Preparation of Blood Agar Plate
Blood agar
 Enriched medium often used to grow fastidious
organisms and to differentiate bacteria based on
their hemolytic properties.
 Differential medium that distinguishes bacterial
species by their ability to break down the red
blood cells included in the media.
 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. It is also
required to detect and differentiate hemolytic
bacteria, especially Streptococcus species.
 Differential media in allowing the detection of
hemolysis (destroying the RBC) by cytolytic
toxins secreted by some bacteria, such as certain
strains of Bacillus, Streptococcus,
Enterococcus, Staphylococcus, and
Aerococcus.
 Usually prepared from Tryptic Soy Agar base
with 5% Sheep blood.
 Blood agar is both enriched and differential.
 About 5% of defibrinated mammalian blood
(human, sheep, or horse) is added to the
autoclaved basal media to prepare blood agar
medium.
 Human blood is discouraged because of the
increased possibility of exposure to human
blood-borne pathogens such as HIV or hepatitis.
 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
 To the base medium, 5% sterile mammalian
(sheep/rabbit or hare/horse) blood is added after
autoclaving and before pouring onto the plates.
Procedure for the Preparation of Blood Agar
1.Suspend 40.0 grams in 1000 ml distilled water or
deionized water. (depends on the manufacturer)
2.Heat to boiling to dissolve the medium completely.
3.Sterilize by autoclaving at 15 lbs pressure (121°C)
for 15 minutes.
4.Cool to 45-50°C and aseptically add 5% sheep
blood. (NOTE: Cooling the agar and warming the
blood are essential steps in this procedure. Hot agar
can damage red blood cells, and cold blood can
cause the agar to gel before pouring.)
5.Mix well and pour into sterile Petri plates. Avoid
formation of air bubbles. You must have warmed the
blood to room temperature at the time of dispensing
to molten agar base.
6.Dispense 15 ml amounts to sterile Petri plates
aseptically
7.Label the medium with the date of preparation and
give it a batch number (if necessary).
Trypticase soy agar/Nutrient agar
Storage and Shelf Life
 Store below 30°C in tightly closed container and
the prepared medium at 2-8°C, preferably in
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
can be visualized on blood agar plates observing
through the bright transmitted light.
 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
provide nitrogen, carbon, amino acids and
vitamins.
 Peptic digest of animal tissue (PDAT) is the
nitrogen source.
 Sodium chloride (NaCl) maintains the osmotic
balance.
Salmonella Typhi on blood agar
Preparation of Chocolate Agar Plate
 Chocolate Agar Plate Chocolate agar is
essentially the same as blood agar except that
during preparation the red blood cells are lysed
when added to molten agar base.
 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.

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

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.