Ast Flashcards
- Challenging the organism with antimicrobial agent in liquid environment
- express in MIC
*kada tube domodouble ‘yong dilution
Broth dilution
It uses small amount of broth
Broth microdilution
0.05 to 0.1 ml - broth volume
48 hours for anaerobes - usage
Microdilution
Equal to or greater than 1 ml
Macrodilution
Temperature, medium and size of the 2 Broth Dilution method
35°C - 37°C for 16 - 24 hours
MH BROTH
5 x 10^5 CFU/ml
5 x 10^6 CFU/ml for anaerobes
- most laboratories use commercially supplied Microdilution in which panels broth already supplement appropriate antimicrobial concentration
- Fresh (16-18 hours)
- 35 - 37°C
Microdilution
Specific concentrations that separates/define the different categories
Breakpoints
Panels that only contain antimicrobial concentrations
Breakpoint panels
Advantage of breakpoint panels
Provide information to both
qualitative and
quantitative results
of your anti microbial agent
Broth microdilution method of
Vancomycin,
Teicoplanin,
Quinn pristin-dalfopristin,
Linezolid,
maxi floxacin
> 0.25 - 32
0.125 - 16
0.125 - 16
0.125 - 16
0.063 - 8
VITEK 2 method
Vancomy
teicoplanin
Quinn pristin-dalfopristin
linezolid
maxofloxacin
> 1-32
0.5 - 32
0.25 - 16
0.5-8
7 0.25-8
- Anti microbial concentration and organism on an agar based medium
- allows examination of for more bacterial isolation per plate
Can use bacterial isolates in one plate
Agar dilution
Standard count of bacteria, advantage
- 1x10^4 CFU/ml
- MIC
- provides a means for determining MIC for
Neisseria gonorrhoea, which does not grow sufficiently in broth
- Suitable only to organisms that grows rapidly overnight
- non-commonly use to fastidious organism, only to non-fastidious
Disk diffusion (kirby-bauer)
- Challenging bacterial isolates with antibiotic disks placed on the surface of an agar plate that has been seeded with a lawn of bacteria.
• If agar is too thick antimicrobial agent diffuses down through the agar as well as outward resulting to a smaller zone sized
• if agar is too thin inhibitor zones are larger molahinton agar
Disk diffusion
Tinitignan Na result Na result sa disk diffusion
Zone of incubation (Z01)
Inoculum preparation (use of pure culture)
- 4-5 colonies inoculate into broth medium incubate for 3-5 hours at 35 - 37°C
- 4-5 colonies, 16 - 24 suspendin broth on 0.9% saline solution
Inoculum preparation
Use of standarized- size of inoculum
Turbidity standard
- 0.5 McFarland standard
- barium salfate suspension
Composition:
- 99.5 ml of 1% and o sulfuric acid
- 0.5 ml of 1.75% barium chloride
Standard size of inoculum:
- 15 x 108 ( FU /ml
Inoculation & incubation
- Surface of the plate is swabbed in 3 directions
- Antibiotics disk are applied within 15 minutes
- Inverted plates - incubate at 35.37°C
- increased CO2 for fastidious bacteria
6.incubation time maybe increased beyond 6 hours’_ for methicillin resistance staphylococci & vancomycin resistance enterococci
Kirby Bauer disc diffusion method
- inoculated agar plate
- Addition of antibiotics disc
- Measurement of the zone of inhibition
Can tolerate high amount of salt
Staphylococci
Can tolerate high amount of salt
Staphylococci
- Commercially, prepared disks 6 mm in diameter should be used.
- Distilled water serves to dissolve most antibiotic powders, but chloramphenicol, rifampicin and erythromycin must first be dissolved in a small amount of ethanol
• nitrofurantoin and sulfonamides in small volume of NaOH solution, trimethoprim in weak acid (acetic or lactic), and amoxicillin and ceftazidime in a small volume of saturated NaHC03
• Disk should be taken out from refrigerator 1-2 hours before applying on the culture medium.
Antibiotic disk
- Commercially, prepared disks 6 mm in diameter should be used.
- Distilled water serves to dissolve most antibiotic powders, but chloramphenicol, rifampicin and erythromycin must first be dissolved in a small amount of ethanol
• nitrofurantoin and sulfonamides in small volume of NaOH solution, trimethoprim in weak acid (acetic or lactic), and amoxicillin and ceftazidime in a small volume of saturated NaHC03
• Disk should be taken out from refrigerator 1-2 hours before applying on the culture medium.
Antibiotic disk
- Examine for the presence of confluent lawn of growth
• a If POOR or NONCONFLUENT - repeat testing
• 2. Examine for purity
• a. If MIXED CULTURES - repeat testing
• 3. A dark background and reflected light are used to examine the plate
• 4. Measure the zone of inhibition in diameters for each antimicrobial agent using a ruler or caliper
• 5. Interpret as “SUSCEPTIBLE”, “INTERMEDIATE” or “RESISTANT”
Reading and interpretation of result
Different categories of sensitivity
Sensitive, intermediate & resistant
Different categories of sensitivity
Sensitive, intermediate & resistance
Staphylococcus species
Cefazolin <14 15-17 >18 clindamycin
Pseudomonas aeruginosa and other non-fermenting gram negative
Amikacin, cefoperazone,, cefotaxine gentamicin
