Microbiology and Cell Biology

• Microbiology studies living things too small for the naked eye.
• Microorganisms are small forms of life not visible without aid.

Groups of Microorganisms

• Bacteria
• Algae
• Fungi
• Protozoa
• Helminths
• Viruses (not cells)
• Prions (not organisms)

Microbe and Disease Transmission

• Microbes spread to humans via:
  • Respiratory droplets
  • Dust
  • Water
  • Food
  • Injury
  • Insect bites
  • Animals
  • Human contact
  • Inanimate objects
• A pathogen is a microbe that causes disease.

Pioneers of Microbiology

• 1665: Robert Hooke used the term "cells."
• 1674: Leeuwenhoek observed microorganisms for the first time.
• 1796: Jenner created a smallpox vaccine through inoculation.
• 1847: Semmelweis found the cause of childbed fever.
• 1854: John Snow studied epidemiology.
• 1895: Pasteur disproved spontaneous generation.
• 1865: Lister introduced antiseptic techniques.
• 1876: Koch developed pure culture on agar.
• 1892: Iwanowski discovered viruses.
• 1894: Ehrlich discovered selective toxicity.
• 1929: Flemming discovered penicillin.
• 1977: Woese classified archae.

Robert Hooke

• Published Micrographia including drawings of cork cells.
• Hooke emphasized looking at small things with a microscope.

Antoni Van Leeuwenhoek

• Sent 300 letters to England's Royal Society of London.
• Described particles he called "animalcules."
• Provided the first description of bacteria.

Spontaneous Generation

• Belief that life forms from inanimate objects.
• Demonstrated that animalcules (microbes) arise from other animalcules in the air.
• Used swan-neck flasks to disprove spontaneous origination.
  • Flasks with intact necks stayed sterile.
  • Flasks with broken necks exposed to air grew microbes.

Louis Pasteur

• Studied yeasts in juice, beer, vinegar, & wine.
• Observed soured wine was full of bacteria that heating the wine could kill.
• The study of soured wine led to the creation of the pasteurization process.
• Formulated the germ theory of disease

Pasteurization

• Kills harmful bacteria in foods and drinks with heat without ruining the taste.

Germ Theory of Disease & Koch's Postulates

• If an animal is sick, scientists can take a sample like blood or tissue and find microbes inside.
• Grow organism in pure culture to make sure it is the only microbe present.
• Scientists inject the cultured microbe into a healthy animal to see if it causes the same disease.
• Injecting cultured microbes into a test animal will cause the same disease.

Gelatin and Agar

• Found pure cultures of bacteria grew best on solid surfaces.
• Used gelatin in dishes to isolate pure cultures.
• 1882: Fanny Hesse suggested using agar instead of gelatin.
• 1887: Julius Petri developed the culture plate (petri dish).

Contributions to Bacteriology

• Pasteur and Koch are two scientists who contributed the new field of bacteriology.
• Koch focused on etiology, or the cause of disease.
  • Koch's lab identified the source of tuberculosis (1882) & cholera (1883).
• Pasteur focused on disease prevention through vaccination.
  • Pasteur's work lead to a vaccine for rabies (1885).

Edward Jenner and Immunity

• Jenner realized dairymaids with mild cowpox didn't get smallpox.
• He thought cowpox made them immune to smallpox.
• Then inoculated a child, who had never had smallpox, with cowpox.
• Resulting in the boy not getting smallpox.

Female Contributions to Microbiology

• Dr. Ruth Ella Moore found a treatment for TB.
• Dr. Jane Hinton co-developed Mueller-Hinton Agar.
• June Almeida first saw Coronavirus in an electron microscope.

Paul Ehrlich and Selective Toxicity

• Selective toxicity means a medicine can kill harmful microbes without hurting the person taking it.
• Drug for syphilis
  • Synthetic drugs
  • Sulfa drugs
  • Antibiotics
  • Penicillin

Modern Bacteriology Terms

• Bacteriology is the study of bacteria
• Virology is the study of viruses
• Mycology is the study of fungus
• Parasitology is the study of protozoa & helminthes
• Immunology is the study of the immune response

Areas of Microbiology

• Medical
• Environmental
• Industrial
• Agricultural

Viruses vs Bacteria Abundance

• Viruses are more abundant than bacteria.

Microbes and the Human Body

• Some microbes are temporary (transient).
• GI tract microbes help prevent infectious disease.
  • GI microbes aid the breakdown of food.
  • GI microbes provides nutrients and vitamins the body cannot make itself.
  • GI microbes communicate with the brain and immune system.
  • GI microbes may influence obesity, asthma, and allergies

Drug-Resistant Pathogens

• Declining effectiveness of antibiotics is caused by:
  • Bacteria evolving through beneficial mutations.
  • Bacteria acquiring resistance genes from other bacteria.
  • Antibiotics abused by professionals & patients leading to superbugs.

Bioterrorism

• Intentional use of biological agents to cause fear/death/disease.
  • Smallpox (virus)
  • Anthrax (bacteria)
  • Plague (bacteria)
  • Botulism (toxin produced by bacteria)

Water as a Solvent

• Cells average 70% water by mass.
• Chemicals, like salts, dissolve in water.
• Dissolved chemicals (solutes) interact in chemical reactions.
• Water can also be a reactant or product in chemical reactions.

Dehydration Synthesis

• When two molecules join to form a larger molecule and lose a water molecule.

Hydrolysis

• When a large molecule breaks into smaller ones with the use of a water molecule.

Cohesion

• Water molecules sticking together via hydrogen bonds.

Acids, Bases, and pH

• Acids increase H+ ions in water.
• Bases donate OH- ions that combine with H+ ions to form water.
• pH measures H+ ions in a solution.
  • Acidic is less than 7 (0-6).
  • Neutral is 7.
  • Basic/Alkaline is more than 7 (8-14).
• Buffers keep pH steady.
  • Buffers soak up excess acid or base.

Proteins

• Contain carbon, hydrogen, oxygen, nitrogen, & sulfur polymers.
• Built from chains of amino acids.
• Amino acids are the building blocks and determine the structure of the protein.

Protein Shape and Function

• Primary structure = polypeptide chain of amino acids.
• Secondary structure = pleated sheets, alpha helices, coils.
• Tertiary structure = bonding between R groups to create a final 3-D shape.
• Quaternary structure = two or more folded polypeptides bonded together.
• Denaturation of a protein is when broken bonds cause it to unfold and lose function > leads to cell death

Nucleic Acids

• Unbranched organic molecules of atoms of carbon, hydrogen, nitrogen, and phosphorous.
• Two forms: DNA and RNA
• Makeup:
  • 5-carbon sugar
  • Phosphate group
  • Nucleobase
  • Adenine, Guanine, Cytosine, Thymine, Uracil
• Rungs are nitrogenous bases held together with hydrogen bonds
• DNA: Double Helix
• Carry units of information called genes arranged on chromosomes (circular or linear)
• A>T
• C>G
• RNA: Single stranded
• Intermediaries in carrying message to construct cellular proteins (mRNA)
• Genetic information unit in some viruses
• Regulate gene activity
• Regulate protein synthesis
  • Makes a good antibiotic target
  • U binds with T instead of A

Microscopy

• Gives a detailed image of objects otherwise too small to see
• Dependent on 3 Factors:
  • Magnification
  • Contrast
  • Resolution

Magnification

• Enlargement of image
• May be fuzzy without contrast & resolution

Contrast

• Is the difference in light intensity
• Usually created with absorption of light
• Stain bacteria to increase contrast

Resolution

• Higher the resolution, the more detailed the picture
• Determined by:
  • Size of objective lens
  • Wavelength of light
  • Refractive index material

Properties of Light

• Light hits an opaque object
  • Rays bounce off the object (Reflection)
• Rays pass through object (Transmission)
  • Requires a clear & transparent object (Glass, water)
• Some light does not pass through (Absorption)
  • Certain wavelengths absorbed
  • Causes different colors
• Bending of light (Refraction)
  • Occurs with objects of different density causing it to slow down.
• Refractive index is the measure of light bending ability.
  • Immersion oil's refractive index is 1.5.
  • Glass's refractive index is also 1.5.
• Using oil keeps the light from bending between the glass and objective lens.

Types of Microscopes

• Compound Light Microscope
  • Compound - more than one lens
  • Visible light

Types of Compound Light Microscopes

- Bright-field
- Dark-field
- Fluorescence
- Phase Contrast

Compound Microscope Parts

- Light Source
- Condenser
    -Direct light through the object
- Stage Mount
    -Holds the specimen
- Objective Lenses
    -Various Magnifying Powers
- Ocular Lens
    -Additional Magnification

Total Magnification

• Objective lens times ocular lens
• Ocular Lens Power X Objective Lens Power = Total Magnification
• Example:
  • Eyepiece (10 x) X Medium power (10 x) = 100 x Total Magnification
  • Low power = 10x
  • High power = 40x
  • Oil immersion = 100x
  • Ocular lens = 10x
  • On low power, total magnification = 100x
  • On high power, total magnification = 400x
  • With oil immersion, total magnification=1000x

Other light microscope

• Phase contrast microscopy
  • Condenser throws light rays out of phase
  • Cellular movement and internal structure of live organisms

Darkfield microscopy

• Light is scattered off object
• Views surface structures

Fluorescence microscopy

• Fluorescent material illuminated by UV light

Electron Microscopes

• Electrons used instead of light rays
• Much greater magnification
• Transmission electron microscope:
  • Electrons pass through specimen
  • Captured on photographic film
  • Ultra-thin specimen
• Scanning electron microscope:
  • Electrons hit specimen causing secondary electrons to eject
  • Captures the surface only

Wet Mount Procedure

• Simple sample preparation for microscopic viewing
• Observe living microorganisms
• Usually not stained
• May use a vital stain

Stains

• Used for increasing the slide contrast
• Simple Stain
  • Use one dye to make cells visible
  • Shows cell size, shape & arrangement
    • Direct stains (basic + charge) = stains the specimen
    • Negative stains (acidic – charge) = stains the background
• Differential stain
  • Distinguishes types of microorganisms
    • Gram Stain
    • Acid-Fast Stain

Special Stains to Identify Cell Features

• Endospore
• Capsule
• Heat fixation: "freezes" cells in place with heat to make them visible.

Types of Dyes

• Basic (positive): safranin, acid fuchsin, crystal violet, methylene blue
• Acidic (negative): eosin, basic fuchsin, congo red

Simple vs Differential Stains

• Simple stains make cells visible with one dye
• Differential stains distinguish between types of microorganisms

The Gram Stain

• 1884: Developed by Danish bacteriologist Christian Gram
• Divides bacteria into two groups
  • Gram-positive
    • Purple/blue.
  • Gram-negative
    • Red

Gram Stain Procedure

1. Heat fix smear: Kills bacteria & sticks it to slide.
2. Crystal Violet: Stains all bacteria purple (both G+ & G-).
3. Iodine (Mordant): Helps purple stain stick.
4. Alcohol (Decolorize): Washes out stain from Gram-negative but not Gram-positive.
5. Safranin (Counterstain): Stains Gram-negative pink, Gram-positive stays purple

• Helps differentiate between G+ (purple) and G- (pink) bacteria.
• Bacterial cell walls use peptidoglycan.
  • Gram-negative walls have one layer of peptidoglycan.
  • Gram-positive walls have many layers of peptidoglycan.

Importance

• Can diagnose infections Bacterial Meningitis -Gram stain spinal fluid Gonorrhea in men -Gram stain penile discharge

Capsule Stain uses Negative Stains

• Highlight capsules surrounding certain bacteria.
• Dark stain (India ink or nigrosin) stains background, leaving capsule clear.
• Counterstaining uses a different color to stain the bacteria making it stand out.

Endospore Stain

• Detects bacterial endospores that are dormant structures.
• Primary stain (Malachite green): Slide heated with malachite green stains the endospores.
• Water decolorizes slide removing green stain from bacterial cells not from endospores.
• Counterstain (Safranin): A red stain colors the rest of the bacteria (not the endospores).
  • Endospores appear green
  • The rest of the bacteria looks red or pink

Acid Fast Stain

• Identifies bacteria with thick, waxy cell, like Mycobacterium (tuberculosis).
• Primary stain (Carbol fuchsin): Heat carbol fuchsin stains bacteria red, and penetrates cell.
• Decolorize (Acid-alcohol): Washes slide with acid-alcohol, removes the red stain from those non-acid-fast. Acid-fast cells hold the stain.
• Counterstain (Methylene blue): Apply a blue stain to non-acid-fast bacteria.
  • Acid-fast bacteria stay red
  • Non-acid-fast bacteria turn blue

Eukaryotic and Prokaryotic Cells

• Homeostasis: organisms attempt to maintain a stable internal state.

Bacteria and Biofilms

• Bacteria are single cell prokaryotic organisms that can come together in Biofilms to live together
• Example: Plaque build up on teeth

Prokaryotic Cells

• Organisms where genetic material (DNA) lacks a cell nucleus. ONLY bacteria (archaea inclduded) are prokaryotic.

Eukaryotic cells

• Organisms which consist of cells containing genetic material within distinct nucleus.
• Include all organisms, but not bacteria (and Archaea).
• Include fungi, algae, protozoa.
• Plants and animals also classified as eukaryotic cells.

Similarities between Eukaryotic and Prokayotic Cells

• All organisms have similar genetic organization where heredity material is expressed
• Both prokaryotic and eukaryotic cells have internal compartments
• Metabolism occurs in cytoplasm.
• Ribosomes perform protein synthesis.
• Can have cell walls for helping maintain water balance
• Both use flagella to move and perform photosynthesis

Eukaryotes VS Prokaryotes:

• Prokaryotic:*
  • No nucleus
  • No membrane-bound organelles
  • Cell wall contains peptidoglycan.
  • Are less than several micrometers
• Eukaryotic:*
  • Nucleus, and Membrane-bound organelles
  • No peptidoglycan, even if cell wall is present
  • May be 10 times larger

Shapes of Prokaryotes:

• Morphology -* Determined by cell wall. Includes:
  • Cocci
  • Rod (bacillus)
  • Spiral

Larger Size cells Eukaryotic > bacterial

Taxonomy

• Science of classification organizing living things into groups.
• mid-1700s: Carolus Linnaeus publishes Systema Naturae to name organisms Ex: Bacillus subtillus (Genus species)

Notice the form of Reproduction for each

Prokaryotic Cell Structures

• Appendages (located outside)*

• -Pili (Straight hair like appendages)

  • attachment
  • sex pili (transfer DNA)
• • Flagella (long thin structures)
  • locomotion motile bacteria

Prokaryotes movement:

• Chemotaxis - away or towards a chemical
• Phototaxis - sense light sensitivity
• Aerotaxis - moving where oxygen concentration is more favorable
• Magnetotaxis - movement along magnetic lines in the soil

Prokaryotes- outermost layer:

• Capsule (glycocalyx).
• Made of polysaccharides.
• Function: Protect against:
• Drying-out
• Phagocytosis (wbc engulfing bacteria cells and digesting it)

Prokaryotic cell Envelope is three layered

-Outer layer - Function: Protection -Cell wall - Function Cell shape -Cytoplasmic membrane or cell membrane - Function Containment * Gram negative - have lipopolysaccharide (LPS) outer membrane * Gram positive - do not have the outer membrane * Mycoplasma - have only the cell membrane

Prokaryotic Cell Wall

• Anchors structures that extend from surface (pili, flagella)
• Prevents osmotic lysis in hypotonic environment: If the outside of the cell has more water than the inside, water rushes in. The cell wall stops the bacteria from bursting like a water balloon. Which is found only in mycoplasmas

Cell membrane

• •Membrane that encloses the cytoplasm of any cell
  • •Function - containment & transport
• * Regulates passage into and out of cell

Membrane proteins-two types:

• Integral Proteins (are stuck inside)
• embedded in the membrane, like a straw stuck through a plastic lid
• Some help things move in and out of the cell, like a tunnel
• Peripheral proteins ( are stuck to the surface
• sit on top or underneath the membrane, like a sticker on a water bottle
• Help support and connect

Cytoplasm

• Functions of membrane proteins:
• Build & break things (like making the cell wall and energy).
• Make energy (ATP) so the cell can work.
• Hold DNA in place when the cell copies itself.
• Sense signals like messages from environment
• Move stuff in & out like doors, carriers, or pumps.
• inside a cell It's like the "goo" that holds everything inside
• Metabolism happens here
• Mostly water (90%)It's like a watery soup with proteins

Key Parts

• Nucleoid - messy ball of DNA that holds the cell's genetic instructions (like a -blueprint). • Ribosomes -tiny factories that make proteins. In prokaryotes, they are 70S -(smaller than eukaryotic 80S ribosomes).

Inclusion Bodies

• tiny storage spots inside the cell with extra nutrients or materials tiny storage bins • Storage Granules holds sugars, fats, or phosphates snack reserves • Gas Vacuoles help bacteria float tiny balloons Chlorosomes •photosynthesis capture light like solar panels • Magnetosomes contain tiny magnetic crystals which help sense direction like a compass

Prokaryotic Endospore

Hardy, resting, non-growing which is produced by some bacteria gram-positive Bacillus Anthrax /Clostridium-botulism, tetanus, gas gangrene Can withstand heat and chemicals

• Sporulation-unequal cell division begins Sporulationis bacteria make spores to survive and grow back when things improve

Prokaryotic Reproduction: Binary Fission

• Growth - increase population size -Asexual Reproduction
• Identical genetic material of parent cell
  • Cell doubles mass
  • DNA replicates
  • Cell Fission Cytokinesis Cell Growth Phaseduring which:
  • Cell mass and size increase -Special enzymes break wall to allow expansion DNA replication phase
  • chromosome is copied

Reproduction Binary Fission

• Cell doubles mass
• Asexual reproduction
• Septum divides cell into two identical daughter cells.
  • Generation Time= Time between binary fissions is a doubling time
• Lag: No cell division, adapting
• Logarithmic/Active: growth – exponential growth
• Stationary: reproduction and death equal
• Decline: death
• Pure culture has has one organism

Steps to Obtaining a Pure Culture

• Sterilize
• Isolate
• streak method of pure culture*

Eukaryotic microbes Appendages

-Flagella - purpose motility

• Cilia - purpose move things across a surface
• Nucleus - location

Eukaryotic: edomembrane system

   - Sorting and transport
    -Includes endoplasm, Reticulum, golgi, lysosomes

• Rough er- ribosomes attached

Golgi apparatus

Flat membrane, stacks and molecules are modified

• Vesicles

Cytoskeleton

Function: -structure, movement, cell division -Three fibrous poteins

Fungi

Fungal cell walls contain chitin is the point

• Fungi do not have chloroplasts
• Two body forms: multicellular and unicellular

Fungal Cell structures:

• Saphrophyte- organism which lives on dead
• Autotrophs – are organisms that prepare for this process photosynthesis
• Heterotrophs- are organisms that depend on others for nutrition

Non-phototrophic:

organisms that lack solar light dependence.

• Fungi are:
• heterotrophic
• Non-phototrophic
• saprophytic
• decompose to obtain nutrients some pathogens
• Mycosis fungal infection Yeastsingle cell that reproduce by Budding Moldsbranch tube like filaments

Fungal interconnected structure

Hyphaeindividual filaments Asexual reproduaction elongation and fragmentation budding division of cells Sexual reproduction

• *Mycoses -**acquired from nature TX difficult
• Toxins - Muscarin
• Amanita muscaria mushroom
• Phalloidin and amanitin
• A. phalloides mushroom
• Ergot Claviceps purpurea-mold on rye
• Aflatoxin Aspergillus spp. Black mold toxin - stachybotrys chartarum Why are most antifungal agents toxic to human cells because the fungus is eukaryotic cells identical and hard to find and treat yeast mold

Prozoa

•Unicellular eukaryotic organisms •The most complex one cell organisms Organelles Four groups- based on motility

• Four groups: -based on motility Flagellate -Mastigophora Amoeboid Sarcodina pseudopods
• Sporozoa
• nonmotile -Cilate -Cilia

Helmniths

• Worms- parasitic and multicellular, flukes, tapeworms, roundworms -tend to chronic rather than fatal because the worms depend on the host for survival

Viruses and Prions

• VIRUSES-* microscopic packet of nucleic acid inprotein Has to DNA OR RNA Viryses NOT CELLS

History- Before germ theory of disease all poisons were called viruses

Viruses VS Bacteria

Both are infectious Viruses cannot be seen with most microscopes Viruses -1/3 smaller than bacteria

• Viruses are 1/10 to 1/3 the size of bacteria
• Some bacteria live inside host cell
• Bacteria are alive
• virion is the intact virus particle
• Capsid is protein coat

Virus structure

proteins may be envelope for storage of genetic either DNA or RNA

what is only cure to virus far?- Hepatitis C

antibiotics do not work because cell wall viruses protected once host cell Used to prevent = vaccines

* Benign: no cancer * Invasive = tissue infection or growths * Malignant = deadly Zoonotic disesases spread by animals to humans Viroids are molecular ssRNA no capsid

*Econom importance plant

Prions

• Proteinacious
• No nucleic acid
• Induces cell changer Aggregated protein results

Test your knowledge of microbiology and cell biology! Topics include the central dogma, microscopy, Gram staining, optical phenomena, virology, and bacterial processes. Review key concepts in molecular biology and microbiology.