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We have developed a new class of chromogenic substrates based on catechol and its derivatives. When these substrates are hydrolysed in the presence of a suitable metal ion, the liberated catechol chelates to form a mainly insoluble coloured precipitate. Catechol-substrates have the following unique combination of properties:

When used in diagnostic microbiology they give discrete highly visible coloured colonies with little or no diffusion into the surrounding medium depending on the substrate - ideal for distinguishing substrate-positive from substrate-negative organisms from within a polymicrobial culture.

They can produce black or dark brown precipitates that can be used to mask the activity of other chromogenic substrates, for instance indoxyl-substrates.

Different colours may be obtained from the same substrate by changing the metal ion in the medium. Colours can vary from yellow or orange through to black.

Catechol-substrates are easy to use and only require the inclusion of a suitable metal salt within the medium. Ferric ammonium citrate and potassium aluminium sulfate have produced good results with the substrates described here.

Most of the catechol-substrates tested, including the new substrates in this section, do not inhibit the growth of a wide variety of micro-organisms.

When produced in the sodium salt form, catechol-substrates are water-soluble and therefore the use of DMF or other organic solvents is not required to prepare the media.

The catechol-substrates included in this catalogue can be autoclaved (116oC for 20 minutes).

Figures 2-8 illustrate the properties and the potential of the new catechol-substrates. All tests were performed on Columbia agar. Positive results were obtained after 18 hours incubation.

Figure 2 shows the formation of discrete brown colonies of Klebsiella pneumoniae on a membrane filter caused by the hydrolysis of DHF-Gal sodium salt [Product code 50045] (400mg/l) in the presence of ferric ammonium citrate (500mg/l). DHF-Gal has the potential to be used to detect the LacZ+ gene in molecular biology. Unlike X-Gal, DHF-Gal is freely soluble in water.
Figure 3 illustrates how the catechol-substrates can be used to distinguish between positive and negative organisms within a mixed culture. Brown-black colonies of Enterobacter cloacae caused by the hydrolysis of DHF-b-D-ribofuranoside sodium salt [Product code 50180] (400mg/l) in a medium containingferric ammonium citrate (500mg/l) are easily differentiated from the colourless colonies of b-D-ribofuranosidase-negative Yersinia enterocolitica.
Figure 4 exemplifies how changing the metal ion produces colonies of a completely different colour from the same substrate. DHF-b-D-ribofuranoside sodium salt (400mg/l), used in Figure 3 with an iron salt to produce brown-black colonies, now generates yellow colonies with E. coli in the presence of an aluminium salt, potassium aluminium sulfate (500mg/l). The b-D-ribofuranosidase-negative Yersinia enterocolitica colonies remain colourless.
Figures 5 and 6 demonstrate the ability of the novel catechol-substrates to mask the colours produced by other chromogenic substrates. In Figure 5, Klebsiella pneumoniae colonies appear rose coloured after hydrolysing the indoxyl-substrate Rose-b-D-glucopyranoside [Product code 60059]. However, when grown on a medium incorporating ferric ammonium citrate and containing Rose-b-D-glucopyranoside and DHF-b-D-ribofuranoside sodium salt (Figure 6), both substrates are hydrolysed and the colonies of K. pneumoniae now appear brown because the iron chelate of DHF masks the rose colour of the indoxyl-substrate.
Figure 7 shows the pink/purple colour generated by hydrolysis of catechol-b-D-ribofuranoside sodium salt [Product code 14180] at a concentration of 300mg/l in the presence of ferric ammonium citrate at 500mg/l. This substrate is hydrolysed by E. coli but not Acinetobacter lwoffii. In terms of the intensity of its colour generation and the extent of colour diffusion, catechol-b-D-ribofuranoside is comparable in its performance to esculetin-based substrates.

Figure 8 provides further evidence of the ability of catechol-derived substrates to form discrete colonies with no diffusion into the surrounding agar. DHF-glucoside sodium salt [Product code 50059] was employed at a concentration of 300mg/l, with ferric ammonium citrate at 500mg/l. The intense black colonies of b-glucosidase-positive Enterococcus faecalis stand out sharply from the cream colonies of Staphylococcus aureus which do not express this enzyme, even where the colonies are practically coincident.

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Glycosynth would like to thank Prof John Perry and Lynne Butterworth, Microbiology Department, Freeman Hospital, The Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, England, who kindly carried out the microbiological testing and provided the photographs used in this section.

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Glycosynth Limited
14 Craven Court, Winwick Quay, Warrington, Cheshire. WA2 8QU England
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