Middlebrook Agar- Composition, Principle, Preparation, Results, Uses

Middlebrook Agar is a type of culture medium that is specially designed for the isolation and cultivation of mycobacteria, which are a group of bacteria that cause diseases such as tuberculosis and leprosy. Mycobacteria are difficult to grow in conventional media because they have a complex cell wall that makes them resistant to many antibiotics and disinfectants. They also require specific nutrients and environmental conditions for optimal growth.

Middlebrook Agar was developed by Dubos and Middlebrook in the 1940s and 1950s, based on their earlier formulations that contained oleic acid and albumin as key ingredients. These components help to protect the mycobacteria from toxic agents and enhance their recovery on primary isolation. Later, Middlebrook and Cohn improved the formulation by adding various inorganic salts, glycerol, malachite green dye, and agar. The resulting medium, known as Middlebrook 7H10 Agar, was found to support faster and more luxuriant growth of mycobacteria than the egg-based media commonly used at that time.

Middlebrook Agar can be supplemented with different additives to enrich the medium or make it more selective for certain types of mycobacteria. For example, Middlebrook OADC Enrichment contains sodium chloride, dextrose, bovine albumin, catalase, and oleic acid, which provide essential electrolytes, carbon source, protection against oxidative stress, and fatty acids for mycobacterial metabolism. Middlebrook ADC Enrichment contains only albumin, dextrose, and catalase. Middlebrook PANTA Enrichment contains polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin, which inhibit the growth of most bacteria other than mycobacteria.

Middlebrook Agar is widely used for the isolation, cultivation, and sensitivity testing of Mycobacterium tuberculosis, the causative agent of tuberculosis. It can also be used for other Mycobacterium species, such as Mycobacterium leprae (leprosy), Mycobacterium avium complex (MAC), Mycobacterium kansasii (lung infections), and Mycobacterium marinum (fish tank granuloma). Middlebrook Agar allows for the observation of colony morphology, pigment production, and cord formation of mycobacteria, which are useful characteristics for identification.

Middlebrook Agar is one of the most important cultural media for mycobacteriology. It has several advantages over other media, such as:

• It supports the growth of both slow- and fast-growing mycobacteria.

• It reduces the contamination by other bacteria due to the presence of malachite green dye.

• It allows for the detection of drug-resistant strains of mycobacteria by incorporating antibiotics into the medium.

• It facilitates the differentiation of mycobacteria based on their phenotypic features.

However, Middlebrook Agar also has some limitations, such as:

• It requires incubation in a 5-10% CO2 atmosphere for optimal growth of mycobacteria.

• It may not recover all types of mycobacteria if the specimens are not properly decontaminated before inoculation.

• It may not provide conclusive identification of mycobacteria without further biochemical, immunological, molecular, or mass spectrometry testing.

In this article, we will discuss the composition, principle, preparation, results, uses, and limitations of Middlebrook Agar in detail. We will also provide some examples of how Middlebrook Agar can be used in clinical microbiology laboratories.

Middlebrook Agar is a solid growth medium that consists of two main components: Middlebrook 7H10 Agar Base and Middlebrook OADC Enrichment. The agar base contains a variety of inorganic salts that provide substances essential for the growth of mycobacteria, such as ammonium sulfate, monopotassium phosphate, disodium phosphate, sodium citrate, magnesium sulfate, calcium chloride, zinc sulfate, copper sulfate, L-glutamic acid, ferric ammonium citrate, pyridoxine hydrochloride, biotin, and malachite green. The malachite green dye acts as a partial inhibitor of bacteria other than mycobacteria. The agar base also contains 15 g/L of agar to solidify the medium. The OADC enrichment contains sodium chloride, dextrose, bovine albumin (fraction V), catalase, and oleic acid. The sodium chloride maintains osmotic equilibrium; the dextrose provides an energy source, the bovine albumin protects the mycobacteria from toxic agents, the catalase destroys peroxides that may be present in the medium, and the oleic acid is a long-chain fatty acid that can be utilized by tubercle bacilli. The OADC enrichment is added to the agar base after autoclaving and cooling to enhance the recovery of mycobacteria on primary isolation.

Middlebrook Agar is a selective and differential medium that supports the growth of mycobacteria and distinguishes them from other bacteria based on their colony morphology and pigment production. The principle of Middlebrook Agar is based on the following features:

• The medium contains a variety of inorganic salts that provide substances essential for the growth of mycobacteria. The component sodium citrate, when converted to citric acid, serves to hold certain inorganic cations in the solution. Glycerol is an abundant source of carbon and energy.

• The medium is supplemented with Middlebrook OADC Enrichment, which contains oleic acid, albumin, dextrose, and catalase. Oleic acid, as well as other long-chain fatty acids, can be utilized by tubercle bacilli and plays an important role in the metabolism of mycobacteria. Albumin aids in the protection of the tubercle bacilli against toxic agents and enhances their recovery in primary isolation. Dextrose is a fermentable carbohydrate and an energy source. Catalase destroys toxic peroxides that may be present in the medium.

• The medium also contains malachite green dye, which acts as a selective agent by inhibiting the growth of most bacteria other than mycobacteria. However, some non-mycobacterial organisms may grow on this medium if specimens are not properly decontaminated before inoculation.

• The medium allows for the differentiation of mycobacteria based on their colony morphology and pigment production. Mycobacteria can be classified into three groups according to their pigment production: nonchromogenic (NC), which produces white, cream, or buff colonies; chromogenic (Ch), which produces lemon, yellow, orange, or red colonies; and scotochromogenic (Sc), which produce pigmented colonies only in the dark. The colony morphology can also vary depending on the species of mycobacteria. For example, Mycobacterium tuberculosis produces rough, dry, and serpentine cord-like colonies; Mycobacterium kansasii produces smooth and yellow colonies; and Mycobacterium avium complex produces smooth and white colonies.

By using Middlebrook Agar, one can isolate and cultivate mycobacteria from clinical specimens and observe their characteristic features that help in their identification. However, it is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

To prepare Middlebrook 7H10 Agar, you will need the following materials and equipment:

• Middlebrook 7H10 Agar powder

• Glycerol

• Purified water

• Autoclave

• Middlebrook OADC Enrichment

• Sterile tubes or bottles

• Water bath

Follow these steps to prepare the medium:

1. Suspend 19 g of the powder in 900 mL of purified water containing 5 mL of glycerol. Mix thoroughly.

1. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.

1. Autoclave at 121°C for 10 minutes.

1. Aseptically add 100 mL of Middlebrook OADC Enrichment to the medium when cooled to 50-55°C.

1. Pour into sterile tubes or bottles.

1. Store at 2-8°C in the dark until use.

To prepare Middlebrook OADC Enrichment, you will need the following materials and equipment:

• Oleic acid

• Albumin (bovine)

• Dextrose

• Catalase

• Sodium chloride

• Purified water

• Sterile filter

Follow these steps to prepare the enrichment:

1. Dissolve 0.6 g of oleic acid in 100 mL of purified water.

1. Add 5 g of albumin (bovine), 2 g of dextrose, and 0.04 g of catalase. Mix well.

1. Add 0.85 g of sodium chloride and adjust the pH to 6.8 +/- 0.2 with sodium hydroxide or hydrochloric acid.

1. Filter sterilize through a 0.22-micron membrane filter.

1. Store at 2-8°C in the dark until use.

To inoculate Middlebrook Agar, you will need the following materials and equipment:

• Specimen (sputum, urine, blood, etc.)

• Digestion and decontamination reagents (such as N-acetyl-L-cysteine and sodium hydroxide)

• Sterile tubes or bottles

• Inoculating loop or needle

• Incubator with 5-10% CO2 atmosphere

Follow these steps to inoculate the medium:

1. Digest and decontaminate the specimen according to standard procedures.

1. Transfer a small amount of the processed specimen to a sterile tube or bottle containing Middlebrook Agar using an inoculating loop or needle.

1. Spread the inoculum evenly over the surface of the agar using a sterile loop or needle.

1. Incubate at 35-37°C in a 5-10% CO2 atmosphere for up to 8 weeks.

Cultures should be read within 5-7 days after inoculation and once a week thereafter for up to 8 weeks. For reading plates or bottles, invert the containers on the stage of a dissecting microscope. Read at 10-60× with transmitted light. Scan rapidly at 10-20× for the presence of colonies. Higher magnification (30-60×) is helpful in observing colony morphology, i.e., serpentine cord-like colonies.

Record the following observations:

• The number of days required for colonies to become macroscopically visible.

• The number of colonies (plates and bottles):

• No colonies = Negative

• Less than 50 colonies = Actual count

• 50-100 colonies = 1+

• 100-200 colonies = 2+

• Almost confluent (200-500) = 3+

• Confluent (more than 500) = 4+

• Pigment production

• White, cream or buff = Nonchromogenic (NC)

• Lemon, yellow, orange, red = Chromogenic (Ch)

The identification of mycobacteria is based on colony morphology, growth rate, pigment production, and biochemical tests. The most common mycobacteria isolated from clinical specimens are Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium kansasii, and Mycobacterium fortuitum complex.

The drug susceptibility testing of mycobacteria is performed by comparing the growth in the drug-containing medium with the growth in the control medium. The LB uses an indirect proportion method for testing M. tuberculosis complex mycobacteria to 12 drugs at 35°C on Middlebrook 7H10 agar. The test requires 1 month to complete. The minimum inhibitory concentration (MIC) of a drug is defined as the lowest concentration that inhibits more than 99% of the bacterial population. The MIC values are used to determine the susceptibility or resistance of the mycobacteria to the drugs.

Middlebrook 7H10 Agar is a solid medium that is widely used for the isolation and cultivation of mycobacteria, especially Mycobacterium tuberculosis, the causative agent of tuberculosis. When supplemented with Middlebrook OADC Enrichment, which contains oleic acid, albumin, dextrose, and catalase, the medium provides optimal conditions for the growth of mycobacteria. The medium also contains malachite green, which inhibits the growth of most other bacteria and fungi.

Middlebrook 7H10 Agar is useful for the following purposes:

• Isolation of mycobacteria from clinical specimens: The medium can be used to inoculate specimens such as sputum, urine, blood, cerebrospinal fluid, etc., that are suspected to contain mycobacteria. The specimens should be properly decontaminated and digested before inoculation to reduce contamination by other microorganisms. The inoculated plates or bottles should be incubated at 35-37°C in a 5-10% CO2 atmosphere for up to 8 weeks. The colonies of mycobacteria can be identified by their morphology, pigmentation, and cord formation under a microscope.

• Cultivation of mycobacteria from pure cultures: The medium can be used to subculture and maintain pure cultures of mycobacteria that have been isolated from other media or sources. The subcultures should be incubated at 35-37°C in a 5-10% CO2 atmosphere for up to 4 weeks. The purity and viability of the cultures can be checked by observing the colony characteristics and performing staining and microscopy.

• Sensitivity testing of mycobacteria: The medium can be used to perform drug susceptibility testing of mycobacteria by using different concentrations of antibiotics or other agents. The inoculated plates or bottles should be incubated at 35-37°C in a 5-10% CO2 atmosphere for up to 4 weeks. The growth or inhibition of the colonies can be compared with control plates or bottles to determine the minimum inhibitory concentration (MIC) or the resistance level of the mycobacteria.

Middlebrook 7H10 Agar is a valuable tool for the diagnosis and treatment of mycobacterial infections, especially tuberculosis. It allows the isolation and cultivation of mycobacteria from various specimens and sources, as well as the determination of their drug susceptibility patterns. However, it also has some limitations that should be considered when using it. These will be discussed in the next point.

Middlebrook Agar is a useful medium for the isolation and cultivation of mycobacteria, but it also has some limitations that should be considered. Some of the limitations are:

• It is not a fully selective medium, so it may allow the growth of other bacteria besides mycobacteria if the specimens are not properly decontaminated before inoculation.

• It requires supplementation with Middlebrook OADC Enrichment, which contains oleic acid, albumin, dextrose, and catalase. These components may interfere with some biochemical or serological tests for mycobacteria identification or susceptibility testing.

• It needs incubation in a 5-10% CO2 atmosphere, which may not be readily available in some laboratories. Moreover, exposure to light or heat may cause the release of formaldehyde in the medium, which may inhibit or kill the mycobacteria.

• It may not detect all cases of mycobacterial infection, as some factors may affect the recovery of the organisms from the specimens. These factors include:

• The quality and quantity of the specimen. For example, saliva instead of sputum may not contain enough mycobacteria to grow on the medium.

• The processing and handling of the specimen. For example, excessive digestion or decontamination may destroy the mycobacteria or reduce their viability.

• The contamination or competition from other microorganisms. For example, bacteria that grow faster or produce substances that inhibit the mycobacteria may prevent their growth in the medium.

Therefore, Middlebrook Agar should be used in conjunction with other methods for the diagnosis and identification of mycobacterial infections. These methods include microscopy, molecular techniques, mass spectrometry, and culture on other media such as Lowenstein-Jensen Agar or liquid media.

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