAS.
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 9 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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(1) Combine any sample volumes remaining after test completion in a 

pool. Record all identifying information, CAS phenol result, and 

expiration date for each sample. Control sample phenol concentration 

is the mean of CAS results for all samples included in the pool. 

(2) Obtain a product produced for use as a control sample. Analyze the 

product a total of three times, mean of these trials must be 0.50 ± 0.04 

% phenol. Expiration date is as indicated on product. Store at 

4°± 10° C.

3. 20% Hydrochloric Acid (HCl): In a 1-L volumetric flask, slowly add 

200 mL hydrochloric acid to 600 mL water; QS to 1 L with water. 

4. 0.1% Methyl Orange: Dissolve 0.1 ± 0.01 g methyl orange in

100 mL water. Filter if necessary. 

5. Silicotungstic acid solution (SAS): Dissolve 60.00 ± 0.5 g 

silicotungstic acid hydrate in 400 mL water in a 500-mL volumetric flask. 

Add 50 mL sulfuric acid. When cool, QS to 500 mL with water. 

6. Clarifying solution (CS): Add 50 mL SAS and 125 mL 20% 

hydrochloric acid to 325 mL water. Prepare fresh prior to each test.

7. "Acid solution" for As2O3 standardization solution: Add 110 mL

hydrochloric acid and 2.5 mL 0.1% methyl orange to 100 mL water. 

8. 0.050 N Arsenic trioxide (As2O3): CAUTION!! Arsenic trioxide is 

extremely toxic. Avoid contact; handle in fume hood using gloves, mask, 

and goggles. Consult the Safety Data Sheet for specific handling 

instructions before proceeding. Dissolve 2.4730 ± 0.001 g anhydrous

arsenic trioxide in 25 mL hot 1 N sodium hydroxide in a 1-L volumetric 

flask. Neutralize solution with 25 mL 1 N sulfuric acid. When cool, QS to 

1 L with water. 

9. 1N Sodium hydroxide: Dissolve 4.00 ± 0.01 g of sodium hydroxide in 

60 mL water in a 100-mL volumetric flask; QS to 100 mL with water. 

10. 1N Sulfuric acid: In a 100-mL volumetric flask, slowly add 

4.904 mL sulfuric acid to 60 mL water; QS to 100 mL with water. 

11. Test fluid (TF): Dissolve 0.30 ± 0.01 g sodium bicarbonate, 1.67 ± 0.01 g potassium 

bromate, and 15.00 ± 0.01 g potassium bromide in water and QS to 1 L with water. CRITICAL 

CONTROL POINT: The test fluid must be standardized as described in 3.3.11(1) prior to use. 

1. Standardization
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Testing Protocol Page 10 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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a. Prepare standardization solution: Add 25 mL 0.050N 

arsenic trioxide to 10 mL “Acid Solution.”

b. Confirm standardization solution by titrating with 

previous lot of TF. It should take 21.3 mL TF to titrate the 

standardization solution.

c. Titrate standardization solution with new lot of TF. The 

required titration volume is 21.3 mL TF. A first time 

titration may require less than 21.3 mL TF, in which case, 

the TF volume must be adjusted by adding the correct 

volume of water to the TF, continue to Step 1d if this is the 

case. If the first time titration is 21.3 ± 0.1 mL TF, continue 

to Step 2.

d. Adjust the TF volume. For this step the calculations are 

shown and an example is used to illustrate.

A = Starting volume of TF (mL)

B = Titration volume of TF (mL)

C = Volume of TF left (mL)

D = Required titration volume (21.3 mL)

E = Adjusted volume of TF (mL)

F = Volume of water to be added to volume of TF 

left to achieve the adjusted volume (mL)

Example: Assume the starting volume of TF is 1000 mL

and the titration volume is 20.5 mL.

• A – B = C

Example: (1000 mL) - (20.5 mL) = 979.5 mL

• (C) (D)

(B) = E

Example: 

(979.5 mL) (21.3 mL)

(20.5 mL) = 1017.7 mL

• E – C = F

Example: (1017.7 mL) - (979.5 mL) = 38.2 mL

• Add the calculated volume of water (F) to the existing 

TF and put any TF remaining in the buret back into 

flask. Continue to Step 2.
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 11 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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2. Repeat Step 1c until three consecutive trials produce an average 

titration volume of 21.3 mL.

3.4 Preparation of the sample

3.4.1 Receipt

Complete sample receipt as described by standard operating procedures.

3.4.2 Preparation 

Licensed or prelicense biologics products are generally received in sealed serum 

bottles and stored at 4°± 10°C prior to testing. Before testing, allow sample vials 

and reagents to come to room temperature.

3.4.3 Clarity Test

Remove label from a sealed bottle of tuberculin. When the bottle has warmed to 

room temperature, make sure the label is dry and peel the label from the bottle 

carefully. Clean the bottle with alcohol and a lint-free towel. 

4. Performance of the Test 

4.1 Hydrogen ion concentration

4.1.1 Place approximately 5.0 mL of tuberculin product in each of two 

disposable beakers.

4.1.2 Flush pH electrode in first beaker of tuberculin by means of dipping it 

several times until the pH readout settles. If using a printer, label this first readout 

“flush.”

4.1.3 Place electrode in second beaker of tuberculin. Wait until pH readout 

becomes stable; record pH.

4.2 Clarity

In an area with subdued light, allow your eyes to adjust. Turn on the spotlight lamp, 

which is positioned upright. Place the unlabeled bottle over the light beam and observe 

for extraneous particles. 
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 12 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

UNCONTROLLED COPY

4.3 Total nitrogen

Analyze duplicate 15 mL samples, consisting of 5 mL from each of three vials. Analyze 

the control, standard and a blank sample in duplicate each time testing is performed. 

4.3.1 Place one Kjeldahl Catalyst tablet, 1.0 mL sample and 3.0 mL sulfuric 

acid into a digestion flask. Follow the same procedure for standard and control, 

use only reagents for the blank.

Caution: HgO is poisonous – Use gloves, mask, and goggles. 

Caution: Concentrated H2SO4 is corrosive--Avoid contact with skin.

4.3.2 Place the digestion flasks in the digestion unit. 

4.3.3 Digest in a method that results in a fully digested product, i.e., 250°C for 

15 minutes, 410°C for 60 minutes, and 500°C for 15 minutes. Final product 

should be clear to white-cloudy.

4.3.4 Cool in digestion flasks, add 6 mL water (Section 2.2.2(2)), mix (a vortex 

may be used), and cool again.

4.3.5 Place digestion flask and a 125-mL Erlenmeyer flask containing 5 mL

saturated boric acid solution and 3 drops 0.1% methyl red into the distillation unit. 

Tilt the flask so the tip of the condenser is immersed in the boric acid.

4.3.6 Add a sufficient amount of 32% sodium hydroxide to make the solution in 

the digestion flask alkaline, i.e., 25 mL. A sodium hydroxide pump may be used 

for this, if the distillation unit is equipped with one. 

4.3.7 Distill for two minutes.

4.3.8 Titrate collected distillate to endpoint color change of yellow to deep rose 

(pH 5.0) with standardized hydrochloric acid. Record the volume of hydrochloric 

acid titrated.

4.4 TCA-ppt nitrogen

Analyze duplicate 15 mL samples, consisting of 5 mL from each of three vials. Analyze 

the control, standard and a blank sample in duplicate each time testing is performed. 
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Testing Protocol Page 13 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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4.4.1 Place 5.0 mL of sample and 5.0 mL 4.0% TCA in a 15 ml, screw-capped 

centrifuge tube. Follow the same procedure for standard and control. Blank 

sample preparation steps begin with Section 4.4.7.

4.4.2 Vigorously shake centrifuge tube for 1 minute, then let stand for 10 

minutes.

4.4.3 Centrifuge tube under conditions that result in pellet formation, i.e., 2,500 

rpm for 10 minutes. Discard supernatant.

4.4.4 Add 3 mL water (Section 2.2.2(2)) and vortex tube until the precipitate at 

bottom mixes. Transfer mix to digestion flask. 

4.4.5 Repeat Section 4.4.4 two more times, each time adding to the original 

digestion flask so the final volume in the digestion flask is approximately 9 mL. 

4.4.6 Add one Kjeldahl Catalyst tablet and 3.0 mL sulfuric acid to the digestion 

flask. 

4.4.7 Proceed to Section 4.3.2 and follow the digestion, distillation and titration 

procedures through Section 4.3.8.

4.5 Phenol

Analyze the control pool and phenol standard in duplicate each time testing is performed. 

Analyze samples in triplicate.

4.5.1 Combine 5 mL sample and 100 mL clarifying solution (CS) to a 250-mL

glass-stoppered flask. Shake 2 minutes. Filter through filter paper and collect 

50 mL of filtrate.

4.5.2 Transfer 50 mL of filtrate to another 250-mL glass-stoppered flask. Add a 

stir bar and place flask on stir plate with buret directly above. Add 1 drop 0.1% 

methyl orange (indicator), stir for a few seconds. Observe the color as pink.

An acceptable alternative to using a stir plate and stir bar would be shaking the 

flask.

4.5.3 Titrate with 2 mL test fluid (TF), stir or shake for a few seconds. Observe 

the color; if pink, repeat. If colorless, go to Section 4.5.4.

4.5.4 Stir or shake 30 seconds. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If colorless for ≤ 10 seconds or if pink, titrate with 1 mL TF, 

and repeat. If colorless for ≥ 10 seconds, go to Section 4.5.5.
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Testing Protocol Page 14 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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4.5.5 Stir or shake 1 minute. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If pink for ≥ 10 seconds, titrate with 0.50 mL TF, and repeat. 

When colorless within 10 seconds, record total volume of TF as the endpoint of 

titration and use this volume for calculation of percent phenol.

5. Interpretation of the Test Results

5.1 Hydrogen ion concentration

No calculation is required.

Satisfactory hydrogen ion concentration*: 7.0 ± 0.3

*This value is to be used unless otherwise noted in the approved Outline of Production 

for the product or 9 CFR 113.406.

5.2 Clarity

No calculation is required.

Satisfactory clarity: Negative (no insoluble particles observed)

5.3 Total nitrogen (Report average of duplicates)

% tttttt nnnn nn nnnn = �mmmmssssssssssss − mmmmbb bbbb�(NN HHHHHH)(1.4007)

1.0 mmmm

mLsample = Volume of standardized HCl required for sample

mLblank = Volume of standardized HCl required for blank

N HCL = Normality of HCl 

1.4007 = Milliequivalent weight of nitrogen x 100

1.0 mL = Volume of sample 

Satisfactory total nitrogen content*: 0.18% ± 0.06%

*This value is to be used unless otherwise noted in the approved Outline of Production 

for the product or 9 CFR 113.406.
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Testing Protocol Page 15 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

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5.4 TCA-ppt nitrogen (Report average of duplicates)

% TT TT − pppp nnnn nn nnnn = �mmmmssssssssssss − mmmmbb bbbb�(NN HHHHHH)(1.4007)

5.0 mmmm

mLsample = Volume of standardized HCl required for sample

mLblank = Volume of standardized HCl required for blank

N HCL = Normality of HCl 

1.4007 = Milliequivalent weight of nitrogen x 100

5.0 mL = Volume of sample 

Satisfactory TCA-ppt nitrogen content*: 0.047% ± 0.01%

*This value is to be used unless otherwise noted in the approved Outline of Production 

for the product or 9 CFR 113.406.

5.5 Phenol (Report average of triplicates)

A correction factor of 0.04 should be subtracted from the final value in the determination 

of phenol in tuberculin. (9 CFR 113.406,(d), (4))

Percent phenol = [(vol of test fluid) x (0.04)] – (0.04)

Satisfactory Phenol Content*: 0.54% ± 0.04%

*This value is to be used unless otherwise noted in the approved Outline of Production 

for the product or 9 CFR 113.409.

5.6 Controls

Results for controls and standards must be within acceptable limits; otherwise repeat 

testing. 

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.406, U.S. Government Printing 

Office, Washington, DC.
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Testing Protocol Page 16 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

UNCONTROLLED COPY

7.2 AOAC Official Method 960.52, Official Methods of Analysis of AOAC 

International, Arlington, Virginia, 16th Edition, Pat Cuniff, Editor (1995), Volume I, 

Chapter 12, page 7.

7.3 ASTM Standard E969, Standard Specification for Glass Volumetric (Transfer) 

Pipets.

7.4 ASTM Standard E288, Standard Specification for Laboratory Glass Volumetric 

Flasks.

7.5 ASTM Standard E694, Standard Specification for Laboratory Glass Volumetric 

Apparatus.

8. Summary of Revisions 

Version .09

• The coversheet has been updated

Version .08

• The Contact information was updated.

• The phenol control information was corrected.

• An additional option to purchase phenol standard was added.

• “Reduced oxygen” under Water was changed to “reverse osmosis.”

Version .07

• The document has been revised to reflect changes in instrumentation, personnel, and 

to provide additional detail. 

• 5.3/5.4: Total and TCA-ppt nitrogen, the calculation was changed to reflect that used 

in AOAC 960.52 taking into account the blank sample. The satisfactory protein 

content level did not change.

• A blank sample requirement has been added to the procedure to reflect AOAC 

Official Method 960.52.
Center for Veterinary Biologics SAM 514.09

Testing Protocol Page 17 of 17

Supplemental Assay Method for the Determination of Hydrogen Ion Concentration, Total Nitrogen, TCA 

Nitrogen, Phenol and Clarity in Intradermic (Filtrate Produced From Cultures of Pn, C, and Dt Strains of 

Mycobacterium tuberculosis) Tuberculin

UNCONTROLLED COPY

• The sampling requirements for total nitrogen and TCA-ppt nitrogen changed from 

“analyze all samples in triplicate” to “analyze duplicate 15 mL samples, consisting of 

5 mL from each of three vials” to reflect the requirements in 9 CFR, Part 113.406, 

(d), (2), (3). 

Version .06

• The document number has been changed from TCSAM0514 to SAM 514.

Version .05

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the name of the contact person has changed.