KONSEP DASAR PRAKTIKUM

MIKROBIOLOGI
Februari, 2016

Referensi
Basic Practical Microbiology A Manual –
Society for General Microbiology, 2006
http://www.microbiologyonline.org.uk/media/transfer/doc/sgm_bas
ic_practical_microbiology_2.pdf
© 2006 SGM
Referensi
 Tortora GJ, Funke BR, Case CL, 2013,
Microbiology an Introduction, 11th edition,
Benjamin Cummings, San Francisco, USA
 Denyer SP, Hodges NA, Gorman SP, 2011, Hugo
and Russell’s: Pharmaceutical Microbiology,
8th edition, Blackwell Science Ltd., Blackwell
Publishing Company, USA
 Cappuccino JG, Sherman N, 2014, Manual
Laboratorium Mikrobiologi (Terjemahan) Edisi 8,
Penerbit Buku Kedokteran EGC, Jakarta
 Anonim, 2005, Merck Microbiology Manual,
12th ed, Merck KGaA, Darmstadt, Germany
Five areas for consideration
 Preparation and sterilisation of equipment
and culture media.
 Preparation of microbial cultures as stock
culture for future investigations and
inoculum for the current investigation.
 Inoculation of the media with the prepared
culture.
 Incubation of cultures and sampling during
growth.
 Sterilisation and safe disposal of all cultures
and decontamination of all contaminated
equipment.
Good Microbiological Laboratory
Practice
To protect:
 practical investigations from becoming
contaminated with microbes from
external sources
 the operators (students, teachers
and technicians)
from the very small
possibility of
infection.
Equipment
Apparatus
Materials
Definition
 Culture medium
a nutrient material used to grow
microorganisms in the laboratory
 Inoculum
microbes that introduced into a culture
medium to initiate growth
 Culture
microbes that grow and multiply in or on a
culture medium
Criteria for a culture medium
 Right nutrient for specific microbes
 A sufficient osmotic balance
 Adjusted pH
 A sufficient O2
 Sterile  contain no living microbes
 The culture should be incubated at
proper temperature
Types of Culture Media
Intend of use
Consistency
Selective
Liquid (Broth) Differential
Semisolid
Enrichment
Solid (Agar)
Preservation
etc.

Nutrient Source
Chemically defined media
Complex media
Example of Media
Nutrient Agar :
Peptone from meat 5.0
Meat extract 3.0
Agar 12.0
Suspend 20 g / 1 L aqua
and autoclave

Nutrient Broth :
Peptone from meat 5.0
Meat extract 3.0
Suspend 8 g / 1 L aqua
and autoclave
Example of Media
PCA : Plate Count Agar
Peptone from casein 5
Yeast extract 2.5
D (+) glucose 1
Agar 14
Suspend 22.5 g / 1 L aqua and
autoclave

SDA : Sabouraud’s Dextrose

Agar
Peptone 10
D (+) glucose 40
Agar 15
Suspend 65 g / 1 L aqua and
autoclave
Storage of dehydrated media in containers

 Monitor entrance and first opening (mark the

date)
 Store under manufacturer’s instructions: keep in
dark and dry place, optimum temp. 15-25°C
 Check the expiry date
 Avoid absorption of water, oxidation and
contamination (check consistency, color,
clumping)
 Discard the media if: clumping or color changed
Weighing and Rehydration of Media
 Prepare the medium in a vessel about 1,2-3 times
the final volume.
 Use distilled or deionized water
 Weigh the powder quickly, accurately, and without
creating ‘clouds of dust’
 After opening, SCREW CONTAINER ASAP and
TIGHTLY
 Rehydrate with correct volumes of water
 Be sure that no residual media covers the wall of
vessels
 Check pH
TAKE CARE NOT TO INHALE POWDER AND
PROLONGED SKIN CONTACT
Sterilization of Media
 Dehydrated media are not free of contaminants
besides heat-resistant microorganisms
 Autoclave only 15 minutes at 121°C;
sterilization duration depends on the
size of load and containers
 Avoid excessive autoclaving to prevent
degradation reactions and breakdown of
nutrient constituents
 Sterilize heat labile substrates by filtration
 Add heat labile supplements with aseptic
precautions to the cooled medium (45°C)
Test for microbial contamination

• The samples to be tested should be at least 1

plates or tube OR 1% of the plates from the end
of a pouring or dispensing process
• The plates or tubes should be incubated for at
least 18 hours at 37°C or under the incubation
conditions which are used routinely for this
medium according to the specific standard
• Discard all sterility samples when the tests have
been complete
Preparation of sterilized media

 Liquid media should be cooled down to room

temperature.
 Agar media should be melted by placing it in a
47-50 ºC waterbath
 Avoid over-heating and remove when it has
melted
 Molten medium should be used ASAP, it’s
recommended that it should not be retained
on waterbath for more than 4 hours
Preparation of media in petri dishes

• Pour the molten agar culture medium into petri

dishes so as to obtain a thickness of at least 2
mm (e.g. for 90 mm diameter dishes, 15 ml of
agar are normally required) or as specified in
the international standard
• Allow the agar to cool and solidify by placing
the plates with lids in place on a cool, horizontal
surface
Storage of media in petridishes

• Use the solidified medium immediately or store

under conditions which prevents its composition
from being modified, i.e. in the dark and or in the
refrigerator at 5°C ± 3°C in the sealed bags
• Label the plates on the base with date of
preparation and or expiry date and identity
• In general, for the surface inoculation of a solid
culture medium, dry the plates, preferably with
the agar surface facing downwards
Shelf life of prepared media
 The shelf life of prepared media varies
considerably.
 It is generally recommended not to exceed 2-4
weeks of storage for plates and 3-6 months for
bottles and tubes,
unless otherwise specified in specific standards
or results of the laboratory shelf life validation
indicate a longer shelf life
Media and Culture disposal

 Both, contaminated and not used culture media

must be disposed in a way which it safe and
meets state or national regulations.
 A heat treatment disinfection using an autoclave
(121°C for 30’) is particularly important before
cleaning or disposal are carried out.
 A chemical disinfection should only be carried
out in exceptional cases
 The MSDS provides detailed information on
disposal of each medium
Media and Culture disposal

• If suitable incinerator is available, the culture can

also be killed and destroyed by burning
• CHEMICAL DISINFECTION is carried out
with appropriate disinfectants.
• ROOMS and equipment can be decontaminated
by fumigation with formaldehyde gas, ozone or
UV radiation
• Wash equipment only after it has been
decontaminated. After washing, rinse all
equipment with deionized water
Material Safety Data Sheet
Chemical hazard of culture media

 Media which contain hazardous and or toxic

agents must be handled with care. The
dispersion of a powder may give allergic or
other reactions to the laboratory personnel
 Selenite (SCB) for Salmonella; will damage liver,
kidney, CNS, allergic
 Lithium Cl, Sodium desoxycholate, malachite
green, fuschin (EA), Tellurite, etc
Chemical Hazard of Culture Media

Irritant (Xi)
Substances with this symbol irritate skin,
eyes and respiratory organs.

(Very) Toxic (T+,T)

Inhalation, swallowing or absorption through
the skin can cause severe illness and in
certain cases death.

Harmful (Xn)
These substances can cause some
discomfort if absorbed into the body.
Sterilization
 Destruction of all forms of microbial life
including endospores
 Sterilizing agent is called sterilant
 Usage in pharmacy: product parenteral
administration
 Sterility of media and tools prior
used are an obligatory
Sterilization methods
 Moist heat
 Dry heat
 Radiation
 Gas
 Filtration
Moist Heat Sterilization
Moist Heat Sterilization
Scale Valve

121°C ~ 249.8°F – 15’

115°C ~ 239°F – 30’
Steps in moist heat sterilization

• Decontaminate, clean, and dry all instruments;

open or unlock all jointed instruments
• If instruments and other items are to be
wrapped before steam sterilization, use two
layers of paper, newsprint, or cotton.
• Arrange all packs in a way that allows steam to
circulate freely; do not overloaded
• Do the sterilization process
• Dry until the water is removed completely
• Storage
Times in moist heat sterilization

• Heating
• Expulsion of the air
• Raising of the temperature
• Equilibrium
• Sterilization / Holding time
• Additional sterilization assurance (0.5 from
equilibrium)
• Cooling
 Equilibrium time

Volume Equilibrium time

(mL) (minutes) Volume Equilibrium time
2.000 20 (mL) (minutes)
501-1.000 20
1.000 15
251-500 15
500 8
101-250 10
200 3
125 2
< 50 1.5 Autoclave 121°C-15’

Autoclave 115º C- 30’

 Moist Heat Sterilization

Pressure Temperature Holding time

kPa Bar/atm (°C) (minutes)
Relationship

172 1.7 115-116 30

202 2.0 121-123 15
242 2.4 126-129 10
304 3.0 134-138 3
 Moist Heat Sterilization

 Steam must contact all surfaces.

 Always sterilize instruments and other items for
the correct amount of time at the correct
pressure and temperature
 Be sure items are completely dry before
removing them from the autoclave
• Dry hypochlorites, or any other strong
oxidizing material, must not be autoclaved with
organic materials such as a paper, cloth, or oil:
POSSIBLE EXPLOSION
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