Microorganisms such as bacteria and fungi require specific nutrients and environmental conditions to grow, which are provided through culture media. Culture media can be liquid (broth) or solid (agar-based). Solid media help in isolating and observing colony morphology, while liquid media are used for growing large volumes of microbial cells or for specific biochemical tests.
Preparing sterile, nutrient-rich media is a fundamental step in microbiology practical work to ensure accurate and uncontaminated microbial growth.
Requirements:
| Apparatus/glassware | Reagents / Chemicals: |
| Conical flask / Media bottles Measuring cylinder pH meter or strips Weighing balance Autoclave / Pressure cooker Petri dishes Test tubes Cotton plugs or screw caps Glass rod or magnetic stirrer Bunsen burner or spirit lamp | Distilled water Peptone Beef extract / Yeast extract Sodium chloride (NaCl) Agar-agar (for solid media) Specific media powders (e.g., Nutrient Agar, Nutrient Broth) pH adjusting agents (NaOH or HCl) |
Principle:
The preparation of culture media is based on providing essential nutrients (carbon, nitrogen, minerals, and growth factors) to support microbial growth. Liquid media such as nutrient broth allow uniform growth throughout the medium, while solid media, which contain agar, help in isolation and differentiation of colonies.
Agar is an inert, non-nutritive solidifying agent derived from seaweed. It melts at about 85°C and solidifies around 42°C, making it suitable for microbial cultivation.
Sterilization via autoclaving is crucial to eliminate any contaminating microorganisms before inoculation.
Procedure of nutrient media preparation:
A. Preparation of Nutrient Broth (Liquid Medium):
Composition of nutrient broth (per 100 ml):
- Peptone – 0.5 g
- Beef extract – 0.3 g
- NaCl – 0.5 g
- Distilled water – 100 ml
Steps for nutrient broth preparation:
- Weigh the ingredients using a balance.
- Dissolve all components in 1000 ml of distilled water in a conical flask.
- Adjust the pH to 7.0 using NaOH or HCl if necessary.
- Dispense into test tubes or flasks.
- Plug with cotton or seal with caps.
- Sterilize by autoclaving at 121°C (15 psi) for 15–20 minutes.
- Cool and store at room temperature or in a refrigerator.
B. Preparation of Nutrient Agar (Solid Medium):
Composition of NAM (nutrient agar media) for 100 ml:
- Peptone – 0.5 g
- Beef extract – 0.3 g
- NaCl – 0.5 g
- Agar – 1.5 g
- Distilled water – 100 ml
Steps for nutrient agar media preparation:
- Dissolve peptone, beef extract, and NaCl in distilled water.
- Heat gently and Add agar to dissolve completely.
- Adjust pH to 7.0.
- Pour into flasks or bottles.
- Sterilize in an autoclave at 121°C for 15–20 minutes.
- Allow it to cool to ~45-50°C before pouring into sterile Petri dishes (for plates) or tubes (for slants) in the laminar air flow or sterile chamber.
- Let it solidify under aseptic conditions.
Observation and Result:
for the Liquid Media (Broth): solution will appear clear light yellow solution before inoculation.
if media appear : Turbidity (cloudiness) indicates microbial growth. this may occur sometime when we skip the media preparation in the class same we mix ingredients.
for the Solid Media (Agar): Appearance: Solid, firm surface in Petri plates or tubes.
Precautions:
- Ensure all glassware and instruments are sterile before use.
- Accurately weigh all ingredients.
- Do not overheat agar to avoid breakdown of nutrients.
- Maintain aseptic conditions while pouring and inoculating media.
- Label all media containers with date, type of media, and pH.
- Cool media to ~45°C before pouring into Petri plates to avoid condensation and burns.
- Use freshly prepared media or store under appropriate conditions to avoid degradation.