Micrb 106

Pasteur

Godfather of modern microbiology

• Wine fermentation

• Germ theory of disease: germs cause certain diseases

• Vaccines

Joseph Lister

Development of antiseptic technique: sterilization technique

Sprayed toxic chemical to presumably kill bacteria

Ignaz Semmelweis

Critical in understanding importance of hand washing

Robert Hook

First person to name a cell found on a cork

Antoni van Leeuwenhoek

Dealt with fabrics with magnifying glass(first microscope)

Romans

One of most Successful civilization

Took cleanliness seriously

Helped in prevention of microbes

Biotechnology

Industrial setting use of microorganisms

Bioremediation

Using microbes to help environmentally: introduce microbes for cleanup

Dr. Robert Koch

• scientist who verified the germ theory of disease

  • set of protocols for assigning a disease-causing organism with its disease state

• Birth of Bacteriology

• Developed methods to grow bacteria in culture

• Studied anthrax: isolated it from an animal specimen and grew it in culture

  • Determined that this bacteria (Bacillus anthracis) could survive in soil to affect animals in future

  • Anthrax is extremely dangerous and causes spores on human skin (coal black)

Dr. Robert Koch growth medium

• Developed a growth medium:

  • stay firm

  • bacteria would not ingest it

  • grow bacteria “in culture”

  • use of agar(breakthrough)

Agar characteristics

• non-toxic to most microbes

• melts at 100 C but gels at ~45 C (a temp most bacteria can survive)

• nontoxic to other forms of life

• stable to sterilization temperatures

• physiologically inert

  • very few bacteria have the necessary enzymes to digest it

Organic molecules vs. Inorganic

Organic: contain C + H

• DNA, table sugar, methane, ethanol

Inorganic:

• carbon dioxide, diamond, silver, table salt

Carbohydrates

Monomer: Monosaccharides

Polymer: Polysaccharides

Purpose

• used for energy production

• to build necessary cellular components

Why we care

function in cell

• 5-6 C, ring structure

• can be linked to form chains

Proteins

• made of chains of amino acids(monomer)

  • amino group, side chain, carboxyl group

• aa link together by peptide bonds

• referred to as peptides or polypeptides(polymer)

• can fold into complex structures, tied to function

4 levels of protein structure

Primary protein Structure

• sequence of a chain of amino acids

Secondary Protein Structure

• Local folding of the polypeptide chain into helices or sheets

Tertiary Protein Structure

• Three-dimensional folding pattern of a protein due to side chain interactions

Quaternary Protein Structure

• Protein consisting of more than one amino acid chain

Hemoglobin (Ex. 1 of protein)

• Not in bacteria

• carries O2 and CO2

• found in RBCs

• Function dependent on proper folding/structure

• Sickle cell anemia: example of disease with improper folding of Hb molecule

Enzymes (Ex. 2 of protein)

• build or break down things/make reactions happen

1. Substrates approach an enzyme

2. The substrates bind to the enzyme

3. The enzyme changes shape and substrates combine to form a product

4. The produce is released and the enzyme returns to its original shape

Lipids

• 3 Classifications

Fatty acids: long chains (C-C-C-C-C-C)

• chain length can differ (4-24C)

• non-polar: will clump in water

• Saturated: no double bond, firmer in room temperature

• Unsaturated: double bond, liquid in room temperature (more space between legs)

Triglycerides

• 3 FA + glycerol

Phospholipid

• 2 FA + Phosphate group

• Ex. cell membranes

Nucleic Acids

DNA + RNA(polymer)

Nucleotide(monomer)

• phosphate group

• sugar

• nitrogenous base

Taxonomy

Describes microorganisms: universal naming

• binomial naming systems

Domain order

Kingdom

Phylum

Class

Order

Family

Genus

Species

Five Is

Inoculation

• Sample is placed into a container of growth medium.

Incubation

• Creates the proper growth conditions with respect to proper temperature and gas requirements.

Isolation

• Once the cultures have grown, they may need to be re-inoculated (and incubated) in such a way that separate species are obtained.

Inspection

• The colonies on agar or the broth cultures are observed macroscopically and microscopically, possibly with the aid of staining.

Identification

• The identity of the of the isolated microbe is determined, usually to the species level. May be enough to identify some microbes, but additional techniques include biochemical tests, immunologic tests, and genetic analysis.

Macroscopic types

Pure level

Mixed culture

Contaminated culture

Categories of Media Classifications

Physical state

Chemical state

Functional type

Selective media

General-purpose non selective medium: all species grow

Selective medium: one species grows

General purpose non differential medium: all species have a similar appearance

Differential medium: all 3 species grow but may have different appearance

Isolation techniques

Streak

• spread species into quadrants to get single colony; sterilization of rod in between

Pour plate

• broth is poured on plate directly, diluting each time for separate colony

Spread

• pour small sample sample into and spread sample evenly over surface

Principles of light microscopy

• Magnification

• Resolution

• Contrast

Resolving power

Def: ability of lens to distinguish between small objects

• lens quality

• Immersion oil

Numerical Aperture

• correlated with resolution

• Higher N.A, better resolution, brighter image

Types of microscopy

1. Brightfield

• simplest, light through specimen

• Specimens typically stained

2. Darkfield

• Creates contrast between object and field

• Background dark

3. Phase contrast

• viewing of living, unstained specimens

• Cell components differ in density

4. Fluorescence microscopy

• species dyes that fluorescence under laser

• Can stain particular components of a cell

5. Confocal microscopy

• eliminates out of focus info

• Produces blur free images of a thick specimen

  • viewing thin slice of object as a whole

6. Electron microscopy

• beam of electrons

• Much better resolution

• Very high mag

Electron microscopy

SEM

• scanning electron microscopy

• 3D View

TEM

• transmission electron microscopy

• Not 3D, just slices

• see very high mag

Positive and Negative stains

positive staining

• Appearance of cell: colored by stain

• Background: not stained(generally white)

• Dyes employed: basic dyes: crystal violet, methylene blue, safranin, malachite green, malachite

• Subtypes of stain: simple stain, differential stain, special

Negative staining

• Appearance of cell: clear and colorless

• Background: stained( dark gray and black)

• Dyes employed: acidic dyes: nigrosin, India ink

• Subtypes: capsule, endospore

Actual cell of gram negative and positive

Simple stains

Specific part of cell

Differential stains

Differentiate different components in cell

Archaea

Prokaryotes

primitive

similar to, but unique from, bacteria

unique anatomy/phys

unique genetics

found living in extreme environments

How bacteria(prokaryotes) differ from eukaryotes

DNA

• location + no histones(protein wheels, DNA is wrapped around)

Cell wall

• composition

• sturdy + peptidoglycans(protein + carb)

Internal structures

• no membrane-bound organelles: still can have organelles

Bacterial Cell

Internal

• cytoplasm

• ribosomes

• inclusions

• nucleoid/chromosome

• cytoskeleton

• endospore

• plasmid

• microcompartments

Boundary(contains)

• cell wall

• cytoplasmic membrane

External

• Appendages

  • flagella

  • pili

  • fimbriae

  • nanowires/nanotubes

• Surface layers

  • S layer

  • Glycocalyx

Cocci Arragnement

Diplo: remain in pairs after dividing

Strepto: remain in chains (beads)

Tetrads: groups of 4

Sarcinae: divide in 3 planes and remain in cubes

Staphylo: divide in multiple planes; grape clusters

Bacilli

Single bacillus

Diplobacilli

Streptobacilli: chain

Coccobacillus: combination of both shapes

Spiral

Vibrio: boomerang shape

Spirillum: single loop

Spirochete

Bacterial Appendages

Motility

• flagella

• axial filaments: part of flagella

Attachment/channels

• fimbriae - adherence (over whole cell, shorter)

• pili - conjugation (fewer in number, longer) - DNA transport

• nanotubes/wires - nutrient transfer

Chemotaxis

Chemicals - to move

Def: bacterial movement using only flagella

Surface Coatings

S layer

• single layers of single protein

• armor

• only produced when in hostile environment (why difficult to grow in labs)

• some use to aid in attachment

Glycocalyx

• coating of repeating polysaccharide/glycoproteins

• protection + adhesion

• slime layer - not well defined - turns to biofilm

• capsule - well organized (protection from system)

• allows bacteria to stick to surfaces and form multi-species collective - biofilm

Cell Envelope

Wall + membranes

Bacteria either gram + or gram -

G+ = cell wall is thick (peptidoglycans)

G- = cell wall is thin and sandwiched between membranes

Cell Wall Structure

Protection of cell membrane in unfavorable conditions

• because are everywhere, need protection

• peptidoglycan layer - long chains of carbs with protein side chains linking them together

point of attach for antibiotics and lysozyme (disrupt layer)

Order of staining gram cells

1. crystal violet

2. Gram’s Iodine

3. Alcohol

4. Safranin (red dye)

Gram + should end purple

Gram - should end pink

Atypical cell walls

Don’t have regular cell walls, many bacteria that cause issues are these types

Mycobacterium

• cell wall has dif composition - thick, protective

• stain can easily identify - acid fast

Mycoplasmas

• no cell wall

• unique membrane resistant to lysis(breaking apart)

• very small, very simple, vary in shape

  • mycoplasma pneumonia

Internal structures of bacteria

DNA - singular, chromosome

can also have circular bits of DNA - plasmids

• antibiotic resistance

• production of unique enzyme

• production of other proteins

• can be shared between cells to transfer ability to other cells

Ribosomes

• 2 pieces come together

• different composition/size

Endospores

prepared by some bacteria during extreme stress

pack important components into small package - survival

medically significant + important to food industry

• very resistant to: heat, chemicals, radiation

• because in soil - found on veggies + animals

  • botulism in canning

  • anthrax in soil where infected animal died

Bacillus and Clostridium - form endospore before die off

• if conditions improve can resume life

• common is soil where endospore can survive decades

• survival mechanism

Eukaryotes

Organelles

• nucleus - genetic info

• ribosomes - proteins

• endoplasmic reticulum - smooth + rough

• mitochondria - glucose to ATP

• Golgi - sorts proteins + delivers to final cell destination

• cilia(numerous) - surface of some cells - similar to structure to flagella

• flagellum - propulsion - different from bacteria

Cell Theory

Living things are made of cells

Cells are the basic units of life

All cells come from other cells

Makes something alive

• maintain homeostasis

• levels of organization

• reproduce

• growth and development

• use E

• respond to stimuli

• adapt to environment

cytoskeleton

network of filaments

• anchoring organelles

• moving rna and vesicles

• allowing shape changes/movement

Endosymbiosis

Last Common Ancestor is likely a large organism with loose boundaries; has RNA as genetic material

Individual cells form'; genetic material becomes DNA, possible through influence of viruses

Thought bacteria cell engulfed most likely by archaea: become mitochondria or chloroplasts in eventual cells