basic notions on lab diagnosis of Gram positive and negative cocci to be used as a support for dentistry students in their 2nd year of study

1. Gram positive and Gram negative cocci
- brief overview of laboratory diagnosis -

2. Gram positive cocci
• Family: Micrococaceae
• Genera:
– Staphylococcus
– Micrococcus
– Stomatococcus
– Planococcus
• Family: Streptococacceae
• Genera:
– Streptococcus
– Enterococcus
– Aerococcus
– Gemella
– Leuconostoc
– Pediococcus
– Lactococcus

3. Genus Staphylococcus
• Cocci:
– Round shape; cluster arrangement (”grape-shaped”)
– Gram positive
– Aerobic growth (+anaerobic)
– Nonsporulated
• Clinically significant microbial species:
– S.aureus – pathogenic
– S.epidermidis – accidentally pathogenic
– S.schleiferi, S.lugdunensis, S.haemolyticus, S.saprophyticus –
low pathogenic potential

4. Staphylococcus aureus
• Community & Hospital acquired infections:
– Skin & subcutaneous tissues: foliculitis, abscesses, furuncles,
carbuncles
– otitis, synusitis, pneumonia
– Osteomyelitis, septic arthritis
– Endocarditis, phlebitis, sepsis
– Food poisoning
– Meningitis, encephalitis

5. Laboratory diagnosis of Staphylococcal Infections:
Collection of specimens
Pus:
Closed lesions (abscesses):
• surgical collection:
– rigurous cleaning and disinfection of skin (iodine)
– Incision and aspiration of pus
Open lesions:
• Cleaning and disinfection of skin around lesion (iodine)
• Collection of pus with sterile swab / loop

6. Staphylococcus aureus: creamy, yellow pus

7. Celulitis with Staphylococcus aureus

8. Laboratory diagnosis of Staphylococcal Infections:
Collection of specimens
Fluid from cavities
e.g. spinal (CSF)/ pleural / pericardic / articular
• Sample collected by punctioning the cavity
• E.g. Lumbar punction (spinal tap)
– patient lies on the side, knees pulled up toward chest,
chin tucked downward
– back cleaned and disinfected + local anesthetic
– spinal needle inserted into lower back area
– needle properly positioned, CSF pressure measured
and sample collected in sterile tube

9. Laboratory diagnosis of Staphylococcal Infections:
Collection of specimens
Pharyngeal, naso-pharingeal exudate
Patient:
– in the morning, before feeding, before brushing teeth;
alternatively: at least 4 hours since last meal & teeth
brushing
– No mouth rinse, no chewing gum!
– No antibiotics during the last 7-10 days
Medical staff:
– Wear gloves, face protection (mask, eye
protection/face shield), protective lab coat

10. Collection of pharyngeal exudate
• Dacron or Rayon swab
• Tongue blade & good light
• Insert swab behind uvula
without touching it
• Swab tonsils, posterior
pharynx + lesions (if any)
• Avoid touching tongue,
cheeks, teeth
• Place swab in sterile tube
• Transport to lab (RT/2-8°C)

11. Collection of pharyngeal exudate

12. Laboratory diagnosis of Staphylococcal Infections:
Gram stained smear
• Gram positive cocci
• Shape: spherical
• aglomerated in clusters / pairs / isolated
• Location: both intra- and extracellular
• Size: 0.5-1 μM
• + WBC (entire & destructed)

13. Staphylococcus: Gram staining biological
product (sputum)

14. Staphylococcus: Gram staining

15. Laboratory diagnosis of Staphylococcal Infections:
Innoculation of culture media
• closed collections / moderately contaminated
collection sites (e.g. nasopharingeal swab)
↓
blood agar
• S.aureus: round colonies, 1-3 mm diameter, smooth,
hemolytic, pigmented (golden-yellow)
• S.epidermidis: white colonies

16. ”Golden” colonies: Staphylococcus aureus

17. Laboratory diagnosis of Staphylococcal Infections:
Innoculation of culture media
• closed collections / moderately contamnated collection
sites (e.g. nasopharingeal swab) → blood agar
• Highly contaminated biological products (e.g. stool)
↓
Chapman agar - selective medium
(high salt content + mannitol + pH indicator)
WHY?:
– A. Inhibit other germs, favour growth of Staphylococcus
– B. Staphylococcal growth →Fermentation of mannitol →colour of
medium changes from pink to yellow (further identification step)

18. Mannitol Salt Agar (Chapman)
- high salt concentration supports growth
of Staphylococcus / inhibits Streptococcus
- mannitol acidification - turn the medium colour to yellow

19. Chapman agar – mannitol acidification

20. Gram positive cocci
• Family: Micrococaceae
• Genera:
– Staphylococcus
– Micrococcus
– Stomatococcus
– Planococcus
• Family:
Streptococacceae
• Genera:
– Streptococcus
– Enterococcus
– Aerococcus
– Gemella
– Leuconostoc
– Pediococcus
– Lactococcus

21. Genus Streptococcus
• Clinically significant microbial species:
– Streptococcus pyogenes: cellulitis, pharyngitis, scarlet
fever + complications: articular (acute rheumatic
fever), cardiac (rheumatic carditis), renal
(glomerulonephritis)
– Streptococcus pneumoniae: pneumonia,
bronchopneumonia, meningitis
– Oral (viridans) streptococci: Streptococcus mutans,
Streptococcus sanguis, Streptococcus anginosus
(dental caries, periodontal disease + septicaemia,
endocarditis)

22. ”Strep throat” – Pharyngitis with Streptococcus pyogenes:
left – petechiae; right – pus deposits

23. Periodontal disease, dental caries

24. Laboratory diagnosis of Streptococcal Infections:
Gram stained smears
• Cocci:
– Round / ovoid shape; arranged in chains / pairs
– Gram positive
– Aerobic growth (+anaerobic)
• + Streptococcus pneumoniae: pairs of
encapsulated cocci

25. Streptococcus pyogenes: Gram stained smear:
ovoid Gram positive cocci, arranged in chains

26. Streptococcus mutans – Gram stained
smear

28. Streptococcus pneumoniae – Gram stained
sputum smear

29. Laboratory diagnosis of Streptococcal Infections:
Innoculation of culture media
Innoculation on blood agar:
• Colonial morphology:
– Str.pyogenes: small, pinpont, 0.5 μM diameter, transparent
– Str.pneumoniae, Str.viridans: small, smooth, flat/depressed
center (autolysis)
• Type of hemolysis:
- β-hemolysis - complete digestion of red blood cell contents
surrounding colony e.g. Streptococcus pyogenes
- α-hemolysis - partial lysis – incomplete hemoglobin digestion →
green or brown (conversion of hemoglobin to methemoglobin)
e.g. Streptococcus viridans, Streptococcus pneumoniae

30. Streptococcus pyogenes on blood agar

31. Streptococcus pneumoniae on blood agar

32. Left: Alpha hemolysis – Streptococcus pneumoniae
Right: Beta hemolysis – Streptococcus pyogenes

33. Staphylococcus / Streptococcus (?)