Blood Agar Base (Infusion Agar)
Intended Use:
Recommended for isolation and cultivation of many fastidious pathogenic micro-organisms like Neisseria, Streptococci
after addition of blood from clinical and non-clinical specimens.
Composition**
Ingredients Gms / Litre
HM peptone B# 10.000
Tryptose 10.000
Sodium chloride 5.000
Agar 15.000
Final pH ( at 25°C) 7.3±0.2
**Formula adjusted, standardized to suit performance parameters
# Equivalent to Beef Heart peptone
Directions
Suspend 40.0 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C and aseptically add 5% v/v sterile defibrinated blood.
Mix well and pour into sterile Petri plates.
Principle And Interpretation
Blood Agar Base is a highly nutritious medium generally used as a basal medium for preparing blood agar by supplementation
with blood. It can also be used as general-purpose media without the addition of blood.
Blood Agar Base media can be used with added phenolphthalein phosphate (1) for the detection of phosphate
producing Staphylococci, with added salt and agar for assessment of surface contamination on equipment and pig carcass
(2) and to determine salinity range of marine Flavobacteria (3). It can also be used for preparation of Salmonella Typhi
antigens (4). Blood Agar Base is recommended by APHA (5) and Standard Methods (6,7) for testing of food samples.
HM peptone B and tryptose provides carbon, nitrogen, amino acids and vitamins. Sodium chloride helps in maintaining the
osmotic equilibrium of the medium. Addition of blood makes the medium more nutritious by providing additional growth
factors required by fastidious organisms. It also helps in visualizing the haemolytic reactions. However, haemolytic reactions
depend on the animal blood used. Sheep blood gives best results for Group A Streptococci (8). But sheep blood fails to
support growth of Haemophilus haemolyticus since sheep blood is deficient in pyridine nucleotides. However when horse
blood is used H. haemolyticus colonies produce haemolysis and mimic Streptococcus pyogenes (9).
Type of specimen
Clinical material : Throat swabs, vaginal secretions and other pathological material; food samples.
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (10,11).
For food samples, follow appropriate techniques for sample collection and processing as per guidelines (5).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear
protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while
handling specimens and culture. Standard precautions as per established guidelines should be followed while handling
clinical specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
1.Addition of sheep blood is recommended to detect haemolysis. This medium does not support the growth of
H.haemolyticus.
2.Addition of Horse blood or rabbit blood to base medium supports growth of H.haemolyticus but resemble beta-haemolytic
Streptococci and hence must be confirmed.
3.Haemolytic pattern varies with the source of blood used.
4.Other tests must be carried out in conjunction for confirmation.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored
at recommended temperature.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Basal medium : Light amber coloured clear to slightly opalescent gel. After addition of 5% v/v sterile defibrinated blood :
Cherry red coloured opaque gel forms in Petri plates.
Reaction
Reaction of 4.0% w/v aqueous solution at 25°C. pH : 7.3±0.2
pH
7.10-7.50
Cultural Response
Cultural characteristics observed with added 5% w/v sterile defibrinated blood,after an incubation at 35-37°C for 18-48
hours.
Organism Inoculum
(CFU)
Growth w/o
blood
Recovery w/o
blood
Growth with
blood
Recovery with
blood
Neisseria meningitidis
ATCC 13090
50-100 fair 40-50% luxuriant =70% none
Staphylococcus aureus
subsp. aureus
ATCC 25923 (00034*)
50-100 good 50-70% luxuriant =70% beta
Staphylococcus epidermidis
ATCC 12228 (00036*)
50-100 good 50-70% luxuriant =70% none
Streptococcus pneumoniae
ATCC 6303
50-100 fair-good 40-50% luxuriant =70% alpha
Streptococcus pyogenes
ATCC 19615
50-100 fair-good 40-50% luxuriant =70% beta
Haemolysis
Key : (*) Corresponding WDCM numbers.
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label.
On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to
the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in
dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after
use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with
clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (10,11).
