KOH mount by iee59083

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									                                                    Combat Support Hospital
                                              Clinical Microbiology, Baghdad, Iraq

                                 Potassium Hydroxide Mount (KOH Prep)

1.    PRINCIPLE:

       The potassium hydroxide (KOH) mount is a rapid method for the direct detection of fungal elements in clinical material.
Direct detection of fungal elements not only provides early information to the provider, it may indicate the need to add fungal
media to the culture battery and helps establish the clinical significance of fungal growth.
       When a specimen is placed in a drop of Potassium Hydroxide, the specimen will dissolve at a faster rate than the fungi.
This is because fungi have chitinous walls offering temporary protection from cell disintegration. KOH facilitates the clearing of
specimens for enhanced microscopic observation without altering the fungal elements.


2.    SPECIMEN:

      The specimen may be any type of clinical material. Fluids such as cerebrospinal fluid generally do not require KOH
treatment.


3.    SUPPLIES AND EQUIPMENT:

      A.     BBL 10% Potassium Hydroxide Reagent Droppers
             1)     Store ampoules at room temperature protected from light
             2)     Each dropper is good for one day’s use after the ampoule has been broken
      B.     Glass microscope slides
      C.     Cover slips
      D.     Slide warmer, dry heating block, 35-37C incubator, or flame


4.    CALIBRATION: N/A


5.    QUALITY CONTROL:

      A.     Perform the following quality control checks each time a KOH mount is examined:
             1)    A drop of KOH is placed on a slide, coverslipped, and screened for contamination by bacteria and fungal
elements.
               2)    Check the reagents prior to use for signs of deterioration. The reagent is sealed in an ampoule which protects
the solution from chemical instability until the expiration date.
                     a)     The reagent should be colorless, but may contain clear glass-like particles floating in the solution.
                     b)     The reagent should not be used if KOH crystals are precipitated onto the wall of the ampule or are
floating in the reagent.
       B.      Smears of hair, skin, or nails treated with 10% KOH should reveal partial cell disintegration.


6.    PROCEDURE:

       A.    Hold the reagent dropper upright and POINT AWAY FROM YOURSELF. Grasp the middle with thumb and
forefinger and squeeze gently to break the ampule inside the dropper.
       Caution: Break ampoule close to center one time only. Do not manipulate the dropper any further as the plastic
may puncture and injury may occur.

      B.     Tap the bottom of the dropper on tabletop a few times, then invert to dispense reagent


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      C.     Place 1-3 drops (depending on thickness and quantity of clinical material) of 10 % KOH onto a clean glass
microscope slide

       D.     Add a small amount of specimen to the KOH

       E.    Coverslip the material and incubate for 5 minutes to facilitate clearing of the specimen. Prior to examination, push
firmly down on top of the coverslip to crush the sample (exception: do not crush hair samples). This will facilitate reading of the
preparation.
             NOTE: keratinous material (hair, skin, nails) require 15-20 minutes for clearing.

       F.     Examine cleared sample under low (100X) and high (400X) power. If clearing is not complete, let smear sit an
additional 5 to 10 minutes.
              1)    Lower the condenser to improve contrast between fungal elements and background material
              2)    Scan the preparation using the 10X objective; use the 40X objective to focus in on suspicious material


7.     CALCULATIONS: N/A


8.     INTERPRETATION:

       Only experienced personnel should interpret direct examinations for fungal elements because background artifacts may be
misleading
       A.      Hyphae appear as relatively large (approximately 2 um in width), branching structures with parallel sides and
internal cross-walls (septa).
       B.      Yeasts are round to oval structures about the size of a red blood cell. In most cases, at least some of the yeast cells
will show budding or will have developed pseudohyphae.
       C.      Dematiaceous hyphae will appear brown or greenish-brown.
       D.      Artifacts may be distinguished from fungal elements when they lack septations, taper at the ends, or exhibit a
significant size difference.


9.     RESULTS REPORTING:

       A.     Negative KOH mount – report “No fungal elements seen.”
       B.     Positive KOH mount
              1)     Yeast – report “yeast present.”
              2)     Hyphae – report “hyphae present.”


10.    PROCEDURE NOTES:

       A.     Cotton swabs should not be used since cotton fibers may resemble hyphae
       B.     Yeast and hyphae should become visible in thick, viscous or opaque specimens following treatment with 10% KOH
       C.     Overheating of the preparation may cause crystallization of the KOH

11.    LIMITATIONS:

       A.     KOH preparations are not permanent; therefore the reagent will eventually destroy the fungi
       B.     Clearing of thicker specimens may require an extended clearing time


12.    REFERENCES:

      A.    BBL 10% Potassium Hydroxide Reagent Droppers Product insert, L-001148. Becton Dickinson and Company,
Sparks, MD. February, 2000.
      B.    Isenberg, Henry D., editor. 2004. Clinical Microbiology Procedures Handbook, American Society for
Microbiology, Washington DC. p.8.3.1-8.3.9.
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