Lecture | Bacterial Identification Flashcards
(178 cards)
1
Q
Microscopy, staining techniques, biochemical test, imumunodiagnosis, molecular diagnosis is for
A
Bacterial identification
2
Q
Used for the detection of microorganisms directly in the clinical specimen and for characterization of organisms grown in culture
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Microscopy
3
Q
Defined as the use of microscope to manify objects
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Microscopy
4
Q
visible light is passed through the specimen and then through a series of lenses that bend the lights
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Bright-field microscopy or Light microscopy
5
Q
Microscopic technique for visual inspection at a time of smear (fixed smear), culture preparation, microscopic examination of gram stain preparation
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Bright-field microscopy or light microscopy
6
Q
Does not use fixed smear and allows to view organism in wet preparation or mark
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Phase-contrast microscopy
7
Q
Uses beam of light passing through the specimen
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Phase-contrast microscopy
8
Q
Deflected light is called
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Refractive index
9
Q
Greater refractive index means
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Decrease light intensity
10
Q
Staining is not part of phase-contrast, this allow identification of viable microorganisms such as
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Fungal identification
11
Q
Fluorescent microscopy uses a dye known as
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Fluorochrome
12
Q
Darkfield microscopy is usually employed for the diagnosis of
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Spirochetes
13
Q
Phase-contrast and dark-field has a similarity of not using any stain or dye to achieve contrast, instead they
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Alter the microscopic technique
14
Q
2 types of Electron microscopy
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SEM & TEM
15
Q
Creates image by detecting reflected or “knock-off” electrons
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Scanning Electron Microscopy
16
Q
Creates 3D information of sample surface and its composition
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SEM
17
Q
Uses transmitted electron
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Transmitted electron Microscopy
18
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Detects the transmitted electron to create an image, offering valuable information in the INNER structure of the sample and morphology inside
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TEM
19
Q
Fixation makes sure that—
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Material would cling to the surface of the microscope slide
20
Q
2 process of fixation
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- Heat
- 95% methanol
21
Q
Preparation : Specimen or Organism Type
(For Gross Examination)
WET PREPARATION : ?
A
Parasites and material >1 mm in size
22
Q
Preparation : Specimen or Organism Type
(For Microscopic Examination)
WET PREPARATION (direct/sedimented) : ?
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Fluids or semi fluid
23
Q
Preparation : Specimen or Organism Type
(For Microscopic Examination)
CYTOCENTRIFUGED (direct/presedimented) : ?
A
Clear or slightly turbid fluid
24
Q
Preparation : Specimen or Organism Type
(For Microscopic Examination)
Smear : ?
A
Clear or slightly turbid fluid
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Preparation : Specimen or Organism Type
(For Microscopic Examination)
? : pus/fluid, tissue homogenate, swab rinse
Drop
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Preparation : Specimen or Organism Type
(For Microscopic Examination)
Pellet : ?
Blood culture and dilute specimen
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Preparation : Specimen or Organism Type
(For Microscopic Examination)
? : swabbed material
Rolled
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Preparation : Specimen or Organism Type
(For Microscopic Examination)
Imprint (touch preparation) : ?
Tissue
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Directed toward coloring the forms and shapes present. Ex: methylene blue
Simple stain
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Directed toward coloring specific components of the elements present. Ex: Gram’s Stain; Acid-Fast Stain
Differential Stain
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Performed to visualize selected tissue element, entities, and microorganisms
Special stains
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Special stain for flagella
Leifson Stain
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Special stain for endospore
Schaeffer-Fulton Stain
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Special stain that uses Malachite green
Schaeffer-Fulton Stain
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A type of stain wherein it’s the background that is stained
Negative stain
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Examples of negative stains
India ink ; Nigrosin
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Use to visualize/examine the presence of Cryptococcus neoformans on Cerebrospinal specimen
India ink
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Principal stain for microscopic examination of bacteria
Gram stain
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Gram stain principle is based on the —?— of the group of microorganisms
Cell wall composition
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Primary stain of Gram Stain
Crystal violet
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Mordant of Gram stain
Gram’s iodine
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Decolorizer of Gram stain
Alcohol/acetone
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Counterstain of gram stain
Safranin
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Gram+ that has an acid that contributes to the ability of gram+ organism to resist alcohol decolorization
Teichoic acid
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Gram+ contains highly cross-linked layer that retains primary dye
Peptidoglycan
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Where peptidoglycan is loosely distributed
Gram-
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Stain utilized for the detection of mycobacterium
Acid-fast stain
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A component of the cell that gives the cell wall the ability to resist decolorization
Mycolic acid
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Acid-fast stain methods
1. Ziehl Neelsen (Hot method)
2. Kinyoun (Cold Method)
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Primary stain of acid-fast
Carbolfuchsin
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Decolorizer of acid-fast stain
1. Acid Alcohol (3% HCL + 95% Ethanol)
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Counter stain of acid-fast
Methylene blue
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Mordant of acid-fast
Phenol ( increased concentration in Kinyoun method)
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A mixture of phenol and the dye basic fuchsin
Carbolfuchsin
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Alternative for acid alcohol
20% Sulfuric Acid
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2 types of fluorochrome
1. Acridine orange
2. Auramine Rhodamine
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A fluorochrome that confirms the presence of bacteria in blood cultures
Acridine orange
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Determination: bacteria will give off bright orange fluorescence
Acridine orange
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Stain that is also used for detection of cell wall-deficient bacteria that are incapable of retaining the dyes used in gram stain
Acridine orange
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Used for the detection of our mycobacteria
Auramine Rhodamine
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Has a very high affinity with the waxing mycolic acid found in the cell wall of mycobacteria
Auramine Rhodamine
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Determination: it would nonspcifically bind to nearly all microbacteria, giving off a bright yellow appearance or orange against a greenish background
Auramine Rhodamine
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Acridine orange detects presence of cell wall deficient bacteria such as
Mycoplasma and ureaplasma
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Enhance detection of micro-bacteria directly from patient specimen. Has higher specificity and sensitivity
Auramine rhodamine
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Colonial morphology is observable after how many hours
18-24 hours of incubation
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Inoculation of the clinical specimen onto laboratory media
Primary plating
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Initial mid culture media we use to plate
Primary media
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Term us to introduce specimen unto the laboratory media
Inoculate
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Interpretation of primary culture
Plate reading
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Comparative examination of the colony morphology
Plate reading
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Media that contain specific nutrients required
Enrichment media
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Used to enhance the growth of organism present in low numbers
Enrichment broth
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Contain nutrient that support growth of most nonfastidious organism
Nutritive media
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Contain nutrients that support growth of most nonfastidious organism
Nutritive media
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Contain 1 or more agents that are inhibitory to all organisms except those “selected” by the specific growth conditiopn or chemical
Selective media
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Based on the ability of bacteria to utilize a substrate that produces a change in ph, detected by a change in the color of a ph indicator
Differential media
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Culture media that could both be differential and selective
Combination media
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Examples of Fastidious organisms that are hard to grow in vitro
Francisella spp. and Brucella spp.
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Break down of RBC
hemolysis
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Type of hemolysis that is partial lysing of rbc in SBA plate around and under the colony; green discoloration
Alpha hemolysis
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Complete clearing of rbc in SBA
Beta hemolysis
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No hemolytic pattern
Gama hemolysis
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Colonies are describe as large, medium, small, or pinpoint
Size
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Describes as smooth, filamentous, rough or rhizoid, or irregular
Form of margin
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Hazy blanket of growth on the surface that extends well beyond the streak lines
Swarming
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Determined by tilting the culture plate and looking at the side of colony
Elevation
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It may be raised, convexed, flat, umbonate,umbilicate
Elevation
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Can be transparent, translucent, opaque
Density
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Determined by touching the colony with a sterile loop. It may be splinters, butyrous, dry or waxy.
Consistency
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Color of colonies may be
White, gray, yellow or buff
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An inherent characteristic of a specific organism confined generally to the colony
Pigmentation
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Enzyme-based test is also called
Single enzyme-based test
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Biochemical test to determine which subsequent identification step should be followed
Enzyme-base test
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Streptococcus can be differentiated from staphylococcus through catalase wherein staphylococcus species are
Catalase positive
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Provide by vital information and is commonly used in schemes for gram-positive identification
Catalyst test
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Catalyzes the release of water and oxygen from hydrogen peroxide
Catalase
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It is the positive result in a catalyst test
Rapid production of bubbles
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Rapid production of bubbles is termed as
Effervescence
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Do not use a bacterial inoculum that is collected from SBA plate because it gives off what type of result
False positive
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Participates in electron transport and in nitrate metabolic pathways of gram negative
Cytochrome Oxidase
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Testing for the presence of oxidase uses a reagent such as
1% tetramethyl-p-phenylenediamine dihydrochloride
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Simpler method/alternative way of performing oxidase test
Rubbing the bacterial colony into a filter paper with reagent
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Rubbing the bacterial colony into a filter paper is a method called
Kovacs method
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Not using an iron containing wire during kovacs method will obtain a result of
False positive
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Do not interpret result during kovacs method after 10 seconds. True or false
True
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End point of carbohydrate fertilization
Production of acid-byproducts
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To detect the the presence of acid by product is through
Change in pH indicator
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When using this pH will turn yellow if acid and remain blue if alkaline
Bromocresol purple and bromothymol blue
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Medium use for oxidative fermentation test is known as o-f medium or also called as
Hugh and Leifson O/F basal medium
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Contain low concentration of peptone and a single carbohydrate substrate such as glucose
O-F medium
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When o/f test are performed 2 tubes of Hugh-Leifson OFBM are inoculated. test tube 1 is ? And test tube 2 is?
Test tube 1 - overlaid with sterile mineral oi
Test tube 2 - without mineral oil overlay
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One tube is overlaid with mineral sterile oil to create
Anaerobic environment (close)
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Test tube is without mineral oil overlay is left
Open or aerobic
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Oxidative requires
Oxygen
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Fermentative requires
Do not require oxygen
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These are test for the presence of Metabolic pathways
1. Carbohydrate oxidation and fermentation
2. Amino acid degradation
3. Single substate
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Oxidase is in color
Purple
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What are the single source nutrition
Citrate, malonate, or acetate
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It determines if organisms can gown in the presence of spa single nutrient or carbon source single substrate
Single substrate utilization
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Citrate test or single substrate utilization detects
Alkaline pH
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Citrate test utilize an agar called
Simmons citrate agar
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Simmons citrate agar contains bromthymol blue. This will turn blue at a pH of
Above 7.6
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The metabolic activity happens and produces a/an
Alkaline pH
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Involves the ability of a bacterial isolate to grow in the presence of 1 or more inhibitory substances
Establishing inhibitor profiles
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A test include Growth in the presence of various NaCl concentrations is for
Identification of enterococci spp. and vibrio spp.
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A test in susceptibility to optochin and solubility in bile is for
Identification of streptococcus pneumoniae
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Test include ability to hydrolyze esculin in the presence of bile
Identification of enterococci spp. in combination with NaCl
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Test include ethanol survival
Identification of Bacillus spp.
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Serotyping
Using sera to identify and differentiate bacteria
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Immunoassay includes
Precipitation assay, agglutination assay, immunofluorescent assay, fluorescein isothiocyanate, enzyme immunoassays
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Involves diffusion of soluble antigen and antibodies
Precipitation assay
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Detecting antigen by means of agglutination with latex bead
Agglutination assay
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Latex agglutination for salmonella typhi
Widal test
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Latex agglutination for treponema pallidum
Hemaaglutination
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Double immunodiffusion is for
Fungal pathogens
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Specific monoclonal or polyclonal antibodies are conjugated with fluorochrome
Immunofluorescent assay
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Labeled antibodies are called ? Because antibody is linked to a label
Conjugate
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Commonly used fluorochrome
Fluorescein isothiocyanate
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Fluorescein isothiocyanate emits
Bright apple-green fluorescence
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Considered more sensitive than DFA
IFA
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Test for T. Pallidum for detecting syphilis
FTA-ABS
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SPIA is used in the detection of
Lyme disease
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Cassette-based membrane-bound ELISA is used to
Test for a single serum
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Cassette-based membrane-bound ELISA is use to test for a single serum, now we refer to it as
Rapid test or rapid kits
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Cassette-based membrane-bound ELISA is rapid because it take how many minutes to get a result
Within 10 minutes
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2 homologous nucleic acid strands that are complementary form a
Double stranded molecule or duplex or hybrid
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Hybridization amplification methods
PCR and amplify a single copy of a nucleic acid target
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Amplification method involves 3 process
Denaturation, annealing, & extension
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1st step in PCR that involves heating at 94 degrees celsius
Denaturation
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In denaturation the double stranded DNA would become
A single strand
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One strand from the duplex formation carries a —?— that consists of a single reporter labeled nucleic acid molecule that is complementary to a nucleic acid target of the suspected pathogen
Probe
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After denaturation is ? Which involves the use of primers
Annealing
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Short single stranded sequence of nucleic acid; oligonucleotide
Primers
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Primers are how many nucleotides long
220 to 30 nucleotides long
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Unlike probes, primers does not have a
Reporter molecule
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Melting temperature of the primer in annealing
50 to 58 degrees celsius
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Addition of the amino acids r nucleic acids into the sequence
Extension
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Added nucleotides to 3 prime terminals of each primer and extended id mediated by
Taq polymerase
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Taq polymerase is obtaine or isolated from
Thermus aquaticus
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Extension happens at a temperature of
72 degrees celsius
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Specific PCR amplification product containing the target nucleic acid
Amplicon
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After amplification, subject PCR mixture to a
Gel electrophoresis
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In gel electrophoresis, a dye is used with the aid of
Ethidium bromide
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Determine genetic relatedness
Genetic fingerprinting
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Based on mutations that accumulate in biological organisms over time
Genetic fingerprinting
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Compare local strains to determine whether local outbreak is caused by a single strain or multiple strain
Strain typing
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It is also used to compare local isolates with worldwide isolates which can show long term spread of strain/s (clonality)
Strain typing
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2 types of genetic fingerprinting
Non-amplified and amplified typin
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What is under non-amplified typing that is one of the 1st method used to type strains of bacteria
Plasmid profile analysis
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Determining the order of nucleotides in fragments of DNA
Sequencing
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Procedure to identify the microbes from clinical sample and confirm identification of bacterial isolate by determining the order of nucleotides in a fragment of DNA
Sequencing
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Grouping at the micron level of DNA molecules attached to a solid support
DNA microarrays
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Gives investigators the potential to evaluate gene expression from an entire organism
DNA microarrays
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Also called as DNA chip or gene chip
DNA microarrays
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Study of proteins on cellular level
Proteomic
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Proteomic is used to determine protein expression in
Disease conditions
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Matrix-assisted laser desorption-ionization time-of-flight
MALDI-TOF mass Spectrometry
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Technology used for RAPIDLY identifying microorganisms, including fungi
MALDI-TOF Mass Spectrometry
