Culturing Microorganisms M 265 General Microbiology Lab

Yasmine Al- shboul

In nature microorganisms exist as mixed population of many different types. However our knowledge of microbiology has come through study of isolated species, grown in environments free from contamination by other living forms. Like all other living forms, microorganisms need suitable nutrients and environments.The culture medium must contain essential nutrients for the growth of a microbial culture ,and it must provide suitable surroundings for growth : the proper Ph, osmotic pressure, atmospheric oxygen, and other factors.

Nutritional Materials in Culture Media:

Water. Carbon Source: Usually glucose. Nitrogen Source: Either inorganic or organic e.g. beef extract or proteins. Buffer System: e.g. carbonate or phosphate buffer. Minerals: Required in small amounts e.g. iron, sulfur, phosphorus, etc . Types of Media

According to composition: Chemically Defined (Synthetic): Exact chemical composition is known e.g. Minimal Salt Medium. Complex (Undefined) Medium: Contains complex materials of biological origin such as Blood, Milk, Yeast, or Beef Extract.

 Synthetic media

 According to Agar (Solidifying Agent) Content:

Agar: Hydrocolloid derived from Red Algae. Physical Properties of Agar: Melts at 100 C and remains liquid until cooled to 40oC, it can't be metabolized by most bacteria. Liquid Media (Broth): Used for growth of pure cultures e.g. Tripticase Soy Broth (TSB), Nutrient Broth (NB) Semi-Solid Media: <1% Agar e.g. Motility Media. Solid Media: 1.5-1.8% Agar, for isolation of pure cultures, colony counting, etc, e.g. Tripticase Soy Agar (TSA), Nutrient Agar (NA).

 Agar agar, popular in Asia, is a gelatinous substance derived from red algae .

 Solid Media Forms:

Deep Agar Position: Used for bacterial storage & for studying the gaseous requirements of organisms. Slanted Agar Position: Used for a variety of purposes. Agar Plate Media (Petri Dishes): Large surface area, used for isolation, studying of pure cultures and for subculture.

Deep solid agar in tube

Slant agar

According to Purpose:
All Purpose Media (Simple): Supports growth of most organisms e.g. NA or NB. Enriched Medium: A base medium + special supplements such as Blood, Vitamins, and Amino Acids e.g. Blood Agar (BA) and Chocolate Agar (CA). Differential Medium: More than one organism can grow, but these organisms can be differentiated from each other based on color change or other criteria e.g. Blood Agar, KIA. Selective Medium: Only allows one or more species to grow and suppresses others by incorporating dyes, antibiotics, bile salts, or by adjusting PH or Temperature e.g. SS Agar. Selective differential Medium: Inhibits growth of unwanted bacteria and differentiates between them e.g. MacConkey Agar (MA), Manitol Salt Agar (MSA). Enrichment-Differential Medium: e.g. Blood Agar: (Alpha), (Beta), (Gamma)Hemolytic.

Chocolate Agar

Blood agar

 Mannitol salt agar

 MacConkey Agar

 SS Agar

Media Preparation: Broth Media: Weigh dehydrated media and dissolve in a specified amount of water. Boil until clear. Dispense in test tubes. Close test tubes. Autoclave at 121oC under 15lbs pressure for 15Min.

Agar Media:
Weigh dehydrated media and dissolve in a specified amount of water. Boil until clear.

For Test tubes: Dispense in test tubes 1/3 or 3/4 full Close test tubes Autoclave at 121C under 15 lbs pressure for 15 Min. Allow test tube to cool at the appropriate position For Petri Dishes: Close flask with cotton and aluminum foil to prevent vaporization. Autoclave at 121oC under 15lbs pressure for 15Min. Cool to 50oC. Remove cap, Flame the mouth, and pour the liquid agar into sterile Petri Dishes. Cover the Petri Dishes and allow cooling

Heat Labile (Sensitive) compounds

that are to be added to the medium should be sterilized by filtration and then added to the medium at 50oC.

Methods of Sterilization: Sterile = Free from viable organisms. Substances are either Sterile or Non-Sterile. Sterilization: is the killing or removal of viable organisms by physical of chemical methods.

Chemical Sterilization Methods

Alcohol 70% Antiseptic and Disinfectant. Formalin (Formaldehyde): Disinfectant. Chlorine (Sodium Hypochlorite): Disinfectant. Ethylene Oxide Gas: used for contaminated medical tools.

Physical Sterilization Methods: 1. Heat:

The common and most important method used. Sterilization requires consideration of temperature, and duration. We should know the heat susceptibility of an organism to know what heat treatment method to apply. Most

Incineration: used for disposable material e.g needles, swabs, gauze. Dry Heat (Hot Air Oven): 160-180oC 1-3 Hours, used for glassware, metal tools, and objects that won't melt. Autoclaving (Steam under pressure or pressure cooker): 121oC/15lbs/15min. Multipurpose. Heat labile substances will be denatured and destroyed. Boiling: 100oC for 30min. Kills everything except some spore forming bacteria. Moist Heat (Tyndalization): Denatures and coagulates proteins, kills vegetative bacteria, yeast and mold. Pasteurization: Kills vegetative forms wile preserving the flavor of foods.

Filtration:
The filter has a pore size smaller than bacteria. Used for heat labile materials such as sugars and urea. e.g. Millipore filter, cellulose acetate disk.

Irradiation (Microwave, UV, X-ray): destroys

microorganisms, many organisms responsible for food storage are easily killed by irradiation.