Transcript 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
deep
Slant
Plate
(Petri dish)
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