Wednesday Notes

Microbial Nutrition & Growth

Elements Essential for Growth:

  • Carbon
  • Oxygen
  • Nitrogen
  • Hydrogen
  • Phosphorus
  • Sulfur
  • + Magensium, Calcium, Iron, Zinc (co-factors for enzymes)
  • + some other trace elements → used in “membrane pumps” (proteins that transfer ions. Also, used to maintain water balance.)

Oxygen Requirement Terms:

Obligate aerobes – must have oxygen in order to conduct metabolic RXNs, such as respiration. (These would be found at the top of a broth tube)

Obligate anaerobes – must live in an environment w/ no oxygen in order to conduct metabolic RXNs (ex. Clostridium botulinum)

Facultative anaerobes – prefer/live best in aerobic environments but can maintain metabolic processes in anaerobic environment. (ex. yeast)

Aerotolerant anaerobes – same as obligate anaerobes but oxygen doesn’t kill them.

Microphiles – Requires a little oxygen.

Toxic Form of Oxygen:

Singlet Oxygen 1O2 : Molecular oxygen that has been boosted to a higher energy state making it extremely reactive.

Superoxide Radical O2 : formed in small amounts during aerobic respiration, highly toxic.

Peroxide anion O2 2-: Produced during RXN beneath. The Superoxide Radical O2 acts as the catalyst for the reaction.

\mathrm{2\ O_2 ^-\ +\ 2\ H^+\ \xrightarrow {SOD}\ \ H_2O_2 +\ O_2}
Formation of hydrogen peroxide by superoxide dismutase (SOD)

Hydroxyl radical: OH

Important enzymes that react with the toxic forms of Oxygen listed above, to detoxify the Oxygen radical/ion.

(Note: Brahce said to focus more on the enzymes that react with the oxygen’s rather than the oxygen structures)

  1. Superoxide dismutase: Detoxifies superoxide radical
  2. Peroxidase: Detoxifies hydrogen peroxide (H2O2) and Superoxide Radical (O2 2¯)
  3. Catalase: Also helps the superoxide dismutase remove the superoxide radical by catalyzing the reaction for peroxide anion. (confusing)

Organism Temperature Requirements:

  1. Psychrophiles – “Cold-loving” (near freezing temps.)
  2. Mesophiles – “Middle-temp-loving” (room temps)
  3. Thermophiles – “Heat-loving” (Hot environments)
  4. Hyperthermophiles – “Extreme-heat-loving” (near boiling temps)

Other Requirements for growth:

pH: most human pathogens grow best around neutral pH (buffers added to growth media to maintain pH)

Note – Fungi grows best in somewhat acidic conditions (6-7 pH range)

Osmotic pressure: most human pathogens grow best in isotonic conditions exceptions include Staphylococcus

◊ Growth of a bacteria population is exponential: meaning as time continues so does cell growth.

Bacteria Life Phases:

Lag Phase: bacterial cells are preparing for growth by replicating chromosomes & making extra cell material (peptidoglycan, etc) for splitting in 1/2 (binary fission)

Log Phase: most of the growth occurs; binary fission occurs (1 cell → 2 cell)

Stationary Phase: amount of cell reproduction is equal to the amount of death.

Death Phase: # of cells dying is > (is greater) than # of cells being produced (this is due to lack of nutrients and waste buildup)

 

 

 

Advertisements

Tuesday Notes

Eukaryotic Cells…

Eukaryotic cells w/ walls:

  • plants (cellulose)
  • algae
  • fungi (chiten)

Membranous Organelles of Eukaryotic cells:

Nuclear envelope – Has 2 membranes w/ pores for communication

Cytoplasmic membrane- composed of phospholipid bilayer

Endoplasmic Reticulum – Rough & Smooth

  • Rough – function is to synthesize (make) proteins & enclose them in vesicles to send out
  • Smooth – makes lipids that help repair cells, also to be sent out in vesicles

Golgi Body – “shipping center” where proteins & lipids get modiified & sent to other locations

  • Cis – “front” location where vesicles are recieved
  • Trans – “back” where vesicles are secreted by the cell enclosed in another membrane.

Mitochandria – respiration …makes ATP (I may have missed some stuff here)

  • Has outer membrane
  • has an inner membrane that is folded into structures called cristae
  • 70s ribosomes, 1 circular chromosome, divides by binary fission

Chloroplasts – Found in plant cells and algae cells (photosynthetic organisms) as the dark green color

  • thylakoid membranes stacked into grana (dense areas)
  • thylakoid contain photosynthetic pigments
  • 70s ribosomes, 1 circular chromosome, divides by binary fission

Free ribosomes – make (synthesize) proteins that will remain in the cell

 

Membranous Sacs:

  1. Vesicles
  2. lysosomes (in animal cells) – digest cell waste
  3. vacuoles (ex. central part of plant) – stores water in plant cells or a contractile vacuole which maintains the water level coming in by pushing out excess  preventing the cell from bursting do to hyper or hypotonic conditions.
  4. peroxisomes – contain enzymes that degrade metabolic wastes such as H & O

 

Eukaryote External Structures: (Not all eukaryotic structures have these structures)

Glycocalyx Functions: (Not part of cells that have a cell wall)

  1. Helps evade the immune system
  2. transmit messages (think biofilms)
  3. helps to adhere to eachother &/or inanimate objects. 

Cytoplasmic membranes –

  1. Some contain steroid lipids such as cholesterol
  2. Most have sugars attached to cell surface : glycolipids (attach to head of phospholipid) or glycoproteins (attach to protein channels) – One function of glycolipids and glycoproteins is self-recognition

Flagella – 

  • Surrounded by cytoplasmic membrane
  • composed of microtubules in a 9+2 arrangement (microtubules contain protein called tubulin)
  • moves in an undulating (wave-like) fashion

Cilia – Short “hair-like” projections, used for motility. Also have 9+2 arrangement of the microtubules (move like oars)

(Prokaryotes do not have cillia)

 

Vesicular Transport

Exocytosis – secretion of material from inside to outside of eukaryotic cells in a vesicule

Endocytosis –  bringing in material from outside to inside the cell in a vesicle.

  • Phagocytosis – “to eat” (larger of dry materials)
  • Pinocytosis – “to drink” (liquids)

Nonmembranous Organelles

Cytoskeleton – composed of various sizes/shapes of protein filaments /tubules for movement & structure

Ribosomes – composed of polypeptides + rRNA & make protein

Centrosomes – Area near nucleus involved in forming the mitotic spindle during mitosis.

Centrioles – found in animal cells withing the centrosome area; bundles of microtubules that are perpendicular to one another.

 

Microbial Growth

Photoautotrophs

  • Plants, algae, cyanobacteria use water to reduce carbon dioxide producing oxygen as a biproduct
  • Green sulfur bacteria & purple nonsulfur bacteria, some archaea

Chemoautotrophs

  • Hydrogen, sulfur, & nitrifying bacteria, some archae

Photoheterotrophs

  • Green nonsulfur bacteria & purple nonsulfur bacteria, some archae

Chemoheterotrophs

  • aerobic respiration, most animals, fungi, protozoa, bacteria & archae

Taxonomy & Images of Organisms

Cyanobacteria…

Domain: Bacteria

Kingdom: N/A

Phylum: Cyanobacteria

 Ocillatoria

Possesses trichomes

Oscillatoria

 Nostoc

Also has trichomes, & a glycocalyx slimelayer

Nostoc

Genus: Gloeocapsa

Single cells with glycocalyx caspules

Gloeocapsa

Genus: Anabaena

Anabaena

Genus: Spirulina

SpirulinaSpirulina

 

 

 

 

 

 

 

Special Structures –

Heterocysts – Contain enzymes for conducting nitrogen fixation. Nitrogen fixation is the process of bacteria capturing N in gas form w/ enzymes in their body.

Akinetes It serves as a survival structure. It is a resting cell of cyanobacteria and unicellular and filamentous green algae.[2] Under magnification, akinetes appear thick walled with granular-looking cytoplasms.

Trichomes – Stacks of cells

Fungi

Eurkaryotic Cells

Most are multicellular but some are unicellular

Nutrition – Release digestive enzymes into environment & then absorb nutrients from broken down organic material.

3 Major groups:

  1. Molds (filamentous)
  2. Fleshy fungi
  3. yeasts (unicellular)

Domain – Eukarya

Kingdom – Fungi

Phylum – Oomycota

Albugo bliti

Albugo blitiAlbugo bliti

Sexual oospores

&

sporangiospores

Saprolegnia

Saprolegnia Saprolegnia

1st image – oogonium w/ sexual oospores

2nd image – asexual sporangiospores

Phylum: Zygomycota

Rhizopus stolonifer

Rhizopus stolonifer – sporangium w/ asexual sporangiospores

Rhizopus stolinifer –  Sexual zygospore

Wednesday Notes

Lecture….

Bacteria: External Structures

Flagella: Long; used for motility

Fimbriae: Used to attache to other cells & inanimate objects

Pili (penis-ish) – Exchanges DNA

Flagella:

Flagellum (latin for whip)

3 Parts

  1. Filament – Composed of a protein called flagellin
  2. Hook – Base of filament near cell wall
  3. Basal Body – Anchors filament & hook to cell wall

Rotates: 360° turns clockwise (tumbles) & counterclockwise (runs)

Arrangements:

  • Petritrichous – flagella cover entire surface
  • Polar – Flagella @ one or both ends. Possible more than one.
  • Axial Filaments (AKA Endofilaments)- flagella at both ends that spiral around cell between cytoplasmic membrane & outer membrane

Taxis (Movement): Bacterial motility in response to environmental stimuli

Positive: Movement toward stimulus 

Negative: Movement away from stimulus

Stimuli:

  • Light – Phototaxis
  • Chemicals – chemotaxis

Bacterial Cells: Fimbriae & Pili the “penis”

Functions:

  • Fimbriae – Attache to other cells & inanimate objects
  • Pili –  Exchanges genetic information. Composed of pilin (protein)

F + … male cell

F – …female cell

Bacterial Cell Walls:

Walls made of peptidoglycan…

Structures:

  • Complex polysaccharides
  • Alternating monosaccharide molecules in chains bonded together by polypeptides
  • N – Acetylglucosamine (NAG) & N – Acetylmuramic Acid (NAM) these alternate

Tertapeptide holds vertical layers & amino acids connects horizontal layers. (look this up, this a poor explanation)

Gram Positive Cell Walls vs. Gram Negative Cell Walls

Gram-Positive cell walls:

  1. Cytoplasmic membrane
  2. multiple layers of peptidoglycan
  3. teichoic acid (contained in the peptidoglycan)
  4. acid gives cell wall negative charge

Gram-Negative Cell Walls:

  1. Cytoplasmic membrane
  2. Few layers of peptidoglycan
  3. Outer membrane (phospholipids) – Contains lipo polysaccharieds & a substance called Lipid A which triggers immune system because it is toxic to mammalian cells

Bacteria Cells: Cystoplasmic Membranes

Structure:

  • Bilayer of phospholipids
  • Proteins: Integral (around); peripheral (inside)

Functions:

  • Semi-permeable membrane (some molecule can pass through, not all)
  • Protective
  • Fluid/semi-fluid

No lab notes today.

Tuesday Notes

Lecture

Nucleic  Acid

General Functions:

  • Genetic Information storage (DNA)
  • Gene Expression (makes product of instructions) (RNA)
  • Energy (ATP)

Contains C, H, O, P

RNA vs. DNA

RNA

DNA

  • Uracil
  • Ribose
  •  Thymine
  • Deoxyribose (de-oxygenated)

Nucleotides (Monomers) – A single nucleotide contains ….

1 phosphate group + 1 five-Carbon sugar + 1 nitrogenous base

Nitrogenous  Bases: Adenine, Thymine (DNA only), Guanine, Cytosine, Uracil (RNA only)

Polynucleotides (Polymers) – a biopolymer composed of 13 or more nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with distinct biological function

  • Phosphodiester bonds- In DNA and RNA, the phosphodiester bond is the linkage between the 3′ carbon atom of one sugar molecule and the 5′ carbon atom of another; the sugar molecules being deoxyribose in DNA and ribose in RNA. (This explanation is slightly confusing…essentially this is the bond the forms between each nucleotide linking them together to form polynucleotides)
  • H – Bonds – Formed between the “teeth” of the polynucleotides connecting one to another polynucleotide to form a helix of DNA. H-bonds allow for DNA to split easily.

ATP – energy molecule for cells, this energy is released when a phosphate group breaks away.

As each phosphate group breaks away it becomes ADP and then AMP (the middle letter stands for the quantity of phosphate groups)

Overview of Cells

Cell Theory:

  • All living things are made of cells Cells are the smallest (basic) units of life. (Theordore Shwann & Jakob Shleiden, 1839)
  • All cells come from preexisting cells. (relates back to biogenesis theory; Rudolph Virchow, 1855)

* = bacteria differs from Archaea

ARCHAEA

BACTERIA

EUKARYA

No True Nucleus No True Nucleus Nucleus
No Membranous Organelles No Membranous Organelles Membranous Organelles
Hami (cell extension) *No Hami No Hami
Cell walls (most) have no peptidoglycan *Cell walls contain peptidoglycan Some contain peptidoglycan (cellulose, chiten)
No Endospores *Endospores (some) No Endospores
70s ribosome’s(s= Svedberg unit; sedimentation rate)

80s ribosome’s (except mitochandria & chloroplasts = 70s)

Single circular chromosome        Single circular chromosome         Most have multiple linear chromosomes.

Bacterial Cells: External Structures

Glycocalyx – Gelatinous material composed of polysaccharides &/or polypeptides

2 Forms:

  • Capsule – Well organized & firmly attached to the cell.
  • Slimelayer (Biofilms) – Loose, less organized & NOT firmly attached to the cell.

Lab

We covered:

Endospore stain

This is a structural stain.

  • Endospore – Small dormant cells produced by many species of bacteria that are super resistant to heat, dry conditions, & toxic chemicals.

Rules of endospores:

  1. Produced in response to stressful environmental conditions (such as those I listed above)
  2. ONE endospore is produced by each cell; location will be central or terminal.
  • Terminal – Will be found at the head/tail of the bacteria (inside)
  • Central – In the center (hence, central) of the bacteria (inside)
  1. Endospores resist staining; but retain the stain once it binds to the cells.

Overall point of this stain is to determine whether your organisms produces endospores or not. We mainly saw exospores today because our samples were too old. Meaning our bacteria cultures had been stressed a few days ago, they created endospores and then released them outside the cell creating exospores. Our cells were vegetative, our exospores & endospores were dormant small dormant cells waiting for good conditions again to blossom in.

Endospore Stain Process…

Stains used:

  • Malachite Green -The green is used to color the endospores, so if you had endospores they should have been green in your slide!!! We reapplied water as necessary not to dry out our paper towels.
  • Safranin – COUNTERSTAIN – colors your vegetative cells pink.

Steps:

  1. Water + Culture smear
  2. Air dry & heat fix (not too much)
  3. Steam
  4. Apply Malachite Green drops to paper towels. (endospore staining)
  5. Keep moist with drops of water as necessary for 15 minutes!
  6. Rinse slide (decoloration step)
  7. Counter stain with Safranin, 60 seconds. (cell staining)
  8. Rinse slide & air dry

Capsule Stain

Capsule Stain: Structural Stain

Capsules – Highly organized structures made of glycocalyx. Capsules protect some of these bacteria from being destroyed by a host’s immune system.

Glycocalyx – Gelatinous material composed of polysaccharide &/or polypeptides. Ex. slime on the outside of a fish is considered a glycocalyx.

Glycocalyx comes in 2 forms:

  1. Capsule – This is the highly organized structure we are testing for.
  2. Slimelayer – Another form (biolfilm) not being tested.

Capsule Stain Process… (non-heat fix)

Stains/Chemicals used: Nigrosin; Isopropyl alcohol; Crystal violet

  1. Prepare smears w/ water & bacteria
  2. While wet add Nigrosin
  3. Air dry
  4. Add Isopropyl acohol; 2 min – this step fixes (kinda) the cells to slide
  5. No rinse…Air dry
  6. Crystal Violet for 2 min
  7. GENTLY rinse with water
  8. Air dry

Results: Cells will be purple and capsules (if present) will be a clear area around cells

Here goes quiz 3…. anyone exhausted yet? I am. I will try to get the practice questions up tonight for quiz tomorrow.

Monday Notes

Lecture

Found a site w/ good pictures http://www.chemistar.com/Biology/organicmolecules.html

Lipids

General Functions:

  • Provide energy (9 cal/gram compared to carbs & proteints at 4cal/gram)
  • Provide structure (ex. cell membranes)
  • Some are hormones. (ex. estrogen, testosterone)

Contain C, H, O

Sources – Fats, oils, waxes, phospholipids (part of cell membranes), steroids.

Fats  are a subgroup of Triglycerides – a typical triglyceride molecule is found in oils (plants) & fats (animals).

ex.      1 glycerol + 3 fatty acids = 1 triglyceride (ester bond- connects glycerol to fatty acids)

Subgroups of Lipids…

Fats:

Saturated: maximum # of Hydrogen attached to each carbon. (no double bonds between Carbon atoms)

Monounsaturated: has only one double bond in the chain. Therefore, fewer Hydrogens & a kink in the chain. (Oleic Acid)

Polyunsaturated:2 + double bonds in the chain. Also, has less Hydrogen & more kinks in the chain. (Linoleic Acid)

Phospholipids:

1 glycerol + 2 fatty acids + 1 phosphate group + 1 organic functional group = phospholipid

Phosphate groups – Interacts with water; because the phosphate head is polar.

Fatty acid ‘legs’ – Non-polar, do not interact with water and align themselves together to be protected from the water while exposing the phosphate groups to the water.

Steroids:

  • Contain 4 fused rings
  • Include cholesterol  (found in cell membranes of animals)

Proteins

General Functions:

  1. Structural
  2. Involved in cell communications (ex. hormones)
  3. Catalysts for chemical RXNs (enzymes)

Contains C, H, N, O (S)

Protein Monomers: Amino Acids – Single amino acids contains a central carbon, carboxyl group(COOH), amino group (NH2) & side group (R-group).

  • R-group: Glutamine; Serine

Polypeptides (Polymers): 2+ amino acids linked together.

  • Peptide Bonds- Protein covalent bonds
  • DNA provides the instructions to make (LARGE CHAINS) proteins; they are made inside the cell linking one amino acid to another.
  • Each protein has a unique combination of amino acids
  • 20 different types of amino acids formed in living things.

Levels of Protein Structure:

  1. Primary – The specific order of amino acids in a polypeptide chain.
  2. Secondary – Hydrogen bonds form between the carboxyl groups & amino groups. This stage forms β -pleated sheets & α-helices throughout the polypeptides.
  3. Tertiary – Additional bonding occurs involving R-groups to make simpe proteins (one peptide chain) functional.
  4. Quaternary – Additional bonding occurs between more than one polypeptide chain to make functional protein (complex)

Ex. of importance of primary structure:

  • Prion that causes Scrapie (sheep) : Alanine is substituted for valine and #136 resulting in a mis-fold of proteins. 
  • Sickle Cell Disease (humans): Valine gets substituted for glutamate @ # 6  in hemoglobin. Valine does not interact well w/ water causing it to crystalize and form a deformed cell.

Protein Denaturation – The loss of tertiary or quaternary structure, resulting in a loss of function. Causes are high heat, alcohol, some metals and/or acids.

Extra Credit: Smith Lemli Opitz Syndrome – an autosomal recessive metabolic and developmental congenital disorder that causes the inability to correctly produce or synthesize cholesterol due to a low occurrence of the 7-DHC reductase enzyme.

Only a few of the symptoms:

  • Small head size (microcephaly)
  • Mental retardation
  • Learning disabilities and behavioral problems
  • Malformations of the heart, lungs, kidneys, gastrointestinal tract, and genitalia
  • Hypocholesterolemia
  • Paleness
  • Low muscle tone (hypotonia)

 Lab

Gram Stain

Q: Is my bacteria gram positive or gram negative?

A: Use a gram stain… Thanks to Hans Gram in 1884 we have a differential stain technique that distinguishes cells based on content of cell walls

Purple colored cells = gram positive

  • Gram Positive Cells – have thick peptidoglycan (multiple layers) that contains teichoic acid

Pink colored cells = gram negative

  • Gram Negative Cells – Thin peptidoglycan (few layers) with outer membrane that contains lipopolysaccharides (toxic to mammalian cells)

KOH Test

Can be used to identify Gram-Positive vs. Gram-Negative cells. Potassium Hydroxide causes Gram – Negative cells to lyse (busrt), releasing a viscous liquid. (It will be ‘snotty-like’) Gram-positive cells do not lyse when mixed with KOH, so the liquid remains thin/non-viscous.

Acid Fast Stain

Q: Does the unknown bacteria have mycolic acid (was like substance) in its cell walls?

A: Use an acid fast stain.

Note: Mycolic acid is only found in gram positive cells, but not all gram positive cells have mycolic acid.