Microscopy for Microbiology

You will identify the components of an optical microscope. You will describe field of view and depth of field. You will view a series of preserved specimens including protozoans, fungi, and bacteria. You will prepare wet mounts of cheek cells and dental tartar.

EXPERIMENT

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Learning Objectives Upon completion of this laboratory, you will be able to:

● Outline the components of an optical microscope.

● Describe field of view and depth of field.

● Calculate the total magnification and field of view for the lenses of an optical microscope.

● Examine prepared slides under scanning, low, high, and oil immersion lenses.

● Prepare wet mounts of cheek cells and dental plaque and examine them under different lenses.

Time Allocation: 3.5 hours

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Experiment Microscopy for Microbiology – Use and Function

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Experiment Microscopy for Microbiology – Use and Function

Materials Student Supplied Materials

Quantity Item Description 1 Camera, digital or smartphone 1 Distilled water 1 Roll of paper towels

HOL Supplied Materials

Quantity Item Description 2 Blank slides 1 Pair of gloves 1 Lens paper (50 sheets) 1 Long thin stem pipet 1 Metric ruler 1 Safety goggles 1 Slide Box:

1 – Slide – Amoeba, whole mount 1 – Slide – Bacteria bacillus form 1 – Slide – Bacteria coccus form 1 – Slide – Bacteria spirillum 1 – Slide – Letter e focusing slide 1 – Slide – Penicillium with Conidia 1 – Slide – Yeast, whole mount

1 Slide cover glass cube 1 Sterile swabs, 2 per pack 1 Student microscope with 100x oil immersion lens*

1 – Immersion Oil

*Microscopes and oil immersion lens are purchased separately from the LabPaq kit.

Note: To fully and accurately complete all lab exercises, you will need access to:

1. A computer to upload digital camera images.

2. Basic photo editing software, such as Microsoft Word® or PowerPoint®, to add labels, leader lines, or text to digital photos.

3. Subject-specific textbook or appropriate reference resources from lecture content or other suggested resources.

Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit.

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Experiment Microscopy for Microbiology – Use and Function

Background Optical Microscopes

A microscope is an optical instrument that uses a lens or a series of lenses to magnify objects that are either too small to be seen by the naked eye, or to view distinct details of an object that are not visible to the naked eye. There are three main categories of microscopes: optical, electron, and scanning probe.

Optical microscopes, also referred to as light microscopes, use reflected visible light and a series of two or more convex lenses to magnify and focus an object. The most common type of light microscope is the compound microscope, which utilizes a lens of short focal length and a second lens of a longer focal length. The short lens forms and focuses the image, and the longer lens magnifies and further focuses the image. Compound microscopes are able to resolve objects to a resolution of approximately 200 nm (0.2 µm). Resolving power is the ability to discern two objects as separate, and is limited by the wavelength of light. As compound microscopes utilize reflected visible light, the resolution of the microscope is limited by the wavelength of the visible light.

The components of a compound microscope work together to create a magnified image. See Figure 1. The description of each of the parts is discussed on the following page.

Figure 1. Main components of a compound microscope. © Picsfive

The ocular lens is located at the top of the microscope. This short lens is termed “ocular” as it the lens that one looks through in order to view the image. An ocular lens is typically 10x or 15x power and is often referred to as the eye piece. A microscope with one ocular lens is called a monocular microscope, a microscope with two ocular lenses is called a binocular microscope (as shown in Figure 1), and a microscope with two ocular lenses and a location for a camera is called a trinocular microscope.

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Experiment Microscopy for Microbiology – Use and Function

The objective lenses of a microscope are found closest to the stage and are held by the turret, or revolving nose piece. The turret allows the lenses to be easily rotated to select the lens that offers the necessary power of magnification. There are typically three to four objective lenses on a microscope, ranging in power from 4x to 100x. The lowest power objective lens (4x) is the scanning lens, the medium strength objective lens (10x) is the low power lens, and the highest strength objective lens (40x) is the high power lens. If a fourth objective lens is present it is typically the 100x oil immersion lens. Oil immersion lenses are typically spring-loaded and retractable, so that the end of the lens will retract if it comes into contact with the object, protecting both the slide and the lens from damage. Additionally, an oil immersion lens is generally sealed, keeping the oil from seeping into the lens. The total magnification factor of a microscope is calculated by multiplying the magnification of the ocular lens with the magnification of the objective lens. For example, if the ocular lens has a magnification of 15x and the objective lens has a total magnification of 40x, the total magnification factor is 600x: 15 x 40 = 600.

Related to the total magnification factor are the field of view and depth of field. The higher the total magnification factor, the smaller the field of view. For example, imagine looking at a book at a distance of 3 feet. At this distance there is a large field of view, or the visible area that can be observed. In this case, the entire book is visible, along with its location relative to other objects. As the distance between your eye and the book is decreased, so is the field of view. If you are holding a book only an inch from your eye, not only is there a loss of dimensionality (from 3D to 2D), only a few words on the cover will be visible instead of the entire book. This is analogous to viewing an object with a microscope: by increasing the magnification, the field of view (or amount of the object seen) is smaller, and more detail is visible.

Depth of field refers to the distance range that is in focus at a given time. This concept is also relevant in photography, where a photographer can adjust the depth of focus to have either a shallow focus that will emphasize a subject, or a greater focus, that will focus to include much of the foreground and background. Like field of view, the depth of view is dependent on the total magnification factor. Compound microscopes have an inherently shallow depth of field, on the order of only a few micrometers. See Figure 2.