Nutrient Broth No. 3

Intended Use

A non-selective medium serves as base for the culture and growth of microorganisms.

Composition**

Final pH ( at 25°C): 7.4±0.2

**Formula adjusted, standardized to suit performance parameters

# - Equivalent to Meat extract

Directions

Suspend 13 grams in 1000 ml purified / distilled water. Heat if necessary to dissolve the medium completely. Dispense into tubes or flasks as desired and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Principle and Interpretation

Nutrient Broth is a general purpose medium used for the cultivation of microorganisms that are not exacting in their nutritive requirements. An infusion of meat and peptone constitute the nutrient composition of many media. Nutrient Broth No. 3 is a basic culture medium used for maintaining microorganisms (1) and for purity checking prior to biochemical or serological testing. It is used for the cultivation and enumeration of bacteria, which are not particularly fastidious. In semisolid form it is used for maintenance or control of standard organisms. Addition of different biological fluids such as horse or sheep blood, serum, egg yolk, etc. makes it suitable for the cultivation of fastidious organisms (2).

Peptone, HM extract and yeast extract provide nitrogen, carbon compounds, long chain amino acids, vitamin B complex and other necessary nutrients for the growth of non-fastidious organisms. Sodium chloride maintains the osmotic equilibrium of the medium.

Type of specimen

Clinical samples - urine, faeces, etc. Food and dairy samples; Water samples

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (3,4).

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (5-7).

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (8).

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic Use. For professional use only. Read the label before opening the container. Wear protective gloves/ protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets

Limitations

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user’s unique requirement.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Cream to yellow homogeneous free flowing powder

Colour and Clarity of prepared medium: Light amber coloured clear solution in tubes

Reaction: Reaction of 1.3% w/v aqueous solution at 25°C. pH : 7.4±0.2

pH: 7.20-7.60

Cultural Response: Cultural characteristics observed after an incubation at 35-37°C for 18-48 hours.

Key : (*) Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 15-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (3,4).

Reference

1. Lapage S., Shelton J. and Mitchell T., 1970, Methods in Microbiology', Norris J. and Ribbons D., (Eds.), Vol. 3A, Academic Press, London.
2. MacFaddin J. F., 2000, Biochemical Tests for Identification of Medical Bacteria, 3rd Ed., Lippincott, Williams and Wilkins, Baltimore.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
4. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
5. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
6. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
7. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
8. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.