Bacterial Cell Size, Morphology, and Arrangement
Bacteria come in many different sizes and shapes (morphology=shape). The sizes of bacteria range from \(<1.0\mu m\) to \(>250\mu m\). However, most bacteria, and the ones you will observe in lab range from about \(1\mu m-15\mu m\). The most common shapes of bacteria, and the ones you will observe are cocci, bacilli, and spirilli.
Bacteria also often grow into different arrangements as the cells divide. Cocci have the most variety in their arrangements, some bacilli may stay in pairs or chains as they divide, but spirilli are found singly. Remember that organisms, and their arrangements, are three-dimensional. Note that sarcina is essentially cuboidal and staphylococci are a cluster formed by cells dividing in an irregular pattern.
Stains are solutions containing a pigmented molecule. The part of the molecule that is colored is called the chromophore. The chromophores carry either a net + or – charge, therefore are attracted to the opposite charge. Stains with a net + charge are called “basic stains”, those with a net – charge are called “acid stains”. Bacterial cells have a net – charge. Thus, basic stains will attach to the cells, while the cell will repel acid stains.
Basic stains (+ charge)-
- Methylene blue
- Basic fuchsin
- Crystal violet
- Malachite green
Acid stains (- charge)-
- Acid fuchsin
100x lens and Oil Immersion
Bacteria are very small of course, so it is necessary to view them at the highest magnification possible with the best resolution possible. In a light microscope this is about 1000x due to optical limitations. The Oil immersion technique is used in order to enhance resolution. This requires a special 100x objective. The 100x lens is immersed in a drop of oil placed on the slide in order to eliminate any air gaps and lossof light due to refraction (bending of the light) as the light passes from glass (slide) → air → glass (objective lens). Immersion oil has the same refractive index of glass. When used, light passes through glass → oil → glass without loss due to refraction.
When the oil immersion lens is in place there are a few things to be aware of and very careful about-
- The working distance is VERY small.
- Depth of focus is also VERY small.
- Use only the fine focus, and focus slowly.
- Increase the light, as the lens opening is very small.
- Adjust the condenser and iris diaphragm as needed.
- Do not drag the 40x lens through the oil, if you need to go to lower power-4x or 10x, rotate the nosepiece in the opposite direction to avoid the 40x lens. Dragging the 40x lens through the oil will damage the lens!
- The immersion oil has the same refractive index as glass, so you may go back to low power (again, not 40x) and still see your specimen.
- Do not make any decisions or assessments about your bacterium until you are focused at 100x.
- If you see the cells clearly with the 4x, 10x, 40x, but don’t see it with 100x, your specimen may be upside down. Ask your instructor for help.
Viewing with the oil immersion 100x objective lens-
- Place a stained slide on the stage in the clips.
- View the slide at low power (4x) and look for staining; for very small cells look for a pattern of regular tiny specks. Once in focus, move to the next objective (10X), focus with fine focus only. Move to 40x, fine focus only (if the 40x objective is not very clear—due to it being repeatedly dragged through the oil, skip it). Make sure that the area you want to see is dead center in the field of view.
- Rotate the objective aside so that you are between it and the 100x objective and let a small drop of oil fall onto the slide where your specimen is. Carefully rotate the 100x objective into place, immersing it into the drop of oil.
- While looking through the oculars, very carefully and slowly use the fine focus to bring the image into sharp focus. If you can’t get it into focus, or have lost what you are looking for you may go back to 4x or 10x and start over (do not remove the oil, it’s fine).
- When finished with your slides wipe the oil from the 100x objective with lens paper (DO NOT use anything else to clean the lens!). The lens retracts into the lens housing. Be sure to push up gently to remove oil that has moved up in the housing (this happens more when one is viewing a slide and over focuses and pushes the lens up into the housing). Then, use lens cleaner and lens paper to completely remove all oil from the lens. Keep the lens paper flat and use a circular motion. Do not wad up the lens paper. Finally, use lens paper to check all the other objectives and parts of the microscope—focus knobs, stage, drips onto the condenser, etc., and clean up any oil on them.
Contributors and Attributions
Kelly C. Burke (College of the Canyons)