Fundamentals Flashcards
Why utilize a diagnostic laboratory?
- to determine if an infectious agent is present (does my patient/herd/flock have an infection?)
- to obtain an etiological diagnosis (what organism am I dealing with?)
- to guide antimicrobial therapy (what is the most appropriate drug to use?)
- THE ONUS IS ON YOU TO UNDERSTAND, & THEREFORE BE ABLE TO EXPLAIN TO YOUR CLIENT, WHY A LABORATORY DIAGNOSTIC TEST IS IMPORTANT!
What are other important functions of diagnostic labs?
- disease surveillance (passive): antimicrobial resistance; trend/outbreak ID
- regulatory: testing for reportable diseases; export/import requirements; public health/food safety
- research: diagnostic submissions are a valuable source of research materials
What is a colony?
blue circles are colonies
- colonies on an agar plate are POPULATIONS of bacteria
- clonal population which descended from a single viable organism (the CFU)
What is a CFU?
colony forming unit: when one cell expands to form entire colony (the ancestor organism)
Assuming a 20 minute generation time, if we start with 1 CFU, how many organisms will we have after 1 day of unimpeded division?
1->2->4->8->16->32->64->128->256->512->1024->2048-> 4096 -> etc.
4.72x10^21
this is just a theoretical b/c would run out of nutrients, etc.
What does a colony theoretically represent & what can it be used to estimate?
- presence of a single viable progenitor organism & is therefore a clonal population
- Can therefore be used to estimate the number of bacteria (CFU) in a sample by making dilutions and plating them until you can count organisms per plate
Why do we care how many organisms are present on a plate?
- establishing clinical significance (from free catch urine, UTI is defined as >50,000 CFU/ml)
- to identify contaminants (identifying dominant organisms in mixed cultures - probably the dominant organism is the one causing the problem)
- to standardize laboratory tests (some assays require testing a specific number of organisms)
How do you streak out plates for isolation?
- 4 streak method
- objective is to get isolated colonies
How is the semi-quantitative method ranked on plates?
- scant = < 10 colonies on 1st streak
- 1+ = > 10 colonies on 1st
- 2+ = > 10 colonies on 2nd
- 3+ = > 10 colonies on 3rd
- 4+ = > 10 colonies on 4th
What is an isolate?
- a pure culture (clonal)
- derived from a single colony
- genetically homogenous
- suitable for additional characterization: identification, susceptibility testing
How are culture media selected?
- selection of a primary media depends on what organisms you are interested in isolating - what is your target pathogen (blood agar & MacConkey are pretty standard for routine cultures)
- introduction of selective & differential media facilitates a presumptive identification on primary culture
What is selective media used for?
- to preferentially isolate particular taxa (contains chemicals to inhibit the growth of non-target organisms)
What does CNA media contain?
Colistin & Nalidixic acid
What does MacConkey media contain?
Bile salts & crystal violet
What does Campy-BAP media contain?
Bacitracin, novobiocin, colistin, cephalothin, polymyxin B
What does CNA media select for?
gram - positives
What does MacConkey media select for?
gram - negative enterics (ex: E. coli)
What does Campy- BAP media select for?
Campylobacter jejuni
What does CNA media select against?
gram-negatives
What does MacConkey media select against?
gram positives
What does Campy-BAP media select against?
most other bacteria (other than Campylobacter jejuni)
What is differential media used for?
- exploits physiological properties of organisms of interest to produce unique colony morphologies (often colourimetric)
What is the differential ingredient in MacConkey media?
lactose
What is the differential ingredient in XLD media?
Ferric ammonium citrate
What is the differential ingredient in CHROMagar (various) media?
proprietary
What does MacConkey media differentiate?
lactose fermenters (pink)
What does XLD media differentiate?
H2S producers (black)
What does CHROMagar (various) media differentiate?
various depending on preparation
What is CHROMagar ESBL?
- selects: 3rd generation cephalosporin resistance
- differentiates: selects spp
- pink colonies, E. coli
- blue colonies, non-E. coli Enterobacteriaceae
- white colonies, Pseudomonas spp.
What is CHROMagar MRSA?
- selects: methicillin resistance
- differentiates: Staphylococcus aureus from other Staphylococci
- pink colonies, MRSA
What is Mannitol Salt Agar?
- selects: NaCl tolerant
- differentiates: mannitol fermentation
- yellow colonies, Staphylococcus aureus
What is Eosin Methylen Blue?
- selects: gram-negatives
- differentiates: lactose fermenters
- metallic green colonies, E. coli
What is liquid media?
- samples are not easily plated onto solid media
- large sample volumes
- research (culturing organisms for food)
- enrichment culture
How do we determine a bacterial species?
- biochemical tests
- matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF)
- NAAT (PCR, RT-PCR, probe based assays)
What is a type of biochemical test?
phenotypic assays: colour change, agglutination, or change in consistency of media (looking for some sort of observable characteristic)
What is MALDI-TOF?
- often treated as “answer box” (v good for most routine ID)
- fast (ID organism from primary culture w/o need for additional overnight incubation! improved turn-around-time)
- inexpensive (VERY low reagent costs, no need to maintain biochemical tests; equipment costs high)
What are biocontainment levels?
- in Canada we use a different system than the USA
- considers facilities, procedures, & equipment required to handle the organism safely
- levels 1-4
1. basic well functioning lab
2. agents require ingestion/inoculation for exposure. requires PPE (labcoats) & primary containment devices (biosafety cabinets)
3. agents which may be airborne. additional primary & secondary barriers: respirator, HEPA filtered lab exhaust
4. maximal precautions. complete isolation of facility, decontamination of lab effluents, positive pressure space suits
What do biological risk groups consider?
- availability of preventative measures (vaccines)
- availability of effective treatments (abx)
- pathogenicity
- infectious dose
- mode of transmission
- host range
What are the different risk groups?
- RG1: low individual, low community
- RG2: moderate individual, low community
- RG3: high individual, low community
- RG4: high individual, high community
What organisms are level 1?
- organisms unlikely to cause disease if healthy
- may encounter infections in immunosuppressed individuals
- ex might include: environmental organisms, lab strains used as experimental controls, plasmid recipients, reference isolates & type strains
What organisms are level 2?
majority of common pathogens:
- Bacteria (Campylobacter jejuni, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus)
- Fungi (Aspergillus fumigatus, Malassezia pachydermatis, Candida albicans)
What organisms are level 3?
includes number of important zoonoses & fungi causing systemic mycoses that you might encounter
- bacteria (Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella abortus)
- fungi (Blastomyces dermatitidis, Coccidioides immitis)
What is level 4?
- These are the scariest pathogens which you will hopefully never encounter
- there are no bacteria or fungi categorized as level 4
- viruses (Ebola, Hendra, Herpes B, Marburg, reconstructed 1918 H1N1, Variola)
BIG PIC: What is a bacterial isolate, how do we go about getting one?
- have CFU, isolate colony, & culture it to create a pure colony
- once you subculture it, you have lots of that pure bacteria (this is the isolate)
BIG PIC: What would you use selective media for? What would you use differential media for?
- have a mix of things in a sample but are only interested in 1 thing, so use selective media to zero in on what we want
- use differential media to allow different species to have different colours or morphological changes to focus on the type you want
