Over the years, many media have been developed for mycobacteria cultivation. Early ones included egg-based formulations such as Lowenstein-Jensen Medium or Petragnani Medium. Later, Dubos, Middlebrook and Middlebrook created a variety of formulations that contained oleic and albumin as key components. These ingredients protect Mycobacterium against toxic agents and allow for the growth and development of tubercle bacteriailli. Cohn and Middlebrook improved the formulations of oleic acids-albumin agar to achieve a faster and more luxurious growth of Mycobacterium strains.
Middlebrook 7H10 Agar base was created as per Middlebrook. Cohn et. al. reformulated the original oleic acids-albumin agar. They observed a rapid and luxuriant increase in Mycobacterium species. Middlebrook 7H10 Agar Base can also be used to isolate, cultivate, and test for M. tuberculosis after enrichment with OADC growth supplement and glycerol. The 7H10 medium is reported to have fewer contaminants than egg-based media used in mycobacteria cultivation.
Principle of Middlebrook Agar
Middlebrook Agar is enriched with inorganic salts, which provide essential nutrients for mycobacteria growth. When converted to citric acid, the component sodium citrate holds certain inorganic compounds in solution. Glycerol is a rich source of energy and carbon. The enriched medium contains sodium chloride, which provides essential electrolytes and helps maintain osmotic equilibrium. Tubercle bacilli can use oleic acid and other long-chain fat acids. This is important for mycobacteria metabolism. The media contains albumin which helps protect tubercle bacteria against harmful agents. This aids in their recovery after primary isolation. Dextrose is a fermentable carbohydrate that can be used as an energy source, while catalase kills any toxic peroxides in the medium. The presence of malachite green color can partially inhibit bacteria.
Composition of Middlebrook Agar
Middlebrook 7H10 Agar Per 900 mL
|Ammonium Sulfate||0.5 g|
|Monopotassium Phosphate||1.5 g|
|Disodium Phosphate||1.5 g|
|Sodium Citrate||0.4 g|
|Magnesium Sulfate||25.0 mg|
|Calcium Chloride||0.5 mg|
|Zinc Sulfate||1.0 mg|
|Copper Sulfate||1.0 mg|
|L-Glutamic Acid (sodium salt)||0.5 g|
|Ferric Ammonium Citrate||0.04 g|
|Pyridoxine Hydrochloride||1.0 mg|
|Malachite Green||250.0 µg|
pH 6.6 +/- 0.2 at 25ºC.
Middlebrook OADC Enrichment
|Sodium Chloride||8.5 g|
|Bovine Albumin (Fraction V)||50.0 g|
|Oleic Acid||0.6 mL|
Preparation of Middlebrook Agar
- In 900 mL purified water with 5 mL glycerol, suspend 19 grams of the powder.
- Mix well.
- To dissolve the powder, heat with frequent stirring and boil for one minute.
- For 10 minutes, autoclave at 121degC
- When the medium is cooled to 50-55degC, add 100mL of Middlebrook OAC Enrichment.
- Put the contents into sterilized tubes.
Results and Interpretation on Middlebrook Agar
- For up to 8 weeks, cultures should be read once per week for up to 7 days.
- Invert the bottles or plates on the stage of the microscope to read them.
- With transmitted light, read at 10-60x
- To find colonies, scan quickly at 10-20x
- Higher magnification (30-60x), is useful for observing colony morphology, i.e. serpentine cord-like colonies.
The time it takes for colonies to become visible macroscopically. The number of colonies (plates or bottles) required to become macroscopically visible.
- 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+
- White, cream or buff = Nonchromogenic (NC)
- Lemon, yellow, orange, red = Chromogenic (Ch)
Uses of Middlebrook Agar
- Middlebrook 7H10 Agar can be used to cultivate mycobacteria in qualitative methods when it is supplemented by Middlebrook OADC enrichment.
- It can be used to isolate, cultivate and test for sensitivity in Mycobacterium tuberculosis.
Limitations of Middlebrook Agar
- For complete identification, it is important to perform biochemical, immunological and molecular testing on colonies grown in pure culture.
- This medium is not selective enough to allow for the growth of bacteria other than mycobacteria if specimens aren’t properly pretreated for decontamination.
- Mycobacteria is a strict aerobe and their growth can be stimulated by higher levels of CO2. Screw caps should be used on tubes and bottles for CO2 exchange.
- Middlebrook 7H10 Aggar requires incubation at 5-10% CO2 in order to recover mycobacteria. Mycobacteria cannot be recovered from candle extinction containers for unknown reasons.
- Exposed media must be kept out of direct sunlight and excessive heat. This can cause the media to release formaldehyde, which can kill or inhibit mycobacteria.
- Mycobacteria active infection can be confirmed by negative culture results. Several factors can cause failure in cultures
- The specimen was not representative for the infectious material, i.e. saliva rather than sputum.
- The mycobacteria were killed during digestion and decontamination.
- Gross contamination was a problem in the growth of mycobacteria.
- Incubation was not conducted in a manner that provided adequate aerobic conditions and an increase in CO2 tension.