microbiology practical goals 1. sterilisation, 2. aseptic technique, 3. preparing broth medium, 4....

Microbiology PracticalMicrobiology Practical

GOALSGOALS1. 1. sterilisation, sterilisation, 2. 2. aseptic technique, aseptic technique, 3. 3. preparing broth medium, preparing broth medium, 4. 4. innoculating with live bacteria, innoculating with live bacteria, 5. 5. The Effects of Chemical Agents on Bacteria II:The Effects of Chemical Agents on Bacteria II:Antimicrobial Agents (Kirby-Bauer Method)Antimicrobial Agents (Kirby-Bauer Method)

Day 1Day 1

HEALTH AND SAFETY IN THE MICROBIOLOGY HEALTH AND SAFETY IN THE MICROBIOLOGY LABORATORYLABORATORY

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

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.


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.


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.


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.


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.)


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.

Protocols (a structured yet simple procedure) 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.

SterilisationSterilisation

Complete destruction of all microorganisms, Complete destruction of all microorganisms, including spores, from a given system (eg. including spores, from a given system (eg. Beaker containg liquid medium)Beaker containg liquid medium)

System must be closed – after sterilisation System must be closed – after sterilisation we must avoid recontaminationwe must avoid recontamination

““Flamming” in a bunsen burner (sterilisation Flamming” in a bunsen burner (sterilisation of loops and tweezer tips)of loops and tweezer tips)

Sterilisation with radiation (UV – wave length Sterilisation with radiation (UV – wave length 200 – 300 nm – non specific ionisation and 200 – 300 nm – non specific ionisation and irreprable damage) irreprable damage) Sterilisation of air and surfaces in a room Sterilisation of air and surfaces in a room

(aspetic boxes and operating theaters...)(aspetic boxes and operating theaters...)


Chemical sterilisationChemical sterilisation 30% peroxyacetic acid, or PAA, used on dry 30% peroxyacetic acid, or PAA, used on dry

objects. Submerged and then left to evaporate.objects. Submerged and then left to evaporate. PAA destroys cell membranesPAA destroys cell membranes


Dry Heat (160°C, 1h)Dry Heat (160°C, 1h) Glassware, bowls, pipettes, steel insturments... Glassware, bowls, pipettes, steel insturments... Vegatative bacteria die within minutes at 100°C, Vegatative bacteria die within minutes at 100°C,

but endospores remain!but endospores remain! Autoclaves are used - pressurisedAutoclaves are used - pressurised



Wet heat Standard pressure 1,5 atm (150 kPa) where

boiling point of water is cca 128 °C

MPA – Meat-Peptone-Agar (nutrient agar (1L)MPA – Meat-Peptone-Agar (nutrient agar (1L) Meat extract (10g)Meat extract (10g) peptone (enpeptone (enzzymatically hydrolysed meat, 10g)ymatically hydrolysed meat, 10g) NaClNaCl + agar (15 g) – 1,5 %+ agar (15 g) – 1,5 % pH 7.2 – 7.4pH 7.2 – 7.4

Day 1 – Media usedDay 1 – Media used

Day 1 - StepsDay 1 - Steps

1.1. Pouring petri dishes for later experimentsPouring petri dishes for later experiments2.2. Preparation of liquid medium, pipettePreparation of liquid medium, pipettess for later for later

experimentsexperiments3.3. Innoculate Innoculate real real samples on prepared agar platessamples on prepared agar plates4.4. The Effects of Chemical Agents on Bacteria II: The Effects of Chemical Agents on Bacteria II:

Antimicrobial Agents (Kirby-Bauer Method)Antimicrobial Agents (Kirby-Bauer Method)

Preparation of broth (Meat peptone broth)Preparation of broth (Meat peptone broth) Goal: learn how to make cotton plugs, prepare Goal: learn how to make cotton plugs, prepare

100100mml of bujon into two conical flasks, put l of bujon into two conical flasks, put cotton stopper in, and seal with aluminium foil cotton stopper in, and seal with aluminium foil (reduces escape of moisture during sterilisation)(reduces escape of moisture during sterilisation)


1.1. Preparation of petri dishes for later Preparation of petri dishes for later experiments:experiments: Goal: learn Goal: learn asepticaseptic technique and how to pour technique and how to pour

agar mediumagar medium


3. Innoculation of samples onto prepared agar3. Innoculation of samples onto prepared agar Finger/Toe print, glasses, handkerchiefFinger/Toe print, glasses, handkerchief Infusion/extract from top soilInfusion/extract from top soil Water (tap, distilled, and from puddle)Water (tap, distilled, and from puddle) Tounge printTounge print Open petri dish on the lab benchOpen petri dish on the lab bench Cleaning sponge Cleaning sponge Coins/moneyCoins/money

Each group will choose one of the above...Each group will choose one of the above...


Innoculating liquid samples onto medium

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

1.AMPICILLIN (beta-lactam antibiotic, cell wall) AMP 2.BACITRACIN (polypeptide, G+ cell wall) B 103.CARBENICILLIN (beta-lactam antibiotic, G-, P. aeruginosa) CAR 1004.ERYTHROMYCIN (macrolide antibiotic, enzyme synthesis) E 155.KANAMYCIN (aminoglycoside antibiotic, enzyme synthesis) K 306.GENTAMICIN (aminoglycoside antibiotic, enzyme synthesis) CN 107.NALIDIXIC ACID (quinolone antibiotic, Escherichia coli, Proteus, Shigella, Enterobacter, and Klebsiella) NA 308.NEOMYCIN (aminoglycoside antibiotic) N 309.PENICILLIN G (beta-lactam antibiotic, cell wall) P 1010.POLYMYXIN B (polypeptide, G-) PB 30011.STREPTOMYCIN (aminoglycoside antibiotic) S 1012.SULPHAMETHOX/TRIMETHOPRIM (COTRIMOXAZOL) SXT 2513.TETRACYCLINE (tetracycline antibiotics, enzyme synthesis) TE 3014.VANCOMYCIN (glycopeptide antibiotic, G+, ant. of last resort) VA 30

Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 10145), and Klebsiella pneumoniae (ATCC e13883)

microbiology practical goals 1. sterilisation, 2. aseptic technique, 3. preparing broth medium, 4....

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