Bacteremia, Septicemia - Laboratory Diagnosis, Culture, Colony Characteristics, Biochemical Tests

Laboratory diagnosis of Bacteremia, Septicemia

Sample

• Blood

* Blood must be collected before antibiotic therapy and when the temperature begins to rise. Usually, 2-3 blood cultures are sufficient to achieve optimum blood culture sensitivity.

* In adults, 10-20ml of blood per culture is recommended while in children 1-5ml of blood per culture is recommended. Although less than 1 ml is not adequate, detection occurs in adults with high levels of bacteremia.

Culture

Dilution of blood and culture medium for bacteremia, and septicemia diagnosis is done at the ratio of 1:10 ratio of blood. However, the 1:5 ratio is used nowadays.

Media

Blood culture media includes:

• Soybean Casein Digest Broth

• Trypticase Soy Broth

• Brain Heart Infusion (BHI) broth

• Supplemented peptone broth

• Thioglycolate broth

• Columbia broth (specialized broth)

• Brucella broth (specialized broth)

Incubation

Incubation of inoculated media is done at 35-37°C. Cultures are examined and sub-cultured daily for 7 days and then twice a week for up to 4 weeks.

Constant agitation of the bottle during the first 24 hours of incubation enhances the growth of most aerobic bacteria.

Interpretation of Blood culture results

Growth is indicated by changes in turbidity, pellicle formation, gas bubble formation, odor, hemolysis, clot formation, etc.

Subculture

A loop full of incubated medium is sub-cultured in MA, BA, and CA for further identification. Incubation of subculture is done at 35°C in blood agar (BA), chocolate agar (CA) in anaerobic conditions, and MacConkey agar (MA) for 24-48 hours in gas.

Colony observation/characteristics

Presumptive identification is done after viewing the characters of colonies on MA, BA, and CA.

* Due to the presence of crystal violet and bile salts, only gram-negative bacteria grow in MA. Thus it is called differential and selective media.

Gram-positive bacteria

Staphylococcus aureus

BA: opaque, golden yellow, mucoid colonies with β-hemolysis

CA: white, moist colony, darkened edges on media

Coagulase Negative Staphylococcus (CoNs)

BA, CA: minute white colony, no hemolysis

Enterococcus spp.

BA, CA: minute paper white colonies, β-hemolysis in some species

Streptococcus pneumoniae

BA, CA: small, grey, moist colony, alpha-hemolysis (green) observed

Streptococcus agalactiae

BA: translucent white colony with β-hemolysis

CA: translucent white, slightly mucoid colony

Streptococcus pyogenes (group A Streptococci)

BA: tiny paper white colonies with large β-hemolysis

CA: flat, opaque, white colonies

Viridans streptococci

BA: α-hemolytic, small transluscent colonies

CA: tiny white colonies showing α-hemolysis

Gram-negative bacteria

Escherichia coli

MA: pink to a dark pink, dry, donut-shaped, dark pink area of precipitated bile salts on the periphery of the colony

BA: smooth, circular, flat colonies, no hemolysis occurs

CA: mucoid, beige, or off-white color with a shiny texture

Pseudomonas aeruginosa

MA: flat, smooth, elliptical-shaped colonies which are 2-3mm in diameter. They are usually colored ranging from blue-green, yellow-green, to red-brown.

BA: dark, translucent, flat, colonies with irregular edges

CA: flat, smooth, dark white colonies with irregular edges

Haemophilus influenzae

MA: No growth

BA: visible growth only by Satellitism test

CA: tiny, flat, smooth white colonies with no hemolysis

Proteus spp.

MA: small, dark, flat colonies with irregular edges, may slightly swarm

BA: translucent colonies with swarming growth (in form of waves), cover the entire surface of an agar plate with no hemolysis

CA: same as in blood agar

Klebsiella pneumoniae

MA: Large, circular, convex, highly mucoid, pink colonies

BA, CA: white, mucoid, convex colonies with no hemolysis

Salmonella spp.

MA: flat, colorless, colonies with irregular edges measuring 2-4mm in diameter

BA, CA: white, flat, smooth, glistering colonies with no hemolysis

Neisseria meningitidis

MA: due to its fastidious nature, does not grow in MA

BA, CA: translucent white, smooth, concave colonies with regular edges, no hemolysis

Confirmatory tests/Biochemical tests

Gram-positive bacteria

Staphylococcus aureus

Catalase: Positive

Oxidase: Negative

Coagulase: Positive

Deoxyribonuclease (DNase): Positive

*can be confirmed by growth on Mannitol Salt Agar

Coagulase Negative Staphylococcus (CoNs)

Catalase: Positive

Oxidase: Negative

Coagulase: Negative

Enterococcus spp.

Catalase: Negative

Oxidase: Negative

Coagulase: Negative

Growth in Bile Esculin Agar: Positive

Streptococcus pneumoniae

Catalase: Negative

Oxidase: Negative

Coagulase: Negative

Optochin test: Sensitive to Optochin

Streptococcus agalactiae

Catalase: Negative

Coagulase: Negative

Oxidase: Negative

CAMP: Positive

Streptococcus pyogenes

Catalase: Negative

Coagulase: Negative

CAMP: Negative

Oxidase: Negative

PYR (Pyrrolidonyl Aminopeptidase) test: Positive

Streptococci viridans

Catalase: Negative

Coagulase: Negative

CAMP: Negative

PYR (Pyrrolidonyl Aminopeptidase) test: Negative

Optochin test: Resistant

Gram-negative bacteria

*All above organisms reduce nitrate

* Catalase and Oxidase are positive for Pseudomonas aeruginosa

* Bacillus spp. (except Bacillus anthracis), Coagulase-negative Staphylococcus (CoNs), Corynebacterium spp., and Propionibacterium acnes are probable contaminants. However, isolation of commercial microbial flora from blood cultures of immunocompromised patients can also be the causative agent of infection.