Atifa AmbreenL-1242

Culturing blood

Bacteraemia The presence of bacteria in the blood is

called bacteraemia. Occurs in the following diseases:• Typhoid fever• Brucellosis• Leptospirosis • Endocarditis.

Septicaemia

Life-threatening bacteraemia in which multiplying bacteria release toxins into the blood stream and trigger the production of cytokines, causing fever, chills, toxicity, tissue anoxia, reduced blood pressure,

and collapse.

Possible pathogens isolated from blood cultures

BACTERIAGram PositiveStaphylococcus aureus Viridance streptococciStreptococcus pneumoniaeStreptococcus pyogenesClostridium perfringensAnaerobic streptococci

Gram NegativeSalmonella TyphiBrucella speciesHaemophilus influenzaePseudomonas aeruginosaEscherichia coliProteus speciesBacteroides fragilisNeisseria meningitidisYersinia pestis

• Also Mycobacterium tuberculosis (HIV-associated tuberculosis), Leptospira species, Borrelia species, rickettsiae, and Bartonella bacilliformis.

• FungiCandida albicans and other yeasts For example:Cryptococcus neoformansHistoplasma capsulatum and other fungi that cause systemic mycoses.

Commensals

Blood does not have a normal microbial flora. Common skin contaminants include coagulase negative staphylococci, viridans streptococci, micrococci, and Corynebacterium species.

Day 1

Blood should be collected• Before antimicrobial treatment.• When the patient has recurring fever.• As soon as possible after receiving the request.Note:• At least two specimens should be cultured.• A strict aseptic technique must be used to

collect the blood.

Aseptic blood collection and dispensing technique

• Wearing gloves, Using 70% ethanol, cleanse an area about 50 mm in diameter. • Allow to air-dry.• Remove the protective cover from top of the

culture bottle.• Wipe the top of the bottle using an ethanol-ether swab.

• Using a sterile syringe and needle, withdraw about 20 ml of blood from an adult or about 2 ml from a young child.

• Insert the needle through the rubber liner of the bottle cap and dispense 10–12 ml of blood into the diphasic culture medium bottle containing 25 ml of broth.

• mix the blood with the broth and mix the blood in the EDTA container.

• Clearly label each bottle with the name and number of the patient, and the date and time of collection.

Choice of culture media

The following media are recommended:• Columbia agar• Columbia broth diphasic medium with added

SPS (sodium polyanethol sulphonate), also called as Liquoid.

Uses of SPS• Prevents the blood from clotting.• Neutralizes complement and other

antibacterial substances in fresh blood.• Enables a greater volume of blood to be

cultured without increasing the volume of broth, i.e. up to 50% of the total volume of medium.

Uses of Columbia Agar and broth

Columbia agar and broth Tryptone soya (tryptic soy) diphasic is recommended:

• For culturing blood but organisms such as S.pneumoniae and N.meningitidis do not grow.

• For the isolation of fastidious organisms. • Brucellae, which grow well in tryptone soya diphasic medium.

Diphasic blood culture medium

• A diphasic (Castenada) medium is one that combines an agar slope with a broth medium. The blood broth is allowed to run over the slope by tipping the bottle at regular intervals. Microbial activity can be seen by growth on the slope (beginning at the broth-agar interface.

• particularly recommended for Brucella species.

Thioglycolate broth

• It consists of nutrient broth Because SPS is inhibitory to anaerobic

streptococci, it is not added to this medium.Use:• Isolate strict anaerobes.

• Diphasic medium• Incubate at 35–37°C for up to 7 days.

• Thioglycollate brothIncubate at 35–37°C for up to 2 weeks

Examine the specimen microscopically

• Centrifuge a sample of EDTA anticoagulated venous blood make smears of the buffy coat

layers. Stain as follows:• Gram smear: To detect Gram positive and Gram negative bacteria,particularly when the patient is an infant or young child.

• Ziehl-Neelsen smear: To detect AFB when the patient has AIDS or suspected HIV disease.

Giemsa or rapid Field’s smear: To detect borreliae, or parasites such as trypanosomes, malaria parasites, and microfilariae.

Day 2

After overnight incubation: • Examine diphasic culture Subculture (even when no growth is seen): –

Blood agar and MacConkey agar. Incubate aerobically.

• Chocolate agar. Incubate in CO2. Examine thioglycollate culture. Subculture and

examine microscopically.• Incubate subculture anaerobically.

• Examine subcultures for likely pathogens.• Perform antimicrobial susceptibility test.

SUMMARY