Bacterial enumeration involves two distinct approaches: total count and viable count. The total count method considers both live and dead cells in a sample, providing an overall estimation of bacterial abundance. On the other hand, the viable count method focuses solely on the number of living cells, offering a measure of the actively replicating and potentially infectious bacterial population. By employing these different approaches, researchers can gain valuable insights into the total bacterial load and the specific subset of viable and possibly pathogenic bacteria.
1. Total count of bacteria
1.1. Counting with a Helber slide
The Helber slide is a specialized counting chamber commonly used in bacterial enumeration. It features a compartment with a precise depth of 0.02 mm, containing numerous small squares, each with an area of 0.0025 square mm. To perform the count, a specific volume of the bacterial suspension (1) is carefully placed within the counting chamber, and the number of bacteria is then observed and recorded using a microscope. This method allows for accurate quantification of bacteria by utilizing the defined area and depth of the chamber, providing researchers with valuable information about the concentration of bacteria in a given sample.
Explanation (1): The term suspension is used when bacteria are in a liquid environment.
Note: this method cannot count suspensions with fewer than 10 million bacteria per millilitre.
1.2. Counting with filter paper
In cases where the number of bacteria is low, a specific volume of liquid containing bacteria is passed over a paper filter, and the bacteria condense on the filter’s surface. Then, the filter’s surface is painted, and the number of bacteria is determined under a microscope.
1.3 spectrophotometer
This method obtains the number of bacteria using a standard curve after turbidity measurement. Here, the standard curve is plotted as bacterial count versus turbidity.
1.4 Coulter counter
This device is an electronic counter. It has two compartments separated by a wall made of insulating material. This wall has a duct whose diameter is almost the same as the cell. Each chamber has an electrode. The sample is poured into a chamber and then pushed to another chamber under pressure, and the suspension passes through the channel. Each bacterial cell that passes through the channel momentarily reduces the electric current, and this change in electric current is recorded as a pulse.
2. Counting live bacteria
2.1. Counting with a Helber slide
Helber’s slide employs a specialized technique for treating the sample bacteria with vital dyes, ensuring that only live bacteria are observable and quantifiable. Staining the bacteria with these dyes allows the cells that retain their vitality to be distinguished and enumerated, allowing for a more accurate assessment of the live bacterial population. This technique provides valuable information about the viability and active status of the bacteria, aiding in understanding their behaviour and potential impact in various applications, such as research, diagnostics, and quality control.
2.2 Plate counting method
The plate count method is a widely used technique for enumerating bacteria in a sample. Also known as the viable count method or colony-forming unit (CFU) assay, estimates the number of viable and culturable bacteria present in a given sample.
The plate count method involves several key steps. First, the sample is appropriately diluted to obtain a manageable number of bacterial colonies on the agar plates. These dilutions ensure the colonies can be easily counted without overcrowding the plates. Next, a portion of each diluted sample is evenly spread across the surface of nutrient agar plates using a sterile spreader or the pour plate method.
The agar plates are then incubated at an optimal temperature for bacterial growth, typically 35 to 37 degrees Celsius. During the incubation period, the viable bacteria in the sample multiply and form visible colonies on the agar surface. Each colony originates from a single viable bacterial cell present in the original sample.
The plate colonies are examined after an appropriate incubation time, usually 24 to 48 hours. Colonies that appear distinguishable in colour, shape, size, or texture are counted manually or using automated colony counters. The number of colonies on the plates is proportional to the number of viable bacteria in the original sample, and results are expressed as CFU per milliliter (CFU/mL) or CFU per gram (CFU/g) of the sample.
The plate count method has certain limitations. It can only enumerate viable, culturable, and able-to-grow bacteria under the specific conditions provided by the growth medium and incubation parameters. Some bacteria may have specific growth requirements not met by the chosen agar medium, leading to an underestimation of their population. Additionally, bacteria forming clusters or chains may give rise to a single colony, resulting in an underestimation of the actual bacterial count.
Despite these limitations, the plate count method remains valuable in microbiology laboratories. It allows researchers to assess the viable bacterial load in various samples, such as food, water, clinical specimens, and environmental samples. This information is valuable for research, quality control, and monitoring the effectiveness of antimicrobial treatments.
