01- Introductory Microbiology, Specimen Processing & Susceptibility Testing (Exam #1)

Question 1

Define prokaryotes.

Answer 1

Organisms without a true nucleus
Size: 0.5-5 µm
(Bacteria)

Question 2

Define eukaryotes.

Answer 2

Organisms with a true nucleus
Size: >10 µm
(Fungi, Parasites)

Question 3

Define facultative anaerobes.

Answer 3

Microorganisms that can grow either with or without oxygen. If oxygen is present, these bacteria will utilize it via aerobic respiration and grow faster than without oxygen.

Question 4

Define obligate (strict) aerobes.

Answer 4

Microorganisms that require oxygen for growth.

Question 5

Define obligate (strict) anaerobes.

Answer 5

Microorganisms that cannot grow in the presence of oxygen. These bacteria must be grown in an atmosphere either devoid of oxygen or with significantly reduced oxygen content.

Question 6

Define flagella.

Answer 6

Exterior protein filaments that rotate and cause bacteria to be motile.

Question 7

Define monotrichous.

Answer 7

Bacteria that have a single flagellum on one end of the bacterial cell.

Question 8

Define lophotrichous.

Answer 8

Bacteria that have multiple flagella located at one end of the bacterial cell.

Question 9

Define amphitrichous.

Answer 9

Bacteria that have a single flagellum on each of the bacterial cell.

Question 10

Define peritrichous.

Answer 10

Bacteria that have many flagella covering the entire surface of the bacterial cell.

Question 11

What type of stain is the Gram Stain?

Answer 11

A Differential stain placing most bacteria into 2 groups- gram-positive and gram-negative.

Question 12

Explain the Heat Fixation Method

Answer 12

To heat fix, you can pass the slide quickly through a flame 2 to 3 times or by placing the slide on a slide warmer at 65ºC for 10 minutes. Allow the slide to cool to room temperature before staining.

Question 13

Explain the Methanol Fixation Method

Answer 13

• Methanol fixation can be performed by applying methanol to the air dried slide for 1 minute, draining off excess by tilting the slide, then allowing it to completely air-dry before staining.

Question 14

Compare the composition of gram-positive and gram-negative cell walls.

Answer 14

Gram-positive bacteria have a thick layer of peptidoglycan and techoic acid (strong negative charge) in their cells walls.

Gram-negative bacteria have a thin layer of peptidoglycan and an outer membrane surrounding their cell walls. The outer membrane is lipid rich due to a compound known as Lipopolysaccharide (LPS).

Question 15

Summarize the Gram stain procedure and its purpose.

Answer 15

1. Fix smear.
2. Apply the primary stain, crystal violet
3. Rinse with tap water.
4. Add mordant, Gram’s Iodine.
5. Rinse with tap water.
6. Decolorize primary stain using decolorizer, acetone.
7. Rinse with tap water.
8. Counterstain using safranin.
9. Rinse with tap water.
10. Blot dry.

• The Gram stain is used to detect bacteria in specimen (direct smear) and to determine the Gram stain of bacteria growing in culture (colony smear). The Gram stain allows for differentiation of bacteria based on their cell walls.

Question 16

Describe

What is the primary stain used in the Gram stain procedure?

Answer 16

Crystal Violet

• The first reagent of the Gram stain is crystal violet. It is an alkaline dye and the first stain applied to a fixed smear.
• During this step, the crystal violet dye is binding to the cell wall of the bacteria.
• If you look at the slide under a microscope at this point, the smear will be purple and all cells will be stained purple.

Question 17

Describe

What is the mordant used in the Gram stain procedure?

Answer 17

Gram’s Iodine

• Gram’s iodine (depending on the kit, this may be called by a different name) is the second reagent used when performing a Gram stain. This is known as the mordant.
• During this step, the iodine binds with the crystal violet to form a crystal violet-iodine, insoluble, complex that binds to the peptidoglycan layer of the bacterial cells.
• After this step, if you look at the slide under a microscope, the smear and all cells will still be purple.

Question 18

Describe

What is the decolorizer used in the Gram stain procedure.

Answer 18

An acetone–alcohol mixture or pure alcohol

• Decolorizer is the third reagent used in the Gram stain procedure. An acetone–alcohol mixture or pure alcohol can be used to perform this step.
• During this step, the decolorizer removes the lipid membrane from Gram negative cells. This results in leaching of the crystal violet–iodine complex, leaving the Gram-negative cells colorless. Gram-positive cells will retain the primary stain; remaining purple in color.
• This step is the most crucial because cells can easily become under–decolorized, if the decolorizer is not left on long enough; or over–decolorized if the decolorizer is left on too long.
• At this stage the smear may appear colorless, but under a microscope the Gram-positive cells will be purple and the Gram-negative cells will be colorless.

Question 19

Describe

What is the counterstain used in the Gram stain procedure?

Answer 19

Safranin

• The fourth and final reagent of the Gram stain is safranin. Safranin is used as a counterstain.
• During this step, the counterstain will stain the colorless cells of Gram-negative bacteria, pink. If you do not add the counterstain, the Gram-negative cells will remain colorless.
• Looking at this slide under a microscope, the Gram-positive cells will be dark purple and the Gram-negative cells will be red to pink in color.

Question 20

When using the Gram stain, what color do gram-positive bacteria stain? Gram-negative bacteria?

Answer 20

• Gram-positive cells will appear dark purple, after the Gram stain procedure, due to retaining the crystal violet–iodine complex within the thick peptidoglycan layer.
• Gram-negative cells will appear red to pink, after the Gram stain procedure, due to the crystal violet–iodine complex leaching out during the decolorization process; then being counterstained with safranin.

Question 21

What are some reasons for gram-variable results?

Answer 21

Some bacteria can produce Gram variable results, in which there will be both Gram-positive and negative cells.

Gram variable outcomes may also be a result of:

• An uneven direct smear (remake and restain slide)
• A direct smear made from an older culture (subculture (transfer) to a new plate and restain)
• Damaged cell walls due to antibiotics (nothing you can do)
• Under–decolorization (restain slide)
• Over–decolorization (restain slide)

Question 22

When gram staining, how would bacteria appear if under decolorized? Over decolorized?

Answer 22

• Under decolorizing your smear can cause gram-negative cells to retain the crystal violet-iodine complex. This will cause the cells to appear purple instead of pink.
• Over decolorization can occur if you leave the decolorizer on the slide too long. This will result in gram-positive bacteria losing the crystal violet–iodine complex, therefore staining pink as seen in the picture to the right.

Question 23

When is the gram stain performed?

Answer 23

It is routinely performed on certain specimen types, especially those collected from normally sterile sites.

Question 24

Why is the gram stain important or helpful?

Answer 24

Gram stain morphology can guide species identification

Question 25

What type of organisms have a **thick peptidoglycan layer in their cell wall** and retain the purple crystal violet/iodine complex during the decolorizer step?

Answer 25

Gram positive organisms

Question 26

What type of organisms have **a thin peptidoglycan cell wall with lipopolysaccaharide outer membranes** and lose the crystal violet/iodine complex during the decolorization step and are visualized using the red safranin counterstain?

Answer 26

Gram negative organisms

Question 27

What type of streak is used to innoculate urine cultures?

Answer 27

Quantitative isolation streak. ## Footnote Primarily used for urine cultures. Plates are inoculated using a calibrated loop to deliver a specified volume. The urine is mixed well, and the calibrated loop (0.01 or 0.001 mL) is vertically inserted into the urine and transferred to the culture medium by making a single streak down the center of the plate. Without flaming, the loop is streaked back and forth through the original inoculum. The number of colonies that grow are multiplied by the dilution factor. (eg, if 0.001mL loop is used, 35 colonies would translate into 35,000 colony forming units. (CFU/mL)

Question 28

What type of streaking is used to innoculate cultures other than urines and to set up transfer plates?

Answer 28

The general-purpose isolation streak ## Footnote The general-purpose isolation streak is useful for most specimens. The relative number of organisms can be estimated based on the extent of growth beyond the original area of inoculum.

Question 29

Define enriched media.

Answer 29

A growth medium that contains added growth factors, such as blood, vitamins, and yeast extract.

Question 30

Define differential media.

Answer 30

Media containing ingredients that allow visualization of metabolic differences between groups or species of bacteria.

Question 31

Define selective media.

Answer 31

Media containing additives such as dyes, bile salts, alcohols, acids, and antimicrobial agents that inhibit the growth of some bacteria but allow others to grow.

Question 32

5% sheep blood agar (BAP)

Answer 32

Enriched, Differential * Included for most specimens * Almost all bacteria & yeast will grow * Sheep blood is useful for distinguishing species that hemolyze the red blood cells around and under the colony Ingredients: Trypticase soy agar; 5% sheep blood Storage: Refrigerate

Question 33

Chocolate Agar (CHOC)

Answer 33

Enriched * Included for most specimens * Almost all bacteria & yeast will grow, including fastidious species. Ingredients: This medium can be made by adding sheep blood while the basal medium is warm enough to lyse the red blood cells, releasing hemoglobin and nicotinamide adenine dinucleotide (NAD). Storage: Refrigerate

Question 34

MacConkey Agar (MAC)

Answer 34

**Selective & Differential** * Included for most specimens * **Gram-negative rods will grow** * **Gram-positive organisms are inhibited** * **Lactose-fermenters are pink; non-lactose fermenters are clear colonies** Ingredients: bile salts & crystal violet inhibit gram-positive organisms; 1% lactose (sole carbohydrate source); neutral red indicator is pink-red if lactose is fermented Storage: Refrigerate

Question 35

What are the optimum atomospheric conditions, temperature, and pH range for most pathogenic bacteria?

Answer 35

Options for atomospheric conditions: Ambient (air): 21 % oxygen, 1% CO2 CO2 -enhanced: 18% oxygen, 5-10% CO2 Microaerophilic: 5%-6% oxygen, 5-10% CO2 Anaerobic atmosphere: <1% oxygen, 5- 10% CO2 Temperature: 35-37 degrees C pH: 7.0-7.5

Question 36

Colistin Nalidixic Acid Agar or Columbia CNA Agar

Answer 36

Enriched, Selective, Differential * Included if gram-positive organisms need to be separated from gram-negative * Gram-negative organisms are inhibited * Hemolytic reactions will be observed Ingredients: Columbia agar base with 5% sheep blood; colistin & nalidixic acid inhibit gram-negative organisms Storage: Refrigerate

Question 37

Phenylethyl Alcohol Agar | (PEA)

Answer 37

Selective * Included if gram-positive organisms need to be separated from gram-negative * Gram-positive bacteria will grow * Facultative gram-negative bacteria are inhibited Ingredients: 5% sheep blood; phenylethyl alcohol inhibits facultative anaerobic gram-negative bacilli; hemolytic reactions are less reliable on PEA than other blood-containing media Storage: Refrigerate

Question 38

Thioglycolate Broth (THIO)

Answer 38

Enriched * Detects a wide range of bacteria, including many anaerobes * Helps to recover organisms in low numbers, especially in normally sterile body sites * When cloudy the broth is subcultured to solid agar for growth & identification of organisms Ingredients: Dextrose, peptone, L-cystine & yeast extract base; thioglycolate consumes oxygen, permitting growth of anaerobes; cystine is a reducing agent. Resazurin is an oxidation-reduction indicator, being pink when oxidized & colorless when reduced. Storage: Refrigerate

Question 39

Minimum inhibitory concentration (MIC)

Answer 39

Minimum concentration of antibiotic required to **INHIBIT** the visible growth of the test organism ## Footnote 10.2 Define terms related to interpretation of susceptibility tests. 10.2.4 Minimum inhibitory concentration (MIC)

Question 40

Minimum bactericidal concentration (MBC)

Answer 40

Minimum concentration of antibiotic required to **KILL** the test organism | (allows less than 0.1% of the original inoculum to survive)

Question 41

Prophylaxis

Answer 41

Antimicrobial agents are administered to prevent infection

Question 42

Treatment

Answer 42

Antimicrobial agents are administered to cure existing or suspected infection

Question 43

Penicillin binding proteins (PBP)

Answer 43

Penicillin binds to a variety of proteins in the bacterial cell membrane and cell wall, called **penicillin-binding proteins (PBPs)**.

Question 44

Nitrocefin disks

Answer 44

Quick screen for beta-lactamase production Nitrocefin is a cephalosporin When you break open the beta lactam ring it changes color

Question 45

Explain the gradient diffusion test (Epsilometer test “Etest")

Answer 45

The strip has a gradient of antibiotics on it, place on bacterial lawn (0.5 McFarland), MIC is the point where the zone of inhibition intersects the strip

Question 46

Explain the broth microdilution procedure (

Answer 46

Quantitative method. 50µl or 100 µl is directly dispensed into each of the 96 wells of the microtiter tray containing doubling dilutions of lyophilized antibacterial agents The trays are examined for growth using a reflected viewing apparatus. The growth from broth alone is used as a comparison (positive control) . The MIC is the lowest concentration that inhibits the visual growth of the organism.

Question 47

What agar plate is used for setting up AST?

Answer 47

Mueller-Hinton

Question 48

Define antibiotic.

Answer 48

* Substance **used to prevent or treat infection** caused by bacteria and other pathogenic microorganisms. * **Selectively inhibits a vital metabolic process** of pathogens such as cell wall, DNA or protein synthesis. * To be clinically useful, the compound needs to reach the site of infection at a sufficient concentration for an adequate length of time.

Question 49

Define zone of Inhibition.

Answer 49

Zone related to disk diffusion testing; a clear area surrounding an antimicrobial disk following overnight incubation; results from diffusion of the antimicrobial molecules into the agar and inhibition of growth of the test bacterium.

Question 50

Define bactericidal

Answer 50

Antimicrobial that **kills a microorganism**.

Question 51

Define bacteriostatic

Answer 51

Antimicrobial that **inhibits bacterial growth but does not kill the bacteria**.

Question 52

Define intrinsic resistance.

Answer 52

* **Resistance that is naturally present in the microorganism**. It is a property controlled by chromosomes and is related to the general physiology of the microorganism. * Certain organisms will **always** be resistant in vivo to certain antibiotics regardless of how they test in vitro. * Therefore this resistance is **predictable once the organism is identified**.

Question 53

Define synergism.

Answer 53

Occurs when the antimicrobial activity of a combination of antimicrobial agents is greater than the activity of the individual agents alone.

Question 54

What McFarland standard is used for antibiotic susceptbility testing? How many CFU/mL does it represent?

Answer 54

0.5 McFarland standard represents a turbidity level for bacterial suspensions. It is used to standardized susceptibility testing and represents 1.5X10 ⁸ colony forming units (CFU/ml)

Question 55

Define acquired resistance.

Answer 55

* Bacteria can utilize plasmids, transposons, and insertion sequences to **transfer resistance genes to another bacterium.** * Can be expressed phenotypically as efflux, modification or acquisition of target sites, and enzymatic inactivation of the antibiotic. * Acquired mechanisms of resistance are caused by changes in the usual genetic makeup of a microorganism, leading to altered cellular physiology and structure. Unlike intrinsic resistance, acquired resistance may be a trait associated with only some strains of a particular species. **Thus the presence of this type of resistance in any of the isolates is unpredictable**.

Question 56

Define susceptible (sensitive) = S

Answer 56

An infection caused by the tested microorganism may be appropriately treated with the usually recommended dose of antibiotics.

Question 57

Define intermediate = I

Answer 57

The isolate may be inhibited by attainable concentrations of certain antibiotics (e.g., the beta-lactam antibiotics) if higher dosages can be safely used or if the infection involves a body site which allows the drug to concentrate (e.g., urinary tract). This category serves as a buffer zone that prevents slight technical artifacts from causing major interpretative discrepancies. (Gray zone)

Question 58

Define resistant = R

Answer 58

**Isolate is not inhibited by the concentration of antimicrobial** agent normally achievable with the recommended dose, indicating specific resistance mechanisms are likely to be present.

Question 59

Explain what the Clinical and Laboratory Standards Institute (CLSI) is.

Answer 59

- Subcommittee of scientists and physicians - Their goal is to establish standard conditions for testing methods based on laboratory investigations and assessment of clinical outcomes

Question 60

List quantitative antibiotic susceptbility testing methods.

Answer 60

* Microbroth dilution * Macrobroth dilution * Agar dilution * Gradient diffusion (E-Test) All result in a Minimum Inhibitory Concentration (micrograms per milliliter) Antimicrobial agents are usually tested at log2 (two-fold serial dilutions ex. 0.5, 1, 2, 4, 8, 16 etc.)

Question 61

List qualitative antibiotic susceptiblity testing methods.

Answer 61

* Agar diffusion (Kirby Bauer) Does not result in a MIC Categorizes an organism as susceptible (S), intermediate (I) or resistant (R) to a particular antimicrobial agent

Question 62

What volume of broth is used in broth macrodilution?

Answer 62

(\>=1 ml broth volume) – Very rarely performed in clinical labs.

Question 63

What volume of broth is used in broth microdilution?

Answer 63

0.1 mL

Question 64

Explain agar dilution.

Answer 64

A series of plates containing various concentrations of each antimicrobial agent are prepared. Test bacteria (0.5 McFarland) are spot-inoculated onto each plate using a multipronged replicating device. After overnight incubation, the MIC is read as the lowest concentration of antimicrobial agent that inhibits the visible growth of the test bacterium

Question 65

Describe the Vitek 2 automated system.

Answer 65

This system facilitates standardized susceptibility testing in a closed environment with validated results and recognition of an organism’s microbial resistance mechanism in 6 to 8 hours for most clinically relevant bacteria. Inoculum is automatically introduced via a filling tube into a miniaturized plastic 64-well, closed card containing specified concentrations (low, mid and high) of antibiotics. Cards are incubated in a temperature-controlled incubator Optical readings are performed every 15 minutes to measure the amount of light transmitted through each well and compared to the growth well without antibiotics. Algorithmic analysis of the growth kinetics in each well is performed by the system’s software to derive the MIC data The MIC results are validated with the Advanced Expert System (AES) software.

Question 66

Explain the principle of disk diffusion (Kirby-Bauer) testing.

Answer 66

Allows categorization of most bacterial isolates as susceptible, intermediate or resistant to a variety of antimicrobial agents. **(Qualitative)** The antibiotic diffuses from the disk, gradually decreasing in concentration as the distance from the disk increases. At a critical point the amount of antibiotic is unable to visibly inhibit the organism being tested; thus, a zone of inhibition develops. The point of this zone of inhibition is directly related to the MIC value.

Question 67

Summarize the disk diffusion (Kirby- Bauer) procedure.

Answer 67

1. A standardized suspension of the organism is spread over the surface of an agar plate (Mueller-Hinton), within 15 minutes of preparation of the inoculum. The plate is swabbed in three directions to ensure even distribution 2. Paper disks impregnated with antibiotics are placed on the agar surface within 15 minutes of inoculation of the agar plate. Disks should be placed at least 24 mm apart. 3. Agar plates are incubated at 35°C within 15 minutes of set-up to prevent prediffuion of the antimicrobial agents into the agar 4. Zone of complete inhibition is measured to the nearest millimeter

Question 68

Summarize how disk diffusion (Kirby Bauer) testing is interpreted.

Answer 68

The diameter of each inhibition zone is measured using a ruler or calipers. Plates are placed a few inches above a black, nonreflecting surface, and zones are examined from the **back side (agar side)** of the plate illuminated with reflected light.

Question 69

# Factors Affecting Size of Zone of Inhibition: Inoculum density of organism

Answer 69

Larger zones with light inoculum and smaller zones with a heavy inoculum

Question 70

# Factors Affecting Size of Zone of Inhibition: Timing of disk application

Answer 70

* The antibiotic disks must be applied to the media within 15 minutes after lawn of growth is made * If after application of disk the plate is kept out longer than 15 minutes at room temperature, small zones may form

Question 71

# Factors Affecting Size of Zone of Inhibition: Temperature of incubation

Answer 71

Larger zones are seen with temps less than 35degC

Question 72

# Factors Affecting Size of Zone of Inhibition: Incubation time

Answer 72

Ideal 16-20 hours - less time does not give reliable results

Question 73

# Factors Affecting Size of Zone of Inhibition: Depth of the agar medium

Answer 73

Standard size is 4mm. Thin media yields excessively large inhibition zones and vice versa

Question 74

# Factors Affecting Size of Zone of Inhibition: Proper spacing of the disks

Answer 74

Avoids overlapping of zones

Question 75

# Factors Affecting Size of Zone of Inhibition: Potency of antibiotic disks

Answer 75

Deterioration in contents leads to reduced size (possibly from condensation)

Question 76

# Factors Affecting Size of Zone of Inhibition: Composition of medium

Answer 76

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

Question 77

# Factors Affecting Size of Zone of Inhibition: Reading of zones

Answer 77

Subjective errors in determining the clear edge

Question 78

Describe 4 mechanisms of intrinsic resistance.

Answer 78

1. **Biofilms** (communities of microorganisms that are irreversibly attached to a solid surface) 2. **Impermeability** (certain antibiotics unable to penetrate the cell wall of particular microorganisms) 3. **Efflux** (function as transporter proteins for the extrusion of toxic substances and antimicrobial agents from the interior of the cell to the external environment.) 4. **Enzymatic Inactivation** (produce enzymes that destroy the antimicrobial agents before they are able to reach their targets.)

Question 79

Describe 4 mechanisms of acquired resistance.

Answer 79

1. **Efflux** (some efflux pump genes have translocated to plasmids, which can be acquired by horizontal gene exchange.) 2. **Target Site Modification** (Modification of a target can reduce the binding affinity of the antimicrobial agent for the target. Modification of target sites occurs primarily by chromosomal mutation) example: vanA gene for VRE 3. **Acquisition of New Targets (**become resistant by acquiring cellular targets with reduced affinity for the antimicrobial agent.) example: mecA gene for MRSA 4. **Enzymatic Inactivation of Antimicrobial Agents (**An existing cellular enzyme is modified to react with the antibiotic in such a way that it no longer affects the microorganism.) Example: CTX-M for ESBL

Question 80

A minimum of how many patient identifiers are required?

Answer 80

2

Question 81

It is best practice to collect how many sets of blood bottles from separate peripheral sites in 24 hours?

Answer 81

At least 2, but no more than 3.

Question 82

What is the purpose of resin in blood bottles?

Answer 82

To chelate antibodies and mediators of the immune system

Question 83

What is the anticoagulant used in blood bottles?

Answer 83

Sodium polyanethole sulfonate (SPS)

Question 84

Describe how CSF is collected and processed?

Answer 84

* Generally, 4 tubes of spinal fluid are collected. The 2nd or 3rd tube should be used for culture. * If the lab only receives enough CSF to inoculate once piece of media, an enriched medium should be used aka CHOC agar

Question 85

When a swab is used for bacteriologic culture, it should be placed in transport media. Name two examples of bacteriologic transport media.

Answer 85

Amies and Stuart

Question 86

What media is used for noninvasive urine cultures?

Answer 86

BAP and MAC inoculated with 0.001 mL of urine.

Question 87

What are some reasons for rejecting specimens for culture?

Answer 87

* Unlabeled or improperly labeled specimen * Improper collection site * Prolonged transit (over 2 hr without preservation) * Improper temperature during transport or storage * Leaking specimens * Specimens in nonsterile containers * Improper transport medium * Culturette ampule not broken, swab dried out * Improper swab, e.g., wood or calcium alginate for viruses or Chlamydia * Syringes with needles attached * Culture for anaerobes requested on inappropriate sources * Specimen received in formalin (other than stool for ova & parasites) * Saliva instead of sputum * Foley catheter tip * Insufficient quantity

Question 88

Describe skin preparation for venipuncture

Answer 88

Disinfect skin with 70%-95% ethanol or isopropyl alcohol and then 2% tincture of iodine or 2% cholhexidine

Question 89

Describe the recommended blood volume and ratio to culture medium for adult blood collections.

Answer 89

* Ideally inoculate each of 2 blood culture bottles (1 set) with 10mL of blood from adults (20mL per set). Keep the ratio of blood to broth 1:10-1:5.

Question 90

Aerotolerant Anaerobes

Answer 90

Microorganisms that can survive in the presence of oxygen but do not use oxygen in metabolism.

Question 91

Microaerophilic

Answer 91

Microorganisms that require a reduced level of oxygen (4-6%) to grow.

Question 92

Capnophilic

Answer 92

Microorganisms that require an atmosphere enriched with carbon dioxide (5% to 10%).

Question 93

Amphilophotrichous

Answer 93

Bacteria that have multiple flagella located at each end of the bacterial cell.

Question 94

Why is methanol prefered over other fixation methods for direct smears?

Answer 94

*** Methanol fixation is the preferred method, as it does not create aerosols and preserves cellular morphology.**

Question 95

In prokaryotes, what is the function of the plasma membrane?

Answer 95

Osmotic barrier and location of the electron transport chain.

Question 96

In prokaryotes, what is the cytoplasm?

Answer 96

Gelatinous liquid that fills the inside of the cell.

Question 97

In prokaryotes, what is the function of the ribosomes?

Answer 97

site of protein synthesis

Question 98

Describe the genome of bacteria.

Answer 98

One circular chromosome with dsDNA

Question 99

In prokaryotes, what is the function of pili/fimbriae?

Answer 99

Proteinaceous appendages used for adhearing to other cells.

Question 100

In prokaryotes, what is the function of the capsule?

Answer 100

Made of polysacchrides and/or proteins and inhibits phagocytosis

Question 101

In prokaryotes, what is the function of the glycocalyx?

Answer 101

Inhibit phagocytosis and aids in adherence to host tissue.

Question 102

In prokaryotes, what is the function of the cell wall?

Answer 102

Maintains shape of the cell and prevents bursting due to high osmotic pressure .

Question 103

In prokaryotes, what is a plasmid?

Answer 103

extrachromomal DNA passed between some bacteria. Often carries genes for aquired antibiotic resistance mechanisms.

Question 104

In prokaryotes, what are endospores?

Answer 104

Internal structure formed during poor environmental conditions. Can be located terminally, centrally, or subterminally.

Question 105

What can mutations lead to in different bacteria?

Answer 105

* Different strains of bacteria within a species that may have different biochemical or susceptbility results from the wild type strain (most common strain)

Question 106

Define opportunistic pathogen.

Answer 106

Microorganisms that do not normally cause disease or damage in a host, but under specific conditions or opportunities causes disease.

Question 107

Define exotoxin.

Answer 107

Toxins created inside the cell and released into the environment.

Question 108

Define endotoxin.

Answer 108

Lipid A- compound found in the cell wall of gram-negatives that is released upon death.

Question 109

Define pathogen.

Answer 109

Microorganism that causes infection.

Question 110

Define Virulence.

Answer 110

An ability of an organism to infect the host and cause a disease.

Question 111

Define polymicrobial infection.

Answer 111

Infection resulting from more than one organism.

Question 112

Define the phases of the growth curve for bacteria

Answer 112

* Lag phase: bacteria preparing for division * Exponential phase bacteria are dividing * Stationary phase:nutrients are becoming limited and the numbers of bacteria remain constant. * Death phase: the number of nonviable cells exceeeds the number of viable.

Question 113

Define the term fastidious.

Answer 113

Any organism that has complex or particular nutritional requirements. In other words, a fastidious organism will only grow when specific nutrients are included in its medium. "picky"

Question 114

What are the atmospheric conditions in a CO2 incubator?

Answer 114

~18% oxygen, and 5-10% CO2

Question 115

What are the atomospheric conditions in a O2 (ambient) incubactor?

Answer 115

~21% oxygen, <1% CO2

Question 116

What are the atmospheric conditions in anaerobic gas pak?

Answer 116

<1 oxygen, 5-10% CO2

Question 117

What is beta-hemolysis?

Answer 117

Complete lysis of red blood cells. Seen as a clear zone around colonies.

Question 118

What is alpha hemolysis?

Answer 118

Partial lysis of red blood cells. Seen as greening around the colony.

Question 119

What is gamma hemolysis?

Answer 119

No lysis of red blood cells.

Question 120

In culture, colonies with capsules appear as?

Answer 120

Mucoid

Question 121

Describe the color of bromcresol purple in acidic and alkaline conditions.

Answer 121

* Acidic=yellow * Alkaline=purple

Question 122

Describe the color of bromthymol blue in acid and alkaline conditions.

Answer 122

* Acidic= yellow * Basic=dark blue

Question 123

Describe the color of methyl red in acid and alkaline conditions.

Answer 123

* Acidic= red * Alkaline: yellow

Question 124

Describe the color of neutral red in acidic and alkaline conditions.

Answer 124

* Acidic= red * Alkaline=yellow

Question 125

Describe the color of phenol red in acidic and alkaline conditions.

Answer 125

* Acidic= yellow * Alkaline= red

Question 126

Describe the color of resazurin in oxidized and reduced conditions.

Answer 126

* Oxidized= pink * Reduced= clear

Question 127

How is media listed in the CLSI M22-A3 document assessed before use?

Answer 127

Assess media for * Moisture * Sterility * Breakage * Appearance

Question 128

As a general rule of thumb, which methods are sterilization processes?

Answer 128

Physical methods and chemical gases.

Question 129

As a general rule of thumb, what type of methods are disinfection techniques?

Answer 129

Chemical methods

Question 130

Define sterilization.

Answer 130

Process that destroys all forms of life including bacterial endospores

Question 131

Define disinfection.

Answer 131

Process that eliminates a defined scope of microorganisms

Question 132

What type of microscope is used to view Gram stained direct smears?

Answer 132

Bright-field. Host cells are quantitated using the 10x objective and microorganisms are quantiated on 100x.

Question 133

When using the acridine orange stain, what part of bacteria does the flurochrome bind?

Answer 133

Nucleic acid of living or dead bacteria. Organisms fluoresce bright orange. Slides are read using a flourescent microscopic and the 20x objective.

Question 134

What is the sensitivity of the Gram stain?

Answer 134

10^4-10^5 CFU/mL

Question 135

Describe examples of work practice and engineering controls.

Answer 135

* Work practice controls are ways we preform tasks to reduce risks. Ex. no food and drink in the lab, washing hands before leaving the lab, not touching your face. * Engineering controls are devices put in place to reduce risks. Ex. BSCs, Fume hoods, bench top shields, sharps containers.

Question 136

When should biological safety cabinets be used?

Answer 136

Processing patient specimens in the microbiology lab

Question 137

When should a fume hood be used?

Answer 137

When working with volatile chemicals.

Question 138

When should face shields be worn?

Answer 138

In situations where biohazardous material could be aerosolized.

Question 139

Define personal protective equipment.

Answer 139

Specialized clothing or equipment worn by an employee for protection against infectious materials.

Question 140

Contrast work practice controls used for working with clinical specimens and actively growing cutlures.

Answer 140

* Clinical specimens = Gloves, lab coat, BSC * Actively growing cutlures = gloves, lab coat

Question 141

Describe safe work practice for the use of sharps.

Answer 141

Discard sharp items into biohazard sharp containers. Do not fill past fill line.

Question 142

Describe possible routes of infection in the microbiology laboratory.

Answer 142

* Hand to mouth/eyes, avoid touching while working * Airborne, work in a BSC * Parenteral, used retractable scaples, and blunt needles.

Question 143

Describe aseptic technique.

Answer 143

Using practices and procedures to prevent contamination from pathogens

Question 144

What two biosaftey levels are used in our lab?

Answer 144

BSL-2 BSL-3

Question 145

Define standard precautions.

Answer 145

* Handwashing must be done after touching blood, body fluids, secretions, excretions, and any items considered contaminated. Hands must be washed after removal of gloves and between patients. * Gloves should be worn when handling blood, body fluids, secretions, excretions, and any items considered contaminated. Clean gloves must be put on before touching mucous membranes and nonintact skin. Hands must be washed after removal of gloves. * Mask, eye protection, or face shield must be worn anytime there is a potential for splashes or sprays of blood, body fluids, secretions, or excretions. * Laboratory coats must be worn to protect skin and clothing when contact with blood, body fluids, secretions, or excretions could occur. * Appropriate sharps disposal must be implemented with care to prevent injuries with sharps, needles, and scalpels. These devices must be placed in appropriate puncture-resistant containers after use. * Environmental control must include procedures for routine care, cleaning, and disinfection of environmental surfaces. These guidelines require that blood and body fluids from all patients be considered infectious and capable of transmitting disease. Blood and all body fluids, including secretions and excretions except sweat, regardless of whether visible blood is present, are considered infectious.

Question 146

Describe PPE for a BSL-3 Laboratory.

Answer 146

Glove, Lab Coat, Respiratory Protection

Question 147

Define the term N95 particulate filter.

Answer 147

a respiratory protective device designed to achieve a very close facial fit and very efficient filtration of airborne particles. Note that the edges of the respirator are designed to form a seal around the nose and mouth. The individual must be fit tested before use.

Question 148

Define the term powered air-purifying respiratory (PAPR)

Answer 148

Battery-powered devices that use a blower to pull air through attached filters (for particles) or cartridges (for gases or vapors) to clean it before delivering it to the breathing zone of the wearer.

Question 149

What is involved in a fit test?

Answer 149

An instrument is used to measure leakage around the face seal while wearing a N95 and doing the following exercises: Normal breathing. Deep breathing. Turning head side to side. Moving head up and down. Talking. Walking in place.

Question 150

What agencies oversee the shipment of clinical specimens and cultures of microorganisms?

Answer 150

* U.S. Department of Transportation * U.S. Postal Service * International Civil Aviation Organization

Question 151

Describe the three layers involved in the packaging of culture isolates.

Answer 151

* The primary receptacle contains the infectious material and must be leakproof. Liquids require packaging with an absorbent material capable of absorbing the entirety of the specimen if a leak occurs. * The secondary container must also be leakproof and encloses the primary receptacle. Cushioning may also be necessary to firmly secure a primary receptacle within the secondary container. Multiple primary receptacles can be placed in the secondary container if the substances are of the same class. If the receptacles are fragile, each must be individually wrapped or supplemented with cushioning to prevent contact. Documentation should be placed between the secondary container and rigid outer packaging. * The third layer, the rigid outer packaging, must protect the secondary container from damage. It must have the appropriate dimensions and strength to contain the primary and secondary containers

Question 152

What guidelines are followed in a biosaftey level 1?

Answer 152

1. Access to the laboratory is limited or restricted, and there must be a biohazard sign posted at the entrance of the laboratory. 2. Employees must wash their hands after they have removed their gloves, after they have handled live organisms, and before they leave the laboratory. 3. Employees must follow basic work practice controls. 4. Employees must follow OSHA guidelines for handling needles and sharps. 5. Work surfaces must be decontaminated after completion of work and any time there has been a spill of potentially infectious material. 6. Laboratory coats and gloves should be worn and other PPE, such as face shields and eye protection, may be needed when there is a potential for splashes or sprays of infectious or other hazardous materials. 7. Cultures and stock material must be decontaminated before disposal. 8. An insect and rodent control planmust be in effect. 9. The laboratory facility should be designed so it can be easily cleaned. Carpets and rugs should not be used. The laboratory must be equipped with handwashing sinks, an eyewash station, and adequate illumination.

Question 153

What guidelines are followed in a biosaftey level 2 laboratory?

Answer 153

BSL-1 guidlines plus: 1. Employees in the laboratory must have specific education in the handling of pathogenic agents and should receive annual updates or additional training as needed. 2. Access to the laboratory is limited when work is being conducted. The laboratory director is ultimately responsible for determining who may enter or work in the laboratory. 3. Laboratorians should receive immunizations or tests for agents handled or for agents that could potentially be in the laboratory environment (e.g., hepatitis B vaccination and the tuberculin skin test). 4. A biosafety manual must be developed and updated. 5. A BSC must be used whenever there is a potential for creating infectious aerosols or splashes or when high concentrations of infectious agents are used. The recommended BSC is class II. 6. All PPE must be worn. Gloves must be worn to protect hands from exposure to hazardous materials. 7. Extreme precautions are taken with contaminated sharp items. The use of needles and syringes is restricted within the laboratory to only times when there is no alternative equipment that can be used.

Question 154

What guidelines are followed in a biosafety level 3 laboratory?

Answer 154

BSL-1 and BSL-2 practices plus: 1. The handling of infectious materials, samples, and cultures must occur within a BSC or other physical containment device. 2. Personnel must wear appropriate PPE. Gloves must be worn to protect hands from exposure to hazardous materials. 3. The BSL-3 laboratory should be separated from the other parts of the building and should be accessed through two self-closing doors. An anteroom may be used for access. 4. The BSL-3 laboratory requires a ducted air ventilation system that must provide sustained directional air flow. This directional air flow pulls air from “clean” areas toward “potentially contaminated” areas (negative air pressure). 5. The ceilings and floors must be solid, and any seams must be sealed. 6. All parts of the laboratory must be constructed for easy cleaning and decontamination.

Question 155

What guidlines are followed in a biosaftey level 4 laboratory?

Answer 155

BSL-1, BSL-2, BSL-3 practices plus: 1. Access is strictly controlled, and the supervisor has the ultimate responsibility for who has access. A logbook is maintained to document all personnel who enter and exit the laboratory. 2. As required by all levels, a biohazard sign must be posted outside the door with the potential hazards; the laboratory director’s name; and any special requirements, such as immunizations, for entering the area. 3. All personnel must demonstrate high proficiency in standard and special microbiology practices. 4. Policies and procedures are established on the collection and storage of serum samples from at-risk personnel. 5. Personnel enter and exit the laboratory through the clothing change room. When personnel leave the laboratory, they must completely change clothes and shower. The clothes are then decontaminated before being laundered. 6. The BSL-4 laboratory has a dedicated nonrecirculating ventilation system, which is filtered through a HEPA filter before being exhausted. 7. All material is decontaminated before leaving the BSL-4 laboratory.

Question 156

For shipping, what is a category A substance?

Answer 156

An infectious substance which is transported in a form that, when exposure occurs can cause permanent disability, life-threatening or fatal disease in otherwise healthy humans or animals. Ex. Mycobacterium tuberculosis

Question 157

For shipping, what is a category B substance?

Answer 157

An infectious substance that is not in a form generally capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposed.

Question 158

Summarize the basic principles of specimen collection handling.

Answer 158

* Specimens should be collected from the appropriate site for recovery of suspected pathogens. * Speicmens should be collected prior to the adminstration of antibiotics. * Collection techniques must be clearly identified and should avoid contamination with normal flora. * The quantity of specimen collected should be adequate for all requested cultures and testing. * Specimens should be transported and processed in a timely manner. (2 hours max between collection and receipt if specimen has not been preserved in transport medium). * Transport media should be utilized to ensure viability of organisms when processing may be delayed. * Specimens should be accurately labeled to include patient identification, date and time of collection, and the source of the specimen. * Laboratory should be notified in advance if an unusual pathogen is suspected to ensure appropraite collection and handling.

Question 159

Which specimen type is boric acid used as preservative?

Answer 159

Urine

Question 160

Why are swabs not an effective way to collect specimen other than those from the upper respiratory tract, external ear, eye, and genital tract?

Answer 160

They are susceptibile to drying out.

Question 161

Why aren't cotton swabs recommended for the collection of specimens for cutlure?

Answer 161

Cotton-tipped swabs tend to have excessive fatty acids that may be toxic to certain bacteria and are not recommended. Instead dacron, rayon, or calcium alginate tipped swabs should be used.

Question 162

What are two types of transport media?

Answer 162

Stuart or Amie transport medium is commonly used.

Question 163

Which specimen type doesn't require a sterile collection container?

Answer 163

Stool

Question 164

What preservative is used for stool?

Answer 164

Cary-Blair transport medium

Question 165

Unpreserved refrigerated urine must be tranported to the lab within

Answer 165

24 hours

Question 166

Preserved urine must be transported to the lab within

Answer 166

48 hours

Question 167

As a general rule of thumb, how can unpreserved nonsterile specimens be preserved if transport is not within 30 mins of collection?

Answer 167

Refrigeration

Question 168

As a general rule of thumb, sterile specimens should never be preserved by

Answer 168

Refrigeration

Question 169

Which specimen type is struck quantitatively?

Answer 169

Urine

Question 170

Define bacteremia.

Answer 170

**Bacteremia: presence of bacteria in the blood stream** * Transient: random bacteria enter the bloodstream, but a cleared with 30 mins. * Intermittent: sporadic release of organisms into the bloodstream from another site of infection. * Continuous: organisms are released into the blood stream at a constant rate

Question 171

Define fungemia.

Answer 171

Presence of fungi in the blood stream.

Question 172

Define septicemia.

Answer 172

Any bacteriemia or fungemia that causes febrile illness.

Question 173

How often are temperature checks performed on temperature-dependent equipment?

Answer 173

Daily

Question 174

How often are GasPak jars and bags check for anerobiosis?

Answer 174

Each use

Question 175

How often are biological safety cabinets checked for proper air flow?

Answer 175

Annually.

Question 176

How often are centrifuge tested for proper revolutions per minute?

Answer 176

Every 6 months.

Question 177

How often are microscopes throughly cleaned and adjusted?

Answer 177

Quarterly.

Question 178

How often are autoclaves tested for proper sterilization?

Answer 178

Weekly using *Geobacillus stearothermophilus* spores at 121 degrees C under 15 psi for 15 mins.

Question 179

How often is quality control performed on staining procedures?

Answer 179

at least daily.

Question 180

What media requires retesting upon arrival in the laboratory?

Answer 180

Media not listed as exempt in the M22-A3 document

Question 181

How should media requiring retesting on arrival in the laboratory be tested?

Answer 181

* All media must be tested before use. * Each medium must be tested with organisms expected to grow as well as organisms expected not to grow * The organisms selected should represent the most fastidious organisms for which the medium was designed. * The medium should be checked for sterility. * Test with dilute suspensions of organisms

Question 182

# What is the mechanism of action for Beta-lactams?

Answer 182

Cell wall synthesis by binding up the enzyme, pencillin-binding protein.

Question 183

# What is the mechanism of action for Glycopeptides?

Answer 183

Cell wall synthesis by binding to the terminal amino acid in the polypeptide chain extending from NAM molecules.

Question 184

# What is the mechanism of action for Lipopeptides?

Answer 184

Disrupting the cell membrane.

Question 185

# What is the mechanism of action for Aminglycosides?

Answer 185

Protein synthesis by binding the 30s ribosome subunit.

Question 186

# What is the mechanism of action for Tetracyclines?

Answer 186

Protein synthesis by binding the 30s ribosome subunit.

Question 187

# What is the mechanism of action for Macrolides?

Answer 187

Protein synthesis by binding the 50s ribosome subunit.

Question 188

# What is the mechanism of action for Oxazolidinones?

Answer 188

Protein synthesis by binding the 50s ribosome subunit.

Question 189

# What is the mechanism of action for Streptogramins?

Answer 189

Protein synthesis by binding the 50s ribosome subunit.

Question 190

# What is the mechanism of action for Phenicols?

Answer 190

Protein synthesis by binding the 50s ribosome subunit.

Question 191

# What is the mechanism of action for Lincosamides?

Answer 191

Protein synthesis by binding the 50s ribosome subunit.

Question 192

# What is the mechanism of action for Fluoroquinolones?

Answer 192

DNA replication by targeting topoisomerases II and IV.

Question 193

# What is the mechanism of action for Sulfonamides?

Answer 193

DNA synthesis by inhibiting folate synthesis.

Question 194

# What is the mechanism of action for Nitronimidazole?

Answer 194

Damages DNA.

Question 195

# What is the mechanism of action for Rifamycins?

Answer 195

RNA synthesis by binding to DNA dependent RNA polymerase.

Question 196

# Which antibiotic class does the following drug(s) fall in? Vancomycin

Answer 196

Glycopeptide

Question 197

# Which antibiotic class does the following drug(s) fall in? Daptomycin Polymixin B Colistin

Answer 197

Lipopeptides

Question 198

# Which antibiotic class does the following drug(s) fall in? Gentamicin Tobramycin Streptomycin Kanamycin

Answer 198

Aminglycosides

Question 199

# Which antibiotic class does the following drug(s) fall in? Tetracycline Doxycycline Minocycline Tigecycline

Answer 199

Tetracyclines

Question 200

# Which antibiotic class does the following drug(s) fall in? Erthromycin Azithromycin Clarithromycin

Answer 200

Macrolides

Question 201

# Which antibiotic class does the following drug(s) fall in? Linezolid Tedizolid

Answer 201

Oxazolidinones

Question 202

# Which antibiotic class does the following drug(s) fall in? Chloramphenicol

Answer 202

Phenicols

Question 203

# Which antibiotic class does the following drug(s) fall in? Dalfopristin Quinupristin

Answer 203

Streptogramins

Question 204

# Which antibiotic class does the following drug(s) fall in? Clindamycin

Answer 204

Lincosamides

Question 205

# Which antibiotic class does the following drug(s) fall in? Ciprofloxacin Levofloxacin Moxifloxacin Nalidixic acid

Answer 205

Fluroquinolones

Question 206

# Which antibiotic class does the following drug(s) fall in? Trimethoprim-Sulfamethoxazole (SXT)

Answer 206

Sulfonamides

Question 207

# Which antibiotic class does the following drug(s) fall in? Metronidazole

Answer 207

Nitroimidazole

Question 208

# Which antibiotic class does the following drug(s) fall in? Rifampin

Answer 208

Rifamycins

Question 209

# Which antibiotic class does the following drug(s) fall in? Pencillin G Pencillin V

Answer 209

Natural Pencillins

Question 210

# Which antibiotic class does the following drug(s) fall in? Ampicillin Amoxicillin

Answer 210

Aminopencillins

Question 211

# Which antibiotic class does the following drug(s) fall in? Oxacillin

Answer 211

Penicillinase-Resistant Pencillins

Question 212

# Which antibiotic class does the following drug(s) fall in? Ticarcillin Carbenicillin

Answer 212

Carboxypencillins

Question 213

# Which antibiotic class does the following drug(s) fall in? Piperacillin

Answer 213

Uredopencillins

Question 214

# Which antibiotic class does the following drug(s) fall in? Cefazolin

Answer 214

1st generation cephalosporin

Question 215

# Which antibiotic class does the following drug(s) fall in? Cefoxitin

Answer 215

2nd generation cephalosporin

Question 216

# Which antibiotic class does the following drug(s) fall in? Ceftriaxone Ceftazidime Cefotaxime

Answer 216

3rd generation cephalosporins

Question 217

# Which antibiotic class does the following drug(s) fall in? Cefepime

Answer 217

4th generation cephalosporins

Question 218

# Which antibiotic class does the following drug(s) fall in? Ceftaroline

Answer 218

5th generation cephalosporins

Question 219

# Which antibiotic class does the following drug(s) fall in? Meropenem Ertapenem Doripenem Imipenem

Answer 219

Carbapenems

Question 220

# Which antibiotic class does the following drug(s) fall in? Aztreonam

Answer 220

Monobactams

Question 221

Sulbactam, Tazobactam, and Clavulanic acid are all

Answer 221

Beta-lactamase inhibitors

Question 222

Define breakpoint

Answer 222

A MIC range used to classify a bug as susceptible, intermediate, or resistant.

Question 223

Define surrogate drug.

Answer 223

Drugs of the same class that when tested will have comparable susceptibility results.

Question 224

Explain the cause of trailing endpoints seen in broth dilution AST testing.

Answer 224

Dilution methods sometimes produce an MIC end point that is not clear-cut, and growth in the wells may demonstrate trailing or skipped wells. Trailing involves heavy growth at lower concentrations followed by one or more wells that show greatly reduced growth in the form of a small button or a light haze. This commonly occurs with sulfonamides, trimethoprim, and trimethoprim-sulfamethoxazole; the mode of action of the agents allows the bacterial cells to grow through several generations before inhibition. In this case the trailing is ignored, and the end point is read as an 80% reduction in growth compared with the growth control. Trailing with most other drugs may represent contamination and should not be ignored unless it is known that trailing commonly occurs with the antimicrobial agent–organism combination.

Question 225

Explain skipped wells seen in broth dilution AST testing.

Answer 225

Skipped wells involve growth at higher concentrations and no growth at one or more of the lower concentrations. This may occur because of contamination, improperly inoculated wells, improper concentrations of antimicrobial agent in the wells, the presence of unusual resistance with the test isolate (e.g., a small resistant subpopulation), or a combination of two or more of these factors. As with trailing, each skipped well occurrence must be evaluated individually to determine whether results are reportable. If any doubt exists about the validity of the results, they should not be reported, and the test should be repeated.

Question 226

What is the biggest difference between broth microdilution and Kirby- bauer tests results?

Answer 226

Broth microdilution is quantitative and results in a MIC where Kirby-bauer is qualitative and results in S,I,or R only.

Question 227

How do blood bottles and other cultures involving liquid broth appear when positive?

Answer 227

Growth = turbidity

Question 228

What is flora found on the epidermidis called?

Answer 228

Normal skin microbiota

Question 229

What is flora found in the mouth, nasopharynx, and oropharynx called?

Answer 229

Normal respiratory microbiota

Question 230

What is flora found in esophagus, stomach, small intestine, and large intestine called?

Answer 230

Normal gastrointestinal microbiota

Question 231

What is flora of the distal urethra and vagina called?

Answer 231

Normal urogenitial microbiota