BLOOD CULTURE:

Blood culture is the most specific test to detect bacteremia. Blood culture is indicated in the diagnosis of following conditions:
1. Enteric fever
2. Bacterial endocarditis
3. Pyrexia of unknown origin (PUO)
4. Bacterial meningitis
5. Brucellosis
6. Septicaemia
7. Pneumonia
8. Epiglottitis

Volume of blood collected:
1-2 ml in neonates
2-3 ml in infants
3-5 ml in children
10-20 ml in adults

Method of collection:
After the vein is located, the overlying skin is cleaned with spirit soaked in cotton. Iodine may be applied for a minute and then cleaned again with spirit. The desired amount is then aseptically drawn with the help of needle and syringe. Vacuum blood collection systems help reduce contamination.

Number and timing of blood specimens:
� Multiple blood specimens are obtained over few hours for endocarditis. 
� Paired specimens may be drawn at the same time but at two different sites. 
� It is rarely necessary to culture more than three sets of samples from each patient.
� Two sets of each sample may be taken; one for aerobic culture and other for anaerobic culture.

Highest success rates are achieved by collecting sample of blood just as the patient's temperature begins to increase. In patients with septicaemia and conditions causing continuous bacteremia, timing is less important.

The purpose of collecting multiple specimens is to:
o Increase the chances of isolation of the bacteria in especially where there is intermittent bacteremia 
o Differentiate contaminants from the pathogens.

In the diagnosis of enteric fever, clotted blood can also be accepted (Clot culture) where the clot is lysed and then inoculated into the blood culture medium.

Blood culture media:
1. Glucose broth: useful in endocarditis
2. Bile broth: useful in enteric fever
3. Brain heart infusion broth: multipurpose medium
4. Trypticase soy broth
5. Columbia Broth

The blood is inoculated into the bottle containing blood culture medium in a blood to broth ratio of 1:10. But, in case of children, where the amount of blood drawn is little a ratio of 1:5 may be achieved.
The commonly used anticoagulants are citrate, oxalate, heparin and sodium polyanethol sulphonate (Liquoid). Liquoid serves to neutralize antibiotics and antibacterial factors such as complement.

The purposes of inoculating blood into the culture medium are:
o Since density of bacteria in the blood is usually very low, this increases the number of organisms introduced into the bottle.
o Dilutes any antibacterial substances, including antibiotic present in the blood
o Presence of blood increases the nutrient content of the medium

Incubation:
The blood culture bottles are incubated at 35-37^oc in the incubator. Cultures for Brucella must be incubated at an environment of 10% CO₂.

Detection of growth in medium:
a) Turbidity in the medium.
b) Hemolysis.
c) Presence of gas.
d) Formation of microcolonies in the sedimented blood cells.

Identification of the growth:
To identify the organism, the blood has to be sub-cultured on to the solid medium. Commonly used media are the McConkey's agar and the blood agar.
In the diagnosis of Brucellosis, a biphasic medium (Castaneda method) is adopted.
If the sub-cultures yield no growth, the blood culture bottles are incubated further for a period of 7 days (or up to 2 weeks when brucellosis is suspected).
The bacterial isolate are then identified based on the Gram's reaction, cultural, biochemical and antigenic properties. The antibiotic sensitivity pattern of the isolate is performed and the report issued to the physician.

Quicker Blood Culture Systems:
� Biphasic Broth-Slide System (Septi-Check)
� Bactec System (automated radiometric system)
� BacT-Alert
� Difco ESP
� Lysis Centrifugation Method

Common pathogens:
In neo-nates: E.coli, Klebsiella, Group B Streptococci, Listeria monocytogenes
In children and adults: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, viridans Streptococci, Salmonella spp, Brucella etc.

Common contaminants:
Staphylococcus epidermidis, Micrococcus, Diptheroides, Propionibacterium acnes, Pseudomonas, Enterobacter are the common contaminants. These organisms can be pathogenic under certain conditions and hence must not be overlooked.