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External Quality Assessment Program / EQAP

http://www.eqap.ir/Bacteriology

KOH (3%) STRING TEST


Babak Valizadeh, DCLS

admin@eqap.ir

The Gram stain reaction is not always truly indicative of the organisms true cell wall structure. Poorly
controlled decolorizing can obviously be a source of misleading results, but even with adequate
technique some organisms are problematic. For example, some strains of Bacillus or Clostridium
consistently stain Gram negative even though they have a Gram positive type cell wall.

The KOH String Test relies on the differential resistance to 3% potassium hydroxide between gram
positive and negative cells, where a portion of a colony is mixed with a small volume of 3% KOH on a
glass slide for no more than 60 seconds. If the cells lyses, the liberated cellular DNA makes the mixture
viscous or “stringy.” The positive string test indicates a gram negative organism

KOH STRING TEST: Confirmation of Gram stain

Principle:

Dilute alkali solutions (3% KOH) lyses gram negative cell walls while the cell walls

Of gram positive bacteria are not disrupted. When gram negative bacteria are lysed (5 to 60

Seconds) the DNA is released causing the mixture to become viscous.

Procedure:

1. Place a drop of 3% KOH on a glass slide.

2. Using a loop remove a visible amount of fresh bacteria from a colony(s) on an agar plate (Blood agar)
incubated for 18-24 hr

3. Stir bacteria into KOH. 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.

4. Frequently raise the loop 1 cm off surface to test if the mixture is becoming viscous and has the

Ability to “string out”.

Interpretation:

Gram negative: Mixture becomes viscous and “strings out”.

Gram positive: After 1 minute the mixture is not viscous and does not string out.

Precautions:

False positive and false negative results may occur.

False positive results can occur from too heavy an inoculum (the solution will appear 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.

This test may be problematic for some anaerobes.


Quality Control:

Known controls should be tested each time the test is performed.

Positive: P. aeruginosa (ATCC 27853)

Negative: S. aureus (ATCC 25923)

References

1. Murray PA et al. Manual of Clinical Microbiology: 7th ed., 1999

2. POTASSIUM HYDROXIDE TEST, BSOP TP 30: NHS, 2010

3. Microbiology Techniques Manual: School of Human Life Sciences, Version 6.0 Jan 2007

4. MICROBIAL DIVERSITY MBIO 3480: LAB MANUAL, 2006

5. Rapid Method That Aids in Distinguishing Gram-Positive from Gram-Negative Anaerobic Bacteria;
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1981, p. 444-448

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