Biofilms + Lab Diagnosis Flashcards
What is a Biofilm
cell community enclosed in slime
- main component : extracellular polymeric substance matrix that is mostly made of polysaccharides that improve adhesion of bacteria
Steps to Biofilm Formation
1) adhesion of planktonic bacteria: some just sticks
2) line up to form monolayer: EPS is made
3) Multilayer forms: micro-colony because more friends join
4) Mature biofilm forms; proliferation and pulls in more friends; form mushroom shape and some leave
What Variables impact bacteria attachment
1) Texture and hydrophobicity of substratum: rougher and more hydrophobic (plastic) == more likely to stick
2) Conditioning film: various polymers make it easier to stick
3) Media/Fluid velocity
4)pH
5) Temperature
6) Cations
What is Quorum sensing
communication in biofilm that is dependent on autoinducers (AI)
- AI are species dependent + self-regulated
**once extracell >intracell —- stop proliferation and AI production
Impact of flow velocity on bacteria attachment
hydrodynamic boundary: layer that forms with no movement over substratum
- as increase speed of bacteria flowing over - hydrodynamic layer gets smaller —- easier for bacteria to stick
If go too fast — no hydrodynamic boundary + bacteria will flow right off
Benefit of biofilms
share nutrients and DNA; shelter each other from harmful stuff
- can share plasmids and Ab R genes
- mutual benefit
Prevention of Biofilm Formation
- prevent adhesion: add coating to substratum
- destruction of EPS matrix: mechanical, laser, heat
- target persister cells: normally stick around and have low levels of activity
Specimen collection
need to reflect the disease process and be collected in high enough amount
- always risk of contaminating bacteria
How to limit sample contamination
collect prior to antibiotic treatment
- sample as directly as possible
- use aseptic technique and PPE
- containers must be sterile and run tests right away
Direct Specimens
collected from normally sterile body fluids (blood, CSF)
- positive finding: diagnostic
Indirect Specimen
specimens of inflammatory exudates (voided urine) that have passed through sites known to be colonized with normal flora
- site of origin is sterile but likely to get contaminated when collecting
Samples from normal flora sites
primary site of infection is area already known to be colonized with other bacteria
- need to make selective examinations of what is normal vs what is causing infection
Why is transport media used
to provide stable conditions and prevent drying out
Enzyme linked immunosorbent assay
use antibodies linked to enzyme to detect Ag/bacteria
1) Direct primary antibody conjugated with detection molecule; primary detects Ag
2) Indirect: use primary antibody specific for Ag, 2nd specific for primary antibody and is conjugated with detection molecule
3) Sandwich: use capture antibody to detect bacteria + capture it , binds to different Ag; then use primary and secondary antibody to detect
—- helps capture specific bacteria with 2 Ag etc
Latex Agglutination
helps screen for specific Ag in sample
1) Positive control: sample that know has Ag
2) negative control: sample known without Ag
3) Sample
** add antibody conjugated with latex and if clump —- Ag present**
PCR: polymerase chain reaction
detection fo DNA particle with specific sequence ; isolate DNA from bacteria and screen for it using primers
Gram staining protocol
1) Add CV: binds to peptidoglycan
2) Add iodine/mortar: helps fix the CV
3) Alcohol wash to decolonize and wash off any unbound CV (wash it off of gram -)
4) Counterstain with safranin
Gram + = purple (thick peptidoglycan)
Gram - = pink (thin peptidoglycan)
What is the most sensitive and specific means of diagnosis
bacterial culture
What is a pure culture
a culture composed of cells arising from single progenitor
- derived from a single cell or group of cells (CFU)
** can use streak plate technique to get it
What is defined media
synthetic or chemically known media
- all parts known and known to have specific conc
- no yeast, animal or plant tissue
Complex media
media derived from plant, animal or yeast products
- made of shit with unknown conc of each thing
- used to culture organisms whose exact nutritional needs are known
ex.. nutrient broth, MacConkey
Selective Media
media that is used to select growth for specific micro-organism
- selective growth; uses specific markers such as Ab to select for R for example
can also use nutritional or salt
Differential Media
anything can grow but allows for visible changes in media or colonies to differentiate bw shit
- differentiate various kinds of bacteria growing on media based on changes
** uses the fact that different bacteria use different things in different ways
Blood Agar
- differential media
- help differentiate bacteria based on hemolytic properties (beta — clearing, gamma- does nothing)
**aka how well are they able to destroy RBCs
