Standard Operating Procedure (SOP) for Blood Culture using Brain Heart Infusion (BHI)Broth

Introduction

This SOP “Standard Operating Procedure (SOP) for Blood Culture using Brain Heart Infusion (BHI)Broth” outlines blood culture preparation, inoculation, incubation, and interpretation. Different microbiological media are used in the laboratory to cultivate and grow various microorganisms.

These broth media are made from a combination of brain and heart tissue infusions from animals, typically cows or pigs, along with peptones. As a result, they are used to support the growth of a wide range of bacteria, fungi, and some fastidious organisms from blood samples.

Examples of these media include Brain Heart Infusion (BHI) broth, Oxoid Signal Bottle, and Cooked Meat Agar.

A blood culture test is usually ordered when septicemia or meningitis is suspected.

Equipment and Materials, “Standard Operating Procedure (SOP) for Blood Culture using Brain Heart Infusion (BHI)Broth”,

• Sterile, wide-mouthed blood culture bottles (containing BHI broth)
• Sterile syringes and needles
• Sterile cotton swabs
• Antiseptic solutions (e.g., 70% alcohol and iodine)
• Incubator (35-37°C)
• Microscope
• Gram stain reagents
• Subculture media: Blood Agar, MacConkey Agar, Chocolate Agar, Sabouraud Dextrose Agar (for fungi, if necessary)
• Autoclave for sterilization
• Laboratory timer

 

 Specimen Collection

• Blood Collection:
  • Blood samples should be collected during the febrile phase when bacteria are likely to be present in the bloodstream.
  • Using a sterile syringe, collect 5–10 mL of blood from adults and 2–5 mL from children. The optimal blood-to-broth ratio should be 1:10.
  • After drawing the blood, disinfect the cap of the blood culture bottle with alcohol before inoculation.

 

• Inoculation:
  • Immediately inoculate the blood sample into the sterile BHI broth.
  • Shake the bottle gently to properly mix the solution.

 

Incubation

• Incubation Conditions:
  • Incubate the inoculated BHI broth at 35-37°C in an incubator.
  • Blood culture bottles should be incubated for 7–10 days. Fastidious organisms may require longer incubation (up to 21 days).
  • Check the bottles daily for signs of bacterial growth, such as turbidity, gas formation, or hemolysis.

 

• Agitation:
  • Gently shake the culture bottles during the incubation period to aerate and encourage growth.

 

Subculture and Identification

• Observation for Growth:
  • If there are visible signs of growth (e.g., turbidity, gas production, or hemolysis), proceed with subculturing.
  • If there are no visible signs after 48 hours, perform a blind subculture onto solid media as a precaution.

 

• Subculturing:
  • Using a sterile syringe, withdraw a small amount of broth from the bottle.
  • Inoculate onto the following solid media:
    • Blood Agar: For general bacterial growth and hemolysis detection.
    • MacConkey Agar: To differentiate between Gram-negative lactose fermenters and non-lactose fermenters.
    • Chocolate Agar: For fastidious organisms (e.g., Haemophilus and Neisseria).
    • Sabouraud Dextrose Agar (if fungal growth is suspected).
  • Incubate the subcultured plates at 35-37°C for 18-24 hours. Fastidious organisms may require CO₂ incubation.

 

• Gram Stain:
  • Perform a direct Gram stain on the sample from the BHI broth or from ths subculture to determine the Gram reaction, morphology, and arrangement of the organisms (e.g., Gram-positive cocci, Gram-negative rods).

 

• Identification of Isolates:
  • Perform biochemical tests (e.g., catalase, coagulase, oxidase) or use automated identification systems to identify the isolated organism.

 

Sensitivity Testing

• Antibiotic Susceptibility Testing:
  • Once bacterial pathogens are isolated, perform antibiotic sensitivity testing using the disc diffusion method on nutrient Agar.
  • Incubate at 35-37°C for 18-24 hours and measure the zones of inhibition.

• Report the organism’s susceptibility (S), intermediate (I), or resistance (R).

 

Interpretation of Results

• Positive Blood Culture:
  • If the blood culture shows bacterial growth in BHI broth or on subculture plates, interpret the result as positive for bacteremia or septicemia.
  • Record the organism(s) identified.

 

• Negative Blood Culture:
  • If no growth is observed after 7–10 days (or longer for fastidious organisms), report the culture as negative.
  • If growth occurs late in the incubation period, consider contamination and perform further testing to confirm the result.

 

Possible pathogens isolated from blood cultures

BACTERIA
Gram positive

• Staphylococcus aureus
• Viridans streptococci
• Streptococcus pneumoniae
• Streptococcus pyogenes
• Enterococcus faecalis
• Clostridium perfringens

Gram negative

• Salmonella Typhi (Other Salmonella serovars)
• Haemophilus influenzae
• Pseudomonas aeruginosa
• Klebsiella strains
• Escherichia coli
• Brucella species
• Proteus species
• Neisseria meningitidis
• Yersinia pestis

References

• Cheesbrough, M. District Laboratory Practice in Tropical Countries, Part 2, 2nd edition. Cambridge University Press, 2006.

 

Meanwhile you can also read this SOP