Comenius Project London Exchange Visit

1. Comenius ProjectLondon Exchange Visit Leyton Sixth Form College Microbiology Aseptic Techniques Looking at the Results

2. Today you will be looking at the results of the seven aseptic techniques carried out on Monday and Tuesday. Microbial growth can be of two types: unicellular organisms (bacteria and the fungus yeast) grow as colonies (usually round although they can have ragged edges and a variety of colours) which consist of thousands of cells which have arisen from one cell multicellular fungi grow into an interconnected mass of hyphae called a mycelium which is a single organism Microbiology at Leyton Introduction Comenius – Leyton Sixth Form College – April 2005

3. This plate should have no growth on it and also very little condensation on the lid. Condensation occurs if the molten agar is too hot when it is poured. Any growth is due to contamination (from a dirty inoculating loop or via the air while the lid was open). Microbiology at Leyton Experiment 10.4 Comenius – Leyton Sixth Form College – April 2005

4. The streak plate should have less and less growth as you look along each of the streaks. Streak plates are used to spread out the culture so that by the end of the third streak there were individual cells which grow into individual colonies. This technique allows us to then separate a mixed culture (several species of microbe) into colonies which consist of one species. Sub-culturing (transferring organisms from one culture to another) can then be used to produce pure cultures. Microbiology at Leyton Experiment 10.5 (a) Comenius – Leyton Sixth Form College – April 2005

5. You should have yeast cells growing along the single streak. Microbiology at Leyton Experiment 10.5 (b) Comenius – Leyton Sixth Form College – April 2005

6. In a pour plate the cells in the inoculum consisting of many cells is spread evenly throughout the molten agar. This either produces an even growth across the whole plate (a lawn) if there were a large number of cells in the inoculum; or a number of individual colonies which can be counted. Pour plates are routinely used to estimate the number of bacteria in a culture by using a known volume of inoculum. Microbiology at Leyton Experiment 10.6 (1) Comenius – Leyton Sixth Form College – April 2005

7. By combining the technique with dilution plating it is possible to find a dilution which gives sufficient (not too many nor too few) colonies on a pour plate for counting. Dilution plating involves making up dilutions of the original culture (1:10, 1:100 etc.) and inoculating plates which each of the dilutions. Remember that each colony came from an individual cell. Microbiology at Leyton Experiment 10.6 (2) Comenius – Leyton Sixth Form College – April 2005

8. Agar slopes are often used for storing or transporting cultures. Bottles/slopes take up less room and are more robust than plates. In addition, there is less exposure to the air and possible contamination when the bottle is opened. Microbiology at Leyton Experiment 10.7 Comenius – Leyton Sixth Form College – April 2005

9. Agar deeps or stabs allow organisms which are anaerobic to be cultured as the deeper down the stab line you go the less oxygen there is. Yeast is a facultative anaerobe which means that it respires aerobically when oxygen is present and anaerobically in the absence of oxygen. You should find yeast growing all the way down the stab line although often it gets rubbed off the needle as it passes down through the agar. Microbiology at Leyton Experiment 10.8 Comenius – Leyton Sixth Form College – April 2005

10. The aim of a spread plate is to produce an even growth (lawn) across the whole plate. This can also be achieved using a pour plate. Lawns are useful for testing the effectiveness of anti-microbial chemicals such as antibiotics. This is done by placing discs soaked in the chemical on the plate immediately after inoculation. The chemical diffuses out into the agar and if it inhibits the growth of the microbe in the inoculum a circular clear area (inhibition zone) where no microbe grows is produced. The larger the diameter of the inhibition zone the more effective the chemical. Microbiology at Leyton Experiment 10.9 Comenius – Leyton Sixth Form College – April 2005

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