micro Learning objectives 1=2 Flashcards
1
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taxonomy
A
the study of systems by which living organisms are placed into a group (taxon)
2
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nomenclature
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the labelling of an organism. each distinct kind of organism is designated a species
3
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constitutive
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metabolically essential processes
4
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inducible
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in response to a stimulus in the environment, either the presence of a substrate or the depletion of a substance which when present, acts as a gene repressor
5
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generation (doubling) time
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replication speed is species specific and dependent on growth conditions ( relative to nutrients and environment)
6
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binary fission
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a single cell elongates, DNA replicates, cross wall forms and two identical daughter cells are formed (mitosis)
7
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plasmid
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small, circular pieces of dsDNA. vary in size, number, and number of genes. loss of plasmids is not detrimental because they don’t code of essential metabolic activities. they can replicate independently and sometimes can be incorporated into the chromosome
8
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transposable elements
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mobile genetic elements, jumping genes…. they don’t exist on their own in the cell and they must be incorporated into a plasmid or chromosome to replicate
9
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sub-clinical disease
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an individual may be infected and develop immunity to the infectious agent without ever having been symptomatically ill
10
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pathogenesis
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biological mechanism that leads to the diseased state. Origin and development of the disease
11
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congenital infection
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infections that are present at birth or often before birth
12
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mutualism
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host and parasite benefit. E. coli in GIT produces vitamin k
13
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parasitism
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one benefits while the other is harmed (roundworms and pinworms)
14
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commensalism
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one benefits but the other is unaffected
15
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colonziation
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occurs when micro-organisms persist on the body surface (skin and mucous membranes)
16
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transient colonization
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survive but do not multiply
17
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nosocomial
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nosocomial colonization refers to transient colonization with hospital strains of organisms
18
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opportunist
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opportunistic infections = introduction of commensals into sites that should be sterile
19
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endogenous
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sources of infection are from within an organism. Normal flora ,,,increased virulence…altered environment….introduced into a habitat it does not belong to
20
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exogenous
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source of infection is external to the host. many reservoirs….humans, animals, soil, water and food, nosocomial…transmitted directly - communicatable (STDs) indirectly - fomite, vectors, inhalation or ingestion)
21
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exotoxin
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exotoxins are released by organism into the environment includes enterotoxin (acts on gut) cytotoxins (destroy cells)neurotoxins (interfere with NS)
22
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endotoxin
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endotoxin is a cell wall component of GN organisms released on bacterial lysis and deaththis results in rash, fever, inflammation and septic shock
23
Q
endemic
A
constant/in cycles/ few individuals and defined areaeither cyclical (expected) or continuousconfined to a defined geographic locationaffects small numbers of the population(ie) California flu….due to yeast in dry soil in the south west USA
24
Q
epidemic
A
temporary, with many individuals and a defined areaepidemic disease attacks many people at the same time in the same location or in the same population for a period of timeCholera epidemics occur sporadically in defined geographic locations
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pandemic
temporary, many individuals, large area, possibly global pandemics are epidemics that have spread over a very wide area. attack majority of the population in a large area, often a country or even globally(ie) typhoid fever, small pox, plague, cholera, influenza, TB
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Describe the areas of study in microbiology
Bacteriology, parasitology, mycology, virology
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parasitology
study of parasites (protozoa, worms)...Giardia lablia
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mycology
study of fungi, moulds, and yeasts. eukaryotic. Candida albicans, tinea pedis (athlete's foot)
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virology
study of viruses. non cellular. influenza, herpies, H1N1
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Special three bacteria? (mycoplasmas, chlamydia, rickettsia vs typical bacteria, how does it affect lab workup?)
mycoplasmas, chlamydia, rickettsia
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(mycoplasmas vs typical bacteria, how does it affect lab workup?)
mycoplamas are bacteria are tri-layered with cell membrane but have no cell wall. cannot be gram stained and are pleomorphicsthey are the smallest known free-living formsfastidious - culture is possible but difficult and time consuming and requires nucleid acid precursors and sterols (special media)
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chlamydia vs typical bacteria, how does it affect lab workup?
the most common STD bacterial pathogenit has an unusual life cyclereproductive reticulate bodyinert, infective elementary body that infects epithelial cells...EB it then becomes RB and then EB then burstcannot be culturedsimilar to GN cell but it cannot be gram stainedobligate intracellular bacteria
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rickettsia vs typical bacteria, how does it affect lab workup?
rickettsia infections - spotted fever, typhus and scrub typhusobligate intracellular parasites and cannot be culturedbiosafety level three organismcell wall structure of GN bacteria, but cannot hold the Gram stain counterstain ( safranin)
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differentiate prokaryotic and eukaryotic cells based on cell wallplasma membranenucleusribosomesDNA arrangementmode of reproduction
Cell wall:o prokaryotes: complex cell walls contain peptidoglycan, with the exception of mycoplasmao eukaryotes: not all have cell wall, some have simple cell wall made of cellulose, phenolic polymers, lignin, chitin, and other glycans Plasma Membrane:o Both prokaryotes and eukaryotes have plasma membraneo Prokaryotes: generally contain no carbohydrates and no sterols, except mycoplasmao Eukaryotes: always have carbohydrate groups present in the membrane- Nucleus:o Prokaryotes - no nucleus: instead there is a nucleoid or nuclear area, no nuclear membrane or nucleolio Eukaryotes - have true nucleus with nuclear membrane and nucleoli- Ribosome:o Prokaryotes – 70S made up of 50S and 30S subunitso Eukaryotes: 80S made of 60S and 40S subunits- DNA Arrangement:o Prokaryotes – have chromosomes in nucleoid in circular form, complexed with RNA, no histone; have extrachromosomal plasmidso Eukaryotes – chromosomes are linear in the nucleus, complexed with histone and other proteins; mitochondria and chloroplasts have their own DNA- Mode of Reproduction:o Prokaryotes – asexual cell division: binary fission, recombination by transfer of DNA fragments, no meiosiso Eukaryotes – both sexual and asexual cell division, with recombination: mitosis and meiosis
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4) Relate the bacterial growth curve and reproduction to best lab practice
Log phase (exponential growth)this is the best stage for biochemical and antimicrobial testing and staining. exponential growth due to change in carbon sourcegrowing cells cells dividing at a constant ratecell size is smallestcell wall are thinnestmost active metabolic time in growth cyclevery susceptible to treatment by chemical and physical agents - good for testing *ie* AST - antibiotic susceptibility testing
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5) Briefly compare and contrast genetic information with respect to location, stability and autonomous replication: chromosomal vs extrachromosomal (plasmid and transposable element)
Chromosomal DNA - Chromosomal DNA is located intracellularly (nucleoid). It is more stable than extrachromosomal DNA. It can also replicate independently.Extrachromosomal DNAPlasmid - It can exist intracellularly or extracellularly. It is less stable compared to chromosomal DNA. It is able to replicate independently.Transposable element - It cannot exist inside a cell by itself, therefore very unstable. It needs to be inserted into a plasmid or chromosome in order for its gene to be replicated
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6) Differentiate “signs and symptoms” with particular reference to the laboratory
Signs of disease: objective evidences of disease, are phenomena that can be detected by someone other than the individual affected by the disease (primarily concern)rashswelltemperaturebloody nosebleedingSymptoms of disease: subjective evidences of disease, are phenomena that are experienced by the individual affected by the diseaseanxietyheadachepainfatigueasthenia
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7) List non-specific surface defenses to infection (host and microbial)
Host: mucus, coughing/peristalsis, epithelial cell turnover, local disinfectant (tears, gastric acid, fatty acids in skin)Microbial: normal flora
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8) describe the process of colonization (from womb to adulthood)
Colonization – when microorganisms persist on the body surfaceAt birth, transient colonization happens when baby come in contact with normal flora from mother’s birth canal, skin, breast milk, and other human contacts → Selection of transient colonizers occurs throughout our lives - some thrive and become normal flora, while others perish, or breach the barrier and become infectious Transient colonization: microorganisms persist on body surface – survive but do not multiplyNosocomial colonization: transient colonization of hospital/institutional strains of microorganismsCarriers: individuals colonized with organisms that are not normal flora, but not presently causing damage (sub-clinical or resolved infections) – reservoir of infection for othersNormal flora: Benign microorganisms establish their population on all body surfaces, would not cause disease to host if stay in its nicheOpportunistic pathogens: normal flora introduced into places that it does not belong and become infectious
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9) Describe the markers of the timeline from exposure to successful resolution of infectious disease
exposureincubationprodromalacutedeclineconvalescence or chronicprodromal - general malaise, symptoms are non-specificacute - symptoms peakdecline - infection declines and disease resolvesconvalescent - patient regains health
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10) List possible reservoirs of exogenous infection and ways in which organisms may be transmitted
Exogenous infections = source is external to the hostreservoirs include:humans: person to person spread from an actively infected individual or a carrieranimals (zoonosis) from bites, hides, carcassessoilwater and foodhospital acquired (nosocomial)ways in which organisms may be transmitteddirectlycommunicable (STDs)zoonotic (animal bite)indirectlyfomite (inanimate objects/substance/clothes/etc → capable of transmitting microbes to one another)vectors (ticks, mosquitos) - lyme disease and malariainhalation (TB)ingestion (Giardia)
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11) Recognize barriers to infection present in healthy individuals including non-specific surface defense (Host and microbial)
Non-specific surface defenseMucousCoughing/PeristalsisHigh rate of epithelial cell sloughing offEnzymes in tears, gastric acid and fatty acids in skinnon-host surface defense: microbial normal floraBarriers to infectionPhysical: Keratin, mucous and flushingChemical: interferon, lysozymes and stomach acidCellular: Cilia, phagocytes and pyogenic responseHumoral: AntibodiesBiological: Normal flora
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12) Briefly describe virulence factors associated with pathogenic organisms
- Adaption- Adherence factors:specific adhesins: lipoteichoic acid and M proteins in Strep. pyogenesnon-specific attractive forces: electrostatic- Enzymes:hemolysins → red blood cellsstreptokinase and staphylokinase → proteins, clotsprotease → immunoglobulincoagulase → converts fibrinogen to fibrin⇒clotsdeoxyribonuclease → phagocyte DNAleukocidins → polymorphonucleocyteshyaluronidase → connective tissuecollagenase → collagen in muscle- Capsule: evades phagocytes- Toxins :exotoxins: released by organisms into the environment, e.g. enterotoxins, cytotoxins, neurotoxinsendotoxin: a cell wall component of GN organisms released on bacterial lysis → rash, fever, inflammation, septic shock
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13) Differentiate endemic, epidemic and pandemic disease
Endemic - constant/ in cycles/ few individuals, defined area disease is either cyclical (expected) or continuousconfined to a defined geographical location…..few individualseffects small numbers of the population(ie) California flu - due to yeast in dry soil in southwest USAEpidemic - temporary, many people, defined areadisease attacks many people at the same timein the same location or in the same population for a period of time(ie) Cholera epidemics happen sporadically in defined geographical locationsPandemic - temporary, many individuals, large area, possibly global spread over a very wide areaattack the majority of the population in a large area (country or globally)(ie) Smallpox, plague
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Define CFU, gram-variable, pleomorphism, palisade, encapsulated, mucoid, acid-fast
CFU: colony-forming unit; a bacterial cell which landed at a specific spot on the plateGram-variable: bacteria that are morphologically similar microscopically but appear gram-positive and gram-negative in the same smearPleomorphism: cells variable in size and shape with some differing markedly from normalPalisade: bacilli shaped like a picket fenceEncapsulated: morphology of bacteria with capsules; the capsule won’t stain and is instead seen as a halo around the bacteria cellMucoid: mucus-like textureAcid-fast: used for organisms that don’t gram-stain; use strong mineral acids and alcohol to force the primary stain into the cell and resist decolourization
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2) State the required concentration of bacteria in a broth culture to be visible to the naked eye
10^6 bacteria/mL must be suspended in the broth for the naked eye to detect growth.
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3) describe components of typical, routine blood agar plates (BAP)
basal media such asMueller-Hinton (MH)columbia agarTryptic soy agar (TSA)plus...5% sheep blood
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explain how these plates are read correctly for macroscopic morphology
Amount +1 to +4Hemolysis BH, AH, GHSize pinpoint, small, medium or largePigment grey, white, is the pigment diffusible?Texture opaque, translucent, transparent, crumbly, mucoidOther odour, shape, pitting
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4) Apply the terms α, α’, β and γ when used to describe hemolysis on a BAP.
α : Alpha Hemolysis.· Partial lysis of SheepRBC results in greenish discolouration around the colony.· Colour due to methemoglobin from hemoglobin breakdown· Ex) “healthy green lawn”β : Beta Hemolysis.· Complete lysis of SheepRBC results in a clear zone around the colony.α’ : Alpha Prime Hemolysis.· Zone of complete hemolysis (β) surrounded by a zone of incomplete hemolysis (α).· Looks like beta but with some green colouring· Seen with some types of organisms as well as ageing cultures of α hemolytic organismsγ : Gamma Hemolysis.· NO hemolysis—NH—preferred abbreviation.
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5) Use BCIT reporting protocols to describe colonial morphology on BAP using the following features: amount, hemolysis, size, colour, texture, other significant and unique features
Amount· semi-quantitative ……..+1 (scant) to +4 (heavy)· quantitative……. 100x106 CFU/LHemolysis· beta (complete lysis)· alpha (partial lysis)· gamma ( non-hemolysis)Size· pinpoint, small, medium or largeColour/ Pigment· often gray, white, is the pigment diffusibleTexture· opaque, translucent/transparent, crumbly, mucoid, waxyOther significant and unique features· odour, shape, pitting
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6) Differentiate anionic, cationic and neutral dyes with respect to Microbiology
Anionic dyes (Acidic Dye)The chromophore is negatively chargedThese dye does not stain the bacteria because the bacteria are also negatively charged. Instead, the dye stains the positively charged background. This is known as “Negative stain” because the object of interest is not stained but appears unstained against a stained background.Example of acidic dye: Nigrosin, a capsular stainCationic dyes (Basic Dye)The chromophore is positively chargedBacteria are basophilic; the protoplasm is acidic, i.e. carries a net negative chargeMost bacteriological stains are in this category: crystal violet, basic fuchsin, safraninNeutral DyesNot used much in routine bacteriology, used in Giemsa for malaria in blood film
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7) Explain the theory of the Gram-stain, i.e. relate the structural differences of Gram-positive and Gram-negative cell envelopes to the Gram-stain method and reaction to the stain technique
There are four steps for Gram-stain, Primary stain( Crystal violet), Mordant( Gram’s iodine), Decolourizer( Acetone-95% ethyl alcohol), and Counter stain( Safranin). It stains the bacterial cytoplasm, but due to the bacterial cell wall peptidoglycan content and cell wall lipid content it has different result. Gram-positive organisms have more peptidoglycan and cross linkages than Gram-negative; Gram-negative organisms have lipid in outer membrane. The decolourizer is a dehydrating and defatting agent. When decolourization, the pores of the peptidoglycan shrink and trap the large dye-iodine complex inside the cells of Gram-positive organisms which will appear purple. On the other hand, the decolourizer removes the outer membrane of Gram-negative organisms and expose the thin peptidoglycan layer which dye-iodine complexes are readily washed out. After decolourization, Gram-positive organisms will stay purple while Gram-negative will be colourless which can be counterstained by Safranin and appear pink.
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8. describe the Gram-stain technique including possible technical issues
Technique:Use crystal violet (basic/cationic dye) & iodine (not a metal but acts as mordant) bind to the negatively charged cytoplasm of the bacteria⇒ stains purpleacetone-alcohol decolourizes Gram-negative cellssafranin counterstains Gram-negative cells pink Technical Issues:gram positive cells may not stain consistently purple due to cells growing old, dividing, organism's own characteristic, or over decolourizationunder decolourization and poor adjustment of microscope can cause gram-negative cells to appear purplesome bacteria are gram-variable and can appear both purple and pink in the same smear due to characteristic of some species, age of cells, or improper staining techniqueGP are purple...but if pink...it can be-technique, mitosis, cell wall type (murein layer number), cells growing old, cell wall breakGN are pink...but if purple its a technical errorstaining technique - under decolourizationpoor adjustment of microscope
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9) Differentiate “direct” smears from “colony” smears (Preparation, purpose)
Direct smear is made directly from patient specimens, before colonies could be isolated from culture plates. It is useful for rapid presumptive identification of an organism and differential diagnosis of disease, since culture plates take a while to grow.After culture plates have yielded “readable” colonies, colony smears are made for each significant colony type found on culture plates. Macroscopic and microscopic morphology of the colony smears will lead to the identification of the specific microbes.
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11) Explain why these variations to an expected Gram-Stain reaction might be seen: a known Gram-positive organism that has stained pink in some in some parts of the colony smear; a known Gram-negative organism that has stained purple in some parts of the colony smear.
If Gram-positive organism has stained pink in some parts of the colony smear it may be due to a loss of stain retention caused by something inherent in the cell (cell undergoing division; cell growing old—cell wall breakage) or due to a human error (artefact). Ex) technical error. If Gram-negative organism has stained purple in some areas then it is a technical error (staining method or microscope). -Gram-negative will always stain pink!
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13) List and briefly describe microscopic techniques/ applications other than Gram-stain: wet mount (including method), darkfield, India ink, methylene blue, lactophenol cotton blue, endospore staining
The Wet MountThis method is quick because the preparation is examined without drying, fixing or staining. It is only suitable for larger cells that can discerned using the high dry objective (40x). Wet mounts can be made directly from a fresh clinical specimen. It is commonly used as a quick check of a colony that macroscopically could be yeast or bacteria. The technique is also useful for checking for motility and the type of motility.Darkfield ExaminationIt is used for the detection of motile spirochetes obtained directly from a syphilitic lesion aspirate. An opaque disk under the condenser lens blocks light from travelling directly to the eye. However, if an organism is present on the slide, it will reflect the light and appear white on a dark background.India InkIt is a negative staining technique used to detect Cryptococcus spp. in cerebrospinal fluid (CSF). The ink particles cannot penetrate the capsule so the organism appears as a clear capsule surrounding the yeast cell against a dark background.Methylene blueMethylene blue is a vital stain that is actively taken up by absorbing cells. Differently structures will absorb the stain differently.Lactophenol Cotton BlueUsed to stain the cell wall of fungi; most often used in “scotch tape preps” from fungal culturesEndospore StainHeat is used to force the stain (malachite green) into the thick-walled spore. The slide is washed and then counterstained with safranin. Endospores appear green within a pink/red bacillus.
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14) Briefly describe the use of fluorescent stains in immunologic and blood culture applications
The fluorescent stains apply antigen-antibody theory, a fluorochrome that binds to protein( fluorescein) may be linked to a monoclonal antibody that has been developed to a specific antigenic component of a cell. The direct fluorescent antibody test is used for detection and identification if culture is not possible, difficult, or slow. Acridine orange which binds to nucleic acid is commonly used for organisms which can be flagged by blood cultures but can’t be Gram-stain.
