1. Introduction
Microorganisms play a crucial role in humans’ lives, but not always in beneficial impact. Microorganisms linked with foods can be characterized as spoilage, useful or pathogenic (1). Intrinsic factors associated with the food itself can affect microorganisms' growth (2)(3). The Intrinsic factors related to food are inherent, and they include water activity, PH, nutrient content, oxidation-reduction potential, etc. (4). Controlling these intrinsic factors can inhibit the growth of pathogens in food such as Escherichia coli K12, and Listeria innocua. These microorganisms cause food-borne illnesses that make it crucial to study them to prevent their growth or include a kill step throw food processing to obtain safe food.
The experiment main objective was to examine the hypothesis of the effect of intrinsic factors such as pH and water activity on Escherichia coli K12, and Listeria innocua growth by using different concentrations of pH and (a w) by making serial dilutions and spread plate technique.
2. Materials and Methods
A. pH effect on microbial growth:
In the first week, 3 ml of a culture of Escherichia coli K12, and Listeria innocua separately, was provided. 0.1 ml of each culture was inoculated in 10 ml Trypticase Soy Broth (TSB) media presented in five tubes having the following pH levels of 3.0, 5.0, 7.0, 9.0, 11.0 at temperature 23 ℃. Then, the tubes were mixed by vortexing and incubated at 37 ℃ for 48 hours. Observations were recorded for growth (turbidity) and no growth (no turbidity) and were reported as (+) or (-) for each.
B. Effect of Water Activity (a w) On Microbial Growth:
3 ml of a culture of Escherichia coli K12, and Listeria innocua separately, was provided. 0.1 ml of each culture was inoculated in 10 ml Trypticase Soy Broth (TSB) media presented in five tubes having sodium chloride (salt) or glucose (sugar).
Salt have the following concentrations in the media 0% (a w 1.00) 1.74% (a w 0.98), 6.57% (a w 0.96), 11.57% (a w 0.94), 14.20% (a w 0.92) at temperature 21 ℃ and for sugar were 0% (a w 1.00), 15.5% (a w 0.98), 35.4% (a w 0.97), 41.0% (a w 0.96), 52.0% (a w 0.94) at temperature 22 ℃. Then, the tubes were mixed by vortexing and incubated at 37 ℃ for 48 hours. Observations were recorded for growth (turbidity) and no growth (no turbidity) and were reported as (+) or (-) for each.
For turbidity checking, the tube sets were observed without shaking it, and results were recorded. For determining the turbidity level, the following scale was used; (No turbidity:(-)0-25% turbidity: (+) 25-50% turbidity: (++) 50-75% turbidity: (+++) 75-100% turbidity: (++++). Then, the inoculated cultures' quantitative analysis was done by performing serial dilutions and spread plating the cultures in duplicate. Tubes of 0.1% peptone were prepared for the specific serial dilutions. Then, 0.1 ml of the inoculum was transferred from the serial dilutions into several Tryptic Soy Agar with Yeast Extract (TSAYE) plates. The Dilutions that were selected to perform spread plating are presented in Tables 1, 2, and 3. From every chosen dilution, spread plating was performed with a clean plastic spreader, and all plates were incubated at 37 ℃ for 48 hours. After the incubation period, the colonies were observed and counted colonies.
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