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Biology of Disease > Practical Notes > Flashcards

Practical Notes Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Describe the appearance of RBCs infected with P.falciparum vs. P.Malariae and P. Knowlesi

A

P.falciparum:
- RBCs not enlarged but distorted.
- Stippling due to Maurer’s clefts
- Infects all RBCs

P.Malariae (old cells only) and P. Knowlesi (all cells):
- RBCs not enlarged or distorted
- No stippling
- Large amoeboid trophozoites

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2
Q

Describe the appearance of RBCs infected with P.vivax vs. P.ovale

A

Both only infect young cells

P.Vivax:
- RBCs enlarged and distorted
- Large amoeboid trophozoites

P.Ovale:
- RBCs fimbriated, enlarged and distorted
- Compact trophozoites
- Stippling as fine Schaffner’s dots

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3
Q

How is malaria diagnosed?

A

Thick and thin blood smear (once asymptomatic):
- Thick determines if parasites are present
- Thin determines parasite type (from morphology and parasitaemia)

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4
Q

What is the morphology of a macrophage infected with leishmania?

A

Infected with many amastigotes (look like mini cells within cell):
- Leishmania nucleus stains purple under Giesma stain
- Kinetoplast in leishmania is rod-shaped circular DNA within mitochondrion closely associated with flagella important as unique

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5
Q

Name a treatment for Leishmania as well as possible side-effects:

A

Glucantime:
- Nephrotoxic (check kidney function)

Miltefosine:
- Teratogenic = do NOT give if pregnant

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6
Q

Name 5 species of schistosoma and describe their unique morphologies:

A

S. Japonicum: Round with very little cytoplasm with a minute knob on surface

S. Mekongi: Round with few surface features (smallest)

S.Mansoni: Oval with distinctive spike on surface

S.intercalatum: Thin and long with central bulge. Stain red with Ziehl-Neelsen to confirm

S.haematobium: elongated oval (quite large) with small terminal spine

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7
Q

Which test allows identification of helminth eggs in faeces?

A

Kato-Katz technique:
- Eggs counted using light microscopy

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8
Q

What are some chemical analysis methods for bacterial identification?

A
  • Chemical composition analysis
  • Mass spectrometry (MALDI-TOF test)
  • Immunochemical analysis
  • “Next generation sequencing”

Metabolic (analytical profile index API):
- Metabolic profiling
- Substrate utilisation tests
- End-product output tests

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8
Q

What are some genetic analysis methods for bacterial identification?

A
  • Specific sequences using PCR
  • GC:AT ratio analysis (useful for evolutionary relationships)
  • DNA hybridisation
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9
Q

Describe the API and what it is used for:

A

Analytical profile index: identifies use of different metabolic substrates by a bacterium (identification purposes):
- Selection of tubes each with a dehydrated media
- Effect of bacterial inoculation observed (colour change) and compared to key.

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10
Q

How does ‘Next generation sequencing’ work?

A
  • Fluorescent markers added to identify bases
  • Transposomes used to chop DNA into smaller pieces (of correct length)
  • Adapters required to run PCR
  • Sections have clustered and multiplexing regions to identify sequence and source of sequence
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11
Q

What are some test to aid morphological identification of bacteria?

A
  • Gram stain
  • Morphological observation
  • Hot malachite green stain (for spores)
  • Coagulase test (staph. species)
  • Albert’s method (diphtheria identification)
  • ELEK plate (diphtheria toxin)
  • Capsule staining
  • Toxin/antitoxin neutralisation
  • Antibiotic response
  • pH change detection
  • Lancefield grouping
  • Gas-liquid chromatography (anaerobic species)
  • Catalase test
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12
Q

How do you perform a Gram’s stain?

A
  1. Cover slide with crystal violet (1 min) and wash off
  2. Cover with Gram’s iodine (1 min) and wash off
  3. Coat with grams decolourise (5-10s); wash off
  4. Cover with Safranin counterstain (30s) and wash
  5. Blot dry
  6. Observe (for structure and colour) – gram +ve = purple; gram -ve = pink

Only Neisseria and B.Fragilis gram -ve (of species in practicals)

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13
Q

Describe the gram stain morphology of common infectious bacteria:

A

Cocci:
- Strep. pyrogenes/pneumoniae and Viridians strep. (all G +ve).
- Strep. pneumoniae individual (and irregular); others in small clumps
- Neisseria in ‘bunch of grapes’ (G -ve)

Rods:
- Diphtheriae (G +ve)
- Clostridium (sporogenes, tetani and perfringens) (G +ve) with decreasing lengths of rod respectively

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14
Q

How is a hot malachite green stain performed?

A
  1. Prepare smears and dry.
  2. Cover with malachite green for 5 mins, leave on hot plate (do not allow stain to dry)
  3. Wash well
  4. Counterstain with safranin for 5 mins
  5. Wash; blot and examine
  6. Spores = green; vegetative cells = pink/red
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15
Q

What are the morphologies of Clostridium species bacteria under green malachite stain?

A

C.tetani:
- Round terminal spores
- Wispy swarms of vegetative cells

C.sporogenes:
- Oval spores
- Spores bulge from vegetative cells

C.perfringenes:
- Oval, central spores

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16
Q

What is a coagulase test and how is it performed?

A

Distinguishes between staph. Aureus and staph. Epidermidis:
- Add sample and plasma
- Place in water bath (37C) for 1.5 hours
- S.Aureus will coagulate (epidermis will not)

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17
Q

Name some tests for C.diphtheriae:

A

Albert’s method:
- Detection of granulated bodies in C.D. (polyphosphate granules stain blue/green)

ELEK plate:
- Tests for toxin production by C.diphtheriae

18
Q

How could Strep.Pneumoniae be identified?

A

Capsule staining:
- Treatment with nigrosin reveals capsule

Morphological appearance:
- Small circular yellow-green matte colonies causing surrounding colour change (green)

19
Q

How can toxin neutralisation technique show the presence of strep.pyrogens?

A

Infection with strep. pyrogens often produces anti-streptolysin O to prevent RBC lysis. This can be tested for:
- Patient serum mixed with horse RBCs and streptolysin O
- Reducing agent added to prevent toxin inactivation by oxidation. Incubated at 37C for 15mins.
- If infected; haemolysis will not occur (remains cloudy); if not-infected then no antibody so haemolysis and clear solution seen

20
Q

Give an example of anti-toxin testing:

A

C.perfringens produces α-toxin (hydrolyses phospholipid lecithin in membranes).
- Growth shown on egg yolk infused agar plate.
- Activity of toxin shown by insoluble region (due to Nagler reaction)

21
Q

How can pH change detection be useful in identification of bacteria?

A

Can be induced by certain secreted enzymes (particularly from resistant bacteria):
- Staph. Aureus and Neisseria convert penicillin to penicilloic acid (e.g. using β-lactamases)

22
Q

What are Lancefield groupings and what do they show?

A

Distinguish different strep species:
- Antibodies for different Strep cell wall molecules tested
- Agglutination suggests a positive result
- Negative and positive controls should be done

23
Q

How can different staphylococci species be distinguished by looking at colony morphology?

A

Aureus:
- Slightly bigger
- ‘golden’ in colour
- No haemolysis
- Facultative aerobe

Epidermidis:
- Smaller
- Off-white
- No Haemolysis
- Obligate aerobe

Viridans strep.(blood plate):
- Very small colonies
- Cream-green
-Translucent
- No haemolysis.

24
Q

How can different streptococci species be distinguished by looking at colony morphology?

A

Pyrogenes:
- Small convex colonies
- Shiny and opaque
- White
- Complete haemolysis

Pneumoniae:
- Small concave colonies
- Matte and translucent
- Yellow-green
- Green surround with incomplete haemolysis

25
Q

How pseudomonas aeruginosa be distinguished by looking at colony morphology?

A
  • Very large (3-4mm) colonies
  • Flat and smooth with undulate edge
  • Cream/green in colour
  • Translucent with darker center
  • Surrounding turns sea-green with complete haemolysis.
26
Q

How can clostridium species be distinguished by colony morphology?

A

Sporogenes:
- Individual “Medusa head colonies”
- Smells like ‘putrefying flesh’ (H2S produced)
- Distorted; shiny; cream and opaque
- Haemolytic

Tetani:
- Huge swarming colony
- Diffuse area
- Flat; rough; off-white and transparent
- No haemolysis

Perfringens:
- Circular, flat and smooth colonies
- Haemolysis

27
Q

How can enterococcia nd staphylococci be told apart?

A

Catalase test:
- Expose bacteria to H2O2
- Bubbling is +ve result showing O2 liberated from H2O2
- Entero are -ve; staph are +ve

28
Q

What is a MacConkey plate? Why is useful?

A

Contains bile salts so shows gastrointestinal bacteria (non-enteric struggle to grow)
- pH indicator shows if bacteria ferment lactose (resulting in low pH due to lactic acid)

Examples:
- Salmonella grow (3-2mm; shiny and yellow plate): no lactose fermentation
- E.Coli grow (4mm; rough with undulate margin and magenta; with pink plate): lactose fermenting
- Enterococcus. Faecalis: grows with small colonies and magenta (lactose fermenting)

29
Q

What is a CLED plate? Why is it useful?

A

Analysis of urine samples:
- Contains pH indicator (differentiates lactose fermenters)
- Blue plate = high pH (e.g. salmonella)
- Yellow plate = low pH (E.Coli; E.Faecelis)

Examples:
- E.Faecelis grows very small colonies
- E.Coli 2-3mm opaque
- Salmonella 3mm translucent colonies

30
Q

What is an H and E stain and what does it show?

A

Haematoxylin = blue/purple stains basic structures (e.g. nucleic acids, DNA (nucleus), ribosomes)

Eosin = pink is acidophilic (proteins, deposited plasma proteins)

30
Q

How might flow cytometry help to differentiate between different types of Leukocytes?

A
  • Cytotoxic T cell = CD3+ CD8+
  • Helper T cell = CD3+ CD4+
  • Treg = CD3+ CD4+ CD25++
31
Q

How do you carry out an ELISA test?

A

Enzyme linked immuno sorbant assay:
1. Add sample to ELISA plate coated with anti-molecule
2. incubate then wash
3. Add secondary antibody with identifying feature
4. Incubate and wash
5. Add indicator/light which shows secondary antibody (e.g. ABTS turns turquoise)

32
Q

Why does haemolytic disease of newborn occur?

A
  • Maternal antibodies for paternally-inherited blood groups (rhesus marker)
  • IgM anti-rhesus cause agglutination while IgG anti-rhesus do not
  • In HDN IgG always observed since IgG only one which can cross placenta)
33
Q

How can viral concentration be estimated?

A
  • Haemagglutination (lowest haemagglutinating concentration = 1 HA unit)
  • Virus assay (count number of particles per unit assay using known number of latex beads)
  • PCR (vague)
    Always Include a negative control (no flu virus included)
34
Q

When haemagglutination is used to identify haemolytic disease of the newborn, why is no agglutination seen before washing?

A

Free unbound antibody compete for binding rabbit serum antibodies - reducing agglutination potential.

35
Q

Why might IgG anti-D antibodies not agglutinate?

A
  • Antibodies cannot overcome repulsion between two cells (both negatively charged)
36
Q

What are the ways that TB could be detected?

A
  • PCR
  • Mantoux test
  • IFN test (quantiferon test)
  • Culture and observe morphology
  • Histology
37
Q

What is the role of CRP and what might raised levels indicate?

A
  • APP released from the liver in response to IL-1, TNF-a, IL-6
  • Can opsonise and activate complement
  • Used clinically to indicate infection
38
Q

What do EIDn per ml numbers represent?

A

Egg infectious dose n where n is the percentage of inoculated samples infected (n = 50 = 50%)

39
Q

How do you calculate the multiplicity of infection?

A

Number of pfu/number of cells

40
Q

Describe a possible progression from atherosclerosis to death

A
  • Plaque formation and growth
  • Plaque rupture
  • Thrombus formation
  • Occlusion of an artery
  • Myocardial infarction
  • Emboli (in brain = death)
41
Q

What is cervical intraepithelial neoplasia?

A

Progression from pre-malignant dysplastic stage to invasive squamous carcinoma
- In squamo-columnar junction (transformation zone)

42
Q

What is Western blotting used for?

A
  • Shows and identifies specific proteins
  • E.g. anti-APC antibody used as tag
  • Normal cells can show two bands (one copy mutated)
  • Tumour cells show one (two copies mutated)

Biology of Disease (7 decks)

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