Cultivation 24 hours, aerobic atmosphere, 37°C. Growth occurred on the nutrient agar and this new growth was also Gram stained. This resulted in acid production as opposed to the negative acid production on the MSA plate. Eosin-methylene blue (EMB) agar plate inoculated with Escherichia coli (a gram-negative coliform bacterium) showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose and acid production which precipitates the green metallic pigment. Mannitol Salt Agar (MSA) is a selective and differential medium. Hardy Diagnostics Nutrient Agar formulations are generalpurpose growth media recommended for use in the isolation andcultivation of nonfastidious microorganisms. Sheep blood is commonly used, but some organisms require rabbit or bovine blood. McDonald, V., Thoele, M., Salsgiver, B., & Gero, S. (2011). By CPR Nashville, TN at January 19, 2014 | 9:14 am | Staphylococcus epidermidis is a gram-positive spherical bacterium that form irregular clusters. American Heart Association CPR Classes, Nashville, Staphylococcus epidermidis | Microbiology Lab Report, Pseudomonas aeruginosa Report | Microbiology Unknown Lab, Causes and Effects of Spondylolisthesis by Caroline Marshak, What Are Hepatocellular Adenomas By Samantha Jespersen, What is Narcolepsy by Clarence Janiszewski, Example of an Unknown Lab Report in Microbiology | BLS-ACLS-PALS Classes, Nashville | AHA CPR Classes, New Articles on Health and Disease | Nashville TN, Healthcare Student Papers | Nursing Students, AED’s – Automated External Defibrillators. EMB Plate Results: Staphylococcus epidermidis (no growth), E. coli (dark purple), Pseudomonas aeruginosa (bottom), Enterobacter aerogenes (left) Bacteria was taken from the MSA plate and inoculated onto a Nutrient Agar plate to grow an isolated colony. The hosts for the Staphylococcus epidermidis are humans and other warm-blooded animals. It is recommended for the detection and enumeration of coagulase-positive Staphylococci in milk, food and other specimens and encourages the growth of a group of certain bacteria while inhibiting the growth of others. The first procedure accomplished was an isolation streak on nutrient agar. Specific S. epidermidis strains have developed antibiotic resistant genes. (2012, Nov 1). FIG. Urea was tested on two separate occasions and both results were negative. Fastidious organisms, such as streptococci, do not grow well on ordinary growth media. This nutrient agar plate was placed in the incubator at 37 degrees Celsius for five days. Microbiology, 7(8), 555-567. Lab Manual (1). The pH indicator phenol red remains red (alkaline). Lab Manual (1) to identify unknown bacteria. Enumeration of Bacteria / Dilutions / Lab Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice. The 7.5% concentration of sodium chloride results in the partial or complete inhibition of bacterial organisms other than staphylococci. Eosin–Methylene Blue (EMB) Agar is a differential medium for the detection of Gram negative enteric bacteria. Bacteria, viruses and fungi have been a leading cause of death in the history of mankind. The lab instructor discussed the validity of the Mannitol Broth over the MSA plate. no. B: Gamma-hemolytic (non-hemolytic) colonies of Staphylococcus epidermidis on sheep blood agar. The growth was also determined to be Gram-positive cocci and this was the sample used to accomplish the remaining tests. Tortora, G., Funke, B., & Case, C. (2013). Otto, M. (2009). Requirements for the Cultivation of Bacteria / AG 5010 Escherichia coli (Gram negative) - No growth. S. epidermidis is a microorganism that demonstrates commensalism with the human body. Since this microorganism mainly lives on the skin, it commonly enters the body through insertion of an indwelling medical device such as a catheter. Microbiology an introduction. It is part of the normal human flora, typically the skin flora, and less commonly the mucosal flora. Both tests displayed a negative result after incubation. S. epidermidis produces exopolymers that help it evade this phagocytosis. S. epidermidis is commonly the cause of infections based on medical devices. S. epidermidis is the species most commonly isolated from human epithelia according to the Microbiology textbook (3). A: Colonies of Staphylococcus epidermidis on sheep blood agar. In 1918, another form of EMB agar was described by Levine. These microorganisms affect us in both a beneficial and harmful way and it is necessary to understand how this works. Cat. After incubation, the nutrient agar plate was examined to determine if isolated growth had been obtained (1). III. This was done in an attempt to grow the Gram-positive bacteria from the original sample. Since MSA is a selective media, which selects for Gram-positive bacteria, a Gram stain was accomplished on this growth to determine if it was the Gram-positive bacteria. This is the most common antibiotic used against Staphylococcus infections. 766-3524 Blood agar is a rich medium that has been supplemented with fresh 5-10% blood. Print. Although S. epidermidis is not usually pathogenic, patients with compromised immune systems are at risk of developing infection. The EMB agar is the preferred one because it is more stable and more sensitive when compared to other types of agars. With this discovery, the original sample was inoculated on to Mannitol Salt Agar (MSA). Biofilms are often resistant to antibiotics and host defenses (5). Wang, R., Kahn, B., Cheung, G., & Bach, T. (2011). Each test was performed in accordance with the lab manual by McDonald et al. This new data pointed to Escherichia coli. Hardy Diagnostics EMB Agar formulations are recommended for use as selective and differential media for the isolation of gram-negative bacilli (including coliform organisms and enteric pathogens) from clinical and nonclinical specimens. Staphylococcus epidermidis is a Gram-positive bacterium, and one of over 40 species belonging to the genus Staphylococcus. It is most frequently found on the head, in the nares and axillae. This results in a heavy burden on the medical community (4). To determine the Gram reaction of the bacterium based on cell wall, Crystal violet, Iodine, Alcohol, Safranin, To determine the ability of the bacterium to ferment Mannitol, To determine the ability of bacterium to use Citrate as sole source of Carbon, Negative result for production of citrate permease, To determine the ability of the bacterium to reduce urea with the enzyme urease, Negative result for production of the enzyme urease, To determine the ability of the bacterium to reduce tryptophan to indole with the enzyme tryptophanase, To determine if bacterium can reduce sulfur to hydrogen sulfide gas with the enzyme thiosulfate reductase, To determine the final products of glucose fermentation, Positive result indicates presence of a mixture of acids, To determine if acetoin is produced from pyruvic acid during glucose fermentation, To determine if bacterium ferments mannitol, Positive result for mannitol fermentation, To determine if bacterium ferments lactose, To determine if bacterium ferments lactose and the strength of acid end products, Positive result for lactose fermentation with strong acid end products, To determine if bacterium reduces nitrate to nitrite, Nitrate Reagent A, Nitrate Reagent B, Zinc, No color change after adding Nitrate Reagent A & B, no color change after adding Zinc, FLOWCHART FOR GRAM NEGATIVE BACTERIA According to the lab Unknown Chart, E. coli should exhibit a negative reaction for nitrate reduction. Table 1: Differential characteristics of the genus Staphylococcus. 2. nitrate reduction test results following growth in nitrate broth. The tests accomplished were from the Unknown Chart distributed by the lab instructor. Once the streak method had been performed, the nutrient agar was incubated at 37 degrees Celsius for 48 hours. Principle of Mannitol Salt Agar. Culture conditions for S. epidermidis are similar to those of S. aureus (see 5.1.1.1), but S. epidermidis grows slowly in medium with 10% NaCl. Staphylococcus epidermidis (growth on MSA white medium) Staphylococcus aureys (growth on MSA yellow medium) How did the results observed on the MSA and EMB agar correlate to the gram rxn of the bacteria? ManConkey agar is a selective and differential agar medium that contains bile salts, lactose and the dye crystal violet that is known to inhibit the growth of Gram positive bacteria. 3. The Gram-negative bacteria was inoculated into Mannitol Broth and incubated. (adsbygoogle = window.adsbygoogle || []).push({}); I. Staphylococcus epidermidis (Gram positive) - growth but no yellow halo. Psuedomonas aeruginosa (Gram negative) - no growth. Streptococci are often classified based on hemolysis which can be seen by their reaction on blood agar. Final pH 7.4 ± 0.2 at 25°C. Biofilm associated infections are difficult to treat and eradicate. The tests that led to the conclusion of a Gram positive (+) bacteria were mannitol, catalase, Blood agar, and Spirit Blue agar tests. There is no mannitol fermentation and, therefore, no acid production. It prefers to grown on a salty or saline medium in order to grow at all. Lab manual for general microbiology. The instructor informed students that each tube contained a Gram positive and Gram Negative bacteria. MATERIALS AND METHODS: The lab instructor distributed a test tube of unknown bacteria labeled 108. Phenylethyl alcohol agar (PEA) is a selective medium that permits the growth of gram-positive cocci while inhibiting most gram-negative organisms. EMB Agar: Composition, uses and colony characteristics EMB Agar: Composition, uses and colony characteristics ... bacterial growth medium. (1). S. epidermidis belongs to a group of coagulase-negative Staphylococci, which is distinguished by a lack of the enzyme coagulase. rwatson@uwyo.edu, The Virtual Edge by http://www.uwyo.edu/virtual_edge/ is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 United States License, Nutritional It is a facultative anaerobic bacteria. The original sample number 108 was inoculated onto an MSA plate since this media selects for Gram-positive bacteria. The nitrate test was performed twice and both times a positive result was achieved. aureus is a lactose fermenter on EMB plate. 1. Some strains of Salmonella and Shigella may fail to grow on EMB Agar. Mannitol Salt Agar (MSA) is used as a selective and differential medium for the isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens. Research now shows it to be an important opportunistic pathogen that is a common cause of nosocomial infections. Cultivation 24 hours in an aerobic atmosphere, 37°C. This time, the agar can differentiate fecal and non-fecal types of coli aerogenes group as well as salmonellae and other types of non-lactose fermenting organisms. After incubation, the MSA plate displayed isolated colonies and turned yellow demonstrating Mannitol fermentation. Staphylococci are commonly found on the skin and in the mucous membranes of humans and other mammals. These infections are rarely life threatening, but they are difficult to treat. The most common resistance seen in hospitals is Methicillin resistance. Mannitol Salt Agar. The relationship between microbes and the human body is a very important topic to study. Some gram-positive bacteria, such as enterococci, staphylococci, and yeast will grow on this medium and usually form pinpoint colonies. Click to … S. epidermidis is the species most commonly isolated from human epithelia according to the Microbiology textbook (3). Staphylococcus aureus is not a fastidious organism and grows well in many different types of media. Retrieved from http://www.microbelibrary.org/library/2-associated-figure-resource/3762-nitrate-reduction-test-results-following-growth-in-nitrate-broth. Staphylococcus aureus (Gram positive) - growth with yellow halo. The observations after the incubation on the Mannitol Salt Agar concluded that the bacterium ferments mannitol. Streak plate of M. luteus colonies growing on TSY agar, 3, 4 & 5. After 48 hours of incubation, the MSA plate that was inoculated with the original sample from tube 108 was interpreted. Lab Manual (1) have been used to identify the unknown bacteria. The appearance of the growing colony was a cloudy, milky white. One test for the Gram-negative bacteria was not consistent with the Unknown Chart results. Blood agar is a type of ... staphylococcus aureus, staphylococcus epidermidis, pseudomonas sp, and Escherichia coli. This growth was then used to inoculate a new nutrient agar plate with the streak method to ensure isolation of one bacterium. One test was not consistent with the Unknown Chart results for Staphylococcus epidermidis. Most nosocomial infections are due to biofilm on medical equipment (3). S. Epidermidis was previously viewed as a common commensal microorganism that produced little harm to its human host. Mar 27, 2015 - EMB plate result - Staphylococcus epidermidis (no growth), E. coli (dark purple), Pseudomonas aeruginosa (bottom), Enterobacter aerogenes (left) Agar (Figure 1). The high concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels.Organisms from other genera may grow, but they typically grow very weakly. Staphylococcus epidermidis is a gram-positive spherical bacterium that form irregular clusters. The genus Streptococcus is a complex group causing a wide range of diseases such as: rheumatic fever, impetigo, pharyngitis, laryngitis, toxic shock syndrome, scarlet fever, and endocarditis. This Gram Stain was then observed at 100X using a compound light microscope and immersion oil. This plate was then incubated at 37 degrees Celsius for five days. Internet research suggests that E. coli does reduce nitrate and should exhibit a positive result to the nitrate test (2). This led to further testing of Methyl Red and Catalase. © 2014 BLS-ACLS-PALS Classes, Nashville | AHA CPR Classes All rights reserved. Although S. epidermidis is not usually pathogenic, patients with compromised immune systems are at risk of developing infection. This plate demonstrated cloudy growth and yellow agar where the growth was present. This bacterium was then Gram stained using the procedures from the McDonald et al. After determining the Gram reaction, specific biochemical tests were accomplished to determine the identity of the Gram-negative rods. A few strains of S. epidermidis also produce an exotoxin that has the ability to lyse human neutrophils. II. Once examined, it was determined that only one species of bacteria had been isolated. Distilled Water = 1000 ml. It is a highly diverse species with 74 different species types. Mannitol broth was then inoculated with the Gram-positive bacteria and a negative result was achieved. The results from these tests concluded that the Gram-positive bacteria were Staphylococcus epidermidis. The hemolytic response can be dependent upon the type of blood. (adsbygoogle = window.adsbygoogle || []).push({}); There were five possibilities of Gram positive (+) bacteria; Bacillus cereus, Bacillus subtitis, Staphylococcus aureus, Straphylococcus epidermidis, and Enterococcus faecalis . Mannitol Salt Agar contains peptones and beef extract, which supply nitrogen, vitamins, minerals and amino acids essential for growth. All of the following tests were performed on this unknown: Table 1 lists the test, purpose, reagents and results for the Gram-negative bacteria. Requirements for the Cultivation of Bacteria, https://docs.google.com/forms/d/1yGbkF0KM92WBSk-IgS-EkjxkTKTQwhzuXmDsVpwRDoU/viewform, Creative Commons Attribution-Noncommercial-Share Alike 3.0 United States License. Incubation, the original sample from tube 108 was inoculated onto an MSA plate since this media selects for bacteria. Gram-Negative bacteria was inoculated on to mannitol Salt agar humans and other mammals immune systems at! 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Manual was used for this procedure because it is possible that there was something wrong with lab... Or saline medium in order to grow on EMB agar is a very important topic to study Stain. Determine the identity of the growing colony was a cloudy, milky..