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MBIO 345 > Module 7 > Flashcards

Module 7 Flashcards

1
Q

When is it adequate to just identify the organism?

A

When the organism is universally susceptible

When standardized susceptibility test methods aren’t available

Ex) anaerobic bacteria

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2
Q

What is the goal of susceptibility/sensitivity testing?

A

Provide a list of effective antimicrobials for that specific organism.

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3
Q

What pharmacological knowledge must be applied to the results of a susceptibility test?

A

Is the antimicrobial effective at the pH of the body site?

Will it penetrate the site of infection?

Is it toxic to the host?

Is it cost effective?

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4
Q

What are the three conventional methods of performing susceptibility testing?

A

Broth dilution

Agar dilution

Disc diffusion

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5
Q

Why is standardized bacteria inoculum used and what is the concentration?

A

Improves accuracy

1.5x10^8 (0.5 MF)

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6
Q

How can a standardized inoculum be prepared?

A

Use 3-10 colonies of the same type in log phase

Indirect- inoculate broth and incubate until turbidity reached

Direct- emulsify colonies in broth to reach turbidity

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7
Q

What are the characteristics of growth medium used for susceptibility testing?

A

Mueller Hinton broth/agar

pH- 7.2-7.4

Ca and Mg close to physiological

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8
Q

Why must Ca and Mg concentrations be close to physiological conditions?

A

They affect the rate of aminoglycoside movement.

Low- false susceptibility

High- false resistance

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9
Q

What is the quality control strain for growth medium?

A

P. aeruginosa

Used with an aminoglycoside antibiotic

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10
Q

How can thymidine affect growth medium?

A

Can interfere with sulfonamide and trimethoprim results.

Can be used to bypass the PABA pathway these compounds inhibit.

Shows false resistance.

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11
Q

What are the two forms of broth dilution susceptibility tests?

A

Macro

Micro

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12
Q

How is the macro broth dilution test performed?

A

Serial doubling dilutions of antimicrobial

Standardized inoculum added to each

Incubated and inspected for growth

Lowest antimicrobial concentration with no growth is the minimal inhibitory concentration (MIC)

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13
Q

What is the growth control for the macro method?

A

One tube gets no inoculum

If there’s growth, test is inaccurate

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14
Q

How does the in vivo level of antimicrobial compare with the in vitro MIC?

A

In vivo should be 2-4x higher

Manufacturers determine what the in vivo level should be

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15
Q

What is the minimal bactericidal concentration (MBC) and how is it determined?

A

Lowest antimicrobial concentration that kills 99.9% of bacteria.

MIC determined

Tubes without growth are subcultured

Plate with 99.9% lack of bacterial growth (death) is the MBC

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16
Q

How is the micro broth dilution test performed?

A

Plates with micro tube wells contain antimicrobials

Each is inoculated

Incubated and examined for growth

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17
Q

What are the controls for the micro method?

A

Sterility- not inoculated, should be no growth

Growth- inoculated, no antimicrobial, should have growth

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18
Q

How can the antimicrobial in micro plates be stored?

A

Frozen (-20)

Lyophilized (4 or room temperature), require more inoculum to rehydrate

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19
Q

What automated system can be used for the micro broth dilution method? Describe it.

A

Vitek

Identifies organisms and performs susceptibility testing

Inoculum is loaded into cassette which is loaded into the reader/incubator

Can be programmed to store data

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20
Q

What are the advantages of automated micro testing?

A

Faster

More accurate

Automatic patient result transfer

Maldi-Tof can perform bacterial identification

Can sample mixed cultures

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21
Q

What should be done to mixed cultures?

A

The inoculum should be subcultured on a plate to check purity.

Must be observed before results are released.

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22
Q

How are agar dilution susceptibility tests performed?

A

Antimicrobials are diluted in agar

Inoculated with standardized inoculum

MIC determine by lowest concentration of antimicrobial that shows no growth in plates (susceptible)

23
Q

How are agar plates inoculated for susceptibility testing?

A

Using Steer’s replicators

Fit multiple wells of standardized inoculum of different bacteria

Multiple probes transferred to each plate

24
Q

What are the advantages of agar dilution testing?

A

Cheap

Test can be customized

Can be adapted for fastidious organisms

25
Q

How is disc diffusion/Kirby Bauer testing performed?

A

Plate is inoculated with standardized broth (usually 0.5 MF)

Antimicrobial discs are placed on the surface

Antimicrobial diffuses out, forms a zone of inhibition where the concentration is great enough to stop bacterial growth

The zones are measured and compared to the interpretive chart provided by CLSI

26
Q

What phase are bacteria chosen for disc diffusion in?

A

Log phase

Stationary/death- growth takes to long, larger zones, falsely sensitive

27
Q

What factors affect antimicrobial disc diffusion and how?

A

Size of molecule- bigger diffuse slower
Agar depth- thin gives falsely large zones
Medium composition- stiff slows diffusion, falsely small zones
Surface moisture
pH
Time between inoculation and disc application- >15min, falsely small zones
Time between disc application and incubation- >15min, falsely large zones
Broth concentration- >1.5x10^8CFU/mL, falsely small zones

28
Q

What is the critical point?

A

The lowest antimicrobial concentration around the disc that inhibits growth.

Corresponds to MIC.

29
Q

What is a regression graph?

A

Prepared for each antimicrobial

Zone size is plotted against MIC

Regression line is drawn

30
Q

How is the break point of an antimicrobial determined from a regression graph?

A

Draw a line that is at half in vivo MIC to the regression line

Draw down to the zone size axis

This value is the break point (lowest concentration that bacteria are susceptible to)

31
Q

What are the incubation standards for disc diffusion?

A

35°C

Ambient air (no extra CO2)

16-24hr

32
Q

How are colonies selected for disc diffusion?

A

More than one colony of the same type

Mixed cultures aren’t used

Purity checked by examining the sensitivity plate or subculturing the broth

33
Q

What are the requirements for Mueller Hinton agar?

A

90-140mm

Depth- 4-6mm

Poured on level surface, flat bottom

Stored in fridge (warmed to room temp before use)

Dry surface

34
Q

How are disc diffusion plates streaked?

A

Three directions

Show confluent growth

35
Q

What is the objective of clinical microbiology?

A

Bacterial identification

Antimicrobial susceptibility

36
Q

What situations require special zone size interpretations?

A

Colonies within zone- resistant subpop, check purity

Swarming into zone- Proteus, ignore

Inner ring- measure inner

Fine veil of growth- sulfonamide or trimethoprim, ignore, measure 80% inhibition

Sharp irregular zone- B-lactams, resistant, B-lactamase action

37
Q

What are intermediate organisms?

A

Difficult to test and produce results of uncertain significance.

38
Q

What are moderately susceptible organisms?

A

An infection that can be treated if higher dosages of the antimicrobial are used.

39
Q

What bacteria can Kirby Bauer tests be used for?

A

Fast growers

Non-fastidious

Able to grow in ambient air

Staphylococcus, enterococci, enteriobacteriaceae, nonfermentative G- rods

40
Q

What are QC organisms used for?

A

To check the accuracy of susceptibility tests.

41
Q

When are QC run for susceptibility tests?

A

When new discs/antimicrobial panels are received

Whenever susceptibility tests are performed

42
Q

What organisms are used for susceptibility test QC?

A

S. aureus, E. coli, P. aeruginosa

Min set required

43
Q

What is a major cause of susceptibility test inaccuracy?

A

Deterioration and loss of antimicrobial potency

Especially B-lactam

44
Q

How should antimicrobial discs be stored?

A

Less than 20°C

Moisture free environment

45
Q

How is MRSA susceptibility detected?

A

Directly prepare inoculum

NaCl to broth and agar (enhance growth)

Incubate at 30-35°C

Disc diffusion- watch for trailing at zone edge

46
Q

What is the oxacillin screen test? How is it performed?

A

Used for MRSA susceptibility

MH agar with 6ug/mL oxacillin and 4% NaCl

Spot inoculate

Any growth- resistance

47
Q

How is penicillin-resistant S. pneumoniae tested for susceptibility?

A

Inoculum streaked on MH agar enriched with blood

1ug oxacillin applied

Incubated in CO2

Zones >20mm indicate susceptibility

48
Q

How is Haemophilus susceptibility testing done?

A

Fastidious bacteria

Modified MH agar, haemophilus test medium (HTM)

Bovine heparin, yeast extract, NAD, thymidine phosphorylase

49
Q

How is N. gonorrhoea susceptibility testing done?

A

Fastidious bacteria

Special medium incubated in CO2

GC agar base enriched to allow growth

50
Q

What is the epsilometer test?

A

A strip with predefined antimicrobial concentration gradient on one side and an MIC scale on the other side

Applied to plate

Creates elliptical inhibitory zone, MIC where the zone intersects with the scale

51
Q

What if during an E-test two different levels are reached on either strip side?

A

The higher MIC is taken

52
Q

What are the advantages of E-testing?

A

Can be used for bacteria that can’t be tested traditionally

Can use a medium that will support group

53
Q

What do the results of an E-test indicate?

A

Low MIC- sensitive

High MIC- resistant

MBIO 345 (11 decks)

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