Quantitative measurement of bacterial growth involves assessing the number of bacteria in a given sample. Two common methods for quantitative measurement are total count and viable count. These methods provide valuable information about the overall bacterial population and the number of viable, living bacteria.
Total Bacterial Count
1. Direct Microscopic Count:
A small known volume of a bacterial suspension is placed on a microscope slide.
Count bacteria using a microscope and extrapolate the count to represent the total number of bacteria in the original sample.
Fast but may include dead or non-viable cells.
2. Serial Dilution and Plate Count:
Serially dilute the sample and plate each dilution onto a solid agar medium. Count the colonies that develop on the plates and calculate the original bacterial count.
Provides an estimate of viable and culturable cells.
Viable Bacterial Count
1. Spread Plate Method:
Spread a known volume of a diluted bacterial sample uniformly over the surface of a solid agar plate.
Count the colonies that form and express the results as colony-forming units (CFUs) per milliliter.
This method provides a count of viable cells capable of forming colonies.
2. Pour Plate Method:
Similar to the spread plate method, mix the bacterial sample with molten agar before pouring it into the plate.
Colonies grow both on the surface and within the agar.
Counts viable cells within the agar as well.
3. Membrane Filtration:
The sample is passed through a membrane filter with known pore size.
Trap bacteria on the filter and place the filter on a solid agar medium for colony development.
Useful for samples with low bacterial counts.
4. Most Probable Number (MPN):
Statistical method used for samples with low bacterial counts.
Inoculate multiple tubes with different dilutions and record the presence or absence of growth.
MPN tables are used to estimate the number of viable cells.
5. Flow Cytometry:
Automated technique that counts and characterizes cells based on their physical and chemical properties.
Can provide rapid and accurate counts of both viable and non-viable cells.
Factors to Consider
1. Viability:
Viable count methods provide information about living, reproducing cells.
Distinguishes between living and dead cells, providing a more accurate representation of the growth potential.
2. Selective Media:
Using selective media helps in counting specific bacterial groups or species.
Allows for a more targeted assessment of microbial populations.
3. Sampling Technique:
Proper sampling is crucial to obtaining representative counts.
Aseptic techniques must be followed to avoid contamination.
4. Incubation Conditions:
Incubation conditions (temperature, atmosphere) should mimic the natural environment of the bacteria being studied.
5. Equipment Calibration:
Equipment such as pipettes and dilution devices must be calibrated for accuracy.
Both total and viable counts are essential for understanding microbial populations and dynamics in various environments. Choose the method based on the study’s specific objectives and the characteristics of the microbial community being investigated.
