Exam 1 Microbiology NWMSU

Study of Archaea

Microbiology

Robert Hooke

First Microscope; identified cells

Antonio van Leeuwenhook

More powerful microscope; identified bacteria drew pictures of microbes

Edward Jenner

Vaccination

Lazzaro Spallanzani

wants to disprove spontaneous generation

Sergei Winogradsky

father of soil microbiology known for his growth chambers, and grew first phototrophic organisms

Martinus Bejerink

proposed that many microbes were ubiquitous (everything is everywhere the environment selects)

Robert Koch

proved that microbes cause disease: Postulates: 1. Microbes present in sick organism must be isolated in pure culture (recognize it on petri dish). When introduced to healthy organism disease is initiated. Reisolate. Demonstrates bacterium can cause disease. Later discovered what caused TB.

Joseph Lister

utilized germ theory in medicine treated medical tools with phenol acid to sterilize them

Louis Pasteur

disproved spontaneous generation by placing nutrient in a long swan neck flask and heated it until all the bacteria were evaporated out. The swan neck of the flask prevents bacteria from getting in.

Alexander Fleming

discovered penicillin while trying to grow bacteria on plates, but the fungal contaminate (penicillium notatum) prevented the bacteria growth

Carl Woese

Founded that there are 3 major domains of life
1. Bacteria
2. Archaebacteria (now called Archaea)
3. Eucarya

What are the 2 major requirements for growing microbes?

Chemical (nutritional)
Physical (environmental)

Nutrient agar

(complex) (NA) bacterial growth

What category of medium does each fall into?

Broth (liquid medium)

Agar/Solid (petri dishes, slants)

What is agar?

Complex polysaccharide from marine (red) algae

Why are microbial cultures incubated at different temperatures?

Cells grow best at their optimum temperatures.

What is the typical size of a bacterial cell?

1.0 micrometers (length), 0.5 micrometers (width)

-Light microscope

(series of lenses, binocular head)

Brightfield:

light directly moves to specimen

Darkfield:

light blocked by a disc and reflects off specimen

Phase-contrast:

light refracts through a specimen; can see internal structure

Differential interference contrast

a prism splits the light into two beams at once; image appears nearly 3-D

Fluorescence

uses some type of fluorescent molecule to light up specimen; can label a cells DNA, protein, etc. if it contains fluorescent genes

Transmission Electron Microscopy(TEM)

beams of electrons gives you details of the internal structure of the cell

How do physical requirements (pH, temperature, oxygen, etc.) impact a microbe?

Distorts the cell whenever it is not in the correct environment.

What are the names of each category of organisms for temperature?

Psychophile: (-10-15C),
Psychrotroph (0-30C),
Mesophile (20-40C),
Thermophile (40-70C),
Hyperthermophile (70C and greater).

What are the names of each category of organisms for Oxygen?

Aerobes
Obligate-Oxygen: love oxygen, need it,
Facultative aerobe: can grow with or without it, but prefers it,
Microaerophile: small amounts of O.
Anaerobes
(Obligate: can't have O),
Aerotolerant anaerobes: doesn't need O, but O doesn't make it grow better.

What are the names of each category of organisms for Osmolarity?

Hypertonic,
hypertonic.
Lysis: cell explosion, plasmolysis: crenation,
osmotic
extremophiles: (high salt concentrations, Halomonas aquamarina), (high sugar concentrations, Aspergillus penicillioides).

What are the names of each category of organisms for pH?

Which elements are needed by a culture to grow successfully?

Carbon 50%
Oxygen 20%
Nitrogen 14%
Hydrogen 8%
Phosphorus 3%
Potassium 2%
Sulfur 1%

Which macromolecules are needed by a culture to grow successfully?

Carbohydrates
Proteins
Lipids
Nucleic acids.

Which elements are provided by each macromolecule?

Carbon=carbs, proteins, lipids, and nucleic acids.
Nitrogen=proteins and nucleic acids.
Sulfur=amino acids and vitamins.
Phosphorus=nucleic acids, ATP, and lipids.

How does a microbiologist supply a culture with each macromolecule?

Using media (liquid or broth), agars (petri dishes, slants)

What categories of culture media do microbiologists use?

Chemically defined medium: all components are known
Complex medium: components unknown
Live medium: when using one type of cell to grow another type of cell (mycoplasm replicate under live cells)

What occurs on a cellular level as a culture of bacterial cells grow?

Cells multiply and increase in number

What is aseptic technique, and why is it important?

Aseptic technique is a process of doing something without Contaminated yourself, the environment, and surroundings. It is important so that a culture stays pure and surrounding environment uninoculated.

What are the purposes in using broth, agar, slants, Petri dishes, etc?

-broth-bacteria suspended,
-petri dishes - streak plate can isolate cultures agar
-slants-solid, less expensive convenient for storage and small scale experiments

What is the difference in a pour and spread plate? Why is each used?

Pour plate: after inoculating a plate, liquid agar is poured over the culture. The culture grow within and on top of the agar. Easy to count, then toss (specific purpose)
Spread Plate: inoculum is spread on a surface of a solidified agar plate, the culture grows on top of agar. (can count and can collect colonies)

What are the major atomic structures?

Protons, neutrons, and electrons

What are the different types of chemical bonds that affect biological organisms?

Hydrogen Bond, covalent bond and ionic bonds

Why is water so important to biological organisms?

-Water acts as a buffer to control pH
-stabilizes body temp.
-Dissociates ions, and it is the medium of transport for many ions and chemicals.
-It is 60%- 80% of body cells.

What is the difference between organic and inorganic molecules?

Organic molecules have carbon and hydrogen ; inorganic molecules don't have both together

How are large, multi-unit macromolecules constructed? How are they broken apart?

Constructed through dehydration synthesis and broken apart by hydrolysis

Carbohydrates

Structure - made of C, H, O (sugars)
Differences
2:1 ratio b/w H and O
Uses - building block of DNA, in cell walls, used for energy

Proteins

Structure - made of amino acids; C, N (O, S)
Differences
Different structures (primary, secondary, tertiary, quaternary)
Uses - catalysts, enzymes, transporters, ATP, involved movement of body, immune system (antibodies)

Lipids

Structure - made of C, H, and O; glycerol molecule attached to fatty acid tail (nonpolar); can contain P, N, and S
Differences
Lack the 2:1 ratio b/w H and O
Uses - energy, osmotic barrier

Nucleic Acids

Structure - C, H, N, O, P; made of nucleotides (N-base, pentose (5 carbon) sugar (deoxyribose or ribose), phosphate group)
Differences - DNA(genetic material) and RNA(protein synthesis) (bases A,T, G, U; RNA has uracil instead of thymine)
Uses - contain genetic material

Why do we see variation in responses to temperature, osmotic pressure, O2, pH, etc?

Different bacteria have different chemical and environmental requirements.

How would you determine the O2 requirements of a bacterium? What types of media and **cultivation would you use?

Rich media, undefined vs. chemically defined, selective (like evolution)/differential (look different by eye)
How would you interpret the results? Agar deep stabs (TSA deep) made with tryptic soy agar (TSA) and gas pack jar (controls oxygen environment).

How do anaerobic jars and thioglycollate media function? How do you interpret their results?

Oxygen is removed during preparation of the medium and then oxygen enters the medium whenever the agar cools and solidifies thus creating an oxygen gradient ranging from aerobic at top to anaerobic to bottom.
After a TSA deep is inoculated with microbes wherever the microbe grows best tells about the microbes desired oxygen.

How are most microbiological media sterilized? Why?

For a test tube, we would hold it at an angle and pass the tip of the tube through a Bunsen burner flame once or twice quickly. This reduces airborne contamination. Using the autoclave, 121C, 15psi, 15 minutes.

Selective Media

suppress growth of unwanted bacteria and encourage growth of desired microbes

Differential Media

allow many microbes to grow, but will change in appearance due to biochemical properties

Fastidious

Microbes are those that require external growth factors, such as certain metals, vitamins, blood. etc

Bacteria and Archaea reproduce asexually though _______?

binary fission

Generation time

The time required for a cell to divide (and its population to double)

What are the 4 phases for bacterial growth

Phase 1- Lag phase ( adjusting to medium, metabolism is "ramping up", making enzymes)
Phase 2- Log phase ( Fastest growth)
Phase 3- Stationary phase ( growth rate slows, nutrients are depleted, metabolic waste builds up)
Phase 4- Death phase

Eukaryotes

-have nucleus
-membrane bound organelles
-cell division is complex; no cell wall;
-genome (DNA) - enclosed in nucleus, broken into multiple chromosomes

Prokaryotes

-don't have nucleus
-no membrane bound organelles
-cell division is simple
-cell wall (PG),
-genome (DNA) - circular, one molecule

Morphology

Spherical = coccus (pl. cocci)
Rod = bacillus (pl. bacilli)
Curved rod = vibrio
Club = coryneform (one end is bigger than the other)
Helical/drill shape = spirillum (pl. spirilla)

Monomorphic

Consistent cell morphology (uniform)

Pleomorphic

Inconsistent cell morphology

Morphology Arrangement

Single cells = solitary
Doubles = diplo + cell morphology
Chains = strepto + cell morphology
Irregular clusters = staphylo + cell morphology
Four (one plane) = tetrads
Four (two planes) = sarcinae

Flagella

long, filamentous appendage used to propel a bacterium; for motility and taxis (chemotaxis - finding nutrients, phototaxis - finding light)

Flagellum Filament

formed from intertwined chains of flagellin protein and hallow core

Basal Body

Anchor flagellum to cell (spins like a propeller powered by proton motive force)

Fimbriae

very short hair like protein fibers; involved in adhesion and flagellum-like

Pili

very very thin, longer than fimbria, made of pilin; involved in twitching motility/gliding (do one or the other), involved in DNA exchange in conjugation (one cell makes pilus, sends tube to other cell, send single strand dna to reciprocating cell to incorporate to genome)

How does a bacterial cell move?

Run and tumble - bacteria, trying to find something (nutrients or light); kick all flagella into gear for a few secs, then go limp like a parachute to sense surroundings; short choppy motion

Cell Wall

complex, semi rigid structure surrounding the cells cytoplasmic membrane

Peptidoglycan

Only found in Bacteria cell wall made up of NAG and NAM ( carbohydrate backbone)

What holds together peptidoglycan cables NAM-NAM?

Polypeptide links

What other bond besides Polypeptide holds together petidoglycan?

glycosidic bonds

2 ways the cell wall can be broken down?

Lysosome- (saliva, tears) breaks the linkages between NAM and NAG
Penicillin- Blocks cell from reforming after cell breaks apart to elongate.

Characteristics of a Gram-positive cell wall

1. Thick layer of petidoglycan covering cytoplasmic membrane
2. teichoic acids and lipoteichoic acids
3. TA link to PG, LA link to plasma membrane
4. negatively charged
5. Can contain waxes (mycolic acids) that make cells acid-fast.
form a layer on top of PG
waxes repel water

Characteristics of Gram negative cell wall

1. Thin layer of PG and outer membrane linked by lipoproteins
2. this leaves a zone called the periplasm (filled with enzymes)
3. No teichoic acids
4. Outer membrane contains the lipopolysaccharide (LPS)
5. LPS replaces most of the phospholilipids in outer portion of the outer membrane

What are the differences between Gram-positive and Gram-negative cell walls?

Gram-Positive: many layers of peptidoglycan (thick, rigid structure), also contains teichoic acid (consists of alcohol and phosphate). Gram-Negative: consists of one or a very few layers of peptidoglycan and an outer membrane, do NOT contain teichoic acids.

How do different cell walls impact the bacterium and other structures?

NAM and NAG (which would be linked by NAM-NAM, etc.)
Gram-positive - 1 basal body
Gram-negative - 2 basal bodies in a flagellum

Which bacteria differ from "typical" cell wall structures?

Acid-fast (mycobacterium) - made of mycolic acid, makes difficult to stain and toxins to penetrate. Downside, difficult to grow and get nutrients; gram positive
No cell wall (mycoplasma) - gram positive because evolutionary history, doesn't stain gram positive; have sterols (other bacteria don't have), makes them sturdier
Bacterial endospores - wrap up DNA in PG, gram positives only

Which functions are carried out by a cytoplasmic membrane (CM)?

Energy production (ATP produced); transport (metabolism - ATP), DNA replication; like a workbench

What is the structure of the CM? How is it constructed?

A lipid bilayer consisting of a polar (phosphate group and glycerol) head and fatty acid tail.

Some cells (e.g. photosynthetic bacteria and archaea) have altered CMs. How are they altered?

Photosynthetic bacteria have thylakoids (folds membranes in, out and in over and over) and chromatophores (light harvesting pigment toward outside edges of cell). Archaea have different phospholipids (the attachment of the heads to tails can differ, but it doesn't affect how it acts); their attached with ether linkage, bacteria and eukaryotes attached with ester linkage. Bacteria only have lipid bilayer (archaea can have two heads at one end).

How do molecules (waste, nutrients, etc.) enter/exit the cell? How are they transported?

Selective permeability/semipermeable (large and charged molecules can't pass through the CM very well unlike small and uncharged molecules); passive transport - requires no energy (simple diffusion - movement down a concentration gradient, facilitated diffusion - helps some molecules down a concentration gradient); active transport - uses energy/ATP to move substances in/out of cell (for ions, amino acids, and sugars)

What linkage is associated with Bacteria and Eukaryotes?

Ester Linkage

What linkage is associated with Archaea?

Ether Linkage

What do you find in bacterial cytoplasm? How is it organized?

Can find lipids, carbohydrates, protein (enzymes), nucleoid (DNA chromosome), RNA, ribosome (translate RNA -> proteins), inclusion bodies (for storage), and iron oxide/magnetosome (fights peroxide), endospore. Unorganized free floating.

What are the differences in prokaryotic and eukaryotic ribosomes?

Prokaryotic: smaller, less dense, 70S ribosomes.
Eukaryotic: bigger, more dense, 80S ribosomes.

Why do bacteria produce endospores? Which bacteria produce them?

For survival (desiccation - very dry, low nutrients, anything that makes the cell unhappy)
Bacillus subtilis, Clostridium sporogenes, and about 100 genera (gram-positive cells, as of now).

What are some characteristics of endospores that are useful for identification?

Spore position (central-middle, terminal-end, subterminal-between middle and end), spore shape (spherical, elliptical), effect cell's shape (mother cell non or swollen)

Gram stain

crystal violet(primary stain), iodine(mordant/fixant), EtOH (decolorizer) > safranin (counter stain)

Capsule Stain

nigrosin and air dry and look at it through oil immersion

Endospore stain

malachite green > water > safranin (secondary stain)

Crystal violet

primary stain

Iodine

mordant/fixant

Ethanol

decolorizer
Negative - rips outer membrane off cell; dehydrate PG layer; crystal violet spills out of cell (like poking holes in), becomes colorless
Positive - Dehydrates PG until drawn tight, traps crystal violet in cell; stays blue

Safranin

secondary stain
positive still appear blue because it covers red; negative sucks up red stain

Know the theory of each technique.

In Gram Stain the thickness of the peptidoglycan determines if the primary stain will be retained after the alcohol rinse. Cells that have thick peptidoglycan are gram positive and cells that have thin peptidoglycan are Gram negative.
Capsule stain: The capsules don't take up stain because they are non-ionic (glycocalyx); so in this method the background is stained.

gram positive

Blue cell

gram negative

Red cell