microbiology project help!

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Name:

Date:

xxx College

Biology 210 – Microbiology

Title: Investigating the Bacterium on the Door Handle

Abstract:

The purpose of this experiment was to investigate the bacteria found on the door handle of a bathroom at xxx College. A sterile swab was taken from a bathroom near the security table, in front of One Stop. After doing an isolation streak, some biochemichemical tests, such as: gram stain, mannitol salt agar (MSA), bile esculin, blood agar plate (BAP), catalase and coagulase were conducted. The bacterium was identified as Staphylococcus aureus (William 2013). This bacterium is responsible for skin infection, respiratory disease, and food poisoning (Mandal 2013).

Introduction:

Bacteria are mostly known for causing disease or harming the health of humans. But not all bacteria should be viewed this way; some are essential to our existence, normal biota, and keep our food chain from collapsing (Williams 2013). Others prove beneficial to humans, such as those that are found naturally in some of our favorite foods (Williams 2013). Most of the times, bacteria can be found at some places we thought was bacteria-free (Williams 2013). In fact, bacteria are everywhere (Williams 2013). The number of estimation of the total number of bacteria on earth is five million trillion trillion, that's a five with 30 zeroes after it (Whitman 1998). With that much numbers, bacteria perform tasks that include everything from causing disease to fixing nitrogen in the soil (Williams 2013). Therefore, a proper identification of bacteria is needed to discover new innovations and possible cures for existing or future diseases (Williams 2013). The investigation of these new bacteria can also help incubate ourselves therefore we could act quickly in response to an invasive strain (Williams 2013). Knowing the advantages of a proper identification of unknown bacteria, this experiment was conducted (Williams 2013). The hypothesis of this experiment is that if everyone washes their hands properly before leaves the bathroom, the door-handle-going-out the bathroom should be pathogenic-bacteria free.

To prove whether or not this hypothesis was true, some biochemichemical tests, such as: gram stain, mannitol salt agar (MSA), bile esculin, blood agar plate (BAP), and coagulase were conducted. Gram stain was needed to determine the morphology and gram status of the bacteria, so appropriate tests could be demonstrated. The first biochemical test was conducted on a MSA plate. MSA has an osmotic pressure created by 7.5% NaCl, while this concentration will inhibit the growth of most other gram-positive and gram-negative bacteria (Williams 2013). Additionally, MSA contains mannitol and uses phenol red as a pH indicator in the medium (Williams 2013). At pH levels below 6.8, the medium is a yellow color (Williams 2013). In the neutral, pH ranges 6.8 to 8.4, the color is red; while above pH 8.4, the color of phenol red is pink (Williams 2013). When mannitol is fermented by a bacterium, acid is produced, which lowers the pH and results in the formation of a yellow area surrounding an isolated colony on MSA (Williams 2013). A nonfermenting bacterium that withstands the high salt concentration would display a red to pink area due to peptone breakdown (Williams 2013).

Bile esculin test was performed next. Bile esculin agar is a selective and differential medium, which is used to identify the ability of an organism to hydrolyze or break down esculin (Williams 2013). Esculin is a glycoside, a sugar molecule bonded by an acetyl linkage to an alcohol, composed of glucose and esculetin (Williams 2013). These linkages are easily hydrolyzed under acidic conditions (Williams 2013). Many bacteria can hydrolyze esculin, but few can do so in the presence of bile (Williams 2013). Organisms that split the esculin molecules and use the liberated glucose to supply energy needs release esculetin into the medium (Williams 2013). The free esculetin reacts with ferric citrate in the medium, which turns the agar slant dark brown to black (Williams 2013).

BAP test was performed next. BAP is a general-purpose enriched medium often used to grow fastidious organisms and to differentiate bacteria based on their hemolytic properties (Williams 2013). Blood agar is usually prepared with 5% Sheep red blood cell (Williams 2013). Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins (Williams 2013). Bacteria can produce different types of hemolysins (Williams 2013).  Beta-hemolysin breaks down the red blood cells and hemoglobin completely (Williams 2013). This leaves a clear zone around the bacterial growth (Williams 2013). Such results are referred to as β-hemolysis (beta hemolysis) (Williams 2013). Alpha-hemolysin partially breaks down the red blood cells (Williams 2013). This is referred to as α-hemolysis (alpha hemolysis) (Williams 2013). If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur (Williams 2013).  This is called γ-hemolysis (gamma hemolysis) (Williams 2013).

Catalase test was performed next. This test detects the enzyme catalase in bacteria (Williams 2013). The catalase enzyme serves to neutralize the bactericidal effects of hydrogen peroxide (Williams 2013). Catalase expedites the breakdown of hydrogen peroxide (H2O2) into water and oxygen (Williams 2013). This reaction is evident by the rapid formation of bubbles (Williams 2013).

Last but not least, coagulase test was performed. The coagulase test identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot (Williams 2013). Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents (Williams 2013). Coagulase test is also used to determine the species of Staphylococcus (Williams 2013). This test differentiates strains of Staphylococcus aureus from S. epidermidis and other coagulase-negative species (Williams 2013).  S. aureus strains are usually capable of coagulating blood plasma in the tube test (Williams 2013).

Methods and Materials:

All protocols are from profession xxx, a professor in microbiology major, 2010.

Aseptic technique was used throughout the experiment.

· Isolation Streak

1. Flame a wire loop and select some colonies from the swab plate.

2. The bacteria will be streaked across the top of the Nutrient Agar plate – this is the first quadrant.

3. Flame the loop and let cool.

4. Pull one time from initial streak, and then squiggle out – this is the second quadrant.

5. Flame the loop and let cool.

6. Pull one time from previous streak, and then squiggle out – this is the third quadrant.

7. Flame the loop and let cool.

8. Pull one time from previous streak, and then squiggle out – this is the fourth quadrant.

9. Flame the loop. Invert the plate and place the plate in the 37oC incubator for 24 hours.

· Gram Staining

1. Prepare a wet mount by dripping one drop of water from spray bottle onto clean glass slide.

2. Flame a wire loop and select bacterial colony from the isolation plate.

3. Transfer the bacteria to the drop of water on slide and spread it around slightly to disperse the cells.

4. Using a clothespin to hold the slide, move slide back and forth over the top of a Bunsen burner flame. Continue until the water has evaporated from the slide.

5. Place slide over gram stain tray and begin the staining steps.

6. Add a few drops of crystal violet onto specimen (add enough stain to cover specimen) – let it sit for 20 seconds.

7. Rinse off stain gently using squirt bottle.

8. Add Gram’s iodine and let it sit for 1 minute – rinse it with water.

9. Decolorize with alcohol (decolorizing agent) – add drops of alcohol until the purple stops coming off of the specimen.

10. Rinse it with water.

11. Add safranin and let it sit for 20 seconds then rinse with water.

12. Let slide dry and then use microscope (up to 100X) to view cells.

13. Observe and record the result in Table 1.

· Making a Lawn

1. Flame a wire loop and select a colony from the isolation streak.

2. Use that colony to inoculate the entire nutrient agar plate.

3. Invert the plate and place the plate in the 37oC incubator for 24 hours.

4. Observe and record the result in Table 1.

· Mannitol Salt Agar (MSA)

1. Flame a wire loop and select a colony from the lawn.

2. Use that colony to inoculate the entire plate of mannitol salt agar.

3. Invert the plate and place the plate in the 37oC incubator for 24 hours.

4. Observe and record the result in Table 1.

· Bile Esculin

1. Flame a wire loop and select a colony from the lawn.

2. Inoculate the slant of the bile esculin.

3. Remove the loop and replace the cap.

4. Incubate for 37oC for 24 hours.

5. Observe and record the result in Table 1.

· Blood Agar Plate (BAP)

1. Flame a wire loop and select a colony from the lawn.

2. Use that colony to inoculate the center of blood agar plate.

5. Invert the plate and place the plate in the 37oC incubator for 24 hours.

6. Observe and record the result in Table 1.

· Catalase

1. Flame a wire loop and select a colony from the lawn.

2. Select a clean glass slide and spread the bacterial colonies on it.

3. Add 2 drops hydrogen peroxide to the colonies on the slide.

4. Observe and record the result in Table 1.

· Coagulase

1. Flame a wire loop and select a colony from the lawn.

2. Mix thoroughly with the 0.5 ml coagulase and then replace the cap on the tube.

3. Incubate at 37oC for 24 hours.

4. Observe and record the result in Table 1.

Results:

Table 1: Investigating an Unknown Bacterium

Test	Purpose	Observations	Results
Gram Stain	Determine the morphology and gram status of the bacterium.	Purple color, grape-like.	Gram (+) cocci
MSA	Distinguish bacteria based on the ability to ferment the sugar mannitol and tolerations of high salt concentration.	White colonies, medium turns yellow.	Mannitol was metabolized.
Bile Esculin	Determine whether a bacterium could break down esculin in the presence of bile.	Dark brown color.	Esculin broke down.
BAP	Differentiate bacteria based on their hemolytic properties.	Complete clearing of the plate.	Beta hemolysis.
Catalase	Determine whether a bacterium has the enzyme catalase.	Formation of bubble.	Catalase found in the bacteria.
Coagulase	Determine whether a bacterium produces coagulase.	Clot was produced.	Coagulase produced.

Discussion:

The results that came from the entire biochemical tests that have been demonstrated show that the unknown bacterium was Staphylococcus aureus (Williams 2013). This bacterium is responsible for skin infection, respiratory disease, and food poisoning (Mandal 2013). The hypothesis of this experiment was proven to be wrong.

Staphylococcus aureus is a facultative anaerobic Gram-positive coccal bacterium (Williams 2013). In medical literature the bacteria is often referred to as S. aureus or Staph aureus (Williams 2013). S. aureus is responsible for many infections but it may also occur as a commensal (Ross 2005) . The presence of S. aureus does not always indicate infection (Ross 2005). S. aureus can survive from hours to weeks, or even months, on dry environmental surfaces, depending on strain (Ryan et al., 2004). S. aureus can infect tissues when the skin or mucosal barriers have been breached (Ryan et al., 2004). This can lead to many different types of infections including furuncles and carbuncles (a collection of furuncles) (Kao & Lee, 2012). S. aureus infections can spread through contact with pus from an infected wound, skin-to-skin contact with an infected person by producing hyaluronidase that destroys tissues, and contact with objects such as towels, sheets, clothing, or athletic equipment used by an infected person (Kao &Lee, 2012). Deeply penetrating S. aureus infections can be severe (Kao & Lee, 2012). Strains of S. aureus can host phages that increase virulence (Kao & Lee, 2012).

Staphylococcus should not be confused with the similarly named and medically relevant genus Streptococcus (Mandal 2013). S. aureus appears as grape-like clusters when viewed through a microscope, and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates (Matthews et al., 1997). S. aureus reproduces asexually by binary fission (Mandal 2013). The two daughter cells do not fully separate and remain attached to one another (Mandal 2013). This is why the cells are observed in clusters (Mandal 2013). S. aureus is catalase-positive (meaning it can produce the enzyme catalase) (Williams 2013). Catalase converts hydrogen peroxide (H
2O
2) to water and oxygen (Williams 2013). Catalase-activity tests are sometimes used to distinguish staphylococci from enterococci and streptococci (Matthews et al., 1997). Previously, S. aureus was differentiated from other staphylococci by the coagulase test (Matthews et al., 1997). However it is now known that not all S. aureus are coagulase-positive and that incorrect species identification can impact effective treatment and control measures (Matthews et al., 1997).

S. aureus is associated in toxic shock syndrome (Todar 2013). It releases toxins that were absorbed into the bloodstream (Todar 2013). Any S. aureus infection can cause the staphylococcal scalded skin syndrome, a cutaneous reaction to exotoxin absorbed into the bloodstream (Todar 2013). The infection can be life-threatening (Todar 2013). Problematically, Methicillin-resistant Staphylococcus aureus (MRSA) has become a major cause of hospital-acquired infections, and is being recognized with increasing frequency in community-acquired infections (Liu et al., 2011).

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several difficult-to-treat infections in humans (Liu et al., 2011). It is also called oxacillin-resistant Staphylococcus aureus (ORSA) (Liu et al., 2011). MRSA is any strain of Staphylococcus aureus that has developed, through the process of natural selection, resistance to beta-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins (Liu et al., 2011). Strains unable to resist these antibiotics are classified as methicillin-sensitive Staphylococcus aureus, or MSSA (Sahebnasagh et al., 2011). The evolution of such resistance does not cause the organism to be more intrinsically virulent than strains of Staphylococcus aureus that have no antibiotic resistance, but resistance does make MRSA infection more difficult to treat with standard types of antibiotics and thus more dangerous (Sahebnasagh et al., 2011).

Although Staphylococcus aureus, could be really hard to get rid off, some prevention of Staphylococcus aureus transmissions are still possible to do (Williams 2013). Clean your hands frequently with soap and warm water or an alcohol-based hand rub will help reduce the chance of infections (Williams 2013). Keep your linens and clothes clean, and do not share personal care items, like razors, towels, or similar items with others (Minnesota Department of Health 2010).

Work Reference

Kao, Lillian S., and Tammy Lee. Surgery: PreTest Self-assessment and Review. 12th ed. New York: McGraw-Hill Medical, 2012. 88. Print.

Liu, C., A. Bayer, S. E. Cosgrove, R. S. Daum, S. K. Fridkin, R. J. Gorwitz, S. L. Kaplan, A. W. Karchmer, D. P. Levine, B. E. Murray, M. J. Rybak, D. A. Talan, and H. F. Chambers. "Clinical Practice Guidelines by the Infectious Diseases Society of America for the Treatment of Methicillin-Resistant Staphylococcus Aureus Infections in Adults and Children: Executive Summary." Clinical Infectious Diseases 52.3 (2011): 285-92. Print.

Mandal, Ananya, Dr. "What Is Staphylococcus Aureus?" What Is Staphylococcus Aureus? News Medical, n.d. Web. 18 Nov. 2013.

Matthews, Karl R., J. Roberson, and B. E. Gillespie. "Identification and Differentiation of Coagulase-Negative Staphylococcus Aureus by Polymerase Chain Reaction." Journal of Food Protection 6 (1997): 686-88. Print.

"Minnesota Department of Health." Preventing Staphylococcus Aureus Transmission. Minnesota Department of Health, 17 Nov. 2010. Web. 27 Nov. 2013.

Ross, Betsy McCaughey. Unnecessary Deaths the Human and Financial Costs of Hospital Infections. 2nd ed. [New York, N.Y.]: Committee to Reduce Infection Deaths, 2005. Print.

Ryan, Kenneth J., C. George Ray, and John C. Sherris. Sherris Medical Microbiology: An Introduction to Infectious Diseases. 4th ed. New York: McGraw-Hill, 2004. Print.

Sahebnasagh R, Saderi H, Owlia P. Detection of methicillin-resistant Staphylococcus aureus strains from clinical samples in Tehran by detection of the mecA and nuc genes. The First Iranian International Congress of Medical Bacteriology; 4–7 September; Tabriz, Iran. 2011. 195 pp.

Todar, Kenneth, Dr. "Staphylococcus Aureus." Staphylococcus Aureus. Online Textbook of Bacteriology, 2005. Web. 24 Nov. 2013.

Whitman, William B. Estimate of Total Bacteria on Earth. First-ever Estimate of Total Bacteria on Earth. University of Georgia, Sept. 1998. Web. 19 Nov. 2013.

Williams, Kim. Introduction to Microbiology. Great Bay Community College. 2013.