How to Detect Bacteria in Food: Total Bacterial Count Procedure

Experimental Principle

The total bacterial count refers to the total number of bacterial colonies contained in 1 g or 1 mL of a food sample after processing and cultivation under specified conditions. It is mainly used as an indicator to assess the degree of contamination in food. This method can also be applied to observe the dynamic growth of bacteria in food, thereby providing a basis for hygienic evaluation of the tested samples. Each type of bacterium has specific physiological characteristics, and different nutritional and physiological conditions (such as temperature, incubation time, pH, and oxygen requirements) must be provided during cultivation to support their growth. However, in practical work, usually only one common method is applied. Therefore, the determination of total bacterial count includes only the colonies of mesophilic aerobic bacteria that are able to grow on nutrient agar.

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Main Experimental Reagents

Nutrient agar medium, sterilized physiological saline (aliquoted in test tubes, 9.0 mL per tube), food test samples.

Main Experimental Instruments and Equipment

Incubator (36 ± 1 °C), constant temperature water bath (46 ± 1 °C), balance, adjustable electric stove, pipettes (1.0 mL, 10.0 mL, graduated in 0.1 mL units), 500 mL wide-mouth bottle with lid, glass beads (diameter 5 mm), Petri dishes (dish bottom diameter 9.0 cm), test tubes, alcohol lamp, test tube rack, mortar and pestle, sterilized knives and scissors, sterilized tweezers, alcohol cotton balls, glass marking pencil.

Experimental Procedure

Step 1: Sample Preparation

Step 2: First Dilution (1:100)

Step 3: Serial Dilutions

Step 4: Selection of Dilution Levels and Plate Preparation

Step 5: Pouring Nutrient Agar and Blank Control

Step 6: Incubation

Step 7: Colony Counting and Calculation

Experimental Result Calculation

1. Selection of Colony Counts on Plates

Plates with colony numbers between 30 and 300 are selected as the standard for total colony count determination. For each dilution, two plates should be used, and the average value of the two plates should be taken. If one plate shows the growth of large spreading colonies, it should not be adopted; instead, the plate without spreading colonies should be used for that dilution. In such cases, half of the plate may be counted and then multiplied to represent the colony number for the whole plate.

2. Selection of Dilution Levels

1) The dilution level with an average colony count between 30 and 300 should be chosen, and the colony number is reported as the average multiplied by the dilution factor (see Table 1, Sample No. 1).

2) If two dilution levels both have colony counts between 30 and 300, the ratio of the two should be considered. If the ratio is<2, their="" average="" should="" be="" if="">2, the smaller number should be reported (see Table 1, Sample No. 2 and 3).

3) If the average colony counts for all dilutions are greater than 300, the average colony count at the highest dilution should be multiplied by the dilution factor and reported (see Table 1, Sample No. 4).

4) If the average colony counts for all dilutions are less than 30, the average colony count at the lowest dilution should be multiplied by the dilution factor and reported (see Table 1, Sample No. 5).

5) If no colonies are observed at all dilutions, the result should be reported as<1 multiplied by the lowest dilution factor (see Table 1, Sample No. 6).

6) If the average colony counts for all dilution factors fall outside the 30–300 range, with some greater than 300 and others less than 30, the average colony count closest to either 30 or 300 should be multiplied by the dilution factor and reported (see Table 1, Sample No. 7).

Table 1 Selection of Dilution Levels and Reporting of Colony Counts

3. Reporting of Colony Counts

When the colony count is within 100, the actual number is reported. For counts less than 100, two significant figures are used, and values beyond the second significant figure are rounded off. To reduce trailing zeros, scientific notation using powers of 10 may also be applied.

Experimental Precautions

Ensure that the operation is carried out under aseptic conditions to avoid contamination that may affect the measurement results.