MicroBiology Lab Quiz #5 – Bacteriological Examination of Water, Staphylococci

Performed at treatment plants to ensure that water is safe for human consumption

If inadequate, human disease-causing pathogens can survive in drinking water

Vibrio cholerae
Salmonella typhi
Shigella dysenteriae
Entamoeba histolytica
Giardia lamblia
Cryptosporidium parvum

Hep A virus
poliovirus

E. coli is a good indicator bacterium for routine water tests

gram-negative

facultative anaerobe

non-endospore forming rods that ferment lactose to produce lactic acid & gas

Found in large quantities in human intestines & NOT in water or soil naturally

can be ID using simple microbiological tests

not as fastidious (grow with certain nutrients); can grow and survive in water more readily than fastidious pathogens

Indicates presence of total coliforms (usually found in envir & post negligible risk) in water sample

From this test, the most probable # (MPN) of coliforms present per 100 mL of water can be determined

3-digit code number is 95% probability of there being between certain amounts of coliforms per 100mL

Indicates lactose-fermenting Gram(-) bacteria are present

Selective medium of Levine EMB (eosin methylene blue) and Endo agar plates allow only growth of G(-) bacteria

Differential medium distinguishing coliforms from non-coliforms

Endo agar produce red lactose fermenters colonies (presence of E. coli), non-fermenters colorless

Selective b/c: Only G(-) grow colonies

Differential b/c: Red colonies for lactose fermenters; non-fermenters are colorless

Selective b/c: Only G(-) grow colonies

Differential b/c: EMB produce green metallic sheen E. coli and inhibit G(+) growth. Other lactose fermenters form brown dark nucleated colonies. Non-fermenters form colorless or lighter colonies

verifies confirmation of coliforms

media include nutrient agar slant & Durham lactose broth

perform IMViC to distinguish E. aerogenes vs E. coli

most probable number

an estimate of the number of coliforms present in 100mL of water

Low to high amounts of coliforms present per 100mL of sample with 95% confidence probability

Allows qualitative ID & quantification of total coliforms

Total coliform count determines potability of water

more rapid & reproducible, and can analyze larger quantities of water compared to MPN test

Filter membrane containing pores 0.45um in diameter b/c bacteria typically can't pass through the pores & remain on the filter

Any coliforms present after membrane filter method will ferment lactose on this Endo broth, producing acids

Acids react with basic fuchsin, dye in medium, causing coliform to have metallic sheen

G(+) inhibited from growing b/c of presence of bile salts & sodium lauryl sulfate, which inhibit these bacteria

If the membrane manufacturing process is not precisely controlled, membranes with wide pore size distribution may result, giving poor separation performance.

Equipment cost can be high

Allow viruses to pass through

May absorb large amts of filtrate and introduce metallic ions into filtrate

Can filter large volumes of fluids rapidly

do not clog easily

can be autoclaved or purchased already sterilized

(total coliforms * dilution factor) / mL

typically divide in more than 1 plane --> Grape-like clusters

pyogenic = produce pus

divide in 1 plane --> diplococci or chains of cells

pyogenic = produce pus

Aerobic bacteria produce H2O2 as byproduct

enables bacteria to metabolize harmful H2O2 --> H2O & O2 (bubbles)

Staphylococcus = Catalase POSITIVE

some S. aureus produce α-toxin - RBC hemolysis

β-hemolysis = clear zone where RBCs have lysed around the colonies

S. epidermis = NEG, no RBC hemolysis

Mannitol salt agar - selective & differential media

contains 7.5% NaCl & selects for staphylococci

if mannitol fermented to form acid, red turns to yellow (differential)

S. aureus = differential POS
S. epidermis = differential NEG

an enzyme that catalyzes the conversion of fibrinogen --> fibrin resulting in clot formation

clumps in plasma

S. aureus = POS
S. epidermis = NEG

diagnostic test on the ability to detect coagulase & protein A from S. aureus

Latex beads from kit coated w/ fibrinogen & IgG

Protein A, attached to cell wall, joins w/ Fc portion of IgG

Both fibrinogen & IgG markers serve to combine w/ products made by S. aureus

The reaction causes "clumping" (agglutination)

Antimicrobial test used to distinguish S. saprophyticus from other coagulase-NEG Staphylococci

S. saprophyticus - RESISTANT to novobiocin
S. epidermis - SUSCEPTIBLE to novobiocin

- S. aureus
- S. epidermis
- S. saprophyticus

Non-motile, non-endospore-forming, able to grow in media containing high salt concs, facultative anaerobes

Most clinically important staphylococcal pathogen

Virulence factor 1: coagulase production, can form clot around staphylococcal infection and protect the bacterium from host defenses

Virulence factor 2: produces α-toxin that causes a wide, clear zone of β-hemolysis on blood agar, important b/c it not only lyses RBCs but also damages leukocytes, heart muscle, & renal tissue

DNAse, a nuclease that digests DNA

produce pigment (staphyloxanthin) that also act as virulence factor, which prevents superoxide (ROS) produced by host immune system from killing the bacteria