Welcome to Measurement Techniques in Microbiology!
Ever wondered how scientists know how many millions of bacteria are hiding in a tiny drop of liquid? Because microorganisms are invisible to the naked eye and reproduce at incredible speeds, we need reliable, scientific methods to estimate their population sizes.
In this chapter of Unit A2 6: Microbiology, we will explore the core techniques used to count and monitor microbial growth. Don't worry if the calculations look daunting at first — we will break down each technique step-by-step so you feel confident tackling every exam question!
---1. Total Count vs. Viable Count: The Big Distinction
Before looking at specific tools, you must understand this fundamental rule:
• Total Count: Measures all cells present in the sample — both living and dead cells.
• Viable Count: Measures only living cells that are capable of reproducing and dividing.
Memory Trick: Think of a Viable count as counting only Vibrant, living cells!
Quick Review:
• Haemocytometer counting = Total Count
• Turbidimetry (cloudiness) = Total Count
• Dilution Plating = Viable Count
2. Direct Counting: The Haemocytometer
A haemocytometer is a specialised, heavy-duty microscope slide with an etched counting grid of known depth and dimensions.
How It Works:
A known volume of liquid culture is loaded into the counting chamber. When viewed under a microscope, you count the cells visible within the grid squares.
The North-West (Top-Left) Rule:
To ensure accuracy and prevent double-counting:
• Count cells that touch or cross the top and left boundary lines.
• Ignore cells that touch the bottom and right boundary lines.
Haemocytometer Calculations:
The volume of the standard counting chamber is \(0.1\text{ mm}^3\). Remember that \(1\text{ ml} = 1,000\text{ mm}^3\) (and \(1\text{ cm}^3 = 1\text{ ml}\)).
Because the chamber holds \(0.1\text{ mm}^3\), there are \(10,000\) such chamber volumes in \(1\text{ ml}\) of liquid (\(1,000 / 0.1 = 10,000\)).
The standard formula to calculate cells per millilitre is:
\(\text{Cells per ml} = \text{Average count in } 0.1\text{ mm}^3\text{ chamber} \times \text{Dilution Factor} \times 10,000\)
Key Takeaway: Haemocytometers give a direct total count (living + dead). Always remember the top-and-left line counting rule!
---3. Viable Counting: Serial Dilution and Plating
If a bacterial culture is too dense, millions of cells spread onto an agar plate will simply merge into an uncounted layer called a "lawn". To count individual colonies, we must first dilute the sample.
Step 1: Serial Dilution
A series of stepwise dilutions is performed (for example, transferring \(1\text{ ml}\) of culture into \(9\text{ ml}\) of sterile broth to create a \(10^{-1}\) or \(1\text{ in } 10\) dilution). This process is repeated down a line of test tubes (\(10^{-2}\), \(10^{-3}\), \(10^{-4}\), etc.) until the bacterial concentration is reduced to a manageable level.
Step 2: Plating and Counting Colony Forming Units (CFUs)
A small volume (such as \(0.1\text{ ml}\) or \(1.0\text{ ml}\)) from chosen dilutions is spread onto agar plates and incubated.
• We operate on the assumption that each single viable cell divides repeatedly to form one visible colony.
• For statistical reliability, choose a plate that has a countable number of colonies — typically between \(20\text{--}200\) or \(30\text{--}300\) colonies.
• Plates with too many colonies overlap (confluent), and plates with too few colonies are not statistically reliable.
Viable Count Calculation:
\(\text{Viable count per ml} = \text{Number of colonies} \times \text{Dilution Factor} \times \text{Volume plated adjustment}\)
Worked Example:
If you plate \(0.1\text{ ml}\) from a \(10^{-4}\) dilution (dilution factor of \(10^4\) or \(10,000\)) and count \(45\) colonies:
1. The count in \(0.1\text{ ml}\) is \(45 \times 10,000 = 450,000\).
2. To find the count in \(1.0\text{ ml}\), multiply by \(10\) (since \(1.0\text{ ml} / 0.1\text{ ml} = 10\)):
\(\text{Viable count per ml} = 45 \times 10,000 \times 10 = 4,500,000\text{ cells per ml}\) (or \(4.5 \times 10^6\text{ CFU/ml}\)).
Key Takeaway: Dilution plating measures only viable cells (CFUs). Be careful to check the volume plated when scaling up to \(1\text{ ml}\)!
---4. Indirect Measurement Methods
Indirect methods estimate microbial populations by measuring a physical property or biological mass rather than counting individual cells directly.
A. Turbidimetry (Optical Density)
• As bacteria reproduce in a liquid broth, the broth becomes cloudier or more turbid.
• A colorimeter or spectrophotometer passes light through the culture tube and measures the amount of light absorbed or transmitted.
• Principle: The higher the cell concentration, the more turbid the broth, and the more light is absorbed (higher absorbance / optical density).
• Calibration Curve (Standard Curve): A colorimeter reading gives absorbance, not cell numbers! To convert absorbance into actual cell numbers, scientists must first construct a standard curve by plotting known cell counts (determined using a haemocytometer) against their corresponding absorbance values.
B. Dry Weight (Biomass)
• Microbial cells are filtered from the broth, thoroughly washed, and dried in an oven until they reach a constant mass.
• This provides a direct measure of total microbial biomass.
Key Takeaway: Turbidimetry is rapid and convenient, but gives a total count and requires a standard curve to find actual cell numbers.
---5. Bacterial Growth Curves
When bacteria are grown in a closed liquid culture, their population growth follows a characteristic four-phase curve when plotting the logarithm of cell numbers vs. time.
Why use a logarithmic scale? Bacterial numbers double rapidly during exponential growth. Plotting raw numbers produces an unwieldy steep curve, whereas a logarithmic scale allows the exponential phase to be plotted and interpreted clearly as a straight line.
The Four Phases:
1. Lag Phase:
• No increase in cell numbers.
• Cells are actively metabolising and synthesising enzymes and proteins needed to adapt to their new environment.
2. Log (Exponential) Phase:
• Rapid, maximum rate of cell division by binary fission.
• Population doubles at regular intervals because nutrients are abundant and toxic wastes are minimal.
3. Stationary Phase:
• The overall population size remains constant.
• The rate of cell division equals the rate of cell death.
• Caused by the depletion of nutrients and the accumulation of toxic metabolic waste products.
4. Death (Decline) Phase:
• The rate of cell death exceeds the rate of cell division.
• Toxic wastes reach lethal concentrations and essential nutrients are exhausted.
6. Summary & Common Exam Pitfalls
Watch out for these common mistakes:
• Total vs. Viable Count: Remember that optical density (turbidimetry) and haemocytometers count dead cells too! Only plating counts living (viable) cells.
• Volume Corrections: If an exam question states that \(0.1\text{ ml}\) was plated, you must multiply by \(10\) to get the count per \(1.0\text{ ml}\). (Remember: \(1\text{ cm}^3 = 1\text{ ml}\)).
• Lag Phase Misconception: Bacteria are not resting in the lag phase; they are intensely active synthesising enzymes!
• Standard Curves: Turbidimeters give absorbance values, not direct cell counts. A standard curve plotted with known haemocytometer counts is required to convert the readings.