Mannitol Salt Agar (MSA)

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Introduction

Mannitol Salt Agar (MSA) is a selective media used for isolation, enumeration and differentiation of pathogenic Staphylococci spp. from samples ranging in the clinical, food, antiseptic, and cosmetic testing industries. 

                          D-Mannitol

                          D-Mannitol

MSA's main component is D-Mannitol which is a fermentable carbohydrate used in detection of acid production via phenol-red. Proteose Peptone supplies the essential growth components needed for bacterial growth while a high concentration of sodium chloride serves as an inhibitory agent against bacteria unable to withstand high salt concentrations. Agar is used as the solidifying agent. The addition of a 5% v/v egg yolk emulsion enables the detection of lipase activity of Staphylococci spp. 

History

Prior to the final development of the media, fermentation of mannitol was found to be used as a means of differentiating pathogenic Staphylococci from non-pathogenic. However, due to constant contamination found in solid media at the time, it was not widely used. 

                            Phenol-Red

                            Phenol-Red

George Chapman and his coworkers at The Clinical Research Laboratory in New York developed a series of medias used for isolation in the 1930-1940s using several staining chemicals.  Through work and re-examination of mannitol fermentation, a combination of bromythymol-blue lactose agar and phenol red mannitol agar was found to be reliable for isolation of pathogenic Staphylococci spp. In 1942, Sodium chloride in media was found to inhibit growth of most organisms except Staphylococci when concentrations were higher than 7.5%. With the additional properties of phenol-red turning yellow in acidic conditions, MSA was developed.  

Selectivity

Staphylococcus spp. are widespread throughout nature, but are found mainly on the skin and mucous membranes of mammals and birds.  Pathogenic Staphylococci are able to ferment mannitol, while non-pathogenic strains are not. When fermented, acid produced as a byproduct changes the acidity of the media, causing a colorimetric change from red to yellow due to the properties of phenol-red. Staphylococci are able to withstand the high concentrations of salt and thus are able to grow on the selective media. 

      Staphylococcus aureus [2]

      Staphylococcus aureus [2]

Staphylococcus aureus is documented as an opportunistic pathogen due to its ability to clot plasma in wound sites. (coagulase-positive). Coagulase-negative strains of S. aureus are typically non-fermenting, however can still grow on media. These colonies will appear pink/red and occasionally be surrounded by red/purple zones depending on the strain used.  The addition of an egg yolk emulsion can be added to the media to visualize the lipase activity of S. aureus.

Other colonies that may appear on the media from the same family include Staphylococcus saprophytic which, while coagulase negative, still ferments mannitol producing a yellow halo that resembles S. aureus colonies.  Other genera such as Micrococcus elutes and Micrococcus roses produce yellow and pink colonies, respectively. Enterococcus faecalis and Enterococcus faceium are also salt tolerant and can ferment mannitol producing yellow colonies on the agar. Catalase tests can help differentiate between Entercococus spp. and Staphylococcus spp.

Reconstitution

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PREPARATION OF Mannitol Salt Agar

  1. Add both dry and wet components to distilled water and boil to dissolve completely. 
  2. Sterilize media by autoclaving at 121ºC for 15 minutes. 
  3. Cool sterilized media to 45-50ºC.
  4. If desired, add 5% v/v egg yolk emulsion and mix well. 
  5. Aseptically dispense into sterile Petri dishes.

STORAGE

Media is both light and temperature sensitive. Store plates away from direct light at 2-8ºC. Plates may be used for one week when stored in a clean sterile area. Media should not be used if any signs of deterioration, color change, contamination, and/or expiration date has passed.  

STERILIZE ALL BIOHAZARD WASTE BEFORE DISPOSAL.


References

  1. Aryal, Sagar, et al.. “Mannitol Salt Agar for the Isolation of Staphylococcus Aureus.” Online Microbiology Notes, 12 June 2018, http://microbiologyinfo.com/mannitol-salt-agar-for-the-isolation-of-staphylococcus-aureus/.
  2. Staphylococcus aureus in Healthcare Settings” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 17 Jan. 2011, http://www.cdc.gov/HAI/organisms/staph.html.
  3. "Mannitol Salt Agar" Himedia Labs. Technical Data M118. http://himedialabs.com/TD/M118.pdf
  4. “Mannitol Salt Agar (MSA): Composition, Uses and Colony Characteristics” Microbe Online, 19 May 2017, http://microbeonline.com/mannitol-salt-agar-msa-composition-uses-and-colony-characteristics/.
  5. “Mannitol Salt Agar (MSA).” Hardy Diagnostics. 18 Oct 2017, http://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MannitolSaltAgar.htm.
  6. Shields, Patricia, and Anne Y. Tsang. “Mannitol Salt Agar Plates Protocols.” ASMscience, American Society of Microbiology, 9 Oct. 2006, http://www.asmscience.org/content/education/protocol/protocol.3034