Chapter 1

how are microorganisms grown and studied?

• Microscopy 

• Culture: cells grown in/on nutrient medium 

  • Very specific needs 

• Medium: liquid/solid mixture containing all required nutrients 

• Growth analysis 

  • Assays

  • Spectrophotometer

  • Etc. 

• Bacterial colonies 

  • Shapes, growth, color, etc. 

• Molecular, biochemical, genetic analysis

what are some key features of microbial cells?

• Create energy 

  • metabolic/anabolic reactions

  • Harness energy 

• Must replicate 

  • Mitosis or binary fission

• Evolution 

  • Goes faster than most species

  • Horizontal gene transfer

    • Can share once alive, not vertically like we do (birth)

what are some additional features microbial cells may have?

• Can become different types of cells

  • Endospores

    • Typically triggered by a crappy environment (too cold, hot, treated incorrectly, etc.)

    • Tough sugary coat and some ribosomes, really tough seed, goes dormant 

    • Better place with better nutrients = new home and can grow 

    • Autoclave is the only way we can destroy it 

• Can talk to each other 

  • Corum sensing 

• Motility 

  • Some move

  • Single flagella, some have multiple

    • Same movement as sperm

cell size of prokaryotes

0.2 to 600 um in diameter

cell size of eukaryotes

5-100 um in length

why is cell size important for microorganisms?

They are small because they are simple, not very complex

Uses diffusion to get rid of waste products and get nutrients

what are some bacteria shapes?

• Coccus (circle)

• Rod, bacillus, bacilli (rod)

• Spirillum (spirula) (squiggle)

• Spirochete (squiggle)

  • Much more rigid in shape than spirilla

• Budding and appendaged (balloon shaped)

• Filamentous (looks like spaghetti)

bacteria

• Prokaryotes 

• Usually undifferentiated single cells 0.5-10 um

• 80+ phylogenetic lineages

archaea

• Prokaryotes 

• Historically associated with extreme environments but not all of them are extremophiles 

  • Thermal vents 

  • Glaciers 

• Lack known parasites or pathogens of plants and animals 

• 12+ phyla

eukarya

• Plants, animals, fungi

• First were unicellular, may have appeared 2 billion years ago

• At least 6 kingdoms

• Varies dramatically in size, shape, physiology 

  • Can change shapes depending on environment

viruses

• Obligate parasites that only replicate within host cell

• Not cells

• Doesn’t carry out metabolism; take over infected cells to replicate

• Have small genomes of double-stranded or single-stranded DNA or RNA

• Classified based on structure, genome composition, and host specificity 

  • Naming is confusing

origin of cell domains

The origin of earth 

• Bacteria, archaea, and eukarya distinguished by 4 BYA

• Descended from last universal common ancestor (LUCA)

• The sun then let them go through photosynthesis (anoxic photosynthesis first, then eventually produces oxygen aka anaerobic (photosynthesis)

what are the good and the bad of microorganisms?

Bad 

• Can cause food spoilage and foodborne disease 

• Fermentation process 

  • Glycolysis into fermentation paths 

Good 

• Improve food safety 

• Preservation

• Yummy foods and drinks

what types of cells are hypothermophiles?

archaea because they like extremely high temperatures

what types of cells are psychrophiles?

bacteria because they like extremely low temperatures

what types of cells are acidophiles?

archaea because their pH are extremely low

what types of cells are alkaliphiles?

archaea because their pH are extremely high

what types of cells are barophiles?

bacteria because they like extremely high pressure

what types of cells are halophile?

archaea because they like extremely high concentrations of salt

light microscope

Ocular lens = 10x magnification

Low power = 4x 

10x x 4x = 40x magnification

10x x 10x = 100x magnification

Oil immersion = 100x 

10x x 100x = 100x magnification

• DON’T USE UNLESS YOU HAVE OIL 

Condenser to control how much light you have in the microscope

When light moves through different medium, it can show very differently due to how fast/slow the light is

phase contrast microscope

converts phase shifts in light passing through a transparent specimen to brightness to change the image

fluorescence microscopy

optical microscope that uses fluorescent light instead of scattering light

differential interference contrast microscopy

uses polarized light and specialized prisms to enhance contrast in unstained samples, which reveals a detailed surface

confocal scanning laser microscopy

uses a laser beam to scan a sample point-by-point, and a pinhole to block out-of-focus light

electron microscope

• Uses electrons instead of light to image cells and structures 

• Electromagnents function as lenses 

• Operates in a vacuum 

transmission electron microscope (TEM)

uses a beam of high-energy electrons that can pass through a thin sample to produce a highly magnified image

scanning electron microscope (SEM)

scans the surface with a focused beam of electrons

magnification

ability to enlarge an image

resolution

the ability to distinguish 2 adjacent objects as distinct and separate

who was the first to describe microbes

Robert Hooke

who was the first to see bacteria?

Antoni van Leeuwenhoek

specimen preparation

No cover slips because it creates too wide of a slide so the oil immersion can’t see anything 

1. Take glass slide and apply culture containing bacteria in center of the slide 

2. Use an inoculating loop to transfer it 

3. Spread very thin

4. We need it to dry completely

   1. If we move too quickly, we will boil the bacteria if we heat fix 

   2. When you boil them, it will burst open and distort what the bacteria looks like 

5. Stain to create contrast so we can visualize microbes when we get to the light microscope

stains to create contrast (positive charge; basic dyes)

1. methylene blue

2. crystal violet

3. safranin

stains to create contrast (negative charge; acidic dyes)

1. nigrosin

2. India ink

what do basic dyes adhere to?

negatively charged proteins

what do acidic dyes adhere to?

sticks to the background of the proteins, repels cells

gram positive bacteria

very thick layer of peptidoglycan

gram negative bacteria

very thin layer of peptidoglycan

procedure for staining gram stains

1. Primary stain in gram stains is crystal violet

   1. Sticks to all cells 

2. All the cells then are purple

3. Rinse with water, then come in with iodine

4. Iodine creates complex with crystal violet, makes it though cell wall 

5. Add 95% ethanol 

   1. Decolorizing step 

   2. Dehydrates some components of peptidoglycan layer 

   3. Creates tougher cell 

   4. Crystal violet and iodine can now sneak out, which makes it colorless 

6. Gram positive cells are purple 

7. Gram negative cells are colorless 

8. We then apply safranin to cells, nothing will happen to the gram-positive cells, but the gram-negative cells will become pink in color

Koch’s postulates

1. When we have an animal with a certain disorder, if we take sample and grow it in culture, we have to identify the pathogen 

2. Create a plate, use inoculating loop to collect sample and streak it across agar plate

   1. Incinerate loop with flame 

   2. Drag initial swabbing 

      1. A lot of colonies on first swipe to just a little on second swipe 

   3. Single colony forms

      1. Arisen from one single cell

   4. Put into broth tube

3. Transfer suspected pathogen to healthy animal 

   1. Has to succumb to a disease as first animal

4. Repeat step 2, see if it is the same 

   1. If it is the same, we can say that the disease is capable of spreading and causing diseases

how can you use molecular biology to study microorganisms

Pasteur and Spontaneous Generation

• Louis Pasteur: chemist and microscopist 

  • Discovered alcoholic fermentation was a biological process, not just a chemical process

  • Developed vaccines for anthrax, fowl cholera, and rabies 

• Pasteur flask: swan-necked flask

  • Pasteur disproved the theory of spontaneous generation 

    • Life arose spontaneously from nonliving matter