PHT 381
Lab# 4
A Culture medium:❊ An artificial preparation which
contains the essential elements and
nutrients needed by the m.o
to grow. (most bacteria &fungi)
❊ Strict intracellular organisms (e.g.,
some bacteria & all viruses)→ only
cultures of living eukaryotic cells.
❊ It may be:
•
Liquid (broth)
•
Solid (containing agar)
•
Semisolid (containing low conc of agar)
Most common ingredients:1. Essential elements and nutrients.
2. Solidifying agents.
Inoculation:
Culturing of sterile media with m.o
[Inoculation loop].
Incubation:
Placing the culture into the incubator
at optimum temperature for growth.
Growth:
Multiplication (↑number) to quantities
sufficient to be seen by naked eye..
Bacterial growth in the lab has 2 main
forms:
1- Development of Colonies ( the
macroscopic products of 20-30 cell
divisions of a single bacterium on solid
media)
2- Turbidity (macroscopic clumps)
of a clear fluid medium.
Bacterial Growth
1- Macroscopical Examination
• (colony morphology):
• Characters of colonies.
• Hemolysis on blood agar.
• Pigment production.
2- Microscopical Examination:
• Examination of wet mount preparation.
• Examination of stained preparation.
3-Biochemical Tests:
(The ability to attack various substances
e.g., carbohydrate breakdown;
or to produce particular metabolic
products e.g., enzymes.
4-Additional Tests:
such as seriological tests
Colony vs. Cell
Colonies morphology
(Macroscopical
examination)
Cells
(Microcopical
examination)
Colony vs. Cell
Colonies morphology
(Macroscopical
examination)
Cells
(Microcopical examination
• Contamination:
Introduction of undesirable m.o.
• Asepsis:
Processes designed to prevent m.o. from
reaching a protected environment.
• Aseptic technique: Practices used by
microbiologists to exclude all organisms from
contaminating media or contacting living
tissues.
• An aseptic technique must be
used when inoculating culture media
to:
1- prevent contamination of cultures
2- prevent infection of laboratory workers
and enviroment.
Isolation of Pure Colonies of
Microorganism
“Streak Plate Method”
In natural environments, bacteria & other m.o
exist in mixed populations.
To study the cultural, morphological, and
physiological characteristics of an individual
organism, it is essential, first of all, that the
organisms are separated from other species
i.e. we must have pure culture of the microorganism.
“Streak Plate Technique”
Streak plate method is one of the most
frequently used methods of getting a pure
culture from a mixed culture.
The individual cells are separated from each
other by certain distance on the surface of
the agar.
After incubation, each single deposited cell
divide many times and finally form visible
mass of growth “COLONY”.
The streak Plate Method
• The culture prepared from a single type
of colony is regarded as a pure culture.
• The streak Plate Method is used for:
Checking the purity of a bacterial culture.
Isolating individual species from a mixture
culture.
The streak Plate Method
• Objective:for isolation of individual species of a mixed
broth culture.
• Materials: Nutrient agar plate.
Mixed broth culture of
Serratia marcescens
aureus.
and Staph.
The streak Plate Method
• Procedure:
Drop of the culture
Flam & Cool
S&S
Flam & Cool
Flam & Cool
Aseptic technique
Invert the plate and
Incubate for 24h at 37℃
The streak Plate Method
The streak Plate Method
Description of Colonies
Sources of Contamination
• Objective: To identify some of the sources
of contamination present in the lab.
✔ in order to avoid them
• Contamination from hands.
• Contamination from breath.
• Contamination from air.
• Contamination from bench.
Sources of Contamination
1. Contamination from hands:
 Sterile nutrient agar plate
a
b
 a- unwashed
b- washed with water
c- disinfected with alcohol
c d
d- control
 incubate the plate Inverted at 37°c for 24
hr.
 Record the appearance of the plate
Sources of Contamination
2. Contamination from breath:
 Take a sterile nutrient agar
plate
 Hold it in front of your mouth
 Cough and breath vigorously
 Invert the plate and incubate
for 1 day at 37 °c
 Record the appearance of the
plate.
Sources of Contamination
3.Contamination from air:
 Expose one sterile nutrient
agar plate on the bench for
30 min
 Invert the plate and incubate
for 1 day at 37 °c
 Record the colonial
appearance
30 min
Sources of Contamination
4.Contamination from bench:
 Take a sterile nutrient agar plate
and mark out 2 sections on its base
 Take a swab from unclean part of
the bench and press it over one
section
 Take another swab from cleaned
part with disinfectant and press it
over the second section
 Invert the plate and incubate for 1
day at 37 °c
 Record the result.
uc
/////////
c
/////////