Technique for Preparing Powdery Mildew Samples for Microscopic Identification

Instruments and Materials Used in Preparation of Microscope Slides for Powdery Mildew Fungi Identification
 

Instruments and Materials Used In Slide Preparation

  • Microscope Slides
  • 5% KOH solution
  • Coverslips
  • Scalpel
  • Sample with Cleistothecia for identification
  • Compound Microscope
  • Dissection Probe
  • Hand lens, or Dissection Microscope

  •            (Recommended)

     

    A Drop of 5% KOH is Applied to the Cleistothecia on the Slide
     
     
     

    Step #1 - Take a clean microscope slide and add 1 drop of 5% KOH to the center of the slide. The 5% KOH solution helps to rehydrate the cleistothecia without rupturing them.
     
     

    Use of a Dissecting Microscope Aids in Selection and Removal of the Cleistothecia from the SampleCleistothecia are Positioned into the Center of the Slide
    Step#2 - Cleistothecium Transfer Technique #1 - The use of a dissection microscope allows you to select desired individual cleistothecia from a collected sample. Under the dissection microscope, gently lift off individual cleistothecia with the scalpel blade and dissection probe and drop onto 5% KOH drop on center of slide. Hint: It may help to have the scalpel tip wet with the 5% KOH solution.
     

    Leaf is Scraped With Blade To Remove Cleistothecia onto Microscope Slide
     

    Cleistothecial Transfer Technique #2 - If a dissecting microscope is unavailable, a hand lens can be used to identify the location of cleistothecia on the sample. This area can be gently scraped over the 5% KOH drop to allow cleistothecia to fall from the sample to the slide.
     
     

    A Coverslip is Applied Over the KOH Drop and Cleistothecium Mixture
     

    Step #3 - Gently place the coverslip over the 5% KOH drop and cleistothecium. Apply the coverslip by placing one edge down first and then lowering the rest of the coverslip down slowly, to reduce the number of air bubbles that will be trapped between the microscope slide and the coverslip.
     
     

    The Slide is Held Briefly 6 inches over an Open Flame to remove Air from the Sample
     
     

    Step #4 - If air bubbles do occur, the prepared microscope slide can be gently waved over an alcohol burner, approximately six inches from the flame for only a few seconds. DO NOT ALLOW THE SAMPLE TO BOIL!!!
     
     

    Step #5 - Place the prepared slide under the compound microscope and use the lowest power magnification to focus in on the sample. Locate the cleistothecia (100x magnification is usually sufficient to do this). Refocus and increase magnification as necessary.

    Step #6 - Observe appendages of the cleistothecia and refer to a key of the Genera of Powdery Mildew.

    ( If identification is necessary through an asci count, remove slide from microscope and gently press coverslip down with dissection probe to rupture the cleistothecia and release the asci. Note: Be careful not to crack the coverslip.)


    Step #7 - Repeat step #5. Locate ruptured cleistothecia and observe asci numbers. Refer back to Genera key of Powdery Mildew for further identification

    Step #8 - Dispose of all glass appropriately.
     


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