Culturing Microorganisms

Culturing Microorganisms Individual microorganisms are invisible to the naked eye. To work with them, scientists must grow them into populations that are large enough to see. Aseptic Technique Many bacteria have similar requirements. Therefore, when growing bacteria, it is essential that the cultures are not contaminated by other unwanted bacteria. Aseptic technique involves practices and procedures to prevent microorganisms from contaminating cultures or escaping into the environment. Although most microorganisms are harmless, some can be dangerous to health. Culture Media The culture medium is the material in which microorganisms are grown. There are two main types of culture medium. Nutrient Broth The microorganisms live in this liquid, which contains essential nutrients. Agar The microorganisms grow and form visible colonies on the surface of this jelly, to which essential nutrients have been added. The culture medium and any container it is put into, such as a petri dish or test tube, must be sterilised before use, to prevent unknown bacteria being transferred into the culture. Similarly, care must be taken when transferring bacteria from a broth culture to an agar 'plate' with an inoculating loop. Follow the steps outlined below to prevent contamination. 1 Pass the loop through a flame. 2 Remove the bung, and flame the mouth of the tube. 3 Dip the loop into the broth to pick up the bacteria. 4 Flame the mouth of the tube, and replace the bung. 5 Streak the loop across the agar surface to inoculate the agar plate with the bacteria. Flaming kills bacteria so no live bacteria enter or leave the tube. Carrying out the technique near a lit Bunsen burner also creates a convection current that carries any bacteria in the air upward, away from the agar plate. Uncontaminated cultures of microorganisms are vital for investigating the action of disinfectants and antibiotics. Incubation Once the bacteria have been transferred to an agar plate, they are then incubated so that the microorganisms can grow. The incubation temperature should not be near 37°C (human body temperature) as this will encourage the growth of dangerous bacteria. In school labs, 25°C is recommended. During incubation, the lid of the Petri dish containing the agar plate should be lightly fastened with sticky tape. When lifting the Petri dish lid, open it very slightly. Do not remove it completely. This will prevent bacteria in the air dropping onto the plate. After incubation, each bacterium will have grown into a circular colony. By counting the colonies, we know how many bacteria were initially placed on the plate. Calculating Colony Areas Bacterial colonies usually grow in a circular form so their areas can be calculated using the formula: Area = πr^2 π = 3.142 r = radius of circle Investigating the Effects of Antiseptics or Antibiotics on Bacterial Growth The area of the inhibition zone indicates the effectiveness of the antiseptic or antibiotic on the bacteria. The larger the area, the more effective the antiseptic or antibiotic. When a sterile antiseptic or antibiotic disc is placed on a bacterial culture, it diffuses out, destroying or inhibiting the growth of bacteria over a certain area, the inhibition zone. The size of the inhibition zone can be calculated by using the area of a circle formula above. The diagram opposite shows the inhibition zone produced by a new antibiotic. Calculate the area of the inhibition zone: Calculate the area of the inhibition zone: Area = πr^2 = 3.142 x 12^2 = 452.4 mm^2