Practical Flashcards
1
Q
What is a plaque?
A
An area of disturbed or lysed cells
Seen as a clearing
2
Q
What is a plaque forming unit (pfu)?
A
That which can infect one cell in a monolayer to give rise to a plaque
3
Q
How is pfu related to virus particle numbers?
A
Relationship is variable fro 1:1 to 1:100
Can be calculated using latex beads and EM microscope
Or calculating pfu/vol and then particle/pfu ratio
4
Q
What is the infectious centre assay?
A
Process of applying pre-infected cells to the monolayer
5
Q
What is the eclipse phase?
A
When virus in uncoated and disassembled as it undergoes transcription and translation
6
Q
What is a monolayer?
A
Single layer of tissue culture cells
If cells are touching each other, this is described as confluent
7
Q
What is the cytopathic effect (cpe)?
A
Disturbed appearance of cells due to pathogen
8
Q
Why is Acyclovir (ACV) added after infection with a virus?
A
ACV works at the level of disrupting DNA replication
So virus needs to be allowed to infect before testing action of ACV
9
Q
Why is acyclovir such as good drug?
A
- Doesn’t interfere with host replication
- Selective as it needs to be activated by thymidine kinase
- Can be used a prophylactic
- Available in various forms, oral and intravenous forms
- Effective against HSV and VZV
10
Q
Describe the Influenza Haemagglutination Assay (HA) and the inhibition assay (HAI)
A
Influenza virus , via its HA molecule, binds to Sialic acid on RBC causing agglutination
In the HAI assay, antibodies bind to HA on influenza, and prevent their binding to sialic acid, preventing agglutination
~ Last well showing agglutination is equal to 1 HA unit
11
Q
What is an adenoma?
A
Tumour with glandular structures
12
Q
What is a teratoma and what does it look like?
A
Tumour formed from all 3 germ cell layers (ectoderm, mesoderm and endoderm)
- Can contain, teeth, hair and muscle
13
Q
What can be done to distinguish between Staphylococcus aureus and other Staph species?
A
- Coagulase test
- S.aureus produces coagulase, causing clotting
- Rest are coagulase negative - Catalase test
- S. aureus is positive, producing the enzyme catalase
- Catalyses the breakdown of hydrogen peroxide into water and oxygen - Mannitol Salt Agar (MSA) test
- S. aureus ferments mannitol resulting in a change in pH, producing yellow colonies.
- Other species produces pink colonies or no colour change is seen - PCR
14
Q
What tests can be done to distinguish between enterococci and Staphylocci?
A
- Catalase test
- Staphylococcus auereus is positive, while enterococcus is not
- Catalase to breakdown H2O2 requires oxygen, and enteric environments are largely anaerobic
15
Q
What is the use of the MacConkey plate?
A
- Whether bacteria can grow in bile salts (present in enteric environment)
- Also checks for presence of β- lactase
- Red colonise, ferment lactose and produce acid e.g. Enterococcus faecalis
- Yellow colonise, don’t ferment lactose e.g. Salmonella
16
Q
What is the use of the CLED plate?
A
- Isolate bacteria found in the urine
- Also can be used as a pH
YELLOW = Acid producing, lactose fermenting e.g. E.coli and Ent. faecalis
PALE GREEN/BLUE = Non-acidic e.g. Salmonella - Plate also inhibits swarming ability of Proteus mirabilis a common bacteria found in UT infections
17
Q
Give examples of 3 bacteria that are obligate anaerobes
A
Clostridium tetani
Clostridium sporogenes
Bacteriodes fragilis
18
Q
What is the difference in morphology of C.tetani and C.sporogenes?
A
C.sporogenes
- Gram positive rod shaped,
- Mini gunshot, slightly raised colonies
C.tetani
- Gram positive rod shaped
- Swarm growth, ruffled edges
19
Q
What gram stain and shape is Bacteriodes fragilis?
A
- Gram negative
- Rod-shaped
20
Q
What is a leiomyoma?
A
- Mesenchymal tumour
- Non-cancerous growth in the smooth muscle cells of the uterus
- Generally benign, non-invasive tumours
- Also known as fibroids
21
Q
What karyotype change can be associated with leiomyomas?
A
Chromosome 12 and 14 translocation
22
Q
Where might a tumour that has metastasised to the lymph node originate from?
A
- Likely from the breast, due to close proximity
- Lung
23
Q
What do you understand by the term moderately differentiated
adenocarcinoma?
A
- Arises from glandular tissue
- Malignant
- Not well differentiated
- Shows little/ some resemblance to parent tissue
24
Q
Where are adenomcarcinomas located?
A
Malignant tumours that originate from grandular tissue
25
In which ventricle is a myocardial infarct more likely to occur in?
Left
26
What types of hypersensitivity can result in ischaemia?
All but especially
Type 1
- rapid inflammation, vasodilation = hypotension
27
What occurs in the development of an atherosclerotic plaque?
1. Endothelial cells damaged by oxidised lipoproteins
2. Smooth muscles secrete/ present collagen
3. Macrophages differentiate and form foam cells
4. Accumulate and form fatty streaks
5. Fibrous cap- Increased deposition of collagen
28
Describe ent faecalis
- Diploccoci
- Gram positive
- Lactose fermentation = Yellow CLED plate
- Red MacConkey (lactose fermentation)
- Catalase negative
29
What are the 3 components of granulation tissue? What does the tissue look like?
1. Fibroblasts
2. Angiogenesis
3. Macrophages
= new vessels thin capillaries, darkly stained cells, some collagen deposition
30
Describe the pathophysiology of sepsis
1. Detection of infection
2. Immune response
3. Dysregulated balance between pro and anti-inflammatory cytokines
4. Endothelial dysfunction
5. Organ damage
31
Describe fibrous scarring
- Fibrin in whorl like structures
- Follows granulation tissue formation
32
What are the components of a structured report for a histology section?
1. Location
2. Description of cells and changes
3. Name pathology e.g. inflammation
4. Diagnosis or process e.g. repair, pneumonia
33
What stimulates angiogenesis?
VEGF and FGF
34
What causes fibroblast recruitment?
TGF-beta
FGF
35
What is a urease test and what is it used for?
Tests whether organism can hydrolyse urea to ammonia and CO2
- Yes leads to pink colour change e.g. H.pylori
36
What differs the white pulp and red pulp areas in the spleen?
White pulp- darker, due to large number of WBC, minimal RBC
Red pulp - contains more RBC, old and new and is where lymphoid follicle with germinal centre forms
37
In what situations are eosinophils commonly found in ?
Allergic response
Type I hypersensitivity
38
What does a toluidine blues stain show?
Mast cells containing
- serotonin, cytokine, and histamines
39
What is miliary TB?
TB that has spread in the blood to other organs
40
What is the significance of a reduction in CD4 T-cell count?
Suggests autoimmune diseases e.g. HIV that targets CD4 via gp120
- Increased risk of opportunistic infections
41
What triggers the smooth muscle cells in the tunica media to migrate
to the tunica intima, proliferate and secrete extracellular matrix?
1. Macrophages become activated by oxidised LDL
2. Secrete factors
3. Recruit smooth muscle
4. Muscle proliferates and secretes collagen
42
What are the histological differences between older and younger thrombi?
Younger
- Clearer laminations
- RBC passively entrapped
- EBC degenerating
Older
- Less visible laminations
43
What is the histological difference between thrombus formed in veins and arteries?
Artery - white thrombi
- Platelets and fibrin
Vein - red thrombi
- caused by slow moving blood
- Greater proportion or RBC
44
What are the histological differences between benign and malignant tumours?
Benign
- Don't invade
- Similar to normal cells
- Well defined borders
Malignant
- Breaches basement membrane or more easily seen muscularis mucosae
- Can metastasise
- Pleomorphic
- Mitotic activity
- Varying degrees of differentiation
45
What is an adenoma?
Non-invasive tumour of glandular tissue
46
What nuclear changes might one see in an adenoma? (4)
1. variation in nuclear size
2. variation in shape
3. variation in chromatin staining pattern (pleomorphism)
and stain darkly (hyperchromasia).
4. A small number of mitotic figures can be seen
47
What is the squamous- columnar junction (SQJ)?
Where the single layered, columnar glandular epithelium of the endocervix meets the squamous epithelium of the ectocervix
48
What are the characteristics of a squamous cell carcinoma?
- Lack of normal architecture
- Keratin deposition (whorls)
- Invasion of basement membrane
49
What are synonymous, nonsense and missense substitutions?
Synonymous - silent mutations, doesn't cause significant change
Missense- causes different amino acid to be encoded
Nonsense - introduction of stop codon
50
What is the common type of mutation found in adenomas and carcinomas?
Nonsense substitutions causing truncations
51
What is the HAI assay used for?
To establish the titre of antibodies to a virus
52
What does a positive result for HAI mean?
No agglutination because serum has antibodies
- Binds to virus and prevents it from agglutinating RBC
53
Why is Acyclovir such a "good" drug?
Host doesn't use ACV or ACV-pi as a nucleotide
However, HSV's DNA polymerase does
= Incorporation disrupts replication
54
How to calculate titre of a virus from an ELISA?
- Last one to agglutinate/ be different = 1 HA unit
- If dilution is 1:128 and 0.2ml is found
then HA/ml is 128x5 = 640
55
What are the likely bacteria if in the gut?
Enteroccocus faecalis
E.coli
Salmonella
Clostridium
Bacteroides fragilis (Anaerobe)
56
What bacteria is in the gut and gram positive?
Enteroccocus faecalis
Clostridium (tetani/sporongenes)
Distinguish by swarm growth on plate (tetani)
- Location of spores using malachite green
- Tetani is terminal, sporongenes is sub-terminal
57
How would you distinguish between Salmonella and E.coli?
- PCR
- CLED and MacConkey plate
= Red and yellow (E.coli), blue CLED and orange MC for Salmonella
- Antigen testing (H, O and V)
58
How would you differentiate between Streptococcus and Enterococcus?
- Lancefield grouping
- Ability to grow in salt environments (entero)
59
How would you distinguish between Staphyloccocus?
aureus - yellow, coagulase positive
epidermidis - white, coagulase negative
60
What are the only gram negative bacteria in the respiratory tract?
- Neisseria pharynges
- Coccus
61
How to differentiate between Strep and Staph?
Catalase test
-ve = Sterp
+ve = Staph
62
How would you differentiate between different Streptococcus? What are some example?
Lancefield grouping
- Streptocoocus pneumoniae (donut)
- Streptococcus pyogenes
- viridans Streptococci
63
What test should be done to check for TB?
Mantoux test and
Ziel- Neelson stain
64
How to structure a histopathology answer?
1. Architecture
2. Cytology (cells)
3. Diagnosis
4. Aetiology
5. Extra tests to check
65
What are the key points to look out for when looking at a thrombus?
1. When it developed
- look for lines of Zahn
2. Source
- Emboli will show little damage to endothelium, as would've traveled from elsewhere
66
What are the key features to mention about a tissue with a tumour?
1. Hyperchromatic due to enlarged nucleus
2. Pleomorphic cells (different shapes)
3. Densely compacted cells
4. Dysplasia
5. Mitotic activity
6. Raised nuclear to plasma ratio
67
What are the key features to look out for when looking at a polyp?
Malignant or benign
- Whether muscularis mucosae is breached or not
68
What is important to determine when looking at cancer in the lymph nodes, and in general?
Squamous epithelium or glandular origin
Squamous
= Keratinised whorls
= Intracellular bridges broken (ladder looking)
Glandular
= contains glands (circular structures)
= No keratin
69
What are common errors in performing a streak plate?
- Failure to spread across a large enough area
- Catching edge of the bacterial loop on the agar
- Using to large of an inoculum
- Moving the loop carelessly into different sections (e.g. D into A)
70
How is Rhesus D incompatibility managed clinically?
RhoGAM (an anti-IgD)
- prevent sensitization (activation) of mother -ve to +ve foetus
71
How are RBCs washed?
Addition of a buffer and repeated centrifugation
- Supernatant removed and pellet of RBC maintained
72
What is the difference between direct and indirect Coombs test?
Direct
- detects the Ab on the surface of RBC
- used for haemolytic disease of the newborn (HDN
Indirect
- detects presence of Ab in serum against donor RBC antigens causing agglutination
- used for transfusion (e.g. same blood group or not)
- used for prenatal testing (Rh antibodies)
73
Describe the difference in appearance between a positive and negative result in a HA assay for blood type
Positive = agglutination, due to antibodies binding and clumping
Negative = sedimentation, lack of Ab binding, so no agglutination
74
What is the Analytical Profile Index (API) test?
1. Ten strips containing substances
2. When bacterial suspension is added, a colour change is seen
75
What are the key things to mention when answering question 2 (3 slides and zoomed in images)?
1. Pathological process
2. Diagnosis
3. Evidence for this suggestion
76
What things need to mentioned in the bacteriology answer?
1. Description of the bacteria
2. Identification of the bacteria
3. Suggest tests to verify identification
(PCR, sequence, Lancefield)
77
How does the appearance of caseous, liquefactive and coagulative necrosis differ?
1. Coagulative - still maintain cell membrane, however, loss of nuclei
2. Liquefactive - liquid looking, no membranes or nuclei
3. Caseous - liquid centre, surrounded by macrophages
78
Describe the appearance of a cell undergoing apoptosis
- Lose connection to surrounding cells
- Shrinking in size
79
Give an example of a secondary Ab (the enzyme attached) and it's substrate
HRP - enzyme
ABTS- substrate
Binds with peroxide to generate colour change
80
What is the significance of a line formation in a precipitation gel?Specifically the presence of multiple lines and a curved line
Line is due to precipitation of Ab that is bound to antigen
Multiple = impure sample, multiple antigens
Curved = share common antigens with neighbour
81
What is the significance of the location of the line in the precipitation gel?
Closer to the well, means less Ab or Antigen from that well.
- As the other has travelled longer distance in greater conc
82
What are the 4 forms of TB?
1. Primary - initial infection at the lungs
2. Secondary - spread to periphery e.g. lymph nodes, initiate a response
3. Miliary - spread in the blood
4. Cavitation - bursting of granuloma
83
Name primary and secondary lymph nodes
Primary
- Bone marrow
- Thymus
Secondary
- Spleen
- Lymph nodes
- Mucosa- Associated Lymphoid Tissue (MALT)
84
What are the two different types of pneumonia?
Broncho - located in bronchi, commonly caused by inspired bacteria
Lobar - exudate in alveoli, due to bacteria found in blood
85
What are the causes and symptoms of sepsis?
Cause
- Bacterial infection causing a chain reaction of inflammation and further damage (cytokine storm)
= Organ failure
Symptoms
- Drop in BP
- Rashes developing
- Inflammation (temp change)
86
What are the components present in inflammatory exudates? (3)
- Neutrophils
- Mast cells
- Fibrin
87
What are the 3 outcomes of inflammation/ damage?
1. Resolution
- short lived injury, limited damage
2. Repair
- formation of granulation tissue
3. chronic inflammation
- function not restored, continual inflammation
88
Describe the components of granulation tissue
1. Fibroblast growth factor (FGF)
2. Fibroblast
- for collagen
3. Angiogenesis
- new vessel formation
4. Macrophages
- to clear debris
89
Name some protein markers that are used to detect T and B cells in flow cytometry
T cells
= CD3 and CD4
B cells
= CD19
90
What are the different characteristics of the spleen and the thymus?
Spleen - abundant in B and T cells
Thymus - more T cells than B cells
91
Describe the function of flow cytometry
1. Sample loaded with fluorophore attached to Ab
2. Cells pushed through a narrow column
3. Excited by laser beams
4. Emit fluorescence
5. Detected by sensor
92
What is forward and side scatter in flow cytometry?
Forward scatter - light scattered forward, indicative of size
Side scatter - light scattered sideways, indicative of complexity and structure
93
What are the predisposing factors (Virchow's triad)?
1. Changes to vessel wall
- Ischaemia
- Infection
- Physical and chemical damage
2. Changes to blood flow
- Disruption to laminar flow
- Arteries - narrowing of vessels can occur
- Veins - stasis of blood and formation of clots
3. Changes to the constituents of blood
- Increase in LDL etc.
94
The most common design of sandwich assay uses two different monoclonal antibodies against the target protein. What differs between the two?
1. Capture antibody
- Immobilizes a specific antigen
2. Detection antibody
- Binds to the antigen, and marks it (has bound fluorophore)
= Binds to different parts (epitopes) of the target antigen, so no competition
95
What is the benefit of an inhibition later flow, compared to a normal lateral flow?
- Not as sensitive to overexpression of proteins
96
What does H& E stain for?
Haemotoxylin = DNA and RNA
Eosin = proteins
97
What does trichrome stain for?
Fibrin = red
RBC = yellow
Connective tissue = blue
98
What does EVG stain for?
Elastin - black/blue
Collagen - pink/red
99
What does MSB stain for?
Stains for fibrin
100
What are the causes and symptoms of glandular fever?
EBV virus infection
Symptoms:
- Swollen lymph nodes
- Enlargement of spleen
- Skin rash
101
Describe the histology associated with glandular fever
SPLEEN
- enlarged white and red pulp
LYMPH
- germinal centres
- hyperplasia
102
How is multiplicity of infection calculated (MOI)?
Number of pfu/ number of cells
103
What are the roles of the controls in HAI assay?
- Serum control to show settle of RBC
- PBS negative control
- Positive control (undiluted virus, to show agglutination) and diluted virus (to ensure that conc is sufficient anyway)`
104
What was the purpose of the antibody stain in cancer sections?
1. Detect different cancers, that present unique different antigens
2. Marker for aggressiveness based on abundance of antigens
e.g. HER2 protein in breast cancer
3. Binds to smooth muscle, demonstrate origin
105
How do the three ABO blood group antigens differ from each other?
Different ABO antigens that are carbohydrate structures found on the surface
O = expresses unmodified H antigen so can be used by A and B patients
A = N-acetylgalactosamine residue
B = galactose residue
106
How can sensitivity be increased in lateral flow?
- Use another molecule for control to ensure that all is flowing through
- Use biotin (bound to streptavidin) small molecule, fixed by carried protein on the control strip (BSA-biotin)
107
What is the effect of Relenza, Tamiflu, Acyclovir and AZT?
Relenza + Tamiflu - targets NA from influenza
AZT - targets HIV reverse transcriptase
Acyclovir- inhibit viral DNA replication, HCMV
108
What molecular techniques could be used to confirm the identity of the virus?
- PCR/qPCR with HCMV specific primers
- DNA sequencing
- Immunohistochemistry (with HCMV specific antibodies)
- ELISA using capture antibodies specific for HCMV proteins
109
What is the purpose of including the metronidazole disc on plate Z?
Testing for bacteroides fragilis
