![Page 1: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/1.jpg)
Medical microbiology Laboratory
Lab 8
Lecturer Maysam A Mezher
Gram-positive cocci Staphylococci and Streptococcia
![Page 2: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/2.jpg)
Gram positive cocci 1-Staphylococcus.
2-Streptococcus.
3-Micrococcus
The medically important genera, are: Staphylococci and
Streptococci. Both of them are non motile and do not form
spores. They are distinguished by two main criteria:
Microscopically: staphylococci appear in grape like
clusters where as streptococci are in chains.
Biochemically: staphylococci produce catalase which
brakes down hydrogen peroxide, where as streptococci do
not.
![Page 3: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/3.jpg)
![Page 4: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/4.jpg)
Staphylococci
![Page 5: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/5.jpg)
Important properties :
Gram-positive cocci, arranged in grape-like clusters
Non spore-forming, non motile and capsule formation is variable.
Aerobic and facultative anaerobic, grow readily on usual culture media, has large ,raised, opaque colonies with smooth entire margin.
Salt tolerant: allows them to tolerate the salt present on human skin.
![Page 6: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/6.jpg)
Clinically important species:
Staphylococcus aureus: most virulent species. It is responsible for awide range of medical illnesses extending from mild localized skin infection to life threatening septicemia.
Staphylococcuse pidermidis: it is normal flora of the skin and mucous membranes,
Staphylococcus saprophyticus: it is free living, common cause of UTI in women
![Page 7: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/7.jpg)
Staphylococcus aureus–General features Coagulase positive
Produces golden yellow pigment
![Page 8: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/8.jpg)
Lab diagnosis –S. aureus Specimens: wound swab, pus, sputum, blood, urine CSF.
Culture
BA: beta hemolysis
MSA: ferment mannitol (yellow colonies).
Microscopy: Gram stain-
Blood agar
![Page 9: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/9.jpg)
Biochemical tests:
Catalse : to differentiate staphylococci from streptococci. This enzyme break down H2O2 to Oxygen and water.
Coagulase: to differentiate S. aureus frorm other staphylococci.
•Slide method: for detection of clumping factor.
•Tube method: for detection of plasma coagulase,
Novobiocin disc: to differentiate S. saprophyticus which is resistant, whereas, other are sensitive.
![Page 10: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/10.jpg)
Coagulase test
![Page 11: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/11.jpg)
![Page 12: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/12.jpg)
Streptococci
![Page 13: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/13.jpg)
General characteristics:
1.Gram-positive cocci,arranged in chains or pairs.
2.Non motile, non spore forming.
3.Some strains are capsulated, which are important in pathogenicity.
4.Catalase-negative.
5.Majority are facultative anaerobes, few are obligate anaerobes.
6.They are fastidious microorganisms grow on enriched media such as blood agar ,have small, pinhead, opaque, circular colonies.
7.Sensitive to drying, heat, and disinfectant.
![Page 14: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/14.jpg)
Classification:
1- Hemolysis: •ẞ-hemolysis: complete destruction of RBCs. e.g. S. pyogenes •α-hemolysis: partial destruction of RBCs e.g. S. mutans, S. pneumoniae. •γ-hemolysis: non-hemolysis.
![Page 15: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/15.jpg)
2- Serology (Lancefield grouping): Streptococci
Lancefield classification
There are differences in the polysaccharide antigens
of the cell wall. Depending on these specific
polysaccharide antigens, streptococci are named as
groups from A-U.
Group A ___S. pyogenes
Group B _____S. agalactiae
Group C _______S. equisimitis
Group D____ Enterococcus
Other groups _______(E-U)
![Page 16: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/16.jpg)
HUMAN STREPTOCOCCAL PATHOGENS
•S. pyogenes •S. agalactiae •Viridans streptococci
•S. pneumoniae Human streptococcal patohogens:
- B -hemolytic
Group A streptococci - S. pyogenes: Most serious streptococcal pathogen
In habits throat, nasopharynx And occasionally skin.
![Page 17: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/17.jpg)
Lab diagnosis–Strep. Pyogenes
Specimens: throat swab, pus, blood
Microscopy :Gram stain -GPC in chains
Culture: BA -beta hemolytic colonies
Identification tests-
Catalase Negative
Bacitracin sensitive
![Page 18: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/18.jpg)
Bacitracin sensitivity Principle:
Bacitracin test Is used for presumptive identification of groupA
To distinguish between S.pyogenes (susceptible to B) & non
group A such as S.agalactiae (Resistant to B)
Bacitracin will inhibit the growth of gp AStrep. Pyogenes giving
zone of inhibition around the disk
Procedure: Inoculate blood agar with heavy suspension of tested organism
After incubation, any zone of inhibition around the disk is
considered as susceptible
![Page 19: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/19.jpg)
B -hemolytic :Group B streptococci- S. agalactiae:
Normal flora of female vaginal tract and cause neonatal
meningitis.
Bacitracin resistant
CAMP test+ve (Christie,
Atkins, Munch-Peterson)
hydrolize sodium hipurate
and give+ve response
in this test
![Page 20: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/20.jpg)
- Alpha hemolytic streptococci Streptococcus pneumoniae (Pneumococcus)
General features:
•Causes 60-70% of all bacterial pneumonias
•Small , lancet-shaped cells arranged in pairs and short chains
•Culture requires blood or chocolate agar, Growth improved by
5-10%CO2
50% of all people carry it as normal flora in the nasopharynx ;
infections are usually endogenousVirulence
![Page 21: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/21.jpg)
![Page 22: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/22.jpg)
Lab. diagnosis: Gram stain: GPC arranged in pairs. (lancet-shaped
diplococci)
Rapid diagnostic test: Quellung test or capsular swelling
reaction for S. pneumoniae: is a Rapid diagnostic test on sputum
or culture. By mixing S. pneumoniae with specific
antipolysaccharide (capsule component) on microscopic slide.
The capsule swells due Ag-Ab
reaction.
Culture: BA-α-hemolytic
Biochemical test:
-optochin sensitivity: sensitive
![Page 23: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/23.jpg)
Optochin Susceptibility Test
Principle:
Optochin(OP) test is presumptive test that is used to identify
S. pneumoniae
S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml)
giving a inhibition zone ≥14 mm in diameter.
Procedure: BAP inoculated with organism to be tested
OP disk is placed on the center of inoculated BAP
After incubation at 37oC for 18 hrs, accurately measure the
diameter of the inhibition zone by the ruler
≥14mm zone of inhibition around the disk is considered as
positive and ≤13 mm is considered negative
S. pneumoniae is positive (S) while S. viridansis
![Page 24: Gram-positive cocci Staphylococci and Streptococciacpha.tu.edu.iq/images/micro_lab_8-gramve_cocci.pdf · Gram-positive cocci, arranged in grape-like clusters Non spore-forming, non](https://reader033.vdocuments.us/reader033/viewer/2022053007/5f0b5d457e708231d4302760/html5/thumbnails/24.jpg)