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Lab 19 New Biochemical Tests

Tests for Gram Positive Rods

Spirit Blue Agar Plate

This agar is used to identify organisms that are capable of producing the enzyme lipase. This enzyme is secreted and hydrolyzes triglycerides to glycerol and three long chain fatty acids. These compounds are small enough to pass through the bacterial cell wall. Glycerol can be converted into a glycolysis intermediate. The fatty acids can be catabolized and their fragments can eventually enter the Kreb’s cycle. Spirit blue agar contains an emulsion of olive oil and spirit blue dye. Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial growth. The Gram-positive rod, Bacillus subtilis is lipase positive (pictured on the right) The plate pictured on the left is lipase negative.

spirit blue negative and positive results

Starch Hydrolysis

This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules, these organisms can not pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (pictured below on the left). The organism shown on the right is negative for starch hydrolysis.

starch positive plate

Tests for Gram Negative Rods

Simmon's Citrate Agar

This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to differentiate between members of Enterobacteriaceae. In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result (the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.

citrate negative and positive results

Rapid KOH string test

Though the Gram stain is a very useful procedure that differentiates between groups of bacteria, it can have several problems. If the culture being stained is old, a false negative — where a Gram-positive bacteria stains like a Gram-negative — can result. There is also the great possibility for human error like missing one of the steps or not staining in order. The mistake that is made most often is over decolorizing with the alcohol, causing a Gram- positive to appear Gram-negative. Since the Gram stain can give such variable results, many clinics have utilized the rapid KOH string test in conjunction with the Gram stain to increase the validity of the results. The KOH string test, done with potassium hydroxide (KOH), should not be the only test done because it does not give any information regarding the morphology of the cells.


1. Collect your inoculating loop, as well as your Gram-positive Unknown A and Gram-negative Unknown B, and proceed to the KOH test station. A solution of 3% KOH should be provided there.

2. Place a drop of 3% KOH on a clean microscope slide from your slide box.

3. Use a sterilized inoculating loop and take a visible amount of fresh cells from one of your working slants.

4. Mix the cells in with the KOH for about 60 seconds.

5. Lift the loop off the slide and look for a slimy string connecting the loop to the slide.

6. Repeat the procedure with the second unknown.

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