Ramesh Sonti
INDICATOR PLATES
Indicator plates have been in use since the early days of bacteriology. The early bacteriologists used indicator plates as diagnostic tools for identifying a bacterial species. Although indicator plates continue to be a part of diagnostic kits, their use has expanded dramatically in recent years. This increased usage can be attributed to the introduction of convenient, transposon based methods for making gene fusions. In many laboratories today, indicator plates are being used in studies on bacterial gene regulation, recombination and chromosome structure.
The principle behind the indicator plate technology is simple. Bacterial colonies that are expressing a certain biochemical activity can be identified by visual examination because they have a unique color on media containing a particular dye. In most cases the dye changes color when it is hydrolysed, or acts as a pH or redox indicator. The following are the indicator plates that are used most commonly in this laboratory.

X-Gal Plates: the chromogen is 5-Bromo-4-Choro-3-Indolyl-B-D-galactoside (X-Gal). This dye is a substrate for B-galactosidase and turns blue when hydrolyzed by this enzyme. X-Gal can be added to minimal medium or nutrient broth; although minimal medium offers the best contrast for distinguishing different shades of blue.
Minimal X-Gal Plates:
Solution A: 12.5g of bacto-agar + 400ml of H2O
Solution B: 400ml of NDEx2
Autoclave solutions A and B separately. Let the solutions cool. To Solution B add the following: 8ml of a 20% solution of the desired carbon source; 0.8ml of 1M MgSO4; and 20mg of X-Gal dissoved in 1ml of N,N-Dimethyl formamide. Mix solutions A and B.
Nutrient broth X-Gal plates:
Solution A: 22.4g of nutrient broth mix + 400ml of H2O
Solution B: 400ml of H2O
Autoclave the two solutions separately and let them cool afterward. Dissolve 20mg of X-Gal in 1 ml of N,N-Dimethyl formamide and add to solution B. Mix solutions A and B.
MacConkey plates: This medium is used with carbon sources that can be fermented by S. typhimurium. Strains that can ferment the carbon source form red colonies while non-fermenting strains form transparent white colonies. The coloration is provided by a pH sensitive dye that is in the MacConkey agar.
Solution A: 32g of Difco basal MacConkey Agar + 400ml of H2O
Solution B: 400ml of H2O
Autoclave solutions A & B separately and allow them to cool. Add 40ml of a 20% solution of the carbon source to solution B. Mix solutions A & B.

Tetrazolium (TTC) plates: The dye is 2-3-5-triphenyl tetrazolium chloride (TTC) which turns red when reduced. This dye is also pH sensitive and the red coloration is observed only if the pH of the medium is above 6 or 7. Since the development of the red color is dependent on reduction of the dye as well as the pH of the medium, TTC can be used as either a redox indicator or a pH indicator. Medium in which TTC serves as a pH indicator was develped by LEDERBERG (1948) and has been modified by WINKLEMAN M and CLARKE (1984). Medium in which TTC server as a redox indicator has been developed by BOCHNER and SAVAGEAU (1977).
Lederberg-Tetrazolium plates: This medium can be used with any carbon source that can be fermented by S. typhimurium. Strains that can ferment the carbon source form light pink colonies, while non-fermenting strains form bright red colonies.
Solution A: 10g tryptone + 1g yeast extract + 15g bacto-agar + 5g NaCl + 500ml H2O
Solution B: 0.05g TTC + 500ml H2O
Autoclave solutions A & B separately and allow them to cool. Add 50ml of a 20% solution of the carbon source to solution B. Mix solution A & B.
Bochner-Tetrazolium plates: This medium contains a buffer to maintain a constant pH in the plates and the color of the dye is determined solely by it's redox state. This medium can be used with any carbon source that can be utilized by S. typhimurium. Strains that can utilize the carbon source form red colonies while non-fermenting strains form whited colonies.
Solution A: 2g bacto agar + 7.2g K2HPO4 . 3H2O +2.4g KH2PO4 + 400ml H2O
Solution B: 12g bacto-agar + 400ml H2O
Autoclave solutions A & B separately and allow them to cool. Add the following to solution B: 8ml of 0.25% TTC, 0.8ml of 1M MgSO4, and carbon source. The final concentration of the added carbon source after mixing solutions A & B should be between 0.2 and 1%.

References:
BACHNER, B. R., and M. A. SAVAGEAU, 1977 Generalized indicator plate for genetic, metabolic and taxonomic studies with microorganisms. App. and Environ. Micorobiol. 33: 434-444.
LEDERBERG, J., 1948 Detection of fermentative variants with tetrazolium. J. Bacteriol. 56: 695.
WINKLEMAN, J.W., and D. P. CLARK, 1984 Proton suicide: general method for direct selection of sugar transport- and fermentation-defective mutants. J. Bacteriol 160: 687-690.

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