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Microbiology Practical

GOALS 1. sterilisation, 2. aseptic technique, 3. preparing broth medium, 4. innoculating with live bacteria, 5. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method). Microbiology Practical. Day 1.

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Microbiology Practical

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  1. GOALS 1. sterilisation, 2. aseptic technique, 3. preparing broth medium, 4. innoculating with live bacteria, 5. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method) Microbiology Practical Day 1

  2. HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORY Access to the lab is only allowed to those who will work there. Upon entering the lab you must remove jackets and place them in the lockers provided. Lab coats should be worn throughout the duration of the practical. Lab coats are used only in the microbiology lab and are not allowed to be used outside of the lab. 1

  3. It is forbbiden to Eat Drink or Smoke in the Lab. It is important to keep good hygine when in contact with bacteria, pathogenic and non-pathogenic strains alike. Always wash hands thoroughly, using provided soap, before leaving the lab. HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORY

  4. Work benches must be disinfected before and after use, using the provided solutions (0,1% Ajatin or 70% Ethanol). During practicals it is important to remember sterile technique. Avoid placing non-sterile objects on the work bench, and to keep the work space tidy and clean. The work area and air in the lab need to be regularly disinfected using UV radiation. This is done overnight when the lab is not in use and is combined with mechanical disinfection using chemicals. HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORY

  5. ALL injuries must be reported to the assistants. Even smaller wounds such as scratches or cuts run the risk of becoming infected if not properly disinfected. After experiments are completed all glassware and insturments used must be properly disposed of. All glassware must be autoclaved for 30mins at 121oC Under NO circumstance is any medium or culture to be disposed of in the sinks or by any other means. HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORY

  6. Broken glassware should be disposed of in the apporpriate recepticle provided. If the glassware has come in contact with microbial cultures, the glass must be disinfected by submerging it into disinfectant with the aid of forceps or tweezers. Under no circumstance should glass shards be cleared by hand – use a dust pan and brush. HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORY

  7. Before leaving the lab it is required that you clean up all things that have been used. Place cultures back into the refridgerator, clean microscope eye pieces, and tidy up the workbench. Make sure that the gas/water/electrical appliances are all switched off (except those that are on perminently -eg. Fridge, shakers etc.)‏ HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORY

  8. 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.

  9. 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.

  10. 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

  11. “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...)‏ Sterilisation

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

  13. 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 Sterilisation

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

  15. 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 Day 1 – Media used

  16. Day 1 - Steps Pouring petri dishes for later experiments Preparation of liquid medium, pipettes for later experiments Innoculate real samples on prepared agar plates The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method)

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

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

  19. 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... Day 1 - Steps Innoculating liquid samples onto medium

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

  21. Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 10145), and Klebsiella pneumoniae (ATCC e13883) 1.AMPICILLIN (beta-lactam antibiotic, cell wall) AMP 2.BACITRACIN (polypeptide, G+ cell wall) B 10 3.CARBENICILLIN (beta-lactam antibiotic, G-, P. aeruginosa) CAR 100 4.ERYTHROMYCIN (macrolide antibiotic, enzyme synthesis) E 15 5.KANAMYCIN (aminoglycoside antibiotic, enzyme synthesis) K 30 6.GENTAMICIN (aminoglycoside antibiotic, enzyme synthesis) CN 10 7.NALIDIXIC ACID (quinolone antibiotic, Escherichia coli, Proteus, Shigella, Enterobacter, and Klebsiella) NA 30 8.NEOMYCIN (aminoglycoside antibiotic) N 30 9.PENICILLIN G (beta-lactam antibiotic, cell wall)P 10 10.POLYMYXIN B (polypeptide, G-) PB 300 11.STREPTOMYCIN (aminoglycoside antibiotic) S 10 12.SULPHAMETHOX/TRIMETHOPRIM (COTRIMOXAZOL) SXT 25 13.TETRACYCLINE (tetracycline antibiotics, enzyme synthesis) TE 30 14.VANCOMYCIN (glycopeptide antibiotic, G+, ant. of last resort) VA 30

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