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