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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
close this folderBlood
View the documentIntroduction
View the documentCauses of bacteraemia
View the documentBlood collection
View the documentBlood-culture media
View the documentProcessing of blood cultures
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Processing of blood cultures

Incubation time

Blood-culture bottles should be incubated at 35-37°C and routinely inspected twice a day (at least for the first 3 days) for signs of microbial growth. A sterile culture usually shows a layer of sedimented red blood covered by a pale yellow transparent broth. Growth is evidenced by:

 

• a floccular deposit on top of the blood layer,
• uniform or subsurface turbidity,
• haemolysis,
• coagulation of the broth,
• a surface pellicle,
• production of gas,
• white grains on the surface or deep in the blood layer.

Whenever visible growth appears, the bottle should be opened aseptically, a small amount of broth removed with a sterile loop or Pasteur pipette, and a Gram-stained smear examined for the presence of microorganisms.

Subcultures are performed by streaking a loopful on appropriate media:

 

• for Gram-negative rods: MacConkey agar, Kligler iron agar, motility-indoleurease (MIU) medium, citrate;

• for staphylococci: blood agar, mannitol salt agar;

• for streptococci: blood agar with optochin, bacitracin, and tellurite discs, sheep blood agar for the CAMP test, and bile-aesculin agar.

For routine examinations, it is not necessary to incubate blood cultures beyond 7 days. In some cases, incubation may be prolonged for an additional 7 days, e.g., if Brucella or other fastidious organisms are suspected, in cases of endocarditis, or if the patient has received antibiotics.

Blind subcultures and final processing

Some microorganisms may grow without producing turbidity or visible alteration of the broth. Other organisms, e.g., pneumococci, tend to undergo autolysis and die very rapidly. For this reason some laboratories perform routine subcultures on chocolate agar after 18-24 hours of incubation. A blind subculture may be made at the end of the 7 days’ incubation, by transferring several drops of the well mixed blood culture (using a sterile Pasteur pipette) into a tube of thioglycollate broth, which in turn is incubated and observed for 3 days.

Antibiogram

When staphylococci or Gram-negative rods are suspected, precious time can be saved by performing a direct, non-standardized antibiogram using the positive broth as an inoculum. A sterile swab is dipped into the turbid broth, excess fluid is expressed, and the swab is used to inoculate Mueller-Hinton medium as in the standard method. A provisional reading can often be made after 6-8 hours of incubation. In 95% of cases the results obtained with this method are in agreement with the standardized test.

Contaminants

Contamination of blood cultures can be avoided by meticulous skin preparation and by adherence to strict aseptic procedures for inoculation and subinoculation. However, even in ideal conditions, 3-5% of blood cultures grow “contaminants” originating from the skin (S. epidermidis, P. acnes, Clostridium spp, diphtheroids) or from the environment (Acinetobacter, Bacillus spp). Such organisms, however, may occasionally behave as pathogens and even cause endocarditis. A true infection should be suspected in the following situations:

 

• if the same organism grows in two bottles of the same blood specimen;
• if the same organism grows in cultures from more than one specimen;
• if growth is rapid (within 48 hours);
• if different isolates of one species show the same biotypes and antibiotic-susceptibility profiles.

All culture results should be reported to the clinician, including the presumed contaminants. However, for the latter no antibiogram need be performed and appropriate mention should be made on the report slip, e.g., Propionibacterium acnes (skin commensal), Staphylococcus epidermidis (probable contaminant). It is to the advantage of all concerned to establish good communication between physicians and laboratory personnel.

Most common causes of bacteraemia or fungaemia

Gram-negative organisms

Gram-positive organisms

Escherichia coli

Staphylococcus aureus

Klebsiella spp

S. epidermidis

Enterobacter spp

α-haemolytic (viridans)

Proteus spp

streptococci

Salmonella typhi

Streptococcus pneumoniae

Salmonella spp other than S. typhi

E. faecalis (group D)

Pseudomonas aeruginosa

S. pyogenes (group A)

Neisseria meningitidis

S. agalactiae (group B)

Haemophilus influenzae

Listeria monocytogenes

Bacteroides fragilis (anaerobe)

Clostridium perfringens

Brucella spp

Peptococcus spp (anaerobes)

Pseudomonas pseudomallei (in certain areas)

Peptostreptococcus spp (anaerobes)

 

Candida albicans and other yeast-like fungi (e.g. Cryptococcus neoformans)

The identification of two or more agents may indicate polymicrobial bacteraemia, which can occur in debilitated patients, but may also be due to contamination. “Anaerobic” bacteraemia is often caused by multiple pathogens; for example, one or more anaerobes may be associated with one or more aerobes in severe fulminating bacteraemia associated with severe trauma or surgery involving the large intestine.

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