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Microbiology Practical

GOALS 1. Pouring Petri dishes for later experiments 2. Preparing broth medium, cotton-plug 3. Innoculating with live bacteria, 4. Streak-plate method 5. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method). Day 1. Microbiology Practical.

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Microbiology Practical

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  1. GOALS 1. Pouring Petri dishes for later experiments 2. Preparing broth medium, cotton-plug 3. Innoculating with live bacteria, 4. Streak-plate method 5. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method) Day 1 Microbiology Practical

  2. Principles of working in Microbiology • In the environment in which we work we are surrounded by a large number of microorganisms that can contaminate, distort, and depreciate our results. • Some of the prepared cultures and/or colonies used were prepared using materials that have originate from animals, this means there is potential for harm to yourself and those around you due to infection. This is why we should always practice: • Aspetic Technique.

  3. Protocols (a structured yet simple procedure) • Name of Project, date • Goal of work • Materials used (name of organism, origin of sample)‏ • Details concerning requirements of cultivation (which medium, incubation temperature, length of incubation period)‏ • Include details of errors in experiment, Even small ones. • All experiments are evaluated graphically or with a table. In the case of using a microscope, include a picture. • End your discussion with results and a conclusion, evaluating the results and methods allowing for further expansion of your experiment and its validation. • Your experiment must be repeatable (and legible) whenever and whoever reads your write-up • A well documented experiment should allow the person perfoming the experiment to locate errors in the experiment.

  4. Sterilisation • Complete destruction of all microorganisms, including spores, from a given system (eg. Beaker containg liquid medium)‏ • System must be closed – after sterilisation we must avoid recontamination

  5. Sterilisation • “Flamming” in a bunsen burner (sterilisation of loops and tweezer tips)‏ • Sterilisation with radiation (UV – wave length 200 – 300 nm – non specific ionisation and irreprable damage) ‏ • Sterilisation of air and surfaces in a room (aspetic boxes and operating theaters...)‏

  6. Sterilisation • Chemical sterilisation • 30% peroxyacetic acid, or PAA, used on dry objects. Submerged and then left to evaporate. • PAA destroys cell membranes

  7. Sterilisation • Dry Heat (160°C, 1h)‏ • Glassware, bowls, pipettes, steel insturments... ‏ • Vegatative bacteria die within minutes at 100°C, but endospores remain! • Autoclaves are used - pressurised

  8. Sterilisation • Wet heat • Standard pressure 1,5 atm (150 kPa) where boiling point of water is cca 128 °C

  9. Day 1 – Media used • MPA – Meat-Peptone-Agar (nutrient agar (1L)‏ • Meat extract (10g)‏ • peptone (enzymatically hydrolysed meat, 10g)‏ • NaCl • + agar (15 g) – 1,5 % • pH 7.2 – 7.4

  10. Day 1 - Steps • 1. Pouring Petri dishes for later experiments • 2. Preparing broth medium, cotton-plug • 3. Innoculating with live bacteria, • 4. Streak-plate method • 5. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method)

  11. Day 1 - Steps Preparation of broth (Meat peptone broth)‏ • Goal: Prepare 100ml of broth into two conical flasks, put cotton stopper in, and seal with aluminium foil (reduces escape of moisture during sterilisation)‏. Learn how to make cotton plugs

  12. Day 1 - Steps • Preparation of petri dishes for later experiments: • Goal: learn aseptic technique and how to pour agar medium

  13. Day 1 - Steps • 3. Innoculation of samples onto prepared agar • Finger/Toe print, glasses, handkerchief • Infusion/extract from top soil • Water (tap, distilled, and from puddle)‏ • Tounge print • Open petri dish on the lab bench • Cleaning sponge • Coins/money • Each group will choose one of the above... Innoculating liquid samples onto medium

  14. 4. Streak-plate method NA (nutrient agar) Group 1-4 (mixture of Micrococcus luteus– yellow, small, Bacillus subtilis– cream white, large) Group 5-8 (mixture of Serratiamarcescens red, small, E. coli, white small-medium size) Goal:Learn the streak plate method EMB agar (selective and differential medium) All groups (mixture of E. coli and Proteus vulgaris)

  15. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method)

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