Online practical 4- microbial detectives

7 things needed for PCR

• DNA sequence

• 2 primers

• tan DNA polymerase

• dNTP’s

• reaction buffered (MgCl2)

• water

• loading buffer for the gel

3 steps for PCR + temps

1. denaturation (90-95)

2. annealing (55)

3. extension (72)

how to make positive control

add 2ul of DNA

how to make a negative control

add 2ul water but no DNA or isolate

what would you see in a negative control PCR

nothing in electrophoresis lane

what would you see in a positive control PCR

fragment should match one of the M lanes in electrophoresis

8 ways to distinguish microbial colonies

• shape

• margin

• elevation

• size

• texture

• appearance

• pigmentation

• optical property

shape

circular, rhizoid, irregular, filamentous, spindle

margin

entire, undulate, lobate, curled, rhizoid, filamentous

elevation

flat, raised, convex, pulvinate, umbonate

how to measure bacteria growth

• spectrophotometer at visible wavelengths at hourly intervals

• viable (dividing) & non viable cells

• need to find just viable cells for quantitative measurements

how to calculate viable number of cells

countable number of colonies * dilution factor

10 biochemical tests to identify bacteria

• gram stain

• shape of cell

• spore (in gram positive)

• motility

• growth in air

• growth anaerobically

• catalase

• oxidase

• glucose utilisation

• carbohydrate (oxidation/fermentation)