Download 226 lab 4

PHT 226 Lab# 4
•Culture media
• Streaking
Culture Media
The culture media is an artificial preparation •
contains the essential element and nutrient
in a proper concentration needed by the
microorganism to grow.
It may be:- •
Liquid (broth) •
Solid (containing agar) •
Semisolid (containing low conc of agar) •
Culture Media
Most common ingredients:-
•
Essential elements and nutrients. .1
Solidifying agents. .2
Objectives of bacterial culture:
bacterial morphology, physiology and To study •
pathogenic properties.
At the clinical level: •
Isolation of bacteria in pure culture .1
Identification .2
Antimicrobial susceptibility .3
Preparation of important bacterial products, e.g., •
toxins, antigens, vaccines … etc.
Essential Elements and Nutrients
A Source of Nitrogen:
(peptone)
meat extract,
yeast extract,
amino acids & in organic compound.
.1
•
•
•
•
Essential Elements and Nutrients
A Source of Carbon: .2
usually supplied in form of carbohydrates. •
Inorganic salts: .3
normally present in peptone but added in •
traces [e.g; Na, K, Ca, Mn, Fe, Co,
Zn…….etc]
NaCl ♙
Essential Elements and Nutrients
4. Buffers:
added to resist any changed in pH of the •
medium.
Blood and milk are naturally occurring 
buffers.
Solidifying Agents
Agar: it is the most common solidifying .1
agent, used in conc. * 1.5 – 2% ( solid
medium).
* 0.2 – 0.5%
(semisolid).
✍ it extracted from certain red marine algae,
and composed of complex carbohydrates
not easily broken down by most common
bacteria.
Solidifying Agents
2. Gelatin:
it is protein derivative added to nutrient broth 
in conc. of 12 -15% as a test for proteolytic
activity of bacteria.
Classification of Media
According to its content:-
•
Non synthetic complex medium; .1
contains extracts or digests of plant or •
animal tissues such as beef, peptone &
soybean digest.
Classification of Media
Synthetic Chemically defined medium;
contains known chemical compounds such
:as
amino acids,
sugar,
Vitamins & salts.
.2
•



Classification of Media
Living medium; .3
it is a living cell of animal or plant in which •
m.o could be cultivated such as viruses
(intracellular tissue culture.)
Types of culture media:
Simple media
Enriched media
Selective media
Indicator media
Selective and indicator media
•
•
•
•
•
Classification of Media
According to the purpose of use:- •
1- Simple [Basic] media;
contain only the essential or basic nutrients •
which support the growth of most common
bacteria.
✎ e.g;
Nutrient agar (plate, slant) •
Nutrient broth •
Classification of Media
2- Enriched media;
in addition to basic nutrients, it contains 
enriched substances such as blood, serum,
vitamins to support the growth of most
pathogens can’t grow on ordinary simple
media.
✎ e.g;
blood agar, 
Chocolate agar, 
Serum agar. 
Blood agar
Chocolate agar
(Neisseria spp.)
C.diphtheria
Classification of Media
3. Selective media;
mostly used in case of mixture. 
Contains substances which selectively enhance the 
growth of certain particular species and inhibit the
growth of undesired bacterial species.
These inhibitory substances include: dyes, heavy 
metals, chemicals, antimicrobial agents …
Examples of Selective Media
Mannitol salt agar; .1
it is selective medium for Staphylococcus •
species because it contains high conc. of
salt (about 7%).
2- Lowenstein Jensen medium
it is selective medium for M. tuberculosis;
because it contains malachite green dye. •
It contains also eggs which solidify the •
medium without agar and make it enriched
medium.
Examples of Selective Media
3- MacConkey agar:
it is selective medium for gram –ve bacteria •
because it contains bile salt which inhibit
the growth of gram +ve bacteria.
Examples of Selective Media
4- Cetrimide agar:
it is highly selective medium for 
pseudomonas species. It contains Cetrimide.
Cetrimide is a surfactant that inhibit the 
growth of all organism except
pseudomonas.
Classification of Media
4- Indicator media:
Contain substances which are affected by •
the growth of organism to produce
characteristic reaction such as indicators
which indicates acid production during sugar
fermentation.
Examples of Diagnostic Media
Mannitol salt agar; .1
It contains mannitol and phenol red indicator. 
S.aureus 
is the only strain of staphylococcus species can
ferment mannitol, leading to production of acid
which can detected by yellow color around the
growth resulting from changing the color of the
indicator.
 Growth with yellow color around the colonies
 Growth without change in the color of the medium
S.aureus
S.epidermidis
Examples of Diagnostic Media
2. MacConkey agar;
Gram –ve bacteria classified to: •
Lactose fermenter
Lactose non-fermenter
It contains neutral red indicator which detect acid •
production due to fermentation of lactose which done by
lactose fermenter gram –ve bacteria such as E-coli and
Klebsiella species to give rose pink colonies.
 Lactose fermenter
Lactose non-fermenter
Pink colonies
Pale colonies
Classification of Media
5. Selective and indicator media;
are both selective and diagnostic since they •
permit the growth of a few desired species
only & contain indicator which enable us to
differentiate between the species which are
able to grow on these media.
e.g.; Mannitol salt agar, •
MacConkey agar.
Media for Fungi
❀ Sabouraud dextrose agar.
similar in composition as those of bacteria
EXCEPT:
They are acidic in nature (PH 3 -6). .1
contain readily fermentable sugars
.2
e.g.; dextrose and maltose.
☞ Culture of Fungi usually incubate at lower
temperature than bacteria (about 25°C).
Anaerobic media
Fluid
thioglycolate:
It is anaerobic medium due to presence of:

Sodium thioglycolate and cystaine which act
as reducing agents.
Cooked
meat medium:
It is anaerobic medium due to presence of:

Meat particles (prepared from heart
muscles)

Hematin& glutathione in meat particles
act as reducing agents.
Anaerobic media
Anaerobic Jar
- The nutrient agar plates employed for routine culturing do
not contain reducing agents.
- They can be used to culture anaerobes if they are placed
inside an anaerobe jar ( a chamber from which the oxygen
is removed).
- Gas generator packets (e.g. GasPak) are placed in the jar
along with plates or tubes containing conventional media
destined to be incubated anaerobically.
- In order to assure that oxygen actually was removed from
the chamber, a strip of paper soaked in methylene blue dye
is included in the jar.
- It is blue when exposed to oxygen but will become
colorless (white) when oxygen is absent.
GAS PAK SYSTEM FOR ANAEROBIC CULTIVATION
Colony vs. Cell
Colonies
Cells
Introduction to Microbiological
Equipments and Materials
Inoculation: Culturing of sterile media with
m.o [Inoculation loop].
Incubation: Placing the culture into the
incubator at optimum temperature for growth.
Sterile: Free from any living microorganism
Contamination: Introduction of undesirable
m.o.
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Description of Colonies
Isolation of Pure Colonies of
Microorganism
“Streak Plate Method”
In natural environments, bacteria &
other m.o exist in mixed population.
“Streak Plate Technique”
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
and staph. aureus.

The streak Plate Method

Procedure:
Drop of the culture
Flame & Cool
S&S
Flame & Cool
Flame & Cool
Aseptic technique
Invert the plate and
Incubate for 24h at 37℃
The streak Plate Method
The streak Plate Method
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.
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Thank you