Finding low-contrast microbiological specimens under the microscope

My microscopy book, Understanding and Using the Light Microscope, has prompted some interesting questions, which we hope to address in later parts of the series. In the meantime, I thought I would share them on my blog.

Q: Do you have a preferred method on focusing on finding the focal plane of micro-biologic specimens when you can’t ‘see’ the specimen with the naked eye?  Do you prefer to begin by focusing on the upper surface of the microscope slide or lettering for instance?

Phase A contrast (a type of Phase contrast)
makes it easier to see very faint, transparent subjects

A: Well, there are several ways I might tackle this. In the absence of knowing what you are looking at, too many answers. But here are some to try:

Practice with a cheek scrape - you will have faint nucleated cheek cells, some of which will have bacterial colonies on them. Use minimal material and a coverslip, so sample mounted very thin in water (well, saliva actually).

1. Either
a) Focus on a known boundary (e.g. coverslip edge, dust on slide, scratch or ink on slide) and use the fine focus to search above or below that point to find your subject. OR

b) With a dried smear. Hold slide up so that you see light reflected on the surface with the sample - see if there is an area which looks less reflective due to more sample having dried there. Start trying to find something to focus on there. Then migrate away to a part of the slide where your sample is more thinly distributed.

2.  Use a method that increases the sample contrast against the background.

a) OBLIQUE ILLUMINATION: Start with a low power objective. Create oblique illumination by cutting off light entering one half of the condenser (a crude but effective way is to use a finger between the lamp and the condenser). Low contrast subjects are thrown in relief and stand out more. Once you find and focus on the subject, zoom in by going through to your higher objectives, again using oblique illumination. When at the highest magnification and focused on the sample, revert to standard optimum lighting.

b) DARKFIELD: Use of a central stop under the condenser creates a black background. Light hitting the samples from the ring around the central stop makes samples glow against the black background. You can create your own stops with ink or card discs on centre of a transparent filter. Many condensers have a filter holder. NOTE that the condenser iris diaphragm has to be opened wider to allow the ring of light outside the field of view to illuminate the sample.

c) If you have it, use PHASE CONTRAST at the higher power. Note that the phase contrast ring in the phase condenser for higher power will act as a darkfield illuminator with your lower power objectives.

d) Consider STAINING. Dilute fountain pen ink (a dye) stains protein (and fingers). Play. Samples stained against background.

e) Consider CONTRAST STAINING with drawing pen or other particulate ink. Ink particles are excluded by subjects, leaving them light against a dark background.

3. If your samples are too sparse and difficult to see - consider creating a new slide with more sample on it.

DO NOT SIMPLY CLOSE THE CONDENSER IRIS DIAPHRAGM TO INCREASE CONTRAST - YOU MAY APPEAR TO GAIN CONTRAST BUT YOU WILL HAVE LOUSY RESOLUTION!

If you need to go above a 40x objective to use an oil immersion one, life is a bit more problematic.

• Find a visible sample using one of the above methods (oblique, dark field or phase contrast) until you get to the 40x objective (bacteria are visible as tiny spots or rods).
• Centre on a good contrast object.
• Move objective away from slide
• Add small drop of immersion oil over sample
• Swing in oil immersion objective (Make sure it is a sprung lens so you do not damage slide, sample or lens if you accidently go too close to sample and touch the slide).
• Lower OI objective to touch oil drop on slide
• Get objective as close as possible to slide/sample by looking from side and lowering carefully with coarse focus.
• Look through eye piece and use fine focus to move objective away from slide.

Note that you should also use an oil immersion bridge between the condenser (if labelled with an NA greater than 1) and underside of slide for optimum resolution.

Do you have a microscopy question? Contact Chris at Chris@miltoncontact.com or visit www.usingthemicroscope.com