Fundamentals

Question 1

Why use a diagnostic lab test?

Answer 1

-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

Question 2

How to grow bacteria?

Answer 2

-broth vs agar
-selective vs differential vs non-selective media

Question 3

Colony forming unit

Answer 3

One cell expands to take up an area/form a colony of bacteria

Question 4

Tests used to ID organisms

Answer 4

-biochemical tests
-MALDI-TOF
-NAAT (nucleic acid amplification test)

Question 5

Colony

Answer 5

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)

Question 6

Why is it important to conduct dilutions and colony counts?

Answer 6

1.Establish clinical significance (# of CFU and whether its important)

2. Identify contaminants (find dominant organisms in mixed cultures)
3. Standardize lab tests (some tests need certain number of organisms to run test)

Question 7

Streak out plates

Answer 7

-use four streak method

-By 4th streak on plate, the objective is to get isolated colonies

Question 8

Define isolate

Answer 8

-A pure culture (clonal)
-comes from a single colony
-genetically homogeneous
-needed for ID and susceptibility testing

Question 9

Selection of culture media

Answer 9

-Culture media used depends on what you are trying to isolate
>use of selective and differential media helps to ID

Question 10

Selective media

Answer 10

-used to preferentially isolate particular taxa since it contains certain chemicals that inhibit the growth of non-target organisms

Question 11

Types of Selective media

Answer 11

1.CNA - selects for gram positives, and against gram negatives

2. MacConkey- selects for gram negative enterics, and against gram positives
3. Campy-BAP- selects for Campylobacter jejuni, and against most other bacteria

Question 12

Differential media

Answer 12

-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

Question 13

Types of differential media

Answer 13

1.MacConkey

2. XLD
3. CHROMager

Question 14

MacConkey as a differential media

Answer 14

Differential ingredient: lactose
Differentiates lactose fermenters

Question 15

XLD as a differential media

Answer 15

Differential ingredient: Ferric ammonium citrate

Differentiates H2S producers

Question 16

CHROMager as a differential media

Answer 16

Differential Ingredient: many
*so differentiates various species

Question 17

CHROMager ESBL

Answer 17

1. blue colonies= non e coli enterobacteriaceae
   white colonies= Pseudomonas spp

OR

2. Selection for 3rd generation cephalosporin resistance. Pink= e coli; Blue= non e coli enterobacteriaceae

Question 18

CHROMager MRSA

Answer 18

Selects for methicillin resistance
Differentiates for Staphylococcus aureus

Pink colonies= MRSA

Question 19

Mannitol salt agar

Answer 19

Selects: NaCl tolerant
Differentiates: mannitol fermentation

Yellow colonies= Staphylococcus aureus

Question 20

Eosin Methylene Blue

Answer 20

Selects: Gram negatives
Differentiates: lactose fermentors

Metallic green= E coli

Question 21

Liquid media

Answer 21

-Used for samples no easily plated on solid media, large volumes, or for culturing organisms from food for research, or using enrichment culture

Question 22

Biochemical tests to ID

Answer 22

-Phenotypic assays- such as colour change, agglutination or change in consistency of media
> compare to tables of known results for different organisms

Question 23

MALDI-TOF

Answer 23

-Very efficient ID, fast, inexpensive

-used to ID organism from primary culture without the need for additional overnight incubation

Question 24

Biocontainment level

Answer 24

-used in Canada
-considers facilities, procedures, and equipment required to handle organism safely

Question 25

Biocontainment levels 1-4

Answer 25

1. A basic well functioning lab
2. Agents requiring ingestion for exposure. Need PPE and containment devices (biosafety cabinets)
3. Agents can become airborne. Additional primary and secondary barriers (respirator, HEPA filtered lab exhaust)
4. Max precautions. Complete isolation of facility, decontamination of lab effluents, positive pressure space suits

Question 26

Biological risk groups

Answer 26

-used in US
-takes into account:
>availability of preventive measures (vaccines)
>availability of effective treatments (antibiotics)
>pathogenicity
>infectious dose
>mode of transmission
>host range

Question 27

Biological risk level 1

Answer 27

-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

Question 28

Biological risk level 2

Answer 28

**moderate individual, low community

-many common bacteria and fungi

Question 29

Biological risk level 3

Answer 29

**high individual, low community

-includes many important zoonoses and fungi causing systemic mycoses

eg. plague found in black-tailed prairie dogs

Question 30

Biological risk level 4

Answer 30

**high individual, high community

-Does not include any bacteria or fungi. Focus is on viruses
Ex. Ebola, Herpes B, reconstructed 1918 H1N1

Question 31

Plasmid

Answer 31

Extrachromasomal, independently replicating DNA molecule

Question 32

PCR

Answer 32

An in vitro technique for the amplification of target DNA sequences

Question 33

RT-PCR

Answer 33

Real time PCR

-An in vitro technique for the simultaneous amplification and detection of target DNA sequences

-may or may not be quantitative