6_Microbial Culture of Clinical Samples: Aerobic Pathogens from Blood

F.B.D.V.N.S

(Featureless, Bull’s eye, Dendritic, Vortex, Non-Swarming, Suppressor)

Types of Swarming

type of hemolysis, presence of enzymes coagulase, catalase and DNase, and the swarming motility

The activity includes showing ____ from test bacteria namely Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli

The activity includes showing type of hemolysis, presence of enzymes coagulase, catalase and DNase, and the swarming motility from test bacteria namely

G.B.B.Y

glucose (1% w/v), bactoagar (0.05% w/v), bactopeptone (0.05% w/v) and yeast extract (0.02% w/v)

Swarming agar consist of

Eppendorf tubes

Hemolysis test:

• Suspend a loopful of each of 24-hr plate culture in __tubes with 1.5ml sterile distilled water. Shake lightly to homogenize the suspension.

Hemolysis test:

4 drops of 3% hydrogen peroxide solution

What is used in catalase test?

1 N HCl

In DNAse, what is used to flood plates with to confirm clearing/DNase production

b-hemolysis

Result of Hemolysis test of S. aureus

y-hemolysis some b-hemolysis

Result of Hemolysis test of E.coli

b-hemolysis in some strains

Result of Hemolysis test of P. aeruginosa

hemolysis test

• assesses bacterial ability to lyse red blood cells on blood agar through secretion of hemolysins.

• types include β-hemolysis (complete lysis with clear zones), α-hemolysis (partial lysis or green discoloration), and γ hemolysis (no lysis).

Hemolysins

• are key virulence factors that facilitate infection by destroying host red blood cells, releasing nutrients such as iron essential for bacterial growth.

• They also promote tissue damage and immune evasion, increasing pathogenicity.

Positive

Coagulase test result of S. aureus

Negative (Coag)

Coagulase test result of E.coli and P. aeruginosa

coagulase test

• detects the presence of coagulase enzyme produced mainly by Staphylococcus aureus

• contributes to virulence by enabling bacteria to form protective fibrin clots around themselves, shielding them from immune defenses such as phagocytosis.

• This enhances bacterial survival, colonization, and dissemination in host tissues, making coagulase-positive strains like S. aureus highly pathogenic and capable of causing serious infections.

Coagulase

converts fibrinogen in plasma to fibrin, resulting in clot formation.

fibrinogen in plasma to fibrin

Coagulase converts ___, resulting in clot formation.

• bound coagulase (clumping factor) attached to bacterial cell wall causing clumping of cells in a slide test, and

• free coagulase that reacts with plasma coagulase reacting factor to form a fibrin clot in tube tests.

There are two types of coagulase:

Non-motile

Swarming result of S. aureus

Negative

Swarming result of E.coli

Positive; shows pronounced swarming motility spreading rapidly over the surface due to flagella and biosurfactants.

Swarming result of P. aeruginosa

swarming motility assay

• detects the ability of certain flagellated bacteria to move rapidly and collectively across solid or semi-solid surfaces.

• This movement is powered by rotating flagella and involves coordinated multicellular groups.

• The assay is typically performed on nutrient agar plates with low agar concentration (0.3–0.7%) to allow surface migration.

Swarming

is a virulence factor that allows bacteria to colonize surfaces rapidly, form biofilms, compete successfully with other microorganisms, and resist host immune responses.

Pseudomonas aeruginosa and Proteus mirabilis

Swarming bacteria often exhibit enhanced antibiotic resistance and evasion from phagocytosis, contributing to persistent and difficult-to-treat infections such as those caused by _____

Positive Catalase

Catalase result of S. aureus, E.coli, P. aeruginosa

catalase test

• detects the presence of the enzyme catalase, which breaks down hydrogen peroxide (H2O2) into water and oxygen, producing visible bubbles (oxygen gas). W

• when a bacterial culture is exposed to H2O2, bubbling indicates a positive __.

Catalase

protects bacteria from oxidative damage caused by reactive oxygen species generated by host phagocytes during the immune response

Positive dnase

DNase test of S. aureus

Negative or weak

DNase test of E. coli

Variable

DNase test of P. aeruginosa

DNase test

• etects bacterial production of deoxyribonuclease (DNase) enzyme, which hydrolyzes DNA.

• Bacteria are cultured on agar containing DNA and a dye such as methyl green.

• DNA hydrolysis by __ results in clear zones around colonies (loss of dye complex), indicating a positive test.

DNase

• acts as a virulence factor by degrading neutrophil extracellular traps (NETs) composed of DNA, which are part of the host’s defense to trap and kill bacteria.

• ___production helps bacteria like Staphylococcus aureus evade these traps, promoting dissemination and severity of infections such as abscesses and pneumonia.