Diagnostics Gram Positive Rods. Classification Gram positive rods Spore formers Aerobes &...

Diagnostics

Gram Positive Rods

Classification

Characteristics of Spore Forming Rods

• All are large Gram positive rods• Make endospores • Mainly found in soil, water and

dust• Highly diverse nutritional

requirements– Use simple and complex carbon

sources– Their spores allow them to resist

extreme conditions

Endospore Morphology

• Location: – Terminal (a, d, e)

– Subterminal (b)

– Central (c, f).

• Shape: – Circular (b, d)

– Ellipsoid (a, c, e, f)

• Spore diameter: – Non-deforming (a, b, c)

– Deforming (d, e, f).

Medically Important Bacilli

• Bacillus– Mostly harmless– A few opportunistic species

• Bacillus cereus & Bacillus subtilis– Food poisoning

– One pathogenic species• Bacillus anthracis

– Anthrax

Medically Important Bacilli

• Clostridium– Several pathogenic species

• Clostridium perfringens– Gas gangrene

• Clostridium tetani– Tetanus

• Clostridium botulinum– Botulism

• Clostridium difficile– Diarrhea

Canned food that has not been sterilized properly; paralytic illness

prolonged contraction of skeletal muscle fibers, neurotoxin produced by the bacteria

Identification: Metabolic Tests

• Phenol red broth– Simple Carbon source:

• Peptone (protein amino acids)• Desired sugar added

– pH indicator• Phenol red

– Yellow acid pH– Orange neutral pH– Red alkaline pH

Phenol Red Broths

• Carbon utilization:– Sugar

• Acid reaction (yellow)

• or neutral (original)

– Protein

• Alkaline reaction (red)

Glucose Fermentation

• Fermentation with acid accumulation:– Glucose pyruvate lactic and/or acetic acid + CO2

• Fermentation with accumulation of neutral products– Glucose pyruvate acetoin 2 butanediol + CO2

Methyl Red Test

• Test for acid accumulation– Carbon Sources: Glucose and proteins– Indicator -methyl red; Added after growth

• MR +: red (pH < 5.2)• MR - : Yellow (pH > 5.2) Neutral Acid

Voges-Proskauer Test

VP + = redVP - = Yellow

Usual results of MR/VP: MR+/VP-; MR-/VP+ MR-/VP-

Reagents VP:

butanediol + -naphthol + KOH + O2 acetoin

Neutral Acid

- +

Acid produced

No acetoin

Neutral Acetoin

Urea Utilization• Enzyme tested

– Urease

• pH Indicator– Phenol red (turns pink)

C O + 2 H2O CO2 + H2O + 2 NH3 (NH4)2CO3

H2N

H2N

Ureaammoniumcarbonate(alkaline)Amino acids

PositiveNegative

Complex Carbon Utilization

• Too large to be transported inside• Requires exocellular enzymes for the external

degradation into smaller units– Polysaccharides

• Starch (amylase)– Lipids (lipase)

• Tributyrin– Proteins (protease)

• Casein (caseinase)

Amylase – Starch AgarBefore iodine addition After iodine addition

Caseinase – Milk Agar

Lipase – Spirit Blue

Aerobic RespirationElectron Transport Chain

3 H2O

H+

2 H+

2 H+

3 H+ + 3 OH-

2 H+

H2O

3 H+ + 1/2 O2

2 e-

2 e-

2 e-

Fp

Fe-S

2 e-

Q

Cyt b

Cyt o

NADH + H+

FADH2

interior

exterior

Oxidase Test

• Cytochrome oxidase catalyzes the reduction of a final electron acceptor, oxygen

• An artifcial e- donor, phenylenediamine, is used to reduce the cytochrome oxidase

• If the enzyme is present, the colorless reagent (reduced state) will turn blue (oxidized state)

phenylenediamine

Catalase

2H2O2 2H2O + O2 catalase

Product of respirationDamaging for DNA

Aerobic metabolism requires catalase

bubbles(O2)

Add 3% H2O2 to bacterial growth

We add this.

Does bacteria make this?Detect bubbles.

Anaerobic Respiration

2 H+

2 H+

3 H+ + 3 OH -

3 H2O

2 H+

NO2- + H2O (N = +3) nitrite

NO3- + 2 H+ (N = +5) nitrate

2 e-

2 e-

2 e-

Fp

Fe-S

2 e-

Q

Cyt b

NADH + H+

FADH2

Nitrate reductase

Interior

Exterior

Final e- acceptor

Nitrate ReductaseNO3

- + 2 H+ + 2 e- H2O + NO2- NO, N2O,

NH2OH, NH3, N2

nitratenitrate nitritenitrite

Step 1: Test for nitriteNO2

- + sulfanilic acid and alpha naphthylamine HNO2

Nitrate is not reduced No Nitrite

Yellow

Nitrate is reducedProduction of Nitrite

Red

Nitrate is reduced to nitrite Nitrite is reduced

No Nitrite Yellow

Nitrate Reductase (Cont’d)NO3

- + 2 H+ + 2 e- H2O + NO2- NO, N2O,

NH2OH, NH3, N2

nitratenitrate nitritenitrite

Step 2: Test for the presence of nitrateNO3

- + Zn (s) NO2-

Nitrate is presentReduction to Nitrite

RedNitrate is absent

Nitrite was reducedYellow

Diagnostics

Gram Positive Cocci

Classification

Characteristics of Gram Positive Cocci

• All are non sporulating• Mainly found amongst the

natural flora of humans and animals

• Fastidious (‘picky’) nutritional requirements– Use simple carbon sources

Cellular Aggregation of Gram Positive Cocci

Micrococcus & Streptococcus

Streptococcus

Micrococcus

Staphylococcus

Gram Positive Cocci of Medical Importance

• Micrococcaceae– Staphylococcus aureus

• Causes several types of infections, food infections and toxic shock (skin and respiratory tract)

– Staphylococcus epidermidis• Cause opportunistic infections (catheters with biofilms)

– Staphylococcus saprophyticus• Major cause of cystitis in women (bladder infection)

Gram Positive Cocci of Medical Importance

• Streptococcaceae– Streptococcus pyogenes

• Strep throat and flesh eating disease– Streptococcus agalactiae

• Genital infections– Streptococcus mutans

• Endocarditis– Streptococcus pneumonia

• Otitis, meningitis, and pneumonia

– Enterococcus spp.• Opportunistic infections

Identification: Metabolic Tests

• Microccocus Vs Staphylococcus• Oxidase test

– Micrococci are +– Staphylococci are –

• Bacitracin (antibiotic)– Micrococci are sensitive– Staphylococci are resistant

Identification: Metabolic Tests

• Differentitation of Staphylococcus species• Coagulase test

– Coagulase positive staphylococci» S. aureus

– Coagulase negative» All the other Staphylococci

• Mannitol fermentation – S. aureus and some S. saprophyticus are positive– S. epidermidis is negative

S. aureusS. epidermidis

Identification: Metabolic Tests

Blood Hemolysis

• Blood agar:– Discrimination according to hemolysis patterns

• Alpha hemolysis – Incomplete hemolysis• Beta hemolysis- Complete hemolysis• Gamma hemolysis – No hemolysis

Identification: Metabolic Tests

• Identification of Streptoccocus pneumoniae– Bile solubilization

• Strep. pneumoniae is positive

• Other Strep. are negative

• Identification of Enterococcus– Bile-esculin test

• Enterococcus is positive

• Other Strep. are negative