Microbio - Exam 3

chpt. 15, 12, 13, 22

phenotypic methods

the observation of the microscopic and macroscopic morphology, physiology, and biochemical properties of a microbe

immunologic methods

the analysis of a microbe using antibodies, or of a patient’s antibodies using prepackage antigens

genotypic methods

the analysis of a microbe’s DNA or RNA

point-of-care diagnosis

tests that can performed at the bedside so that patient care can continue without delay

viable nonculturable (VNC) microbes

microbes that cannot be grown in the lab and are currently being identified through studies such as the Human Microbiome Project

saliva and sputum

samples (2) taken from the mouth, nasopharynx, or throat (via swab)

aseptically via catheter or “clean catch” midstream

2 methods of urine collection from the bladder

swabbing or scraping

2 methods of collection from a skin lesion

swabbing or using a punch biopsy tool

2 methods of collection from a wound

sterile needle aspiration

method of collection for fluids such as blood, cerebrospinal fluid, and tissue fluid

1. nonnutritive maintenance media
2. buffering system
3. anaerobic environment

devices maintain collected samples in stable conditions through 3 methods

selective media

media type used in cases where the suspected pathogen is present in small numbers or is easily overgrown by normal biota

differential media

media type used to identify definitive characteristics such as reactions in blood and fermentation patterns

dichotomous key

a flowchart with two choices at each fork in the chart that is used for microbial identification

biochemical testing

a phenotypic testing method based on enzyme-mediated metabolic reactions that is visualized by a color change

antimicrobial susceptibility testing

a phenotypic testing method that incorporates a panel of commonly used antimicrobials and tests their susceptibility while identifying the pathogen

phage typing

a phenotypic testing method that relies on bacteriophages and is useful in identifying some bacteria (ex: *Salmonella*) and is used to trace strains in epidemics

1. culturing takes a minimum of 18-24 hours
2. many infectious conditions may be caused by nonculturable organisms (meaning the cultured organism is a bystander)

2 major drawbacks of phenotypic methods

serology

the branch of immunology that deals with *in vitro* diagnostic testing of serum

based on the principle that when a particular antigen is exposed to its specific antibody, it will fit perfectly

**false**, we test all kinds of body samples including urine, cerebrospinal fluid, whole tissues, and saliva

**T/F**: serology involves only the testing of sera

agglutination reactions

an immunologic testing method where the antigens are whole cells or organisms (ex: RBCs, bacteria, or viruses displaying surface antigens)

also used to determine blood compatibility and to diagnose rickettsial infections

precipiation reactions

an immunologic testing method where the antigen examined is a soluble molecule

immunochromatography

the use of devices with antigens or antibodies embedded in matrices that detect the presence of antigens or antibodies in a sample

ex: drugstore pregnancy tests

aka “lateral flow test”

antibody titers

an antigen-antibody reaction in liquid that reveals the concentration of antibodies in a sample

determined by serially diluting serum into test tubes/wells of a microtiter plate

used to diagnose autoimmune disorders (ex: lupus) and determine past disease exposure (ex: rubella)

titer

the highest dilution of serum that still produces agglutination

(the more a sample can be diluted and still react with antigen = greater antibody concentration = greater _____ )

serotyping

an antigen-antibody technique to identify, classify, and subgroup certain bacteria into categories (serotypes)

uses antisera against cell antigens (ex: capsule, flagellum, cell wall)

used to identify *Salmonella* species and strains

Western blot test

an immunologic testing method for separating and identifying antigen or antibody mixtures by 2D electrophoresis in polyacrylamide gel, followed by immune labeling

direct testing (of FAbs)

an immunofluorescent testing method in which an unknown specimen or antigen is fixed to a slide and exposed to a FAb solution of known composition

^^+ if antibody-antigen complexes form and remain bound to the sample^^

used to identify/locate microbial antigens on cell surfaces/in tissues and identify causative agents of syphilis, gonorrhea, meningitis, etc.

indirect testing (of FAbs)

an immunofluorescent testing method that recognizes the Fc region of antibodies, known antigens are added to the test serum and the binding of fluorescent antibody is visualized through fluorescence microscopy

enzyme-linked immunosorbent assay (ELISA) test

an immunologic testing method that uses an enzyme-linked indicator antibody to visualize antigen-antibody reactions

used to detect antibodies in diseases such as AIDS

indirect ELISA test

ELISA test that detects microbe-specific antibodies in patient sera

used for antibody screening of HIV, various rickettsial species, hepatitis A & C, and *Helicobacter*

direct ELISA test

ELISA test that detects antigens and may be performed using the sandwich test or utilizing computer chips

*in vivo* testing

an immunologic testing method where an antigen is introduced into a patient to elicit some sort of visible reaction

tuberculin reaction

a type of *in vivo* test in which PPD is injected superficially under the skin and the area of reaction is measured

aka *Mantoux test*

specificity and sensitivity

the most effective diagnostic tests have a high degree of these two characteristics

specificity

the property of a test to focus on only a certain antigen/antibody and not react with unrelated or distantly related ones

(low false-positive rate)

sensitivty

the property of a test which refers to the detection of even minute quantities of antigens/antibodies in a specimen

(low false-negative rate)

polymerase chain reaction (PCR)

a genotypic testing method that results in the production of numerous identical copies of DNA or RNA molecules within hours

can amplify the nucleic acid of bacteria, viruses, protozoa, and fungi

real-time PCR (qPCR)

a PCR method that uses fluorescent labeling and the fluorescence is measured in real time as the reaction is running

multiplex PCR

a PCR method that contains primers for multiple organisms instead of just one

transcription-mediated amplification

a genotypic testing method that does not require temperature changes and instead uses 2 enzymes (reverse transcriptase and RNA polymerase)

hybridization

a genotypic testing method that matches complementary strands of nucleic acid and is used to locate a specific site or type of nucleic acid

probes

small fragments of single-stranded DNA or RNA that are known to be complementary to the specific sequences of DNA or RNA being studied

fluorescent *in situ* hybridization (FISH)

a genotypic testing method that involves the application of fluorescently labeled probes to intact cells within a patient specimen or an environmental sample

microarrays

a genotypic testing method that uses “chips” containing gene sequences from thousands of different possible infectious agents

whole-genome sequencing

a genotypic testing method that is particularly useful for rapid analysis of outbreaks and drug-resistant organisms

pulsed-field gel electrophoresis (PFGE)

a genotypic testing method that involves the separation of DNA fragments that are too large for conventional gel electrophoresis methods

often used in acute outbreaks of foodborne, etc. infections

mass spectrometry

an additional diagnostic technology that has been utilized to determine the structure and composition of various chemical compounds and biological molecules to provide highly accurate microbial identification within minutes

first line of defense

any barrier (physical, chemical, or microbiota) that blocks invasion at the portal of entry or limits access to internal tissues that is general in action

second line of defense

“innate immunity.” the internal system of cells, fluids, and processes (ex: inflammation and phagocytosis) that acts rapidly at the local and systemic level once the first line of defense is overcome

third line of defense

“adaptive immunity,” highly specific system that is only acquired as each foreign substance is encountered by lymphocytes; provides long-term immunity

1. body **surveillance**
2. **recognition** of foreign material
3. **destruction** of foreign-demmed entities

roles of the immune system (3)

markers

any trait or factor of a cell/virus/molecule that makes it distinct and recognizable

aka *antigens*

self

natural markers of the body that are recognized by the immune system (no threat)

nonself

molecules recognized by the immune system as containing foreign markers indicating a need for immune response

pathogen-associated molecular patterns (PAMPs)

molecules on the surface of microbes that are not present on host cells that mark the microbes as foreign

pattern recognition receptors (PRRs)

molecules on the surface of host defense cells that recognize PAMPs

mononuclear phagocyte system (MPS)

collection of monocytes and macrophages that are scattered through the extracellular spaces that function to engulf and degrade foreign molecules

lymphatic system

the compartmentalized network of cells/vessels/organs that serve as sites for the development of immune cells and reactions

includes: spleen, thymus, lymph nodes, etc.

lymphatic, circulatory

body systems that participate in immunity (2)

lymphatic fluid

plasma-like liquid formed when certain blood components move out of the blood vessels into extracellular spaces and diffuse & migrate into the lymphatic capillaries

transports WBCs, fats, cellular debris, etc.

lymphatic vessels

structures similar in appearance to thin-walled veins of the circulatory system through which lymph flows from the extremities to the heart and moves by the contraction of skeletal muscles

primary lymphatic organs

organs that are the sites of immune cell birth & maturation

includes: red bone marrow, thymus

secondary lymphatic organs

organs that are where immune cells are activated, reside, or carry out their functions

includes: SALT, MALT, GALT, lymph nodes, spleen, tonsils, Peyer’s patches

red bone marrow

the site of blood cell production, located in flat bones and the ends of long bones

thymus

butterfly-shaped organ near the tip of the sternum that is the site of T cell maturation

lymph nodes

bean-shaped organs that are clustered along lymphatic channels and large blood vessels of the thoracic & abdominal cavities that filter out materials in the lymph and provide cells for immune reactions

cortex

the outer rim of lymph nodes

paracortical area

the inner area of lymph nodes that contains T lymphocytes

medullary sinus

the inner area of lymph nodes that contains B lymphocytes and macrophages

spleen

the lymphoid organ in the upper left quadrant of the abdominal cavity that filters pathogens from the blood

skin-associated lymphoid tissue (SALT)

contains B & T cells and underlies some skin surfaces

mucosa-associated lymphoid tissue (MALT)

contains B & T cells and underlies most mucosal surfaces

gut-associated lymphoid tissue (GALT)

a collection of lymphoid tissue in the GI tract

includes: appendix, lacteals, Peyer’s patches

tonsils

a ring of lymphoid tissue in the pharynx that provides an active source of lymphocytes

Peyer’s patches

compact aggregations of lymphocytes in the ileum of the small intestine

whole blood

liquid connective tissue consisting of RBCs and WBCs suspended in plasma

hematopoiesis

the production of blood cells

differentiation

the process by which stem cells change by being subjected to a variety of growth factors and hormones

leukocytes

WBCs, primarily infection-fighting

granulocytes

leukocytes that have dark staining granules

includes: neutrophils, eosinophils, basophils, mast cells

agranulocytes

leukocytes that do __not__ have granules but have large nuclei

includes: B & T cells, macrophages, monocytes

cytokine

a regulatory chemical, released by the immune system cells, that serves as a signal between different cells

pro-inflammatory cytokine

cytokine category that encourages adaptive and innate immune responses

anti-inflammatory cytokine

cytokine category that discourages adaptive and innate immune responses

vasodilators/vasoconstrictors

cytokine categories (2) that change the diameter of blood vessels or their permeability

growth factor

cytokine category that regulates lymphocyte growth or activation

T cell

lymphocyte with cell-mediated immunity that assist B cells

B cell

antigen-presenting lymphocyte that differentiates into plasma cells and releases antibody

respiratory, urinary, digestive, integumentary

body systems (4) that participate in the first line of defense

phagocytosis, fever, inflammation, antimicrobial products

the major categories (4) of the second line of defense

neutrophils

general purpose phagocytes that are found in high numbers in bacterial infections

macrophages

WBC derived from monocytes that leaves circulation and goes to the tissues; important in nonspecific phagocytosis, the regulation/stimulation/cleanup after immune responses

chemotaxis

1st step of phagocytosis

phagocytes migrate to the region of inflammation due to attraction by a gradient of stimulant products from parasite and host tissue at the site of injury

adhesion

2nd step of phagocytosis

phagocytes use their PRRs to recognize PAMPs, causing the 2 to stick together

engulfment

3rd step of phagocytosis

phagocytes make contact with their “prey” and extend pseudopods that enclose the cells/particles in a pocket

phagosome formation

4th step of phagocytosis

a vacuole (phagosome) internalizes the cells/particles and secretes more cytokines to further amplify the innate response

phagolysosome formation and killing

5th step of phagocytosis

lysosomes migrate to the phagosome and fuse with it to create a phagolysosome

granules with antimicrobial chemicals are released into the phagolysosome to poison & dismantle the material inside

destruction

6th step of phagocytosis

involves 2 systems of chemicals that act on the microbes in the phagolysosome

* oxygen-dependent system (reactive oxygen products)
* enzymes

elimination

7th step of phagocytosis

small bits of undigestible debris are released via exocytosis