Method for detecting microbial growth - Database & Sql Blog Articles

Microorganisms are not only widely distributed in nature but also live in mixed communities. To study or utilize a specific microorganism, it is essential to separate the mixed microbial population to obtain a culture containing only one species. In microbiology, this process is known as pure culture, where offspring are derived from a single cell or a 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 measures the amount of hyphae present in a given volume of culture. A sample (e.g., 10 ml) is centrifuged for a set time and speed (e.g., 5 minutes at 5000 rpm). After removing the supernatant, the mycelial concentration is calculated as (10 - v)/10, where v is the volume of the supernatant. This technique is commonly used in industrial fermentation to monitor microbial growth.

2. Dry Weight Method: This involves centrifugation or filtration followed by washing and drying. The dry weight is typically 10–20% of the wet weight. The sample is dried in an oven (105°C or 100°C), under infrared light, or in a vacuum at 80°C or 40°C, then weighed. It's a reliable way to estimate biomass.

3. Turbidimetry: Microbial growth increases the turbidity of the culture. Using a UV spectrophotometer, the absorbance at a specific wavelength (e.g., 600 nm) is measured. For real-time monitoring, a special flask with a side arm can be used to insert into a colorimeter without removing the sample. This method is widely used in bacterial fermentation processes.

4. Hyphae Length Measurement: For filamentous fungi and actinomycetes, the length of hyphae can be measured on agar plates or using a scaled glass tube. The growth length is recorded after inoculation, providing an indirect measure of growth.

2. Physiological Index Method

1. Nitrogen Content Determination: Most bacteria contain 12.5% nitrogen by dry weight, while yeast has 7.5%, and mold has 6%. Crude protein can be estimated by multiplying the nitrogen content by 6.25. Methods include Dumas’ gas method, which involves heating the sample with CuO to produce nitrogen gas and measuring its volume.

2. Carbon Content Analysis: A small amount of biological material is mixed with a K₂Cr₂O₇ solution and heated. The remaining chromium is measured spectrophotometrically at 580 nm to estimate carbon content. This method helps assess microbial metabolism.

3. Reducing Sugar Assay: Reducing sugars like monosaccharides and oligosaccharides are directly utilized by microbes. The Fehling’s reagent method is commonly used, involving boiling the sample, acidifying, and titrating with Na₂S₂O₃ to determine sugar content.

4. Amino Nitrogen Determination: By titrating the supernatant with NaOH after adding formaldehyde, the amino nitrogen content can be calculated. This reflects the metabolic activity of the microorganism.

5. Other Physiological Indicators: Measuring P, DNA, RNA, ATP, NAM, COâ‚‚ production, oxygen consumption, and other factors can also indicate microbial growth. In industrial settings, dissolved oxygen and pH changes are often monitored to track bacterial growth in real-time, such as in BMP-2 fermentation.