Chapter 3 The Tools of Microbiology Microscopy n

Chapter 3 The Tools of Microbiology

Microscopy n 1. Light microscopy n Structure of the light microscope ¨ n Image formation ¨ n Resolution: the ability to distinguish between two objects that are very close together. Working distance ¨ n Uses glass or plastic lenses to focus the image Resolution and determining resolving power ¨ n Two lenses – Ocular and Objective The distance from the end of the objective lens to the slide Oil-immersion microscopy ¨ Prevents further refraction of light because the oil has the same refractive index as the slide – therefore increasing resolution

Fig. 03. 01

The Five I’s of Microbiology n n n Inoculation – Obtaining a specimen and placing it in media Incubation – Placing specimen at optimal for growth Isolation – separating one organisms from other Inspection – viewing object under microscope Identification – using differential staining, biochemical and genetic testing to identify the organisms

Brightfield Microscopy

n 3. Dark-field and phase-contrast microscopy ¨ Use of star diaphragm - refracts light around the object – slide is dark, objects that are 3 dimensional are light ¨ Phase contrast – special objective lenses put the image slightly out of “phase” to give more resolution n 4. Fluorescent and electron microscopy ¨ The n fluorescent antibody technique – fluorescent antibody will attach to a specific structure and the pigment will fluoresce so that the specific structure can be distinguished from other structures.

Darkfield

Fluorescent pigments attached to antibodies bind to particular structures (ie. Microtubules, Nucleic Acid, Cell wall Structures, etc… used to identify structures

Confocal – many layers in focus

Light vs. Electron Microscopes

Resolution differences

Electron Microscopy n Transmission electron microscopy ¨ Transmission of electrons through the object – gives a 2 -D look at the inside of a cell – uses magnets to focus the electron beam. Photographic film collects the beams that go through the object – giving various areas of “light” and “dark” depending on whether the electrons exposed the film or not. n Scanning electron microscopy ¨ Bounces electrons off of the object – gives a 3 -D look at the surface of objects – works like “SONAR” but uses electron beam instead of sound waves to detect the shape of objects. – uses magnets to focus the electron beam – collects information to form a “picture” through a television monitor or computer.

TEM – Transmission Electron Microscope

SEM – Scanning Electron Microscope

2. Staining techniques n Simple stain technique ¨ n Negative stain technique ¨ n Basic dyes – pigment is on the positive charged ions attracted to the negatively charged cell § Stain cell Acidic dyes – pigment is on the negative charged ions and are repelled by the negatively charged cell § Stain background Gram stain technique § § Primary Dye – Crystal Violet Mordant – Iodine Decolorizer – Ethanol Secondary Dye - Safranin

Simple Stain

Gram Stain

Acid Fast Stain

Capsule Stain

Spore Stain

Fluorescent Microscopy