Preparing a peripheral blood drop slide for examination for nematodes.

Method 1

This is the simplest way to get a clear view of any likely specimens but only uses one drop of blood, so if specimens are scarce the chances of finding one are reduced.

Put a drop of water on the slide and mix a drop of blood into it.

Drop a coverslip on to the sample and gently blot the edges with paper towel.

ALTERNATIVELY: Only a blood drop is placed on the slide and the coverslip put in place.

If a good specimen is found but it is obscured by blood cells surrounding it; a syringe or pipette can be used to put a few drops of water next to the edge of the coverslip. Capillary action will suck the water under the slip and often this will clear the specimen.

Method 2

This method can be useful when specimens are few as it allows several drops of blood to be examined on a single slide.

A 40 or 50mm coverslip (coverglass) is useful to provide a good area.

A 1-2mm deep bead of rubber cement (I use 'Bostik All Purpose Adhesive') is applied to the slide roughly matching the perimeter of the coverslip.

The cavity this provides is filled with distilled water so that the water stands above the adhesive.

A few drops of blood are thoroughly mixed into the water. The aim is for a light pink coloured sample.

When the coverslip is placed there will be leakage so this must be done on a cleanable surface. A bubble in the sample is not a major problem but can lead to annoying movement in the sample so try to avoid this. Once the slip is gently bedded down the bottom of the slide and the coverslip can be blotted and cleaned if necessary.

The final sample depth is around .5 -1.5mm deep.

After a few minutes the red blood cells will become transparent due to the distilled water and subjects will stand out clearly. 100x or 200x magnification allows for a quick initial scan of the slide and will easily show nematodes, some larva and cuticle debris.

After several hours the sample will contain a lot of debris from damaged cells making observation difficult so it is only really suitable for immediate inspection.

A 20x objective and 20x eyepiece provides an excellent combination for revealing details with low risk of touching the coverslip with the objective. The 40x objective with additional camera zoom (if available) should provide ample magnification for most subjects; though in deep samples the 40x might not be able to focus on the surface of the slide without touching the coverslip. An oil immersion objective is not generally practical for this type of sample.

Due to the high degree of transparency of many specimens, a small condenser aperture may improve contrast (unless phase-contrast is available). Polarized light works very well to bring out details. A pair of simple linear polarizing filters (or discs cut from polarized plastic sheet) attached to the light source (or condenser filter holder) and eyepiece can reveal much more detail. I got some linear polarized plastic sheet from Instrument Plastics Ltd which worked better than I could have imagined. The plastic is easy to cut into circles that can be stuck to the eyepiece with BluTack or plastacine. For the lightsource - especially if halogen is used - a disk placed in the condenser filter tray should tolerate the heat from the light. In general, simply rotating the eyepiece provides sufficient effect for examining a specimen so a rotating stage and light filter are not essential.

Method 3

Make a normal smear slide:

1. place a tiny drop of blood near one end of the slide and draw a second slide towards it

2. when the second slide makes contact with the blood drop wait for a moment while the blood runs along the edge of the second slide

3. with a smooth movement push the second slide along the surface of the specimen slide.

The work must be done quickly to prevent clotting. A perfect smear should result in all the blood cells spaced out evenly, though for this subject it is not essential.

I usually prepare 3 slides at a time as the small amount of blood reduces the chances of finding a specimen.

This type of slide can be examined immediately using a magnifying glass or low magnification microscope. Possible nematode specimens are easily seen.

If a likely specimen is found a tiny drop of water can be dropped over the specimen with a syringe or pipette and a coverslip droped into place. I find a 15mm circular coverslip ideal for this.

When the specimen is observed at high power, many of the blood cells around and on the specimen will be gone providing an excellent view.

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