Culturing Microorganisms

Binary Fission

Mean Division Time

The mean division time is the average time it takes for one bacterial cell to divide once.

Mean division time can be used to work out how many times a cell has divided and therefore the number of cells produced.

\text{Number of divisions} =\dfrac{\text{Time spent dividing}}{\text{Mean division time}}

\text{Number of cells produced} = 2^\text{ Number of Divisions}

Example: A cell has a mean division time of 20 minutes. How many cells will it have produced after 3 hours.

\text{Number of divisions} =\dfrac{180}{20} = 9 \text{ divisions}

\text{Number of cells produced} = 2^9 = 512 \text{ cells}

Aseptic Techniques

Bacteria can be grown in labs in petri dishes that contain a growth medium. The growth medium contains all the molecules needed for the cells to grow and can either be a nutrient broth solution or solid agar jelly. Bacteria grown on agar gel plates will often form colonies on the surface.

As bacteria are found all around, it is important to reduce contamination when studying bacterial growth to prevent unwanted pathogens forming that can affect your investigations. these unwanted pathogens can also pose a risk to your health.

To create uncontaminated cultures when growing bacteria it is important to use aseptic techniques:

• Wash your hands and work surfaces before beginning to prevent contamination.
• Growth media must be heated before use to kill any bacteria living in it.
• Growth media must be added to a sterile petri dish.
• All work should be done in the presence of a Bunsen burner on a yellow flame to create a convection current above the bench and prevent contamination from the air.
• The Bunsen burner can also be used to sterilise equipment such as the inoculating loop that is used to transfer bacteria. Passing it through the flame will kill any bacteria.
• The lid of the petri dish must be taped on and opened as little as possible to prevent contamination from microorganisms in the air.
• The petri dish should be stored upside down to stop condensation falling on to the agar.
• The dish should incubated at 25°C to restrict the growth of harmful pathogens.

Required Practical

Investigating the effects of antiseptics and antibiotics on bacterial growth.

Preparing the petri dish

Remember to use aseptic techniques mentioned above to prevent contamination.

1. Pour hot agar into the sterile petri dish and leave to cool and set. Then use a sterile dropping pipette and spreader to evenly spread the bacteria (this step may have already been done for you before you started the experiment).
2. Soak paper discs in different types or concentrations of antibiotics and antiseptics for the same length of time and place them, evenly distributed, on the agar plate with the bacterial covering. This allows the antibiotic or antiseptic to diffuse into the agar. 
3. Place a disc that has been soaked in sterile water onto the plate as a control.
4. Tape the lid onto the petri dish and incubate upside down for 48 hours. 

Analysing the results

• There should be clear areas surrounding some of the discs of paper which is where the bacteria have been killed. These areas are called inhibition zones.
• The more effective the treatment is at killing bacteria, the larger the inhibition zone.
• Increasing the concentration of the solution increases the size of the inhibition zone.
• Some bacteria may be antibiotic-resistant and will be able to grow in the presence of the treatment so there will be no inhibition zone. 
• You should notice that there is no inhibition zone around the control disc (water) and therefore it is the antibiotic/antiseptic that kills the bacteria.

Measuring inhibition zones

To truly compare how effective the different treatments are, you can calculate the area of the inhibition zones.

1. Measure the diameter of zones of inhibition using a ruler.
2. Half the diameter to get the length of the radius.
3. Use \text{Area of a circle} =\pi\text{r}^2 to find the area of the inhibition zone.

Example: Find the area of inhibition of antibiotics X and Y to 2 decimal places and use them to decide which antibiotic is most effective against the bacteria in the plate.

\text{Radius of X} =\dfrac{16}{2} = 8

\text{Area of X} =\pi\times\text{8}^2 = 201.06\text{ (2.d.p.)}

\text{Radius of Y} =\dfrac{12}{2} = 6

\text{Area of Y} =\pi\times\text{6}^2 = 113.10\text{ (2.d.p.)}

 

Antibiotic X is more effective than antibiotic Y because

201.06>113.10

 

This method can also be used to find the area of a colony on the surface of an agar plate.