ch3 micro

Microscope

  • The microscope is an essential tool for studying microorganisms due to their small size.

  • Various types of microscopes differ in magnification and price, including light, electron, and atomic force microscopes.

  • Microbiology labs commonly use light microscopes.

Compound Light Microscope

  • Uses visible light for illumination.

  • Called compound because it uses two sets of magnifying lenses:

    • Ocular lens (eyepiece): Magnifies 10x.

    • Objective lenses: Vary from 4x to 100x (typically 10x, 40x, and 100x in a microbiology lab).

Parts of the Microscope and Their Functions:

  • Ocular Lens (Eyepiece):

    • What you look into; magnifies (typically 10x).

  • Stage:

    • Holds the slide.

    • Stage clips keep the slide in place.

  • Condenser:

    • Focuses light from the illuminator (light source) onto the specimen.

  • Iris Diaphragm:

    • A lever within the condenser that controls the amount of light reaching the specimen (like a shutter).

  • Objective Lenses:

    • Magnify at different powers.

  • Rheostat:

    • Controls the amount of light (usually located on the side of the microscope).

  • Coarse Focus Knob:

    • Used on low power (10x) to bring the image into focus.

  • Fine Focus Knob:

    • Used on 40x and 100x to fine-tune focus because the slide is very close to the objective lens at these powers.

How the Microscope Works:

  • Light from the light source passes through the condenser, through the specimen, and then through the objective and ocular lenses to form an image.

Concepts for Forming an Image:

  • Magnification:

    • Increase in the size of the specimen.

    • Dependent on both objective and ocular lenses.

    • Calculate total magnification (TM) by multiplying the power of the objective lens by the power of the ocular lens.

    • TM=Objective Lens Power×Ocular Lens PowerTM = \text{Objective Lens Power} \times \text{Ocular Lens Power}

    • Example: 100x objective lens with a 10x ocular lens results in a total magnification of 1,000x.

    • Most bacteria are visible only with the 100x (oil immersion) lens; immersion oil is required.

  • Resolution (Resolving Power):

    • The ability to distinguish two objects that are very close together as separate entities.

    • Dependent on the type of lens, wavelength of light (shorter wavelengths give sharper images), and specimen preparation.

    • how sharp image is

    • Maximum resolving power of a light microscope is 0.2 microns.

    • Objects closer than 0.2 microns will appear to blend together.

    • Immersion oil is used with the 100x objective to increase resolution.

Resolution and the use of immersion oil

*   Light bends when it passes from one medium to another (e.g., from glass slide to air).
*   The objective lens needs to capture the light that goes through a specimen to form an image.
*   Immersion oil has a refractive index similar to glass, so light does not bend when passing from the slide into the oil, allowing more light to enter the objective lens and increase resolution.

  • Contrast:

    • How easily the organism can be seen against the background.

    • Bacteria are often transparent and have low contrast against a clear background.

    • Contrast can be increased by:

      • Staining the bacteria.

      • Using different types of microscopy, such as phase contrast or dark field.

Different Types of Microscopes

Light Microscopy

  • Uses visible light.

  • Maximum magnification is 1,000x.

  • Different types:

    • Bright Field:

      • Background is bright.

      • Best for colored or stained specimens.

      • Transparent organisms are hard to see because of lack of contrast.

    • Dark Field:

      • Background is black (dark).

      • Transparent organisms appear light against the dark background.

      • Used to view transparent live organisms

    • Phase Contrast:

      • Uses special optics to amplify differences between dark and light regions.

      • Background is grayish.

      • Good for viewing internal structures of live organisms due to variations in lighting.

Fluorescence Microscopy

  • Uses UV light.

  • Specimens are stained with fluorescent dyes that emit visible light when exposed to UV light.

  • Epifluorescence:

    • Uses fluorescent dyes attached to antibodies to detect specific pathogens.

    • If the antibody binds to the organism in the sample, fluorescence indicates the presence of that particular bacteria.

Scanning Laser Microscope

  • Allows for detailed views and greater magnification than light or fluorescence microscopes.

  • Used to study biofilms and locate specific structures within cells.

  • Specimens are stained with fluorescent dyes

  • tagged bind specifically to certain internal compounds & to detect specific structures.

  • Allow detailed views of interior of intact cells

  • Marks there location

Electron Microscopy

  • Uses electron beams instead of light, resulting in shorter wavelengths and greater resolving power.

  • Magnification up to 100,000x.

  • Two types:

    • Scanning Electron Microscope (SEM):

      • Looks at the surface of the cell to create a 3D image.

    • Transmission Electron Microscope (TEM):

      • Looks at the internal structures of cells.

      • Specimens are embedded in plastic, sliced thinly with a diamond knife, and then viewed.

Scanning Probe Microscopes

  • Use metal probes for high resolution images.

  • Example: Atomic Force Microscope

  • Resolving power much greater than that of electron microscope (EM)

  • Can be used to view detailed images of parts of cells, such as nucleic acids and proteins.

Specimen Preparation for Light Microscopy

  • Can examine live or stained specimens.

  • Live bacteria are hard to see due to lack of contrast.

  • Dark field and phase contrast microscopy can improve visualization of live specimens.

  • Higher contrast is achieved by specimen staining (makes bacteria pop out) & different types of microscopy

preparing Staining Specimens

  • Increases contrast by coloring the bacteria.

  • Stains Start with a smear which is a thin layer of bacteria on a slide.

  • Fix the bacteria to the slide using heat.

  • Stain with different dyes:

    Bacteria: slightly negatively charged

    • Basic Dyes:

      • Positively charged (cationic).

      • Bind to the negatively charged bacterial cell.

      • Bond to cell & stain it

      • Examples: methylene blue, crystal violet, safranin.

    • Acidic Dyes:

      • Negatively charged (anionic).

      • Repelled by the negatively charged bacterial cell.

      • Stain the background, making the bacteria stand out.

      • Since this dye is negative and bacteria is negatively charged, they repel each other

      • Commonly stain background, but not the cell

Staining Procedures

  • Simple Stain:

    • Uses one stain to color either the cell or the background.

    • Increases contrast to observe cell size, shape , and arrangement.

    • ALL cells stained the same color

    • Basic dyes stain the cell.

    • Acidic dyes stain the background (e.g., nigrosin).

  • Differential Stains:

    • Used to Distinguish between different types of bacteria.

    • Use a series of reagents and stains.

    • Two common examples:

      • Gram Stain.

      • Acid-Fast Stain.

Gram Stain

  • Developed by Hans Christian Gram.

  • Most widely used procedure for staining bacteria

  • Separates bacteria into two groups based on cell wall structure:

    • Gram-positive: Appear purple.

    • Gram-negative: Appear pink to red.

  • Gram Stain Procedure:

    1. Prepare a smear, air dry, and heat fix.

    2. Apply crystal violet (primary stain) to stain cells purple.

    3. Rinse with water.

    4. Apply iodine (mordant) to form crystal violet-iodine complex, making it harder to wash out the dye.

    5. Rinse with water.

    6. Decolorize with alcohol, which removes the dye from gram-negative cells.

    7. Rinse with water.

    8. Counterstain with safranin, which stains the decolorized gram-negative cells red.

  • Results:

    • Gram-positive cells retain crystal violet and appear purple.

    • Gram-negative cells lose crystal violet and are stained by safranin, appearing pink to red.

Important Note

  • The terms "positive" and "negative" in Gram stain refer to whether they retain the primary dye and does not have to do with charge.

Acid-Fast Stain

  • Used to detect members of the genus Mycobacterium *

  • includes causative agents of tuberculosis and leprosy*

  • Cell wall contains high concentrations of Mycolic acid

  • Waxy fatty acid that prevents uptake of dyes

  • Mycobacterium have waxy cell walls with high concentrations of mycolic acid, which repels Gram stain dyes.

  • Acid-Fast Stain Procedure:

    1. Apply carbol fuchin (primary stain) to stain acid fast bacteria red;

    2. Decolorize with acid alcohol to remove stain from non-acid-fast bacteria.

    3. Counterstain with methylene blue to stain non-acid-fast bacteria blue.

  • Results:

    • Acid-fast cells retain carbolfuchsin and appear red.

    • Non-acid-fast cells lose carbolfuchsin and are stained by methylene blue, appearing blue.

Acid fast refers to its ability to withstand acid wash to stay with the primary dye.

Special Stains

  • Used to identify specific structures.

    • Capsule Stain

      • Capsule is a gel-like polysaccharide layer surrounding cell

      • Stains poorly, negative stains often used – use acidic dyes

      • Capsule helps bacteria from host defenses, helps them attach to surfaces and protect from desiccation and also as a nutrient.

      • Allows capsule to stand out around organism

      • Stain the background to create a halo effect around the cell.

        • Or, stain the background and the bacterial cell, leaving the capsule unstained.

    • Endospore Stain:

      • Endospore is a resistant, dormant structure formed by species of (Bacillus, Clostridium)

        • Resists Gram stain, often appears as clear object

          Endospore stain

      • Use heat facilitate uptake of the primary dye malachite

        green by endospore

      • Counterstain with safranin to stain the vegetative cells.

        • Endospores appear green, and vegetative cells appear red.

    • Flagella Stain:

      • Flagella used for prokaryotic motility.

      • STAIN: Add dye to strengthen/thicken the flagella, making them visible under microscope

      • Useful for identifying bacteria based on the number and location of flagella.

Bacterial Shape (Morphology)

  • Coccus: Spherical.

  • Rod (Bacillus): rod or Cylindrical.

Other shapes:

  • Spirals:

    • Vibrio: curved rod

    • Spirillum: Spiral-shaped.

    • Spirochete: Helical, corkscrew-shaped.

  • Pleomorphic: many different shapes (usually bacteria without cell walls).

Bacterial Groupings (Arrangements)

  • Refers to how cells are arranged relative to each other.

  • Most prokaryotes divide by binary fission.

  • Form characteristic groupings or arrangements depending on plan of division

    Division along a single plane form:

  • Pairs = diplo cocci (2 spherical bacteria)

  • Forms chains = streptococci (species of Streptococcus)

Division in 2 or 3 perpendicular planes:

  • Forms cubicle Packets

  • Example: Sarcina genus

*For packets of 4. forms tetrads.

Division along several random planes form grape like structures

Example: species of Staphylococcus (grape-like clusters of cells =staphylo)

  • Single bacillus rods are often single rod.

  • 2 rods together are diplobacilli.

  • Chains of rods is called streptobacilli.

Parts of the Bacterial Cell

  • Common structures:

    • Cytoplasm.

    • Chromosome (located in the nucleoid region).

    • Ribosomes.

    • Plasma membrane.

    • Cell wall.

  • Optional structures:

    • Flagella.

    • Capsule.

    • Pili (fimbriae).

Structures External to Cell Wall

  1. Capsule : organized, attached to cell wall

  2. Slime layers:unorganized, loose

• Composed of sugar (glycocalyx) and/or polypeptides

• Excreted by organism, not always present

•Function

• Protection from host defenses ( phagocytosis)

• Attachment to surfaces

• Protection against drying (desiccation)

• Reserve of nutrients

(Increases virulence of pathogens)

Capsule of slime layer, both serve the same function.

  • Protects bacteria from your host defenses, protect them for dry mouth, and a reserve of nutrients.

Glycocalyx

  • Dental plaques: oral streptococci (chain circular) use capsular slime to adhere to surfaces of teeth and gums → biofilm