Biphasic Medium in Microbiology: Use, Composition and Advantage

Use of Biphasic Medium

Biphasic medium or diphasic medium is called so because of containing medium both solid and liquid phases in a single bottle. It uses mainly for minimizing contamination and spread of infections. It applies to cultures of highly infectious agents like Brucella and Mycobacterium.

Isolation of Brucella

It requires a selective medium: It contains bacitracin, nalidixic acid, cycloheximide, nystatin, polymyxin B, and vancomycin in a serum dextrose agar base. Bone marrow culture -criterion standard samples of any body fluid, or tissue can be cultured.
blood – most common
Incubated at 37 ⁰ C in air enriched with 5-10 % CO₂.
Typical virulent brucella: Colonies are smooth and translucent.
Merit
Gold Standard
Demerit
Hazardous bacterium
level 3 laboratories
labor-intensive
time- consuming procedure

Casteneda Method: Diphasic Medium for Brucella

It is a diphasic medium, one being agar and another broth. It allows the subculture to be done without opening the bottle by tilting the bottle and allowing the broth to flow over agar at intervals of times. Agar surface is examined for colonies.

Trypticase soy agar

Composition of Trypticase soy agar
Pancreatic Digest of Casein: 15.0 g
Papaic Digest of Soybean Meal: 5.0 g
Sodium Chloride: 5.0 g
Agar: 15.0 g

Distilled water: 1000 ml

Trypticase soy broth

The composition is the same as trypticase soy agar except for agar which is missing in this medium.

Inoculate specimen
Incubate at 37°C or 5-10% CO₂

Advantages of Biphasic Medium

Minimizes materials
Minimizes inoculation
Reduces contamination
Reduces risk of infection to lab workers

Isolation of Mycobacterium tuberculosis

The biphasic system for Mycobacterium tuberculosis is Middlebrook 7H11 agar slant+Middlebrook 9H broth in comparison with Lowenstein-Jensen (LJ) medium. The biphasic system has a high recovery rate of 97.05% as against 79.41% on LJ on incubation. The biphasic system shows a lower contamination rate. Biphasic medium is superior to LJ medium in isolation of M. tuberculosis.

Composition of Middlebrook 7H11 agar

Glycerol: 5.0 ml
Disodium Phosphate: 1.5 gm
Monopotassium Phosphate: 1.5 gm
Pancreatic Digest of Casein: 1.0 gm
L-Glutamic Acid: 0.5 gm
Ammonium Sulfate; 0.5 gm
Sodium Citrate: 0.4 gm
Magnesium Sulfate : 50.0 mg
Ferric Ammonium Citrate: 40.0 mg
Malachite Green: 1.0 mg
Pyridoxine: 1.0 mg
Biotin: 0.5 mg
Agar : 15.0 gm
Distilled or deionized water (D.W): 900 ml

OADC Enrichment

Bovine Albumin: 5.0 gm
Beef Catalase: 4.0 mg
Dextrose: 2.0 gm
Sodium Chloride: 0.85 gm
Oleic Acid: 50.0 mg

Middlebrook 7H11 Selective Agar contains the following additional selective ingredients per liter-

Carbenicillin: 50.0 mg
Polymyxin B: 25.0 mg
Trimethoprim :20.0 mg
Amphotericin B: 10.0 mg
Final pH 6.8 +/- 0.3 at 25°C

Compostion of Middlebrook 7H11 broth

The composition is the same as Middlebrook 7H11 agar except for agar which is missing in this medium.

Keynotes on Diphasic/Biphasic Medium

Common diphasic media are for Brucella- Castaneda’s biphasic medium consisting of brain heart infusion agar and broth and Castaneda’s biphasic medium consisting of Trypticase soy agar and broth, for Mycobacterium tuberculosis-Middlebrook 7H11 Selective Agar and broth, while for fastidious parasites like Leishmania and Trypanosoma-Novy-MacNeal-Nicolle medium.
NNN medium is also a diphasic medium and it stands for Novy-MacNeal-Nicolle medium which was developed by Novy, McNeal, and modified by Nicolle. It uses to grow parasites, Leishmania and Trypanosoma. Culture is sensitive than smear and thus used to grow– needed when the amastigotes are not found in sufficient quantities in smear. NNN medium consists of a blood agar base and an overlay medium (Locke’s solution). The blood agar base is a highly nutritious medium that helps the growth of fastidious parasites like Leishmania and Trypanosoma. The specimens are inoculated into the liquid phase of this NNN diphasic medium and incubated. This supports the development of organisms in the insect vector. The amastigotes transform into promastigotes and are hence also called phase converter media.

universe84a