Monday, December 10, 2007



Name: Wong Fei Hong (Case 5)
Sex: Male
Age: 37 years old
Type of Specimen: Wound swab

Clinical diagnosis
Symptoms: Fever, swelling around operation wound
Diagnosis: Wound infection

Approach for the diagnosis of wound infection [1]

  1. A wound swab was obtained

  2. The specimen is gram-stained. The shape, size, arrangement and whether they are gram-positive or gram-negative should be observed.

  3. The specimen is cultured on appropriate media, to obtain ‘pure culture’. Plates should be incubated in aerobic or anaerobic environment.

  4. Identification of the organism by means of biochemical tests.
  5. Perform antibiotic susceptibility tests.

    Note: Wound infection could be due to multiple organisms, so it’s important to culture the specimen on several different media under different atmospheric conditions e.g. aerobic or anaerobic. Gram staining provides useful information about the range of possible causative agents. [1]
Short listed bacteria [1, 2]:
Gram-positive cocci:
1) Streptococci Species (Facultative anaerobes)
- Enterococcus faecalis
- Streptococcus pyogenes

2) Staphylococcus aureus (Facultative anaerobes)

Gram-positive rod:
1) Bacillus anthracis (Strict aerobes; spore formation)

2) Clostridium perfringens (Stric anaerobes; spore formation)

Gram-negative rod:
1) Pseudomonas aeruginosa (Strict aerobes)

2) Enterobacteriaceae species (Facultative anaerobes)
- Escherichia coli
- Enterobacter cloacae
- Serratia marcescens
- Proteus mirabilis
- Klebsiella pneumoniae

Differential diagnosis for gram-positive cocci:

Streptococci Species
1] Enterococcus faecalis
2] Streptococcus pyogenes

Sheep blood agar:[1]

- Beta-hemolytic colonies: Streptococcus pyogenes
- Gamma-hemolytic or lack of change in the medium around colonies: Enterococcus faecalis

Catalase test: Both Negative [2]
This test differentiates Streptococci Species and Staphylococcus aureus.

- A drop of hydrogen peroxide is added to a sample from the bacterial colony
- Presence of catalase converts hydrogen peroxide into water and oxygen
- Results in the production of visible bubbles

Bile-esculi agar: [2]

- Positive: E. faecalis
- Negative: Strep. Pyogenes

E. faecalis hydrolyse esculin, which results in black discoloration of the agar.

Hypertonic (6.5%) NaCl solution:

- E. faecalis grows, while Strep. Pyogenes doesn’t

Antibody Susceptibility: [1]
1] Enterococcus faecalis:
- Penicillin
- Erythromycin
- Bacitracin
- Tetracycline

2] Streptococcus pyogenes:
- Penicillin
- Erythromycin
- Bacitracin
- Tetracycline

Staphylococcus aureus [1]

Sheep blood agar:
- Beta-hemolytic colonies indicate Staphylococcus aureus

Catalase test: Positive

Coagulase test: Positive

Coagulase test differentiates S. aureus, from other staphylococcus species such as S. epidermidis present in the normal flora.

Mannitol Salt Agar: Yellow colonies

Differential test for mannitol fermentors and non-fermentators; S. aureus ferments mannitol.

Antibody susceptiblity:
- Vancomycin
- Gentamicin
- Nafcillin
- Methicillin

Picture 1: Beta-hemolysis represented by beta sign and gamma-hemolysis represented by gamma sign
Picture: micrsweb>lab3grampostitive>lab3introtomethods.html/">rsweb>lab3grampostitive>lab3introtomethods.html/">>micrsweb>lab3grampostitive>lab3introtomethods.html

Picture 2: Catalase positive reaction indicating S. aureus infection

Differential diagnosis for spore forming gram-positive rods:

Bacillus anthracis: [1, 2]

Blood agar (Culture aerobically): Gamma-hemolytic ;large and irregular colonies

Egg yolk agar: Lecithinase negative

In addition, it can be diagnosed by means of antigen and molecular tests.

Antibody susceptibility: [1]
- Ciprofloxacin
- Doxycycline

Clostridium perfringens: [2]

Blood agar (Culture anaerobically): Double zone of beta-hemolysis

Egg yolk agar: Lecithinase positive

Antibody susceptibility: [1]
- Penicillin

Picture 3: Left plate: Positive test for lecithinase; Right plate: Negative for lecithinase
Picture: kkiser>lecithpn.jpg/">kkiser>lecithpn.jpg/">kkiser>lecithpn.jpg/">>kkiser>Lecithpn.jpg

Differential diagnosis for gram-negative rods:[1]

MacConkey agar: selective agar used for the isolation and differentiation of lactose-fermenting and non-fermenting gram-negative rods. Alternatively, Eosin Methylene Blue Agar (EMB) can be used.

Lactose fermenters: Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae; form red to pink colonies.

Non-fermenters: Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens; form colourless colonies

Oxidase test: [1]
- Positive: Pseudomonas aeruginosa
- Negative: Enterobacteriaceae species

This test determines the presence of oxidase enzyme. An oxidase paper is touched onto colony, in the presence of oxidase, paper changes to purple (Pseudomonas aeruginosa).

Triple sugar iron agar: [1]

Biochemcial tests: Indole test, Methyl red test, Voges-Proskauer reaction test and Citrate utilization test (IMViC test)

Pseudomonas aeruginosa: [1]
- Colonies: Fluoresce under ultraviolet light; give fruity odor and grow at 42° C
- Indole test: Negative
- Methyl red test: Positive
- Voges-Proskauer reaction test: Negative
- Citrate utilization test: Positive
- Urease test: Negative

Antibody susceptibility: [1]
- Penicillin e.g. ticarcillin or piperacillin
- Aminoglyoside e.g. gentamicin or amikacin

Enterobacteriaceae species:

1] Escherichia coli [1]
- Motile
- Indole test: Negative
- Methyl red test: Positive
- Voges-Proskauer reaction test: Negative
- Citrate utilization test: Negative
- Urease test: Negative

Antibody susceptibility: [1]
- Cefotaxime
- Gentamicin
- Ampicillin
- Loperamide

2] Enterobacter cloacae [1]
- Indole test:Negative
- Methyl red test: Negative
- Voges-Proskauer reaction test: Positive
- Citrate utilization test: Positive
- Urease test: Negative

Antibody susceptibility: [1]
- Imipenem
- Gentamicin
- Cefotaxime

3] Serratia marcescens [1]
- Indole test: Positive
- Methyl red test: Negative
- Voges-Proskauer reaction test: Positive
- Citrate utilization test: Positive
- Urease test: Negative

Antibody susceptibility: [1]
- Imipenem
- Gentamicin
- Cefotaxime

4] Proteus mirabilis [1]
- Indole test: Positive
- Methyl red test: Positive
- Voges-Proskauer reaction test: Negative
- Citrate utilization test: Positive
- Urease test: Positive

Antibody susceptibility: [1]
- Ampicillin
- Cefotaxime

5] Klebsiella pneumoniae [1]
- Indole test: Positive
- Methyl red test: Positive
- Voges-Proskauer reaction test: Positive
- Citrate utilization test: Positive
- Urease test: Positive

Antibody susceptibility: [1]
- Imipenem
- Gentamicin
- Cefotaxime


1)Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division


Foo Yong Yang

Corrections: So far I have found, in my experience, that E. Coli is Indole Positive, Klebsiella Pneumoniae is Indole Negative, Klebsiella Oxytoca is Indole Positive, Proteus mirabilis is Indole Negative, Proteus Vulgaris is Indole Positive. In this article it has the opposite of some of the above..
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