Micro Lab practical

Fluid Thioglycollate- growth on top and bottom away from stab
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Fluid Thioglycollate- growth on top and bottom away from stab
facultative anaerobe (motile or nonmotile)
-24 H to 1 Week
Fluid Thioglycollate- growth on just top
obligate anaerobe
-24H to 1 week
age of gram stain cell culture
20-24 H
Fluid thioglycollate medium
specially designed to understand o2 requirements of bacteria
.25% agar, rich broth, reducing agents, resazurin: an oxygen indicator dye
pink band: presence o2
colorless: absence of oxygen
MacKonkey agar
-selective for gram -
-lactic acid fermentation
-yellow: no acid production (can't ferment lactose)
-red: can ferment lactose
-pH indicator: neutral red (maN)
-24 H to 1 week
MSA (Mannitol Salt Agar)
-selective and differential
-bacteria that can grow in high salt environment (7.5%)
-No growth: not salt tolerant
-Mannitol and Phenol Red
-24 h to 1 week
Sugar fermentation test
-color change for gas production (tiny tube)
-red: basic (produces ammonia)
-Pink: no fermentation
-yellow: fermentation (produces acid)--> lactose or other sugars
-24-48H
citrate utlization
-tests for citrate utilization for carbon source
-green: negative
-blue: citrate positive
-sodium citrate, ammonium dihydrogen phosphate
-indicator: Bromothymol blue
-24 H - 1 week
MR-VP(methyl red)
-color change for acid production
-High acid production (won't change until pH 4.4)
-Red: high acid production from fermentation
-48H-1 week
MR-VP (Voges Prosauker)
-tests for alcohol production
-Barritt's A (alphanapthol)
-Barritt's B (40% potassium hydroxide)
-Red color indicates strong alcohol production
-carbon source: glucose degraded by pyruvic acid and converted to acetone and 2,3-butanediol and EtOH
-48 H to 1 week
Indole test-Big tube -LB (tryptone broth -rich in tryptophan) -tyrptophanase breaks down tryptophan to indole, pyruvic acid, and ammonia -add 5 drops Kovack's reagent (toxic) -red: indole present -no color change: no indole (or tryptophanase) -24 H to 1 weekUrease test-tiny tube -tests for urease -urea broth -urease hydrolyzes urea into ammonia and CO2 -either produce urease or don't use urea in broth -indicator Phenol Red -Pink: basic (urease --> ammonia) -orange (neutral)Phenylalanine Deaminase Test-agar slant- stab streak -ability to breakdown phenylaline (deaminase) -add 10 drops 10% ferric chloride after incubation -24 H-1 week -yellow: no phenylalanine deaminase -green: phenylalanine's presentCatalase test-conversion of toxic H2O2 to H2O and O2 using catalase enzyme -positive= bubbling present -negative= no bubbling -reagent: 3% H2O2 -requires fresh culture -toothpick of bacteria -Must be alive 24-36 HOxidase test-Tests for the presence of cytochrome c -dark blue: when adding oxidase reagent (phenylendediamine dihydrochloride)--> organism produces oxidase -no color change: no cyt C oxidase -indophenol blue: cyt C present -requires fresh culture -Must be aliveCoagulase test-Rabbit plasma -looking for clot or liquid -liquid: no coagulase -clot: coagulase -Coagulase: converts fibrinogen to fibrinHemolytic test-differential -blood agar plates -alpha: partial hemolysis (green) -beta: large hemolysis and extreme clearing -gamma: no clearing -hold into the light -24-48 HCAMP test-s.aures and other organism -triangle of clearing= CAMP -increases hemolytic activity -Group B streptococciSnyder-differential and selective -pH indicator: Bromocrystal green -Very soft -selecting organisms for high acid tolerance -test for glucose fermentation -growth of plate: organism can tolerate strong acidic environment (no fermentation) -no growth: not acid tolerant - if yellow and growth?:acid tolerant and fermentation of glucose (not likely) -24-48 HTiterT=N/(DF*V) CFU/mLincreasing the magnification of the objective lense decreases theworking distanceworking distancedistance between objective lens and specimenHow do prokaryotes and eukaryotes differ in size?prokaryote: .1-5 um eukaryote: 10-100 imWhat is the iris diaphragm?Located below the stage, it control the amount of light through the confessor and allowed onto the slide -makes the sample brighterWhen is the final adjustment knob used? and for what?after coarse adjustment -moves stage to make image more clearfine adjustment knobMoves the stage slightly to sharpen the imageWhat should you write down in your microscope observations?shape, arrangement, total magnification, endospore, motility, and colorWhat bacterial shapes do we observe in lab?coccus, bacillus, spirochete, filamentouscoccussphericalbacillusRod shaped bacteriaspirochetespiralfilamentousWhat arrangements do we use in lab to classify bacteria?diploid, chain(strepto), tetrad, cluster(Staphylo)SAM-EMCshape arrangement total magnitude endospore motility colorHow many dyes are used in a simple stain? What charge do they posses?one (+)Give an example of a dye that can be used for simple staining?crystal violetWhat type of dye can be used to stain a bacterial structure such as flagella? Why can we see this?simple stain dye coats the bacterial flagella, precipitating Lang the structure and increasing the flagellawhat charge do negative stains carry? how do they interact with the cell wall?negative; repel from negatively charged cell wall -stain backgroundExample of negative stain and advantage?india ink no need for heat fixingWhat is a differential stain used for?More than one stain is used to distinguish different groups of bacteriaHow many dyes are used in gram staining?3 + 1 decolorizerGram + and Gram - reaction to decolorizergram +: decreases permeability of PTG causing the crystal violet complexes to be trapped gram -: alcohol dissolves outer membrane of the cells uncovering the larger pores in the thin PTG layer allowing the crystal violet complexes to leak outPurpose of Gram's iodine?forms complexes of crystal violet and iodine--> more difficult to remove from cell walls and cytoplasm than crystal violet moleculesproblems from thick smears?outer edges will get decolorized but not the insideWhat type if stain is used for Acid fast stains? and with what?carbol fuschin applied with heatWhat primary stain is used in acid-fast staining?carbol fuschinwhat is the secondary stain used in acid-fast staining?methylene bluewhat is used to destain some of the bacterial cells (acid fast)acid ethanolacid fast vs non acid fast bacteria react to acid alcoholacid fast: retain carbon fuschin in cell wall lipids whereas non acid fast cells quickly lose stainsPink bacteria in acid fast stainacid fastblue bacteria in acid fast stainnon acid fastwhat structure does a spore stain make visible?endosporeswhat is the primary stain in a spore stainmalachite green--> greenwhat is the secondary stain in the spore stainsafranin--> pinkwhat is green and what is red in a spore stain?green: endospore Pink: vegetable cellWhat was the purpose of the wet mound experiment?observe motility or movement of an organismWhat are the 2 different types of fungi?parasitic: chitin based cell wall saprophytic: live on dead materialWhat are the 2 types of bacteria?Heterotrophic: motile/nonmotile with cilia/flagella Autotrophic: nonmotileAre protozoa motile?motile via flagellaHow do protozoa retrieve nutrients1. Phagocytosis 2. PinocytosisWhat are algae?archaeplastida (eukaryotes)--> oxygenic photosynthetic organismHow do algae move?motile/nonmotile via flagella or gliding motilityWhat is bacterial isolation?separate a specific bacteria of a single speciesWhy do we want a pure culture?only cells of a single bacterial species growing on an isolated environmentCHESTcolor, height, edges, size, textureWhat di you label your agar plate with?name, division, date, media, and treatmentWhat was the treatment added to our bacterial isolation plates in lab?SCI mixWhat is the difference between transient and resident microorganisms?transient: external source. but do not live in the environment resident: live in environmentWhat are two methods to test for microorganisms present on surfaces?contact plate and swab plateWhy should we work aseptically?introduction of a new bacteria could alter results and outcomes form contaminationWhat does sterile mean?no living microbeswhat are ways to sterilize objects in the lab?treat media at high temps/ pressurewhat is turbidity?cloudiness of tube--> cells are evenly suspendeddifferent types of turbidity?flocculent (flocs/clumps), pellicle (thick layer on top), sediment (cells sink to bottom)4 methods of determining sample densitycell mass, turbidity, direct cell count, dilution to extinction, and quantitative cell countsWhy do we record CFU/mLCFU only counts viable cellsselective mediacontains one or more components that supresses the growth of one bacteria without altering the effect of growth of anotherhow can a media be selectiveadding dyes, abx, salts, or metabolic inhibitorsdifferential mediaallows distinction b/t 2 closely related organismCan media be both selective and differential?yesdifferential media examplesMacConkey's: lactose and neutral red to differentiate bt lactose fermenters and non lactose fermenters MSA: mannitol and phenol red to differential between mannitol fermenters (yellow) and non-mannitol fermenters (pink) EMB: lactose and 2 pH indicators (eosin Y and methylene blue) differentiates bt vigorous fermentors (purple) and weak fermenters (pink) and non-lactose fermenters (no color) Snyder: contains glucose and bromocrescol green to differentiate glucose fermenters (yellow) and non-glucose fermenters (green) Blood agar: 5% sheep blood to differentiate between hemolytic organisms (clearing), weak hemolysis (greenish) and non-hemolytic (no clearing)selective media examplesabx media: inhibits the growth of non-abx resistance bacteria MacConkeys: contains bile salts and crystal violet (inhibits growth of Gram +) MSA: contains 7.5% NaCl (allows for growth of bacteria in high salt concentration) Eosin methylene blue: contains eosin Y and methylene blue to inhibit Gram + Snyder: low pH inhibits growth of bacteria sensitive to low pHobligate aerobesstrictly require oxygen for growthobligate anaerobesCannot survive in the presence of oxygen--> toxicaerotolerant anaerobesnot affected by the presence of oxygen but they do not use oxygen for their growthfacultative anaerobescan grow in the absence of oxygen as they have the ability to ferment ; presence of oxygen they tend to grow betterMicroaerophilesrequire oxygen, but at low concentrations--> toxic