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/microorganisms/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 : Sabourauds 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 manufacturers instructions: keep in dark and dry place, optimum temp. 15-25C 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 121C; 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 (45C)

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 37C 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 C waterbath Avoid over-heating and remove when it has melted Molten medium should be used ASAP, its 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 5C 3C 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 (121C 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

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

Scale Valve

121C ~ 249.8F 15
115C ~ 239F 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)

2.000 1.000 500 200 125 < 50

20 15 8 3 2 1.5

Volume (mL) 501-1.000

Equilibrium time (minutes) 20

251-500
101-250

15
10

Autoclave 121C-15

Autoclave 115 C- 30

Moist Heat Sterilization

Pressure

Relationship

kPa 172 202 242 304

Bar/atm 1.7 2.0 2.4 3.0

Temperature (C) 115-116 121-123 126-129 134-138

Holding time (minutes) 30 15 10 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