PREPARATION OF DIFFERENTIAL AND SELECTIVE MEDIUM

 

Preparation of Differential Medium and Selective Medium

AIM: To prepare differential medium (e.g., MacConkey Agar) and selective medium (e.g., Mannitol Salt Agar) for the isolation and identification of bacteria based on their growth characteristics and biochemical properties.

PRINCIPLE:

Differential Medium: A differential medium contains specific ingredients (dyes, sugars) that allow different bacterial species to be distinguished from one another based on visible changes in the medium — such as colour change, colony appearance, precipitate formation — even though multiple organisms may grow on it.

Selective Medium: A selective medium contains specific agents (antibiotics, dyes, high salt concentration, or bile salts) that inhibit the growth of unwanted/competing organisms while allowing the desired organism to grow. For example, Mannitol Salt Agar contains 7.5% NaCl, which inhibits most bacteria except salt-tolerant organisms like Staphylococcus aureus.

MATERIALS REQUIRED

Category	Items
Media/Chemicals	Nutrient agar base, MacConkey agar powder,  Mannitol salt agar powder, Distilled water
Glassware	Conical flask, Petri dishes, Measuring cylinder, Glass rod
Equipment	Weighing balance, Autoclave, Hot air oven / Laminar air flow, Water bath, pH meter/pH paper
Others	Cotton plugs/non-absorbent cotton, Aluminium foil/kraft paper, Inoculating loop, Bunsen burner/spirit lamp, Test cultures (e.g., E. coli, Staphylococcus aureus, Salmonella sp.), Labels/marker

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PROCEDURE

1. Weigh the required quantity of dehydrated medium as per manufacturer's instructions.
2. Dissolve completely in distilled water by heating; adjust pH if necessary.
3. Sterilize by autoclaving at 121°C, 15 lbs pressure for 15 minutes.
4. Cool to 45–50°C and Pour into sterile Petri dishes under aseptic conditions (laminar air flow) and allow to solidify.
5. Inoculate test organisms by streak plate method and incubate at 37°C for 24 hours.

 

 

 RESULT

Medium	Organism	Observation
MacConkey Agar (Selective + Differential)	E. coli	Pink/red colonies (lactose fermenter)
MacConkey Agar (Selective + Differential)	Salmonella sp.	Pale/colourless colonies (non-lactose fermenter)
Mannitol Salt Agar (Selective)	Staphylococcus aureus	Yellow colonies with yellow zone (mannitol fermented)
Mannitol Salt Agar (Selective)	Staphylococcus epidermidis	Pink/red colonies, no colour change (mannitol not fermented)

INTERPRETATION

• The differential medium helped distinguish between different bacterial species growing together based on visible biochemical reactions (fermentation-based colour change), aiding in preliminary identification.
• The selective medium successfully inhibited the growth of unwanted/competing flora and allowed only the specific target organism to grow, aiding in the isolation of that organism from a mixed sample.
• These media are extensively used in clinical microbiology laboratories for the isolation and presumptive identification of pathogens directly from clinical specimens (e.g., stool, throat swab, urine) without prior pure culture isolation.

 

 

WINE PREPARATION

 

Preparation of Wine by Fermentation

Aim: To prepare wine from grape juice by alcoholic fermentation using Saccharomyces cerevisiae.

Principle: Wine is prepared by alcoholic fermentation of sugars present in fruit juice by yeast (Saccharomyces cerevisiae) under anaerobic conditions. The yeast converts fermentable sugars such as glucose and fructose into ethanol and carbon dioxide.

The lab-scale production of wine is a study of anaerobic metabolism.

·         Inoculum: While wild fermentation is possible via "bloom" (natural yeast on skins), lab practicals use a standardized pure culture of Saccharomyces cerevisiae (e.g., strain EC-1118) to ensure predictable kinetics.

·         Selective Inhibition: We use Potassium Metabisulfite (K₂S₂O₅). It releases Sulfur Dioxide ($SO_2$), which inhibits wild yeasts and acetic acid bacteria while the inoculated S. cerevisiae (which is sulfite-tolerant) takes over.

·         Substrate Chemistry: The "Must" must be balanced. If the sugar concentration is too high (hypertonic), it can cause osmotic stress on the yeast, leading to a "stuck" fermentation.

 

Chemical Reaction: C₆​H₁₂​O₆​→2C₂​H₅​OH+2CO₂​+Energy

Requirements: Materials:

• Fresh grapes – 1 kg
• Sucrose 20 g
• Distilled water – as required
• Active dry yeast (Saccharomyces cerevisiae) – 2–5 g
• Potassium metabisulphite – 0.1% (optional)

Equipment:

• Beakers, Conical flask, Measuring cylinder, Muslin cloth, Fermentation bottle with airlock, pH paper. Incubator (25–30°C)

Procedure:

1. Wash the grapes thoroughly with distilled water.
2. Crush the grapes to extract juice 500 ml. (must).
3. Filter the juice using muslin cloth.
4. Add a small quantity of potassium metabisulphite and allow to stand for 30 minutes to reduce contamination.
5. Activate yeast in water for 5–10 minutes.
6. Inoculate the 50 ml of yeast culture (10%) and 20 ml of sucrose into the grape juice and mix well.
7. Transfer the mixture into a fermentation bottle fitted with an airlock.
8. Incubate at 25–30°C for 7–14 days under anaerobic conditions.
9. After completion of fermentation (no bubble formation), filter and transfer the clear wine into a clean bottle for aging.

Observations:

• Formation of CO₂ bubbles during fermentation
• Alcoholic odor detected
• Sediment formation at the bottom
• Decrease in pH (acidic nature)

Result:

Wine was successfully prepared from grape juice by alcoholic fermentation using Saccharomyces cerevisiae.

 

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