BIOS 44 Microbiology - LECTURE 1 Microscopy

EXAM 1 LECTURE 1

Microbial World

Split into 2 categories

1. Organisms

2. Acellular Infectious Agents

Cellular Organisms

Living organisms containing cellular structures. They can be split into 2 categories: Prokaryotes and Eukaryotes.

1. Bacteria

2. Archaea

3. Eukarya

Prokaryotes

A simple cell containing no membrane bound organelles, such as a nucleus. They are unicellular. Prokaryotes consist of:

1. Bacteria

2. Archaea

Eukaryotes

Advanced cells containing membrane bound organelles, and have a nucleus. They can be unicellular or multicellular. THey consist of:

1. Protists

   • Algae (uni/multi)

   • Protozoa (uni/multi)

2. Fungi

3. Helminths

Acellular Infectious Agents

A non cellular organism- no nucleus, not considered a cell. Viruses consist of genetic material (DNA/RNA) that is nonfunctional without a host. When the host is hijacked, it replicates.

1. Viruses

2. Viroids

3. Prions

Binomial Nomenclature

A system used to name living organisms- NOT including viruses.

E.g. E. choli, Escherichia coli

1. Genus name 1st, always capitalized

2. Species name 2nd, always lowercase

Parts of a Light Microscope

Rheostat knob: Light intensity

Condenser: Focus light onto specimen

Iris Diaphragm lever: Adjusts light for better contrast

Objective lenses: 4x, 10x, 40x, 100x

Ocular lenses: Always 10x magnification

Coarse/Fine focus knob: Focuses speciment

Microscope Resolution

The ability of a microscope to distinguish 2 closely spaced objects as separate entities. It determines how much detail a microscope can reveal.

Or, the minimum DISTANCE where 2 separate objects can be distinguished.

Oil immersion can increase resolution at a 100x objective on light microscopes; it prevents refraction and loss of light rays.

Types of Microscopes

1. Light Microscope

   • Brightfield

   • Darkfield

   • Florescence

2. Electron Microscope

   • TEM 2D

   • SEM 3D

3. Atomic Force Microscope

Light Microscope

Uses light and glass lenses and magnify up to 1000x. Resolution is 0.2 micrometers.

3 Types of Light Microscopes:

1. Bright-field

   • Evenly illuminates the FOV, white bg

2. Dark-field

   • Black background for contrast

3. Florescence

   • UV Lights shot at specimen which are either naturally florescent or have florescent staining

Electron Microscope

Uses electrons/electromagnetic lenses and magnify up to 100,000x. Resolution is 0.3 nanometers.

2 Types of Electron Microscopes:

1. Transmission Electron Microscope

   • 2D images

2. Scanning electron Microscope

   • 3D images

Special preparation is needed to operate an electron microscope.

Atomic Force Microscope

Uses a probe to create a topographic map; able to see molecules, and requires no special preparation to use/operate.

Staining Steps

BEFORE ANYTHING: Basic prep.

1. Smear

2. Air dry (no heat at this stage)

3. Fix with heat

4. Stain!

Types of Stains

1. Positive / Basic / Simple

2. Negative / Acidic

3. Differential / Gram Stain

4. Acid Fast Stain / Ziehl-Neelsen Stain

Positive Stains

Consists of ONE STAIN.

AKA basic or simple stain, utilizes a pH>7 stain to stain the cells

Stains cells, not the background.

Negative Stains

Consists of ONE STAIN.

AKA acidic stain, utilizes a pH<7 stain that cannot penetrate cells, but stain the surrounding area.

Stains background, not the cell.

Differential/Gram Stain

Consists of 2 STAINS; a violet, and a pink/red color. Gram stains help differentiate 2 different types of bacteria.

Gram positive: Containing a thick peptide wall, and takes on a violet color.

Gram Negative: Containing a thin peptide wall, taking on a clear and then pink/red color.

Gram Staining Steps

1. Crystal violet stain, stains all cells purple.

2. Iodine as a mordant, to hold the stain in the cell walls.

3. Alcohol as a decolorizer, to differentiate the gram pos/neg cells. Gram negative cells will lose color. This is due to their cellular structure, having thin peptide walls.

4. Safranin, a counterstain, to re-stain the gram negative cells PINK. Because gram positive cells already contain the primary stain, they cannot pick up any more color.

Acid-Fast Stain/Ziehl Neelsen Stain

2 Types of Acid Fast Stains:

Consists of ONE STAIN.

When gram staining does not stain the cells, we can use an acid-fast stain. Acid fast stains help identify mycobacterium, which stain pink.

These cells contain mycolic acid, that creates a waxy outer layer that doesn’t take up a gram stain.

1. Acid-Fast

   • Pink/Red

     • Mycobacterium

1. Non Acid Fast

   • Blue/green

Important Historical Microbiology Figures

Anton van Leeuwenhoek

• “Father of microbiology”; created and used the earliest microscopes

• Other Dutch spectacle/lens makers included

Hans Gram Christian

• Invented gram stain

Franz Ziehl and Friedrich Neelsen:

• Invented Acid-Fast stain