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Diagnostics
Gram Positive Rods
Classification
Aerobes &
facultative
aerobes
Bacillus
Strict anaerobes
Clostridium
Aerobes
Listeria
Spore formers
Gram positive
rods
Non spore
formers
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
Aerobes &
facultative
aerobes
Bacillus
Strict anaerobes
Clostridium
Aerobes
Listeria
Spore formers
Gram positive
rods
Non spore
formers
Medically Important Bacilli
• Clostridium
– Several pathogenic species
• Clostridium perfringens
– Gas gangrene
• Clostridium tetani
– Tetanus
• Clostridium botulinum
– Botulism
• Clostridium difficile
– Diarrhea
prolonged contraction of skeletal
muscle fibers, neurotoxin
produced by the bacteria
Canned food that has not been
sterilized properly; paralytic illness
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
Reagents VP:
butanediol + -naphthol + KOH + O2  acetoin
VP + = red
VP - = Yellow
Usual results of MR/VP:
MR+/VP-; MR-/VP+ MR-/VPAcid
produced
No
acetoin
Neutral Acetoin
- +
Neutral
Acid
Urea Utilization
• Enzyme tested
– Urease
Negative
Positive
• pH Indicator
– Phenol red (turns pink)
H2 N
H2 N
C O + 2 H2O  CO2 + H2O + 2 NH3  (NH4)2CO3
Urea
Amino acids
ammonium
carbonate
(alkaline)
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 Agar
Before iodine addition
After iodine addition
Caseinase – Milk Agar
Lipase – Spirit Blue
Aerobic Respiration
Electron Transport Chain
2 H+
2 e-
Fe-S
2 H+
exterior
Fp
interior
2 e-
Q
NADH + H+
FADH2
2 e-
Cyt b
3 H+ + 3 OH-
2 e-
H+
2 H+
Cyt o
3 H+ + 1/2 O2
H2O
3 H2O
Oxidase Test
phenylenediamine
• 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)
Catalase
Does bacteria make this?
2H2O2
We add this.
catalase
2H2O + O2 
Detect bubbles.
Product of respiration
Damaging for DNA
Add 3% H2O2 to
bacterial growth
bubbles
(O2)
Aerobic metabolism requires catalase
Anaerobic Respiration
2 H+
2 e-
Exterior
Fp
Fe-S
2 H+
Interior
2 e-
Q
NADH + H+
FADH2
2 e-
Cyt
b
2 H+
3 H+ + 3 OH -
2 e-
Nitrate
reductase
3 H2O
NO3- + 2 H+ (N = +5) nitrate
Final e- acceptor
NO2- + H2O (N = +3) nitrite
Nitrate Reductase
NO3- + 2 H+ + 2 e-  H2O +
nitrate
NO2-
nitrite

NO, N2O,
NH2OH,
NH3, N2
Step 1: Test for nitrite
NO2- + sulfanilic acid and alpha naphthylamine  HNO2
Nitrate is reduced
Production of Nitrite
Red
Nitrate is reduced to nitrite
Nitrite is reduced
No Nitrite
Yellow
Nitrate is not reduced
No Nitrite
Yellow
Nitrate Reductase (Cont’d)
NO3- + 2 H+ + 2 e-  H2O +
nitrate
NO2-
nitrite

NO, N2O,
NH2OH,
NH3, N2
Step 2: Test for the presence of nitrate
NO3- + Zn (s)  NO2-
Nitrate is present
Reduction to Nitrite
Red
Nitrate is absent
Nitrite was reduced
Yellow
Diagnostics
Gram Positive Cocci
Classification
Streptococcaceae
Catalase -
Aerobes &
facultative
anaerobes
Gram positive
cocci
Streptococcus
Micrococcus
Micrococcaceae
Catalase +
Aerobes
Staphylococcus
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. epidermidis
S. aureus
Identification: Metabolic Tests
• Differentiation of Streptoccocus species
• Serological grouping according to Lancefield
– Based on the type of carbohydrate in their cell wall
» 8 groups (A-H and K-U)
» Group A: β-hemolytic - Streptococcus pyogenes
» Group B: β-hemolytic - S. agalactiae
» Group C: α or γ hemolytic- S. viridans
» Group D: γ –hemolytic – Enterococcus
» Does not belong to any Lancfield group
» α hemolytic- S. pneumoniae and S. mutans
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