Microbiology lab #1

Pathogen 

disease causing agent 

Medium

a collection of nutrients used for cultivating microorganisms

• sugar, vitamins, minerals, proteins

Agar

a gelatin-like sugar made from seaweed extract

• used as a thickening agent in mediums

incubation

growing microbes under the appropriate conditions

Aerobic

microbes grown in the presence of oxygen

Anaerobic 

microbes grown without oxygen 

generation time

time required for a cell to grow and divide

• aka “the doubling time”

colony

aggregate population of microbes visible to the naked eye

streak-plate method - isolating colonies

a sterile inoculation loop is used to spread an inoculum across the surface of a solid medium in petri dishes.

aseptic (sterile) technique 

accepted laboratory geared toward preventing contamination of cultures and the laboratory. It covers procedures regarding lab preparation, transfers of cultures, and cleanup/disposal

aseptic (sterile) technique

1. disinfect work area with alcohol. Wash your hands with soap and water.

2. flame the opening of test tubes before opening and closing the lid

• use your non-dominant hand and hold the tube at a 45 degree angle

3. hold the lid over the petri dish to prevent contamination

4. disinfect work area again when finished and wash your hands

5. dispose of cultures in a safe manner. sterilize with autoclave or bleach

simple stain

stain composed of a single dye

• specimens will be one color

• useful in determining size and shape of cells

differential stain

stain using more than one dye so that different cells or structures can be distinguished

• ex. acid fast stain, endospore stain, H and E, gram stain(the most common)

gram stain results 

purple cells are gram-positive

pink cells are gram-negative 

acid-fast stain results

red cells are acid-fast

blue cells are non-acid-fast

spore stain results

showing endospores (red)

vegetative cells (blue)

gram stain is used

first method used to identify pathogens

Gram-positive 

prokaryotic cell with a thick peptidoglycan wall appear purple after gram-staining 

gram negative

prokaryotic cell with a thin peptidoglycan wall and an outer membrane composed of phospholipids, proteins, and lipopolysaccharides appear pink after gram-staining

DNA extraction process involves 4 parts

1. put the cells into a solution

2. use a detergent to break apart the cell membrane

3. place in hot water bath to denature nuclease enzymes

4. use 95% ethanol to take out the DNA from the lysate

bacterial suspension medium 

used to make a solution of the cells 

SDS

a detergent that lyses the cell by removing the lipids from the cell membrane

lysate

preparation containing the lysed contents of a cell

contents of the lysate after shaking, rotating and inverting the tube

1. DNA

2. Nucleases

• enzymes that digest DNA

what happens to the lysate next

the lysate is then placed in a hot water bath set at 60-65 degrees celsius. Why? enzymes denature at high temperature. The hot water bath destroys the damaging nucleases in the lysate. 

why is 95% ethanol used

causes the DNA to precipitate out of the lysate

True or False DNA is insoluble in ethanol

True

DNA extraction technique

• By drawing the lysate up into the alcohol layer, the DNA comes in contact with the ethanol, becoming visible to the naked eye.

restriction enzymes 

cuts DNA  at specific nucleotide sequences 

gel electrophoresis

separates DNA fragments by size and electric charge

agarose

sugar derived from seaweed

TBE buffer

maintains pH to protect to the DNA and conducts electricity

DNA fingerprinting 

technique using restriction enzymes and gel electrophoresis to compare DNA of different individuals  

PCR- polymerase chain reaction to amplify multiply DNA

gel electrophoresis

technique used to separate DNA fragments

shorter fragments

travel more rapidly toward + pole

what does gel electrophoresis allow us to do

determine the size of the fragments of known size are used as a standard