Measuring Bacterial Growth: Direct & Indirect Methods
Hi, I’m Kathy with Love Larn! In this video, I’ll be breaking down how to measure bacterial growth using both direct and indirect methods.
By the end of the video, you'll have a clear understanding of these techniques, and I’ll even include a quiz to test your knowledge—so stay tuned!
If you have our Level Up RN Microbiology Flashcards, grab them now to follow along. These flashcards are also available on Flashable, which provides personalized learning and progress tracking.
Why Measure Bacterial Growth?
Microbiologists use bacterial growth measurements to:
✔️ Determine infection severity in clinical samples.
✔️ Assess water and food contamination levels.
✔️ Optimize bacterial cultures for industrial and pharmaceutical applications.
✔️ Monitor the effectiveness of antibiotics and disinfectants.
Now, let's explore the direct and indirect methods used to measure bacterial growth.
Direct Methods of Measuring Bacterial Growth
Direct methods provide a quantitative count of bacterial cells, either by counting individual cells or estimating viable colonies on a growth medium.
1. Direct Cell Count
Direct cell counting involves measuring the total number of bacterial cells in a sample—whether alive or dead.
A. Microscopic Cell Counting (Hemocytometer Method)
A hemocytometer (calibrated counting chamber) is used to manually count bacteria under a microscope.
🔹 Process:
- A bacterial suspension is placed on a hemocytometer, which has a grid with known dimensions.
- The number of bacteria in a selected grid square is counted.
- The bacterial count is multiplied by a conversion factor to determine cells per milliliter (cells/mL).
⚠️ Limitation:
- This method does not distinguish between live and dead cells unless a viability stain (e.g., Trypan Blue) is used.
B. Electronic Cell Counters (Coulter Counter Method)
A Coulter counter is an automated device that rapidly counts bacterial cells.
🔹 How It Works:
- A bacterial suspension is passed through a narrow aperture.
- As each cell passes through, it disrupts an electrical current, allowing the device to count them.
✅ Advantage: Faster and more accurate than manual counting.
⚠️ Limitation: Still cannot distinguish between live and dead cells.
2. Plate Count (Viable Cell Counting)
Unlike direct cell counting, the plate count method only measures viable (living) bacteria by counting colony-forming units (CFUs).
A. Why Count CFUs Instead of Individual Cells?
Bacteria often form clusters, chains, or pairs. For example:
- Staphylococcus aureus forms grape-like clusters.
- Streptococcus pyogenes grows in chains.
- Neisseria gonorrhoeae exists as diplococci (pairs of cells).
Since one CFU may originate from multiple cells, we express results as CFU/mL, not cells/mL.
B. The Serial Dilution & Plate Count Process
💡 Goal: Reduce bacterial concentration to obtain a plate with a countable number of colonies (30–300 CFUs).
🔹 Steps:
- Serial Dilution: A bacterial sample is diluted stepwise (1:10, 1:100, 1:1,000, etc.).
- Plating: 1 mL from each dilution is spread onto an agar plate using the spread plate or pour plate method.
- Incubation: Plates are incubated at 37°C for 24–48 hours.
- Counting CFUs: The plate with 30–300 colonies is selected for CFU calculation.
Example Calculation:
A plate with a 1:10,000 dilution has 47 CFUs.
⚠️ Limitations of Plate Counts:
- Bacteria that form biofilms or are difficult to culture may be undercounted.
- Some fastidious bacteria require specific nutrients that may not be present in standard agar.
3. Membrane Filtration (For Low Bacterial Concentration)
Used to estimate bacterial numbers in very dilute samples, such as drinking water or lake water.
🔹 Steps:
- A known volume (e.g., 100 mL) is vacuum-filtered through a membrane filter (pore size: 0.45 μm).
- The membrane is transferred onto an agar plate.
- After incubation, CFUs are counted to determine bacterial concentration per 100 mL of liquid.
4. Most Probable Number (MPN) Method
A statistical method for estimating bacterial numbers in samples where direct counting is impractical, such as contaminated water.
🔹 Process:
- Diluted samples are inoculated into nutrient broth tubes.
- Tubes are incubated and observed for bacterial growth indicators:
- Color change (pH shift due to fermentation).
- Gas production (seen in Durham tubes).
- Increased turbidity (cloudiness).
- Results are compared with an MPN table to estimate bacterial concentration per 100 mL.
Indirect Methods of Measuring Bacterial Growth
Indirect methods do not count bacteria directly but estimate growth using turbidity, biomass, or metabolic activity.
1. Turbidity Measurement (Spectrophotometry)
A spectrophotometer measures light absorption to estimate bacterial growth.
🔹 How It Works:
- A bacterial suspension is placed in a cuvette.
- Light is passed through the sample.
- The amount of light absorbed (optical density, OD) is recorded.
✅ Advantage: Rapid & non-destructive.
⚠️ Limitation: Cannot distinguish live from dead cells.
2. Dry Weight Measurement
Used for bacteria that form filaments or biofilms, such as Actinomyces.
🔹 Process:
- Bacteria are harvested, washed, dried, and weighed.
- Growth is determined based on biomass weight.
✅ Best for filamentous bacteria that do not form discrete colonies.
3. Measuring Metabolic Activity
Microbial growth can also be estimated by measuring nutrient consumption or waste product accumulation.
💡 Example: Methylene Blue Reduction Test (Milk Quality Test)
- Methylene blue dye is blue in oxygen.
- As bacteria consume oxygen, the dye loses color.
- The faster the decolorization, the higher the bacterial contamination.
Quiz Time!
Question 1:
Why is a Coulter counter limited in bacterial counting?
✅ It cannot distinguish between live and dead cells.
Question 2:
A plate has 120 colonies from a 1:100 dilution.
How many CFU/mL in the original sample?
✅ 120 × 100 = 12,000 CFU/mL
Question 3:
Which of the following are indirect bacterial growth measurement methods?
✅ A. Metabolic activity
✅ D. Dry weight
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