1. Learning Objectives
i Understand the basic aseptic techniques used in culturing organisms.
ii Understand the principles and techniques involved in culturing microorganisms.
iii Understand the use of different media (broth cultures, agar and selective media).
iv Understand the different methods of measuring the growth of a bacterial culture as illustrated by cell counts, dilution plating, mass and optical methods (turbidity).
v Understand the different phases of a bacterial growth curve (lag phase, log phase, stationary phase and death phase) and calculate exponential growth rate constants

2. Starter
On the boards, write out the main ways of measuring bacterial growth – what are the advantages and disadvantages of these methods?

3. Draw a line under last lesson’s work and write today’s title in your book...
Microbial technqiues
22/09/2018
c/w
Check homework!!!
Question booklets completed…

4. Aseptic Technique:
TO PREVENT CONTAMINATION TO THE ENVIRONMENT by the bacteria being used in the experiments.
Sterilise the work surface before and after an experiment using a disinfectant, e.g. Lysol used at a 3% solution.
Use the CORRECT HANDLING TECHNIQUES to prevent the contamination of personnel and the immediate environment by the organisms being cultured; for example, when carrying out the process of INNOCULATION.

5. Aseptic Technique:
Secure the lid with adhesive tape. Use two pieces to fasten the lid, but do not seal all the way round (this could create anaerobic conditions and encourage the growth of possible pathogenic microbes.
CORRECT HANDLING TECHNIQUES
Grasp the culture bottle in one hand; remove the cap with the little finger of the other hand – do not place the cap on the work surface.
Lift the lid of the Petri dish just enough to allow entry of the inoculating loop.
Flame the mouth of the bottle for 2 or 3 seconds.
Incubate at around 25oC (cultures should not be cultured at 37oC as this is an ideal temperature for the growth of many pathogenic species.)
Pass the inoculating loop through the flame until red hot.
Dip the loop into the culture and streak over the agar plate.
Do not open the Petri dish after incubation.

7. Sterilisation: AUTOCLAVE:
In the lab., the preferred method of sterilisation is to use an AUTOCLAVE.
This is a sealed container in which glass and metal equipment is heated at 121oC in steam under pressure for 15 minutes after the required pressure has been reached.
This ensures that any resistant endospores are destroyed e.g. gram positive bacteria such as Clostridium are resistant to boiling.
Disposable materials, such as plastic Petri dishes, can be sealed inside autoclavable plastic bags and placed in a dustbin after having been autoclaved.
RADIATION:
E.g. gamma rays, is used commercially to sterilise plastic equipment.

8. Methods of Measuring Growth:
Estimating the growth of bacteria is extremely important. Why?
Environmental health officers regularly inspect food premises and taker samples for analysis.
Water boards check water supplies daily.
Many products are produced using bacteria grown in fermenters.
Measuring their growth is an important part of the process.
Total and viable counts.
Dilution plating.
Haemocytometer
Measuring turbidity

9. Measuring the Size of a Population:
The size of a population of microorganisms in liquid culture may be measured by counting cells directly, or by taking some indirect method such as turbidity (cloudiness) of the culture.
TOTAL COUNTS – include both living and dead cells.
VIABLE COUNTS – count living cells only.
In practice, it is never possible to count whole populations of microorganisms.
Cells in a very small SAMPLE are counted.
Result is multiplied up to give a population density with a unit of: organisms per cm3 of culture.
Even then, the population density is likely to be so high that cell counts are usually made in known dilutions of the culture, usually in 10-fold steps. This is known as SERIAL DILUTION.

11. Measuring the Growth of a Population:
Rough estimates of growth rates can be made by regularly measuring the diameter of a bacterial or fungal colony as it spreads from a central point to cover the surface of a solid growth medium.
DILUTION PLATING technique:
A sample, 1cm3, is streaked onto a sterile agar plate, which is then placed in an incubator at 25oC for two days.
After all the streaks have been allowed to grow, the dilution at which the colonies are distinct and separate is counted.
If the dilution is insufficient then colonies will merge, referred to as ‘clumping’ and counting is inaccurate.
The separate colonies of bacteria are counted with the assumption being made that each colony has arisen from a single cell, which has divided asexually, from the original medium.
To find the total viable cell count the number of colonies is multiplied by the appropriate dilution factor. This method makes no allowances for clumping of cells so may cause an underestimate of numbers.

12. Measuring the Growth of a Population:
Using a HAEMOCYTOMETER:
This is a more accurate method and is a specialised microscope slide originally used to count red blood cells.
Using a haemocytometer gives TOTAL CELL COUNTS as it is not possible to distinguish between living and dead cells.
The TURBIDIMETRY method:
This involves using a colorimeter to measure the cloudiness or turbidity of the culture as cell numbers increase.
Results are derived by comparison with a standard graph of light absorbance plotted against known cell numbers.

14. Evaluate = Strengths and Weaknesses of.
*(b)  Evaluate the use of dilution plating and optical methods for determining the number of bacterial cells in a culture. (6)
Evaluate = Strengths and Weaknesses of.

16. Complete Haemocytometer activity