Fundamentals Flashcards
Why use a diagnostic lab test?
-to determine if an infectious agent is present
-to obtain etiological diagnosis (what organism is the issue)
-to guide antimicrobial therapy (what drug is best)
-also used for disease surveillance (resistance, outbreaks), regulation (reportable diseases, public health), research
How to grow bacteria?
-broth vs agar
-selective vs differential vs non-selective media
Colony forming unit
One cell expands to take up an area/form a colony of bacteria
Tests used to ID organisms
-biochemical tests
-MALDI-TOF
-NAAT (nucleic acid amplification test)
Colony
A clonal population on an agar plate of bacteria that formed from a single viable organism (bacterium)
-form quickly- but does depend on generation time of the organism AND limiting factors (nutrition/resources)
Why is it important to conduct dilutions and colony counts?
1.Establish clinical significance (# of CFU and whether its important)
- Identify contaminants (find dominant organisms in mixed cultures)
- Standardize lab tests (some tests need certain number of organisms to run test)
Streak out plates
-use four streak method
-By 4th streak on plate, the objective is to get isolated colonies
Define isolate
-A pure culture (clonal)
-comes from a single colony
-genetically homogeneous
-needed for ID and susceptibility testing
Selection of culture media
-Culture media used depends on what you are trying to isolate
>use of selective and differential media helps to ID
Selective media
-used to preferentially isolate particular taxa since it contains certain chemicals that inhibit the growth of non-target organisms
Types of Selective media
1.CNA - selects for gram positives, and against gram negatives
- MacConkey- selects for gram negative enterics, and against gram positives
- Campy-BAP- selects for Campylobacter jejuni, and against most other bacteria
Differential media
-Exploits physiological properties of organisms of interest to produce unique colony morphologies
*has something in it that certain bacteria need to survive and will result in a characteristic difference from the other bacteria present
Types of differential media
1.MacConkey
- XLD
- CHROMager
MacConkey as a differential media
Differential ingredient: lactose
Differentiates lactose fermenters
XLD as a differential media
Differential ingredient: Ferric ammonium citrate
Differentiates H2S producers
CHROMager as a differential media
Differential Ingredient: many
*so differentiates various species
CHROMager ESBL
- blue colonies= non e coli enterobacteriaceae
white colonies= Pseudomonas spp
OR
- Selection for 3rd generation cephalosporin resistance. Pink= e coli; Blue= non e coli enterobacteriaceae
CHROMager MRSA
Selects for methicillin resistance
Differentiates for Staphylococcus aureus
Pink colonies= MRSA
Mannitol salt agar
Selects: NaCl tolerant
Differentiates: mannitol fermentation
Yellow colonies= Staphylococcus aureus
Eosin Methylene Blue
Selects: Gram negatives
Differentiates: lactose fermentors
Metallic green= E coli
Liquid media
-Used for samples no easily plated on solid media, large volumes, or for culturing organisms from food for research, or using enrichment culture
Biochemical tests to ID
-Phenotypic assays- such as colour change, agglutination or change in consistency of media
> compare to tables of known results for different organisms
MALDI-TOF
-Very efficient ID, fast, inexpensive
-used to ID organism from primary culture without the need for additional overnight incubation
Biocontainment level
-used in Canada
-considers facilities, procedures, and equipment required to handle organism safely
Biocontainment levels 1-4
- A basic well functioning lab
- Agents requiring ingestion for exposure. Need PPE and containment devices (biosafety cabinets)
- Agents can become airborne. Additional primary and secondary barriers (respirator, HEPA filtered lab exhaust)
- Max precautions. Complete isolation of facility, decontamination of lab effluents, positive pressure space suits
Biological risk groups
-used in US
-takes into account:
>availability of preventive measures (vaccines)
>availability of effective treatments (antibiotics)
>pathogenicity
>infectious dose
>mode of transmission
>host range
Biological risk level 1
-organisms unlikely to cause disease if healthy (may in immunosuppressed individuals)
**low individual, low community
-Examples:
>environmental organisms
>lab strains used as experiment controls
> plasmid recipients
>reference isolates and type strains
Biological risk level 2
**moderate individual, low community
-many common bacteria and fungi
Biological risk level 3
**high individual, low community
-includes many important zoonoses and fungi causing systemic mycoses
eg. plague found in black-tailed prairie dogs
Biological risk level 4
**high individual, high community
-Does not include any bacteria or fungi. Focus is on viruses
Ex. Ebola, Herpes B, reconstructed 1918 H1N1
Plasmid
Extrachromasomal, independently replicating DNA molecule
PCR
An in vitro technique for the amplification of target DNA sequences
RT-PCR
Real time PCR
-An in vitro technique for the simultaneous amplification and detection of target DNA sequences
-may or may not be quantitative
