Microbiology+-+techniques+and+materials

Techniques used
During our investigation, we will need to use a number of specialist microbiological techniques. Have a look at the methods below if your unsure. Everthing we carry out should be done using **Aseptic technique**. This is not a particular method or test - it's a way of working. It means that you are not contaminating any of your samples with bugs you don't want and that your bugs aren't contaminating everything else.

To work aseptically you need to:
 * //Wash your hands.// Do this before you start and when you finish work.
 * //Flame your loop.// This means heating up yuor wire loop until it glows - this will kill off any microbes that may be on the loop before you start. You should do this everytime you pick up your loop and everytime you put it down.
 * //Light a fire.// Actually this means you should just work next to a lit bunsen burner. The heat will create an updraft, keeping microbes away from your work.
 * //Keep a lid on it.// Make sure that you only remove the lids from your agar plates when you really need to. Any time you leave the lid off is a chance for contamination.

Make sure you stick to this way of working to protect yourself - and others - from biohazards. We use these techniques during microbiological investigations.

**Affect of nutrients**
During our investigation we will be looking at the effect of different butrients on the growth of bacteria. We will do this by using different growth media (agar plates) that contain different ingredients. We may use any of the agars below. The following information was retrieved and adapted from wikipedia on 18/03/09.

Nutrient agar typically contains: 0.5g peptone 0.3g beef extract1.5g agar100ml Distilled water.
 * Nutrient agar** is a microbiological growth medium commonly used for the routine cultivation of bacteria. Nutrient agar is a solid medium. Very few microbes can degrade agar, so the agar remains solid during microbial growth.

During fermentation of the sugar, acid is formed and the pH of the agar drops, changing the color of the pH indicator. For example, lactose fermenters turn a deep red when this pH indicator is used. Those bacteria that are unable to ferment lactose, often referred to as non-lactose fermenters, or NLFs for short, use the peptone in the medium. This releases ammonia, which raises the pH of the medium. Although some authors refer to NLFs as being colourless, in reality they turn neutral red a buff-ish color.
 * MacConkey Agar** has been used to distinguish those bacteria that ferment a sugar called lactose from those that do not. This is important because gut bacteria, such as //Escherichia coli// can typically ferment lactose, while important gut pathogens such as Salmonella sp. and most shigellas are unable to ferment lactose.


 * Mannitol salt agar** or **MSA** is a commonly used growth medium in microbiology. It contains a high concentration (~7.5%-10%) of salt (NaCl), making it selective for //Staphylococcus// since this level of sodium chloride is inhibitory to most other bacteria. It is also a differential medium, containing a sugar called mannitol and a pH indicator. Acid production as a result of bacteria fermenting mannitol, a feature of several clinically significant species such as //Staphylococcus aureus//, will result in the agar's normal red color changing to yellow. Mannitol fermenters produce a yellow colony while non-mannitol fermenters will produce a redish/purple colony.

Incubation conditions
The term incubation is used to describe the conditions your plates are left in to allow the microbes to grow. Different favour different temperatures, so by incubating your plates at a certain temperature, you are encouraging certain bacteria to grow and discouraging others.
 * Remember** - we NEVER incubate our plates at 37°C in school. This is the temperature of the human body, so by incubating at this temperature, we would be encouraging potential pathogens to grow. Only use temperatures above or below this.

Incubation time can also affect your results. Bacteria take a while to grow, so if you incubate for too short a period, you won't have any colonies to look at. Incubate for too long, and your plate could be overgrown. The usual period that plates are incubated for is 48 hours.

Other factors can also affect incubation, such as the amount of moisture in the air (hummidity) and the proportion of different gases (some bacteria are killed by oxygen for example).


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