Streptococcus and Staphylococcus are common bacterial genera that can cause infections. The document outlines the steps for laboratory diagnosis of infections caused by these bacteria, including specimen collection, culture, staining, and biochemical and antimicrobial testing. Gram staining reveals Gram-positive cocci arranged in clusters for Staphylococcus or chains for Streptococcus. Culture on blood agar shows hemolytic patterns. Biochemical tests help identify pathogenic species like S. aureus and S. pyogenes. Antibiotic susceptibility testing is also important for treatment.

1. Bacteriological diagnosis of infections
caused by bacteria of
Staphylococcus and Streptococcus genera
- Part One -
http://www.slideshare.net/DanaSinzianaBreharCi/
staphylococcus-streptococcus-bacteriological-diagnosisi

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 i.e. purple (violet)
– 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
- clinical significance -
• Community & Hospital acquired infections:
– Skin & subcutaneous tissues: foliculitis, abscesses, furuncles,
carbuncles
– otitis, synusitis, pneumonia
– Osteomyelitis, septic arthritis
– Gentio-Urinary: cystitis, prostatitis, pielonephritis, renal
abscesses
– Cardiovascular: endocarditis, phlebitis, sepsis
– Digestive: Food poisoning
– Nervous system: meningitis, encephalitis

5. Genus Staphylococcus:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
urine, stool, etc)
• Macroscopic examination
• Microscopic examination
• Inoculation of culture media
• Pathogenicity tests
• Biochemical tests
• Agglutination tests
• Antimicrobial susceptibility tests (antibiogram)

6. 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

7. Staphylococcus aureus: creamy, yellow pus

8. Celulitis with Staphylococcus aureus

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. Genus Staphylococcus:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
urine, stool, etc)
• Macroscopic examination
• Microscopic examination
• Inoculation of culture media
• Pathogenicity tests
• Biochemical tests
• Agglutination tests
• Antimicrobial susceptibility tests (antibiogram)

13. Laboratory diagnosis of Staphylococcal Infections:
Gram stained smear from specimen (e.g. pus)
• White blood cells + Gram positive cocci
• Shape: spherical
• aglomerated in clusters / + pairs / + isolated
• Location: both intra- and extracellular
• Size: 0.5-1 µM

14. Staphylococcus: Gram staining biological
product (sputum)

15. Staphylococcus: Gram staining

16. Genus Staphylococcus:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
urine, stool, etc)
• Macroscopic examination
• Microscopic examination
• Inoculation of culture media
• Pathogenicity tests
• Biochemical tests
• Agglutination tests
• Antimicrobial susceptibility tests (antibiogram)

17. Laboratory diagnosis of Staphylococcal Infections:
Inoculation 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, non-hemolytic

18. ”Golden” colonies: Staphylococcus aureus

19. S.aureus golden, hemolytic colonies on
blood agar

20. 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 →S.aureus: Fermentation of mannitol
→colour of medium changes from pink to yellow (further
identification step) – difference between S.aureus and other
staphylococci

21. Mannitol Salt Agar (Chapman)
- high salt concentration supports growth
of Staphylococcus / inhibits Streptococcus
- S.aureus: mannitol fermentation – changes the colour of
the medium from pink to yellow

22. Chapman agar – mannitol fermentation
(yellow) and no fermentation (pink)

23. Genus Staphylococcus:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
urine, stool, etc)
• Macroscopic examination
• Microscopic examination
• Inoculation of culture media
• Pathogenicity & Biochemical tests
• Agglutination tests
• Antimicrobial susceptibility tests (antibiogram)

24. Laboratory diagnosis of Staphylococcal Infections:
Pathogenicity & biochemical tests
• Hemolysins – already discussed
• Mannitol fermentation – already discussed
• Catalase
• Coagulase
• Fibrinolysin
• Biochemical tests
• Bacteriophage typing

25. The Catalase test
• Principle: the enzyme catalase decomposes hydrogen
peroxide (H2O2) into water and oxygen:
2H2O2 →2H2O + O2 (gas bubbles)
• 2-3 drops of hydrogen peroxide placed directly on a
colony
• POSITIVE TEST: rapid effervescence
• differentiates Staphylococcus (+) / Streptococcus (-)

26. The Slide Coagulase test
• Principle: the coagulase of Staphylococcus aureus (aka
”clumping factor”) converts fibrinogen into fibrin →clot
• Procedure:
– 2 drops of saline solution in 2 circles drawn on glass slide
– Emulsify colony in each of the 2 circles
– 1 drop of plasma (rabbit plasma with EDTA) in one circle
– 1 drop of water in the other circle (control)
– Rock slide back and forth & observe agglutination
• POSITIVE TEST: white precipitate & agglutination in 10-
15 sec (control should remain smooth)

27. Slide and tube coagulase test

28. The fibrinolysin test
• Principle: fibrinolytic enzymes (e.g. the staphylokinase of
S.aureus) can dissolve fibrin clots
• Procedure:
• 3 tubes: CaCl2 + plasma → fibrin clot
• Tube 1: add nothing else (clotting control)
• Tube 2: inoculum of S. epidermidis strain; homogenize
with the loop, incubate at 37o C, 1-4 hours – fibrin clot
not dissolved = NEGATIVE test
• Tube 3: inoculum of (suspected) S.aureus strain;
homogenize inoculum with loop, incubate; clot slowly
lysed = POSITIVE test

29. Testing for Enzyme Systems
• Final characterization of unknown bacterial isolate by
testing for characteristic enzyme systems
• Method: re-inoculation of isolated colony (primary
culture) into a series of culture media containing specific
substrates and chemical indicators
• Principle: detection of
– pH changes produced by utilization of substrates /
– colour changes produced by specific by-products
• Challenge: Selection of appropriate sets of
characteristics to allow bacterial group identification

30. Biochemical tests (testing for enzyme
systems)
• The API Staph System (bioMerieux) – identification of 23 species of
staphylococci
• 19 microampules containing dehydrated substrates and/or nutrient
media
Procedure:
- make a saline suspension of the organism from isolated colony
- place staph strip in a tray with small amount of water to provide
humidity during incubation
- dispense 2-3 drops of bacterial suspension in each microampule
with sterile pipette
- cover tray and incubate aerobically for 18-24 hours at 35-37
degrees Celsius
- seven-digit profile number obtained and used to determine the
identity of the organism (match to profile numbers from database)

31. Staphylococcus spp – biochemical tests

32. Genus Staphylococcus:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
urine, stool, etc)
• Macroscopic examination
• Microscopic examination
• Inoculation of culture media
• Pathogenicity & Biochemical tests
• Agglutination tests
• Antimicrobial susceptibility tests (antibiogram)

33. Rapid agglutination tests – detection of
clumping factor of S.aureus

34. (Bacterio)phage typing – identification of
strains causing epidemic clusters
• (Bacterio)phage = virus that
infects bacterium – specificity
allows id. of bacterial strains
• Bacterial culture+Grid drawn
on lid of Petri dish
• 1 drop of different phage
cultures in each quadrant &
incubation
• Bacterial culture dissolved by
respective bacteriophage –
epidemiologic utility
(identify source of
epidemics)

35. Antimicrobial susceptibility
• 1950-1960: emergence of strains resistant to antibiotics
• MRSA (Methicillin Resistant S.aureus)
• Mechanisms:
– enzyme which destroys β-lactam antibiotics (β-lactamase)
– decrease of bacterial wall permeability
• Methicillin resistance = resistance to ALL β-lactam
antibiotics (penicillins, cephalosporins, monobactams
and carbapenems)

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

37. Classification of streptococci
Criteria:
• I. Type of hemolysis produced by bacterial growth on
blood agar
• II. Antigenic structure (Lancefield)

38. Classification of streptococci according to
type of hemolysis
• β-hemolytic streptococci:
– Complete, clear hemolysis (medium around the colony is
transparent = bacterial growth produced complete digestion of
red blood cells in the blood agar) e.g. Streptococcus pyogenes
• α-hemolytic streptococci:
– partial hemolysis (medium around the colony is translucent and
greenish = bacterial growth produced incomplete digestion of
hemoglobin in the blood agar (conversion of hemoglobin to
methemoglobin) e.g. Streptococcus viridans, Streptococcus
pneumoniae)
• Non-hemolytic streptococci

39. Blood agar:
Enterococcus fecalis (non-hemolytic/variable) and
Streptococcus pyogenes (hemolytic)

40. Classification of streptococci according to
antigenic structure (Lancefield grouping)
Rebecca Lancefield (1895-1981)
(American microbiologist at the
Rockefeller Institute for Medical
Research)
• based upon the C polysacharidic antigen (group-specific) in
bacterial wall → groups A – H and K-V
• based upon M and T proteins (type specific) → over 80 types of
group A streptococci
• !! Lancefield grouping does not include streptococci lacking group
antigens e.g. Str.pneumoniae, Str.viridans, etc.)

41. 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)

42. Streptococcus pyogenes
- clinical significance -
• Acute, respiratory infections: pharyngitis, scarlet fever +
complications: articular (acute rheumatic fever), cardiac
(rheumatic carditis), renal (glomerulonephritis)
• Skin infections: erysipelas, impetigo, intertrigo, pemfigus,
celulitis, abscesses + complications: sepsis

43. Streptococcus pyogenes:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
content of vesicles, CSF, urine, etc)
• Microscopic examination
• Inoculation of culture media
• Biochemical tests
• Agglutination tests
• Antimicrobial susceptibility tests (antibiogram)

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

45. Erysipelas – streptococcal infection of the
dermis and superficial lymph vessels

46. Impetigo – non-bulous and bulous

47. Streptococcus pyogenes
– Microscopic examination -
Gram stained smears:
• Cocci:
– Round / ovoid shape; arranged in chains /
pairs
– Gram positive
– Aerobic growth (+anaerobic)

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

49. Streptococcus – Gram stained smear

50. Streptococcus pyogenes
- Cultivation & isolation -
• Blood containing media e.g. blood agar, Todd-Hewit
broth, SSP (selective medium for streptococci and
pneumococci)
• Most frequently:
– (Initial inoculation of selective medium (Pick) – favours growth
and multiplication of streptococci and inhibits other bacterial
species)
– ↓
– Reinoculation on 5% sheep blood agar

51. Streptococcus pyogenes
- identification -
• Colonial characters:
– small, pinpont, 0.5 µM diameter, transparent
– β-hemolysis - complete digestion of red blood cell contents
surrounding colony
• Group identification:
– bacitracin sensitivity test – group A streptococci are bacitracin
sensitive / other streptococci are resistant

52. Bacitracin sensitivity test
• used to determine the effect of
a small amount of bacitracin
(0.04 U) on an organism.
• Streptococcus pyogenes
(group A) is inhibited
(minimum 10 mm inhibition
diameter) by the small amount
of bacitracin in the disk; other
beta-hemolytic streptococci
usually are not

53. Blood agar plates
Left: Staphylococcus; Right: Streptococcus

54. Staph. aureus - mannitol fermentation (left side, left plate)
Staph.epidermidis - no mannitol fermentation (right side, left plate)
Streptococcus – plate on the right

55. To be continued....