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Microbiology Exam 3 > 2 - Labs Tools and Techniques > Flashcards

2 - Labs Tools and Techniques Flashcards

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1
Q

Overview of Microorganism Recovery and Identification

A
  1. Culture and biochemical tests

a. Type of culture and testing depends on anticipated pathogen and testing conditions (e.g. time,
reagents, instruments)

b. Distinguish between screening/presumptive tests and definitive tests.
(1) Screening/Presumptive – rapid, sensitive but possibly less specific, less expensive
(2) Definitive/Confirmatory – less rapid, more specific, more expensive

  1. Microscopy
  2. Immunodiagnostic tests
    a. Antibody in patient’s serum
    b. Antigen in patient’s specimen
  3. Detection of microbial genes
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2
Q

Distinguish between screening/presumptive tests and definitive tests.

A

(1) Screening/Presumptive – rapid, sensitive but possibly less specific, less expensive
(2) Definitive/Confirmatory – less rapid, more specific, more expensive

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3
Q

Immunodiagnostic tests

two mentioned?

A

a. Antibody in patient’s serum

b. Antigen in patient’s specimen

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4
Q

Recovery and Identification of Bacteria

1. Laboratory isolation of bacteria

A 
a. Isolation: To dilute the specimen in order to obtain colonies which are physically separated from
each other (streak the specimen across agar culture media in a petri dish).

b. Pure culture: A single type of bacteria which is maintained free from other bacteria. Pure culture is required for biochemical testing.
c. Streaking technique (Four Quadrant Method) [See Fig.1]
d. Subculture technique for purity (picking a colony) [See Fig. 1]

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5
Q

a. Isolation:

A

To dilute the specimen in order to obtain colonies which are physically separated from each other (streak the specimen across agar culture media in a petri dish).

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6
Q

b. Pure culture:

A

A single type of bacteria which is maintained free from other bacteria. Pure culture is required for biochemical testing.

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7
Q

c. Streaking technique is:

d. Subculture technique for:

A

(Four Quadrant Method) [See Fig.1]

purity (picking a colony) [See Fig. 1]

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8
Q

Bacterial culture media:

a. Major Types:

A

(1) Nutrient (general) – provides general nutrients required for growth of most common bacteria.
(2) Enriched – provides general nutrients plus various enrichments required for growth of common and more fastidious bacteria (i.e. those requiring special nutrients).
(3) Selective – contains ingredients that restrict the growth of certain types of bacteria (e.g. antibiotics, inhibitory chemicals, etc.).
(4) Differential – contains ingredients that visually indicate certain chemical reactions caused by bacterial growth (e.g. detects pH change, H2S production, metabolism of specific chemicals).

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9
Q

Bacterial culture media:
a. Major Types:

(1) Nutrient (general) –

A

provides general nutrients required for growth of most common bacteria.

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10
Q

Bacterial culture media:
a. Major Types:

(2) Enriched –

A

provides general nutrients plus various enrichments required for growth of common and more fastidious bacteria (i.e. those requiring special nutrients).

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11
Q

Bacterial culture media:
a. Major Types:

(3) Selective –

A

contains ingredients that restrict the growth of certain types of bacteria (e.g. antibiotics, inhibitory chemicals, etc.).

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12
Q

Bacterial culture media:
a. Major Types:

(4) Differential –

A

contains ingredients that visually indicate certain chemical reactions caused by bacterial growth (e.g. detects pH change, H2S production, metabolism of specific chemicals).

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13
Q

Bacterial culture media:

a. Major Types:

A
  1. Nutrient
  2. Enriched
  3. Selective
  4. Differential
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14
Q

Bacterial culture media:

Examples:

A
  1. Blood Agar Plate (BAP)
  2. Supplemented Chocolate Sugar
  3. MacConkey Agar (MAC)
  4. Broth media (liquid)
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15
Q

(1) Blood Agar Plate (BAP) (usually sheep blood) –

A

moderately enriched medium on which colonies can be differentiated by hemolytic pattern – grows almost all bacteria, except Neisseria gonorrhoeae, Haemophilus influenzae, and a few others which require SCA. Permits visual observation of blood cell hemolysis (alpha, beta, gamma –seen #3 below).
Can be made to be selective with the addition of antibiotics.

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16
Q

(2) Supplemented Chocolate Agar (SCA) –

A

highly enriched media, grows most medically significant bacteria, but without visual display of certain colony characteristics. Example of SCA made selective with the addition of antibiotics: Thayer-Martin (TM) medium – inhibits most bacteria while allowing pathogenic Neisseria species to grow.

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17
Q

(3) MacConkey Agar (MAC) –

A

Selective & differential media for gram-negative bacilli – Contain inhibitors for gram-positive cocci/bacilli and gram-negative cocci

(a) Differentiates colony types based on utilization of ingredients; observe pH changes

(b) Others examples of similar media: Xylose-Lactose-Desoxycholate (XLD), Hektoen (HEK),
Salmonella-Shigella (SS), Eosin Methylene Blue (EMB).

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18
Q

(4) Broth media (liquid) –

A

such as Thioglycollate or Trypticase Soy Broth can have various enrichments; used for blood culture & other specimens; may or may not be selective for certain organisms.

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19
Q
  1. Bacterial colony morphology – usually interpreted at _____. – Growth is observed for colony
    morphology (size, color & other distinguishing features

a. Significance of colony morphology – enables visual differentiation of many bacteria (e.g. hemolysis, carbohydrate utilization, general appearance)

b. Size: from less than ____ to more than _____
c. What else are we assessing?
d. What else are we assessing?
e. Hemolysis (only on blood agar) – due to _____
(1) Alpha – ___ ____ around the colony
(2) Beta – ___ ____ around the colony
(3) Gamma – ____ ____ in the red blood cells around the colony

A

18-24 h

1 mm diameter to more than 4 mm

assessing 3rd: Surface texture: e.g. smooth, rough, wrinkled, etc.

Assessing 4th: Color: natural pigment or due to pH indicator

bacterial production of enzymes which alter the
hemoglobin of red blood cells
(1) Alpha – green zone around the colony
(2) Beta – clear zone around the colony
(3) Gamma – no change in the red blood cells around the colony

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20
Q

bacterial colony morphology interpreted at

5 things we observe?

A

18-24 hrs

Distinguishing features
Size
Surface texture
Color
Hemolysis (only on blood agar)
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21
Q

e. Hemolysis (only on blood agar) – due to _____

(1) Alpha – ___ ____ around the colony
(2) Beta – ___ ____ around the colony
(3) Gamma – ____ ____ in the red blood cells around the colony

A

bacterial production of enzymes which alter the
hemoglobin of red blood cells

(1) Alpha – green zone around the colony
(2) Beta – clear zone around the colony
(3) Gamma – no change in the red blood cells around the colony

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22
Q

Recovery and Identification of Bacteria

  1. Microscopic exam - look for?
  2. Biochemical Identification Tests
    a. Principle – Tests for presence or production of certain enzymes or metabolic products
    b. Examples – test tube version, kits (API), instrumentation (MicroScan)
    c. Identification of most bacteria complete in about 1 to 2 days
    d. Identification of mycobacteria (tuberculosis) ready in 2 to 6 weeks
  3. Bacterial Identification (all are usually required for genus and species identification)
    a. Gram stain
    b. Culture characteristics
    c. Biochemical tests
    d. Time to ID – 6-48 hr (time for slow-growing bacteria such as tuberculosis may be 2 weeks or
    more)
A

(Gram stain or various wet mount preparations) – cellular morphology and staining characteristics are useful

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23
Q

(Gram stain or various wet mount preparations) – 4. Microscopic exam - look for?

A

cellular morphology and staining characteristics are useful

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24
Q
  1. Biochemical Identification Tests
    a. Principle – Tests for presence or production of _____
    b. Examples –
    c. Identification of most bacteria complete in about ____
    d. Identification of mycobacteria (tuberculosis) ready in _______
A

a. certain enzymes or metabolic products
b. test tube version, kits (API), instrumentation (MicroScan)
c. Most bacteria: 1 to 2 days
d. mycobacteria (tuberculosis): 2 to 6 weeks

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25
Q
  1. Bacterial Identification (all are usually required for _____)
    a. Gram stain
    b. Culture characteristics
    c. Biochemical tests
    d. Time to ID – ______
A

genus and species identification

a. Gram stain
b. Culture Chracteristics
c. Biochemical Tests
d. Time to ID - 6-48 hr (time for slow-growing bacteria such as tuberculosis may be 2 weeks or more)

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26
Q

Recovery and Identification of Other Microbes
2. Molds

a. Cultivation – grow on media with ____which inhibit the growth of bacteria. Mycelial masses (colonies) will be visible in ____
b. Identification – direct microscopic exam of ____; microscopic exam of ____; few biochemical tests; few antigen detection tests

A

Molds

a. antibiotics (e.g. Sabouraud with antibiotics); 1 to 4 weeks

b. ID - mainly direct microscope of specimen or culture
(few tests available)

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27
Q

Recovery and Identification of Other Microbes

  1. Viruses and some atypical bacteria
    a. Cultivation – grow in _____ culture cells – ____ to grow
    b. Identification – ____ detection; ____ detection; gene probes
A

Viruses and some atypical bacteria

a. Cultivation – grow in living tissue culture cells – 2 to 21 days to grow
b. Identification – antibody detection; antigen detection; gene probes

28
Q

Immunological Diagnostic Tests

  1. Introduction to Antigen-Antibody Reactions
    a. Reaction is based on specific Antigen-Antibody match-up.
A

(1) Epitopes must be detectable – sufficient quantity needed
(2) Epitopes must be highly specific in order to produce
accurate test result. Some antigens/epitopes are very
similar between different microbes, and are difficult to
distinguish
(3) Possibility of Cross-reactive – some Ab Fab sites will bind with very similar, but incorrect
antigens – causes false positive results (Sometimes cross-reactivity of multiple strains or
species of the same microbe may be desirable for a screening test.)

29
Q

Immunological Diagnostic Tests

  1. Introduction to Antigen-Antibody Reactions

b. Sensitivity – the ability of the test to detect ____
present.
(1) Test kits are designed to ensure that ______

(2) Expressed as (Limit of Detection) and (2) Percent Accuracy
(3) Tests with high sensitivity may be used as ______

A

Sensitivity – the ability of the test to detect very low levels of antigen (or antibody) when it is present

Test kits are designed to ensure that all true-positives are detected, even if that means a low percentage of false-positives are included.

Tests with high sensitivity may be used as initial screening or presumptive tests.

30
Q

Immunological Diagnostic Tests

  1. Introduction to Antigen-Antibody Reactions
    c. Specificity – the ability of the test to accurately detect _____
    (1) Distinguish _______ and ____-positive results from ____ positives.
A

c. Specificity – the ability of the test to accurately detect only the correct antigen or antibody while not reacting with incorrect antigen or antibody.
(1) Distinguish cross-reactive and false-positive results from true positives.

31
Q

Immunological Diagnostic Tests

  1. Introduction to Antigen-Antibody Reactions

f. Specimen
(1) Usually test serum – tests for _____

(2) Microorganism in lab culture or body specimen – tests for _____

A

Specimen

(1) Usually test serum – tests for presence of antibodies in the patient blood
(2) Microorganism in lab culture or body specimen – tests for presence of antigen

32
Q

Immunological Diagnostic Tests

  1. Introduction to Antigen-Antibody Reactions

f. Specimen
(1) Usually test serum – tests for _____

(2) Microorganism in lab culture or body specimen – tests for _____

A

Specimen

(1) Usually test serum – tests for presence of antibodies in the patient blood
(2) Microorganism in lab culture or body specimen – tests for presence of antigen

33
Q

Summary of Immunoassay Reaction Procedures

a. Antigens bind with specific antibodies
b. Remove or Wash off * unbound antibody, otherwise a false-positive reaction will result

A

Summary of Immunoassay Reaction Procedures

a. Antigens bind with specific antibodies
b. Remove or Wash off * unbound antibody, otherwise a false-positive reaction will result

34
Q

Summary of Immunoassay Reaction Procedures

a. Antigens bind with ___ _____
b. Remove or Wash off * unbound antibody, otherwise a _______________________

A

Summary of Immunoassay Reaction Procedures

a. Antigens bind with specific antibodies
b. Remove or Wash off * unbound antibody, otherwise a false-positive reaction will result

35
Q

The term “wash off” refers to

A

some means of removing or blocking the unbound reactants so that unintended binding will not occur.]

36
Q

. Summary of Immunoassay Reaction Procedures

c. Detect antigen-antibody binding. Examples:

A

(1) LFI (Lateral Flow Immunoassay): Capture molecule – captures Ag or Ab, and detector reaction makes it visible
(2) Agglutination is visible clumping without label
(3) Immunofluorescence: Fluorescent molecule produces light of detectible wavelength when excited by UV light
(4) ELISA: Color-producing enzyme reacts with substrate

37
Q

Lateral Flow Immunoassay

a. ____ molecule and _____molecules are embedded in (attached to) a membrane
b. ______ is deposited on one end of the membrane and a ___ ___ draws the fluid containing the specimen and reagents through the membrane; thus bringing them into contact.
c. ___ - ____ binding (reaction) is visible by deposit of the bound molecules at the “test line”.

A

Lateral Flow Immunoassay
a. Known molecule and detection molecules are embedded in (attached to) a membrane
b. Specimen is deposited on one end of the membrane and a wicking pad draws the fluid
containing the specimen and reagents through the membrane; thus bringing them into contact.
c. Antigen-Antibody binding (reaction) is visible by deposit of the bound molecules at the “test line”.

38
Q

Agglutination Method –

A

The reaction of a particle-sized antigen (e.g. bacteria or red blood cell or antigen on a latex particle) with its corresponding antibody resulting in a macroscopic (visible) clumping. This is commonly performed on a glass or paper slide and thus termed “slide agglutination”. The specimen may have to be treated or reacted with a reagent prior to the agglutination step.

a. Time to results – Direct agglutination tests – results in 2 to 20 min.
b. Relatively low sensitivity (i.e. requires greater quantity of Ag or Ab in the unknown sample)

c. Uses of agglutination tests
(1) Specimen screening in clinic or early in lab workup
(2) Confirm cultural and biochemical identification

39
Q

Agglutination Method – reaction of particle-sized antigen (e.g. bacteria or red blood cell or antigen on a latex particle) with its corresponding antibody resulting in a macroscopic (visible) clumping. This is commonly performed on a glass or paper slide and thus termed “slide agglutination”. The specimen may have to be treated or reacted with a reagent prior to the agglutination step.

a. Time to results – Direct agglutination tests – results in ______.
b. Relatively low _____

c. Uses of agglutination tests
(1) Specimen screening in clinic or early in lab workup
(2) Confirm cultural and biochemical identification

A

Direct agglutination tests –2 to 20 min

b. Relatively low sensitivity (i.e. requires greater quantity of Ag or Ab in the unknown sample)

c. Uses of agglutination tests
(1) Specimen screening in clinic or early in lab workup
(2) Confirm cultural and biochemical identification

40
Q

Immunofluorescence Method

a. Antibody is labeled (tagged) with a _____
b. Labeled antibody is reacted with ____ and then ____ is washed off
c. The reaction of the bound, labeled antibody with its corresponding antigen is made visible by exposure to ____
d. Emitted light is detected by an instrument or by using a _____ ____ ____. NOTE: Some fluorescence tests are not antibody-related; rather, the chemical reaction is fluorescent.
e. Results available from _____
f. Very ___ and ____; especially useful for certain types of agents

A

Immunofluorescence Method

a. Antibody is labeled (tagged) with a fluorescent substance which gives off a particular color of light at a specified wavelength of ultraviolet light
b. Labeled antibody is reacted with unknown antigen and then unbound antibody is washed off
c. The reaction of the bound, labeled antibody with its corresponding antigen is made visible by exposure to ultraviolet light
d. Emitted light is detected by an instrument or by using a ultraviolet light microscope. NOTE: Some fluorescence tests are not antibody-related; rather, the chemical reaction is fluorescent.
e. Results available from “real-time” (as the reaction occurs) to 1 hour to 2 days after specimen is collected, depending on the nature of the specimen, the antigen, and test procedure.
f. Very sensitive and specific; especially useful for certain types of agents

41
Q

Enzyme Immunoassay (EIA) or Enzyme Linked Immunosorbent Assay (ELISA) Method

a. ____ is labeled with a color-producing enzyme
b. Labeled antibody is reacted with ___ ____and ___ _____ is washed off (NOTE: this may use a known antigen & unknown antibody, and create a sandwich layering effect)
c. The reacted antigens and antibodies are exposed to a ______ to make the antigen-antibody reaction visible.

d. This is a very sensitive and accurate test, and is usually read by an instrument; however, some
products are membrane-bound (lateral flow) and read by the naked eye.

e. Results available from a few minutes to 2 days after specimen is collected, depending on the nature of the specimen and the test; typical is 1 to 4 hours.

A

a. Antibody (known) is labeled with a color-producing enzyme
b. Labeled antibody is reacted with unknown antigen and unbound antibody is washed off (NOTE: this may use a known antigen & unknown antibody, and create a sandwich layering effect)
c. The reacted antigens and antibodies are exposed to a color-producing substrate to make the antigen-antibody reaction visible.

d. This is a very sensitive and accurate test, and is usually read by an instrument; however, some
products are membrane-bound (lateral flow) and read by the naked eye.

e. Results available from a few minutes to 2 days after specimen is collected, depending on the nature of the specimen and the test; typical is 1 to 4 hours.

42
Q

– test kits used to detect the presence of significant gene sequences

A

Gene probes

43
Q

– increase the number of copies of significant microbial gene sequences so they can be detected, e.g. polymerase chain reaction (PCR)

A

Gene amplification

44
Q

Major Components of the Microscope

A

Base, arm, substage, stage, body

a. Base
(1) Light source, power switch, & variable intensity control
(2) Neutral density filter & control knob
(3) Field diaphragm & control knob
b. Arm – supports the upper parts of the microscope
(1) Coarse focus adjustment knob
(2) Fine focus adjustment knob
c. Substage
(1) Holds Abbe condenser (or darkfield condenser)
(2) Knob adjusts height
d. Stage
(1) Clips hold microscope slide in place
(2) Mechanical stage controls slide movement
e. Body
(1) Nose piece (rotating) – holds objective lenses
(2) Head – holds adjustable eye pieces

45
Q

Lens magnification system

(a) Low power: ___
(b) High (dry) power: _____
(c) Oil immersion high power: _____

(2) Eyepiece (ocular) lens – usually __
(3) Total magnification = the power (magnification) of the objective lens times the power (magnification) of the ocular lens.
(4) Examples:
(a) Low Power – 10X objective X 10X eyepiece = 100X total
(b) High Dry Power – 45X objective X 10X eyepiece = 450X total
(c) Oil Immersion – 100X objective X 10X eyepiece = 1000X total

A

(1) Commonly used Objectives

(a) Low power: 10X
(b) High (dry) power: 40 to 45X
(c) Oil immersion high power: 90 to 100X

(2) Eyepiece (ocular) lens – usually 10X
(3) Total magnification = the power (magnification) of the objective lens times the power (magnification) of the ocular lens.
(4) Examples:
(a) Low Power – 10X objective X 10X eyepiece = 100X total
(b) High Dry Power – 45X objective X 10X eyepiece = 450X total
(c) Oil Immersion – 100X objective X 10X eyepiece = 1000X total

46
Q

Lens magnification system

A

(5) Match-up of lens and lighting
(a) Low power lens – use low light intensity
(b) High dry power lens – use low to medium light
(c) Oil immersion high power lens – use high light intensity

47
Q

(6) Match-up of microbes with lens and magnification for final, detailed study/observation
(a) Gram stain of bacteria – _____
(b) KOH preparation for molds – _____
(c) Other wet mount preparations – _____

A

(a) Gram stain of bacteria – oil immersion high power
(b) KOH preparation for molds – low power
(c) Other wet mount preparations – low or high dry power (not oil immersion because that would press on the fluid specimen)

48
Q
  1. Operation of the Binocular Microscope
A

a. Use lens paper to clean the exposed lenses
b. Prepare and mount the specimen on a microscope slide
c. Using low power lens and low light intensity, focus down toward the slide by turning the coarse focus adjustment.
d. When the specimen is in focus and higher magnification is desired, rotate the objective lens turrent so the desired lens is in position.

e. Place a drop of immersion oil onto the slide before turning the oil-immersion lens into place.
NOTE: Oil immersion lenses should not be used for wet mounts.

f. Adjust the fine focus knob so the images are clear.
g. Higher light intensity will be required for higher magnification.

49
Q

Purpose of Gram Stain

A

Purpose
(1) Staining enables bacterial cells to be seen using brightfield microscopy (unstained cells are invisible)

(2) Enables bacterial differentiation based on morphologic and staining differences (different cell wall composition)

50
Q

Principle of Gram Stain

A

Bacteria stain either gram-positive or gram-negative on the basis of differences in their cell wall composition

51
Q

Staining Procedure for Gram Stain

Smear Preparation explained next:
Stain
Examine

A

(1) Smear preparation
(a) Smear the specimen onto a clean glass microscope slide
(b) Allow the smear to dry in the air without heating
(c) Fix the smear to the slide by flooding the dry smear with methanol for about 1 minute

(Less favorable alternate: Heat the dried smear slightly to fix the bacteria to the slide)

52
Q

Staining Procedure for Gram Stain

Smear Preparation
Stain (explained next slide)
Examine

A

(2) Staining steps
(a) Primary stain (Crystal Violet): 1 minute, then rinse with tap water
(b) Mordant (Gram’s Iodine): 1 minute, then rinse with water
(c) Decolorizer (Acetone & Alcohol): 2 to 5 seconds, then rinse with water
(d) Counterstain (Safranin): 30 to 60 seconds, then rinse with water and allow to dry

53
Q

Staining Procedure for Gram Stain

Smear Preparation
Stain
Examine (explained next side)

A

(a) Allow the slide to dry without heating (careful blotting without wiping is acceptable)
(b) Focus on objects using the low power lens and low light intensity; use coarse focus adjustment
(c) Change to oil immersion lens to observe details of bacteria; add a drop of oil to the slide; increase the light intensity; focus by using the fine focus knob

54
Q

Gram stain

Interpretation – usually reported within _____ if necessary

A

30 to 60 minutes

55
Q

Wet Mount Microscopic Examinations

a. Principle: Direct examination of certain

A

unfixed (i.e. “wet”) specimens to permit examination of
cells in their “natural” state

b. General procedure
(1) Place a portion of the specimen and any reagents required on a clean slide and cover with a thin coverglass
(2) Observe using low and/or high-dry power objectives and using reduced light intensity

56
Q

c. Microscopic examination types and purposes – some of these exams are performed in the clinic. When performed in the lab, results usually available shortly after procedure is performed.

A

(1) Potassium Hydroxide Wet Preparation (KOH) – 10% KOH dissolves (clears) epithelial cells permitting visualization of fungal mycelial fragments in skin scrapings; performed by the clinician without about 10 minutes after specimen is collected
(2) Yeast cells with capsules in cerebrospinal fluid (CSF) are revealed by the addition of India Ink to the CSF
(3) Saline wet preparation of vaginal exudate reveals yeast cells and motile Trichomonas organisms

57
Q

Antimicrobic Susceptibility Testing (AST)

1. Purpose & Principles of Antimicrobial Susceptibility Testing –

A

To determine if the pathogen is Susceptible or Resistant to a particular set of antimicrobics

a. Decision to perform AST is based on clinical significance of the pathogen
(1) Some bacteria in the specimen are likely to be non-pathogens which do not need to be tested
(2) Some pathogens can be treated by empiric therapy (i.e. treat with a specific antibiotic without testing) because the appropriate antibiotic is known and these pathogens are very unlikely to have resistant strains
(3) Test the pathogens that have increased likelihood of having resistant strains. (when tested under defined conditions

b. Each antimicrobic has a maximum concentration (dosage) that can be administered safely.
This dosage leads to a specific concentration in the blood (or urine or tissue).

c. Bacteria can tolerate (resist) only a certain (low) concentration of the drug. Each antimicrobic
can be safely administered only up to its maximum safe concentration (dosage).

58
Q

Antimicrobic Susceptibility Testing (AST)

Definitions:

(1) Resistant – The microorganism is _____
(2) Susceptible – The microorganism is _____
(3) Intermediate (susceptible dose dependent) – the microbe ____

It may be possible to use this antibiotic for treatment under very carefully defined situations and controlled conditions.

e. Each drug has a concentration range at which it is effective and safe. Concentrations vary among antimicrobics. Test determines if the antimicrobic is effective within its safe dosage.

A

(1) Resistant – The microorganism is NOT INHIBITED (can tolerate) by the maximum safe dose/concentration of the antimicrobic. Therefore, this drug should not be selected for therapy of this infection.
(2) Susceptible – The microorganism IS INHIBITED (or killed) by the maximum safe dose/concentration of the antimicrobic. This antimicrobic is a candidate for use in treating this infection.
(3) Intermediate (susceptible dose dependent) – the microbe MAY BE inhibited by a high dosage that very near the resistant point. This is typically interpreted as if it is “resistant”.

It may be possible to use this antibiotic for treatment under very carefully defined situations and controlled conditions.

e. Each drug has a concentration range at which it is effective and safe. Concentrations vary among antimicrobics. Test determines if the antimicrobic is effective within its safe dosage.

59
Q

Disk diffusion testing method

a. Purpose – Determine which ____
b. Principle and Method - Technique is useful only for _________
(1) ___ ___ containing known, standard concentrations of antimicrobics (each type of disk contains a particular antimicrobic) are applied to an inoculated agar plate using strict standardized procedures.
(2) The antimicrobic diffuses out of the impregnated paper disk into the agar at a known rate. Higher concentration of antimicrobic exists nearer the disk. The molecular size of the antimicrobic is a major factor in diffusion (i.e. large molecule diffuses more slowly), thus affecting zone size.

(3) A zone of inhibition (no growth) will occur where the antimicrobic concentration is sufficiently high. A large zone of inhibited growth indicates that greater concentration of antibiotic has migrated farther away from the disk AND/OR the bacteria is killed at a low
concentration.

(4) Measurement of size of zone of inhibition of growth is interpreted using a detailed chart that converts zone size (millimeters) into “Susceptible”, “Intermediate” or “Resistant”.

A

a. Purpose – Determine which antimicrobics are effective against a particular bacterium at the ‘breakpoint’ concentration of each antimicrobic.
Very rigid procedures and quality control

b. Principle and Method - Technique is useful only for rapidly growing aerobic & facultative bacteria
(1) Paper disks containing known, standard concentrations of antimicrobics (each type of disk contains a particular antimicrobic) are applied to an inoculated agar plate using strict standardized procedures.
(2) The antimicrobic diffuses out of the impregnated paper disk into the agar at a known rate. Higher concentration of antimicrobic exists nearer the disk. The molecular size of the antimicrobic is a major factor in diffusion (i.e. large molecule diffuses more slowly), thus affecting zone size.

(3) A zone of inhibition (no growth) will occur where the antimicrobic concentration is sufficiently high. A large zone of inhibited growth indicates that greater concentration of antibiotic has migrated farther away from the disk AND/OR the bacteria is killed at a low
concentration.

(4) Measurement of size of zone of inhibition of growth is interpreted using a detailed chart that converts zone size (millimeters) into “Susceptible”, “Intermediate” or “Resistant”.

60
Q

Disk diffusion testing method - PURPOSE?

A

Purpose – Determine which antimicrobics are effective against a particular bacterium at the ‘breakpoint’ concentration of each antimicrobic.

Very rigid procedures and quality control

61
Q

Disk diffusion testing method -

Principle and Method - Technique is useful only for __________

A

rapidly growing aerobic & facultative bacteria

62
Q

Minimum Inhibitory Concentration

a. Purpose –

A

Determines the minimum concentration of each antimicrobic agent that is effective against the bacterial pathogen. Because this information permits more precise selection of antimicrobic and dosage, this is especially useful when using one of the more TOXIC
antimicrobics, when treating the more resistant bacteria, or the patient has complications

63
Q

Minimum Inhibitory Concentration

b. Principle
(1) The bacterium is tested against various _____ of each antimicrobic

(2) For each antimicrobic tested, the minimum ____ required to inhibit the bacteria (no
growth in the test), the ___ value (µg per mL), is reported. Additionally, the susceptible, intermediate, or resistant interpretation is included.

A

concentrations

concentrations

MIC value

64
Q

c. Interpretation and Clinical Application

(1) The achievable concentration of antimicrobic at the site of infection varies with the site/tissue and the ________. Example: Most antimicrobics can be
administered in higher doses via intravenous vs oral route.

(2) A periodic dosing schedule leads to peaks and troughs in actual antimicrobic concentration in the tissue.
(3) “Susceptible” interpretation means the antibiotic concentration of at least 2 times the MIC can be achieved at the site of infection when administered by an appropriate route.
(4) “Resistant” and “Intermediate” – antibiotic concentration of 2X the MIC cannot be achieved even with the maximum safe dosage.
(5) Select the most effective “Susceptible” drug based on site of infection, toxicity, route of administration, etc.
(a) Concentration in certain tissues/organs will be lower
(b) Kidneys increase concentration of certain antibiotics

A

route of administration

65
Q

DO THE LEARNING EXERCISE!!!!

A

Good job nerd

Microbiology Exam 3 (15 decks)

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