Lecture 1: Exercises 1 & 2

Antonie Van Leeuwenhoek

Antonie Van Leeuwenhoek

First observed microbes and called them animalcules

Magnification

The capacity to enlarge image (objective & ocular)

total magnification = magnification of objective lens x magnification of ocular lens

Resolution

Ability to distinguish 2 adjacent objects as distinct & separate, the light-gathering ability of the objective lens

Numerical Aperture

A measure of the lens’ ability to collect light

When the wavelength of the light source is decreased, the resolution of the lens increases

Light Microscopy

• Any kind of microscope that uses visible light to observe specimens

• Visible light refracts to magnify image - refraction enlarges the image

• 0.2 μm limit of resolution

Brightfield Microscope

• Two lens systems (ocular & objective lens), specimen is illuminated by beam of light that is focused by a Abbe condenser. Result is a specimen that appears dark on a bright background

• Microscope used in lab

Immersion Oil

• Results in light bending that is less than when the light is passed through air. This is because immersion oil has the same refractive index as glass

• Used with 100x objective to improve resolution of an image by increasing the numerical aperture

• Don't let other objectives touch oil, use ethanol to remove oil from stage

Darkfield Microscope

• Condenser is modified so specimen is not illuminated directly and light is deflected/scattered, specimen will appear bright on dark background

  • Opaque disks blocks most of the light from the illuminator

• Better for living specimens

Phase-Contrast Microscope

• Unstained, visualize cellular components based on differences in refractive indexes bc of variations in density/thickness, specimen appears dark on light background

Fluorescent Microscope

• Visualize specimens that have been chemically tagged with fluorescent dye, illumination source is UV light. Ocular lens has filter to block harmful short wavelengths. Ultraviolet radiations are absorbed by the fluorescent label, and the energy is re-emitted in the form of a different wavelength in the visible light range.

  • Different microbes have autofluorescence and don’t require dye

• Used for antigen-antibody detection (antibodies are conjugated with fluorescent dye)

Electron Microscope

increased magnification, visualization of submicroscopic cellular particles & viral agents. Specimen is illuminated by beam of electrons instead of light & focused with electromagnets

Transmission Electron Microscope requires thin fixed specimens for visualizing internal cellular structures

Scanning Electron Microscope visualizes surface characteristics via scanning a narrow beam of electrons back and forth (produces 3D image)

Culture Medium

a solution containing adequate supply of nutrients (low molecular weight substances) & favorable growth environment

Broth

liquid medium that lacks solidifying agent, fresh culture

Solid Medium

hardened surface that microbes can grow on with specialized techniques to isolate discrete colonies, only medium used in petri dishes

Agar

extract of seaweed, complex carbohydrate composed mainly of galactose, has no nutritional value. Excellent solidifying agent because liquifies at 100°C and solidifies at 40°C (means organisms can be cultivated at 37.5°C without the medium liquifying)

Agar Slant

while in the liquefied state, solid media can be placed in test tubes, which are then allowed to cool and harden in a slanted position, helpful for maintaining pure cultures

Agar Slant Growth Forms

filiform - threadlike with smooth edges echinulate -threadlike with rough edges beaded,

effuse - thin spreading growth

arborescent - treelike

rhizoid - rootlike

Agar Deep Tube

harden in the upright position, used to study gaseous requirements of microorganisms

when liquified, poured into plates for Agar plates

Agar Plate Growth

Forms:

• circular - unbroken, peripheral edge

• irregular - indented peripheral edge

• rhizoid - rootlike spreading growth

Margin: the outer edge

• entire - sharply defined, even

• lobate - marked indentations

• undulate - wavy indentations

• serrate - toothlike

• filamentous - threadlike

Elevation

• flat

• raised

• convex - dome-shaped

• umbonate - raised, with elevated convex central region

Subculturing

when microorganisms are transferred from one vessel to another or from stock cultures to various media for maintenance and study

Tools for Sterile Transfers

Wire loops & needles - sterilized by incineration with Bunsen burner flame

• Use needle to inoculate deep tube with stab inoculation, loop to inoculate slant

Pipette - calibrated to deliver different volumes depending on requirements

Aseptic Technique

• Practices procedures to prevent contamination from pathogens & minimize the risk of infection

• Sterilize inoculating loop/needle by holding it in the hottest portion of bunsen burner at 45° until it is red hot. Do not put down after flaming, hold in hand and cool for 10-20 seconds.

• Hold stock culture tube and tube to be inoculated with in the palm of other hand. Remove caps and flame necks of tubes, keep caps in the hand holding inoculating tool (do not place on bench bc of contamination). Following inoculation re-flame necks of tubes and inoculating tool until red hot

• Always cool inoculating tool

• Work quickly (lower exposure time) and stay within 6 in of bunsen burner

Pure Culture

A culture that contains only the growth of a single unadulterated bacterial species

Once there are discrete colonies on agar plate, we can pick them up with a sterile needle and transfer to an agar slant to obtain a pure culture

Spread Plate

Requires a previously diluted liquid mixture of microorganisms, cells are spread over the surface of a solid agar medium with a sterile L-shaped bent rod (bent glass rod) while the plate is spun on a "lazy-Susan" turntable

Keep bent glass rod in ethanol beaker, then flame and cool

To use the rod: place a slight amount of pressure with the rod at the edge of the agar and move it toward the center of the agar surface while spinning the turntable

Streak Plate

Rapid qualitative isolation method/dilution technique that involves spreading a loopful of culture over the surface of an agar plate from one quadrant to another (1, flame, 1 to 2, flame, 2 to 3, flame, then 3 to 4, flame)

Done to separate the cells of a mixed culture to isolate discrete colonies