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.

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