Classification, Identification & Molecular Diagnostics

Question 1

Bacterial classification used for?

Answer 1

describing & classifying bacteria & bacterial taxonomy

Question 2

Naming is based on

Answer 2

Based on morphology, biochemical reaction & genetic characteristics

Question 3

16s rRNA use

Answer 3

16s rRNA in bacteria always unique to the genus & species

Question 4

what is 16s rRNA

Answer 4

16S rRNA is a component of the small subunit of prokaryotic ribosomes

Question 5

Function of 16S rRNA over time

Answer 5

is not changed

Question 6

what is needed for 16s rRNA to change

Answer 6

1. for it to change, it is needs to go through lots of evolutionary process
2. even acquiring plasmid will not change 16s rRNA

Question 7

Importance of sampling

Answer 7

• needs to be right and obtained and placed in the right media for proper culture
  
  • to make sure that there are no contaminants when obtaining sample

Question 8

Importance of transport

Answer 8

Transport needs to have ideal temperature to prevent microbes from replicating

Question 9

Purpose of transport media

Answer 9

provides enough nourishment for bacteria so they don’t die
Holds bacteria in suspended animation until arrival at the dx lab

Question 10

components of transport media

Answer 10

non- nutritional, semi-nutritional, semi-solid agar containing salts & buffer

Question 11

condition of transport media

Answer 11

keep sample at 4oC

Question 12

eg of transport media

Answer 12

• Stuart’s transport medium
• Arnies transport medium (charcoal)

Question 13

Objective of bacteria ID

Answer 13

to ID an unknown organism to species level

Question 14

Definition of a species

Answer 14

closely related bacteria displaying a high degree of genetic & phenotypic similarity that differ from related organisms in many characteristics

Question 15

definition of strain

Answer 15

a line of bacteria descended from a single ancestor through various subcultures

Question 16

definition of Isolate:

Answer 16

each primary culture isolated from a natural source

• An isolate may become a strain after it is characterised

Question 17

Describe pure cultures

Answer 17

• important
• enables you to work with the colonies to know what the organism is
• If culture is contaminated, you don’t know what you’re dealing with

Question 18

define single colonies

Answer 18

individual bacteria from a single cell

Question 19

subculture define

Answer 19

isolate the organsims that you’re after

Question 20

what iS used to isolate pure cultures

Answer 20

blood agar

Question 21

Key steps for bacterial ID in vet lab

Answer 21

1.Correct sampling procedure & provision of adequate hx

2. Transport to the lab to ensure viability of the suspect organism & minimal contamination
3. Growth, ID & abx sensitivity testing of aetiological agent

Question 22

Cold chain & right media role

Answer 22

to prevent minimal contamination from other organisms

Question 23

Cold chain prevents

Answer 23

prevents bacteria replication as there can be contaminants from elsewhere which can mask the presence of the pathogen

Question 24

Right media for?

Answer 24

for strict anaerobes or others

Question 25

Choice of media for Mucosal Infection

Answer 25

Blood agar & MacConkey Agar

Question 26

Choice of media for Body Fluid

Answer 26

Additional blood agar incubated anaerobically

Question 27

Choice of media for Suspect fastidious organism

Answer 27

Chocolate agar or specialised agar

Question 28

Choice of media for Fungal pathogens

Answer 28

Sabouraud’s agar + chloramphenicol

Question 29

ID steps

Answer 29

1. Choice of media +right temp 1. Plate out and incubate 2. Examine plates every 24h 3. Colony morphology on solid media 1. important; gives clues to determine what is causing the infection based on the growth 4. Obtain a pure culture 1. Blood agar, gram or other staining & morphology 5. Selective biochemical tests 1. Substrate utilisation, motility, end products, enzyme detection 6. +/- Antigenic characteristics (serology) 7. Aids to diagnosis: 1. species specific PCR. 16s rRNA

Question 30

Role of gram stain

Answer 30

- what is seen in smear is what will be grown - help to choose right abx - not always practical but always encouraged

Question 31

role of Ziehl-Neelsen Stain

Answer 31

- Acid fast organisms - mycolic acids in cell wall

Question 32

use of biochemical tests

Answer 32

1. detection of enzymatic activity 2. Substrate utilisation via detection of end products

Question 33

Serological tests

Answer 33

antibodies raised in animals (mice or rabbits) by vax using bacterial antigens (highly immunoreactive protein or CHO epitopes

Question 34

Serotypes/Serovars used for?

Answer 34

commonly used for typing organisms like E.coli or salmonella where one can see various strains that produce various characteristics

Question 35

What can be typed?

Answer 35

Surface structures: flagella, LPS , capsular outside capsules & fimbriae all can be typed because they could lead to immune response & particular type lead to various antigen antibody complexes

Question 36

Lancefield’s Grouping (A-G) for streptococci used for?

Answer 36

heavily used for streptococci ID though it is replaced by MALDI (faster & quicker)

Question 37

Grp A strep

Answer 37

S. pyogenes

Question 38

Grp C strep:

Answer 38

S. equi

Question 39

Grp G strep:

Answer 39

S. canis

Question 40

Grp A strep: S. pyogenes

Answer 40

- Causes strep throat in humans - necrotising fasciitis: humans & possums

Question 41

Grp C strep: S. equi

Answer 41

causes strangles in horses

Question 42

Grp G strep: S. canis

Answer 42

Necrotising fasciitis in dogs

Question 43

MADLI what is it

Answer 43

- is a spectrometry - allows ID what organism is based on the profile - gives answer within 2 minutes - Cost effective

Question 44

Molecular Diagnostics use

Answer 44

- heavily used in virology but also certain bacteria - cannot grow; use PCR - serotype them, ID them based on PCR - Serotyping is heavily used in molecular PCR

Question 45

PCR based detection use

Answer 45

16S rRNA detection via direct PCR use 16S rRNA & do PCR from mixed culture like viruses from organisms that are highly pathogenic or will not grow in the lab