Research Methodology

Nasal-Carriage and Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) among hospital Nurses of Tuguegarao City, Cagayan: A Cross-Sectional Study

LABORATORY ANALYSIS A. Preparation of Culture Media Mannitol Salt Agar (MSA) and Mueller Hinton (MH) Medium will be prepared exactly according to the package inserts. Generally, the media as well as the transport tube broth will be made by dissolving a specified amount of powder in water and will be boiled. This will be allowed to cool down and be sterilized to minimize or eliminate contamination of specimen. B. Isolation and Identification Nasal swabs will be screened for Staphylococcus aureus using Mannitol Salt Agar. After inoculation, the plates will be incubated at 35 2C in an aerobic atmosphere for 24-48 hours. Coagulase-positive staphylococci will produce growth of yellow colonies with yellow zones. Coagulase negative staphylococci will produce small red colonies with no color change to the medium. C. Biochemical Tests 1. Catalase Test A small amount of the yellow colony growth from MSA will be transferred to the surface of a clean, dry glass slide using an inoculating loop. Thirty percent (30%) of hydrogen peroxide (H2O2) will be mixed to the medium. The production of copious oxygen bubbles will indicate positive result, and no or few bubbles will indicate negative result. 2. Tube Coagulase Test Colony growth will be emulsified in 0.5 mL of rabbit plasma (with EDTA) which will give a milky suspension. The tube will be incubated at 35 C in ambient air for 4 hours. Clot formation will indicate positive result. If negative at 4 hours, the tube will be incubated at room temperature overnight and checked again for any clot formation. Note: Tests can be can be positive at 4 hours and then revert to negative after 24 hours. D. Antimicrobial Susceptibility Testing A direct colony suspension, equivalent to 0.5 McFarland standard will be prepared. Three to five colonies will be transferred with an inoculation needle or loop into a 4-5 mL of Trypticase Soy Broth. The broth culture will be incubated at 35 C for 2-6 hours to develop a turbidity
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Research Methodology
Nasal-Carriage and Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) among hospital Nurses of Tuguegarao City, Cagayan: A Cross-Sectional Study equivalent to 0.5 McFarland standard (approximately 1 to 2 x 108 CFU/mL). Dilute, if required to obtain turbidity equivalent to 0.5 McFarland turbidity standard using broth or saline. Within 15 minutes, a sterile cotton swab will be dipped into the inoculums and rotated firmly several times against the upper inside wall of the tube to express excess fluid. The entire agar surface of a Mueller Hinton Agar plate will be streaked three times, turning the plate 60 between streakings to obtain even inoculation. The lid may be left ajar for 3 5 min, but no more than 15 min, to allow for any surface moisture to be absorbed before applying the drugimpregnated discs. The resistance of S. aureus will be detected through Kirby-Bauer disk diffusion method. A panel of antibiotics will be used particularly Cefoxitin, Vancomycin, Oxacillin, and Clindamycin. Antibiotics were chosen accordingly from performance standards for antimicrobial susceptibility testing of CLSI (2007). The discs will be impregnated to the agar using aseptic precautions using forceps if the discs are not self-despensed. The discs will be deposited so that the centers are at least 24 mm apart. Within 15 min, the plates will be positioned side up in 33 to 35C incubator. Note: testing at temperatures above 35C may not detect Methicillin-Resistant Staphylococci (MRS). A full 24 h of incubation is recommended for MRSA detection. The diameters of the zones of complete inhibition are measured, as determined by gross visual inspection using vernier caliper. Zones are measured to the nearest whole millimeter. Results will be interpreted according to the Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement by CLSI (2007).

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Research Methodology
Nasal-Carriage and Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) among hospital Nurses of Tuguegarao City, Cagayan: A Cross-Sectional Study

METHODOLOGICAL FLOWCHART

Nasal Swab in cotton applicator stick moistened with saline Transport Medium (Trypticase Soy Broth with 10% NaCl) Mannitol Salt Agar
Yellow Colony

24 hrs
Red Colony

24 hrs

Discard

Catalase Test

4 to 24 hrs

Tube Coagulase Test

Discard

24 hrs

Direct Colony Suspension (Trypticase Soy Broth)

Discard

24 hrs

Antibiotic Susceptibility Testing (Mueller-Hinton agar)

Cefoxitin

Oxacillin

Clindamycin n

Vancomycin

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