Potassium Hydroxide String Test – Principle, Procedure, Uses and Result interpretation

 

Introduction

The potassium hydroxide string test is a very simple, easy to perform and also inexpensive rapid test could be used in addition to the Gram staining for differentiate between Gram positive and negative isolates, where a Gram -negative cell wall are lyses when it’s mixed with a small volume of 3% KOH and releasing viscid chromosomal material from cell, makes the bacterial mixture suspension to become thick viscous or “stringy.” The positive string test indicates a gram negative organism. Hence the alternative name for the test is “String Test”. Some organism like Bacillus and Clostridium sp. appear Gram negative on staining resulting in possible misidentification because of they have lost some of the integrity of their cell wall.

Purpose

Distinguishing between Gram -negative and Gram -positive organisms

Principle

In the presence of 3% potassium hydroxide, the cell wall of Gram-negative is broken down. KOH easily dissolves the thin layer of cell walls (Peptidoglycan) of the gram-negative bacteria. When the bacterial cell wall is disrupted, the cell wall lyses and the chromosomal material is released. As a result, a viscous chromosomal material is released, making the bacterial suspension thick and viscous. This reaction, which is viscous and the solution sticks to the loop when touched, this reaction characterizes Gram-negative bacteria. On other hand, Gram-positive bacteria have a thicker peptidoglycan layer in their cell wall, Unaffected by KOH.

Required equipment and reagents

Equipment:

Light microscope 

Biological safety cabinet

Reagents:

3% KOH (aqueous)

Microscopic slides

Procedure

1.    Add one drop of 3% potassium hydroxide solution to a clean microscope slide.

2.    Emulsify 2-3 colonies of the organisms in a drop of potassium hydroxide until a thick suspension is obtained.

3.    Stir constantly for 30 seconds and then gently pull the loop out of the suspension to form a mucoid string within 30 seconds. The bacterial solution is viscous

Results and Interpretation

Positive: The formation of mucoid string within the 30seconds. The solution with the bacteria will be viscous. This is Gram Negative bacteria

Negative: The absence of stringing reaction. This is Gram-positive bacteria.

Quality Control

Quality control should be performed daily or immediately prior to use.

Positive control: Escherichia coli ATCC 25922

Negative control: Staphylococcus aureus ATCC 25923

  

Positive Reaction of String test - Gram Negative bacteria

Uses

1. This test may be used in addition to Gram stain for preliminary differentiation in laboratories dealing with large numbers of cultures has to be processed.

2.    This is a useful complement to the Gram stain test.

Limitations

1. A negative test is useful but does not conclusively prove that a microorganism is Gram-positive.

2. Older cultures (>48hr) may become positive after 30 seconds after mixing the bacteria with KOH solution, giving unreliable results. These include Achromobacter species, Brucella melitensis, Pseudomonas paucimobilis, Moraxella species, etc. it is common in with certain species such as

3.   Using too heavy an inoculum (the solution will appear to gel, but not string), or inoculation with mucoid colonies, may give false-positive results.

4.  Using too little inoculums or too much KOH (DNA-induced viscosity not noticeable), may give false negative results.

References

1.    Suslow, T. V., M. N Schroth, and M. Isaka. 1982. Application of a rapid method for Gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathology 72: 917 918.