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Year : 2004  |  Volume : 22  |  Issue : 1  |  Page : 69-70
 

Simple broth-disk technique for the detection of methicillin resistance in staphylococcus aureus


Department of Microbiology, Kasturba Medical College, Mangalore - 575 001, Karnataka, India

Correspondence Address:
Department of Microbiology, Kasturba Medical College, Mangalore - 575 001, Karnataka, India



How to cite this article:
Nishi V, Raman B R, Yengkokpam C, Bhat G K. Simple broth-disk technique for the detection of methicillin resistance in staphylococcus aureus. Indian J Med Microbiol 2004;22:69-70


How to cite this URL:
Nishi V, Raman B R, Yengkokpam C, Bhat G K. Simple broth-disk technique for the detection of methicillin resistance in staphylococcus aureus. Indian J Med Microbiol [serial online] 2004 [cited 2019 Aug 22];22:69-70. Available from: http://www.ijmm.org/text.asp?2004/22/1/69/8071


Dear Editor,
Methicillin resistant Staphylococcus aureus   (MRSA) is an important nosocomial pathogen.[1] Since most of these strains are multidrug resistant, management of infections is difficult. Rapid and accurate detection of MRSA is important for proper management and prevention of transmission. Heterogeneous nature of methicillin resistance is a limitation to accurate detection of MRSA by routine testing methods.[1] We report here a simple broth-disk technique for the detection of methicillin resistance in S. aureus. This method is a modification of a similar procedure described for anaerobic bacteria.[2]
Eighty strains of S. aureus isolated from clinical samples in the Department of microbiology, Kasturba Medical College, Mangalore, were studied for methicillin resistance with disk diffusion,[3] agar screen,[4] agar dilution[5] and broth-disk methods.[2]
Disks of diameter 6.1 mm were prepared using Whatman no. 1 filter paper and sterilized in hot air oven at 140C for 1 hour. Oxacillin was diluted in distilled water to get solutions of concentration 400 and 200 mg/mL. Antibiotic solution (0.01 mL per disk) was added to disks to prepare disks of potency 4 and 2 mg oxacillin. The disks were stored at 4C.
For each test, two tubes each containing 1mL Mueller Hinton broth with 2% NaCl was taken. One disk each of potency 4 mg and 2 mg was added separately to each tube and shaken well. To each tube, 0.01 mL of 1 in 200 diluted overnight broth culture of the test organism was added. An inoculated broth containing no antibiotic was used as growth control and a tube of uninoculated broth was included as sterility control. All the tubes were incubated at 35C for 24 hours and observed for growth. S.aureus that could grow in broth containing 2 and 4 mg/mL oxacillin was considered methicillin resistant. S.aureus ATCC 25923 and ATCC 29213 were used as controls in disk diffusion and dilution methods respectively.
Of 80 S. aureus isolates tested, 28 (35%) were scored methicillin resistant by disk diffusion whereas 32 (40%) were scored methicillin resistant by broth-disk method. All the strains which were methicillin resistant in broth-disk method were positive by agar screen and agar dilution, indicating reliability of the broth-disk technique [Table].
The concentrations of oxacillin used were selected based on breakpoint level for S. aureus (> 4 mg/mL-resistant and < 2 mg/mL -susceptible).[5] The procedure is simple and cost-effective. The potency of the disk can be maintained for 2 months if stored at 4C. Considering the above advantages, we feel that the broth-disk method can be conveniently used in laboratories to detect MRSA. 

 ~ References Top

1.Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Antimicrobial susceptibility testing, chapter 15. In: Color Atlas and Textbook of Diagnostic Microbiology, 5th edition (Lippincott, Philadelphia) 1997: 785.  Back to cited text no. 1    
2.Matsen JM. Antimicrobial susceptibility test - laboratory testing in support of antimicrobial therapy. In: Gradwohl's clinical laboratory methods and diagnosis, vol.2, 8th edition, Sonnenwirth AC, Jarett L. Eds. (BI Publications, New Delhi) 1990:1937-1970.  Back to cited text no. 2    
3.National Committee for Clinical Laboratory Standards: Performance standards for Antimicrobial Disk susceptibility Tests. Approved standards (M2-A5) Villanova, Pa, 1993.  Back to cited text no. 3    
4.Chamber HF. Methicillin resistance in Staphylococci: Molecular and biochemical basis and implications. Clin Microbiol Rev 1997;10:781-791.  Back to cited text no. 4    
5.National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Approved standards (M100-59) Wayne, PA 1999.  Back to cited text no. 5    
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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04