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Enumeration of Microorganisms: Total and Viable Counts

1. Aim

To distinguish a direct total count from a colony-forming viable count and calculate a teaching example. Sample type, dilution, and counting method are specified in your college lab manual and instructor's method sheet.

2. Requirements

Instructor-supplied count images or approved low-risk cultures, counting chamber or image data, approved agar plates or colony-count records, dilution log, pipettes, counter, protective equipment, and waste stream. Plate volume, dilution scheme, incubation, and countability criterion are specified in your college lab manual and instructor's method sheet.

3. Theory

A direct count estimates all visible cells in a measured field or chamber, including cells that may not grow. A viable plate count estimates colony-forming units, abbreviated CFU, because a colony can arise from one cell or a cell cluster. Dilution and plated volume must be tracked to express CFU per millilitre. A viable count may be lower than the total count because non-growing cells are not represented. Countable-range rules depend on the method and must be checked rather than guessed.

4. Procedure

1. Confirm the risk assessment and choose supplied count images or an approved low-risk sample. Record the actual counting method.
2. For a direct count, record the number of counted chamber squares or image area and the instrument volume factor.
3. For a viable count, record plate identifier, dilution, plated volume, and colonies from a method-accepted plate.
4. Apply the appropriate formula, keeping cells per volume separate from CFU per volume.
5. Document controls, replicates, and limitations before reporting the sample estimate.

5. Calculation, graph, or images as appropriate

Assumed values for practice only: assume an assumed count of 80 colonies on an assumed plate from a 10⁻⁴ dilution, with assumed 0.10 mL plated. These invented values are not actual culture data or a plating instruction.

1. Illustrative viable concentration = assumed 80 ÷ (10⁻⁴ × assumed 0.10 mL) = 8.0 × 10⁶ CFU/mL.
2. An actual total cell count cannot be derived from these plate data and must be recorded here from a separate direct count.
3. Actual viable count must be recorded here until the real dilution log and plate record are supplied.

6. Result

The actual result and any identification or count must be recorded here until the verified method and observed record are available. Safety note: Counting data are preferred for this teaching page. Any live culture work requires approved organisms, containment, and supervised decontamination.

7. References

1. Pharmacy Council of India, Pharm.D. Regulations, 2008.
2. OpenStax Microbiology, How Microbes Grow.
3. American Society for Microbiology, Biosafety Guidelines for Teaching Laboratories.

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Theory notes: Pharmaceutical Microbiology