Part IV: Introduction Flashcards
gram staining technique for fungi
Hucker’s modification
Most CRITICAL step in Gram staining:
DECOLORIZATION
Fungi are reported gram (+)
gram (+)
Size of AFB smear
2by3 cm
FITE-FARACO modification of acid fast stain is used to demonstrate
M. leprae
FITE FARACO Uses HEMATOXYLIN as
counter stain
Carried out to determine susceptibility or resistance of
organisms to antimicrobial agents
ANTIBIOTIC SUSCEPTIBILITY TESTING
Antimicrobials may be classified as
- Bactericidal
- Bacteriostatic
- Narrow and Broad Spectrum
ANTIBIOTICS
Cell wall inhibitors
- beta lactams
- Glycopeptides
ANTIBIOTICS
Cell wall inhibitors - Beta Lactams (3)
Penicillin
Carbapenems
Cephalosporin
ANTIBIOTICS
Cell wall inhibitors - Glycopeptides
Vancomycin
ANTIBIOTICS
Protein Synthesis inhibitor (5)
2C-MAT
Chloramphenicol
Clindamycin
Macrolides
Aminoglycoside
Tetracycline
ANTIBIOTICS
Protein Synthesis inhibitor - Macrolides (1)
Erythromycin
ANTIBIOTICS
PROTEIN SYNTHESIS
INHIBITORS - Aminoglycoside (2)
Gentamicin
Tobramycin
ANTIBIOTICS
PROTEIN SYNTHESIS
INHIBITORS - Tetracyclines (1)
Doxycycline
ANTIBIOTICS
BETA LACTAMASE INHIBITORS
Tazobactam
Sulbactam
Cluvanilic Acid
SULFONAMIDES
SXT
trimetophrim sulfamethoxazole
Drug which can be used as treatment for UTI
NITROFURANTOIN
NITROFURANTOIN action
Inhibits bacterial enzyme
inhibition of DNA activity
QUINOLONES
example of quinolones
Levoflaxin
Ciproflaxin
KIRBY BAUER DISK DIFFUSION
Qualitative
E-TEST DILUTION
Quantitative
Kirby Bauer Disk Diffusion
Media used
Mueller Hinton Agar
Kirby Bauer Disk Diffusion
pH
7.2-7.4
Kirby Bauer Disk Diffusion
Depth of agar
4 mm
Kirby Bauer Disk Diffusion
Inoculum size
1.5 x 108 CFU/ml
Kirby Bauer Disk Diffusion
Use of 0.5 Mac Farland standard is composed of
99.5 ml 1% sulfuric acid
1.175% barium chloride
Kirby Bauer Disk Diffusion
Manner of streaking using
Sterile cotton swab
Kirby Bauer Disk Diffusion
Streaking Method
Overlapping
Kirby Bauer Disk Diffusion
light inoculum, very dry agar, thin agar
False SUSCEPTIBLE
Kirby Bauer Disk Diffusion
heavy inoculum, too much moisture in agar, thick agar
False RESISTANT
Kirby Bauer Disk Diffusion
Size of filter paper disk
6mm
Kirby Bauer Disk Diffusion
Working supply
2-8 degC
Kirby Bauer Disk Diffusion
Incubation requirements
Incubate at 35-37 degC for 16-18 hrs in ambient air
If plate size is 100 mm
no more than 5 disks
If plate size is 150 mm
no more than 12 disks
Distance of disk:
From center
24 mm
Distance of disk
Between 2 disks
15 mm
Waiting time prior to placement of disks
3-5 mins
Use _______ when measuring ZONE OF INHIBITION
RULER OR CALIPER
SWARMING by PROTEUS
Ignore
Sulfonamides like SXT, may result into 2 concentric zones of inhibition, measure the
OUTER ZONE
Possible Results In Ast:
Resistant
Intermediate
Susceptible
Uses a strip with single antibiotic of decreasing concentrations along its length
E-TEST
E-TEST is a dilution method for fastidious organisms like
Haemophilus
E-TEST (+) Result
ELLIPSE OF GROWTH INHIBITION
detect the ability of organism to produce the enzyme B-lactamase
BETA - LACTAMASE TEST (Cephalosporinase Test)
enzyme that makes organism beta lactam resistant
B-lactamase
BETA - LACTAMASE TEST (Cephalosporinase Test)
Disk used
CEFINASE DISK
BETA - LACTAMASE TEST
Substrate
NITROCEFIN
BETA - LACTAMASE TEST
+ result:
pink to red coloration of the disk
BETA LACTAMS/PENICILLINASE RESISTANT DRUGS:
Methicillin and Oxacylin are used to treat
CAGI
S. aureus
H. influenza
N. gonorrhoeae
M. catarrhalis
some S. aureus have resistance to penicillinase resistant drugs and are called
MRSA
Methicillin Resistant S. aureus (MRSA)
MRSA is caused by the presence of
PBP2a
Penicillin Binding Protein 2a
PBP2a is encoded by
mec A gene
MRSA DETECTION
Media use (3)
Chromogenic agar
Cefoxitin Disk Test
Oxacillin Screen Agar
MRSA DETECTION
Positive and Negative Result
Pos: Red/mauve
Negative: Blue colonies
MRSA DETECTION
Oxacillin Screen agar is composed of
6 ug Oxacillin
4% NaCI
MRSA DETECTION
Gold standard
PCR
Detect inducible clindamycin resistance among strains of
S. aureus
D-TEST
D-test is done if the results is discrepant
true
different results
are obtained from same action antibiotics: discrepant result must be when
Clindamycin susceptible and erythromycin resistant
D-test uses
15 ug erythromycin
2 ug clindamycin
D-test
antibiotics are positioned
15 mm apart
(+) RESULT: blunting or flattening of clindamycin zone to produce
D - pattern
D-test
Interpretation of Result
(+) D-test: Clindamycin -
(-) D-test: Clindamycin -
(+) D-test: Clindamycin - RESISTANT
(-) D-test: Clindamycin - SENSITIVE
One way to detect S. pneumoniae
PENICILLIN RESISTANCE
S. pneumoniae
Hemolysis:
Optochin:
Alpha hemolytic
Optochin susceptible
PENICILLIN RESISTANCE
Culture Media used
MHA supplemented with sheep’s blood + 1 ug oxacillin disk
PENICILLIN RESISTANCE
o Greater than 20 mm -
o Less than 20 mm -
o Greater than 20 mm SENSITIVE
o Less than 20 mm RESISTANT
Screening test to detect ability of organism to produce the enzyme CARBAPENEMASE
MODIFIED HODGE TEST
Enzyme that make beta lactam antibiotic INEFFECTIVE
CARBAPENEMASE
MODIFIED HODGE TEST
(+) RESULT -
clover leaf leaf-likelike pattern
SEMI-AUTOMATED METHODS FOR BACTERIAL ID that uses microtubes with dehydrated substrates
API
SEMI-AUTOMATED METHODS FOR BACTERIAL ID
involves one step inoculation , no
need to add reagent and oil
BBL crystal ID
AUTOMATED SYSTEM
automation program for bacterial identificationand susceptibility
VITEK
AUTOMATED SYSTEM
same with VITEK but faster
MALDI-TOF