Microscope slide preparation methods

Aerobiolgy & forensic microscopy lab procedures:

How to prepare microscope slides for lab analysis. This is a collection of aerobiology and mold testing lab standard operating procedures - SOP's and slide preparation recipes useful for identification of mold, pollen, animal dander, skin cells, mite fecals, mouse dander, and other airborne allergens and bioaerosols. Comments and suggestions are invited.

Slide Preparation Procedures & Guidelines

Our photo (left) shows particle traces from two different air sampling cassettes prepared on microscope slides for further evaluation.

Our study comparing the performance of these cassettes is found at AIRBORNE PARTICLE TEST SAMPLING CASSETTE STUDY.

Article Series Contents:

• MICROSCOPE SLIDE PREPARATION
  • Slide Preparation Procedure
  • Slide Stain & Media Recipes
  • Permanent Slide Mounts
  • Gelvatol
• MICROSCOPE SLIDE BULK SAMPLE PREP - tips for preparing successful microscope slides from bulk material samples such as wood chips & scrapings collected during environmental sampling
• MICROSCOPE SLIDE PREP - PERMANENT MOUNTS: Cargille meltmount™ - slides for rapid field or laboratory use, how to prepare - microscopy lab procedures
• MICROSCOPE TECHNIQUES: Microscopy - Microscope sources, different types of microscopes, microscope techniques and laboratory procedures for indoor air quality, toxic mold identification, & bioaerosol investigations - microscopy lab procedures

Microscope Slide Stain and Media Recipes

How to Prepare Permanent Microscope Slide Mounts

Here is a list of some candidates. WARNING: some of these involve dangerous chemicals, possibly carcinogenic (cancer-causing) and should be used only with expert advice..

• Canada Balsam - traditional, excellent slide mounting media, generally not available. See Cargille's "Meltmount" alternative.
• Clear nail polish - experimental -DF
• Damar Varnish - see Brinkworth and Smith article below
• Fructose Larry Legg's recipe
• Glycerin or glycerine jelly. DF note: we've been disappointed that even after adding phenol or other preservatives some of our glycerine jelly slides become cloudy or even opaque with age. We no longer recommend this mountant for permanent slide mounts.
• Gum Arabic (Acacia) or similar water soluble gums
• Hyrax / Dirax high refractive index mountants - Northern Biological Supplies
• Lactic acid combined with phenol (carbolic acid), polyvinyl alcohol, glycerine, water. (Not for caliciferous materials - you'll find that materials like drywall dust, gypsum, some plaster contents, will dissolve and then recrystalize in this media)
• Loctite glass bond - local auto supply stores, needs UV light to cure - experimental, Brinkworth and Smith [DF note: We have had good results using this slide preparation media for permanent mounts of certain particles; we need to research the refractive index but in general that's not been a problem with paint samples nor with fungal spores or other common indoor building dust particles. We use a halogen lamp to generate the UV needed to cure the mountant.
• Meltmount - see CARGILLE MELTMOUNT PROCEDURE
• Mowiol - available from Burkard as a replacement for Gelvatol - burkard.com
• NBS Bioseal Two
• Numount - from Brunel microscopes & Northern Biological Supplies
• The Stains File - stainsfile.info/StainsFile/jindex.html This is the most extensive list we have found of slide preparation stains.
  
  However it's quite shy on stains for pollen and spores. Above are listed stains and recipes of particular interest to aerobiologists and for which I have detail or alternatives beyond the information at the Stains File.
• Turtox - no longer available, formula sought. See Howey's article
• Water soluble gums - gum Arabic (Acacia)

How to Use Gelvatol to Prepare Microscope Slides - A Detailed Step by Step Procedure

Recipe for Gelvatol Slide Mount Medium

Gelvatol may be used as a permanent slide mounting medium for mold spore or pollen slides. Gelvatol has been replaced by Mowiol from Burkard - see Mowiol below.

• 100 ml H2O (I would use distilled water)
• 35g Gelvatol (or Mowiol from Burkard)
• Polyvinyl alcohol (from Fisher Scientific, or other sources)

Heat to dissolve, not to much. Then add:

• 50 ml Glycerine (Glycerol, Fisher Cat. No. ?)
• 2g Phenol (as a preservative if needed)(Phenol smells a lot and it is no good for health, it gets yellow with time, I do not use it)

Add

• ACID fuchsin (as a colorant for SPORES if needed - Fisher Cat. No. AC22790-0250) Just use a few crystals, mix and wait. Add more if wanted, do not use too much as it will hide the structures.

OR BASIC fuchsin (as a colorant for POLLEN if needed - Fisher Cat. No. NC9653986)

OR no colorant

Keep closed tight because it gets harder as it gets in contact with air.

Source: Burkard company; I was told that they call the "polyvinyl alcohol" Gelvatol, a product available from Burkard.

Reader Question: getting rid of air bubbles trapped in the Gelvatol?

(Apr 4, 2014) Anonymous said:
I've got a lot of tiny air bubbles trapped in the gelvatol I prepared. I left it for a while and remove them, but more bubbles form when touch with a glass rod. How can I get rid of them?

Reply:

Anon. I would try heating the gelvatol - not hot enough to make it bubble or you'll have to start all over. Gently heat and watch it. When viscosity is pretty low, try tapping the container on a surface.

Also I find that one or two small bubbles in the mountant can be easily pushed to one side when I put on the cover slip. Or if there is not enough mountant under the cover slip I use a pipette to introduce a bit more mountant from one edge, later wiping that edge clean.

If more bubbles are forming when you push down on the cover slip I suspect something else is going on, possibly a chemical reaction with the substance being examined, or or you are pushing out air that was trapped in the mounted particles. For example, gypsum (drywall dust) in some indoor dust samples will froth many mountants, and flower or insect parts or large samples of fairly large or disparate particles will sometimes trap air that needs to be expelled.

Often after the cover slip is in place I will press down on it gently with the non-pinching end of a small pair of tweezers over which I've mounted a rubber dropper bulb. The bulb on tweezers gives me a dual -purpose tool since the rubber allows me to press gently without risking breaking the cover slip, and it also makes easier the slight adjustment of the cover slip position.

Finally, watch out for using slide mountant chemicals that react with some of the particles present. For excample using an acidic slide mountant with particles containing drywall dust will lead to bubble formation.

...

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