Microscopes

Image Formation and Magnification

Bright-field microscopes create images through the interaction of light waves with the object and the lenses. While the physics behind image formation is complex, understanding how to generate high-quality images is more pertinent for this course. When light passes through an object, it interacts with different media (e.g., air, water, oil, cytoplasm) and changes speed or direction, leading to variations in brightness and contrast in the resulting image. Thicker or denser parts of a specimen, such as a cell nucleus, will appear darker, while thinner regions will appear lighter.

Microscopes use a combination of lenses to magnify the objects under observation, allowing us to see details that are otherwise invisible to the naked eye. The two main types of lenses used in a compound microscope are the ocular lenses (or eyepieces) and the objective lenses.  Located at the top of the microscope, the ocular lenses are where you place your eyes to view the magnified image. Most microscopes have binocular oculars, meaning there are two eyepieces, each containing a lens that typically provides 10x magnification. This means the ocular lens alone magnifies the image ten times its actual size. These are the lenses closest to the specimen. Microscopes usually have multiple objective lenses with varying magnification powers, such as 4x (scanning), 10x (low power), 40x (high power), and 100x (oil immersion). These lenses are attached to a rotating nosepiece, allowing you to switch between different magnifications as needed. The objective lens magnifies the specimen and produces a real image, which is further magnified by the ocular lens to produce the final virtual image that you see. The objective lens magnifies the object to create a real image, which is then further magnified by the ocular lens to produce a virtual image visible to the observer. The total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens (typically 10x). For example, using a high power objective lens (40x) would result in a total magnification of 400x when combined with a 10x ocular lens.  Some labs replace the 10X ocular with higher powered oculars (15x, 20x, 25x).  With a 25x ocular the total magnification with the high power objective lens (40x) would result in a total magnification of 1000x.

Magnification with different objective lenses when paired with a 10x ocular lens

·         Scanning Lens (4x objective): 10 x 4 = 40x total magnification

·         Low Power Lens (10x objective): 10 x 10 = 100x total magnification

·         High Power Lens (40x objective): 10 x 40 = 400x total magnification

·         Oil Immersion Lens (100x objective): 10 x 100 = 1000x total magnification

Magnification with different objective lenses when paired with a 25x ocular lens

·         Scanning Lens (4x objective): 25 x 4 = 100x total magnification

·         Low Power Lens (10x objective): 25 x 10 = 250x total magnification

·         High Power Lens (40x objective): 25 x 40 = 1000x total magnification

·         Oil Immersion Lens (100x objective): 25 x 100 = 2500x total magnification

While microscopes allow us to enlarge images of small objects, there is a limit to how much magnification is useful. Resolution is the ability of a lens to distinguish between two close objects as separate entities. It is ultimately limited by the diffraction of light waves, with most microscopes having a practical resolution limit of about 0.2 μm. Beyond this, magnifying the image further leads to "empty magnification," where the image becomes larger but blurry and lacks detail.

To achieve the highest possible resolution, particularly when using the 100x objective lens (wet lens), immersion oil is used. The oil has a refractive index similar to that of glass, reducing the refraction (bending) of light as it passes from the slide to the lens. This allows more light to enter the lens, improving the clarity and sharpness of the image at high magnifications.

Using the Microscope: Practical Guidelines

For those new to microscopy, achieving high-quality images can be challenging. A general rule is that smaller specimens require higher magnification. For instance, bacteria, which are typically only a few micrometers in size, are best observed with high-power or oil immersion objectives.  When switching between objectives, you may need to adjust the light intensity, condenser position, and iris diaphragm to achieve the best image. Once an object is focused with a lower power lens, it should remain nearly in focus when switching to a higher power lens.  The concept of remaining in focus while magnifying is called parfocal.

In addition to observing specimens under the microscope, it is essential to document your findings through micrography (photographing microscopic images).

Micrographs are photographic images captured through a microscope, providing a permanent visual record of the microscopic structures or organisms observed. These images are essential for documentation, analysis, and sharing of microscopic findings in research, education, and clinical settings. Traditionally, micrographs were captured using dedicated cameras attached to the microscope, but with advancements in technology, both ocular cameras and smartphones have become popular tools for this purpose.  An ocular camera is a specialized digital camera designed to fit into the eyepiece (ocular) of a microscope. These cameras are optimized for capturing images and videos of specimens observed under the microscope.

With the increasing quality of smartphone cameras, cell phones have become a convenient and accessible alternative for capturing micrographs. To capture a micrograph with a cell phone, the phone’s camera is aligned with the microscope's eyepiece. This can be done either by hand or with the help of an adapter designed to hold the phone steady over the eyepiece. The camera's autofocus and exposure settings adjust automatically to capture the image seen through the microscope. The primary advantage of using a cell phone is its accessibility. Almost everyone has a smartphone, making it easy to quickly capture and share micrographs without needing specialized equipment. Modern smartphones also have high-resolution cameras capable of producing detailed images, especially when paired with microscopes of good optical quality. One of the challenges of using a smartphone is ensuring proper alignment with the eyepiece, as any misalignment can result in vignetting (dark corners) or blurred images. Additionally, without an adapter, holding the phone steady can be difficult, leading to potential camera shake and blurred images. Cell phone micrography is particularly useful for fieldwork, quick documentation, or situations where a dedicated ocular camera is not available. It is also popular in educational settings, where students can easily capture and share their observations.

Colony Morphology

Colony morphology refers to the visible characteristics of a bacterial or fungal colony when it is cultured on a solid medium, such as agar in a petri dish. These characteristics are crucial in microbiology for the preliminary identification and differentiation of microbial species. The morphology of a colony can provide valuable insights into the organism's identity, including its genus or species, pathogenicity, and even its metabolic capabilities. See Figure 10.

Size

The size of a colony can range from tiny pinpoint colonies (known as "punctiform") to large, expansive growths. The size often depends on the species of the microorganism and the conditions of incubation, including temperature, medium composition, and incubation time.

Shape

·         Coccus or Cocci can appear spherical or ball-shaped bacteria. 

·         Bacillus are rod-shaped bacteria.

·         Coccobacillus are oval-shaped bacteria.

·         Vibrio are comma-shaped or curved rod bacteria.

·         Spirochete are thin, flexible spiral-shaped bacteria. Spirochetes move in a corkscrew motion due to their flexible structure.

·         Spirillum are rigid, spiral-shaped bacteria. Unlike spirochetes, spirilla have a more rigid structure and a few spirals.

Bacterial Arrangements

·         Diplo, describes pairs of bacteria. This arrangement occurs when bacteria divide in one plane and remain attached in pairs and are called diplococci.

·         Staphylo, describes grape-like clusters of bacteria. Staphylococci form clusters because they divide in multiple planes.

·         Strepto, describes chains of bacteria. This arrangement occurs when bacteria divide in one plane and remain attached, forming chains such as streptobacillus (chains of linked rods).

·         Tetrads are a group of four cocci arranged in a square. Tetrads form when bacteria divide in two planes perpendicular to each other. (Figure 11)

Figure 11 Bacterial Arrangements

Edge (Margin)

The edge or margin of a colony can also vary significantly and used to help identify the bacteria.

·         Entire: Smooth and even.

·         Undulate: Wavy or slightly bumpy.

·         Lobate: Marked with deep, rounded lobes.

·         Filamentous: Thin, hair-like projections.

·         Curled: Wavy, tightly packed curls.

Elevation

The elevation describes how the colony rises above the surface of the agar. See Figure 12.

·         Flat: The colony is level with the surface.

·         Raised: Slightly elevated above the surface.

·         Convex: Dome-shaped, smoothly curving upwards.

·         Umbonate: Raised with a central bump.

·         Crateriform: Central depression resembling a crater.

Figure 12 Colony Characteristics

Color (Pigmentation)

Some colonies may be white, cream, or yellow, while others can exhibit bright colors like red, green, or blue, depending on the pigment produced by the microorganism. Pigmentation can also change based on environmental conditions, such as light exposure or medium composition.

Surface Texture

The surface texture of a colony can be described as:

·         Smooth: Even and shiny.

·         Rough: Coarse and irregular.

·         Wrinkled (Rugose): Covered with folds or ridges.

·         Mucoid: Slimy or sticky, often due to the production of extracellular polysaccharides.

Lab Exercise

Objective

The microscope is an essential tool, allowing us to observe organisms such as bacteria, fungi, and other single-celled entities that are otherwise invisible to the naked eye. Microscopes utilize lenses to magnify these tiny organisms, making it possible to study their form and structure in detail. To maximize the effectiveness of this critical equipment, it's important to understand how a microscope functions. This begins with familiarizing yourself with the names and roles of its various components, as terms like "ocular lenses" and "condenser" frequently arise when discussing ways to enhance microscopic images.

Materials

·         Prepared mouth cell slides

·         Microscope slides and coverslips

·         Sterile cotton swabs

·         Sterile water

·         Commercial yogurt sample

·         Homemade yogurt sample

·         Methylene blue stain

·         Markers and labels

·         Bunsen burner

Procedure

Identifying Microscope Parts

Match the names of the major microscope components with their description of their functions:

A.   Ocular Lenses

B.   Head Piece

C.   Objective Lenses

D.  Revolving Nosepiece

E.   Stage

F.   Coarse Focus Knobs

G.  Fine Focus Knobs

H.  Condenser Lens

I.         Iris Diaphragm

J.        Stage Adjustment Knobs

K.   Condenser Adjustment Knob

_____  These are the lenses you look through at the top of the microscope.

_____  The upper part of the microscope that houses the ocular lenses and connects them to the objective lenses.

_____  These are the primary lenses that vary in magnification (e.g., 4x, 10x, 40x, 100x) and are used to focus on the specimen.

_____  The rotating part that holds the objective lenses. It allows you to switch between different objective lenses.

_____  The flat platform where the slide is placed.

_____  These large knobs move the stage up and down rapidly to bring the specimen into general focus. Used primarily with low-power objectives.

_____  These smaller knobs move the stage slightly. They are used to sharpen the image under higher magnification.

_____  Located beneath the stage, this lens focuses light from the light source onto the specimen to enhance clarity and resolution.

_____  A rotating disk under the stage that adjusts the amount of light passing through the specimen, controlling the contrast and brightness of the image.

_____  These knobs allow you to move the stage left, right, (x axis) and forward, or backward (y axis) to position the slide and focus on different areas of the specimen.

_____  This knob is used to adjust the focus of the light on the specimen for better image quality.

Mouth Smear

The human mouth harbors numerous microbes that persist despite regular brushing and mouthwash use. Obtain a prepared slide labeled "mouth smear" to observe these microorganisms. Begin with the low power objective to locate the large squamous epithelial cells and then switch to high power and oil immersion lenses to identify the smaller bacterial cells.  Then compare the prepared slide with a mouth smear you create from your own mouth.

1.   Look at prepared mouth slides and observe the shapes (cocci, bacilli, etc.) and arrangements (diplo-, staphylo-, strepto-) of bacteria present.

2.   Take a micrograph

Describe the bacterial shapes and arrangements observed. ______________

1.   Create a wet mount mouth slide by using a sterile cotton swab to gently scrape the inside of your cheek.

2.   Roll the swab onto a clean microscope slide with drop of water on it.

3.   Heat fix the bacteria to the slide by passing it over a flame from a Bunsen burner.

4.   Add a drop of methylene blue stain to enhance visibility, let set for 60 seconds.

5.   Gently wash the stain off with sterile water.

6.   Gently place a coverslip over the sample, avoiding air bubbles.

7.   Take a micrograph of the stained slide

Observe and describe the bacteria present, focusing on morphology and arrangement. _______________

Yogurt Slides

Prepared Slide

1.   Obtain a prepared yogurt slide.

2.   Observe the slide under the microscope at 40x magnification.

3.   Capture micrographs of the bacterial shapes and arrangements.

4.   Perform 10 bacterial counts by randomly selecting 10 different fields of view and counting the number of bacteria in each field.

o    Record the average count below.

Prepared Fields   |    Number of Bacteria Counted

1

2

3

4

5

6

7

8

9

10

Average

Describe the colony morphology observed in the prepared yogurt slide.

__________________________________________________________________________________________________________________________________________________________________________________________

Commercial Yogurt

1.   Create a wet mount slide with commercial yogurt

2.   Use a dropper to place a small amount of commercial yogurt onto the edge of a clean microscope slide.

3.   Using a second slide positioned at approximately 45 degrees, drag it across the slide with the yogurt to smear a thin layer across the entire slide.

4.   Heat fix the bacteria to the slide by passing it over a flame from a Bunsen burner.

5.   Add a drop of methylene blue stain to enhance visibility, let set for 60 seconds.

6.   Gently wash the stain off with sterile water.

7.   Place a coverslip over the sample.

8.   Observe at 40x magnification, focusing on bacterial morphology.

9.   Capture micrographs of the bacteria.

10. Perform 10 bacterial counts by randomly selecting 10 different fields of view and counting the number of bacteria in each field.

o    Record the average count below.

Commercial Fields    |    Number of Bacteria Counted

1

2

3

4

5

6

7

8

9

10

Average

Describe the colony morphology observed in the commercial yogurt slide.

__________________________________________________________________________________________________________________________________________________________________________________________

Homemade Yogurt

1.   Repeat the commercial yogurt steps using a sample of homemade yogurt.

2.   Observe at 40x and 100x magnification, focusing on bacterial morphology.

3.   Capture micrographs of the bacteria.

4.   Perform 10 bacterial counts as described above.

o    Record the average count below.

Homemade Fields  |  Number of Bacteria Counted

1

2

3

4

5

6

7

8

9

10

Average

Describe the colony morphology observed in the homemade yogurt slide.

__________________________________________________________________________________________________________________________________________________________________________________________

Compare the bacterial morphology and counts between the prepared slides, wet mounts, commercial yogurt, and homemade yogurt samples. Discuss any observed differences in bacterial morphology and colony arrangements between commercial and homemade yogurt.

__________________________________________________________________________________________________________________________________________________________________________________________