Morphologic Unknown, Cultivation

of Bacteria, Microbes in the

Environment, Transfer of
Bacteria:Aseptic Technique,
Isolation of Bacteria by Dilution
Mic. Lab

Cultivation of Bacteria
Bacteria needs carbon as sources of energy,
nitrogen, minerals and growth factors (organic
compounds such as vitamins or amino acids)
Two types of medium;
Broth (liquid)
Agar (solid)

Microbes in the Environment

MEDIA
Chemically defined medium: A medium whose exact
chemical composition is known. Glucose-Minimal salts
broth (Glucose,NaCl,NH4Cl,KH2PO4 etc.)
Complex media: Media for which the exact chemical
composition varies slightly from batch to batch.
Nutrient broth and agar (Nitrogen,Peptone,Yeast
Extract etc.) are commonly used complex media
Agar: An extract from marine red algae. Few microbes can
degrade agar so it remains solid during microbial growth.
Agar is not a nutrition for microorganism!

Microbes in the Environment

STERILIZATION
Steam Sterilization(Autoclav)

121 oC at 15 pound of pressure (15psi) for 15 min

Transfer of Bacteria: Aseptic

technique

Isolation of Bacteria by Dilution

Techniques
Three inoculation methods;

Spread Plate

Streak Plate

Pour Plate

Isolation of Bacteria by Dilution

Techniques
The spread and pour plate techniques are quantitative techniques that
allow determination of the number of bacteria in a sample
To determine the number of bacteria in the original sample, a plate with
between 25 and 250 colonies is selected. The number of the bacteria in
the original sample is calculated using the following equation;
Colony-forming units (per ml) =
Number of colonies/ (Dilution X Amount plated)

Disadvantages of Pour Plate:

Agar may be hot than estimated so it can
damage the microorganism
Bacteria can grow both on the surface and
inside of the plate. It will be hard to count and
strictly aerobe microorganisms may not have
the chance to get enough oxygen

Experiment-1
Take a Nutrient Agar and put anywhere in your
house.
Agars will be observed next week for
determining morphologi of microorganisms.

Experiment-2
Streak Plate Technique
Take a NA and streak a piece loop of bacteria
from original petri as described in previous

Experiment-3
Spread Plate Technique
Dilute the original stock up to 5 times.
Take 100 m from each sample and spread on
NA.Observe after incubation.

Experiment-4
Pour Plate Technique
Take 1 ml bacteria from original stock to sterile
petri plate and pour a warm NA on bacteria,
shake gently. Observe after incubation