Download Isolation and Identification

Aerobic GramPositive Cocci
Catalase
Negative
Positive
Streptococci
See Streptococci identification
chart
Staphylococci
See Staphylococci Identification
chart
streptococci
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Gram-positive cocci
Facultatively anaerobic
Occur in pairs and chains of varying length.
Catalase negative
They are usually classified based on their
hemolytic properties on blood agar and
according to their serologic groups.
• Hemolysis refers to is the lysis of the red
blood cells in the agar surrounding bacterial
colonies and is a result of bacterial enzymes
called hemolysins.
Types of hemolysis
1. Beta hemolysis refers to colorless zone surrounding
the colony, where a complete lysis of the red blood
cells.
2. Alpha hemolysis appears as a zone of partial hemolysis
surrounding the colony, often accompanied by a
greenish discoloration of the agar.
3. Gamma reaction refers to no hemolysis or discoloration
of the agar surrounding the colony.
Lancefield system
• Many of the streptococci can also be classified under
the Lancefield system.
• In this case, they are divided into a number of distinct
serologic groups on the basis of carbohydrate
antigens in their cell wall.
• These antigenic groups are designated by the letters A
through T.
• Lancefield serologic groups A, B, C, D, F, and G are
the ones that normally infect humans.
• Not all pathogenic streptococci can be identified by
Lancefield typing (e.g., Streptococcus pneumoniae).
Beta hemolytic streptococci
1. Group A Streptococcus pyogenes: have some
virulence factors like:
A. Lipoteichoic acid (which allows adherence to target
host cells).
B. M-protein (which resists phagocytic engulfment).
C. Tissue damaging toxins such as leukocidin (kills
leukocytes) and erythrogenic toxin (damages
endothelium).
D. Tissue damaging enzymes such as proteinase,
hyaluronidase, and Dnase (thought to be especially
important in infections of the skin and soft tissues as
well as the rapid spread of the organism through the
lymphatics).
Isolation and identification
• Usually isolated on Blood agar.
• There are two streptococcal hemolysins,
– streptolysin S and streptolysin O.
– Streptolysin O can be inactivated by oxygen so more distinct
hemolysis can be seen by stabbing the agar several times.
• Only Streptococcus pyogenes among the group A beta
streptococci is sensitive to bacitracin, as shown by a
zone of inhibition
B
Bacitracin Resistant
Non group A β-hemolytic Streptococci
B
Bacitracin Sensitive
S.Pyogenes
Group B Streptococcus agalactiae
• Usually show a small zone of beta hemolysis on
Blood agar, although some strains are non-hemolytic.
• They are often found in the genital and intestinal
tracts of healthy adults with from 5% to 40%,
depending on the population.
• These organisms cause neonatal meningitis and
septicemia after transmission from the normal vaginal
flora of the mother.
Isolation and identification
• The organism can be identified on the basis of
beta hemolysis.
• Hydrolysis of hippurate and CAMP reaction
• CAMP is an abbreviation for the names of the four
individuals who originally described the test.
• Group B streptococci produce a factor that
increases beta hemolysis of an S. aureus indicator
strain.
CAMP Test
Streptococcus agalactiae ”PMAC" eht dellac nietorp a seterces
factor and when it comes into contact with a beta hemolysin
produced by a specific strain of Staphylococcus aureus a sesuac
.sisylomeh decnahne ,citsigrenys
The test is performed by streaking the S. aureus na nwod niarts
.doolb peehs gniniatnoc etalp raga
Then placing the unknown beta hemolytic strep perpendicular to the
staph.gnihcuot ton tub kaerts
After overnight incubation if the strep a si group B strep eht ,
dna hpats eht morf snisylomehstrep will synergistically produce a
large "arrowhead" of hemolysis.
Hippurate Test
• The organism hydrolyzes hippurate to its basic
components, benzoic acid and the amino acid
glycine.
• Detection of the glycine is accomplished by
oxidation with ninhydrin which produces a deep
purple color end product.
• The hippurate test is also used to identify unrelated
bacterial species like Campylobacter jejuni,
Gardnerella vaginalis and Listeria monocytogenes.
Group D Streptococci
• Streptococcus bovis and Enterococcus faecalis.
• Both are normal flora of the intestinal tract.
• Both organisms are among the most common
organisms causing endocarditis.
• Most group D streptococci show alpha
hemolysis or no hemolysis on blood agar,
although some strains are beta hemolytic.
Isolation and identification
• Streptococcus [Enterococcus] faecalis broth (SFB):
• selective medium for the detection of fecal
streptococci (group D) and enterococci from water,
milk and other materials.
• Growth of all other cocci is inhibited.
• Fermentation of glucose is indicated by a color
change in the broth.
 Sodium azide is the inhibitory agent.
 Bromcresol purple is the indicator.
Uninoculated tube of SF broth. pH
indicator is violet at a neutral pH .
Enterococcus faecalis growing in SF broth.
Note yellow-brown color indicating acid
Bile Esculin agar
• Selective medium for the detection of fecal
streptococci (group D) and enterococci.
• Tests ability of the organism to hydrolyze esculin
to esculetin.
• The esculetin reacts with ferric ammonium citrate
to form a blackish precipitate.
 Oxgall (bile) is inhibitory agent.
 Ferric citrate is indicator
Streptococcus pneumoniae
• Gram-positive usually appearing as a
diplococcus, but occasionally appearing singularly
or in short chains.
• Alpha-hemolytic bacterium.
• Fastidious, growing best in 5% carbon dioxide.
• Pneumococci are frequently found as normal flora
of the nasopharynx of healthy carriers.
Isolation and identification
• Isolation on Blood agar (They are surrounded
by a zone of alpha hemolysis).
• Optochin sensitivity
Optochin sensitive
S.pneumoniae
Optochin resistant
Viridans Streptococci
• Staphylococci are often found in the human nasal cavity (and on
other mucous membranes) as well as on the skin.
• They are gram-positive cocci and occur singly, in pairs, in short
chains, and most commonly, in irregular grape-like clusters.
• The staphylococci are strongly catalase positive.
• Reduce nitrates to nitrites.
• Generally tolerate relatively high concentrations of sodium
chloride (7.5-10%).
• There are five species of staphylococci commonly associated
with clinical infections:
• Staphylococcus aureus.
• S. epidermidis.
• S. haemolyticus.
• S. saprophyticus.
Staphylococci
Use API STAPH if Necessary
Coagulase
+
-
Novobiocin
Staphylococcus aureus
Sensitive
Resistant
Staph. Saprophyticus
Staph. epidermidis
Staphylococcus aureus
• Gram-positive, cluster-forming coccus.
• Nonmotile, nonsporeforming and facultative anaerobe
• Fermentation of glucose produces mainly lactic acid
ferments mannitol (distinguishes from S. epidermidis).
• Catalase positive and coagulase positive.
• Given golden yellow colony on agar.
• Normal flora of humans found on nasal passages, skin
and mucous membranes.
• Pathogen of humans, causes a wide range of
suppurative infections, as well as food poisoning and
toxic shock syndrome.
Virulence factors
• Leukocidin (kills leukocytes).
• Alpha and delta toxins (damage tissue membranes).
• Microcapsules (resist phagocytic engulfment and
destruction).
• Coagulase and protein A (both help resist phagocytic
engulfment).
• Some strains also produce TSST-1 (toxic shock
syndrome toxin-1) and cause toxic shock syndrome.
Isolation and Identification
• Blood agar with a novobiocin:
 Hemolysis: Usually beta.
 Pigment: Often creamy gold.
• Mannitol fermentation on Mannitol Salt agar (MSA)
 Staphylococci are able to tolerate the high salt concentration found in
mannitol Salt agar and thus grow readily.
 If mannitol is fermented, the acid produced turns the phenol red pH
indicator from red (alkaline) to yellow (acid).
• Production of deoxyribonuclease (DNase) on DNase agar:
Positive
• Cell wall clumping factor: Positive