Lab Skills Flashcards
What does EMIT stand for ?
Enzyme-Multiplied Immunoassay Technique
What is the purpose of EMIT ?
To detect & quantify small molecules like drugs, hormones & metabolites in biological fluids
What are the key principles of EMIT ?
- sample contains target molecule
- a specific antibody bind to target molecule
- enzyme-labeled target molcule compete with sample target molecules for antibody binding
- only unbound enzyme-labelled molecules produce a signal as antibody binding inhibits enzyme activity
What is EMIT commonly used for ?
- therapeutic drug monitoring
What does ELISA stand for ?
Enzyme-Linked Immunosorbent Assay
What is the purpose of ELISA?
- Detect & quantify proteins, such as antigens, antibodies, hormones & cytokines
What are some different types of ELISA ?
- Direct = antigen is detected with an enzyme- conjugated antibody
- indirect
- sandwich
- completive
What are 2 main applications of ELISA?
- diagnostic tests for infections
- detection of biomarkers In research or clinical settings
What is the purpose of the Miles Misra technique ?
- used to quantify viable microorganisms in a liquid sample by counting colony-forming units
Describe the procedure of the Miles Misra Technique
- a series of dilutions of the microbial suspension are prepared
- small amount of dilution are pipetted onto agar in separate labelled sections
- drops are allowed to dry, plate is incubated under appropriate conditions for growth
- after incubation colonies are counted within each drop zone
What are some advantages of Miles Misra ?
- economical & require minimal material
- allows rapid determination of viable counts without need for larger plates
What are some applications of Miles Misra ?
- used in microbiology labs for bacterial enumeration
- quality control in food, water & pharmaceutical microbiology
Describe the order of gram staining
- apply culture to drop of water
- heat fix
- apply crystal violet
rinse - apply iodine
- rinse with alcohol
- apply safranin
rinse
What is the key principle of gram staining ?
- based on the differences in bacterial cell wall structures
What is the importance of applying the iodine to the slide ?
- allows for crystal violet-iodine complex to form, which helps the stain adhere to the bacterial cell wall
What is the purpose of rinsing with alcohol ?
- 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
Describe the why gram-positive bacteria appear purple
- thick peptidoglycan layer
- the CV-I complex becomes trapped in thick layer
- alcohol dehydrates the peptidoglycan, causing it to shrink & retain purple stain
Why do gram-negative bacteria appear pink ?
- 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
Why is gram staining important ?
- aids in clinical diagnosis & antibiotic selection
- morphological identification - rerelease shapes & arrangements
Describe the API antibiotic system
- 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
How does the API system work ?
- 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
What are the advantages of the API system ?
- provides rapid identification of susceptibility
- can test for a wide range of antibiotics
- pre-manufactured strips save time & reduce manual prep
Describe the process of the Oxidase test
- drop of oxidase reagent on slide
- using a sterile applicator, transfer a colony to the reagent
- observe a colour change
Describe a positive & negative result for an oxidase test
Positive = dark purple/blue colour (indicates presence of cytochrome C oxidase)
Negative = no colour change
What is the purpose of the oxidase test ?
Identifies bacteria that produce the enzyme cytochrome c oxidase, part of the electron transport chain
What is the theory behind the oxidase test ?
- cytochrome c oxidase transfers electrons from cytochrome c –> oxygen
- oxidase reagent serves as an artificial electron donor, changing colour when oxidised by enzyme
What is the purpose of the Catalase test ?
Determines if bacteria produce the enzyme catalase, which breaks down hydrogen peroxide into water & oxygen
Describe the procedure of the catalase test
- a drop of hydrogen peroxide on a slide
- add a small amount of bacterial culture using a sterile loop
- observe for immediate bubble formation
Describe a positive and negative result for the catalase test
positive = vigorous bubbling
negative = no bubbles
What is the theory behind the catalase test ?
- catalase protects bacteria from oxidative damage by decomposing hydrogen peroxide
- this test is particularly useful in differentiating gram-negative species
Describe the growth characteristics of Staphylococci
- grow as clusters/ grape like arrangements
- colonies are typically larger, circular & opaque
Describe the growth characteristics of Streptococci
- forms chains or pairs
- colonies are smaller & translucent
How do growth patterns reflect differences in cellular arrangement ?
- staphylococci divides along multiple planes forming clusters
- streptococci divide along a single plane, forming chains
