SUGAR (GLUCOSE) UTILIZATION TEST

Sugar (glucose) utilization test (also known as carbohydrate fermentation test) is used to detect bacteria that ferment various sugars (e.g. glucose) as well as convert pyruvate (the end product of glycolysis) into gaseous by-products (e.g. hydrogen and CO2). Bacteria in an effort to generate energy can ferment various simple sugars including glucose, sucrose, mannitol, and lactose, and this serves as basis for their identification in the laboratory. This test is performed in broth medium containing different fermentable carbohydrate or sugar, and also very special tubes known as Durham tubes. The sugars are added to a peptone medium to which a pH indicator is also added.

The Durham tubes are known to collect the gases produced from the sugar fermentation. Sugar utilization test is a differential test which is specifically used to distinguish Gram negative enterobacteria based on their ability to ferment glucose and produce gas at the same time. All Gram negative enteric bacteria are glucose fermenters but not all of them produce gas. Gram negative enterobacteria that show gas production are able to convert pyruvic acid to formic acid which is further broken-down by an enzyme to produce H2 and CO2. The gases produced (i.e. H2 and CO2) are trapped in the Durham tube(s), and they appear as bubbles at the top of the tube. Proteus mirabilis and Escherichia coli are both glucose fermenters and gas producers.

Procedure

  1. Prepare the baseline medium such as peptone water according to the manufacturer’s instructions.
  2. Dispense 9 ml portion of the basal medium into clean test tubes or bijou bottles.
  3. Add about 2-3 ml drops of 1 % Andrade’s indicator or any other pH indicator (e.g. bromothymol blue and phenol red) into the basal medium.
  4. Carefully and aseptically insert clean Durham tube(s) into all the aliquoted portions of the basal medium, taking care to avoid the inclusion of air bubbles into the Durham tube(s).
  5. Sterilize the tubes in the autoclave at 121oC for 15 mins and cool to normal room temperature.
  6. Prepare 1 % aqueous solution of the test sugar.
  7. Sterilize test sugar at 115oC for 15 mins in the autoclave. Membrane filtration technique can also be used to sterilize the test sugar.
  8. Aseptically introduce 1 ml of the 1 % sugar into each 9 ml portion of the basal medium containing the Durham tubes.
  9. Inoculate all the portions with the test bacteria while leaving one tube uninoculated. This last tube without the test bacteria will serve as the negative control tube.
  10. Incubate all inoculated tubes overnight at 37o
  11. Examine tubes for sugar utilization and gas production after incubation. A change in the colour of the indicator used signifies substrate (sugar) utilization with acid production: Andrade’s indicator to pink; phenol red to yellow; and bromothymol blue to yellow. Correspondingly, the presence of gas bubbles in the Durham tube(s) indicates gas production.

Illustration of glucose/sugar (carbohydrate) utilization test

From left to right: Tube 1 is a negative control and uninoculated tube, tube 2 shows acid production, tube 3 shows  gas and acid production, tube 4 shows a negative glucose fermentation test, and tube 5 shows a positive glucose fermentation test with acid and gas production as indicated by the change in colour from red to yellow. The sharp change in colour is indicative of a drop in the pH of the medium, and this confirms that the test bacterium has fermented the carbohydrate source in the test tube (in this case glucose).

REFERENCES

Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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