The Gram staining reaction is not always effective in the indications of the organism’s cell wall structure. Poorly controlled decolorizing, wrong solution dilution and negligence of the technician can obviously be a source of errors resulting during gram staining, but even with adequate technique, some bacteria are problematic. For example, some strains of Bacillus, Clostridium and aged bacteria consistently stain Gram negative even though they have a Gram-positive type cell wall.
The KOH String Test relies on the differential resistance of these bacteria to 3% potassium hydroxide between gram positive and negative cells, which involves the mixture of a portion of a colony with a small volume of 3% KOH on a glass slide for no more than 60 seconds. If the cells lyses, this releases cellular DNA that makes the mixture viscous or “stringy.” The positive string test indicates gram-negative bacteria. This method is an alternative to Gram stain technique.
Principle of KOH String test
Based on the differences in the cell wall composition of gram-positive and gram-negative bacteria. Gram-negative bacteria cells wall are lysed by diluted alkali solutions of (3% KOH), which has no effect on the cell walls of gram-positive bacteria due to the presence of a thick peptidoglycan. When gram-negative bacteria are lysed (5 to 60 Seconds) the cellular DNA is released causing the mixture to become viscous or stringy.
- Prepare a 3% KOH solution.
- A 24-48 hour bacterial culture from solid media.
Procedure of KOH string Test
- Place a drop of 3% KOH on a glass slide.
- Using a loop remove a visible amount of fresh bacteria from a colony(s) on an agar plate (Blood agar, Nutrient agar, CLED AGAR) incubated for 18-24 hr
- Stir bacteria into KOH on the glass slide. Mix continuously in a 1 to 2 cm area on the glass slide for a maximum of 1 Minute (60 sec) and by slowly lifting the loop, observe for the formation of a string.
- Frequently raise the loop 1 cm off surface to test if the mixture is becoming viscous and has the Ability to “string out”.
Interpretation of KOH string test
- Gram-negative bacteria: Mixture becomes viscous and “strings out”.
- Gram-positive bacteria: After 1 minute the mixture is not viscous and does not string out.
Precautions when carrying KOH string test
The following may lead to false positive and false negative results
- False positive results can occur from too heavy inoculums. This will make the solution to gel, but not string, or inoculation with mucoid colonies.
- False negative results can occur in the test by using too little inoculums or too much KOH (DNA-induced viscosity not noticeable).
This test has the advantage of simplicity, and it can be performed on older cultures. The test may be problematic for some anaerobic bacteria.
In order to maintain and provide accurate and reliable result, Quality control is recommended for this test. Organism such as Gram-negative (E. coli) and Gram-positive (Staphylococcus aureus) can be use for quality control
- Murray PA et al. Manual of Clinical Microbiology: 7th ed., 1999 2
- POTASSIUM HYDROXIDE TEST, BSOP TP 30: NHS, 2010
- Microbiology Techniques Manual: School of Human Life Sciences, Version 6.0 Jan 2007
- MICROBIAL DIVERSITY MBIO 3480: LAB MANUAL, 2006
- Rapid Method That Aids in Distinguishing Gram-Positive from Gram-Negative Anaerobic Bacteria; JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1981, p. 444-448