Bacterial enumeration involves two distinct approaches: total count and viable count. The total count method considers both live and dead cells present 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 potentially pathogenic bacteria.
1. Total count of bacteria
1.1. Counting with a Helber slide
The Helber slide is a type of specialized counting chamber commonly used in bacterial enumeration. It features a compartment with a precise depth of 0.02 mm, containing numerous small squares, where each square has 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): When bacteria are in a liquid environment, the term suspension is used.
Note: suspensions that have less than 10 million bacteria per millilitre cannot be counted with this method.
1.2. Counting with filter paper
In cases where the number of bacteria is low, a certain volume of liquid containing bacteria is passed over a paper filter, and the bacteria condense on the surface of the filter. Then the surface of the filter is painted and the number of bacteria is determined under a microscope.
1.3 spectrophotometer
In this method, after turbidity measurement, the number of bacteria is obtained using a standard curve. 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 that are 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
In Helber’s slide, a specialized technique is employed where the sample bacteria are treated with vital dyes, ensuring that only live bacteria are observable and quantifiable. By staining the bacteria with these dyes, the cells that retain their vitality can 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 the understanding of 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, it provides an estimate of 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 that 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 a technique called the pour plate method.
The agar plates are then incubated at an optimal temperature for bacterial growth, which typically ranges from 35 to 37 degrees Celsius. During the incubation period, the viable bacteria present in the sample multiply and form visible colonies on the agar surface. Each colony originates from a single viable bacterial cell that was present in the original sample.
After an appropriate incubation time, usually 24 to 48 hours, the colonies on the plates are examined. Colonies that appear distinguishable in terms of 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 millilitre (CFU/mL) or CFU per gram (CFU/g) of the sample.
The plate count method has certain limitations. It can only enumerate bacteria that are viable, culturable, and able to grow under the specific conditions provided by the growth medium and incubation parameters. Some bacteria may have specific growth requirements that are not met by the chosen agar medium, leading to an underestimation of their population. Additionally, bacteria that form 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 a valuable tool in microbiology laboratories, allowing researchers to assess the viable bacterial load in various samples, such as food, water, clinical specimens, and environmental samples. It provides valuable information for research, quality control, and monitoring the effectiveness of antimicrobial treatments.
