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Immunology & Medical Microbiology > Lab Skills > Flashcards

Lab Skills Flashcards

1
Q

What does EMIT stand for ?

A

Enzyme-Multiplied Immunoassay Technique

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2
Q

What is the purpose of EMIT ?

A

To detect & quantify small molecules like drugs, hormones & metabolites in biological fluids

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3
Q

What are the key principles of EMIT ?

A
  1. sample contains target molecule
  2. a specific antibody bind to target molecule
  3. enzyme-labeled target molcule compete with sample target molecules for antibody binding
  4. only unbound enzyme-labelled molecules produce a signal as antibody binding inhibits enzyme activity
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4
Q

What is EMIT commonly used for ?

A
  • therapeutic drug monitoring
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5
Q

What does ELISA stand for ?

A

Enzyme-Linked Immunosorbent Assay

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6
Q

What is the purpose of ELISA?

A
  • Detect & quantify proteins, such as antigens, antibodies, hormones & cytokines
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7
Q

What are some different types of ELISA ?

A
  • Direct = antigen is detected with an enzyme- conjugated antibody
  • indirect
  • sandwich
  • completive
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8
Q

What are 2 main applications of ELISA?

A
  • diagnostic tests for infections
  • detection of biomarkers In research or clinical settings
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9
Q

What is the purpose of the Miles Misra technique ?

A
  • used to quantify viable microorganisms in a liquid sample by counting colony-forming units
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10
Q

Describe the procedure of the Miles Misra Technique

A
  1. a series of dilutions of the microbial suspension are prepared
  2. small amount of dilution are pipetted onto agar in separate labelled sections
  3. drops are allowed to dry, plate is incubated under appropriate conditions for growth
  4. after incubation colonies are counted within each drop zone
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11
Q

What are some advantages of Miles Misra ?

A
  • economical & require minimal material
  • allows rapid determination of viable counts without need for larger plates
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12
Q

What are some applications of Miles Misra ?

A
  • used in microbiology labs for bacterial enumeration
  • quality control in food, water & pharmaceutical microbiology
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13
Q

Describe the order of gram staining

A
  1. apply culture to drop of water
  2. heat fix
  3. apply crystal violet
    rinse
  4. apply iodine
  5. rinse with alcohol
  6. apply safranin
    rinse
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14
Q

What is the key principle of gram staining ?

A
  • based on the differences in bacterial cell wall structures
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15
Q

What is the importance of applying the iodine to the slide ?

A
  • allows for crystal violet-iodine complex to form, which helps the stain adhere to the bacterial cell wall
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16
Q

What is the purpose of rinsing with alcohol ?

A
  • removes the CV-I complex from gram-negative cells due to thin peptidoglycan layer
  • gram-positive cells retain the CV-I complex as their thick peptidoglycan layer resists decolourisation
17
Q

Describe the why gram-positive bacteria appear purple

A
  • thick peptidoglycan layer
  • the CV-I complex becomes trapped in thick layer
  • alcohol dehydrates the peptidoglycan, causing it to shrink & retain purple stain
18
Q

Why do gram-negative bacteria appear pink ?

A
  • thin peptidoglycan layer & outer membrane containing lipopolysaccharides
  • CV-I complex is washed out by alcohol as the outer membrane is disrupted, this exposes the thin peptidoglycan which cannot retain the stain
  • safranin counterstains these cells pink
19
Q

Why is gram staining important ?

A
  • aids in clinical diagnosis & antibiotic selection
  • morphological identification - rerelease shapes & arrangements
20
Q

Describe the API antibiotic system

A
  • diagnostic tool used to identify & classify bacterial species based on their susceptibility to various antibiotics
  • pre-prepared panels containing various test wells each with different antibiotics
21
Q

How does the API system work ?

A
  • bacterial sample is isolates & cultured
  • suspension is prepared & inoculated into wells
  • strip is incubated under appropriate conditions
  • if the bacteria are susceptible to antibiotics bacteria won’t grow = no visible growth
  • bacteria resistant = growth in well
  • pattern of growth in each well is recorded
  • results are compared to standard chart/database that matches pattern of susceptibility to specific bacterial species
22
Q

What are the advantages of the API system ?

A
  • provides rapid identification of susceptibility
  • can test for a wide range of antibiotics
  • pre-manufactured strips save time & reduce manual prep
23
Q

Describe the process of the Oxidase test

A
  1. drop of oxidase reagent on slide
  2. using a sterile applicator, transfer a colony to the reagent
  3. observe a colour change
24
Q

Describe a positive & negative result for an oxidase test

A

Positive = dark purple/blue colour (indicates presence of cytochrome C oxidase)
Negative = no colour change

25
Q

What is the purpose of the oxidase test ?

A

Identifies bacteria that produce the enzyme cytochrome c oxidase, part of the electron transport chain

26
Q

What is the theory behind the oxidase test ?

A
  • cytochrome c oxidase transfers electrons from cytochrome c –> oxygen
  • oxidase reagent serves as an artificial electron donor, changing colour when oxidised by enzyme
27
Q

What is the purpose of the Catalase test ?

A

Determines if bacteria produce the enzyme catalase, which breaks down hydrogen peroxide into water & oxygen

28
Q

Describe the procedure of the catalase test

A
  1. a drop of hydrogen peroxide on a slide
  2. add a small amount of bacterial culture using a sterile loop
  3. observe for immediate bubble formation
29
Q

Describe a positive and negative result for the catalase test

A

positive = vigorous bubbling
negative = no bubbles

30
Q

What is the theory behind the catalase test ?

A
  • catalase protects bacteria from oxidative damage by decomposing hydrogen peroxide
  • this test is particularly useful in differentiating gram-negative species
31
Q

Describe the growth characteristics of Staphylococci

A
  • grow as clusters/ grape like arrangements
  • colonies are typically larger, circular & opaque
32
Q

Describe the growth characteristics of Streptococci

A
  • forms chains or pairs
  • colonies are smaller & translucent
33
Q

How do growth patterns reflect differences in cellular arrangement ?

A
  • staphylococci divides along multiple planes forming clusters
  • streptococci divide along a single plane, forming chains

Immunology & Medical Microbiology (26 decks)

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