Microbiology Practical
Day 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)
HEALTH AND SAFETY IN THE MICROBIOLOGY
LABORATORY
1  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.
HEALTH AND SAFETY IN THE MICROBIOLOGY
LABORATORY
 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 nonpathogenic strains alike.
 Always wash hands thoroughly, using
provided soap, before leaving the lab.
HEALTH AND SAFETY IN THE MICROBIOLOGY
LABORATORY
 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
 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
 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
 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)
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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.
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
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...)
Sterilisation
 Chemical sterilisation
 30% peroxyacetic acid, or PAA, used on dry
objects. Submerged and then left to evaporate.
 PAA destroys cell membranes
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
Sterilisation
 Wet heat
 Standard pressure 1,5 atm (150 kPa) where
boiling point of water is cca 128 °C
Day 1 – Media used
 MPA – Meat-Peptone-Agar (nutrient agar (1L)
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Meat extract (10g)
peptone (enzymatically hydrolysed meat, 10g)
NaCl
+ agar (15 g) – 1,5 %
pH 7.2 – 7.4
Day 1 - Steps
1.
2.
Pouring petri dishes for later experiments
Preparation of liquid medium, pipettes for later
experiments
3. Innoculate real samples on prepared agar plates
4. The Effects of Chemical Agents on Bacteria II:
Antimicrobial Agents (Kirby-Bauer Method)
Day 1 - Steps
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
1.Preparation of petri dishes for later
experiments:
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Goal: learn aseptic technique and how to pour
agar medium
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
The Effects of Chemical Agents on Bacteria II:
Antimicrobial Agents (Kirby-Bauer Method)
Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 10145), and Klebsiella
pneumoniae (ATCC e13883)
1.AMPICILLIN (beta-lactam antibiotic, cell wall)
2.BACITRACIN (polypeptide, G+ cell wall)
3.CARBENICILLIN (beta-lactam antibiotic, G-, P. aeruginosa)
4.ERYTHROMYCIN (macrolide antibiotic, enzyme synthesis)
5.KANAMYCIN (aminoglycoside antibiotic, enzyme synthesis)
6.GENTAMICIN (aminoglycoside antibiotic, enzyme synthesis)
7.NALIDIXIC ACID (quinolone antibiotic, Escherichia coli, Proteus, Shigella, Enterobacter, and Klebsiella)
8.NEOMYCIN (aminoglycoside antibiotic)
9.PENICILLIN G (beta-lactam antibiotic, cell wall)
10.POLYMYXIN B (polypeptide, G-)
11.STREPTOMYCIN (aminoglycoside antibiotic)
12.SULPHAMETHOX/TRIMETHOPRIM (COTRIMOXAZOL)
13.TETRACYCLINE (tetracycline antibiotics, enzyme synthesis)
14.VANCOMYCIN (glycopeptide antibiotic, G+, ant. of last resort)
AMP
B 10
CAR 100
E 15
K 30
CN 10
NA 30
N 30
P 10
PB 300
S 10
SXT 25
TE 30
VA 30