Microb Lab Flashcards
CSF volume of neonates?
10-60 ml
CSF volume of adults?
85-150 ml
What is CSF?
“Selective” ultrafiltrate of plasma
Adult rate of formation?
500 ml/day
Turnover of CSF?
20 ml/hour
CSF functions?
1) CNS protection
2) Waste management
3) Lubrication
4) Nutrition
5) Reduction of real weight of brain (1.5 kg)
Site of lumbar puncture for adults?
Between L3/L4
Site of lumbar puncture for infants?
Between L4/L5
Lumbar puncture procedure?
1) Positioned in a fetal position
2) Skin of lower back is prepared using antiseptic solution and local anasthetic is injected
3) Spinal needle is inserted between two lower lumbar vertebrae into subarachnoid space
4) After CSF is collected needle is withdrawn and bandage is placed
Precautions for lumbar puncture
1) Spinal needle must be connected to manometer to measure CSF pressure
2) If the pressure is normal (50-180 mm H2O), CSF can be collected
3) Up to 20 ml of CSF can be taken from adults
3 sterile tubes for CSF sample
1) Chemistry lab -> to measure protein and glucose levels
2) Microbiology lab -> to study if we have bacterial or viral infections
3) Hematology lab -> to count lymphocytes, neutrophils
Small volume of CSF sample?
Sent to microbiology lab first (sterile issue)
Storage of 3 sterile tubes
1) Freeze it after centrifugation
2) Keep at room temperature (avoid autolysis of H. influenzae, N. meningiditis)
3) Refrigerate for only 24 hours
Normal CSF appearance?
Crystal clear & colorless
Pleocytosis
Increased CSF cell numbers
1) WBC > 200 cells/µ L
2) RBC > 400 cells/µ L
Cloudy or turbid CSF
High concentration of protein or neutrophils
Bloody CSF
RBCs
Clot CSF
TB meningitis
Viscous CSF
Cryptococcosis
Xanthochromic
Orange or pinky supernatant after centrifuge of CSF
Difference between traumatic tap and CNS hemorrhage
1) Maximum amount of blood in 3 samples:
- Traumatic tap -> progressive decrease in subsequent sample tubes
- CNS hemorrhage -> blood evenly mixed in all tubes
2) After centrifugation:
- Traumatic tap -> Clear supernatant
- CNS hemorrhage -> Xanthochromic supernatant
Blood clot in CSF sample
Very bloody traumatic tap (not usually associated with CNS hemorrhage)
CSF glucose
50-80 mg/dl
CSF protein
20-45 mg/dl
CSF pressure
50-180 mm. H20
Leukocyte count in normal CSF
1) Adults -> 0-5 cell/ml
2) Neonates -> 0-30 cell/ml
CSF analysis for virus
1) Normal pressure
2) Clear
3) Lymphocyte
4) Protein increased slightly
5) CSF sugar normal
6) Special lab
CSF analysis for bacteria
1) Normal, increased pressure (increased protein concentration)
2) Turbid
3) PMN (neutrophils)
4) Protein increased significantly
5) CSF sugar decreased
6) Gram stain, culture, CIE, latex agglutination
Microbiology examination procedure
1) Centrifuge must be done if volume more than 1 mL
2) Remove supernatant (sterile pipette) and leave about 0.5 mL over sediment to concentrate suspension
3) Take 1-2 drops of suspension for culture media
Types of culture media
1) Chocolate agar
2) 5% Sheep Blood agar
3) Modified Thayer-Martin media
4) MacConkey agar
5) Lowenstein-Jensen agar
Enriched medium
Blood and Chocolate agar
Selective, differential for gram negative bacteria
MacConkey agar
Selective for Neisseria spp.
Modified Thayer-Martin media
Used for culture of TB
Lowenstein-Jensen agar
Organisms most frequently encountered in infants
1) E. coli
2) Klebsiella pneumonia
3) K. oxytoca
4) Group B streptococci (Streptococcus agalactiae)
5) Listeria monocytogenes
Organisms most frequently encountered in children (6 months-3 years)
1) Haemophilus influenzae
2) Neisseria meningitidis
3) Streptococcus pneumoniae
Organisms most frequently encountered in adults (5-29 years)
Neisseria meningitidis
Organisms most frequently encountered in old adults (30-60 years)
Streptococcus pneumoniae
Organisms most frequently encountered in elderly (>60 years)
1) E. coli
2) Listeria monocytogenes
1) MacConkey agar -> Dry, pink (lactose positive) colonies with surrounding pink areas
2) EMB agar -> Metallic green sheen colonies
3) Motile bacteria
4) IMViC ++–
5) kIA test A/A (glucose lactose fermenter)
E. coli
Klebsiella spp.
1) Lactose positive
2) Most urease positive
3) Non-motile
1) MacConkey agar -> Pink (lactose fermentation) and mucoid (large colonies, honey appearance because it’s encapsulated bacteria)
2) IMViC –++
3) kIA test A/A glucose lactose fermenter
Klebsiella pneumoniae
1) Klebsiella spp.
2) Differs from others by being Indole Test + IMViC +-++
Klebsiella oxytoca
1) G+ diplococci, lancet-shaped, crystal violet
2) Blood agar -> Narrow zone of β-hemolysis (complete reduction of RBCs)
3) Whitish colonies
4) Most bacitracin resistant
5) Positive CAMP test
Group B Streptococci (Streptococcus agalactiae)
Catalase test for Streptococcus
Negative (-)
Catalase test for Staphylococcus
Positive (+)
CAMP test
1) Specific for S. agalactiae (Group B)
2) CAMP act synergistically with staphylococcal β-lysin (lysis of RBCs)
How to differentiate Streptococcus and Staphylococcus?
Catalase test
How to differentiate β-hemolytic streptococci?
1) Lancefield Classification
2) Bacitracin susceptibility test (Bacitracin Disk):
- Group A (pyogenes) -> sensitive (zone of inhibition)
- Group B (agalactiae) -> non-sensitive/resistant (no zone of
inhibition)
Procedure of CAMP test
1) 2 streaks of Streptococcus spp. and S. aureus perpendicular to each other
2) 3-5 mm distance between the 2 streaks
3) After incubation an arrowhead-shaped zone shows complete hemolysis
4) S. agalactiae is CAMP test-positive (other streptococci negative)
1) 5% sheep blood agar -> α-hemolysis (partial reduction of RBCs)
2) Sensitivity to optochin (inhibition)
Streptococcus pneumoniae
How to differentiate α-hemolytic Streptococci?
Optochin Susceptibility test (S. pneumoniae sensitive, S. viridans non-sensitive)
1) Facultative anaerobic, Gram-negative coccobacilli
2) Small short-rod, pleomorphism
3) Non-motile
4) Grows on Chocolate agar only (Grayish mucoid colonies)
5) Blood-loving
6) Need X and V factor in Chocolate agar
Haemophilus influenzae
Special characteristic of H. influenzae
Satellitism (Can grow in Blood agar (X-factor only) when streaked in the middle with a line of S. aureus (produce V-factor from RBCs)
1) Aerobic
2) Gram-negative cocci arranged in pairs (diplococci) with adjacent side flattened (coffee beans)
3) Oxidase positive
4) Non-motile
Neisseria meningiditis (Meningococcus)
How to diagnose Neisseria meningitidis?
1) Direct smear from CSF -> Intra- & extracellular Gram-negative diplococci
2) Sheep Blood agar & Chocolate agar in CO2 (must be incubated at 35°C in 3-5% CO2 or candle jar)
3) Modified Thayer-Martin (MTM) agar -> small, gray, translucent, raised colonies
4) Oxidase test -> Positive (Violet) other Enterobacteriacae negative
1) Aerobic and facultatively anaerobic
2) Motile, β-hemolytic, non-spore forming
3) Gram-positive rod, regular, short
4) Singly or in short chains
Listeria monocytgenes
“Umbrella” growth in semi-solid medium (motility test)?
Listeria monocytogenes
How to diagnose Tuberculosis meningitis?
1) Acid-fast Positive Stain (Ziehl-Neelsen stain) -> Bright red stain (Acid-fast bacilli)
2) Lowenstein-Jensen media -> Puff, rough and tough colonies like bread crumbs (21 days to grow on this media)
3) We don’t depend on culture because it takes 4-6 weeks to grow
1) Encapsulated yeast
2) Grows as yeast (unicellular) and replicates by budding
Cryptococcus neoformans (fungus)
How to diagnose Cryptococcus neoformans?
1) India Ink preparation (negrosin, eosin) -> Colorless halo (stained background leaving halo representing distinct, wide and gelatinous capsule)
