What are the methods that can be used for counting viable bacteria?
Posted January 30, 2024
Answer
The plate count method, the spread plate method, the pour plate method, and spectrophotometric (turbidimetric) analysis can be used for counting viable bacteria.
- In the plate count or spread plate method, the sample is diluted in a series of steps (1:10, 1:100, etc.) and these dilutions are plated onto agar dishes. The agar provides nutrients for bacterial growth, and the viable cells form visible colonies that can be counted. It's vital to arrange dilutions to ensure there are between 30 to 300 colonies per plate; too few or too many colonies can affect the accuracy of the count. Different types of agar media, such as Plate Count Agar or MacConkey Agar, are used to support the growth of specific bacteria and resist the growth of unwanted organisms. After sealing and incubating the plates at different temperatures (usually 22°C and 37°C), colonies develop. They are then counted manually without needing a microscope.
- The pour plate method is used when studying bacteria that grow poorly in air. Serial dilutions of the sample are mixed with liquid nutrient agar and poured into bottles. After incubation, colonies that grow within the agar are counted, and this method provides the Total Viable Count (TVC) expressed in cfu/ml (colony forming units per milliliter) related to the original sample.
- A spectrophotometer is used to measure the turbidity of the culture and indirectly measures all bacteria. As the number of bacterial cells rises, less light passes through the culture and the spectrophotometer detects this decrease in transmitted light. This decrease is then converted into an electrical signal that's displayed on a galvanometer. The measurement obtained is known as absorbance or optical density and indirectly indicates the quantity of bacteria present in the culture. This method is quicker compared to traditional plate counting methods. However, its effectiveness is limited because it can only accurately measure bacterial suspensions with a concentration of around 10^7 cells or higher.
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