Total count versus viable count.
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Total Count vs. Viable Count
These are two fundamental ways to measure microbial concentrations in a culture or sample. They measure different things and serve different purposes.
Definitions
| Parameter | What it measures |
|---|---|
| Total count | All cells present - alive, dead, and sometimes non-cellular debris |
| Viable count | Only living cells capable of reproduction |
Microbial concentrations can be expressed as cell concentration (number of viable cells per unit volume) or biomass concentration (dry weight per unit volume). These are not always equivalent because average cell dry weight varies at different stages of a culture.
- Jawetz, Melnick & Adelberg's Medical Microbiology, 28e, p. 60-61
Methods
Viable Count - Plate Count Method
The viable cell count is typically the standard measure of cell concentration. The procedure:
- Remove 1 mL from a bacterial suspension.
- Serially dilute 10-fold (e.g., 10^-1 through 10^-5).
- Plate 0.1 mL aliquots on a suitable agar medium.
- Each single invisible bacterium (or clump of bacteria) grows into a visible, countable colony.
- For statistical accuracy, plates containing 30-300 colonies give the most reliable data.
Formula:
CFU/mL = Plate count × Dilution factor × 10
Key point: Dead bacteria do NOT contribute to the final count. Only metabolically active cells that can reproduce are counted. Each unit measured is called a colony-forming unit (CFU).
Example from Table 4-1 (Jawetz, p. 61):
| Dilution | Colonies |
|---|---|
| Undiluted | Too many to count |
| 10^-1 | Too many to count |
| 10^-2 | 510 (too high) |
| 10^-3 | 72 ✓ (in range) |
| 10^-4 | 6 (too low) |
| 10^-5 | 1 (too low) |
Using the 10^-3 dilution: 72 × 10^3 × 10 = 7.2 × 10^5 CFU/mL
Total Count - Turbidity / Spectrophotometry
Turbidity (cloudiness of a culture) is measured by photoelectric means (spectrophotometer/colorimeter). A standard curve correlates turbidity (optical density) with viable count. Rough visual estimates:
- Barely turbid E. coli suspension ≈ 10^7 cells/mL
- Fairly turbid suspension ≈ 10^8 cells/mL
Critical limitation: Turbidity measures ALL particles (living + dead). Cells may lose viability without any loss in turbidity - so turbidity can overestimate live cell numbers, especially during culture decline. - Jawetz, p. 61
Total Count - Direct Microscopic Count (Petroff-Hausser Chamber)
A counting chamber with a known volume grid is used under a microscope. All cells (live + dead) are counted. This gives the total cell number but cannot distinguish viable from non-viable cells.
Biomass Concentration
Measured by:
- Direct: Dry weight of washed culture cells
- Indirect: Measuring a cellular component (e.g., protein content) or cell settling volume
Biomass is significant in biochemical and nutritional studies, whereas cell concentration (viable count) is more relevant in genetics and microbial inactivation studies.
Key Differences at a Glance
| Feature | Total Count | Viable Count |
|---|---|---|
| Cells counted | All (living + dead) | Only living/reproducing |
| Primary method | Turbidimetry, direct microscopy | Plate count (CFU) |
| Unit | cells/mL | CFU/mL |
| Speed | Fast (minutes) | Slow (hours to days for colonies) |
| Accuracy | Overestimates live population | Accurate for cultureable organisms |
| Affected by dead cells | Yes | No |
| VBNC organisms counted? | Yes | No (cannot culture) |
| Use case | Biomass, growth curves | Infection inoculum, sterilization efficacy |
Practical Importance
-
Turbidity vs. viability discordance: A culture in decline may remain turbid while most cells are dead. Turbidity (total count proxy) will read high even when the viable count has dropped sharply. This is why plate counts are used when viability matters (e.g., antibiotic kill curves).
-
Viable-but-non-culturable (VBNC) state: Some bacteria enter a dormant, metabolically minimal state and will not form colonies on standard media. The viable count underestimates the true live population in environmental samples; methods like flow cytometry with live/dead staining bridge this gap.
-
Clinical relevance: Urine cultures (UTI diagnosis) use plate count to report CFU/mL. Significant bacteriuria is defined as ≥10^5 CFU/mL - a viable count threshold. A total count method alone would be insufficient here.
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