Mannitol Information and Courses from MediaLab, Inc.

Key reactions for the identification of Clostridium septicum are demonstrated in the two quadrant plates shown in the images to the right. Included in the upper image are reactions for milk (casein) proteolysis (12 o'clock quadrant), glucose fermentation, DNAse hydrolysis, and starch hydrolysis respectively reading clockwise. The media in the quadrant plate shown in the lower image include gelatin hydrolysis (2 o'clock quadrant) and fermentation of each of mannitol, lactose, and rhamnose respectively, reading clockwise. Milk (casein) hydrolysis and glucose fermentation are key reactions for the identification in the upper plate, including no proteolysis of milk, fermentation of glucose (yellow red color along the inoculation streak), positive DNAse (reddish clearing around the streak) ,and a negative reaction for starch. Key reactions in the lower plate include hydrolysis of gelatin, fermentation of lactose (yellow pigment), and negative reactions for mannitol and rhamnose (no pigment). Most strains of C. perfringens hydrolyze starch and produce proteolysins of milk, the key reactions that distinguish C. septicum (negative). Reactions to the other tests do not distinguish between the two.

The ability to grow in 6.5% NaCl and to produce acid from mannitol was once considered sufficient to identify S. aureus. Although other Staphylococcus species have these characteristics, mannitol salt agar, as shown here, can still be used for epidemiologic studies in suspected cases of S. aureus outbreaks. As shown in the image, the presence of colonies indicates the ability to grow on 6.5% NaCl; and, the yellow pigment of the colonies indicates acid production from mannitol.

Traditional culture methods for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) employing mannitol salt and/or blood agar for cultivation, can take up to 72 hours for isolation and identification, depending on the identification procedures utilized. Concurrent with the development of molecular assays, improvements in culture methods have also been achieved. CHROmagar™ media, specific for MRSA, are employed by many laboratories. These media are both selective and differential, containing chromogenic substrates. MRSA strains utilize the substrates to produce colonies of a specific and characteristic color, minimizing the need for additional identification procedures.Initially these agars required 48 hours of incubation; newer formulations require only 24 hours incubation.Given the reduced incubation and identification requirements, what are the pros and cons of the molecular assays? Cost per test will be greater with the molecular assays as compared to culture methods. Will molecular methods provide for a more efficient workflow and significant improvement in availability of results? To some extent, this will depend on how they can be implemented within each different laboratory setting. Both of the previously described molecular assays require manual specimen preparation and extraction before the sample is placed into the instrument. This hands-on work may actually be greater than the effort expended in swabbing and streaking a culture plate. How much an obstacle this is for implementation will depend on both the volume of testing and the staff available. In a high volume setting, this will be a greater factor.Will tests be performed as specimens come in, or will specimens be accumulated and batched? If controls are required with each run, batching is desirable to reduce this cost. If testing will occur in batches, how many batches can be performed in one day? This will be heavily influenced by the capacity of the instrument. (For example, a single Smart Cycler unit can run up to 16 samples; multiple units would be needed in a high volume lab.) Can they be set up on more than one shift? The greater the number and frequency of batches that can be run, the greater improvement in turnaround time can be realized. Given these variables, implementation of a molecular assay for MRSA is not a given in each laboratory.

Surveillance is a critical component of any program for controlling multi-drug resistant organisms. Many institutions are using active surveillance cultures to identify patients who are colonized with a targeted MDRO. With respect to MRSA, an increasing number of hospitals are screening patients upon admission and on a periodic basis (usually weekly). The anterior nares is the primary site that is swabbed for screening.There are several selective and/or differential media that can be used for this purpose.Baird Parker Agar is a selective medium for the isolation of S. aureus; on this medium S. aureus produces black colonies with a clear halo.Mannitol Salt Agar is also a selective medium; S. aureus produces yellow colonies which contrast with the red color of the medium.Chromogenic agars have been developed for the isolation and presumptive identification of different species of bacteria and yeast. The media are formulated so that as different organisms utilize various substrates in the media, the organism of interest produce colonies with a unique color. Chromogenic agars specifically designed for the detection of MRSA are commercially available.In addition to culture methods, there are now commercially available, FDA approved methodologies for screening for MRSA by PCR. Although equipment and cost factors may not make these a viable option for every laboratory, they may offer greater sensitivity and improved turnaround times.