Intro to Clinicals

true pathogen

a microorganisms capable of causing an infectious disease in a healthy person

opportunistic pathogen

organisms (usually normal flora) that cause an infection when their environment is changed or the host immune system is compromised

basic guidlines for specimen collection

collect early in the infection to get the best chance of isolation

use proper technique to minimize contamination from normal flora

use proper container that will maintain viability

label the specimen with pt. info and anatomical site

transport within 2 hrs and store in proper environment to prevent degradation

why do we inculate specimens onto the media in a specific order (ex: do you inculate media or prepare a slide first if you only have one swab)

if only one swab s avaialble you would inoculate the media first and then use the swab on a sldie because a slide is not sterile

proper isolation technique

first sterilize the loop then inculate the media onto the plate and make the first streak, then sterilize again and make the second streak, then repeat for the last streak

aerobe

grow in ambienet air or with oxygen

facultative aerobes

can grow in the prescence or abscence of oxygen

strict/obligate anaerobes

can not grow in the prescence of oxygen

what temperature are most routine bacterial cultures incubated at

between 35-37 degrees celcius

why do we have a CO2 incubator and an ambient air incubator

some bacteria need different environments in order to grow, including environments with more CO2 and some with more oxygen

what is hemolysis

the breakdown/lysing of RBCs

what is the difference between beta, alpha, and gamma hemolysis

alpha: turns a green/brown color (partial hemolysis)

beta: turns clear (hemolysis)

gamma: no change in color

how do you write an organisms name using binomal nomeclature

you’ll need the genus and species name and the genus name will be capitalized while and species is lowercase

ex: Streptococcus pyogenes

cell wall of gram positive bacteria

thick peptidoglycan layer

has teichoic and lipoteichoic acids

cell wall of gram negative bacteria

thin peptidoglycan layer

has an additional outer layer composed of proteins, phospholipids, and lipopolysaccharides

there is a space between the outer and inner memebrane called the periplasmic space that obtains nutrients and degrades large molecules

what is a capsule

a polysacchardie layer that surrounds the bacteria and acts as a virulence factor that helps the bacteria cause disease

princple of the gram stain

1. cyrstal violet penetrates the cell wall and stains all bacteria a blue-purple color

2. iodine causes large complexes to forms with the crystal violet

3. the decolorizer damages the outer memebrane of gram-negative organisms leaving it’s thin peptidoglycan layer exposed causing the crystal violet to be washed away while the crystal violet in gram-positive organsims doesn’t due to it’s thick peptidoglycan layer

4. safranin stains the gram-negative organsims pink and has ne effect on gram-positive organisms

list the steps of a gram stain

1. crystal violet

2. iodine (mordent)

3. alcohol wash (decolorizer)

4. safranin

if you forget to apply the iodine during your gram stain, what color would the gram positive organism appear

pink-red (due to the large complexes not forming, causing the crystal violet stain to wash out when decolorizer is added)

if you walked away and left your decolorizer on for 5 minutes, what color would the gram-positive organisms appear

pink-red (the decolorizer would remove the crystal violet stain if left on for too long)

if you forgot to apply the decolorizer, what color would gram-negative organisms appear

blue-purple (due to the crystal violet stain not being removed before the safranin is added)

what is the final electron acceptor in aerobic respiration

oxygen

what is fermentation

when bacteria user other sugars such as carbohydrates (not glucose) to produce energy to grow

do all bacteria metabolize glucose in the same way

no, depending on the abscence or prescence of oxygen metabolism is done differently (this is why it can be helpful for identification)

what are standard precautions

preventative measures applied to all patients that are designed to reduce the risk of infection in a healthcare setting

treating all patient speciman as if they are transmittable infections

PPE

barrier protections we use to protect ourselves from pathogens

we choose to use these to protect ourselves

engineering controls

products made to help protect us from pathogens, for example, biosafety levels and biological safety cabinets to protect against aerosols

primary disinfectant ingrediant in bleach

sodium hypochlorite

what is the most common biological safety cabinet class used in clinical micro lab

class IIA

generally, what temperature, pressure, and time is used to autoclave

121 degrees celcius, 15 psi for 1 hour

what is the difference between sterilizer, disinfectant, and antiseptic

sterilizer: the physical or chemical process that kills ALL micororganisms, including endospores

disinfectant: used on non-living surfaces to destroy microbes but not endospores

antiseptic: used on the skin to destroy microbes but not endospores

nosocomial infection

a hospital acquired infection

endogenous infection

a microbe from the host’s normal flora causes an infection

exogenous infection

a microbe from outside of the hosts environment enters the host and causes an infection

what are some ways that bacteria avoid phagocytosis

capsules prevent recognition and binding of complement proteins

cell wall proteins interfere with phagocytosis

toxins released kill phagocytes

exotoxins are produced by

gram negative and positive bacteria

the toxin from exotoxins are found

inside of the bacterial cell and then excreted upon lysis of the cell

exotoxins need a _________ dose to be fatal

low

endotoxins are produced by

gram-negative bacteria

what is the actual toxic part of an endotoxin

the lipopolysaccharide

what does the LPS cause in endotoxins

fever, hypotension, septic shock, activates complement, and initates coagulation

exotoxins are species ___________

specific

endotoxins are species ____________

non-specific

endotoxins require a __________ dose to be fatal

higher

components if the innate immune system

physical and chemical barriers (skin, mucousal secretions, pH, enzymes)

normal flora

phagocytosis (not effective against intracellular pathogens)

inflammation (from cytokines)

natural killer cells (recognize infected cells and pop them, effective against intracellular pathogens)

what occurs to bacteria in the humoral immune response

B-cells bind to bacterial antigen, they engulf the bacteria and display the bacterias antigen on their surface via MHC-II

helper T-cells will then bind to the antigen presented on the B-cell which causes it to release cytokines leading to the proliferationa nd activation of B-cells that differentiate into plasma cells

IgG charcteristics

small so it can cross the placenta barrier

appear after IgM in primary exposure

predominant in secondary exposures

IgM characteristics

large so it can’t cross the placenta barrier

appears first in initial exposures

minor amounts in secondary exposures

which antibody is predominant in primary exposure

IgM

which antibody is predominant in secondary exposure

IgG

which type of microbes is the cell mediated adaptive immune response particularly important

intracellular bacterial and viral infections

paired sera antibody titer

a test used to measure the concentration of specific antibodies in the blood at two different times (the acute phase (when disease is first suspected) and the convalescent phase (2 weeks after acute phase))

when would it be okay to test a single serum specimen for antibody titer

when assessing immune status (vaccinations)

testing for IgM in congenital infections (newborns)

for infections that are rare

precipitation test

when soluble antigen and antibody diffuse through agar, combine, and a visible precipiate forms

principle of an agglutination test

the clumping of particles coated with antigen-antibody complexes

1st stage: antigen combines with antibody

2nd stage: clumping occurs and is visualized

direct fluorescent antibody test

patient antigen is applied to a slide and a fluorescent-labeled antibody attaches

indirect fluorescent anitbody test

manufactured antigen is applied to a slide that patient antibody attached to and then a fluorescent labeled antibody attaches to the patient antibody

princples of lateral flow enzyme immunoassay test

a sample that contains the target analyte is applied to a test strip found in a cartridge, the sample moves through the test strip via capillary action, the sample interacts with the molecules dried onto the test strip, if the target analyte is present it will bind to the dried molecules and form a complex (or a strip of color you can see) this then continues onto the test line indicating if the target analyte is present

what does MALDI-TOF stand for

matrix-assisted laser desorption/ionization time of flight

how does MALDI_TOF identify microbes

a small amount of sample is mixed with a matrix (chemical) which is then placed on a metal plate that’s hit with a laser

the energy from the laser causes the sample to ionize (turn into charged particles)

the charged particles are then accelerated in an electric field and the time it takes them to reach the detector is measured (time of flight)

time of flight is used to create a mass spectrum (unique pattern of peaks) that is compared to a database of known spectra to ID the mcirobe

where is the bacterial genome located in most bacteria

the nucleoid

how do bacteria undergo genetic change

they multiple via binary fission, recombination (trasnfer of DNA from one organism to another), and mutations

transformation

bacteria taking free DNA and integrating it into their chromosome

transduction

bacteriophage infects donor bacteria and mistakenly package up bacterial DNA into their viral particles and then go an infect and spread the donor bacteria DNA

conjugation

donor bacteria transfers plasmid (extrachromosomal DNA) via a pilus

replication

the production of two identical DNA strands from one original DNA molecule

transcription

the process of DNA being copied to RNA

translation

mRNA being decoded into a sequence of amino acids

principle of PCR

process that amplified a target sequence of DNA into and exponential amount

denature (heat to seperate strand), annealing (freeze to hold strands so primer bind), and extension (synthesize new strand)

what is qPCR

real time PCR that measures the amount of amplified product during each cycle

what objective do we use to numerate host cells

10x

what objective do we use to numerate microorganisms

100x

what color are the nuclei of WBCs on a gram stain

pink

what does a squamous epithelial cell look like on a gram stain

pink, bigger than other cells, pink nucleus, might be folded onto itself

what are the criteria used to reject suboptimal sputum for culture

if there's more than 10 squamous cell/low power field OR

more than 25 squamous cells/low power field

some labs compare the amount of WBCs vs epithelial cells

how do you prepare a body fluiid slide for gram stain

use a sterile slide and cytospin (flings the sample onto the slide) to concetrate the cells on the slide

where is blood made

in the bone marrow (spongy material in the center of the bone)

what are stem cells (aka hemocytoblasts)

cells in the bone marrow that form the elements that make up the blood

list the five areas where blood cell development takes place

vertebrae, ribs, sternum, pelvis, and skull

what organs are involved in the hematopoietic system

bone marrow, liver, spleen, lymph nodes, and thymus

which stem cell series do lymphocytes generate from

the lymphoid series

which stem cell series do RBCs generate from

the myeloid series

what are the two ways the immune system maintains equilibrium

1.      Recognition of foreign organisms

2.      Clearing foreign organisms and preventing them from spreading

what are the two types of responses the immune system has

innate and adaptive

can the innate and adaptive responses trigger cellular and humoral responses

yes, they can both trigger cellular and humoral responses

describe the innate response

includes your physical barriers (skin, mucosal lining) and phagocytic cells

it’s the first to respond and is nonspecific so it reacts the same way every time

no memory

not always successful which leads to the adaptive response

describe the adaptive response

is a targeted/specific response that leads to long-term immunity against antigens

has memory so it has developed antibodies against antigens

which type of lymphocyte releases antibodies

B-lymphocytes

which lymphocytes releases lymphokines

T-lymphocytes

which lymphocytes matures in the bone marrow

B-lymphocytes

which lymphocytes matures in the thymus

T-lymphocytes

which lymphocyte produces cytokines

T-lymphocytes

what cells can mediate a cellular response

cytotoxic t-cells, dendritic cells, and macrophages

what two cells can mediate a humoral response

B-cells (produce antibodies) and helper t-cells (activate b-cells to produce antibodies)

can antigenic substances cause an adaptive immune response

yes

what are clusters of differentiation (CD)

what is used to ID different types of immune cells by looking at their membrane markers

what are the five types of CD cells

B-cells, T-cells, MHC, HLA, and APC

what is the CD marker called on T-helper cells

CD4

what is the CD marker called on T-cytotoxic cells

CD8

what is the purpose of antigen presenting cells (APC)

they are crucial for initiating the adaptive immune responses by engulfing the pathogen and breaking them down, then presenting that antigen on their surface to allow T-cells to connect to the specific antigen on the surface so they can respond