1st practical midterm Q-A Flashcards
Structure of gram + wall, result of gram staining (color)
- Cytoplasmic lipid membrane
- Thick peptidoglycan layer
** Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve as chelating agents, and also for certain types of adherence.
**A much smaller volume of periplasm than that in gram-negative bacteria.
Peptidoglycan structure: N-acteylglucosamine (NAG) bound to N-Acetyl-muramic acid (NAM)
NAM is bound to a tetramer (L-Ala-D-glu-P3-D-alanine) which is cross bridged to a glycine pentamer to another tetramer sequence.
Gram + stain gives a dark blue / purple color.
Gram - Gives a red pink color.
Alpha and beta hemolysis with examples
• Beta Hemolysis (complete hemolysis): complete dissolution of the RBCs around the colonies results in a colorless zone.
Examples- Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus
• Alpha Hemolysis (incomplete hemolysis): incomplete decomposition of RBCs around the colonies results in coloring the media green.
Examples- Streptococcus pneumoniae, viridant streptococci
**Alpha hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green methemoglobin.
what is the gas pak method?
Gas-pak is a method used in the production of an anaerobic environment. It is used to culture bacteria which die or fail to grow in presence of oxygen (anaerobes).
transport media- definition and types.
Transport media are special media formulated to preserve a specimen and minimize bacterial overgrowth from the time of collection to the time it is received at the laboratory to be processed. Depending on the type of organisms suspected in the sample, transport media may vary.
Types- 1) Transport swabs example: Stuart medium bacteria only survive for maximum 48 hrs, but do not grow ! 0,2% agar storage at RT! anaerobes also survive.
2) Transport plates
Bacteria multiply, by the time they arrive in the lab
examples: Uriline, Uricult, Gonoline, etc.
Both sides of a plastic sheet 2-3 different media (including selectives)
Disinfection, those affecting the cell wall
Disinfection = decrease of germ count (pathogens)
Those affecting the cell wall- 1) Detergents (surface active agents) A) Anionic: soaps effective in acidic environment low anti-bacterial effect B) Cationic: invert soaps, quaterner ammonium compounds, benzalconium cloride effective in alkaline environment good anti-bacterial effect
2) Alcohols
70% ethanol, i-propanol
disorganisation of lipid membrane, denaturation of proteins, dehydrating effect
3) Phenol containing organic solvents more effective if used with soaps strongly bactericid alkil-derivative: crezol (Lister!) cloro-derivatives: hexaclorophene, clorhexidine
chemotherapeutic index
the ratio of the minimal effective dose (in Latin: dosis
curativa minima) of a chemotherapeutic agent to the maximal tolerated dose (dosis tolerata
maxima). CI=DTM/DCM, the higher is this ratio, is the safer to use the substance.
major modes of antibiotics and examples
1) Inhibition of cell-wall synthesis
inhibition of peptidoglycan cross-linking (beta-lactams)
inhibition of peptidoglycan synthesis (vancomycin)
2) Disruption of cell membrane
polymyxins
3) Inhibition of protein synthesis
at 30S ribosomal subunit (aminoglycosides, tetracyclines)
at 50S ribosomal subunit (macrolides, chloramphenicol)
4) Inhibition of nucleic acid
inhibition of folic acid synthesis (sulphonamides, trimethoprim)
inhibition of DNA gyrase (fluoroquinolones)
inhibition of RNA synthesis (rifampin)
characteristic of endotoxin
Endotoxin = lipid A of Gram – bacteria (it is released only when bacteria are lysed)
-> general symptoms:
fever
hypotension
DIC (diss. iv. coagulatio)
- > heat- and enzyme resistant
- > poor antigen (LPS)
-> can not be attenuated to toxoid
agglutination (what is the antigen, what do we see… etc)
Cellular:
Gram- cell wall antigen „O”
capsular antigen „K”
pili antigen „H”
All produce agglutination, which you see “clumping”….
etc I have no idea what to say besides the different procedures-
1) Slide agglutination
2) Passive agglutination
3) Tube agglutination.
Steps of gram staining
- Fixation - degrasion og slide (5xflame), smear the bacteria suspension (if not liquid then use a small drop of water), then do the fixation from below so that the bacteria becomes white.
- Crystal violet staining for 1-2 min
- Lugol solution stain for 1 min
- wash with Alcohol
- Safranine staining
- wash with water
- Let it dry
- look at it under microscope.
(just remember CLAS)
Preparation of blood agar plates
First make normal agar plates with bouillon (made from meat) this is made hard with agar agar (functions like gelatin), when this is around 50 degrees we can add the roomtemperated blood.
Haemoculture
Cultivating blood or CSF. Incubation time is 7-10 days, but we usually can see the result after 24h.
When to use:
- meningitis, endocarditis, peritonitis, fever after implantation…
Imp rules! - At least 2 different samples ( diff. location and diff. time) - NOT from venous canule!! - best at the onest of fever - enough amount is needed. Sterility! During antibiotic treatment use ab-binding reasons Aerobic or anaerobic jars.
Autoclave (significane, parameters)
It uses high pressure steam to sterilize equipment. The efficacy depends on the level of steam saturation.
+1Atm –> 121 degrees for 20-30 min
+2 Atm –> 134 degrees for 10-20 min
Sterilization methods other than heat
By filtration By irradiation (UV + gamma ) By gas (formaldehide gases. ethylene-oxid, beta-propiolactone) Mechanical harm - freezing, boiling, ultrasonication
Sterilization methods other than heat
By filtration By irradiation (UV + gamma ) By gas (formaldehide gases. ethylene-oxid, beta-propiolactone) Mechanical harm - freezing, boiling, ultrasonication With chemicals (formaldehyde)
MIC (definition, determination)
MIC = minimal inhibitory concentration
Can be determinated either by:
1. Diffusion. Use the E-test (concentration-gradient strip)
2. Dillution. By agar dilution (antibiotic serial dilution mixed into the agar medium) OR by broth dilution (in tubes, or micro dilution in plates)
Major antibiotic resistance mechanisms
A. Natural resistance
- against the antibiotic produced by themselves
- cell wall barrier, or lack it it.
- lack of transport system
- lack of receptors
B. Acquired resistance
- Vertical - spontaneous mutations
- Horizontal- giving resistance genes to other bacterias
1.Enzymatic inactivation
2. Alteration of target by mutation
- decreased or no affinity
3. Efflux pump
- removal of antibiotic, not very effective
4. Overproduction of targets
5. Metabolite by-pass
6. Change of membrane permeability
- blocking active transport
7. Decreased modification to active component
CAMP test
is a test used to identify group B beta-streptococci based of their formation of a substance (CAMP factor) that enlarges the area formed by beta hemolysis from staphylococcus aureus. It is frequently used to identify group B street.
ELISA
=enzyme linked immunosorbent assay
Steps:
1.first component is bound to solid surface
2. incubation with every component again, to enable binding
3- Washing after every step (to remove unbound components)
4. Detection of signal - adding the substrate.
The enzyme cleaves the substrate, causing color change. (color density correlates with quantity)
Exotoxins
Exotoxins are mainly produced by the gram + bacterias.
- characteristic symptoms
- heat and enzyme sensitive
- good antigen
- can be attenuated to toxoid (vaccines)
- e.g Tetanus, diphtheria, cholera.
We have different mechanism
- Cytolytic effect - they form a pore on the membrane
- inhibition of protein synthesis
- hyper secretion of ions
- neurotoxins (botulinum toxin, tetanus toxin)
- Superantigens - Activation of a large number of T cells –> Cytokin storm (IL-1, IL-1, TNF alpha… ). Its a life-threatening autoimmune-like response. E.g Toxic shock syndrome.
Antibiotics disrupting protein synthesis
chloramphenicol, aminoglycosidans and tetracyclins
Antibiotics inhibit the cell wall synthesis
Beta-lactams
Vancomycin
Complement fixation test
- antibody detection from patient. e.g in syphilis diagnostics.
- complement: lyses immunocomplex after binding to it.
we make it visible with the indicator system (RBC + hemolysis).
First add complenet, then the antibody. Then we add the competing antigen and the competing antibody if these binds then there will be no hemolysis and we have a positive test.
How can we avoid developing resistance in bacterias to antibiotic?
By following the prescription of the doctor (take the dose and the days)
Not use so much antibiotics in animal farming.
Hygiene at the hospital, so that we don’t spread the resistant hospital strains.
