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Clinical Bacteriology (Laboratory) > Antibiotic Susceptibility Testing (Kirby-Bauer Method | M) > Flashcards

Antibiotic Susceptibility Testing (Kirby-Bauer Method | M) Flashcards

1
Q

What is the meaning of AST?

A

Antimicrobial Susceptibility Testing

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2
Q

What are the 2 main purposes of susceptibility test (antimicrobial susceptibility testing)?

A

1) As a guide for treatment
- > Sensitivity of a given bacteria to known concentration of drugs
- > Its concentration in body fluids or tissues
2) As an epidemiological tool
- > The emergence of resistant strains of major pathogens
- > Continued surveillance of the susceptibility pattern of the prevalent strains

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3
Q

What are the 2 methods of AST?

A

1) Dilution method

2) Diffusion method

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4
Q

What are the principles of dilution method (of AST)?

A

1) Vary amt of antimicrobial substances incorporated into liquid or solid media
2) Followed by inoculation of test bacteria

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5
Q

What is the principle (or process) of diffusion method (of AST)?

A

Put a filter disc, or a porous cup / a bottomless cylinder containing a measured quantity of drugs on the solid medium that has been seeded with test bacteria

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6
Q

What are control strains (in AST)?

A

1) These are organisms obtained from the American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC) for UK, and Philippine Network of Microbial Culture Collections (PNMCC)
2) They should be grown in standard conditions
3) They have a known recorded sensitivity to antibiotics

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7
Q

Where does the control strains for UK come from?

A

From National Collection of Type Cultures (NCTC)

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8
Q

Where does the control strains for Philippines come from?

A

From Philippine Network of Microbial Culture Collections (PNMCC)

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9
Q

What is Muller Hinton medium and what is its principle?

A

1) It is a special media for sensitivity testing
2) It does not interfere with test results because it has a:
- > standard pH
- > standard electrolytes

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10
Q

Why does Muller Hinton medium not interfere w/ test results?

A

Because it has a:

1) Standard pH
2) Standard electrolytes

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11
Q

What is a standard inoculum size?

A

It is a std concentration of bacterial cells to be inoculated

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12
Q

What is the std inoculum size?

A

Std inoculum should have a turbidity equivalent to 0.5 MacFarland standard

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13
Q

What is the meaning of BaCl2?

A

Barium chloride

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14
Q

What is the meaning of H2SO4?

A

Sulfuric acid

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15
Q

What is being observed during preparation of inoculum?

A

Turbidity

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16
Q

What should be the turbidity of a std inoculum size?

A

Turbidity of 1X10^5

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17
Q

What is the requirement for std inoculum size?

A

It should be from a freshly overnight growth

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18
Q

What are the ways on how to prepare antibiotic discs?

A

1) Commercially prepared discs 6 mm in diameter

2) Can be prepared from Whatman filter paper no. 1, sterilized in hot air oven

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19
Q

If the antibiotic discs are commercially prepared, what is its size (in diameter)?

A

6 mm

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20
Q

What filter paper is used to prepare antibiotic discs (other way of preparation)?

A

Whatman filter paper no. 1

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21
Q

How are antibiotic discs (via the use of Whatman filter paper no. 1) sterilized?

A

Sterilized in hot air oven

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22
Q

True or False.

Kirby-Bauer Disc Diffusion method is not a conventional testing method (of AST)

A

False, because it is a conventional testing method (of AST)

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23
Q

What are the principles of Kirby Bauer Disc Diffusion method?

A

1) Impregnated disc absorbs moisture from the agar and the antibiotic diffuses into the agar medium
2) As distance from disc increases, antibiotic concentration decreases
3) Visible growth of bacteria occurs on the surface of agar where the concentration of antibiotic falls below the inhibitory level for the test strain
4) Concentration of diffused antibiotic at the interface of growing and inhibited bacteria approximates to MIC obtained in dilution tests

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24
Q

True or False.

As distance from disc increases, antibiotic concentration increases as well

A

False, because as the distance from disc increased, antibiotic concentration decreases

It should be inversely proportional and not directly proportional

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25
Q

What happens if the concentration of antibiotic falls below the inhibitory lvl for the test strain (of a sp bacteria or inoculum)?

A

Visibile growth of bacteria occurs on the surface of agar

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26
Q

What is the meaning of MIC?

A

Minimum inhibitory concentrations

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27
Q

How is MIC obtained?

A

Via dilution tests

28
Q

What is the process (or steps) of Modified Kirby-Bauer Method as per National Committee for Clinical Laboratory Standards (NCCLS)?

A

1) Prepare applx. (approximately?) 10^8 CFU/mL bacterial inoculum in a saline or tryptic soy broth tube (TSB) or Mueller-Hinton broth (5 mL)
- > pick 3 - 5 isolated colonies from plate
- > adjust the turbidity to the same as the McFarland No. 0.5 standard
2) Streak the swab on the surface of the Mueller-Hinton agar (3 times in 3 quadrants)
3) Leave 5 - 10 mins to dry the surface of agar
4) Place the appropriate drug-impregnated disc on the surface of the inoculated agar plate
5) Invert the plates and incubate them at 35 DC, o/n (18 - 24 hrs)
6) Measure the diameters of inhibition zone in mm

29
Q

What should be the concentration of the inoculum that will be embedded to the medium?

A

10^8 CFU/mL bacterial inoculum

30
Q

What are the media (broths) where the bacterial inoculum is embedded or putted?

A

1) Saline broth
2) Tryptic soy broth tube (TSB)
3) Mueller-Hinton broth

31
Q

If Mueller-Hinton broth is the medium that is being inoculated with the inoculum, what is the concentration (or how many) should be the inoculum?

A

5 mL

32
Q

How many isolated colonies should be picked up from the medium?

A

3 - 5 isolated colonies

33
Q

What should be the turbidity of the medium (in broth)?

A

Turbidity should be adjusted to the same as the McFarland No. 0.5 std

34
Q

After obtaining the inoculum from the broth and after adjusting the broth to the proper concentration of turbidity, what should be done next?

A

Streak the swab on the surface of the Mueller-Hinton agar

35
Q

What is the proper way of streaking (to Mueller-Hinton agar from broth)?

A

Streak the swab 3 times in 3 quadrants

36
Q

What is the time duration for leaving the surface of agar to air dry?

A

5 - 10 mins

37
Q

What should be done before incubating the agar medium?

A

Invert the agar medium first

38
Q

What is the proper temp for incubation of the agar medium?

A

35 DC

39
Q

What is the proper time duration for incubation of the agar medium?

A

18 - 24 hrs

40
Q

What should be the unit in measuring the inhibition zone (or zone of inhibition)?

A

mm

41
Q

What is the process (or steps) of simplified Kirby-Bauer Disc Diffusion method?

A

1) Select colonies
2) Prepare inoculum suspension
3) Standardize inoculum suspension
4) Mix well
5) Remove sample
6) Swab plate
7) Add disks (or discs)
8) Incubate overnight
9) Measure zones

42
Q

What are the 3 interpretations for results of AST?

A

1) Sensitive
2) Intermediate
3) Resistant

43
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Ampicillin belong?
b) What is the disk content of Ampicillin?
c) What is the zone diameter (resistance) of Ampicillin?
d) What is the zone diameter (intermediate) of Ampicillin?
e) What is the zone diameter (sensitive) of Ampicillin?

A

a) Penicillin
b) 10 ug
c) < or equal to 13 mm
d) 14 - 16 mm
e) > or equal to 17 mm

44
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Amoxicillin-clavulanic acid belong?
b) What is the disk content of Amoxicillin-clavulanic acid ?
c) What is the zone diameter (resistance) of Amoxicillin-clavulanic acid ?
d) What is the zone diameter (intermediate) of Amoxicillin-clavulanic acid ?
e) What is the zone diameter (sensitive) of Amoxicillin-clavulanic acid?

A

a) Beta (insert sign)-lactamase inhibitor combination
b) 20/10 ug
c) < or equal to 13 mm
d) 14 - 17 mm
e) > or equal to 18 mm

45
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Ceftriaxone belong?
b) What is the disk content of Ceftriaxone?
c) What is the zone diameter (resistance) of Ceftriaxone?
d) What is the zone diameter (intermediate) of Ceftriaxone?
e) What is the zone diameter (sensitive) of Ceftriaxone?

A

a) Cephems
b) 30 ug
c) < or equal to 13 mm
d) 14 - 20 mm
e) > or equal to 21 mm

46
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Gentamicin belong?
b) What is the disk content of Gentamicin?
c) What is the zone diameter (resistance) of Gentamicin?
d) What is the zone diameter (intermediate) of Gentamicin?
e) What is the zone diameter (sensitive) of Gentamicin?

A

a) Amino glycosides (or Aminoglycosides)
b) 10 ug
c) < or equal to 12 mm
d) 13 - 14 mm
e) > or equal to 15 mm

47
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Tetracycline belong?
b) What is the disk content of Tetracycline?
c) What is the zone diameter (resistance) of Tetracycline?
d) What is the zone diameter (intermediate) of Tetracycline?
e) What is the zone diameter (sensitive) of Tetracycline?

A

a) Tetraxycline
b) 30 ug
c) < or equal to 11 mm
d) 12 - 14 mm
e) > or equal to 15 mm

48
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Doxycycline belong?
b) What is the disk content of Doxycycline?
c) What is the zone diameter (resistance) of Doxycycline?
d) What is the zone diameter (intermediate) of Doxycycline?
e) What is the zone diameter (sensitive) of Doxycycline?

A

a) Tetracycline
b) 30 ug
c) < or equal to 10 mm
d) 11 - 13 mm
e) > or equal to 14 mm

49
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Ciprofloxacin belong?
b) What is the disk content of Ciprofloxacin?
c) What is the zone diameter (resistance) of Ciprofloxacin?
d) What is the zone diameter (intermediate) of Ciprofloxacin?
e) What is the zone diameter (sensitive) of Ciprofloxacin?

A

a) Fluoroquinolones
b) 5 ug
c) < or equal to 15 mm
d) 16 - 20 mm
e) > or equal to 21 mm

50
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Nalidixic acid belong?
b) What is the disk content of Nalidixic acid?
c) What is the zone diameter (resistance) of Nalidixic acid?
d) What is the zone diameter (intermediate) of Nalidixic acid?
e) What is the zone diameter (sensitive) of Nalidixic acid?

A

a) Quinolones
b) 30 ug
c) < or equal to 13 mm
d) 14 - 18 mm
e) > or equal to 19 mm

51
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Trimethoprim-sulfamethoxazole belong?
b) What is the disk content of Trimethoprim-sulfamethoxazole?
c) What is the zone diameter (resistance) of Trimethoprim-sulfamethoxazole?
d) What is the zone diameter (intermediate) of Trimethoprim-sulfamethoxazole?
e) What is the zone diameter (sensitive) of Trimethoprim-sulfamethoxazole?

A

a) Folate pathway inhibitor
b) 1.25/23.7 ug
c) < or equal to 10 mm
d) 11 - 15 mm
e) > or equal to 16 mm

52
Q

Answer the following questions (concentrations and zone diameter interpretative stds for E. coli):

a) At what grp of antimicrobial agents does Chloramphenicol belong?
b) What is the disk content of Chloramphenicol?
c) What is the zone diameter (resistance) of Chloramphenicol?
d) What is the zone diameter (intermediate) of Chloramphenicol?
e) What is the zone diameter (sensitive) of Chloramphenicol?

A

a) Phenicoles
b) 30 ug
c) < or equal to 12 mm
d) 13 - 17 mm
e) > or equal to 18 mm

53
Q

What are the factors affecting size of inhibition?

A

1) Inoculum density
2) Timing of disc application
3) Temperature of incubation
4) Incubation time
5) Potency of antibiotic discs
6) Composition of medium
7) Acidic pH of medium
8) Alkaline pH of medium
9) Reading of zones
10) Size of the plate
11) Depth of the agar medium (4 mm)
12) Proper spacing of the discs (2.5 cm)

54
Q

What is the principle for inoculum density (as factor affecting size of zone of inhibition)?

A

Larger zones w/ light inoculum and vice versa

55
Q

What is the principle for timing of disc application (as factor affecting size of zone of inhibition)?

A

If after application of disc, the plate is kept for longer time at room temp, small zones may form

56
Q

What is the principle for temperature of incubation (as factor affecting size of zone of inhibition)?

A

Larger zones are seen w/ temps < 35 DC

57
Q

What is the principle for incubation time (as factor affecting size of zone of inhibition)?

A

Ideal 16 - 18 hrs; less time does not give reliable results

58
Q

What is the principle for potency of antibiotic discs (as factor affecting size of zone of inhibition)?

A

Deterioration in contents leads to reduced size

59
Q

What is the principle for composition of medium (as factor affecting size of zone of inhibition)?

A

Affects rate of growth, diffusion of antibiotics and activity of antibiotics

60
Q

What is the principle for acidic pH of medium (as factor affecting size of zone of inhibition)?

A

Tetracycline, novobiocin, methicillin zones are larger

61
Q

What is the principle for alkaline pH of medium (as factor affecting size of zone of inhibition)?

A

Aminoglycosides and erythromycin zones are larger

62
Q

What is the principle for reading of zones (as factor affecting size of zone of inhibition)?

A

Subjective errors in determining the clear edge

63
Q

What is the principle for size of the plate (as factor affecting size of zone of inhibition)?

A

Smaller plates accommodate less number of discs

64
Q

What is the principle for depth of the agar medium (as factor affecting size of zone of inhibition)?

A

Thin media yield excessively large inhibition zones and vice versa

65
Q

What is the proper depth of the agar medium?

A

4 mm

66
Q

What is the principle for proper spacing of the discs (as factor affecting size of zone of inhibition)?

A

Avoids overlapping of zones

67
Q

What is the proper spacing of the discs?

A

2.5 cm

Clinical Bacteriology (Laboratory) (24 decks)

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