Chapter 10: Identification Of Microorganisms Flashcards
Taxonomy
Science of classifying organisms
Differential staining
Gram stain and acid-fast stain
Not useful for wall-less bacteria
Classification and identification
Microbe identification: biochemical tests
identify microorganisms by detecting the presence or absence of enzymes. These tests are quick (enzymatic activity is used to differentiate bacteria)
-catalase test
-coagulase test
-oxidase test
Tests that determine carb fermentation include enterotube
(Used in identification not classification)
Serology
Study of antigen and antibody reactions
-for identification not classification
-microorganisms are antigenic and cause antibody production by host
-rapid testing
-combine known antiserum (solution where antibodies can be found) + unknown bacterium
-slide agglutination tests
-clumping =+ test
-Salmonella serotyping
-serogroups of Salmonella: A, B, C, etc.
-cell wall antigen
-gastroenteritisi
-antibody titer
-fluorescent antibody (FA) techniques (immune fluorescence)
-ELISA
-Western blot
Antibody
Defense proteins made in response to a specific antigen
Antigen
Foreign microbial cell
Rapid identification: enterotube 2
-24-48hrs at 37 degrees Celsius
1) 1 tube containing media for 15 biochemical tests is inoculated w/ an unknown enteric bacterium
2)after incubation, the tube is observed for results
3) the valve for each (+) test is circled, and the #’s from each group for tests are added to give code #
4) comparing the resultant code w/ a computerized listing shows that the organism in the tube is Citrobacter freundi
Mass spectrophotometry
-detects cellular proteins
-highly sensitive analytical technique that measures the mass-to-charge ratio of ionized particles, like atoms and molecules
Agglutination tests
-antigen on a cell combine w/ antibodies
-ex. Salmonella serotyping
-reaction between antibodies and bacterial antigens that causes bacterial cells to clump together
Antibody titer
The concentration of antibodies against a particular antigen
-ex. Immune status of a pt- determine if booster is needed or follow the immune response to infection or follow effectiveness of treatment
Fluorescent antibody techniques (direct)
Detect antigens
-ex. Group of Streptococcu from pt’s throat + fluorescent dye- labeled antibodies to group A Streptoccoci ➡️ fluorescent Streptococci
-antigen and antibodies combine to form a complex
-false (+) and false (-) possible- secondary confirmatory test
-24hrs
-penicillin right away
Fluorescent antibody techniques (indirect)
Detect antibodies
-ex. Treponema pallidum: T. Pallidum from lab stock + specific antibodies in serum of pt (state of public health lab; heat fix) ➡️ antibodies binding to T. Pallidium (specific) + fluorescent dye- labeled antibodies-human immune serum globulin (will react w/ an immunoglobulin)➡️ fluorescent spirochetes
ELISA test- enzyme linked immunosorbent assay
-known antibodies are placed in wells (or membrane) and unknown bacteria is added
-ex. Pregnancy test, COVID test
Direct ELISA- (screening)
Antibody on membrane
Detects antigen
1) antibody is absorbed to we’ll
2) pt sample is added; complementary antigen binds to antibody
3) enzyme-linked antibody specific for test antigen is added and bind to antigen; forming sandwich (made in animal- antibody)
-buffer washes of false (+) or false (-)
4) enzyme’s substrate is added and reaction produces a product that causes a visible color change- (+) test
Indirect ELISA
Detects antibodies made in response to specific antigens
Western Blot
-separate proteins in serum by gel electrophoresis (motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. Electrophoresis is used in labs to separate (rem proteins from) macromolecules based on their charges)/ seperates proteins in a polycrylamide gel based upon their charge and size
-based on size and charge
-(indirect) 1st ELISA test (+) or equivocal result
-2nd west blot
-transfer proteins to filter by blotting (via blotting paper)
-Borrelia burgdorferi
-wash pt’s serum over filter
-antibodies present?
-wash anti-human (antibody) serum linked to enzyme over filter
-wash again
-add substrate
Nucleic acid hybridization
DNA probe used to identify bacteria
-for identification and classification
1) Salmonella DNA fragments is cloned in E. coli (specific piece of genetic material from a Salmonella bacteria has been inserted into the DNA of an Escherichia coli (E. coli) bacteria, allowing the E. coli to replicate and express that specific Salmonella DNA fragment in large quantities, essentially making copies of the Salmonella DNA within the E. coli cell)
2) cloned DNA fragments are marked w/ fluorescent dye and separated into single strands, forming DNA probes
3) unknown bacteria are collected on a filter
4) cells are later and the DNA is released
-centrifuge
5) DNA is separated into single strands
6) DNA probes are added to the DNA from the unknown bacteria
7) DNA probes hydribize w/ (complementary DNA) Salmonella DNA from the sample. Then the excess probes is washed off. Flourescence indicated presence of Salmonella
Polymerase chain reaction
-for classification and identification
1) incubate target DNA at 94 degrees Celsius for 1 min to separate the strands
2) add primers, nucleotides (deoxynucleotides), and DNA polymerase
3) primers attach to single-stranded DNA during incubation at 60 degrees Celsius
4) incubate at 72 degrees Celsius for 1 min; during this time, 2 copies of the target DNA are formed
5) repeat the cycle of heating and cooling to make 2 or more copies of the target DNA
Ex. MRSA (nares), chlamydia, measles, herpes simplex virus
Nucleic acid amplification tests (NAATs)
Use PCR to amplify DNA of an unknown microorganism that cannot be cultured
-can produce microbial DNA to be analyzed by gel electriphoresis
-NAATs include PCR (polymerase chain reaction)
Methods of classification
-differential staining
-nucleus acid hybridization
-NAATs (PCR)
-DNA base composition
-rRNA sequencing
Eukarya
Single celled (yeast, algae, and amoebas) or multicellular (fungi, plants, and animals
Contain membrane-bound sub compartments that separate specific functions, particularly the nucleus
Division includes microscopic organisms previously belonging to the Monera kingdom
Antibiotic sensitivity: No
No cell wall
Bacteria
Simple-celled
Prokaryotic (no nucleus or organelles) organisms
Contain peptidoglycan (carb and protein complex) in cell walls
3micrometers
Reproduce asexually via binary fission
Shape: coccus, bacillus, or spiral-shape
-important in dx
Genetic info: DNA
Lipid bilayer: present
Ribosome: small, unique
RNA polymerase: simple, 5 subunits
Grow w/o outside help: yes
Max # of cells: 1
First cells on earth
Contain over 50 different phyla (major groups)
Most abundant form of life- no matter bacteria growing in/on humans than they have human cells
Antibiotic sensitivity: Yes
Nutrition source: organic chemical (derived from dead or living organisms); photosynthesis; inorganic substances
Motile: flagella
Archae
Most common in extreme environments
-combine feature of prokaryotes and eukaryotes
-extremophiles; some will grow when other organisms cannot
-halophiles: salt loving
-sulfophiles: sulfur loving
-thermotropes: heat loving
-lithotrophs: rock loving
-methanophiles: methane loving
Antibiotic sensitivity: No
Does not cause disease in humans
Classification
Placing organisms in groups (taxonomic categories) related to species
-list of characteristics of known organisms
Identification
Matching characteristics of an “unknown” organism to lists of unknown organisms
-determining the specific species or strain of a microbe by analyzing its phenotypic or genotypic traits
Explain the purpose of Bergey’s manual
to identify and classify bacteria
-provided phylogenetic (evolutionary info) and identification info on bacteria
-classifies and identifies based on:
-cell morphology
-differential staining (gram-stain, acid-fast)
-O2 requirements
-motility
-nutrition
-metabolic properties
Immunofluorenscence
technique used for bacterial identification by utilizing fluorescently labeled antibodies that bind to specific bacterial antigens, allowing for visualization of the bacteria under a fluorescence microscope, essentially “lighting up” the targeted bacteria within a sample; this method is particularly useful for rapid identification of certain bacterial pathogens.
Enzyme-linked immunosorbent assays (ELISA)
laboratory technique that leverages specific antibodies to detect the presence of certain bacterial antigens in a sample, allowing for the identification of specific bacterial species by measuring the binding reaction between the antibody and the bacterial antigen, often visualized through a color change reaction on a plate.
Western blotting
technique used to identify specific proteins within a sample, making it useful for confirming the presence of a specific bacterial protein, thus aiding in bacterial identification, but it is not the primary method for identifying bacteria as it only detects proteins, not the whole bacteria itself; other methods like Gram staining or biochemical tests are typically used for initial bacterial identification.
Morphology
-used in identification (not classification)
-structural characteristics include shape or the presence of endospores, flagella
-simple stains tell little about evolutionary relationships
Molecular methods
1) nucleic acid hybridization
2) polymerase chain reaction (PCR)
Methods used primarily for classification
1) differential staining
2) DNA base composition
-the percentage of G-C pairs in the nucleic acid of cells can be used in the classification of organisms
3) single strands of DNA, or of DNA and RNA, from related organisms will hydrogen bond to form a 2x-stranded molecule; this bonding is called Nucleic Acid Hybridization; DNA probes for rapid identification of bacteria
4) Ribosomal RNA sequencing
5) Polymerase chain reaction (PCR) detects small amounts of microbial DNA in organism w/o culturing. It makes multiple copies of a DNA template
