The Bacterial Specific Growth Medium - Lab Report Example

Culture Media: Introduction: Microorganisms require carbon, nitrogen, oxygen, phosphorous and nitrogen source for their growth and multiplication. Under laboratory conditions, medium is used for the growth of micro organisms such as solid media (simple and complex) and liquid media. These media are developed such that only specific microorganisms will grow in the media. (Selective and culture media). Methods: The bacterial specific growth medium for Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa were inoculated in the Synthetic medium, SF medium and Tryptic soy broth respectively and incubated at 32 °C for 3 – 4 days. The petriplate of Mac Conkey Agar was divided into five and Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa, Salmonella enteric and Enterococcus faecalis were inoculated in the sections. The plates were then incubated at 32 °C for 3 days and the results were recorded. The presence of enteric, lactose fermenting Gram positive and Gram negative cultures were identified using Mac Conkey Agar. Results: Organism Growth in Synthetic Medium Growth in Tryptic Soy broth Escherichia coli + + Enterococcus faecalis - + Pseudomonas aeruginosa + + Organism Growth in SF Broth Growth on Macconkey Agar and Color change Growth on Nutrient Agar and glucose Staphylococcus aureus Ve purple Enterococcus faecalis + ve yellow - + Salmonoella enterica - + Pseudomonas aeruginosa - + Esherichia coli + + Bacillus subtilis - + Discussion: Escherichia coli and Pseudomonas aeruginosa grows well in both the synthetic and Tryptic soy broth. Enterococcus faecalis was not able to grow in the synthetic medium and grew well in SF broth. All the bacteria grew well in Nutrient agar medium with glucose and only Escherichia coli grew in the Mac conkey agar medium. Conclusion: Bacteria are of two types based on Grams staining. Bacillus subtilis and Enterococcus faecalis are Gram positive and Escherichia Coli, Pseudomonas aeruginosa and Salmonella enterica are Gram negative. Mac Conkey Agar is the specific medium for Escherichia coli and only it grew in the medium. Thus using the synthetic and specific medium, the bacteria were identified. Enzymes: Introduction: Bacteria can be differentiated based on the enzymes secreted by them. Some enzymes are secreted out by the micro organisms enabling simple biochemical tests. The major exo enzymes present in bacteria are amylases, caseinase, gelatinase (hydrolytic enzymes), oxidase and catalase. Since most of the exoenzymes are hydrolytic enzymes which break down complex substances into simpler molecules, they are used as identifiers for the bacteria. Amylases hydrolyse the starch, gelatinase hydrolyses gelatin and caesinase converts casein into peptides and amino acids. Catalase converts hydrogen peroxide into oxygen and water and oxidases oxidizes dichlorophenol indophenols from colorless to blue or red. Method: Two plates of starch, casein and TSA media were taken and one set of the plates were inoculated with Bacillus subtilis and the other set of plates were divided in to 5 and the four cultures were inoculated and the last section was left as control. Spread plate technique was used to inoculate the culture and inverted and incubated for 24 – 48 hours at 32 °C. Organism Test Oxidase Amylase Caseinase Gelatinase Catalase Pseudomonas aeruginosa + - + + + Bacillus subtilis + + + + + Staphylococcus aureus - - - + + Enterococcus faecalis - - - - - Esherichia coli - - + - + Discussion: When the starch medium was flooded with Iodine solution, it was found that only bacillus subtilis was able to hydrolyse the starch indicating positive results. Pseudomonas aeruginosa was positive for all the enzymes except amylase. Enterococcus faecalis was negative for all the tests and concludes that it requires some other distinct methods for identification. Escherichia coli were positive for caesinase and catalase enzymes. Staphylococcus aureus was positive for gelatinase and caesinase. Conclusion: Biochemical tests helps to identify the bacteria which are morphologically similar and have similar physical characters. Biochemical tests are based on the exoenzymes synthesized by the bacteria. Bacteria such as Escherichia coli are positive for most of the basic tests and the differentiation between the enterobacteria and other gram positive bacteria are possible only through biochemical tests. Fermentation Introduction: Fermentation is nothing but the anaerobic oxidation of the substrates by the micro organisms. Sugars are fermented by most of the micro organisms. Sucrose, glucose and lactose were fermented by many micro organisms. The conversion of lactose into carbon-di- oxide and water is done by many bacteria. This can be tested using the Durham’s tube method. Eschrichia coli, Enterobacter aerogenes, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis were tested for their ability to ferment glucose, sucrose amd lactose with phenol red and Durham tubes. Method: All the six bacteria were inoculated in glucose, lactose and sucrose fermentation tubes for 3 days at 32 degree Celsius. The tubes were looked upon for the changes in the color and for the rise of the Durham tube.Result: Organism Growth Fermentation Glucose Sucrose Lactose Glucose Sucrose Lactose 2 – 4 days 2 – 4 days 2 – 4 days Escherichia coli + + + AG SA Acidic gas Enterobacter aerogenes + + + AG -A Acidic gas Salmonella enterica + - + AG - - NC Pseudomonas aeruginosa - + - R- N- No acid no gas Staphylococcus aureus + - + A- - Acidic no gas Enterococcus faecalis + + + A- A- Slightly acidic no gas Discussion: The presence of acidic gas was present in Escherichia coli and Enterobacter aerogenes bacteria. They were able to ferment all the three carbon sources. Salmonella enterica was positive only for lactose test and there was no color change in the inoculated medium. Enterobacter faecalis was also able to ferment all the carbon sources but there was no gas formation. Similarly Pseudomonas aeruginosa showed positive result only for sucrose and Staphylococcus aureus was positive for glucose and lactose. Conclusion: The fermentation of glucose, sucrose and lactose into carbon di oxide and water are performed only by some specific bacteria. The isolation of the bacteria was found to be very easy using the fermentation experiment and the further classification of the bacteria under different species. Motility: Introduction: The motility of the bacteria is found by wet mount and motility agar tests. Bacteria move with the help of flagella or through Brownian movement. Wet mount is the quick and easy method to identify the motility of the bacteria under a microscope but this method has greater chance for false results as the oxygen may deplete quickly and the bacteria may die or the medium dries of quick and the risk is more for the pathogenic bacteria. To overcome these difficulties hanging drop method is used. Motility agar medium is prepared and kept in the test tubes, after the inoculation of the medium, if the bacteria metabolize the dye triphenyl tetrazoniuim chloride in to simple products, the medium changes from colorless to red. If the bacteria are motile, then we can easily observe the path travelled by it in the medium. Methods: Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus were inoculated in the TSB broth and Glucose agar cultures present in the test tube and incubate for 1 – 2 days. Wet mount is prepared for each bacterial culture and the motility of the bacteria was observed under the microscope. Result: Organism Motility Wet mount Hanging drop slide Motility agar Bacillus subtilis Not moving Not moving Growth Obligate aerobic Pseudomonas aeruginosa Moving Moving Growth not obligate aerobic Staphylococcus aureus Not moving Not moving Growth not obligate aerobic Discussion: In the wet mount, Hanging drop slide and motility agar tests, it was found that Pseudomonas aeruginosa was the only bacteria which was motile. The results are same in all the tests. Bacillus subtilis and Staphylococcus aureus are not motile. Conclusion: The bacteria move from one place to another using the flagella and some Brownian movement. Pseudomonas aeruginosa is the motile bacteria and also aerobic. It is a non obligate aerobic bacteria. Differentiation of enteric bacteria: Introduction: Enterobacteriaceae family consists of Gram negative, non-spore forming facultative anerobic rods. This family is responsible for the diseases at the gastrointestinal tract. The family includes Escherichia coli, Enterobacter, Salmonella spp and Shigella spp. They are differentiated based on their ability to ferment lactose. The members of the Enterobacteriaceae are easily identified and differentiated using the Enterotube II. Method: The given bacterial cultures were inoculated in each of the Enterotube as per the procedure and incubated at 37 degree Celsius for 24 hours. Result: Test Organisms B. Subtilis C. glutamicum E.coli S. aureus E. faecalis E. aerogenes S.enterica P. aeruginosa Glucose + - + + + + + Slight - Lysine - - + - - + + - Ornithine - - + - - + + - H2S - - - - - - - - Adonitol - - - - - + - - Lactose - - + + - + - - Arabinose + - + - - + + + Sorbitol + - + - + + + - Dulcitol - - + Slight + - - Slight + - PA - - + - - - + - Urea + pinkish + - + slight - - pinkish - + Citrate - + - - - + + + Discussion: Bacillus subtilis was positive for Glucose, arabinose, sorbitol and Urea. Corynebacterium glutamicum was positive for Urea and Citrate test. Escherichia coli were positive for glucose, lysine, ornithine, and lactose, Arabinose, Sorbitol, Dulcitol and PA. Staphylococcus aureus was positive for glucose, lactose, Dulcitol and urea. Enterococcus faecaliswas positive for glucose and sorbitol. Enterobacter aerogenes was positive for almost all tests except Dulcitol, PA and H2S. Salmonella enterica was positive for Glucose, lysine, ornithine, Arabinose, Sorbitol, Dulcitol, PA, urea and Citrate. Pseudomonas aeruginosa was positive for urea, citrate and arabinose. Conclusion: Enterotube II test differentiates the Enterobacteria based on their ability to ferment specific substrate. By using the test eight different enterobacteria were identified and differentiated clearly. (Benson, 2001). References: Benson, HJ., 2001, Microbiological application: laboratory manual in general microbiology, McGraw –Hill. Read More