Practical Microbiology& Immunology

Practical Microbiology& Immunology

Microbiology • Microbiology is the science dealing with microorganisms • We are going to study microorganisms: Microscopically Macroscopically

Our session today • Culture media • Aseptic technique • Sources of contamination

Culture Medium • Definition: It is a medium used for growing microorganisms, such as bacteria or yeast. • Forms: liquid, solid, semi solid

Essential requirements in culture media • Any culture medium must contains: -A source of energy -Sources of carbon, nitrogen, sulfur, phosphorus -Minerals, e.g., Ca2+, Mg2+, Na+ -Vitamins and growth factors - Water

Classification of culture media according to chemical composition • Synthetic media (chemically defined): exact chemical composition is known • Complex media: Contains ingredients whose exact chemical composition is not known Usually, bacteria are grown in complex media

Examples of ingredients added in complex media • Beef extract : concentrate of hot aqueous infusion of fresh beef • Peptone: Spray dried hydrolysate of various proteins • Yeast extract: Spray dried water soluble autolysed yeast cells

For solid medium, A solidifying agent is incorporated in the media: Agar (1-2%) • Agar agar:It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae or seaweed. • Not digested by M.O • Agar melts in a water bath at 98 C, and solidifies at 42 C

Basic media • These are media which may be used for cultivation of most ordinary microorganisms • May be in liquid form ex: Nutrient broth : composed of beef ext+ Peptone+ Nacl • May be in a solid form ex: Nutrient agar: similar to nutrient broth but supplemented with 1-2% agar

Solid culture media Plate (Petri dish) deep Slant

Inoculation • Inoculation: • Surface:Streaking • using an inoculation loop • Seed: Suspension of M.O is put in the plate Then agar is poured or M.O is mixed with agar at suitable temperature.

Incubation • plate is incubated, usually for 24 to 36 hours, to allow the bacteria to reproduce. At the end of incubation there should be enough bacteria to form visible colonies in the areas touched by the inoculation loop.

M.O are widely distributed around us How to avoid contamination of our work with pure culture?

Aseptic technique • the procedures used by microbiologists to prevent microbial contamination of themselves, which may result in infection, contamination of the environment they are working in and contamination of the specimen they are working on, which is especially important when a pure culture is desired.

Aseptic technique involves: 1- working in 20 cm diameter around a blue flame (sterile zone) 2-Never leave a culture dish open, even for a short time ,When it is necessary to open a dish, keep the lid close to the dish, and keep the lid between your face and the agar surface. 3- For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum. 4-Flame a loop or needle to red-hot just prior to use, burning off any organic material ,Cool the loop by touching the sterile agar or liquid surface prior to touching a culture

Aseptic technique • Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking culture from it • Use sterile glass wares • Use sterile culture media

Practical work Sources of contamination • Materials one sterile petri dish one molten nutrient agar tube • Procedure 1- Pour the nutrient agar tube at the suitable temperature aseptically into the provided plate 2- Leave the nutrient agar plate to solidify 3- Expose the agar plate to one of the following source of contamination:

Practical work Sources of contamination - Air - Forced air - Skin touch - cough - a piece of hair or cloth - drop of water 4- A student in each bench will perform a control plate 5- Label the cover of the plate with your name, nb, source of contamination 6- Incubate the plate inverted , except for water containing plates

Pouring agar

Practical Microbiology& Immunology