Observing with the Microscope

Microscope Setup

A drop of immersion oil goes on the coverslip over the specimen. For darkfield put a drop either on top of the condenser or on the underneath of the slide. On cavity slides this drop is above the edge of the cavity. The drop will spread as the slide is moved and can remain on the coverslip for days and rarely needs replenishing.

The slide is placed on the stage and held in place by the spring arm.

The condenser is moved upwards until it almost touches the bottom of the slide. Once the specimen is in focus the condenser may need adjusting again to optimise illumination of the subject.

BRIGHTFIELD: With the microscope light set to its brightest, the aperture on the condenser is closed to the smallest size that allows enough light. The purpose of the tiny aperture is to give the maximum possible contrast. This is not a pleasant effect for normal viewing but bacteria are so small that this is needed to make them visible. With very strong illumination (as with the LEDs - see:LED Illumination for Microscopy) a pinhole apperture can be made and placed into the filter holder beneath the condenser. This modification can increase contrast even further.

The stage and focus controls are used to lower the 100x objective lens into the oil and adjusted until the specimen is in focus.

I almost exclusively use the camera connected to the computer for observing - and this shows more detail than I can see with my eye. I have heard others remark that they can see more with their eye than their camera shows so this is an area where individual preference and setup will determine what method is used.

On a newly made slide the blood cells often seem motionless at first but gradually start to move until the whole sample appears to be twitching. Much of the subtle movement is due to 'Brownian Motion'. A Google search will show explanations of this phenomenon.

Sometimes strange artifacts can be seen, these might be the remains of blood cells that have ruptured, other bits of cellular debris, crystals of cholesterol, fibrin, other bacteria or contamination from the slide preparation. In fact preparing slides in this way is almost certain to result in some type of contamination and damage to the sample.

A blood slide can actually contain so much debris that I find it better to look for moving organisms to observe. Subjects that are moving actually seem to become more visible. Their motion helps to define their outline. If you play some of my videos and pause them, you can see that individual frames are not very distinct, but when the video is playing more detail can be discerned. You might notice that in my videos I usually try to include a red blood cell in the frame to give scale to the subject.

I hope you enjoyed these few pages about my microscopy hobby - thanks for looking.

UPDATE

Here are a few tips I've found useful with DARKFIELD microscopy for bacteria:

If the view seems washed out, first check for bubbles on the condenser or coverslip. A minute bubble on the condenser can ruin the illumination. Raising and lowering the condenser a few times and moving the slide with the stage controls can push bubbles out of the light path.

Remember you may need to adjust the vertical position of the condenser. When setting up the slide I often find that raising the condenser till it makes contact with the slide then backing it off a fraction is fine. However sometimes a minute adjustment to the condenser can make an improvement in resolution.

If you can see coccoid bacteria down to below .5uM it does not follow that the thinnest long bacteria will be visible. spherical/round bacteria can have a lens like effect making them especially visible so they can be a misleading guide to how good the resolution is.

Don't adjust the setup for a black background. It may look impressive but a lot of tiny subjects remain invisible, especially if the sample is relatively deep - i.e., >20uM. Adjust the objective diaphragm/camera to give a slightly overexposed view. If the view is excessively bright contrast will be lost but if the view is too dark the thinnest bacteria may not be visible. This is because their brightness levels are sometimes little more than the darkest colours visible.

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