Pharmaceutical

Microbiology

-Lab.Work-

Culture Media

References

Tortora GJ, Funke BR, Case CL, 2007, Microbiology an

Introduction, 9th edition, Benjamin Cummings, San

Francisco, CA 94111, USA

Madigan MT, Martinko JM, 2006, Brock Biology of

Microorganisms, 11th edition, Pearson Education Inc.,

USA

Merck, Merck Microbiology Manual 12th ed, Merck.,

Europe

Cabri, 1998, Laboratory Procedures for

Microorganisms, Available from:

http://www.cabri.org/guidelines/micro-

organisms/M203Ap1.html

Merck, 2007, Safety in Microbiology Laboratory,

Surabaya, December 18th

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

Consistency

Liquid (Broth)

Semisolid

Solid (Agar)

Intend of use

Selective

Differential

Enrichment

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

• 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

Media and Culture disposal

Material Safety Data Sheet

Material Safety Data Sheet

Staphylococcus aureus - Material Safety

Data Sheets (MSDS).htm

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

Valve Scale

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

Autoclave 115º C- 30’

Autoclave 121°C-15’

Volume

(mL)

Equilibrium time

(minutes)

2.000 20

1.000 15

500 8

200 3

125 2

< 50 1.5

Volume

(mL)

Equilibrium time

(minutes)

501-1.000 20

251-500 15

101-250 10

Moist Heat Sterilization

Pressure Temperature

(°C)

Holding time

(minutes) kPa Bar/atm

172 1.7 115-116 30

202 2.0 121-123 15

242 2.4 126-129 10

304 3.0 134-138 3

Rela

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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

Moist Heat Sterilization