Microbiology 2060

Note in here: SI units

Main Principle of Modern Science

all natural phenomena have natural causes

Jansen

• late 1500s

• developed first compound microscope but it had poor magnification

Hooke

improved upon Jansen’s design using better lenses, he used reflective light

Van Leeuwenhoek

• introduced transmitted and focused light

• crafted lenses that could magnify up to 270x

• believed to be the first human to observe individual bacteria

  • called them animalcules

Van Leeuwenhoek’s Observation

1. “animalcules” from biological scrappings

2. “animalcules’” division

   • bacterial reproduction

Abiogenesis

aka Spontaneous Generation

• the ability of “lesser organisms” to spontaneously form out of nonliving material

• relies on the belief of “Vital Force” of abiotic structures that allow production of organisms

• Not supported by modern science

Vitalism

the belief that there are vital energy forces that govern our health and well-being

Disproving Abiogenesis: Redi’s Jar

• Francesco Redi Experiment:

  • jar containing raw meat and no lid

    • “produce” flies

  • jar containing raw meat and lid

    • does not “produce” flies

  • jar containing raw meat with a porous cloth covering

    • flies appear on cloth outside the jar but not on meat

was not enough to sway public opinion

Infusoria Community

the collections of microbes produced by soaking HAY in water

microorganisms from the hay are flushed into the water

a highly diverse microbial community

Used to disprove/study VITALISM

Infusion

the liquid produced by soaking plant material in water

ex. tea

John Needham Infusoria Experiment

poured infusion into a flask and boiled it (killing microbes) and then sealed it

Result: RETURN of Infusoria

Larazzo Spallanzani Infusoria Experiment

poured infusion into flask, sealed it and then boiled it (killing microbes)

Result: continued ABSENCE of infusoria

Louis Pasteur Infusoria Experiment

• poured beef broth into long necked flask

• heated the neck and bent it into and S-shape

• boiled the bottom

Result: no microorganisms in cooled solution

finally and fully disproved abiogenesis and vitalism

S-Flask Experiment Details

the curve of the neck prevents reinfection of the broth by blocking dust from easily transporting microbes to the broth

Germ Theory

“that microorganisms are the causative agent of infectious disease”

• disproves belief that disease is caused by karma, possession or the Wrath of God

• first suggested by VARRO REATINUS around 80 BCE Rome

  • couldn’t be proven/demonstrated

Agostino Bassi

first formed the connection between microbes and the health of organisms via SILKWORMS

demonstrated mold contributed to silkworm health

Ignaz Semmelweis

made the connection between microbes and humans (indirectly)

• “the elimination of microbes eliminated disease”

directed his nurse WASH their HANDS prior to attending labouring patients

• reduced childbirth fever deaths drastically

Joseph Lister

extended Semmelweis’ idea by CLEANING SURGICAL INSTRUMENTS in a phenol solution prior to operations

Direct Connection between Microbes and Humans

LOUIS PASTEUR + ROBERT KOCH

• developed Koch’s Postulates

• concluded that a specific disease is caused by a specific microbe

• etiology is the first step to treatment and prevention

Koch’s Postulates

A series of steps, as follows:

1. isolate microorganisms from a suspended diseased or dead animal

2. grow a pure culture of said microorganism

   • identify microorganism

3. inject microorganism into a healthy laboratory animal

   • the disease will reproduce in lab animal

4. repeat steps 1 and 2 using sample from laboratory animal

5. microorganism of concern is identified

are used to establish proof of direct connection between microbes and health

Edward Jenner

developed the first live “vaccine”

• observed those infected by cowpox are resistant to smallpox

• began infecting people with cowpox to prevent severe infection of smallpox

• this worked due to similar enough structure for compatible antibodies

Louis Pasteur

developed the first “intention” vaccine

• observed that old microbes would lose their ability to cause disease while retaining ability to induce resistance to disease they cause

his beta test Rabies vaccine on a child

Paul Ehrlich

1910

• developed a synthetic compound called SALAVARSAN to treat syphillis and sleeping sickness

Alexander Flemming

discovered that the Penicillium fungus secreted an antibiotic compound (penicillin)

Antimicrobial Resistance

overuse and improper uses of antibiotics has led to many pathogenic microbes being resistant to treatment

bacteria can achieve resistance via a variety of ways

Molecular Biology in Microbiology

• genetic profiles are now key in testing for the presence of certain microbes

  • also used for identification

• microbes can be easily genetically modified using Recombinant DNA

Paul Berg

1960s

• developed basis for modifying microbes

Herb Boyer + Stanley Cohen

1973

• founded first biotech company:

  • Genentech

3 Factors in Microscopy Image Quality

1. Magnification

2. Contrast

3. Resolution

Magnification: BENDING LIGHT

two types:

• reflection

• refraction

when light enters a specimen it will both reflect and refract

• occurrence due to light passing from one medium to another when the mediums have different indecies

human eye perceives the refracted light differently

• usually enough to create sufficient contrast to see microbes

Resolution: RESOLVING POWER (d)

the ability of a lens system to allow you to see two points as being distinct

• the lower the value of ‘d’ the better the resolving power

maximizing resolving power/reducing d value can be done by:

1. using a shorter wavelength of light (reduce wavelength)

   • reduces contrast

2. using a higher magnification

   • shorter focal length of lens = closer

   • increase angle (larger angle)

3. altering refractive index

   • make it more closely match the specimen to capture more refracted light

     • oil immersion

       • has a refractive index closer to water in specimen

Resolving Power Formulas

NA = numerical apperature

n = refractive index between specimen and lens

u = angle from specimen to outer edge of objective lens

wavelength is the wave thingy

Oil Immersion

• can only be done a higher magnification (100x objective)

• oil bust be held in place by surface tension (oil replace air)

• lens must have short focal length

  • only 100x lens short enough

Focal Length

the space between the specimen and objective lens

short = close to specimen

long = far from specimen

Contrast: Staining

• live cells are transparent with no significant contrast with surrounding medium

• contrast can be added via:

  • changing illumination

  • adding colour (stain)

• Biological stains have 2 main categories

  • basic

  • acid

Biological Stains: BASIC STAINS

• the coloured portion (CHROMOPHORE) is cationic

• colour binds to cells’ negative charge

• simple stains use only basic stains

example → crystal violet

Biological Stain: ACIDIC STAINS

• the coloured portion (CHROMOPHORE) is anionic

• colour is repelled by cells’ negative charge

  • around the cells - stars in night sky

• negative stains use acidic dyes - does not need heat fix or wash

example → nigrosin

Basic Slide Preparation

1. smear the sample onto a slide

   • needs to be a thin layer, not too thick/heavy

2. apply a LITTLE but of heat

   • this heat fixes specimen to the slide

3. apply appropriate staining technique

Differential/Special Stain Protocols

• target specific features that differ between different bacterial types

• includes use of

  • mordants (enhance stains)

  • counterstains

  • heat

• examples:

  • Gram Stain

  • Acid-Fast Stain

  • Endospore Stain

Important Staining Techniques

need to know:

1. Gram Stain

2. Acid-Fast Stain

also good to know:

1. Endospore Stain

2. Flagella Stain

Gram Stain Protocol

• targets an important difference in bacterial cell walls

  • gram⁺ or gram^-

• 4 steps:

  1. stain the fixed seam with CRYSTAL VIOLET

     • wash away excess

  2. apply a MORDANT (iodine for example)

     • this binds to the crystal violet to form a larger complex (iodine-crystal violet complex) that will help distinguish cells

     • wash away excess

  3. apply DECOLOURIZER

     • this removes the ICV complex from gram^- cells

  4. apply COUNTERSTAIN (safranin for example)

     • allows us to see gram^- cells/washed cells

     • different colour

Iodine-Crystal Violet Complex

too big to pass through gram positive cells walls (stuck inside) but are able to pass through gram negative cell walls

Acid-Fast Stain Protocol

• oriented toward bacteria with WAXY MYCOLIC ACIDS in their cell walls

  • often Mycobacteria

• 4 Steps:

  1. stain bacteria with CARBOL FUSCHIN

     • if the cell wall contains mycolic acids it will bind to the Carbol Fuschin tightly

  2. heat the slide

     • allows enhanced penetration of dye in cell wall

  3. apply an ACID-ALCOHOL. RINSE

     • cells with mycolic acid will remain dyed and remove dye from non-mycolic acid cells

  4. apply COUNTERSTAIN

     • allows you to see the unstained cells

Endospore Stain Protocol

• looks/targets the formation of endospores

• uses MALACHTE GREEN

• 4 Steps:

  1. apply MALACHTE GREEN to the heat fixed slide

  2. add additional heat to allow dye to penetrate endospore wall

  3. rinse with water to remove excess dye in non-endospore cells

  4. apply COUNTERSTAIN

     • so you can see vegetative cells

Endospores

• are immobile form of cells that are created in response to environmental pressure

  • only two Genera do this

    • Bacillus

    • Clostridium

Steps to Endospore Formation

1. newly replicated DNA along with a small amount of cytoplasm are isolated

2. membrane closes around DNA package

3. spore septum surrounds package forming FORESPORE

4. peptidoglycan and spore coat form

5. endospore is freed from the cell

Flagella Stain Protocol

• goal is to make flagella thicker so they can be seen

  • to do this we use a MORDANT

    • careful not to add too much, could cause breakage

• once thickened you carefully stain the flagella

Types of Microscopy

1. Bright Field

2. Dark Field

3. Fluorescence

4. Confocal

5. Electron

   1. Scanning

   2. Transmission

Brightfield Microscopy

• light from the source is focused on the specimen

• image is produced by refracted light

  • often lacks significant contrast between refracted and background (unrefracted) light

Darkfield Microscopy

• an opaque disk is placed in the path of the sources light cause the light to form a ring shape

  • causes the light that does not refract or reflect to miss the objective lenses

• creates and image only using the light reflected/refracted via the specimen

  • no background light

• usually poor resolution

Fluorescence Microscopy

• often used to identify specific types of microorganisms in a mixed background

• does not use visible light

  • instead uses Ultra Violet to detect antibodies

• antibodies are combined with fluorochrome then introduced and stick to the specific surface proteins

Confocal Microscopy

• the manipulation of specific wavelengths of light that are focused on a specimen at different depths of the specimen

  • specimen is treated so different parts are stained differently

• essentially creates slices you can reassemble digitally

• produces a 3D image of specimen

Electron Microscopy

• uses an electron beam instead of light

  • beam has VERY short wavelength (0.0005nm)

  • which has a very high frequency giving it amazing resolving power and magnification (100x better)

two types

Scanning Electron Microscopy (SEM)

surface of specimen is coated in very thin layer of “electron dense medium” (usually gold)

electron beam knocks electrons off the surface of the specimen, they are collected by detectors to create an image

lots of surface detail - electron beam cannot penetrate surface

Transmission Electron Microscopy (TEM)

the specimen is fixed in resin and cut VERY thin

• a time consuming and finicky process that can produce artifacts

special stains can be used to increase contrast

detailed image of internal structures

Prokaryotic Plasma Membrane Functions

• permeability and transport

• respiratory reactions

• synthesis of the cell wall

• photosynthetic reactions

Peptidoglycan

the material used to create the bacterial cell wall

2 primary structural components

• long polysaccharide stands

  • NAG and NAM amino sugars

• short peptides

  • join the NAG/NAM strands

  • composed of side chains and cross-bridges

N - acetylglucosamine (NAG)

amino sugar

modified amino group on #2 carbon

joined by glycosidic bond

N - Acetylmuramic Acid (NAM)

amino sugar

modified amino group on #2 carbon

joined by glycosidic bond

Gram Positive Bacteria

THICK layer of peptidoglycan

contiain TEICHOIC ACID in its membrane (substance regulates cell wall growth)

Gram Negative Bacteria

THIN layer of peptidoglycan

have a second membrane (outer membrane)

contains:

• Lipopolysaccharides

• Pourin Proteins

Lipopolysaccharides (LPs)

a large complex molecule that contains lipids and carbohydrates

3 major components:

• Lipid A

• O-polysaccharide

• Core Polysaccharide

Lipid A

the lipid portion of LPs embedded in the top layer of the outer membrane

O-Polysaccharide

extends outward from the core polysaccharide in LPs

functions as an antigen (sugar based)

Core Polysaccharide

attach to Lipid A and contains unusual sugars

provides stability and structure to LPs

Antibiotics and How They Work

5 main types:

1. CELL WALL INHIBITOR

   • prevents the synthesis of the cell wall/peptidoglycan subunit

2. PROTEIN SYNTHESIS INHIBITOR

   • occurs inside the cell typically during mRNA translation

3. NUCLEIC ACID INHIBITOR

   • prevents proper nucleic acid (DNA/RNA) replication and transcription

4. PLASMA MEMBRANE ATTACKER

   • damage the plasma membrane to make in dysfunctional (starves the cell out by preventing photosynthesis)

5. METABOLIC INHIBITOR

   • act as a competitive or non-competitive inhibitor to vital enzymes in important metabolic pathways