TYPES
OF BACTERIOLOGICAL MEDIA USED TO CULTURE SAMPLES
- · Cystine lactose electrolyte deficient (CLED) agar.
- ·
Chocolate
Agar
- ·
Blood
Agar
- ·
MacConkey
Agar
- · Mueller – Hinton agar (MHA)
- · Cary- Blair medium
- · Gonococci agar
PREPARATION
OF BACTERIAL AGAR PLATES
1 1. Blood agar
This is a general purpose medium capable of growing a
range of microorganisms of clinical significance. The medium gives good
colonial appearances, hemolysis patterns and pigment production.
Procedure
a)
0.5g of blood agar base was suspended in 1 liter of
distilled water.
b)
The mixture was boiled to dissolve completely.
c)
The solution was sterilized by autoclaving at 1210c
for 15 minutes.
d) The base was
cooled to 500c and sterile human blood was added to make a
concentration of 7%.
e)
The solution was mixed with gentle rotation and poured
into the petri dishes.
f) A flame was
carried with the Bunsen burner over the surface of the agar to remove air
bubbles as a final step.
Results and observation
|
Positive
controls: |
Expected
results |
|
Staphylococcus aureus |
Good growth;
grey white colored colonies |
|
Streptococcus pyogenes |
Good growth;
pale colonies; beta hemolysis |
|
Streptococcus pneumoniae |
Good growth;
green grey colored colonies; alpha hemolysis |
|
|
|
|
Negative control: |
|
|
Uninoculated
plate |
No change |
2.
Chocolate Agar
This medium is
suitable for the growth of fastidious organisms such as Haemophilus influenzae, Pneumococci and Neisseriae. During heating
the red cells are ruptured and nutrients are liberated.
Procedure
a)
0.5g of blood agar base was suspended in 1 liter of
distilled water.
b)
The mixture was boiled to dissolve completely.
c)
The solution was sterilized by autoclaving at 1210c
for 15 minutes.
d)
The base was cooled to 500c and sterile human blood
was added to make a concentration of 7%.
e)
The solution is then heated it in a water bath to 800C
with frequent mixing until the medium has a chocolate color.
f)
The agar is then poured into petri dishes.
g)
A flame is run over the surface of the agar to remove
air bubbles as a final step.
Results and observation
|
Positive control: |
Expected results |
|
Haemophilus influenzae |
Good growth |
|
Negative control: |
|
|
Uninoculated
plate |
No change |
Figure 1.3: Bacterial growth in Chocolate agar
3.
Cystine lactose electrolyte deficient (CLED)
agar
CLED medium is a valuable non inhibitory diagnostic
medium for urinary bacteria as it supports the growth of all urinary pathogens.
It is recommended for diagnostic urinary bacteria. It gives good colonial differentiation
and clear diagnostic characteristics. The absence of electrolytes inhibits the
swarming of Proteus species. Cystine
is added for growth of organisms that require it. Differentiation of lactose
fermenters and non-lactose fermenters is achieved by using Bromothymol blue as
a pH indicator.
Procedure
a)
10.8g of the medium is suspended in 1 liter of
distilled water.
b)
The mix was boiled to dissolve completely.
c)
Sterilized by autoclaving at 1210C for 15 minutes.
d) The solution
was poured into petri dishes.
e)
A flame was moved over the surface of the agar to
remove air bubbles.
Results and observation
|
Positive controls: |
Expected results |
|
Proteus mirabilis |
Good growth;
blue colonies; no swarming |
|
Staphylococcus aureus |
Good growth;
yellow colonies |
|
|
|
|
Negative control: |
|
|
Uninoculated
medium |
No change |
Figure: bacterial
growth in CLED agar
4.
MacConkey Agar (M1)
This is a useful
selective, differential primary plating medium for the cultivation of
enterobacteria. It contains
bile salt to inhibit non
intestinal bacteria and lactose with
neutral red to distinguish lactose fermenting from non-lactose fermenting organisms. The omission of NaCl from the medium
prevents the spreading of Proteus species.
Procedure
a)
50g of the medium is suspended in 1 litre of distilled water.
b)
The solution is boiled to dissolve completely.
c)
Sterilized by autoclaving at 1210C for 15 minutes.
d)
Prepared agar is then poured into petri dishes.
e)
A flame is moved over the surface of the agar to
remove air bubbles.
Results and observation
|
Positive controls: |
Expected results |
|
Enterococcus faecalis |
Good growth;
red colored colonies |
|
Staphylococcus aureus |
Good growth;
pale pink colored colonies |
|
|
|
|
Negative control: |
|
|
Uninoculated
medium |
No change |
Figure: Bacterial growth in MacConkey agar
5)
Mueller-Hinton agar (MHA)
Muller-Hinton agar is a transparent medium that is
recommended for antimicrobial susceptibility testing. The composition of the
medium may vary from batch to batch which could affect zones of inhibition
Procedure
a) 38g of the
medium is suspended in 1 litre of distilled
water.
b) The mixture is
boiled to dissolve completely.
c)
The solution is then sterilized by autoclaving at 1210C
for 15 minutes.
d) The agar is
poured into petri dishes). The depth of the medium should be 4mm.
e) A flame is
moved over the surface of the agar to remove air bubbles.
Results and
observation
|
Positive controls: |
Expected results |
|
Escherichia coli |
Good growth;
pale straw colored colonies |
|
Pseudomonas aeruginosa |
Good growth;
straw colored colonies |
|
Staphylococcus aureus |
Good growth;
cream colored colonies |
|
|
|
|
Negative
control: |
|
|
Uninoculated
medium |
No change |
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