Laboratory diagnosis gram positive and gram negative cocci

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 (?)