Exam 1

Prokaryotic cells

Lack a true membrane-delimited nucleus

(this is not absolute)

Eukaryotic cells

Have a membrane-enclosed nucleus; more morphologically complex

What is the definition of life?

• all life is represented by cells

  • all cells have similar organization

• central dogma

• react to changes in the environment

• growth, development, death

• subject for evolution

• energy and metabolism

• homeostasis and regulation

• reproduction

How have the classification schemes evolved over time?

The three classification schemes are based on a comparison of ribosomal RNA genes by Carl Woese

Domain Bacteria

• usually single-celled

• majority have cell wall with peptidoglycan

• most lack a membrane-bound nucleus

• found everywhere and some live in extreme environments

• cyanobacteria produce significant amounts of oxygen

Domain Archaea

• distinguished from Bacteria by unique rRNA sequences

• lack peptidoglycan in cell walls

• have unique membrane lipids

• some have unusual metabolic characteristics

• many live in extreme environments

Domain Eukarya

• Protists: generally larger than Bacteria & Archaea

  • Algae: photosynthetic

  • Protozoa: may be motile, “hunters, grazers”

  • Slime molds: two life cycle stages

  • Water molds: devastating disease in plants

• Fungi

  • Yeast: unicellular

  • Molds and mushrooms: multicellular

Acellular Infectious Agents

• Viruses

  • smallest of all microbes

  • requires host cell to replicate

  • cause range of diseases, some cancers

• Viroids

  • infectious agents composed of RNA

• Satellites

  • nucleic acid enclosed in protein shell

  • most coinfect a hose cell with a virus to complete life cycle

• Prions: infectious proteins

What experiment did Stanley Miller conduct?

He formed organic molecules from a primordial soup

What is the purpose of the ribozyme in evolution?

Ribozymes: RNA molecules with the ability to catalyze reactions, form peptide bonds, and perform cellular work and replication.

RNA could serve the dual purpose of genetic information storage AND catalyzing reactions, therefore it is believed the earliest cells may have been RNA surrounded by liposomes.

What is the endosymbiotic theory?

Primitive prokaryotic microbes ingested other microbes, beginning a symbiotic relationship, which formed the first basic eukaryote.

What are some hypothesis from the endosymbiotic theory?

• Ingested microbes that could use oxygen for a respiratory process to produce chemical energy became mitochondria.

• Ingested microbes that could fix carbon dioxide into organic molecules using light energy became chloroplasts.

How to correctly name a microorganism

Binomial (scientific) nomenclature

Genus species

Microbial ecology

How microbes interact with the world around them

• microbes can help in biogeochemical cycling by turning inorganic molecules into organic molecules

• live in diverse groups in nature

• examples: microbiome, plague on teeth, slime on rocks on beaches

Microbial Biotechnology

Studying the genetics of microbes, which can help us to use them to benefit humans

• we can mass produce molecules that humans want by altering the genomes of microbes

• example: production of human insulin by inserting the gene into E. coli cells

Medical microbiology

Disease of humans and animals

Public health microbiology

Control and spread of communicable diseases

Immunology

How the immune system protects a host from pathogens

Microbial ecology

The relationships of organisms with their environment

Agricultural microbiology

The impact of microorganisms on food production

Food microbiology

Microbes used to make food and beverages as well as spoilage microbes

Industrial microbiology

Penicillin and other antibiotics, vaccines, steroids, vitamins, etc.

Microbial physiology

Studies metabolic pathways of microorganisms

Microbial genetics, molecular biology, bioinformatics

Studies the nature of genetic information and how it regulates the development and function of cells and organisms

Synthetic microbiology

Microbes area model system of genomies

How does RNA play a role in the evolution of life?

RNA is believed to be a precursor to double stranded DNA and can regulate gene expression. rRNA genes were found to show bacterial lineage in the evolution of prokaryotes to eukaryotes. The earliest cells may have been RNA surrounded by liposomes.

How was early metabolism done?

Early energy sources were under harsh conditions and not many organic molecules were available. Microbes turned inorganic molecules into organic molecules

Photosynthesis was also used

Chemoorganotrophs

Using organic molecules as a source of energy

ex. glucose, acetate

O2 → CO2 + H2O

Chemolithotrophs

Using inorganic molecules as a source of energy

ex. NH4, Fe

H2 + O2 → H2O

Fermentation

Anaerobic respiration; does not yield much energy for microbes

Aerobic respiration

Requires oxygen; yields much more energy

Resolution

The ability of a microscope to distinguish details of a sample

Does electron microscopy positively or negatively affect resolution?

Positively; it is more precise and allow for microbial structure study in more details

How does wavelength effect resolution?

Shorter wavelength = higher resolution

Longer wavelength = lower resolution

ex. light wavelength is longer than electron beam, therefore electron microscopy has a higher resolution

Purpose of immersion oil

Use of immersion oil between lens and sample can increase the refractive index, allowing more light to pass through lense, increasing the numerical aperature, increasing resolution

Numerical aperature

The light-collecting power of an objective lense

Numerical aperature effect on resolution

The higher the numerical aperature, the better the resolution

Determining total magnification of a microscope

objective x optical lense

Electron microscopy

• Electrons replace light as the illuminating beam

• Allows for study of microbial morphology in great detail

• Electron beam > light beam

Transmission electron microscope (TEM)

• Point electron beam at sample → electrons scatter when they pass through thin sections of a specimen, the electrons are under vacuum which reduces scatter → produces an image

• 2D image

• Denser regions in specimen scatter more electrons and appear darker

Disadvantages of TEM

• Electrons can only penetrate thin specimen

• Only gives 2D image

• Specimens must be viewed under high vacuum

• Specimens are dead or artifacts

Scanning electron microscope (SEM)

• Uses electrons excited from the surface of a specimen to created a detailed image

• 3D image

• Can determine actual in situ location of microorganisms in ecological niches

Electron cryotomography

Rapid freezing technique using steel slices to create extremely high resolution

Scanning probe microscopy

Based on the atomic force interaction, allows us to see atoms!

Creates a sort of shape by placing a thing probe close to the surface and casting an electric current through the landscape

The current is constant and the probe moves based on atom size, which creates a picture

Scanning tunneling microscope

Steady current is maintained between microscope probe and specimen and creates image of surface of specimen

Atomic force microscope

Sharp probe moves over surface of specimen at a constant distance and creates an image

Light vs Electron microscope

• Resolution: light < electron

• Magnification: light < electron

• Medium of travel

  • Light: air

  • Electron: high vacuum

Fluorescent imaging

Your specimen must iether be naturally fluorescent, or labeled with a fluorescent dye/fluorescently labeled antibody

Bright-Field microscope

• Allows us to see live specimen → most organisms are colorless so we stain to see

• Most standard microscope

• Source of life comes from beneath the sample

• Produces a dark image against a brighter background

Refractive index

How much a substance bends a light ray

Dark-Field Microscope

Image is formed by light reflected or refracted by specimen → produces a bright image of the object against a dark background

• Used to observe living, unstained preparations

• Light source comes from the side

• Uses a hollow cone of light so that only light that has been reflected or refracted by the specimen enters the lens

Phase-Contrast Microscope

Uses slight differences in refractive index and cell density

Uses hollow cone of light → cone of light passes through a specimen, some is out of phase → light passes through phase plate and bringing it back to phase

Excellent way to observe unstained, living cells

Each compound reflects slightly differently

2D image

Differential Interference Contrast Microscope (DIC)

Similar to phase-contrast → creates image by detecting differences in refractive index and thickness of diff parts of specimen

Uses two beams of polarized light to create 3D image of specimen

Excellent way to observe living cells

Fluorescence Microscope

• 1st step up in detection power

• Exposes specimen to UV, violet, or blue light

  • Shine one wavelength and object reflects different wavelength

• Specimens stained with fluorochromes (fluorescent dyes), labeled with fluorescent antibodies, or naturally fluorescent

• Shows a bright image of the object on a dark background

• Can mix organisms with artificial fluorescence so sample does not get killed

Immuno-Fluorescence

Antibodies with fluorochromes make molecule glow → can be placed on bacterium to see under fluorescent microscope

Confocal Microscopy

• Step up from fluoro

• Creates sharp, composite 3D image of specimens by using laser beam instead of light

• Specimen is usually fluorescently stained

Why do we prepare and stain specimen?

Increases visibility, accentuates specific morphological features, preserves specimens

Fixation/Smear Preparation

Preserves internal and external structures and fixes them in position

1. Prep smear

2. Stain it

Organisms usually killed and firmly attached to microscope slide

Heat fixation

Expose glass to high heat, slightly melting the sugars in sample to glass and glues it

Used with bacteria and archaea

Preserves overall morphology but not internal structures

Chemical fixation

Used with larger, more delicate organisms

Protects fine cellular substructure and morphology

What are dyes useful?

Make internal and external structures of cell more visible by increasing contrast with background

Ionizable dyes

Basic dyes = positive charges

• Bind to negatively charged molecules (nucleic acids, proteins, surfaces of bacterial and archaeal cells)

• ex. methylene blue, crystal violet

Acidic dyes = negative charges

• Bind to positively charged cell structures

• ex. eosin, rose bengal

Simple staining

A single stain is used

Can determine size, shape, and arrangement of bacteria

Use any dye with positive charge

Negative staining

Stain everything BUT cells (colorful background with empty spot)

Differential staining

Divides microorganisms into groups based on their staining properties

Gram staining

Divides bacteria into two groups: Gram Positive & Gram negative

• Based on differences in cell wall structure

Steps of gram staining

1. Simple stain with crystal violet for 1 minute. Water rinse. (Cells stain purple)

2. Add iodine to act as a binder and holds crystal violet together. Let sit for one minute. Water rinse. (Cells remain purple)

3. Add ethanol (alcohol) as a decolorizer for 10-30 seconds. Water rinse.

   • Gram positive cells = remain purple

   • Gram negative cells = become colorless

4. Add safranin (counter stain) for 30-60 seconds. Water rinse. Blot dry.

   • Gram positive cells = remain purple

   • Gram negative cells = appear red

Acid-fast staining

Determines what bacteria has in cell wall based on result

Acid-fast cells = red cells

Non-acid-fast = blue cells

High lipid content in cell walls is responsible for their staining

Capsule staining

Used to visualize polysaccharide capsules surrounding bacteria (most external layer of bacteria)

Capsules may be colorless against a stained background

Flagella staining

Mordant applied to increase thickness of flagella for visualization

Endospore staining

Endospores ensure survival of the bacteria and reside within the cell

Dyed with malachite green

Negative staining

Used for the study of viruses and cellular microbes

Only the background is stained while the cells remain unstained

Great tool for visualizing intact structures of microorganisms without disturbing the cell

Shadowing

Coating specimen with a thin film of a heavy metal on only one side

Useful for virus particle morphology, flagella, and DNA

Freeze-etching

Freeze specimen, causing it to fracture/crack along the lines of greatest weakness → exposes different structures within cell

Allows for 3D observation of intracellular structures

Coccus

Spherical

Bacillus

Rod

Coccobacillus

Very short and plump

Vibrio

Gently curved

Spirillum

Helical, comma, twisted rod (think spiral)

Spirochete

Spring-like

Pleomorphism

Variation in cell shape and size within a single species

Diplo

In pairs

Tetrads

Groups of four

Strepto

Chain

Staphilo

Cluster

Macronutrients

Required in large quantities; play principal roles in cell structure and metabolism

ex. C, N, O

Micronutrients

Required in small amounts; involved in enzyme function and maintenance of protein structure

ex. Mn, Zn, Ni

Heterotroph

Must obtain carbon in an organic form made by other living organisms such as proteins, carbohydrates, lipids and nucleic aicds

Autotroph

An organism that uses CO2 (inorganic gas) as its carbon source; not nutritionally dependent on other things

Chemotroph

Gain energy from chemical compounds

Phototrophs

Gain energy through photosynthesis

Photoautotrophs

Oxygenic photosynthesis → byproduct of splitting H2O

Anoxygenic photosynthesis → uses energy of light and uses protons to directly produce gradient

Chemoautotrophs

Survive totally on inorganic substances; very slow

Methanogens

Type of chemoautotroph; produce methane gas under anaerobic conditions

Chemoheterotrophs

Aerobic respiration

Saprobes

Free-living microorganisms that feed on organic detritus from dead organisms

Opportunistic pathogen

No harm until host is weakened

Facultative parasite

Prefer not to be a parasite, but will be if they must

Parasite

Derive nutrients from host

What is the importance of nitrogen?

It is a macronutrients that is essential for amino acids, purines & pyrimidines, some carbohydrates, lipids, etc.

It can be supplied through metabolism of amino acids, nitrates, ammonia, and nitrogen fixation.