PHT 313 Lab (3) Streptococci

Staphylococci Streptococci Enterococci NeisseriaCorynbacterium Clostridum Bacillus Enterobacteriaceae Pseudomonas. Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve Cocci Bacilli

Streptococci

Classification Schemes (Taxonomy):Taxonomy Based on Hemolysis on blood agar plates – β-hemolytic : S. pyogenes – α-hemolytic : Viridans streptococci (incomplete,) – γ-hemolytic : Enterococci (no hemolysis) α -hemolytic Sterptococci β -hemolytic Sterptococci

 Aerobic streptococci  Anaerobic streptococci (Peptostreptococci):  These are normal flora in bowel and vagina.  They are often participate in mixed anaerobic infections (Abscesses) in the abdomen, brain, pelvis, lung and uterus.  They are sensitive to penicillin and metronidazol. Based on O₂ requirements

Major pathogens: Group A Streptococci: Streptococcus pyogenes Group B Streptococci: S. agalactiae Enterococcus faecalis was previously classified as Group D streptococci. Based on serology: Lancefield grouping based on group specific carbohydrate antigens. Lancefield serological groups (A-W with the exception of I & J). N.B. Streptococci lacking group-specific antigens (Viridans streptococci & S.pneumoniae ) Gamma or alpha-hemolysis

Microscopic morphology: - Gram positive cocci, - arranged in chains or pairs, - non-motile, - non-spore forming. streptococci

StreptococcusStaphylococcus

Culture Characters: Environment: Facultative anaerobes Temp.: 37 °C pH: 7.2 Media: Blood agar (Fastidious organism): Complex nutritional requirements, Require blood- or serum- enriched media; S. pyogenes 1-β-hemolytic Sterptococci (S. pyogenes ). S.pneumonia, viridans streptococci 2-α-hemolytic Sterptococci (S.pneumonia, viridans streptococci). 3-Non-hemolytic streptococci. β-hemolytic Non-hemolyticα-hemolytic

B.R.:  Catalase:  Fermentation of glucose without the production of gas, produces mainly lactic acid, ethanol, acetate - ve

Blood culture Definition: It is microbiological culture of blood. It is employed to detect infections that are spreading through the bloodstream (Bacteremia & Septicemia). A sample of blood is put in a special laboratory preparation and is incubated in a controlled environment for 1 to 7 days.

Procedures: Blood is drawn from a person and put directly into a blood culture bottle containing a nutritional broth (Aerobic and anaerobic). After the laboratory receives the blood culture bottle, several processes must be completed: provide an environment for the bacteria to grow detect the growth when it occurs identify the bacteria that grow test the bacteria against certain antibiotics to determine which antibiotic will be effective Uses: The detection of microorganisms in a patient’s blood has diagnostic and prognostic importance. Blood cultures are done to detect the presence of bacteria or yeasts, which may have spread from a specific site in the body into the bloodstream.bacteriayeasts,

Collecting blood cultures: by vein puncture Fill the anaerobic bottle first, so that any air in the syringe is not inoculated into that bottle.

Blood culture bottles should be placed in any ward incubator until collected by the porters for transport to the laboratory. The type of system used may be, manual or automated

Manual technique The bottles are placed in an incubator and kept at body temperature. They are watched daily for signs of growth, including cloudiness or a color change in the broth, gas bubbles, or clumps of bacteria. When there is evidence of growth, the laboratory does a gram stain and a subculture. To do the gram stain, a drop of blood is removed from the bottle and placed on a microscope slide. To do the subculture, a drop of blood is placed on a culture plate, spread over the surface, and placed in an incubator. If there is no immediate visible evidence of growth in the bottles, the laboratory looks for bacteria by doing gram stains and subcultures. These steps are repeated daily for the first several days and periodically after that.

blood culture machine ( The blood culture machine (BACTEC 9000) are designed for the rapid detection of microorganisms in clinical specimens. The sample to be tested is inoculated into the vial which is entered into the Blood culture instrument for incubation and periodic reading. Each vial contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption of oxygen needed for the growth of microorgnisms. The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the increasing amount of CO2 or the decreasing amount of O2 present in the vial. A positive reading indicates the presumptive presence of viable microorganisms in the vial. Automated technique

When a bottle is registered as “positive”, it is removed from the machine and a portion is placed on a glass slide, gram-stained and examined under the light microscope.

It’s also used to inoculate agar plates on which most bacteria will grow. The inoculum is also spread over a susceptibility agar plate, on which are placed antibiotic discs appropriate for the organism seen on the gram stain. This will give the “direct” sensitivity results.

Catalase test Gram’s +ve Cocci Staphylococci Streptococci + ve - ve

Streptococci Growth on Blood agar α-Haemolysisβ-Haemolysis Optochin Test Bacitracin Test S R S R S.pneumoniae Viridans Streptococci S. pyogenes S. agalactiae

β-hemolytic Sterptococci  Example: S. pyogenes (group A β-hemolytic Strept.) S.agalactiae (group B β-hemolytic Strept. =Non group A β-hemolytic strept.) = Non group A β-hemolytic strept.),………………………….. To differentiate between S.pyogenes & Non group A β-hemolytic streptococci,………………………….. Bacitracin Sensitivity Test:

Bacitracin Sensitivity Test Procedure: 1. Inoculate blood agar plate with the test organism. B 2. Aseptically apply Bacitracin disc onto the center of the streaked area. 3. Incubate the plate at 35 o C for 18 hrs. Principle: A low conc. of Bacitracin (0.04 units) will selectively inhibit the growth of S.pyogenes giving a zone of inhibition around the disc

B Results: Positive test: any zone of inhibition around the disc. B Bacitracin ResistantBacitracin Sensitive S.pyogenes β-hemolytic Streptococci Non group A

CAMP test CAMP reaction: The hemolysis is enhanced in the vicinity of the ß -lysin producing S.aureus (Arrow head-shaped zone of ß-hemolysis). S.agalactiae. Group B streptococci (S.agalactiae. ) produce a factor that increases beta hemolysis of an S. aureus indicator strain. CAMP+veS.agalactiae CAMP-ve

 It causes: 1. Zone of greenish discolouration around the colonies. 2. It is due to peroxidase release. α-hemolytic Sterptococci  Example: S.pneumonia, viridans streptococci. S.pneumonia, viridans streptococci, ……… To differentiate between S.pneumonia, viridans streptococci, ……… 1) Optochin sensitivity. 2) Bile solubility test

Procedure: 1. Inoculate blood agar plate with the test organism. O 2. Aseptically apply Optochin disc onto the center of the streaked area. 3. Incubate the plate at 35 o C for 18 hrs. 4. Accurately measure the diameter of the inhibition zone around the disc. Optochin Sensitivity Test S.pneumoniae is inhibited by less than 5µg/ml Optochin reagent giving a zone of inhibition more than 15 mm in diameter. Principle:

Optochin sensitive S.pneumoniae Optochin resistant Viridans Streptococci Results: Positive test: inhibition zone more than 15 mm in diameter.

Bile Solubility Test: S.pneumoniae produces a self-lysing enzyme to depress the growth of old colonies. The presence of bile salt accelerate this process. Principle: Procedure: 1)Add 1 ml 10% bile salt solution to 10 ml broth culture of the test organism 2)Incubate at 37 o C for 15 min 3)Observe for the visible clearing of the turbid culture Visible clearance S.Pneumoniae Remain turbid Viridans Streptococci Results

 Gram-positive cocci, arranged singly, in pairs, and in short chains.  Catalase negative  Most strains react with Lancefield group D antisera.  E.faecalis is the most important species.  It has no effect on RBCs (Non hemolytic)  Enterococcus faecalis is the only gram-positive cocci which can grow on MacConkey’s agar giving pink colonies. Enterococci Enterococcus faecalis

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Gram’s Stain Catalase test Growth on blood agar Gram’s +ve Cocci arranged in chains - ve Complete hemolysis Greenish discoloration Non hemolytic

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Inhibition zone >15mm Optochin sensitive S.pneumoniae No zone or <15mm Optochin resistant Viridans Streptococci Optochin sensitivity Bile Solubility Visibile clearance Remain turbid (+ve) (-ve)

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Growth on MacConkey’s Agar No Growth Growth of pink colonies Enterococcus faecalis