CP 12 Flashcards
CV
● Nutrients in the Broth
● Time Left
How can a colorimeter be used to measure the rate
of growth of microorganisms?
As microorganisms increase in solution, the turbidness of the solution increases, less light passes through and absorbance increases.
PROCEDURE
-an optical method so the more bacterial cells in a culture, the more turbid the culture hence less light is able to pass through the sample. You can use a colorimeter to measure turbidity in samples taken at regular intervals.
● Follow aseptic technique
● Wash your hands with soap and disinfect your bench area, leaving it to soak for 10 minutes if using Virkon®, before wiping it down. Light a Bunsen burner set to a safety flame on the disinfected working area.
● Collect a culture vessel containing sterile nutrient broth. Using aseptic technique, inoculate your culture vessel. Using a measuring cylinder, add 2 cm3 bacterial culture for every 100 cm3 of nutrient broth. Swirl to mix.
● Place the culture vessel on the magnetic stirrer and stopper with cotton wool and aluminium foil. This is to prevent contamination whilst maintaining oxygenation of the culture.
● Place sterile culture medium into a cuvette and use this as a blank to set the absorbance of the colourimeter to zero.
Keep this reference medium (the blank) in a refrigerator between measurements.
● Measure 3 cm3 of the bacterial culture using a clean pipette then transfer to a cuvette, which should be just over half full.
● Measure the absorbance of the sample you have just withdrawn.
● Return the culture to the magnetic stirrer, stopper with cotton wool and incubate at no more than 30 °C.
EVALUATION
● Contamination of the sample could lead to inaccurate readings
● Magnetic stirrer stopping = culture no longer evenly maintained, can lead to advanced cell death due to lack of nutrients & O2
● Accuracy lost in absorbances above 2
● This method does not distinguish between live cells and other particles in suspension such as dead cells, spores or cell fragments
● To find actual cell numbers, a calibration curve is needed. To produce a calibration curve
plot turbidity readings against either a haemocytometer reading or a viable count reading of the same culture.
How is the growth rate of the microorganism
measured?
Transfer a sample from the microorganisms culture to a cuvette at intervals, or use a datalogger to continuously record absorbance.
As the age of the culture increases, how does the
absorbance vary?
The absorbance increases to a
maximum, then remains constant.
How can a cell count be found?
Stain the yeast suspension with methylene blue and view in a haemocytometer to find the cell density.
Multiply cell density by total volume for
cell count.
State the hazards and safety precautions involved in
this practical.
Yeast may cause an allergic reaction.
Avoid contact with skin, use aseptic
techniques.
Why was the magnetic stirrer used?
The magnetic stirrer keeps cells in suspension and ensures that nutrients are evenly
distributed.
It also oxygenates the culture to allow aerobic respiration.
The simple optical method for examining growth gives no measure of the actual numbers of cells. How can this method be improved to provide such information?
Calibrate OD readings using either dilution plating or direct counting (using a haemocytometer).
Produce a calibration curve for a range of OD readings and use the graph to estimate the number of microorganisms present.
Comment on the validity of using turbidity measurements to follow the growth of populations of
microorganisms
The method is valid because most of the change in turbidity will be a result of microorganism
cells in suspension.
However, the method does not distinguish between live cells and other particles in suspension such as dead cells, spores or cell fragments, which reduces the validity.
