Biofilm Formation

Question 1

What is biofilm

Answer 1

Microbial communities that form a slime layer (ECM) via quorom sensing

Question 2

Define quorom sensing

Answer 2

cell-cell communication that allows bacteria to share info on cell density –> and adjust gene expression accordingly

Question 3

How does quorom sensing work

Answer 3

1. Bacteria produces autoinducers AI (extracellular < intracellular)
2. Different bacteria can interpret different types of autoinducers
3. AI signals attract other bacteria cells –> produce more AI
4. Once extracellular conc of AI > intracellular –> bacteria self-downregulates to stop making AI

Question 4

What are 3 strategies to overcome biofilm

Answer 4

1. Prevent adhesion of biofilm
2. Destroy the biofilm matrix
3. Action on dormant bacteria
   - dormant bacteria will stop quorum sensing during antibacterial treatment
   (most vulnerable)

Question 5

Explain Direct ELISA (antigen detecting, enzyme linked immunosorbent assay)

Answer 5

Antigen binds to primary antibody conjugate –> binds directly to detection molecule
- Good for looking for PURE ANTIGEN

Question 6

Explain Indirect ELISA

Answer 6

Antigen binds to primary antibody conjugate –> binds to secondary antibody conjugate –> binds to detection molecule
- DOES NOT RELY ON ANTIGEN BINDING, only presence of primary antibody conjugate

Question 7

Explain Sandwich ELISA

Answer 7

like indirect ELISA, but capture antibody is needed to secure antigen to secondary antibody conjugate

Question 8

What are the steps for Latex agglutination? (5)

Answer 8

1. One drop of positive control is added to circle #1
2. One drop of negative control is added to circle #2
3. One drop of latex reagent is added to 3 circles
4. 2 complete colonies are quickly emulsified into reagent circle #3
5. The slide is rocked by hand for 45 secs and placed on a slide rotator for another 45 secs

Question 9

Explain polymerase chain reaction PCR test

Answer 9

Small amount of DNA needed
- DNA is detected + amplified, analyzed
- selective amplification of bacterial genes to minimize false positive

Question 10

What is the most important aspect of specimen collection

Answer 10

Selection + collection

Question 11

What are the 5 rules of specimen collection?

Answer 11

1. Enough quantity of material used
2. Sample should be drawn from site of infection
3. Use sterile equipment
4. Examination must occur PROMPTLY afer collection
5. Specimens needed to diagnose bacterial infections should be secured before antimicrobial drugs are given

Question 12

Explain direct specimens

Answer 12

Sample collected from sterile body fluids
eg. (CSF, blood)

Question 13

Explain indirect specimens

Answer 13

Sample that has passed through body parts that contain bacteria
- need to differentiate between the good and bad
eg. coughed sputum, stool sample, voided urine

Question 14

What happens if the gram-pos bacteria contains endospores in gram-staining?

Answer 14

Will stain clear

Question 15

What is the most sensitive and specific method for diagnosing biofilm?

Answer 15

Bacterial cultures

Question 16

Why are bacterial cultures used?

Answer 16

Most bacteria can be grown in artificial media
- HOWEVER strict intracellular microorganisms (chlamydia, rickettsia) can only be isolated in living eukaryotic cells

Question 17

In Disk diffusion antibiotic suceptibility test, explain the following areas
Cloudy
Clear
No zone of inhibition

Answer 17

Cloudy = bacterial growth
Clear = bacterial death (due to release of antibiotics)
No zone of inhibition: too low dose, resistance, not effective antibiotic

Question 18

Define colony-forming unit CFU

Answer 18

The origin of a colony in a pure culture (single or multiple cells)

Question 19

Explain the streak plate technique

Answer 19

Sterile inoculating loop is used to apply CFUs to culture plate
- observe streaks after some time –> see where bacteria grew

Question 20

Explain Defined media

Answer 20

You know the exact chemical composition of the media

Question 21

Explain complex media. give example

Answer 21

You don’t know exact composition.
- Contains more nutrients and growth
eg. yeast, trypticase soy agar, macconkey agar

Yeast breaks things down into smaller components, therefore the final components of the media are not exactly known

Question 22

When is blood added in a complex media

Answer 22

To provide additional growth factors for fastidious microorganisms

Question 23

Explain selective media. Provide an example

Answer 23

Contains substances that favour the growth of particular microorganisms OR inhibit the growth of unwanted ones

Eg. Inhibit gram-pos, grow gram negative: Eosin, methylene blue, crystal violet dies, bile salts

Question 24

Explain differential media. Give an example

Answer 24

Allow growth of multiple bacteria types –> cause visible changes to help differentiate between various bacteria

Blood agar
- used to differentiate bacteria based on their hemolytic properties
- can also be considered an enriched medium for fastidious organisms

Question 25

MacConkey agar is what type of media? Why?

Answer 25

Complex media
Selective
- Kills gram-pos, allow gram-neg to grow

Differential media
- Lactose fermenting colonies (also gram neg) will turn red/pink
- nonlactose fermenters are colourless

Question 26

How to culture anaerobic bacteria using reducing agents? Eg?

Answer 26

Combine with free oxygen to remove it from the medium
- forms water molecules
Eg. Sodium thioglycolate, palladium

Question 27

How to culture anaerobic bacteria using stab culture? Eg?

Answer 27

Introduce anaerobes with straight inoculating wire into anoxic (no oxygen) depths of a solid media to form a “stab culture”.
- Deep = no oxygen