Transcript Document
Diagnostics
Gram Positive Rods
Classification
Gram positive rods Spore formers Non spore formers Aerobes & facultative aerobes Strict anaerobes Aerobes
Bacillus Clostridium Listeria
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 Gram positive rods Spore formers Non spore formers Aerobes & facultative aerobes Strict anaerobes Aerobes
Bacillus Clostridium Listeria
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 + CO 2 • Fermentation with accumulation of neutral
products
– Glucose pyruvate
acetoin
2 butanediol + CO 2
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 + O 2 acetoin VP + = red VP - = Yellow Usual results of MR/VP: MR+/VP-; MR-/VP+ MR-/VP - + Neutral Acid Acid produced No acetoin Neutral Acetoin
Negative
Urea Utilization
• Enzyme tested – Urease Positive • pH Indicator – Phenol red (turns pink)
H 2 N
C O + 2 H 2 O
H 2 N
Urea CO 2 + H 2 O + 2 NH 3 Amino acids (NH 4 ) 2 CO 3 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 +
exterior
H + 2 H 2 e + Cyt
b
Cyt
o
2 e Q 2 e 2 e Fe-S 2 H + 3 H + + 3 OH
-
3 H + + 1/2 O 2 Fp H 2 O
interior
NADH + H + FADH 2 3 H 2 O
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
We add this.
2H 2 O 2
Does bacteria make this?
catalase
2H 2 O + O 2
Detect bubbles.
Product of respiration Damaging for DNA Add 3% H 2 O 2 to bacterial growth bubbles (O 2 ) Aerobic metabolism requires catalase
Anaerobic Respiration
2 H + Exterior 2 H + 2 e Cyt
b
2 e Q 2 e 2 e Fe-S 2 H + 3 H + + 3 OH Fp Nitrate reductase NADH + H FADH 3 H 2 2 NO 3 + 2 H + (N = +5) nitrate Final e- acceptor O +
Interior
NO 2 + H 2 O (N = +3) nitrite
Nitrate Reductase
NO 3 nitrate + 2 H + + 2 e H 2 O + NO 2 nitrite NO, N 2 O, NH 2 OH, NH 3 , N 2 Step 1: Test for nitrite NO 2 + sulfanilic acid and alpha naphthylamine HNO 2 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)
NO 3 nitrate + 2 H + + 2 e H 2 O + NO 2 nitrite NO, N 2 O, NH 2 OH, NH 3 , N 2 Step 2: Test for the presence of nitrate NO 3 + Zn (s) NO 2 Nitrate is present Reduction to Nitrite Red Nitrate is absent Nitrite was reduced Yellow
Diagnostics
Gram Positive Cocci
Classification
Gram positive cocci
Streptococcaceae
Catalase -
Micrococcaceae
Catalase + Aerobes & facultative anaerobes Aerobes
Streptococcus Micrococcus 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