LABORATORY # 4
MRSA
Lab# 4: Determination of Methicillin Resistance
Points= 10
Objective:
At the completion of this activity, the student should be able to:
1. Be able to distinguish methicillin resistant Staphylococcus aureus from other
Staphylococcus species.
2. State the characteristics of methicillin resistant Staphylococcus aureus
colonies.
3. List the biochemicals in the medium that select for methicillin resistant
Staphylococcus aureus.
4. Name the specimen of choice to screen for MRSA.
5. Define MRSA.
6. Determine whether a patient should be placed in isolation, based on MRSA
CHROMagar testing results.
Materials:
1 CHROMagar MRSA plate
1 Unknown swab
Bacti-Incinerator
Inoculating Loop
Staphylococcus aureus ATCC 25923/29213
Staphylococcus aureus ATCC 43300
Microscope
Immersion Oil
References:
1. BD BBLTM CHROMagar TMpackage insert 8022632 2006/5
Discussion:
MRSA are a major cause of nosocomial and life-threatening infections. MRSA are known
to have a higher morbidity, mortality and cost than non-MRSA bacteria. Isolation of
these organisms has been the greatest in the healthcare setting; however, MRSA are
becoming more prevalent in the community. Results from CHROMagar MRSA should be
used as an adjunct to nosocomial infection control efforts to identify patients needing
enhanced precautions. This test is not intended to identify patients with staphylococcal
infection. This device can be used to identify patients for isolation or removal from
isolation to control nosocomial transmission of MRSA.
MLAB 2534- Laboratory 4 – Page 1
LABORATORY # 4
MRSA
Principle:
CHROMagar medium permits the direct detection and identification of MRSA through
the incorporation of specific chromogenic substrates and cefoxitin. MRSA strains will
grow in the presence of cefoxitin and produce mauve-colored colonies resulting from
the hydrolysis of the chromogenic substrate. Additional selective agents are
incorporated for the suppression of gram-negative organisms, yeasts and some gram
positive cocci. Bacteria other than MRSA may utilize other chromogenic substrates in
the medium resulting in a blue to blue-green colored colonies or if no chromogenic
substrates are utilized, the colonies will appear as white or colorless.
Specimen Collection and Handling:
The primary specimen of choice is the anterior nares.
Procedure:
1. Instructor will demonstrate how to set-up quality control for this laboratory. For
future use, the instructions are listed in the “Technical Notes” section at the end
of this lab. Record the results of the quality control on your report form at both
24 hours and 48 hours.
2. Observe aseptic techniques. The agar surface should be smooth and moist, but
without excessive moisture. Allow the media to warm to room temperature in
the dark before inoculation.
3. Select an unknown from the rack. Document the name and identification
number of your patient on your report sheet. Inoculate the specimen onto a BBL
Chromagar MRSA plate and streak for isolation. Incubate plates aerobically, in a
non-CO2 incubator at 35-37o C for 24+ 4 hours in an inverted position. Do not
incubate in CO2. Avoid exposure to light during the incubation (>4 hours) as light
may result in reduced recovery and/or coloration of isolates. Exposure to light is
permissible after colony color develops.
4. After 24 hours, examine the plate(s) for the presence of mauve colonies. The
plates should be read against a white background. The presence of mauve
colonies indicates a positive result. Other organisms (non-MRSA) will be inhibited
or produce colorless, white, blue or blue/green colonies.
5. Record your result as either “MRSA detected” or “No MRSA detected.”
Technical Notes:
 Minimize exposure (>4 hours) of CHROMagar MRSA to light both before
and during incubation. Prolonged exposure may result in reduced
recovery and/or coloration of isolates.
 Quality Control will be performed by the instructor for this lab, using
Staphylococcus aureus ATCC 25923/29213 strain and Staphylococcus
aureus ATCC 43300 strain. Both Staphylococcus aureus ATCC
25923/29213 and Staphylococcus aureus ATCC 43300 will be directly
MLAB 2534- Laboratory 4 – Page 2
LABORATORY # 4
MRSA
streaked onto the CHROMagar plate to determine the growth capacity of
the medium and the performance of the chromogenic reaction.
Interpretation
Test Strain
Staphylococcus aureus ATCC
25923/29213
Staphylococcus aureus ATCC
43300
Expected Results
No growth
Growth with mauve colonies
MLAB 2534- Laboratory 4 – Page 3
LABORATORY # 4
MRSA
Name:_____________
Date:______________
Laboratory #4: Determination of Methicillin Resistance
Lab Report Sheet
Points= 10
**Note for quality control strains, the student will observe and record the reactions from the instructor demonstration.
24 Hour Incubation:
Patient Name and ID
Colony Morphology/
Color
Final Report/Interpretation
Staphylococcus
aureus ATCC
25923 **
Staphylococcus
aureus ATCC
43300 **
Unknown
MLAB 2534- Laboratory 4 – Page 4