BI438 Microbiology Lab Practical 1

A.

Binocular Eyepiece

B.

Nosepiece

C.

Objective Lens

D.

Stage

E.

Illuminator

F.

Base

G.

Arm

H.

Coarse focus knob

I.

Fine focus knob

J.

Stage adjustment

K.

Light adjustment

Ocular Lens/Eye piece

The lens at the top of the microscope. Can be removed and used as a hand magnifier. Magnification is 10x (produces image 10 diameters larger than actual size)

Objective Lens

Separated from the ocular lens by the body tube and located on a revolving nosepiece. Four objectives (4x scanning,10x, 40x 100x oil immersion).

Total Magnification

10 (objective lens) times the magnification of objective lens

Focus knobs

Moves the objectives up or down to focus the microscope

Aperature

The hole in the stage that light travels through

Transverse knobs

Move the microscope slide smoothly

Iris Diaphragm

Controls the size of the opening of light passing through the condenser and aids in adjustment of contrast

Condenser

The third lens is positioned under the stage. Collects the light rays from the illuminator and focuses them to a point in the same optical plane as the specimen. Allows more or less light to enter the microscope optics.

Abbe Condenser

Iris diaphragm and condenser coupled into a single structure

Why does the oil immersion lens require oil?

Oil prevents light loss from light rays bending when light travels through the gap between glass slide and objective lens

Resolution

the ability to distinguish detail in the magnified image

Bright-field Light Microscopy

Produces image with light transmitted through a specimen. Biological specimens often require stain (stains often require fixing and killing the specimen). Technically can be used on live specimen.

Advantages: versatile, natural colors, easy preparation

Disadvantages: often requires fixing/staining, cannot see very small specimen, light must be able to pass through specimen for visualization

Dark-field Light Microscopy

Similar to light-field but uses special condenser so only light reflected off the specimen will enter the objective lens. Used to enhance contrast of unstained samples.

Advantages: high contrast, view live organisms

Disadvantages: required high intensity light that can damage specimen, halo effects, sensitive to artifacts and debris

Phase-contrast Light Microscope

Designed to enhance contrast of unstained specimen. Utilizes light transmission and it less damaging to specimens. The STANDARD for viewing live cultures.

Advantages: high contrast, view live organisms, internal structures, minimally invasive to life

Disadvantages: halo effects, some signal loss

Fluorescence Light Microscopy

Uses fluorescent dyes or stains to illuminate specific features

Advantages: ability to label/stain specific features, high signal- to-noise ration, multiplexed imaging

Disadvantages: photobleaching, dyes/stains are invasive, phototoxicity

Electron Microscopy

Uses electrons to transmit through or off specimen rather than light. Capable of greater levels of resolution. Must prepare specimens in heavy metal stain or coating

Advantages: higher resolution

Disadvantages: require fixed specimen, lack of color

Transmission Electron Microscope (TEM)

Produces 2D image by transmitting electrons through a think slice of sample,

Advantages: very high resolution cross-sectional images

Disadvantages: fixing is invasive, no color

Scanning Electron Microscope (SEM)

Creates 3D image of the surface of the specimen by examining the reflective pattern of electrons targeting the surface

Advantages: very high-resolution surface images,

Disadvantages: fixing is invasive, no color

Abbe Diffraction Limit

the maximum resolution of a light microscope is constrained by the wavelength of light used and the numerical aperture of the objective lens (about ½ of the wavelength of visible light)

Stain

A biological reagent or preparation which is ready to use and made of one or more dyes. A stain is a dye in solution. Stains are organic compounds containing a chromophore

Dye

chemical compounds which impart color or specific visible feature to a stain

Auxochrome

part of a dye which gives the dye its charge and enables binding to molecular targets. Auxochromes makes it possible for a chromophore to undergo ionization and therefore act as a stain

Chromophore

The chemical group that gives dyes their color by absorbing light. Must be undergo ionization with assistance of auxochrome to act as a stain

Acidic dyes

anionic (negative charge ) used to stain positively charged components of the cell. Typically stain the background, not the cells

ex: sodium, potassium, calcium, ammonium salts.

Basic Dyes

cationic (positively charged), stain negative charged components. More likely to be used to stain cells directly

ex: methylene blue and crystal violet

Smear

A think layer of cells spread over the surface of a slide, done before staining. Can be prepared with liquid broth (dont add water) or solid cultures (add water first).

Simple stain

Used to improve contrast of the specimen

Differential stains

Like a gram stain, colors identify aspects of the specimen

Tryptic soy agar (TSA)

non-selective and can grow wide variety of microbial species such as bacteria and fungi. Ingredients: enzymatic digest of casein and soybean meal (amino acids), glucose (sugar), sodium chloride (salt), amd agar

Elevation?

Flat

Elevation?

Raised

Elevation?

Crateriform

Form?

Round

Form?

Filamentous

Form?

Irregular

Form?

Rhizoid

Form?

Punctiform

Margin

Entire

Margin

Lobate

Margin

Scalloped

Margin

Filiform

Margin

Undulate

Margin

Serrate

Margin

Curled