Abstract: This experiment is directed to investigate the various physical factors affecting the growth of microbes: temperature, pH of the extracellular environment and the requirement of atmospheric oxygen. Consequently this experiment consists of three parts: 1st part is about investigating the effect of temperature on microbial growth by varying the temperature range: 4oC, 20oC, 37oC and 60oC; 2nd part is directed to determine the effect of pH on growth of microorganisms by varying the pH ranges: pH3, pH6, pH7, pH9; the last part is about effect of oxygen requirement on growth of microbes, where the microorganisms grow depending on oxygen demand. Introduction: Microorganisms are classified depending on growth requirements. In this laboratory the microorganisms are divided due to effect of temperature, pH and oxygen demand. The microorganisms are divided into three groups depending on temperature ranges they live: psychrophiles, mesophiles and thermophiles; inhabiting cold environment with optimal temperature for reproduction below 20oC, living in temperature ranges between 20oC to 45oC and the last growing optimally at temperature above 45oC respectively.
The optimal temperature is the temperature that is best condition for growth of microorganisms as the enzymes they contain act most effectively at this temperature. The second group of microorganisms investigated in this experiment is divided due to effect of pH on microbial growth. They divided to acidophiles(acid-loving) inhabiting the regions with pH below 4, neutrophiles growing in pH range between 6.5-7.5 and the alkalophiles (base-loving) grow at pH range above 9. Also the term optimal pH is used here as mircoorganisms reproduce effectively at narrow pH range depending on activity of enzymes responsible for replication of DNA.
The last group of microorganism is divided into three categories depending on oxygen requirement into anaerobes and aerobes. The anaerobes are subdivided further into facultative anaerobes, obligatory anaerobes and aerotolerant anaerobes: facultative are those that can reproduce in the absence of oxygen but grow effectively at the presence of oxygen, obligatory anaerobes are microbes that cannot reproduce in the presence of oxygen, even it is harmful for them and aerotolerant anaerobes are species that can grow in presence of oxygen, but do not benefit from it. The microaerophilic anaerobes are types of microorganisms that require oxygen for reproduction at low concentrations compared to obligate aerobes.
Materials and Methods:
24- to 48-hour nutrient broth cultures of Esherichia coli, Bacillus coagulans, Pseudomonas savastanoi, Serratia marcescens, and Sabouraud broth culture of Saccharomyces cerevisiae.
Per designated student group: four Trypticase soy agar plates and four Sabouraud broth tubes
Bunsen burner, inoculating loop, refrigerator set at 40C, two incubators set at 370C and 600C, automatic pipette (20-200 µl), sterile tips for automatic pipette, test tube rack, and glassware marking pencil.
Saline suspensions of 24-hour nutrient broth cultures, adjusted to an absorbance (A) of 0,05 at a wavelength of 600 nm, of Alcaligenes faecalis, Escherichia coli, and Saccharomyces cerevisiae.
Per designated student group: 12 Trypticase soy broth (TSB) tubes, three at each of the following pH designations: 3, 6, 7, and 9. The pH has been adjusted with 1 N NaOH or 1 N HCl
Bunsen burner, sterile 1-ml tips, automatic pipette (1000 µl), spectrophotometer, test tube rack, and glassware marking pencil.
24- to 48- hour nutrient broth cultures of Staphylococcus aureus, Alcaligenes faecalis; 48- to 72- hour Sabouraud broth cultures of Saccharomyces cerevisiae
Three brain heart infusion agar deep tubes per student group.
Bunsen burner, waterbath, iced waterbath, thermometer, sterile automatic pipettes (200 µl and 1000 µl), test tube rack, and glassware marking pencil.
Part 1. Temperature.
1) All plates were divided into 4 quadrants with marker and labeled with name of appropriate organism. Each plate was labeled with definite temperature (40C, 200C, 370C, or 600C) 2) Each of the species: E. coli, B. coagulans, P. savastanoi, and S. marcescens were inoculated into each plate using aseptic techniques. 3) Foure Sabouraud broth tubes were labeled including temperature of incubation as indicated in step 2. 4) The S. cerevisiae culture was shaken to suspend organisms. 50 µl of the culture were transferred by automatic pipette into each of the four tubes of broth media. 5. All plates were incubated in an inverted position and the broth cultures at each of the four experimental temperatures (40C, 200C, 370C, or 600C) for 24 to 48 hours.
Part 2. pH
1. Using a pipette, a series of the appropriately labeled TSB tubes of media were inoculated, pH values of 3, 6, 7, and 9, with E. coli by adding 0,1 ml of the saline culture to each. 2. Step 1 for the inoculation of A. faecalis and S. cerevisiae was repaeated, using a new sterile tip each time. 3. The A. faecalis and E. coli cultures for 24 to 48 hours at 370C and the S. cerevisiae cultures were incubate for 48 to 72 hours at 250C.
1. Using the spectrophotometer the absorbance of all cultures determined. The readings where recorded in the chart provided in the Lab Report. 2. In the second chart provided in the Lab Report, results were summarized to the overall range and optimum pH of each organism studied.
Part 3. Oxygen
1. Using aseptic technique, each experimental organism was inoculated by introducing two drop of the culture from a sterile Pasteur pipette into the appropriate labeled tubes of molten agar. 2. The freshly inoculated molten infusion agar was vigorously rotated between the palms of the hands to distribute the organisms. 3. Inoculated test tubes were placed in an upright position in the iced waterbath to solidify the medium rapidly. 4. The S. aureus and A. faecalis cultures were incubated for 24 to 48 hours at 370C and the S. cerevisiae cultures for 48 to 72 hours at 250C.
The experimental results are presented in three parts according to the physical factor temperature, pH and oxygen requirement. From the results in table 1 it can be noticed that all of the microorganisms investigated are mesophiles indicating that they grow at temperature range of between 20oC to 37oC. Also this results were expected as the mesophiles predominate in nature and this temperature range is the optimal range for cellular reactions as enzymatic activity is most efficient in this range. The enzymatic activity increases with an increase in temperature until its 3D structural shape changes, as it is highly ordered molecule involving active sites that have to be ideally permanent for successful reaction. Since, at higher temperatures the enzymes denature. The cardinal temperatures for microorganisms determined are minimal, maximal and optimal temperatures. Consequently the all microorganisms investigated appeared to be mesophiles with optimal temperature range within 20oC to 37oC. The Serratia marcescens’ activity due to temperature range is provided in table 1, where it can be noticed that the pigmentation rate is maximum at 20oC and decreases with increase in temperature.
This species produce specific red pigment called prodigiosin during reproduction and as a result this molecule is responsible to the degree of pigmentation. It was concluded that prodigiosin was produced at 20oC and the red pigmentation appeared, but at 37 oC its pigmentation rate is decreased as the pigment denatures due to relatively higher temperature. Serratia marcescens was classified as mesophile due to growth in temperature range between 20oC to 37oC. The next species of Pseudomonas savastanoi and Escherichia coli show abundant growth at 20oC to 37oC temperature range and were classified as mesophiles as the pigmentation degree was high and growth rate was abundant. Bacillus coagulans and Saccharomyces cerevisiae were also classified as mesophile as growth was identified in temperature range of 20oC to 37oC, however the growth rate was relatively lower in comparison with above described species with no pigmentation appeared. The only species that shown growth at 60oC are Saccharomyces cerevisiae.
Therefore the conclusion of this specie as mesophile is questionable as it might appear as thermophile also. However due to fact that results show the greater amount of growth at 20oC and the enzymatic activity was most efficient at this optimal temperature it was concluded that Saccharomyces cerevisiae is mesophile. Next, the microorganisms were investigated for effect of pH on microbial growth at different pH ranges:3,6,7 and 9. The incorporation of the buffers into the media is significant as due to metabolic activities the concentration of H+ and OH- ions change resulting in pH changes. The buffers are solutions that resist the significant shift in pH range. The table 2 summarizes the absorbance readings of the species made by spectrophotometer and table 3 illustrates related summary of pH for species. It is clear that the optimum pH for all species is varies among species. The species of E. coli have an optimum pH of 7, A. faecalis show the largest range varying from 4.6 to 9 and the last S. cerevisae have an optimum pH of 6.
The last factor is the requirement of oxygen. The species were classified as following: S. aureus is obligatory aerobe, because it shown the growth only at the top of the tube, consequently they require oxygen and can reproduce only in presence of oxygen. While the next species A. faecalis shows the growth distributed along the tube and more abundant at the top of tube. Since, it is obvious that this species are facultative anaerobes, those that are able to grow in the absence of oxygen by fermentation processes. However reproduce effectively at the presence of oxygen, due to fact that aerobically ATP is synthesized that is the best energy carrier rather than products of fermentation. The reason for abundant growth at the top is the growth using oxygen, as a result more effective, while the growth distributed along the tube is lower in amount.
The last species are S. cerevisae, those shown the abundant growth near the top, indicating that these species are microaerophilic which require oxygen but at lower concentrations in comparison with obligate aerobes or facultative anaerobes. As a result, due to fact that agar was solidified preventing oxygen supply the area at the bottom of the tube contains oxygen at low concentrations. Since it is obvious that microaerophilic species, those that utilize oxygen at lower conectrations shown growth distribution near the top, where the oxygen concentration is lower.
Conclusion: The purpose of the experiment was to investigate the physical factors affecting the microbial growth of species. From the results in tables it can be concluded that the expected results were obtained. The reliability and validity of the results were ensured by repeating the unsuccessful steps and following the procedure in manual. Also most of the time provided was used ensuring the precision and accuracy of the results.
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