Microbiology Midterm review

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History is about

consequences, not just names

What are microorganisms

"Microscopic forms of life"
I.E. can be seen with a microscope, but not with the naked eye

"Microbes"

are what microorganisms are sometimes referred to as

Why study "Microbes"

they are essential for all life: produce usable nitrogen for plants which can then be used for the rest of the food chain

Microbes aids/functions

digest cellulose in cows and sheep, ecology: recycling, industrial processes, disease aents, models for study of basic biology

1st Discovery of microorganisms

Antoni Van Leeuwenhoek (1685)

Antoni Van Leeuwenhoek

built simple microscope and observed protozoa and bacteria. He called them "animalcules" (sort of like the term molecules)

What Leeuwenhoek saw

"numerous, fantastic, cavorting creatures"

Simple microscope

only having one lens

Compound microscope

was developed around 1820 but it took awhile for it to be refined. It uses more than one lens

How are microbes classified?

fungi: mold & yeast, protozoa, algae, prokaryotes, and viruses (viruses are NOT "organisms", cannot grow on their own)

golden age of Microbiology

1850-1920

Key questions of the golden age

1. Is spontaneous generation possible? (life created from nothing)
2. What causes fermentation?
3. What causes disease?
4. How can infection and disease be prevented?

Spontaneous generation

Can life arise from dead organic matter. Theory was first compiled by Aristotle.

Redi's Experiment (1668)

Used flies and dead meat to test the theory of spontaneous generation. Results: larvae appeared if flies in contact with decaying mean. No larvae was produced if the flies were kept out. Therefore, spontaneous generation was not supported.

Redi's Experiment results

Unsealed-->larvae
Sealed--> No larvae
Covered with Gauze-->

Needham (1745)/ Spallanzani (1768) also tested for spontaneous generation:

Broth in air-tight flasks
Boiled and incubated
N: growth--used cork to seal (but it was leaky!)
S: No growth if HERMETICALLY sealed. Growth formed if air was allowed.
These experiments appear to refute spontaneous generation
OBJECTION: Air contains "vitalizing power" needed for life

Spallanzani

also famous for the idea of artificial insemenation

How to deal with the "air" problem in disproving spontaneous generation?

Louis Pasteur's experiment did this

Louis Pasteur's experiment

broth is in swan-necked flasks
Boil-->incubate-->no growth (germs trapped)
Tilted falsk-->incubated-->growth

Pasteur's experiment showed that

air problem was solved, no growth if the flask was kept upright, but there was growth if the flask was tilted. Therefore the concept of spontaneous generation was finally laid to rest.

Pasteur showed that growth was due to

Contamination from the air! In his elegant (simple but beautiful) approach, no "vitalizing power" would be destroyed.

Pasteur also showed that fermentation was caused by

yeast.

What causes infectious disease?

Germ theory of disease developed by Pasteur

Koch's germ theory of disease

Isolation of "pure culture"
1. Host-several different types of bacteria present in the host
2. Grow the bacteria from host in liquid medium
3. Separate individual types by spreading bacteria on solid media (agar plate). Then incubate
4. Distinct colonies of bacteria on agar plate after incubation.
5. Pick colony of a particular type. Check cell type by microscopy. Grow in broth. Spread on agar. Repeat cycle at least 3 times: pure culture

Pure culture steps were

not around before Robert Koch discovered them

pure culture

represents a genetically PURE stain because all the bacteria in a colony came from a single bacterial cell.

Koch's lab was separated from others because

first use of agar (better than potato slices)
distinct colony types
allowed isolation of specific microbes: "pure culture"
Pure cultures are essential to determine whether a particular microbe causes a specific disease

Koch's Experiment (CA. 1876)

He used 6 types of bacteria on 6 types of mice. Objective was to show that each type of bacterium caused a SPECIFIC disease. Koch argued that FOUR conditions must be met to prove this

Koch's Postulates

conditions that need to be met to prove that a bacterium caused a specific disease
1. Causative agent found in EVERY case of disease; absent from healthy hosts
2. Putative (thought to be like this or cause) agent can be isolated and can be grown outside the host
3. Disease forms when agent is introduced to healthy host (tentative evidence)
4. Same agent is RE-ISOLATED from diseased experimental host. (complete proof)

Impact of Koch's work

etiology (cause) of many diseases established by his group and later by otehr labs. This list includes: anthrax, malaria, tuberculosis, cholera, tetanus, tobacco mosaic disease, bubonic plague, and yellow fever

Anthrax

bacillus anthracis

malaria

plasmodium malariae (protozoan)

tuberculosis

mycobacterium

cholera

vibrio

tetanus

clostridium

tobacco mosaic disease

tobamovirus

bubonic plague

yersinia

yellow fever

flavivirus

Prevention of infection/ disease

first realized by Ignaz Semmelweis

Ignaz Semmelweis

studied Puerperal sepsis: post partum disease. He observed that medical students would work from the morgue to the maternity ward without washing their hands. He theorized that "cadaver particles" (today, streptococcus) was what caused these mothers to die. He advised people to wash their hands and came up with a bleach solution. He argued that mortality rates would decrease from 18 to 1%. People called him crazy initially, but he was eventually deemed right.

Joseph Lister

a fan of Pasteur. He treated surgical instruments with boiling water. He also sprayed wounds and dressing with carbolic acid (phenol). This reduced the mortality rate by 70%

Florence Nightingale (during the Crimean War 1854-1856)

introduced hygiene and antiseptic practice. She helped to reduce the transfer of infection. Calculated statistically correlating sanitation vs. low mortality. She also suggested that bathroom facilities be in a different room or area which also helped to prevent infection. When she went back to England, she campaigned to reform hospitals and public health policy. Also, founded the Nightingale school of Nurses (1st of its kind)

Other pioneers in disease prevention

Snow: infection control (cholera in London)
Jenner: small pox vaccine
Ehrlich: chemotherapy (sulfadrugs; organoarsenics; Nobel prize in 1908)

Summary of the Golden age

living things com from other living things
microbes cause fermentation & disease
certain procedures and chemicals can prevent or cure infectious disease
Excellent examples of the scientific method as applied to biological sciences

technological developments of the Golden Age

Agar
Nutrient broth (beef extract, NaCl, peptone, water)
Compound microscope
Staining methods
Sterilization & disinfection
Antiseptics

Modern Age of Microbiology: Basic

1. Microbe centered-bacteriology, mycology, protozoology, virology, parasitology
2. Process centered- Metabolism, genetics, ecology, origin of life (Extra-terrestial microbes?)

Modern Age of Microbiology: Applied

1. Medical-immunology, epidemiology, chemotherapy, gene therapy
2. Environment-pollution, public health, agriculture, transgenic crops (Genetically Modified crops)
3. Industrial-food, pharmaceuticals, recDNA, products AKA genetic engineering technology

recDNA

recombinant DNA technology came from microbiology and affects all areas of biology today. It is defined as DNA sequences that result from the use of lab methods to bring together genetic material from multiple sources.

Cell structure

characteristics of life:
growth, reproduction, responsiveness, metabolism

Two basic cell types

prokaryotic and eukaryotic

Eukaryotic cell types

contain a nucleus, have membrane-bound organelles, have complex chromosomes, and are 10-100 um diameter

Prokaryotic cell types

have no nucleus, no membrane-bound organelles, have simple chromosomes, and are 0.2-2.0 um diameter

Nucleoids

used to describe the region where the DNA is

Glycocalyx

glue-like substance that makes up the capsule

Prokaryotic structures: Flagella

helical filament made of subunits: single protein species. rigid
Linked via the hook to: basal structure (motor.
Hook has 90 degree bend which allows the helix to point directly away from the cell.

Arrangement of flagella

1. Polar: at one or both ends of the cell
-monotrichous: single flagellum
-lophotrichous: tuft at the end
-amphitrichous: flagella at both ends
2. Peritrichous: all over the cell

Flagella and motility

prokaryotic flagella rotates. There is no whip-like motion but it is rigid. Induces motility: "runs" and "tumbles"

"Runs"

refers to a darting motion

"tumbles"

is actually referring to tumbling like motion

-CCW

would go through a "run" motion (1 sec)

+CW

would go through a "tumbles" motion (0.1 sec)

Total motion of flagella

10 lengths/sec

Why do flagella appear on Bacteria?

chemotaxis: attraction to chemicals
phototaxis: attraction to light
sensors in cell membranes
more "runs" when there is attractant
more "tumbles" when it is repellant

Fimbriae aka Pilli

short protein structures which adhesion to tissues via the tips; it has a twitching motility which attach and retract. Other pili racking motility is incremental motility unlike flagella. Gliding motility (100/cell)

Sex pilli

refers to cell to cell DNA transfer. Longer hollow tubes than fimbriae/pilli (1-10/cell)

Capsule (or slimy layer)

gelatinous sticky substance called glycocalyx (sugar cup) which is a feature of numberous pathogenic bacteria; e.g. Streptococcus. This enables oral bacteria to attach to teeth. This structure allows bacteria to avoid host defense cells

Cell Wall

provides structure and shape to cells, partially protects the cell from osmotic forces, key component in a net-like structure called peptidoglycan

Peptido part of peptidoglycan

amino acid crosslinks

Gram Stain

two fundamental types of prokaryotic cells
based on cell wall structure

Gram+

stains purple

Gram-

stains pink

Gram positive

thick cell wall is made up of peptidoglycan (90%) and techoic acid fibers

Gram negative

thin cell wall with an LPS layer

Cell membrane

contains a phospholipid bilayer (40%) and protein (60%)
Functions in:
harnessing energy (metabolism) electro chemical gradient across membrane
solute transport into cells
chemical censors

Solute transport

may be "passive" or "active" (requires energy

diffusion

across membrane

Non-specific facilitated diffusion

for various solutes

Specific facilitated diffusion

carrier proteins needed for specific solutes

Osmosis

diffusion of H20 across membrane

Prokaryotic cell wall

protects against LYSIS, does not protect against PLASMOLYSIS (used in preservation of foods with salt/sugar), MYCOPLASMA do NOT have a cell wall

Solute Transport: Active

Uphill:against a concentration gradient
requires energy: ATP
specific for particular solutes
uses carrier proteins

Internal structures of cells

cytoplasm: gel-like; 80% water
chromosome: circular DNA (single
ribosomes: protein synthesis
Inclusion: e.g. Storage granules (NOT membrane-bound!)

Microscope types

light microscopy (LM), confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), scanning electron microscopy (SEM)

Size units

Milimeters mm 10^-3 meter
Micrometers um 10^-6 meter (microns
Nanometers nm 10^-9 meter

Light microscopy (LM)

compound: 2+lens
2000 X mag
oil immersion
bright-field
phase contrast
Other-dark field; differential interference

Light is diluted by________ & lost due to _________.

magnification; refraction

Regular light microscopy

glass to air: light refracted (bent)
Less light enters lens
dim image beyond 1000 X

Oil Immersion LM

glass to oil: light not refracted
more light enters lens
bright image beyond 1000 X

Simple stain

only one stain is used

gram stain

based on cell wall structure; cells older than 24 hours appear to gram-ve (pink) when it is actually gram+ve (purple)

Capsule stain

performing a negative stain to perform/highlight the capsule

Fluorescent Antibody (FAb) Staining

An antigen is a foreign substance (usually a protein) that can trigger the production of specific antibody molecules in an animal. (Ag) Antibodies are made of proteins and react specifically with antigens. UV light: dye flows green. We use this system because it is effective in showing that the antibody is specific to the antigen.

Phase contrast microscopy

Light rays passing through specimen shifted by half wavelength. Rays in phase are brighter; rays out of phase are dimmer. Phase changes due to properties of the medium-which results in contrast. This gives you much better visual detail because of the contrast.

Confocal (laser-scanning) microscopy, CLSM

cells are stained with protein or antibody TAGGED with fluorescent dye. Illumination will be visible with UV laser. Dye glows. Small aperture removes blue:
in focus: bright
out-of-focus: black. Each image is an optical slice down to ~0.5 um. It scans specific planes; builds 3D images
Resolution: 0.4 um
Can observe living organisms in complex communities (biofilms), etc.

Transmission Electron Microscopy (TEM)

rays: electron beams
focused by magnetic lenses
tissue: electron transparent
Metallic stain: electron opague

TEM

stains binds differentially to tissue, electrons are transmitted or stopped. If transmitted, the screen glows. If not, the screen is black. Grayscale image is formed

Scanning EM (SEM)

specimen is coated with metal. focuses electron beam and scans the specimen. The computer reconsctructs images from reflections which has a 3D effect.

Looking at molecules

Atomic Force Microscopy (AFM): 1-10 nm: protein, DNA
Scanning (Quantum) Tunneling Microscopy: 0.5nm-10nm: Amino acids, protein, and DNA

Metabolism overview

organisms harness energy by breaking down molecules. This energy is utilized to build larger molecules and to perform other cellular functions. The chain of reactions used for the above are called "pathways." Enzymes are needed to carry out these reactions under physiological conditions and in a specific manner.

Reactions that release energy

EXERGONIC (spontaneous reaction)
A--> B+C
\
\
Energy

Reactions that require energy are

ENDERGONIC (do not do actions on their own)
\ energy is added in
\
D+E--------------->F

Reactions that release energy DRIVE

reactions that require energy
A--> B+C
Energy
D+E---------->F

ATP

adenosine triphosphate; formed by a base sugar and three phosphates. It is a high energy molecule. Breaking of phosphate bonds releases energy
A-P~P~P-->A-P~P+P+Energy

Making of P~P bond

requires energy
ADP+P+energy--->ATP

ATP stores

energy and provides energy when needed. It is considered to be energy currency (money)

Metabolism=

catabolism+anabolism

Catabolism

larger molecules are broken down with release of energy into smaller molecules

Anabolism

smaller molecules use energy to build larger molecules and requires energy

Pathway

series of chains of reactions

Oxidation/Reduction

are critical in metabolism

Oxidation

is the loss of an electron by a molecule

Reduction

gain of an electron

OIL RIG

oxidation is lost
reduction is a gain

Redox reactions must occur in pairs

because electrons cannot exist ALONE in water.

Oxidation/Reduction may also involve the transfer of

hydrogen

Oxidation (Hydrogen)

loss of hydrogen (or gain of oxygen)

Reduction (hydrogen)

gain of hydrogen

Metabolic pathways are carried out by

enzymes

Enzymes

allow reactions to occur at physiological temperatures, speed up reactions (catalysts), all are mostly made of protein (sometimes RNA), specific for molecules on which they act, and can be recycled

Enzymes facilitate reactions by

lowering the "activation energy"

Energy hurdle

normally must heat mixture to start a reaction

Lowering the activation energy allows

reactions to occur at physiological temperature by enzymes.

How enzymes work

1. Substrate-the molecule on which the enzyme acts
2. Active sites-specific for shape of substrate
3. Action: cuts or joins substates
4. Produces end products and enzymes are recycled

Enzyme activity is affected by:

temperature, pH levels and substrate concentration.

In catabolism, glycolysis is where

glucose is broken down
many steps...ATP input (energy investment)
Input: 2 NAD (Electron carrier)

Each glucose molecule leads to net-production of:

2 NADH (Reduced NAD)
2 ATP
2 Pyruvic Acids

Glycolysis

converting glucose to pyruvic acid. NAD (oxidized form) is used to make NADH (reduced form). It needs NAD back to keep glycolysis going

How do we get NAD back?

fermentation or respiration aka reactions that release/require energy

Fermentation

Glucose
NAD ->NADH | ADP -> ATP
Pyruvic Acid
NADH-> NAD |
Lactic Acid
In this process, pyruvic acid is reduced to lactic acid (or other acids/alcohols)-with the help of NADH which loses H to yield ATP. Thus, NAD is available again and glycolysis can go on...

Fermentation allows

microbes to grow in the absence of oxygen

Fermentation is inefficient

compared to respiration (2 ATP yield per glucose vs. 38 for prokaryotes)

But growth under fermentation

can be fast and plentiful IF substrate (e.g. sugar) is not limiting

When does fermentation occur?

it doesn't happen everytime there is glycolysis. Facultative anaerobes may undergo respiration or fermentation depending on oxygen availability. Only some organisms are facultative; others are either aerobic/anaerobic.

Types of Fermentation

Depending on type of bacteria, it may yield
Lactic acid: Homolactic
Lactic acid + other acids: heterolactic
Alcohols: alcoholic

End products of metabolism

give "personality" to species. Important for identification of bacterial: Medical diagnosis.

Respiration

breakdown of glucose to CO2 and H2O which gives a maximum release of energy
Glucose
|
Pyruvic Acid
|
Krebs Cycle
|
Electron Transport
Chain
ATP production=substrate level phosphorylation

Krebs cycle

Pyruvic acd converted to Acetyl Co-A. Acetyl Co-A enters the krebs cycle: 4-6 carbon molecules; undergoing oxidation.
Products: NADH, FADH2, ATP, CO2 (by product)

Key products of the Krebs cycle are fed into the...

Electron transport chain

Electron transport chain

electrons and protons from NADH and FADH2 are transported via a series of REDOX reactions. Electrons are lost/gained in redox reactions. Final electron acceptor is O2 (usually

NADH e- negative redox potential
from Krebs -----> | Redox reactions in the
Cycle ----> H | electron transport chain
FADH2 |
Protons<------| Positive redox potential
O2 final electron
acceptor 1/2 O2 + 2H+ 2e -----> H2O

Electron transport

occurs in the cell membrane (bacteria). Electrons move along the membrane via redox reactions

What governs the electron transport process?

redox potential of reactions and position of proteins in the membrane

Results of the electron transport chains

Protons (H) moved across the membrane to the outerside
Hydroxyl ions (OH-) accumulate the inner side of the membrane
Formation of charge gradient across the cell membrane: "battery"
Energy is released when protons move back in a process called chemiosmosis!

Charge gradient holds energy

Proton motive force (PMF)

PMF is used to

make ATP from ADP for membrane transport, for movement (flagella)

NADH

molecule that contains a lot of potential chemical energy

Catabolism outline

glucose broken down via: glycolysis/(fermentation), Krebs cycle, electron transport chain
to yield: PMF: ATP (oxidative phosphorylation)
Transport motion
Enzymes and special proteins are essential for key steps

Anabolism

biosynthesis, building up, requires energy (ATP)
ATP--->ADP+ P exergonic
energy
D+E------>F endergonic

What is a PRECURSOR?

in metabolism, these are a substance from which another more mature substance is formed (opposite of breakdown product)

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