Name: ___________________________________________________

Subject & Section: __________________________ Date & Time: ________________________ Group:________

Date Performed: ________________________

Activity 3: Culture Media Preparation

Microorganisms need nutrient and certain environmental conditions to grow and reproduce. In the
environment, these microorganisms have adapted to the habitats most suitable to their needs. In the laboratory,
medium is basically an aqueous solution to which all the necessary nutrients have been added. Depending on
the type, physical properties, and combination of nutrient, different classification of culture media can be made.

I. Objectives:

At the end of the activity, the student should be able to:

1. explain the principles employed in the preparation of commonly used culture media.

2. learn the correct procedures for preparing and sterilizing culture media.

3. reinforce and practice aseptic technique in the preparation of culture media.

II. Materials

Preparation of solid medium:

nutrient agar (follow the formulation on the bottle)

4L distilled water
2pcs Erlenmeyer flask (with cotton plug; 250mL or 500mL)
250 mL graduated cylinder
stirring rod or hot plate (with stirrer)
16 pcs Petri dishes

Preparation of phosphate buffer solution:

1.5mL stock phosphate buffer solution

5.0 mL magnesium chloride solution
1L distilled water
1L beaker
4 pcs. dilution bottles (160mL; screw-capped or cotton plugged)
glass pipette (1mL and 10mL)

Pour plate and spread plate:

L-shaped glass spreader

alcohol lamp
denatured alcohol
5 pcs glass pipette (10mL; for each dilution and sample)

III. Procedure

A. Work area

Clean the bench top with a disinfectant before starting any procedures below. Use bleach (10mL
zonrox + 900mL water; you can used 1L mineral water bottle) or alcohol (70%).

B. Preparation of culture medium:

 1. Measure medium following manufacturer’s recommendation.

2. Mix thoroughly using the glass rod or magnetic stirrer of the hot plate.

3. Capped with cotton plug and put in an autoclavable cellophane.

4. Sterilize at 121°C for 15min.

C. Preparation of dilution buffer

1. In a 1L beaker, add 1.25mL stock phosphate buffer solution and 5.0mL magnesium chloride solution.

2. Add 1L distilled water.

3. Dispense 90mL to each dilution bottles.

4. Autoclave at 121°C for 15min.

D. Sample collection: Water

1. Collect samples in clean, sterilized, wide-mouth, nonreactive borosilicate glass or plastic bottles.

2. Collect samples that are representative of the water being tested.

3. Collect 100mL water samples, give enough head space for proper mixing.

4. Remove any attachments from the tap because they may harbor bacteria that do not reflect the
source’s water quality.

5. Open water tap fully and let water run to waste for 2 to 3 mins. Reduce water flow so bottle can be
filled without splashing.

E. Sample collection: Food

1. Dissolve 8.5 g of NaCl in 1L water. Autoclave 15 min. at 121°C.

2. Grind food with or mortar and pestle and measure 30g.

3. Mix thoroughly 30g of food sample to 970mL saline solution.

4. Measure 10mL of liquid from this mixture to use for the dilution process.

F. Serial dilutions

1. Label each tube with the dilution factor, such as 10⁻¹, 10⁻², 10⁻³, and 10⁻4, according to the dilution
you are performing.

2. Add 10 mL of the original sample solution to the first tube labeled 10⁻¹ (1/10 dilution). You now have a
total of 10 mL in this tube. Mix thoroughly by vortexing or shaking to ensure even distribution of the
sample in the diluent.

3. Using a fresh pipette tip, take 1 mL from the 10⁻¹ dilution tube and add it to the next tube labeled 10⁻².
Mix thoroughly by vortexing.

4. Continue this process, transferring 1 mL from each tube to the next tube containing 90 mL of dilution
buffer, until you reach the final desired dilution (10⁻4).

G. Pour plate method:

1. Prepare the nutrient agar and maintain melted medium in a water batch between 44 and 46°C.

2. Prepare two Petri dishes for each dilution and label accordingly.

3. In each plate, add 1mL of sample. Do this aseptically.

 4. After adding sample to Petri dish, gently lift cover just high enough to pour at least 10mL liquefied
medium (maintained at 44 to 46°C) in each dish.

5. As each plate is poured, mix melted medium thoroughly with test portions in Petri dish—taking care
not to splash mixture over the edge—by rotating the dish clockwise and counterclockwise, or by
rotating and tilting.

6. Let plates solidify (within 10 min) on a level surface.

7. After medium solidifies, invert plates and place in incubator. Incubate at 35°C for 24 to 48hrs.

H. Spread plate method.

1. Pour 10mL of medium into sterile Petri dishes, let agar solidify. Store these plates in a sterile
environment at ref temperature.

2. Prepare two Petri dishes for each dilution and label accordingly.

3. Pipet 0.1 mL sample onto surface of a pre-dried agar plate.

4. Using a sterile bent glass, distribute inoculum over surface of medium by rotating plate manually.

5. Let inoculum absorb completely into medium before inverting and incubating.

6. Incubate at 35°C for 24 to 48hrs.

I. Counting and Reporting.

1. Count all colonies on selected plates promptly after incubation. If counting must be delayed
temporarily, store plates refrigerated for ≤24 h, but avoid this as routine practice.

2. Count colonies manually using a colony counter.

3. Use only plate containing 30 to 300 colonies when determining plate count.

4. Compute bacterial count per milliliter as follows:

5. The term colony-forming unit(s) (CFU) is descriptive of the methods used; therefore, report all counts
as CFUs.
IV. Documentation and Guide Questions (use the back portion of this activity, label accordingly)

A. Documentation

1. Preparation of dilution bottles.

2. Sample collection.

3. Serial dilutions.

4. Plating methods: Pour and Spread Plates

5. Colony counting.

B. Guide Questions:

1. What is the purpose of adding phosphate buffer solution and magnesium chloride when preparing the
dilution buffer?

2. Explain why it is important to sterilize culture media and dilution buffers before use.

3. During sample collection, why must the water tap be left running for 2 to 3 minutes before collecting a
sample?

4. Describe the aseptic techniques you used during this activity and explain their importance in microbiology.

5. What are the key differences between the pour plate and spread plate methods, and when might each
method be preferred?

6. Why is it essential to mix the sample thoroughly at each step during serial dilutions?

7. When counting colonies, why are plates with 30 to 300 colonies considered ideal for calculating colony -
forming units (CFU)?

8. Explain how you would calculate the bacterial count per milliliter if you had 50 colonies on a plate from a
10⁻⁴ dilution.

9. Why is it recommended to invert plates during incubation?

10. What might cause variation in colony counts between plates inoculated with the same dilution, and how
can these variations be minimized?