Method for detecting microbial growth - Database & Sql Blog Articles

Microorganisms are not only widespread in nature but also live together in mixed populations. To study or utilize a specific microorganism, it is necessary to isolate it from the mixed population to obtain a culture containing only that single organism. In microbiology, this process is known as obtaining a pure culture, which refers to offspring propagated from one cell or a single cell population under controlled experimental conditions. There are several methods used to achieve this goal.

1. Growth Assay

1. Volume Measurement Method (Mycelial Concentration): This method involves measuring the amount of mycelium in a given volume of culture solution. For example, take 10 ml of the culture, centrifuge it for 5 minutes at 5000 rpm, then measure the supernatant volume. The mycelial concentration is calculated as (10 - v)/10, where v is the supernatant volume. This technique is commonly used in industrial fermentation to monitor microbial growth effectively.

2. Dry Weight Method: This method determines biomass by centrifugation or filtration. Typically, dry weight accounts for about 10–20% of wet weight. After centrifugation, the pellet is washed, dried in an oven at 105°C or 100°C, or under vacuum at 80°C or 40°C, and then weighed. It’s a reliable way to estimate microbial biomass.

3. Turbidimetry: As microorganisms grow, they increase the turbidity of the culture. Using a spectrophotometer, the absorbance at a specific wavelength (e.g., 600 nm) can be measured to determine the growth state. This method is widely used in bacterial fermentation, such as monitoring E. coli growth with a UNICO UV-Vis spectrophotometer.

4. Mycelial Length Measurement: This method is suitable for filamentous fungi and actinomycetes. By inoculating the microorganism into a scaled glass tube and measuring the length of mycelial growth over time, you can assess the growth of filamentous microbes effectively.

2. Physiological Index Methods

1. Nitrogen Content Determination: Most bacteria contain 12.5% nitrogen by dry weight, while yeast has 7.5%, and mold has 6%. Crude protein content can be estimated using the formula: nitrogen content × 6.25. Common methods include the Dumas method, which measures nitrogen gas produced after combustion in CO₂.

2. Carbon Content Determination: A small sample of biological material is treated with K₂Cr₂O₇, heated, and the absorbance at 580 nm is measured to estimate carbon content. This is useful for monitoring microbial metabolism during fermentation.

3. Reducing Sugar Assay: Reducing sugars like monosaccharides and oligosaccharides are directly utilized by microbes. This method helps monitor microbial growth in large-scale fermentations. Steps include centrifugation, adding reagents, boiling, and titrating with Na₂S₂O₃ to calculate sugar content.

4. Amino Nitrogen Determination: This method involves measuring the amino nitrogen content in the culture. The process includes centrifugation, acid-base titration, and calculation based on NaOH consumption. It provides an indirect measure of microbial growth.

5. Other Physiological Substances: Measuring substances like P, DNA, RNA, ATP, NAM, and tracking gas production, COâ‚‚, oxygen consumption, and heat generation can help assess microbial activity. These indicators are crucial in industrial processes, such as monitoring dissolved oxygen and pH changes during BMP-2 fermentation to track bacterial growth effectively.