Fundamentals practicals

What information do you put on a Petri dish?

• Name

• Date

• Organism

• Bench number

On which part of the Petri dish do you write the information?

The base (with the agar in it)

How do you prevent contamination using aseptic technique?

• Work near a blue Bunsen flame

• Keep vessels open for the minimum length of time possible

• Flame the neck of bottles when opening and closing them

• Use sterile equipment

• Flame the inoculating loop in the flame before use

What are the two types of agar used in the aseptic technique practical?

MacConkey and nutrient agar

How do you prepare a streak plate?

• Flame the inoculating loop and cool

• Collect a single colony off the sample plate

• Zig-zag the colony over a small section of the test plate

• Flame and cool the inoculating loop

• Streak 4-5 times from the beginning colony onto fresh agar

• Flame and cool the inoculating loop

• Turn the plate 90° and streak from the second set of lines

• Flame and cool the inoculating loop

• Repeat until most of the plate is covered (3/5)

• Flame and cool the loop

• Use a single zig-zag motion to pull colonies into the last part of the plate

• Replace the lid, invert and incubate

How do you prepare a spread plate?

• Pipette 100μl of the organism onto the agar

• Dispose of the pipette tip

• Remove the sterile disposable spreader, touching only the handle

• Carefully spread the bacteria across the whole plate

• Replace the lid and leave to allow the bacteria solution to adsorb onto the agar

• Invert and incubate

Why are lids of universal bottles loosened when disposing?

Prevents pressure build-up and explosions in the autoclave

How do you calculate the CFUs in an original sample from a dilution?

number of CFUs in the dilution / amount plated

How do you calculate the number of CFUs in a given sample?

Count the number of colonies in a dilution

How do you set up a microscope for bright field viewing?

• Plug the microscope into mains power, ensure the plug is switched on

• Switch on the power until three blue lights are seen on the side

• Lower the stage using the coarse focus dial

• Place the slide onto the stage and position it in the specimen holder

• Centre the slide using mechanical stage controls

• Adjust the intraocular distance until comfortable

• Ensure the x10 objective lens is switched in

• Raise the stage using the coarse focus dial until the slide comes into focus

• Use the fine focus dial to perfect the focus

• Close the field diaphragm until visible

• Adjust the condenser height until the diaphragm edges are in focus

• Centre the diaphragm onto the subject

• Open the diaphragm until the edges disappear from view

• Adjust the condenser diaphragm position with the lever to match the objective or until the desired contract is reached

Which two organisms were used for the microscopy viewing?

Sachharomyces sp. and Chlamydomonas

How do you prepare a stock solution?

• Weigh out the desired weight of the compound in a weighing boat

• Transfer to a beaker and wash out the weighing boat with deionised water

• Add a small amount of water to dissolve the compound, mixing with a spatula

• When dissolved, transfer to a volumetric flask through a funnel

• Wash the beaker, spatula and funnel with deionised water

• Add deionised water until the line on the volumetric flask (when viewed at eye level)

• Add the stopper and invert a few times to ensure the solution is evenly mixed

How do you plot a graph of concentration and absorbance?

• X-axis: concentration - units in M

• Y-axis: absorbance - no units

How do you set up a gel for agarose gel electrophoresis?

• Remove the well-combs from the gel and the autoclave tape from the ends of the gel former

• Place the gel in the electrophoresis tank with the wells near the black electrode (negative)

• Cover the gel with Tris-Borate-EDTA (TBE) buffer until it is just submerged

• Place the tank next to the electrophoresis power pack

• Add 10μl of the molecular weight ladder to the first lane

• Add the sample mixture into the following lane, marking its position

• Connect the power pack cables to the gel by putting on the lid

• Leave the samples to run for 60 minutes

How do you calibrate a pH electrode?

• Add the pH electrode to a solution of known pH 7 to calibrate it

• Test the calibration by adding to other solutions of known pHs