When first learning to identify pathogens and non-pathogenic items under the microscope, it’s important to do a few things first to help you sort
out the vast variety of items you may see on a slide. There’s a whole world there to be explored!

As discussed on the previous page, knowing your microscope is the first step.  If it is new to you, practice using it on typical household items
and articles like a piece of your own hair or pet hair, sugar or salt granules, fine powdered items like cinnamon or flour.  Take a small sample
of every food and/or supplement you give your birds and view them under the scope so you will recognize them when you see them in the
droppings.  Look at them both wet and dry.  A single drop of saline solution is often all you need for a slide sample.  

If your scope is capable, try both the dark and light field views.  Adjust the light while focusing – often times the light intensity will make a huge
difference in what you can and cannot see!  If you have a movable condenser, play with the adjustments.  Get comfortable with your scope so
that when a sick bird situation arises, you are ready.

Look at each item dry, take note of what you see, then add a bit of saline or distilled water and watch how those items change.  Many items,
like yeast or spirulina, will “bloom” or expand when liquid is added.  For loose and/or powdered items, I choose to place the item in a small
bowl, add enough saline to make it watery, and then use a sterile glass dropper to take up enough of the solution to place a single drop on my
slide.  

If you have a digital camera, take photographs through the eyepiece and keep the photos for future reference.

Important: Be sure to view everything you’ll use to create your slides as well, such as fuzz from a cotton bud, pieces of toothpick, your saline
solution or distilled water, and immersion oil as well as any stains you may use.  Occasionally, bits of these items may show up on a slide and
confuse the issue.  Threads or fuzz can look like thread worms, saline can dry leaving crystals on your slide, immersion oil – if not kept sealed
and stored properly – can pick up bits of sediment, etc. Try to view everything possible before creating a slide!  It will save you heartache in
the future!

Practice makes perfect!
Depending on the foods you are feeding your birds, plant material (anything from seed to greens and natural nesting materials) can look like
some pretty scary stuff.  But it can also look like art!  The crystallized saline shown above right is lovely, though there is little benefit to a
crystallized sample! However, these crystals are something you may see in a sample that has dried out, or one that does not have the
necessary amount of saline.

The plant material shown at immediate right is probably
from seed. At the time this sample was taken, my birds
were being fed  their austerity diet which consists of only
seed and water.  No other plant material or supplements
were being given at this time.

Spirulina, seen as a green macaroni to the far right, is a
very distinctive shape.  While it may not always appear
green depending on the degradation of the sample, the
shape will always give it away as a non-pathogen!

This little dickens was the very first foodstuff I every
looked at under the scope - and the item that convinced
me that viewing all foodstuffs and implements was first
and foremost when viewing samples.  

I thought it was worms due to the clear color of the original sample.  By the second or third sample, it was obvious by the color that it was not a
pathogen.  I had to literally sit back and think about what I feed my birds that is green!  Nearly knocked myself out of my chair smacking myself
in the forehead when I realized that I had wasted months trying to find this "worm" in my research!

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Using a Microscope - Page 2
Terms
Pathogen – the bad guys. Parasites and their eggs, oocysts, coccidia, yeast, bad bacteria such as E-coli and Salmonella

Artifact -  Non-Pathogenic, normal food stuffs or items that may have been ingested that do not affect the health of the bird.
Before you begin...
To properly prepare a slide, you will need the following tools:

  • Glass Slides – plastic tends to have minute scratches and when heated to create a “fixed” slide will melt
  • Glass Cover slips – I prefer the longer, rectangular covers instead of the small square ones. These allow a larger sample area.
  • Cotton Swabs and/or Toothpicks
  • Sterile Saline Solution (some folks choose to use distilled water)
  • Heat Source – preferably one that won’t leave residue on your slide such as a hand-held torch or lighter
  • Microscope with light source and magnification up to at least 400x (40x objective x 10x eyepiece)
  • Digital camera
  • Notepad and writing utensils
  • Sharpie marker for labeling slides if necessary
  • Alcohol  wipes
  • Crystal Violet (blue), Saffranin (yellow) or other stain - for fixed slides - these stains are the standard for Gram Stains.  There are many
    others, but they are for more advanced users!  I'll discuss those in a later chapter!
  • Immersion Oil – for immersion oil objectives only! Using immersion oil with an objective that is not specifically created for that function will
    damage the objective.

Make certain to begin with spotlessly clean slides.  Use an alcohol wipe to clean a slide if there is any residue on it.  DO NOT use your shirt
to dry the slide – you may leave traces of your own skin cells (epithelials) on the slide that could throw off your results.
Before we discuss "poop", I'd like to walk you through a few steps for
preparing slides and viewing them.  I prefer to have my students start out
with the foodstuffs their birds eat and the implements you'll use such as
cotton buds, toothpicks, saline, etc.  

Once you familiarize yourself these items, you'll be less likely to panic
when you see them in the droppings.  Many foodstuffs can look like
pathogens - as can paper, fabric fibers and epithelial cells - so it is
important to know what you should and shouldn't be seeing first!!
Plant Material - notice the circular cells
Crystalized Saline - 100x
Spirulina - notice the color and shape
Just poop!  Plant material at 100x magnification.
Spirulina particles at 100x magnification.
Creating a Slide with Foodstuffs...
You should attempt to view everything you feed your birds - seed, greens, egg food, brewers yeast, kelp or spirulina powder,
grit, eggshell or oyster shell, dried supplements of every kind.  You also want to take a sample of every "liquid" supplement
you give your birds in their water.  Mix the supplements as you would to give them to the birds then, using a glass eyedropper,
add 1 or 2 drops to a clean glass slide, add a cover slip and view.  If you have a digital camera, or camera on your scope,
photograph each of these items so you can refer back to them when viewing actual fecal samples.
For this demonstration, we'll use Brewer's Yeast to show how to create a foodstuff sample.

First, we take a small amount of the Brewer's Yeast and add it to a shallow dish.  We then add just enough saline to make a soupy thin liquid
that can be brought up in a glass eyedropper.

Collect up a small amount of the liquid in your glass eyedropper, then place one or two drops on a clean glass slide.  Place your cover slip
over the sample and use a toothpick to work the airbubbles out of the samples.
Prepare your work area - tools to set up BEFORE collecting samples...
Practice Makes Perfect
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