What does a bacterium look like? Internal Structures: cytoplasm nucleoid ribosomes Boundaries: cell membrane cell wall capsule Appendages: flagellum pili Facts About Bacteria

Shapes of Bacteria

Bacteria Identification Criteria Shapes Bacilli (rod) Cocci (round) Sprilli (spiral) Growth Patterns “Prefixes describe pattern” “strepto…. Means in chains “staphylo…Means in clusters Examples: Streptococcus pneumoniae

Bacterial Growth Patterns: Streptococcus

Bacterial Growth Patterns: Staphylococcus

DNA Extraction Lab Step by step procedure that weakens the outer boundaries of a cell and lyses it to release the DNA for future study.

DNA Extraction Process Part I Put the cells into a solution. Use Bacterial suspension solution to put the dry cells into a solution. Use SDS (a detergent) solution to disrupt the cell membrane’s structure The three steps are responsible for the lysis (bursting) of the bacteria cell to release the DNA and cell contents.

Previously, the bacteria pictured below was used in the DNA extraction lab. Note its Spherical appearance and the color after gram staining. The gram stain specifically stains the cell wall. According to this picture, they are gram positive bacteria at 1000x. The official name of this bacteria is Micrococcus luteus

Today’s lab will use the bacteria Escherichia coli Today’s lab will use the bacteria Escherichia coli. It is a gram negative bacteria as seen 1000x magnified.

Time to Re-read the Protocol Steps 1-4 were done on Day 1 Steps 5-9 were done on Day 2

Extraction Solutions: Bacterial Suspension Medium – used to make a solution of the cells Lysozyme – enzyme that hydrolyses specific bonds that hold the cell wall SDS – a detergent that lyses the cell by removing the lipids from the cell membrane By adding these solutions to a test tube of bacteria, a lysate is made that contains DNA.

The Lysate Contents of the lysate after shaking, rotating and inverting the tube: 1. DNA 2. Nucleases

The lysate is then placed in a hot water bath set at 60-65 degrees Celsius. Why? Enzymes denature at high temperatures. Nucleases are enzymes that digest DNA so the hot water bath destroys the damaging nucleases in the lysate.

DNA Extraction Part II Need to make the dissolved DNA in the lysate (yellow liquid) visible. 95% Ethanol is the chemical that will cause the DNA to become visible. Carefully place ethanol on top of the lysate to form two distinct layers of liquids. Insert the glass spooling rod through both layers and then twirl the rod. The twirling with cause the DNA to go into the alcohol and become visible (called precipitation).

Precipitated DNA ethanol In this picture, the student used 95% ethanol to cause the DNA to precipitate out of the lysate. DNA is soluble in the lysate but insoluble in ethanol. By drawing the lysate up into the alcohol layer, the DNA comes in contact with the ethanol, thus, becoming visible to the naked eye. DNA lysate

Analysis Questions Describe three obserservations made after the addition of bacterial suspension solution. After the addition of SDS, the contents became more viscous or thicker. What caused this? Explain. Protein will denature at 60-65oC, while DNA denatures at 80oC. Research why this occurs and explain. What are nucleases? Why would you need to remove these from the contents of the DNA extraction test tube? How was this accomplished during the preparation steps? What chemical is used to extract the DNA from the lysate (contents of cell and chemicals)? Describe the appearance of extracted DNA after adding the ethanol? What happened as the spooling rod was rotated in the aqueous-ethanol interface? What are you thoughts about extracting DNA?

What do you do with extracted DNA?