www.FrancheskaCamilo.com

Laboratory Exercise for Medical Microbiology:

Bacterial Identification of Gram – Positive Cocci

Prepared by: Francheska Camilo González

November 10, 2013

English version: February 1, 2014

i

ABSTRACT

Pathogenic bacteria such as Staphylococcus aureus, Streptococcus pyogenes and Streptococcus

pneumoniae belong to the group of gram-positive cocci, causing disease and nosocomial

infection that is increasingly affecting healthy individuals in the community. This has led to the

clinical and microbiological testing laboratory to be used consecutively to find treatments for

resistant bacterial strains and to identify the bacteria causing a specific disease and thus give the

antibiotic or treatment. Through this lab exercise is conducted the catalase test, Gram stain,

hemolysis, and susceptibility test to bacitracin and optochin in pathogenic bacteria

(Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes). It was

determined that the Staphylococcus aureus is a catalase positive, Streptococcus pneumoniae is

susceptible to Optochin, and Streptococcus pyogenes is susceptible to bacitracin. It also was

found that Staphylococcus aureus and Streptococcus pyogenes are beta hemolysis, whereas,

Streptococcus pneumoniae is alpha hemolytic and that the three bacteria are gram-positive.

Keywords: Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes,

Microbiology Laboratory, laboratory tests, the gram-positive cocci

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CONTENT Prepared by: Francheska Camilo González ......................................................................................................... i

November 10, 2013 ........................................................................................................................................... i

English version: February 1, 2014 ....................................................................................................................... i

ABSTRACT ....................................................................................................................................................... i

ILLUSTRATIONS ............................................................................................................................................ iii

INTRODUCTION ............................................................................................................................................. 1

STAPHYLOCOCCUS ........................................................................................................................................ 2

STAPHYLOCOCCUS AUREUS .......................................................................................................................... 3

Figure 1: Staphylococcus aureus ........................................................................................................... 4

Figure 2: Cutaneous abscess located on the back and caused by Methicillin-resistant Staphylococcus

aureus bacteria (MRSA). ....................................................................................................................... 5

STREPTOCOCCUS........................................................................................................................................... 6

Figure 3: Streptococcus pneumoniae ................................................................................................... 6

STREPTOCOCCUS PYOGENES ........................................................................................................................ 7

Figure 4: Pharyngitis by Streptococcus pyogenes ................................................................................. 7

STREPTOCOCCUS PNEUMONIAE ................................................................................................................... 8

Figure 5: Pneumoonia-infected right lung by Streptococcus pneumoniae .......................................... 8

Table 1: Diseases and infections by Staphylococcus aureus, Streptococcus pyogenes, and

Streptococcus pneumoniae .................................................................................................................. 9

Part 2: Practice of Medical Microbiology Laboratory to identify gram-positive cocci ............................... 11

Laboratory group: Kelvin Garced , José Noy , Francheska Camilo .............. 11

BACKGROUND ............................................................................................................................................. 11

MATERIALS / METHODS .............................................................................................. 12

Figure 6: Hemolysis Beta (β) for the bacteria Streptococcus pyogenes and Staphylococcus aureus in

blood agar plate (Laboratory exercise: plate 3) .................................................................................. 13

RESULTS ...................................................................................................................................................... 14

CONCLUSION ............................................................................................................................................... 16

REFERENCES ................................................................................................................................................ 18

iii

ILLUSTRATIONS

Figure 1: Staphylococcus aureus ……. ………………. 4

Figure 2: Cutaneous abscess located on the back and caused by Methicillin-resistant

Staphylococcus aureus bacteria (MRSA) …… 5

Figure 3: Streptococcus pneumoniae …… 8

Figure 4: Pharyngitis by Streptococcus pyogenes…… 9

Figure 5: Pneumoonia-infected right lung by Streptococcus pneumoniae…… 11

Figure 6: Hemolysis Beta (β) for the bacteria Streptococcus pyogenes and Staphylococcus

aureus in blood agar plate (Laboratory exercise: plate 3) …… 15

1

INTRODUCTION

The presence of microorganisms such as viruses, fungi, parasites, and pathogenic bacteria,

causes the use of microbiological techniques is needed to identify these infectious agents in the

urine, feces, blood, sputum, and in other body fluids, in persons with compromised immune

systems or diseases related to these microorganisms. Microbiologists consistently perform

immunological tests and other experiments chemical, genetic and microbiological, where

methods and research techniques are used to identify pathogens. The sputum culture, stool test,

urine culture, sensitivity of burns and wounds, and blood cultures are examples of

microbiological tests. Microbiologists also use microbiological tests such as Gram stain, catalase

test , and test sensitivity to optochin and bacitracin, among other experimental methods of

analysis to identify bacteria in a group composed of different species, as an example, our

laboratory exercise, where we made an analysis using these experimental tests to identify

bacteria Cocci Gram-positive (Streptococcus pneumoniae, Streptococcus pyogenes and

Staphylococcus aureus), which cause diseases and serious infections .

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Part 1: What are the Gram-positive Cocci?

Gram - positive cocci are spherical bacterial cells. These bacteria are characterized by reaction to

Gram staining, and the absence of endospores [1]

. In this group of bacteria was analyzed

Staphylococcus aureus species of genus Staphylococcus, and species Streptococcus pneumoniae

and Streptococcus pyogenes, of genus Streptococcus.

STAPHYLOCOCCUS

Staphylococcus, also known as staphylococci, is a bacterial genus of gram - positive cocci that is

located in the normal flora in the skin and mucosal surfaces. These bacteria can be classified as a

pathogenic group that can cause infections in the skin, soft tissues, bone and genitourinary tract.

These cells have a pattern like a bunch of grapes, but in samples at the clinical level, these

microorganisms can occur as single cells, in pairs, or short chains. Most of these bacteria are

facultative anaerobic (can increase in anaerobic or aerobic environments), and are able to grow

in media with elevated concentrations in salt, and in temperatures of 18- 40°C .

3

STAPHYLOCOCCUS AUREUS

Staphylococcus aureus is a bacterium that is gram-positive, catalase-positive, and is

characterized by presence of a thick layer of peptidoglycan. These bacteria without outer

membrane can be found in the normal flora of the skin, mucosal surfaces, and spread is from

contact between people or with contaminated surfaces. Staphylococcus aureus can survive in dry

areas for long periods of time. The virulence factors of this bacterium are the structural

components (Capsule, Layer of extracellular polysaccharides, peptidoglycan, teichoic acid,

protein A), toxins (Cytotoxins, exfoliative toxins (ETA, ETB), enterotoxins, Toxin 1 TSS), and

enzymes (Coagulase, Hyaluronidase, Fibrinolysin, lipases, and nucleases ). Risk factors are the

presence of foreign bodies (prosthesis, catheter), previous surgery, and the use of antibiotics that

inhibit the normal microbial flora necessary in the human body. MRSA (Staphylococcus aureus

resistant to methicillin) is a bacterium that causes severe nosocomial infections, and extra-

hospital infections in children and adults previously healthy .

Antibiotics that should be used to Staphylococcus aureus are

Oxacillin (or other penicillinase-resistant penicillins)

Vancomycin (for strains that are resistant to oxacillin)

Trimethoprim-sulfamethoxazole, clindamycin, linezolid, quinupristin-dalfopristin or

daptomycin (All antibiotics mentioned are alternatives that may be used as antibiotics to

treat infections caused by MRSA).

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Staphylococcus aureus Resistant to Methicillin (MRSA) is a major problem worldwide, as this

has become one of the causes of skin infection, and in more recent cases, is the cause of invasive

infections in children and healthy adults in the community In Research Publication,

"Vancomycin versus linezolid in the treatment of methicillin -resistant Staphylococcus aureus

meningitis in an experimental rabbit model", is compared the antibacterial efficacy of treatments

such as linezolid and vancomycin in an experimental rabbit model (MRSA) meningitis. In this

study, meningitis was induced by intracisternal inoculation using the ATCC 43300 strain. After

16 hours of incubation and development of meningitis, the control group received no antibiotic,

the experimental group "vancomycin" received (vancomycin 20mg/kg) every 12 hours, the

experimental group "linezolid 10" received (linezolid 10mg / kg) every 12 hours and the

experimental group "linezolid 20" received (linezolid 20mg/kg) every 12 hours. He proceeded to

count bacteria from cerebrospinal fluid to 16 hours of incubation and 24 hours after treatment,

and found that at 16 hours, the bacterial count was similar in all experimental groups, but at the

end of treatment (24 hours) in the vancomycin group the value obtained was 2 logs higher than

obtained for the linezolid group 20 (p> 0.05), and 4 logs higher than obtained for the linezolid

Figure 1: Staphylococcus aureus Source: Samaritan Infectious Disease page.

<http://www.samaritanid.com/staphylococcus_aureus.html> Accessed: 11/8/13.

5

group 10 (p=0.037). Obtaining the full or partial bacteriological response was greater in

vancomycin vs. "Linezolid-10", but not in the comparison of the results obtained for vancomycin

versus "linezolid-20". Therefore, by this research is suggests that treatment "linezolid" is not

statistically inferior to treatment with vancomycin, when these were tested in the treatment

(MRSA) meningitis, using an experimental model of rabbit at a dose of linezolid 20 mg / kg, but

the treatment was less for a dose of linezolid when being used 10 mg / kg .

Figure 2: Cutaneous abscess located on the back and caused by

Methicillin-resistant Staphylococcus aureus bacteria (MRSA). Photo credit: Gregory Moran, M.D.

Source: Centers for Disease Control and Prevention page.

<http://www.cdc.gov/mrsa/community/photos/photo-mrsa-10.html>

Accessed: 11/8/13.

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STREPTOCOCCUS

The Streptococcus is bacteria that are gram-positive cocci, which are arranged in pairs or chains.

Most are facultative anaerobes, but some only grow in an atmosphere enriched with carbon

dioxide (CO2). Streptococci are bacteria that for be isolated need an enriched blood or serum,

and they can ferment carbohydrates and generate lactic acid by this process. The species of

streptococci are catalase-negative, and these stand out as pathogenic bacteria to humans. Species

of this genus are classified by the following three systems: 1) the serological properties:

Lancefield groups (A - W); 2) hemolytic patterns: complete hemolysis (beta [β]), incomplete

hemolysis (alpha [α]) and absence of hemolysis (gamma [γ]); and 3) the biochemical properties

(physiological) .

Figure 3: Streptococcus pneumoniae Source: International Society of Microbial Resistance page.

<http://www.microresistance.org/amdr.cfm?CEProgramID=95&HeaderCEPr

ogramContentID=85> Accessed: 11/8/13.

7

STREPTOCOCCUS PYOGENES

Streptococcus pyogenes is a species of the genus Streptococcus. These bacteria are gram-positive

cocci that are arranged in chains and have rapid growth. In the interior of the cell wall of this

bacteria is located the antigen group A of Lancefield and the protein M, which is associated with

the virulent streptococci. Streptococcus pyogenes is spread by the contact between people for

respiratory droplets (pharyngitis) and the direct contact with an infected person in a skin rupture.

This bacterium is identified because is positive for PYR and susceptible to bacitracin, whereby

the general treatment used for patients with this bacterium is penicillin .

People most at risk of developing a disease by Streptococcus pyogenes are:

Children 5 to 15 years (pharyngitis)

Children between 2-5 years with poor hygiene (pyoderma)

Patients with soft tissue infection (streptococcal toxic shock syndrome)

Patients with a history of pharyngitis (rheumatic fever, glomerulonephritis)

Figure 4: Pharyngitis by Streptococcus pyogenes Source: Grupo de Infecciosas SoMaMFYC page.

<http://grupoinfeccsomamfyc.wordpress.com/category/microorganismos/bacterias/strept

ococcus-pyogenes> Accessed: 11/8/13.

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STREPTOCOCCUS PNEUMONIAE

Streptococcus pneumoniae is a species of the genus Streptococcus, which is formed by gram-

positive cocci, which have the form of "lancet" and are arranged in pairs (diplococci), or short

chains. These bacteria have teichoic acid rich in polysaccharide C (phosphocholine) and an

autolytic enzyme in the cell wall. The virulence

of Streptococcus pneumoniae is because these

bacteria can colonize the oropharynx, and

extend to sterile tissue, being capable of inciting

the local inflammatory response (peptidoglycan

fragments, teichoic acid, pneumolysin) and

escape the phagocytosis. Streptococcus

pneumoniae infections are transmitted from

person to person and are caused by the

endogenous spread from the nasopharynx to

distant regions (lungs, ears, blood, meninges

and sinuses). Colonization is highest in young children, and diseases caused by this organism are

more common in the colder months, because people stay home and tend to have more contact in

confined spaces. This bacterial strain can be identified because is detectable in laboratory test

like catalase-negative, susceptible to optochin and soluble in bile. The general treatment is

penicillin and immunization methods (conjugate vaccine of 7 serotypes for children of 2 years of

age, and the polysaccharide vaccine of 23 serotypes for adults at risk of acquire the disease

caused by Streptococcus pneumoniae)

Figure 5: Pneumoonia-infected

right lung by Streptococcus

pneumoniae Source: University of Michigan page.

<http://sitemaker.umich.edu/mc2/pneumonia> Accessed: 11/10/13.

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Table 1: Diseases and infections by Staphylococcus aureus, Streptococcus pyogenes,

and Streptococcus pneumoniae

Diseases

Diseases caused by Staphylococcus aureus

Disease Description

Scalded skin syndrome

It is a toxin-mediated disease, in which is disseminated epithelial desquamation in infants

Toxic shock Toxin-mediated disease in which occurs multisystem poisoning

Impetigo Cutaneous suppurative infection characterized by the presence of pus-filled vesicles on an erythematous base

Boils Suppurative infection in which large pus-filled skin nodules are observed

Carbuncles Suppurative infection characterized by the union of boils, which extend to the subcutaneous tissues

Bacteriemia and endocarditis

Suppurative infection characterized by the spread of bacteria into the blood from a focus of infection, where endocarditis causes damage to endothelial lining of the heart

Pneumonia and empyema

Suppurative infection where abscess formation in the lungs occurs

Osteomyelitis Suppurative infection where occurs bone destructions.

Septic arthritis Suppurative infection in which is observed a erythematous joint painful with accumulation of purulent material in the joint space

Diseases caused by Streptococcus pyogenes

Disease Description

Pharyngitis Suppurative infection that causes a red pharynx with presence of exudates

Cellulitis Cutaneous suppurative infection that affect the subcutaneous tissues

Necrotizing fasciitis Suppurative skin infection that causes destruction of muscle and adipose tissue layers

Streptococcal toxic shock syndrome

Multiorgan suppurative infection similar to staphylococcal toxic shock syndrome, and patients present bacteremia and signs of fasciitis

Rheumatic fever Nonsuppurative infection characterized by inflammatory changes of the heart, joints from arthralgia to arthritis, blood vessels and subcutaneous tissues

Acute glomerulonephritis

Nonsuppurative infection in which occurs acute inflammation of the renal glomeruli with edema, hematuria, hypertension and proteinuria

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Diseases caused by Streptococcus pneumoniae

Disease Description

Pneumonia Disease with clinical features of severe chills, persistent fever, and cough with bloody sputum

Meningitis Serious illness or infection that affects the meninges and presents with headache, fever and septicemia, and can cause neurological deficits for survivors

Bacteremia Disease that occurs most often in patients with meningitis than in patients with pneumonia, otitis media or sinusitis

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Part 2: Practice of Medical Microbiology

Laboratory to identify gram-positive cocci

Laboratory group: Kelvin Garced , José Noy , Francheska Camilo

Method and laboratory exercise: Livier González , William Arias

Medical Microbiology’s Professor at the Interamerican University of Puerto Rico.

Microbiology’s Professor at the Interamerican University of Puerto Rico.

Students of the InterAmerican University of Puerto Rico.

BACKGROUND

The aim of this study was to identify bacteria gram-positive cocci in microbiology's tests (Gram

staining, catalase test, and susceptibility to bacitracin and optochin).

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MATERIALS / METHODS

For the analysis, the procedure of Practical exercise was used: Streptococcus by Dr. Livier

González . The bacteria used in the experiment were: Staphylococcus aureus, Streptococcus

pyogenes and Streptococcus pneumoniae. For the lab exercise, we proceeded to divide four

blood agar plates in the center (with a Sharpie) to prepare individual crops of gram-positive cocci

bacteria. For the experimental practice, we conducted a simple striated for the boards (3A, 4A),

and a confluent striated for the blood agar plates (3B, 4B), to do the susceptibility test to

bacitracin and optochin. The blood agar plates were inoculated as follows: the plate 3A

(Streptococcus pyogenes and Staphylococcus aureus), the plate 4A (Streptococcus pneumoniae

and Staphylococcus aureus), the plate 3B (Streptococcus pyogenes and Staphylococcus aureus)

and the plate 4B (Streptococcus pneumoniae and Staphylococcus aureus).

We proceeded to add a bacitracin disk for each bacteria in plaque 3B, and an optochin disk each

bacteria in plaque 4B. Then we proceeded to incubate the four plates at 37 ° C using a jar with

CO2 for a period of 24 hours. For this exercise laboratory, we also conducted a test to determine

the production of the enzyme catalase. For this, we proceeded to prepare three microscope slide

laminae with one or two drops of 3% peroxide and a colony of one bacteria (Staphylococcus

aureus, Streptococcus pyogenes and Streptococcus pneumoniae) for each slide, and thus to

observe and analyze the production of bubbles (O2). In the next lab period (after incubating the

blood agar plates for a period of time> 24 hours), we prepared the Gram stain with each

bacterium using the procedure Laboratory # 5: Gram stain, by Dr. William Arias . The

procedures for the Gram stain were carried out as follows: prepare smears, drying this at room

temperature, fix with heat, cover with crystal violet dye for 1 minute, rinse with water for 30

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seconds, cover with iodine solution (mordant) for 1 minute, rinse with water for 30 seconds,

discolor with alcohol (95% ethanol) for 15-30 seconds, rinse with water for 30 seconds, dye with

safranin (counterstain) for 1 minute, rinse with water for 30 seconds, dry with bibulous paper

(blot dry), and then we observed each slide prepared in the microscope using the oil

immersion After completing our laboratory exercise, we proceeded to collect and analyze the

results for the catalase test, Gram stain, hemolysis and susceptibility testing using bacitracin and

optochin.

Figure 6: Hemolysis Beta (β) for the bacteria Streptococcus

pyogenes and Staphylococcus aureus in blood agar plate

(Laboratory exercise: plate 3)

Photo credit: Kelvin Garced – , Jose Noy – Francheska Camilo

Source: Medical Microbiology Laboratory's Exercise for identify bacteria coccus

gram positive.

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RESULTS

In the exercise of Laboratory of Medical Microbiology "identification of gram-positive cocci

bacteria", was performed, the catalase test, hemolysis, Gram stain and susceptibility using

optochin and bacitracin in samples prepared with the bacteria Staphylococcus aureus,

Streptococcus pneumoniae and Streptococcus pyogenes. In the catalase test, it was found that

Staphylococcus aureus bacteria are catalase-positive, whereas Streptococcus pneumoniae and

Streptococcus pyogenes are catalase - negative. For the susceptibility test to optochin and

bacitracin, we find that the Streptococcus pneumoniae is susceptible to optochin, and

Streptococcus pyogenes is susceptible to bacitracin. In this laboratory exercise, was obtained that

the Streptococcus pyogenes and Staphylococcus aureus are hemolysis beta [β], while

Streptococcus pneumoniae is alpha hemolytic [α].

Table 2: Results for the Laboratory Exercise of Medical Microbiology to identified the

bacteria cocci gram-positive

Bacteria Gram

staining Hemolysis Catalase test

Susceptibility test to

bacitracin

Susceptibility test to optochin

Staphylococcus aureus

Gram positive

Beta [β] + - -

Streptococcus pyogenes

Gram positive

Beta [β] - + -

Streptococcus pneumoniae

Gram positive

Alfa [α] - - +

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RECOMMENDATIONS

The Microbiology requires using different experimental tests to identify the bacteria that can

cause disease or infection. The procedure must be aseptic, using appropriate methods that

promote the protection of the skin to prevent infection or transmission of diseases caused by

these opportunistic bacteria. In the laboratory, the tests should be performed more than once to

obtain reliable results.

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CONCLUSION

Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae, belong to the

group of gram-positive cocci bacteria, which have high importance for clinical and

microbiological industry, as these bacteria are considered pathogenic, causing diseases and

nosocomial infections in healthy individuals or the community. Staphylococcus aureus is a

bacterium can be founded in the flora of the skin and mucosal surface. This bacterium that

exhibits resistance to penicillin and can cause impetigo or other infections and disease such as

bacteremia, that cause diffusion of bacteria in the blood, and endocarditis, a disease that can

damage the endothelial lining of the heart. Streptococcus pyogenes is important in research,

because it is easily transmitted from person to person through respiratory droplets or skin

wounds by contact with an infected person, causing diseases as pharyngitis and rheumatic fever.

Streptococcus pneumoniae is a bacterium which causes pneumonia, a disease in which a patient

can develop lung disease. In this laboratory study the catalase test, hemolysis, Gram staining and

susceptibility testing to optochin and bacitracin was performed. Through these laboratory tests,

was obtained that the Staphylococcus aureus is catalase-positive, Streptococcus pneumoniae is

susceptible to optochin, and Streptococcus pyogenes is susceptible to bacitracin. Furthermore,

when these bacteria were cultured on blood agar, we determined that the Streptococcus

pneumoniae is alpha-hemolytic, during the Streptococcus pyogenes and Staphylococcus aureus

have complete hemolysis (Beta Hemolysis). Therefore, by the analysis of results, was

determined that it is necessary to perform different tests for accurate the identification of bacteria

causing disease or infection, to be able to assign an effective treatment. To distinguish in

different genera (Staphylococcus and Streptococcus), or to identify different species in the

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bacterial genus (Streptococcus pyogenes and Streptococcus pneumoniae), is necessary to use

different experimental methods. We conclude that a bacteria may not react equal to other in the

same test, causing necessary perform several test to confirm the presence of a bacterium with

accuracy.

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REFERENCES

1. Murray, P. & Rosenthal, K. & Pfaller, M. (2009). Microbiología Medica.209-

223p.Staphylococcus y cocos grampositivos relacionados. ISBN 978-84-8086-465-7.

2. Murray, P. & Rosenthal, K. & Pfaller, M. (2009). Microbiología Medica.224-

242p.Streptococcus. ISBN 978-84-8086-465-7.

3. Facultas Ciencias de la Salud, Universidad del Cauca. Cocos Gram Positivos.

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vos.pdf> Available: November 10, 2013

4. Gorwitz R, Jernigan D, Powers J, Jernigan J and Participants in the Centers for Disease

Control and Prevention-Convened Experts’ Meeting on Management of MRSA in the

Community. (2006). Strategies for Clinical Management of MRSA in the Community:

Summary of an Experts’ Meeting Convened by the Centers for Disease Control and

Prevention.

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5. Calik S, Turhan T, Yurtseven T, Sipahi OR, Buke C. Vancomycin versus linezolid in the

treatment of methicillin-resistant Staphylococcus aureus meningitis in an experimental

rabbit model. Med Sci Monit. 2012; 18(11): SC5–SC8.

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6. Amador, C. Laboratorio No. 5: tinción Gram, del Dr. William Arias.

<http://www.studyblue.com/#file/view/4596192 > Available: November 10, 2013

7. NIH (National Institutes of Health). Neumonía.

<http://www.nlm.nih.gov/medlineplus/spanish/pneumonia.html >

Available: November 10, 2013