Preparation of Blood Agar Plate

Blood agar
 Enriched medium often used to grow fastidious
organisms and to differentiate bacteria based on
their hemolytic properties. Procedure for the Preparation of Blood Agar
 Differential medium that distinguishes bacterial
species by their ability to break down the red 1.Suspend 40.0 grams in 1000 ml distilled water or
blood cells included in the media. deionized water. (depends on the manufacturer)
 Used to grow a wide range of pathogens
2.Heat to boiling to dissolve the medium completely.
particularly those that are more difficult to grow
such as Haemophilus influenzae, Streptococcus 3.Sterilize by autoclaving at 15 lbs pressure (121°C)
pneumoniae and Neisseria species. It is also for 15 minutes.
required to detect and differentiate hemolytic
bacteria, especially Streptococcus species. 4.Cool to 45-50°C and aseptically add 5% sheep
 Differential media in allowing the detection of blood. (NOTE: Cooling the agar and warming the
hemolysis (destroying the RBC) by cytolytic blood are essential steps in this procedure. Hot agar
toxins secreted by some bacteria, such as certain can damage red blood cells, and cold blood can
strains of Bacillus, Streptococcus, cause the agar to gel before pouring.)
Enterococcus, Staphylococcus, and
Aerococcus. 5.Mix well and pour into sterile Petri plates. Avoid
 Usually prepared from Tryptic Soy Agar base formation of air bubbles. You must have warmed the
with 5% Sheep blood. blood to room temperature at the time of dispensing
 Blood agar is both enriched and differential. to molten agar base.
 About 5% of defibrinated mammalian blood
(human, sheep, or horse) is added to the 6.Dispense 15 ml amounts to sterile Petri plates
autoclaved basal media to prepare blood agar aseptically
medium. 7.Label the medium with the date of preparation and
 Human blood is discouraged because of the
give it a batch number (if necessary).
increased possibility of exposure to human
blood-borne pathogens such as HIV or hepatitis. Trypticase soy agar/Nutrient agar
 Blood Agar is used to grow a wide range of
pathogens particularly those that are more
difficult to grow such as Haemophilus
influenzae, Streptococcus pneumoniae and
Neisseria species.

Composition of Sheep Blood Agar Base

S.N Ingredients Gram/liter
1. Peptone 10.0 Storage and Shelf Life
2. Tryptose 10.0
3. Sodium chloride 5.0  Store below 30°C in tightly closed container and
4. Agar 15.0 the prepared medium at 2-8°C, preferably in
Final pH at 25°C: 7.3 ±0.2 sealed plastic bags to prevent loss of moisture.
The shelf life of thus prepared blood agar is up
to four weeks. Use before expiry date on the
label.
Principle and Interpretation

 Hemolysins are exotoxins produced by bacteria

that lyse red blood cells. The hemolytic reaction
 To the base medium, 5% sterile mammalian can be visualized on blood agar plates observing
(sheep/rabbit or hare/horse) blood is added after through the bright transmitted light.
autoclaving and before pouring onto the plates.
  Beta hemolysis is defined by a clear zone of
hemolysis under and around the colonies when
grown on blood agar. The clear zone appears as
a result of the complete lysis of the red blood
cells present in the medium, causing
denaturation of hemoglobin to form colorless
products.
 Other types of bacteria can reduce hemoglobin
to methemoglobin which produces a greenish
zone around the colonies and is called alpha
hemolysis.
 Gamma hemolysis is lacking haemolysis where
no change in the medium is observed.

Principle and Interpretation

 Sheep blood agar base with added sheep blood

was developed to allow maximum recovery of
organisms without interfering with their
haemolytic reactions. Sheep blood agar base was
formulated to be compatible with sheep blood
and give improved haemolytic reactions of
organisms.
 Casein enzymic hydrolysate and yeast extract
Preparation of Chocolate Agar Plate
provide nitrogen, carbon, amino acids and
vitamins.  Chocolate Agar Plate Chocolate agar is
 Peptic digest of animal tissue (PDAT) is the essentially the same as blood agar except that
nitrogen source. during preparation the red blood cells are lysed
 Sodium chloride (NaCl) maintains the osmotic when added to molten agar base.
balance.  As a result, the cell lysis releases intracellular
nutrients such as hemoglobin, hemin (“X”
factor), and the coenzyme nicotinamide adenine
dinucleotide (NAD or “V” factor) into the agar
which is utilized by fastidious bacteria.
 Red blood cell lysis gives the medium a
chocolate-brown coloration when prepared from
which the agar gets its name.
 The most common bacterial pathogens that
require this enriched medium for growth include
Neisseria gonorrhoeae and Haemophilus
species.
Salmonella Typhi on blood agar
 as amino acids, nitrogenous nutrients, and other vital
elements for the organism’s growth.
Purpose:
 Chocolate Agar Base, with the addition of
 Chocolate agar is used to isolate and cultivate
supplements, gives excellent growth of the
fastidious microorganisms such as Neisseria
fastidious organisms without overgrowth by
species and Haemophilus species.
contaminating organisms.
 A chocolate agar is also useful in isolating N.
 Casein and animal tissue digest that provides the
gonorrheae from both acute and chronic cases of
organism with nitrogenous nutrients, amino
gonococcal infections.
acids, and other elements essential for the
Composition of chocolate agar growth of the organisms.
 As Neisseria species are highly sensitive to toxic
1.Blood agar base- which is the solidifying agent substances such as fatty acids, hence the
2.Beef/meat extract – which provide sources of nitrogen, addition of corn starch helps neutralize possible
carbon, and vitamins toxic metabolites, while potassium phosphate
helps maintain a uniform pH during growth.
3.pH – which is usually adjusted to 7.3 at 25 °C (or 77  Sodium chloride maintains osmotic equilibrium
°F) thereby maintaining the integrity of cells.
4.Peptone – which serves as source of vitamins  Chocolate agar is also considered as a variant of
the blood agar plate, containing red blood cells
5.Sodium chloride – for maintaining a balanced that have been lysed by slowly heating to 80°C.
physiological environment as obtainable in living The heat inactivates enzymes which could
systems otherwise degrade NAD. The added
supplements provide the necessary X factor
NOTE: Chocolate agar is prepared from molten blood
(from Haemoglobin) and V factor (from Growth
agar, with only a slight modification in the protocol for
Supplement) required by the fastidious
preparing blood agar.
organisms.
Procedure of media preparation
Modification of Chocolate Agar
 Same as to preparation of BLOOD AGAR but
 Thayer-Martin agar: It is used for the selective
the preparation of chocolate agar is done by
isolation of N. gonorrhoeae and N. meningitidis.
slowly heating the blood agar medium
Thayer-Martin Media is a chocolate agar
containing red blood cells to a temperature of 80
supplemented with vancomycin, nystatin, and
C for initiating RBC lysis.
colistin to inhibit the normal flora, including non
 GC Agar supplemented with hemoglobin and
pathogenic Neisseria.
IsoVitaleX Enrichment (in place of yeast
 Chocolate Agar with bacitracin: CAP with
concentrate) provides an improved medium for
bacitracin is a selective medium used to improve
the isolation of microorganisms
the primary isolation of H. influenzae from
 Enrichment provides V factor (nicotinamide
specimens containing a mixed flora of bacteria
adenine dinucleotide, NAD) for Haemophilus
and/or fungi.
species
 Chocolate agar with GC base and growth
 Also provides vitamins, amino acids,
supplement: It is a medium that supports the
coenzymes, dextrose, ferric ions and other
special growth requirements (hemin and NAD)
factors which improve the growth of pathogenic
needed for the isolation of fastidious organisms,
Neisseria
such as H. influenzae, when incubated at 35-
Principle and interpretation 37°C in a 5% CO2 atmosphere
 Chocolate agar with TSA and growth
*If a supplement is added on a chocolate agar base, it supplements: It is a medium that supports the
would be a perfect growth place for fastidious special growth requirements (hemin and NAD)
organisms. For the organism to grow, various nutrients needed for the isolation of fastidious organisms,
are added such as casein and animal tissue, which serves such as H. influenzae, when incubated at 35-
37°C in a 5% CO2 atmosphere.
Colony characteristics in chocolate agar
Neisseria meningitidis Ingredients Grams/Litre
Casein enzymic hydrolysate 14.000
 Round, large, smooth, convex colorless to grey
Peptic digest of animal tissue 4.500
colony.
Yeast extract 4.500
 The colonies appear opaque on the plate with no Sodium chloride 5.000
obvious medium discoloration. Agar 12.500
 The colonies glistened with a defined edge Sheep Blood 5.000
Final pH [at 25° C] 7.3±2

Neisseria gonorrhoeae

 Pinkish to brownish translucent colonies.

 They have a smooth consistency with a defined
margin.

Haemophilus influenza

 The colonies appear to be non-hemolytic with

opaque cream to grey in color.

Streptococcus pneumoniae

 The chocolate agar plate has small greyish to

greenish colonies with the alpha-hemolysis
zone.
 S.N Organism Growth Colony Morphology Hemolysis
1. Neisseria meningiditis Good-luxuriant Grey and unpigmented colonies Non-hemolytic or γ-
that appear round, smooth, hemolytic.
moist, glistening, and convex,
with a clearly defined edge.

2. Salmonella Typhi Good-luxuriant Smooth colorless colonies that Non-hemolytic or γ-

are smooth, moist, and flat with hemolytic.
the diameter range of 2-4 mm.

3. Staphylococcus aureus Luxuriant Golden yellow colored circular, β-hemolytic.

convex and smooth colonies of
the diameter range of 2-4 mm;
opaque colonies with a zone of
hemolysis.

4. Staphylococcus Luxuriant Circular, colonies of the size 1-4 Non-hemolytic or γ-

epidermidis mm in diameter; grey to white- hemolytic.
colored with low convex
elevation; moist, glistening
colonies.

5. Streptococcus Luxuriant White-greyish-colored colonies β-hemolytic.

pyogenes with a diameter of > 0.5 mm; the
colonies are surrounded by a
zone of β-hemolysis that is often
two to four times as large as the
colony diameter.

6. Streptococcus Luxuriant small, grey, moist (sometimes α-hemolytic.

pneumonia mucoidal in encapsulated
virulent strains), colonies with
the characteristic zone of alpha-
hemolysis (green); due to
autolysis, often produces a
dimple-like zone of hemolysis
than the typical crater-like
appearance.
7. Pseudomonas Good-luxuriant Large colonies of the size 2-5mm β-hemolytic
aeruginosa in diameter; flat colonies that are
  grey to white-colored with an
undulate margin with a zone of
β-hemolysis.