A Brief on Bacterial Investigation

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Introduction

Mayonnaise when left outside for a long period of time will spoil. Spoilage of mayonnaise results from a variety of causes including growth of microorganisms or contamination. Yeast and bacteria are the main causes for the spoilage of mayonnaise. Previous studies on Staphylococci and Salmonela found in mayonnaise done by Wethington and Fabian (1) found that higher acid content inhibits microbial growth. Similary, Appleman et al (2) found Bacilus subtilis to be abundant in mayonnaise. Other studies suggested that initial pH of the product as well as storage temperature plays an important role in spoilage of mayonnaise (3). Unsanitary handling and preparation of foods in home kitchens and foodservice operations pose the greatest threat of bacterial contamination of food. In our previous experiment Staphylococcus epidermidis was isolated from a sample of spoiled mayonnaise. However same sample of mayonnaise contained Staphylococcus lentus, Bacilus badius, Bacilus laterosporus, Micrococcus varians. S.epidermidis is part of our skin flora and due to contamination is one of the most common bacteria found in laboratory tests. In order to further analyze S. epidermidis additional tests were performed in the laboratory including Gram staining, Kirby-Bauer test for antibiotic susceptibility, Biofilm formation and Antibiotic production. Based on previous academic research we hypothesize that S. epidermidis will be resistant to most of the antibiotics including: Ampicilin, Erytromycin, Chloramphenicol, Penicilin, Streptomycin, Tetracycline, and will be sensitive to Sulfamethoxazole. Also S. epidermidis will form biofilm.

Material and Methods
A sample of mayonnaise bought at a grocery stored was left outside for 24 hours. Staphylococcus epidermidis was isolated from the sample and used for further testing. Muller Hinton agar in 150mm petri plate along with Antibiotic impregnated disks:(Ampicillin , Erythromycin,Chloramphenicol, Penicilin, Streptomycin, Sulfamethoxazole-trimethoprin , Tetracycline); LB medium , Filtered Crystal Violet , 1xPBS; 95%Ethanol; TSA plate, culture of E.coli, S.aureus, B.subtilis, and antibiotic solution. The CSU Fullerton Microbiology laboratory supplied all the materials. Gram Staining

This method was used to prove that S.epidermidis is a gram-positive bacterium as determined on our previous experiment. A smear of S.epidermidis was prepared on a clear slide, the smear was covered with Crystal violet for one minute and then rinsed with water; gram’s iodine was added to interact for one minute and rinsed with water; 95%ethanol was applied for 10-20 seconds and rinsed with water; and Safranin was added for 20 seconds and then rinsed with water. The slide was observed with a Light Microscope set in Kohler Illumination. Kirby-Bauer

This method was used to measure antibiotic susceptibility.
A known concentration of Streptococcus epidermidis was spread across a plate containing Mueller Hinton agar. Antibiotic impregnated disks were placed on the plate and the plates were incubated for 24 hours. Biofilm Formation

Method used to determine if Streptococcus epidermidis will grow a biofilm and measure the biofilm associated bacteria. 2 ml of LB medium was added into the well plate, 200uL of Streptococcus epidermidis was inoculated into duplicate wells; two wells were used as control. The well plate was incubated at 30C˚. The liquid portion was aspirated and each well was stained with 1ml Crystal Violet and let sit for 30min. The stain was aspirated and the well was rinsed four times with 1xPBS. 95%Ethanol was added (6ml) and let sit for 5minutes to dissolves the biofilm. A spec tube was prepared using 3mL from well and OD was read at 590nm. Antibiotic production

This method was used to determine if Streptococcus epidermidis produce any antibiotics in presence of other bacteria. On a TSA plate E. coli, S. aureus and B. subtilis were streaked parallel at equal distance from each other...
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