Seven H11 Agars

Intended Use

These media are used in qualitative procedures for isolation and cultivation of mycobacteria, especially Mycobacterium tuberculosis, from clinical and nonclinical specimens.

Summary and Explanation

Seven H11 Agar (also referred to as Middlebrook 7H11 Agar) was developed by Cohn et al. by the addition of casein hydrolysate to 7H10 Agar.¹ Seven H11 Agar provides enhancedgrowth of fastidious, drug-resistant strains of M. tuberculosis that grow poorly (or not at all) on 7H10 Agar or other widelyused media.^1,2

The Selective Seven H11 Agar is 7H11 Agar modified by the addition of four antimicrobial agents: polymyxin B, carbenicillin, amphotericin B and trimethoprim lactate. Mitchison et al. initially developed the medium to reduce the need for decontamination procedures.³ They found that the alkaline agents used to reduce the growth of contaminating organisms inhibited some species of mycobacteria. McClatchy recommended reducing the concentration of carbenicillin used by Mitchison et al. to make the medium less inhibitory to mycobacteria.⁴

The addition of pyruvate to Seven H11 Agar has been recommended for specimens suspected of containing Mycobacterium bovis. The addition of aspartic acid has been recommended to enhance the production of niacin.⁵
Deep-filled plates are available to reduce the effects of drying during prolonged incubation.

Principles of the Procedure

Middlebrook 7H10 Agar is a defined medium consisting of oleic acid-albumin enrichment, glycerol, dextrose and inorganic compounds to supply the nutrients necessary to support the growth of mycobacterial species. Catalase destroys toxic peroxides that may be present in the medium. Malachite green acts as an inhibitory agent to provide partial inhibition of contaminating bacteria.

Seven H11 Agar consists of 7H10 Agar supplemented with pancreatic digest of casein to enhance the growth of fastidious strains of M. tuberculosis.

The addition of antimicrobial agents to Seven H11 Agar improves the recovery of mycobacteria from specimens containing mixed flora.2 Polymyxin B is a polypeptide antibiotic that selectively inhibits most species of gramnegative bacilli, including Pseudomonas, but not Proteus species.6 Carbenicillin is a semi-synthetic penicillin effective against gram-positive and gram-negative bacteria, including strains of Escherichia coli resistant to other antimicrobial agents.⁶ Amphotericin B is an antifungal antibiotic, and trimethoprim lactate is a synthetic antimicrobial agent that inhibits both grampositive and gram-negative bacteria, including Proteus species.

With Seven H11 Agar containing aspartic acid and pyruvate, aspartic acid serves as a precursor for niacin synthesis by M. tuberculosis and M. bovis. All mycobacteria produce nicotinic acid (niacin). Because of a blocked metabolic pathway for the conversion of free niacin to nicotinic mononucleotide, M. tuberculosis accumulates niacin and excretes it into the culture medium, a function that differentiates it from most other mycobacterial species. Pyruvate enhances the recovery of M. bovis.

User Quality Control

Identity Specifications
Seven H11 Agar Base
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       19 g/900 mL solution, soluble in purified
                                   water with 0.5% glycerol. Solution is
                                   colorless to pale tan to light tan-green,
                                   slightly hazy to hazy.
Prepared Appearance:    Colorless to pale tan to light tan-green,
                                   slightly hazy to hazy.
Reaction of 19 g/900 mL
with 0.5% Glycerol
Solution at 25°C:           pH 6.6 ± 0.2
Cultural Response
Seven H11 Agar Base
Prepare the medium per label directions with added Middlebrook OADC enrichment. Inoculate and incubate at 35 ± 2°C with 3-5% and up to 10% CO₂ for up to 21 days.

Formula

Seven H11 Agar Base
Approximate Formula* Per 900 mL
Pancreatic Digest of Casein............................................ 1.0 g
Monosodium Glutamate................................................. 0.5 g
Sodium Citrate.............................................................. 0.4 g
Pyridoxine................................................................. 1.0 mg
Biotin........................................................................ 0.5 mg
Ferric Ammonium Citrate.............................................. 0.04 g
Ammonium Sulfate........................................................ 0.5 g
Disodium Phosphate...................................................... 1.5 g
Monopotassium Phosphate............................................. 1.5 g
Magnesium Sulfate...................................................... 0.05 g
Agar........................................................................... 13.5 g
Malachite Green........................................................ 0.25 mg
Zinc Sulfate................................................................ 1.0 mg
Copper Sulfate............................................................ 1.0 mg
Calcium Chloride......................................................... 0.5 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Precaution⁷

Biosafety Level 2 practices and procedures, containment equipment and facilities are required for non-aerosol-producing manipulations of clinical specimens such as preparation of acid-fast smears. All aerosol-generating activities must be conducted in a Class I or II biological safety cabinet. Biosafety Level 3 practices, containment equipment and facilities are required for laboratory activities in the propagation and manipulation of cultures of M. tuberculosis and M. bovis. Animal studies also require special procedures.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 900 mL of purified water containing 5 mL of glycerol:
    Seven H11 Agar Base – 19 g.
2. Swirl to obtain a smooth suspension. For the Difco base, boil if necessary to completely dissolve the powder. For the BBL base do not boil.
3. Autoclave at 121°C for 15 minutes for the Difco base and 10 minutes for the BBL base.
4. Aseptically add 100 mL of Middlebrook OADC Enrichment to the medium when cooled to 50-55°C. Mix well.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

The test procedures are those recommended by the Centers for Disease Control and Prevention (CDC) for primary isolation from specimens containing mycobacteria. N-Acetyl-L-cysteinesodium hydroxide (NALC-NaOH) solution is recommended as a gentle but effective digesting and decontaminating agent. These reagents are provided in the BBL™ MycoPrep™ Mycobacterial Specimen Digestion/Decontamination Kit. For detailed decontamination and culturing instructions, consult an appropriate reference.^8-11

Following inoculation keep tubes shielded from light and place them in a suitable system providing an aerobic atmosphere enriched with carbon dioxide. Incubate at 35 ± 2°C. Slanted media should be incubated in a horizontal plane until the inoculum is absorbed. Tubes should have screw caps loose for the first 3 weeks to permit circulation of carbon dioxide for the initiation of growth. Thereafter, to prevent dehydration, tighten caps; loosen briefly once a week. Stand tubes upright if space is a problem.

NOTE: Cultures from skin lesions suspected to be M. marinum or M. ulcerans should be incubated at 25-33°C for primary isolation; cultures suspected to contain M. avium or M. xenopi exhibit optimum growth at 40-42°C.⁴ Incubate a duplicate culture at 35-37°C.

For information on the niacin test, consult the BBL™ Quality Control and Product Information Manual for Plated and Tubed Media and other appropriate references.^2,8-11 BBL™ Taxo™ TB Niacin Test Reagents (strips and control) may be used instead of the test reagents.

Expected Results

Cultures on Seven H11 Agar should be read within 5-7 days after incubation and once a week thereafter for up to 8 weeks.
Record Observations:⁸
1. Number of days required for colonies to become macroscopically visible. Rapid growers have mature colonies within 7 days. Slow growers require more than 7 days for mature colony forms.
2. Pigment production
White, cream or buff = Nonchromogenic (NC)
Lemon, yellow, orange, red = Chromogenic (Ch)

Stained smears may show acid-fast bacilli, which are reported only as “acid-fast bacilli” unless definitive tests are performed.
Test all nonchromogenic mycobacteria on Seven H11 Agar with Aspartic Acid and Sodium Pyruvate for niacin production; only the rough nonchromogenic strains need to be tested for niacin. A culture must have at least 50-100 colonies with growth 3-4 weeks old. M. tuberculosis and the more rare M. simiae are usually niacin positive. Most other mycobacteria are niacin negative.

Limitations of the Procedure

1. Negative culture results do not rule-out active infection by mycobacteria. Some factors that are responsible for unsuccessful cultures are:
    • The specimen was not representative of the infectious material; i.e., saliva instead of sputum.
    • The mycobacteria were destroyed during digestion and decontamination of the specimen.
    • Gross contamination interfered with the growth of the mycobacteria.
    • Proper aerobic conditions and increased CO₂ tension were not provided during incubation.
2. Mycobacteria are strict aerobes and growth is stimulated by increased levels of CO₂. Screw caps on tubes or bottles should be handled as directed for exchange of CO₂.

*Store at 2-8° C.

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