Ch. 3 - Observing Microbes

Compared to 1 meter, how much is a millimeter?

10^(-3)

Compared to 1 meter, how much is a micrometer?

10^(-6)

Compared to 1 meter, how much is a nanometer?

10^(-9)

Compared to 1 meter, how much is a picometer?

10^(-12)

What microscopes use light to observe specimens?

• Compound

• Darkfield

• Phase-contrast

• Fluorescence

How do you find the total magnification?

objective lens magnification x ocular lens magnification

What does resolution in a microscope do?

Distinguishes the differences in details & structures

What will provide a greater resolution in a microscope?

Shorter wavelengths

Describe a brightfield microscopy

• Direct light enters objective lens

• Dark objects become visible against the light reflection

• Staining increases contrast

Describe a darkfield microscopy

• Opaque disk placed between light & specimen

• Only light diffracted by specimen reaches eyepiece

• Emphasize edges of structure

Describe fluorescence microscopy

• Uses UV light (short wavelength)

• Absorb UV light & emit visible light (longer wavelength)

• Stained with antibodies containing fluorochrome

What is the color of the fluorochrome stained on a microorganism?

Ultraviolet

Describe electron microscopy & what are the kinds?

• Transmission & Scanning electron microscopy

• Uses a beam of electrons (short wavelength)

• B&W images unless artificially colored

• Stained with metal salts or antibodies

Describe transmission electron microscopy

• Ultrathin sections of specimen

• Uses magnets instead of an objective lens

• Produces 2D images of internal structure

• Magnification of 10,000-100,000

• Resolution = 2.5

Describe scanning electron microscopy

• Scans the surface of specimen, no internal structures

• Produces 3D image

• Magnification of 1,000-10,000,00

• Resolution = 20nm

What are electron microscopes able to see?

Small details (flagella) & viruses

Order of preparation of specimens for light microscopy

1. Smear (organism spread on slide)

2. Fixing (pass slide through flame)

3. Staining

What is the purpose of “fixing” the slide

• Kills the bacteria

• Attaches the bacteria to the slide

• Preserves the structures the bacteria originally had

pH of a bacteria cell

pH 7 — slightly acidic

How does the pH of a bacteria cell relate to a body cell?

Plasma membranes are negatively charged

What are stains?

Salts (NaCl) where once of the ions are colored

Describe a basic dye

• Most common

• Chromophore is a cation

• Crystal violet, methylene blue, safranin

Describe an acidic dye

• Chromophore is an anion

• Used in negative staining (colorless bacteria, stains background)

Why do we stain cells?

Determines the size, shape, & arrangement of the cells

Purpose of a simple stain

Increase contrast highlighting the structures

Purpose of a differential stain

Using multiple dyes to differentiate between organism or structure

Purpose of special stains

Used to differentiate structures such as flagella, capsules, or endosphores

What is the Gram stain procedure?

1. Smear is covered in basic crystal violet dye

2. Cover in iodine (mordant) & wash it off

3. Wash with alcohol & rinse off

4. Stain with basic safranin dye & wash off

How is a gram-positive bacteria determined?

If the bacteria remains purple after decolorization

How is a gram-negative bacteria determined?

If the bacteria loses the purple & becomes red or pink after decolorization

What are some gram-positive bacteria?

• Staphylococcus aureus

• Streptococcus pyogenes

• Streptococcus pneumoniae

What are some gram-negative bacteria?

• Escherichia coli

• Neisseria gonorrhoeae

Describe the acid-fast stain

• Bacteria stains red

• Thick outer lipid layer containing mycolic acid

Acid-fast stain is used for what bacteria?

• Mycobacterium tuberculosis

• Mycobacterium leprae

Process of the acid-fast stain

1. Primary stain — carbolfuchsin (red dye)

2. Decolorized (alcohol)

3. Counterstain — methylene blue

4. Non acid-fast stain becomes blue