Chapter 4: Microscopy, staining, classification

2 characteristics of staining for electron microscopy

- Chemicals containing heavy metals used for transmission electron microscopy

- Stains may bind molecules in specimens or the background

4 general principles of microscopy

1. Wavelength

2. Magnification

3. Resolution

4. Contrast

Resolution

Ability to distinguish two points that are close together

T o F: The better the resolution, the harder is it to distinguish two nearby objects from one another

False, the better the resolution, the EASIER it is to distinguish the two near objects

Contrast

Difference in intensity between two objects or between an object and its background

2 types of field within light microscopy

1. Bright field microscopes

2. Dark field microscopes

Bright field microscope visuals/appearance

Colored or clear specimen against bright background

1 multiple choice option

Dark field microscope visuals/appearance

Bright specimen against dark background

1 multiple choice option

Bright field microscope special feature

Stained specimens often required

1 multiple choice option

Dark field microscope special feature

Only light scattered by the specimen is visible to observe living, colorless, unstained organisms

1 multiple choice option

Bright field microscope typical uses

To observe killed stained specimens and naturally colored live ones; also used to count microorganisms

1 multiple choice option

Dark field microscope typical uses

To observe living, colorless, unstained organisms

1 multiple choice option

2 types of bright-field microscopes

1. Simple

2. Compound

Total magnification

Objective lens x ocular lens

3 types of dark-field microscopes

1. Phase

2. Fluorescence

3. Confocal

Phase microscope use

Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining

Fluorescence microscope use

Uses UV light source at specimen

Confocal microscope use

Uses UV layers to illuminate fluorescent chemicals in a single pore with fluorescent dyes

Electron microscopy

Magnifies objects 10,000x to 100,000x

2 types of electron microscopes

1. Transmission

2. Scanning

Probe microscopy

Magnifies more then 100,000,000x

2 types of probe microscopes

1. Scanning tunneling

2/. Atomic force

Staining

Coloring the microbe with a dye that emphasizes certain structures

4 Principles of staining

- Dyes used as stains are usually salts

- Chromophore is the colored portion of the dye

- Acidic dyes stain alkaline structures

- Basic dyes stain acidic structures

T o F: Basic dyes are more common because most cells are negatively charged

True

Acidic dyes stain

Alkaline structures

Basic dyes stain

Acidic structures

4 types of staining methods

1. Simple

2. Differential

3. Histological

4. Special

Simple stains

Single basic dye is used to determine size, shape, and arrangement of cells

3 common simple stains

1. Crystal violet

2. Safranin

3. Methylene blue

Differential stains

More then one dye is used to distinguish between different cells, chemicals, or structures

4 common differential stain methods

1. Gram stain

2. Acid-fast stain

3. Endospore stain

4. Histological stains

Gram stain

Distinguishes between two different kinds of bacterial cell walls

Step 1 in Gram staining

After smear preparation, the slide is flooded with crystal violet, which stains all bacterial cells purple

Step 2 in Gram staining

Iodine is added, forming a complex with the crystal violet, which helps the stain adhere to the cell wall

Step 3 in Gram staining

A decolorizing agent (alcohol or acetone) is applied. This is the most crucial step. Gram-positive bacteria retain the crystal violet-iodine complex, while Gram-negative bacteria lose the stain due to their thinner peptidoglycan layer and outer membrane

Step 4 in Gram staining

Safranin is added, which stains the decolorized Gram-negative bacteria pink or red

Step 5 in Gram staining

The slide is blotted dry and examined under a microscope. Gram-positive bacteria will appear purple, and Gram-negative bacteria will appear pink or red

Acid-fast stain

Used for identifying bacteria that have a waxy cell wall

Endospore stain

Used to detect the presence and location of spores in bacterial cells

Histological stain

Used to color tissue sections to highlight and differentiate structures under a microscope

2 types of histological stains

1. Gomori methenamine silver

2. Hematoxylin and eosin

Special stains

Simple stains used to identify specific microbial structure

3 types of special stains

1. Negative stains

2. Flagellar stains

3. Fluorescent stains

Why is a gram-negative bacterium colorless but a gram-positive bacterium is purple after it is rinsed with decolorizer?

Because the thick peptidoglycan layer in Gram-positive bacteria retains the crystal violet-iodine complex, while the thin peptidoglycan layer and outer membrane of Gram-negative bacteria do not, so the dye is washed out by the decolorizer, leaving them colorless

Taxonomy

Consists of classification, nomenclature, and identification of organisms

8 levels of taxonomy

1. Domain

2. Kingdom

3. Phylum

4. Class

5. Order

6. Family

7. Genus

8. Species

Domain

Broad category, groups all life based on major cell differences. Includes Bacteria, archaea, eukarya

Kingdom

Large groups within domains. Includes animals, plants, fungi, protists

Phylum

Groups organisms based on major body plans or organization. Includes chordata for animals with a backbone

Class

Groups organisms based on breaking down phyla into groups like mammalia

Order

Groups similar families together. Includes primates like monkeys, apes, humans

Family

More closely related organisms. Include Hominidae, the great apes and humans

Genus

Very closely related species grouped together (Homo)

Species

Most specific level; groups organisms that can interbreed and produce fertile offspring like homo sapiens

5 kingdoms

1. Animalia

2. Plantae

3. Fungi

4. Protista

5. Prokaryote

Phylogenic hierarchy

All taxonomic categories

Greater emphasis on comparisons of organism's genetic material led to proposal of adding ________

Domains

______ _______ was known for comparing nucleotide sequences of rRNA subunits which led to proposal of three more domains

Carl woese

6 taxonomic identifying characteristics

1. Physical characteristics

2. Biochemical tests

3. Serological tests

4. Phage typing

5. MALDI/TOF Mass spectrometry

6. Analysis of nucleic acids

Physical characteristics can be used to identify microorganisms through

Their morphology

4 microorganisms that can be identified based only on their morphology

1. Protozoa

2. Fungi

3. Algae

4. Parasitic worms

Biochemical tests can be used to identify microorganisms through

A prokaryotes ability to utilize to produce certain chemicals

Biochemical tests are usually used for

Identifying pathogens

Serological tests can be used to identify microorganisms through

The study of antigen-antibody reactions in lab settings

Phage typing can be used to identify microorganisms through

Through their susceptibility to specific bacteriophages (viruses that infect bacteria)

MALDI/TOF Mass spectrometry can be used to identify microorganisms through

Through analyzing their unique protein "fingerprint" (mainly ribosomal proteins)

MALDI/TOF Mass spectrometry is used for

Identifying species, predicting resistance to antibiotics, and to rapidly diagnose diseases

Analysis of nucleic acids can be used to identify microorganisms through

Through detecting and comparing their unique DNA or RNA sequences

Taxonomic keys or Dichotomous keys

Series of paired statements where only one of two "either/or" choices applies to any particular organism