Bacteriology • Laboratory Manual • Digital Learning Chapter
Procedure for Preparation of Culture Media
A structured laboratory guide covering the preparation of common culture media and biochemical media used in routine microbiological laboratory work.
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Culture media are nutrient preparations used
for the cultivation, isolation, identification and maintenance
of microorganisms.
The preparation of culture media generally involves accurately weighing the dehydrated medium, dissolving it in distilled or purified water, adjusting the final volume and sterilizing the medium using an appropriate method.
Laboratory note:
Always follow the manufacturer's instructions for the particular
commercial dehydrated medium because composition, concentration,
sterilization requirements and cooling temperatures may differ
among manufacturers.
General-purpose medium
Preparation
- Suspend 37 g of dehydrated Nutrient Agar in 1 L distilled water.
- Heat or boil with mixing until the medium is completely dissolved.
- Sterilize by autoclaving at 121 °C, 15 psi for 15 minutes, according to the original M4S procedure.
- Cool appropriately before dispensing or pouring plates.
Use: General cultivation and maintenance of
many non-fastidious bacteria.
Liquid medium
Preparation
- Dissolve 13 g of dehydrated Nutrient Broth in 1 L distilled water.
- Mix thoroughly until completely dissolved.
- Autoclave at 121 °C for 15 minutes, following the original M4S procedure.
Use: Cultivation of microorganisms in liquid
culture and preparation of bacterial inocula.
Selective + Differential
Preparation
- Suspend 55 g of medium in 1 L water.
- Heat or boil until the medium is completely dissolved.
- Autoclave at 121 °C, 15 psi for 15 minutes according to the original procedure.
- Cool and dispense aseptically as appropriate.
Use: Isolation and differentiation of
Gram-negative enteric bacteria based on lactose fermentation.
Enriched + Differential
Preparation
- Suspend 42.5 g of blood agar base in 1 L water.
- Sterilize at 121 °C for 15 minutes.
- Cool the sterilized base to approximately 40–50 °C.
- Add 50 mL sterile sheep blood and mix gently.
- Pour aseptically into sterile Petri plates.
Use: Isolation of many clinically important
bacteria and observation of hemolytic reactions.
Enriched medium
Preparation
- Prepare sterilized blood agar base.
- The original M4S procedure describes heating the blood-containing medium at approximately 75 °C for 30 minutes.
- The red blood cells lyse and the medium develops a characteristic chocolate-brown appearance.
Use: Isolation of fastidious organisms such as
species of Haemophilus and Neisseria.
Important: In routine clinical laboratories,
preparation parameters should follow the validated SOP and
manufacturer's instructions for the specific medium.
AST medium
Preparation
- Suspend 38 g of medium in 1 L water.
- Warm with mixing until the medium dissolves.
- The original M4S page describes dispensing approximately 10 mL into tubes and heating in a water bath for 10 minutes.
Use: Mueller-Hinton Agar is widely used for
antimicrobial susceptibility testing by standardized methods.
Biochemical medium
Preparation
- Suspend 9.4 g in 1 L cold water.
- Boil to dissolve.
- Autoclave at 121 °C, 15 psi for 15 minutes.
- Aseptically add 10 mL sterile dextrose per 100 mL medium, according to the original M4S procedure.
- Dispense into sterile tubes.
Principle: The O/F test helps determine whether
an organism metabolizes carbohydrates oxidatively, fermentatively,
or neither.
Biochemical medium
Preparation
- Suspend 36 g of medium in 1 L water.
- Dissolve completely.
- Dispense into tubes to approximately 3 inches depth.
- Sterilize by autoclaving according to the validated laboratory procedure.
Use: Detection of hydrogen sulfide production,
indole production and bacterial motility.
Biochemical medium
Preparation
- Dissolve 17 g of medium in 1 L water.
- Dispense approximately 3 mL per tube.
- Autoclave at 121 °C for 15 minutes.
Use: Used for the Methyl Red and Voges-Proskauer
reactions in the identification of enteric bacteria.
Biochemical medium
Preparation
- Dissolve 24.2 g in 1 L water.
- Dispense approximately 3 mL into tubes.
- Autoclave at 121 °C for 15 minutes.
- Tilt the tubes while cooling to form agar slants.
Use: Determines whether an organism can utilize
citrate as a carbon source under the test conditions.
Biochemical medium
Preparation
- Suspend 65 g in 1 L water.
- Sterilize at 121 °C, 15 psi for 15 minutes.
- Dispense into tubes to produce an agar slant with a butt of approximately 1 inch.
Use: TSI Agar is used to assess glucose,
lactose and/or sucrose fermentation, gas production and
hydrogen sulfide production.
Biochemical medium
Preparation
- Dissolve 24 g in 950 mL water.
- Autoclave at 121 °C for 15 minutes.
- Cool the medium to approximately 45 °C.
- Add 50 mL of 40% urea, following the original M4S procedure.
- Dispense into tubes as slants.
Use: Detection of urease activity based on
alkaline changes produced by urea hydrolysis.
| Medium | Original quantity | Volume | Main application |
|---|---|---|---|
| Nutrient Agar | 37 g | 1 L | General bacterial cultivation |
| Nutrient Broth | 13 g | 1 L | Liquid bacterial cultivation |
| MacConkey Agar | 55 g | 1 L | Gram-negative enteric bacteria |
| Blood Agar | 42.5 g + 50 mL blood | 1 L base | Enrichment and hemolysis |
| Chocolate Agar | Prepared from blood-containing medium | — | Fastidious bacteria |
| Mueller-Hinton Agar | 38 g | 1 L | Antimicrobial susceptibility testing |
| O/F Medium | 9.4 g | 1 L | Oxidative/fermentative metabolism |
| SIM | 36 g | 1 L | H₂S, indole and motility |
| MR-VP | 17 g | 1 L | MR and VP reactions |
| Simmons Citrate | 24.2 g | 1 L | Citrate utilization |
| TSI Agar | 65 g | 1 L | Sugar fermentation and H₂S |
| Christensen Urea Agar | 24 g + urea | 950 mL + 50 mL | Urease detection |
The original M4S page contains a visual reference section showing common agar plates.
Image note:
Replace the three image URLs above with the original Blogger
image URLs if this HTML is being used outside the original post.
- M4S — Microbiology for Students: Procedure for Preparation of Culture Media, original publication dated December 1, 2016.
- Manufacturer's instructions for the specific dehydrated culture medium should be consulted before laboratory use.
- Laboratory SOPs and applicable standards should be followed for preparation, sterilization, quality control and storage.
Quality-control reminder:
Medium preparation should be performed using validated laboratory
procedures. Sterility, appearance, pH, volume, storage conditions
and appropriate QC organisms should be checked according to the
laboratory's quality-management system.
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