Post on 30-Dec-2015
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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