01- Introductory Microbiology, Specimen Processing & Susceptibility Testing (Exam #1) Flashcards
Define prokaryotes.
Organisms without a true nucleus
Size: 0.5-5 µm
(Bacteria)
Define eukaryotes.
Organisms with a true nucleus
Size: >10 µm
(Fungi, Parasites)
Define facultative anaerobes.
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.
Define obligate (strict) aerobes.
Microorganisms that require oxygen for growth.
Define obligate (strict) anaerobes.
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.
Define flagella.
Exterior protein filaments that rotate and cause bacteria to be motile.
Define monotrichous.
Bacteria that have a single flagellum on one end of the bacterial cell.
Define lophotrichous.
Bacteria that have multiple flagella located at one end of the bacterial cell.
Define amphitrichous.
Bacteria that have a single flagellum on each of the bacterial cell.
Define peritrichous.
Bacteria that have many flagella covering the entire surface of the bacterial cell.
What type of stain is the Gram Stain?
A Differential stain placing most bacteria into 2 groups- gram-positive and gram-negative.
Explain the Heat Fixation Method
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.
Explain the Methanol Fixation Method
- 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.
Compare the composition of gram-positive and gram-negative cell walls.
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).
Summarize the Gram stain procedure and its purpose.
- Fix smear.
- Apply the primary stain, crystal violet
- Rinse with tap water.
- Add mordant, Gram’s Iodine.
- Rinse with tap water.
- Decolorize primary stain using decolorizer, acetone.
- Rinse with tap water.
- Counterstain using safranin.
- Rinse with tap water.
- 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.
Describe
What is the primary stain used in the Gram stain procedure?
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.
Describe
What is the mordant used in the Gram stain procedure?
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.
Describe
What is the decolorizer used in the Gram stain procedure.
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.
Describe
What is the counterstain used in the Gram stain procedure?
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.
When using the Gram stain, what color do gram-positive bacteria stain? Gram-negative bacteria?
- 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.
What are some reasons for gram-variable results?
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)
When gram staining, how would bacteria appear if under decolorized? Over decolorized?
- 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.
When is the gram stain performed?
It is routinely performed on certain specimen types, especially those collected from normally sterile sites.
Why is the gram stain important or helpful?
Gram stain morphology can guide species identification
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?
Gram positive organisms
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?
Gram negative organisms
What type of streak is used to innoculate urine cultures?
Quantitative isolation streak.
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)
What type of streaking is used to innoculate cultures other than urines and to set up transfer plates?
The general-purpose isolation streak
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.
Define enriched media.
A growth medium that contains added growth factors, such as blood, vitamins, and yeast extract.
Define differential media.
Media containing ingredients that allow visualization of metabolic differences between groups or species of bacteria.
Define selective media.
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.
5% sheep blood agar
(BAP)
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
Chocolate Agar
(CHOC)
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
MacConkey Agar
(MAC)
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
What are the optimum atomospheric conditions, temperature, and pH range for most pathogenic bacteria?
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
Colistin Nalidixic Acid Agar or Columbia CNA Agar
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
Phenylethyl Alcohol Agar
(PEA)
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
Thioglycolate Broth (THIO)
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
Minimum inhibitory concentration (MIC)
Minimum concentration of antibiotic required to INHIBIT the visible growth of the test organism
10.2 Define terms related to interpretation of susceptibility tests.
10.2.4 Minimum inhibitory concentration (MIC)
Minimum bactericidal concentration (MBC)
Minimum concentration of antibiotic required to KILL the test organism
(allows less than 0.1% of the original inoculum to survive)
Prophylaxis
Antimicrobial agents are administered to prevent infection
Treatment
Antimicrobial agents are administered to cure existing or suspected infection
Penicillin binding proteins (PBP)
Penicillin binds to a variety of proteins in the bacterial cell membrane and cell wall, called penicillin-binding proteins (PBPs).
Nitrocefin disks
Quick screen for beta-lactamase production
Nitrocefin is a cephalosporin
When you break open the beta lactam ring it changes color
Explain the gradient diffusion test (Epsilometer test “Etest”)
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
Explain the broth microdilution procedure (</=0.1mL broth volume).
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.
What agar plate is used for setting up AST?
Mueller-Hinton
Define antibiotic.
- 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.
Define zone of Inhibition.
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.
Define bactericidal
Antimicrobial that kills a microorganism.
Define bacteriostatic
Antimicrobial that inhibits bacterial growth but does not kill the bacteria.
Define intrinsic resistance.
- 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.
Define synergism.
Occurs when the antimicrobial activity of a combination of antimicrobial agents is greater than the activity of the individual agents alone.
What McFarland standard is used for antibiotic susceptbility testing? How many CFU/mL does it represent?
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)
Define acquired resistance.
- 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.
Define susceptible (sensitive) = S
An infection caused by the tested microorganism may be appropriately treated with the usually recommended dose of antibiotics.
Define intermediate = I
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)
Define resistant = R
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.
Explain what the Clinical and Laboratory Standards Institute (CLSI) is.
- Subcommittee of scientists and physicians
- Their goal is to establish standard conditions for testing methods based on laboratory investigations and assessment of clinical outcomes
List quantitative antibiotic susceptbility testing methods.
- 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.)
List qualitative antibiotic susceptiblity testing methods.
- 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
What volume of broth is used in broth macrodilution?
(>=1 ml broth volume) – Very rarely performed in clinical labs.
What volume of broth is used in broth microdilution?
0.1 mL
Explain agar dilution.
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
Describe the Vitek 2 automated system.
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.
Explain the principle of disk diffusion (Kirby-Bauer) testing.
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.
Summarize the disk diffusion (Kirby- Bauer) procedure.
- 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
- 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.
- Agar plates are incubated at 35°C within 15 minutes of set-up to prevent prediffuion of the antimicrobial agents into the agar
- Zone of complete inhibition is measured to the nearest millimeter
Summarize how disk diffusion (Kirby Bauer) testing is interpreted.
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.
Factors Affecting Size of Zone of Inhibition:
Inoculum density of organism
Larger zones with light inoculum and smaller zones with a heavy inoculum
Factors Affecting Size of Zone of Inhibition:
Timing of disk application
- 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
Factors Affecting Size of Zone of Inhibition:
Temperature of incubation
Larger zones are seen with temps less than 35degC
Factors Affecting Size of Zone of Inhibition:
Incubation time
Ideal 16-20 hours - less time does not give reliable results
Factors Affecting Size of Zone of Inhibition:
Depth of the agar medium
Standard size is 4mm.
Thin media yields excessively large inhibition zones and vice versa
Factors Affecting Size of Zone of Inhibition:
Proper spacing of the disks
Avoids overlapping of zones
Factors Affecting Size of Zone of Inhibition:
Potency of antibiotic disks
Deterioration in contents leads to reduced size (possibly from condensation)
Factors Affecting Size of Zone of Inhibition:
Composition of medium
Affects rate of growth, diffusion of antibiotics and activity of antibiotics
Factors Affecting Size of Zone of Inhibition:
Reading of zones
Subjective errors in determining the clear edge
Describe 4 mechanisms of intrinsic resistance.
- Biofilms (communities of microorganisms that are irreversibly attached to a solid surface)
- Impermeability (certain antibiotics unable to penetrate the cell wall of particular microorganisms)
- Efflux (function as transporter proteins for the extrusion of toxic substances and antimicrobial agents from the interior of the cell to the external environment.)
- Enzymatic Inactivation (produce enzymes that destroy the antimicrobial agents before they are able to reach their targets.)
Describe 4 mechanisms of acquired resistance.
- Efflux (some efflux pump genes have translocated to plasmids, which can be acquired by horizontal gene exchange.)
- 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
- Acquisition of New Targets (become resistant by acquiring cellular targets with reduced affinity for the antimicrobial agent.) example: mecA gene for MRSA
- 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
A minimum of how many patient identifiers are required?
2
It is best practice to collect how many sets of blood bottles from separate peripheral sites in 24 hours?
Atleast 2, but no more than 3.
What is the purpose of resin in blood bottles?
To chelate antibodies and mediators of the immune system
What is the anticoagulant used in blood bottles?
Sodium polyanethole sulfonate (SPS)
Describe how CSF is collected and processed?
- 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
When a swab is used for bacteriologic culture, it should be placed in transport media. Name two examples of bacteriologic transport media.
Amies and Stuart
What media is used for noninvasive urine cultures?
BAP and MAC inoculated with 0.001 mL of urine.
What are some reasons for rejecting specimens for culture?
- 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
Describe skin preparation for venipuncture
Disinfect skin with 70%-95% ethanol or isopropyl alcohol and then 2% tincture of iodine or 2% cholhexidine
Describe the recommended blood volume and ratio to culture medium for adult blood collections.
- 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.
Aerotolerant Anaerobes
Microorganisms that can survive in the presence of oxygen but do not use oxygen in metabolism.
Microaerophilic
Microorganisms that require a reduced level of oxygen (4-6%) to grow.
Capnophilic
Microorganisms that require an atmosphere enriched with carbon dioxide (5% to 10%).
