6.4 Core Practical 13: Rate of Growth of Microorganisms

• There are several ways to investigate the growth of microorganisms
• In this example the change in turbidity of a yeast solution will be used as a measure of the growth of microorganisms in a liquid culture over time
  • Turbidity = cloudiness of a solution

Apparatus

• Cloths and antimicrobial solution
• Conical flask
• Glucose solution 
• Dried baker's yeast
• Bunsen burner
• Cotton wool
• Magnetic stirrer
• Aluminium foil
• Colorimeter
• Dropper pipette
• Cuvettes
• Optional
  • Microscope
  • Microscope slides with cover slips and graph paper photocopied on to acetate OR a haemocytometer

Method

1. Use antimicrobial solution to sterilise the work area
2. Place 250 cm³ glucose solution into a conical flask
   • This solution will be the liquid culture medium
3. Inoculate the glucose solution with 1.25 g dried yeast using aseptic techniques
   • Work next to a Bunsen flame to ensure that microorganisms in the air are drawn away from the nutrient broth
4. Seal the flask using a cotton wool stopper immediately after inoculation is complete to prevent contamination
5. Swirl to mix the yeast with the glucose solution and place on a magnetic stirrer
6. While stirring continuously, loosely cover the stopper with aluminium foil and incubate the flask at 20 °C
7. Fill a cuvette with plain glucose solution and use this as a blank to calibrate the colorimeter
   • This ensures that the colorimeter is reset to zero before each measurement is taken
8. Transfer about 3 cm³ of the yeast suspension into a cuvette using a dropping pipette
9. Use the colorimeter to measure the absorbance and record this in a suitable table against time
10. Repeat steps 7-9 at intervals over a 12 hour period
    • E.g. this could involve taking samples every 30 minutes for the first two hours and then every 2-3 hours after this
11. Plot a graph of absorbance against time

• The temperature or concentration of the glucose solution can be changed and the experiment repeated to investigate the effect these changes would have on the growth of the yeast

Optional extension to practical

• Turbidity measurements are useful as the turbidity of the medium is mainly a measure of the number of living cells in suspension; however, the measurement can be affected by the presence of dead cells and other suspended particles, so other methods of cell counting can be used to back up the turbidity findings, e.g.
  • Use a haemocytometer to estimate the number of yeast cells in the glucose solution
  • Calculate the area of the microscope's field of view using graph paper copied on to acetate, and use the number of cells in the field of view to estimate the number of cells in the glucose solution
• If using the area of the microscope's field of view the following steps can be used to estimate the number of cells in the growth medium
  • Count the number of squares of acetate graph paper visible when using the x4 objective lens and use this to calculate the area of the field of view under a x40 objective lens; this will be 0.01 of the area of the x4 objective lens
  • Stain the yeast suspension with methylene blue and place one drop of the suspension on a microscope slide
  • View under the x40 objective lens and count the number of yeast cells in the field of view
  • Calculate the volume of this drop by measuring the volume of 10 drops and dividing by 10
  • To calculate the volume under the field of view when using the x40 objective lens, use the following formula
    

volume = (area of field of view of x40 objective lens ÷ area of cover slip) x volume of one drop

• • To calculate the number of yeast cells in 1 mm³ of medium the following formula can be used

number of cells per mm³ = average cell count in field of view ÷ volume of field of view

• • Note that if many cells overlap when viewed under x40 objective lens a serial dilution of the yeast suspension will be required

Safety

• Eye protection should be worn
• Care should be taken around Bunsen burners
• Aseptic techniques should be used when transferring microorganisms
• Cultures should be incubated at a safe temperature for school laboratory work; 20 °C and not 37 °C
• Work spaces and hands should be thoroughly disinfected at the end of the experiment
• Cultures should be safely destroyed at the end of the experiment