Lab Practical Flashcards
Slide created by adding bacterial suspension to one slide and then mixing loopful of given dye in suspension. The suspension is then spread using an additional slide. Slide is then air-dried (neither rinsed or heat-fixed)
What type of stain is this? What is it used for? What dye(s) typically used?
Negative Stain (of Bacillus) - stains background
Sufficient in determing morphology & size
Nigrosin (black anionic dye) &/or India Ink (insoluble dye)
Slide created by adding bacteria then heat fixing. Then single stain added & then rinsed with water until excess stain is removed.
What type of stain? What is purpose of stain? What dye(s) typicalled used?
Simple stain (crystal violet)
Detects Morphology & size (negative stain better at detecting size)
Cationic dyes - Methylene blue &/or Crystal Violet
Slide created using 2 different stains causing cells to stain differently based on properties of cell?
What type of stain is this? Stain differentiates between what characteristics of cell? Primary Stain/Mordant/Decolorizer/Secondary Stain? How to read results? Typical microbes for each result?
Gram Stain
Differentiates between cell wall layers - Gm (+) have higher peptidoglycan & lower lipid conent than gm (-)
Primary = Crystal Violet/ Mordant = Iodine/ Decolorizer = Ethanol/ Secondary = Safranin
Gm (+) = purple/ Gm (-) = pink
- Ethanol dissolves high lipid cell wall (of gm neg. microbes)
Gm (+) = Staphylcoccus; Streptococcus/ Gm(-) = Escherichia coli
Slide created using 2 different stains causing cells to stain differently based on properties of cell.
What type of stain is this? Stain differentiates between what properties of cell? Primary stain/ Mordant/ Decolorizer/ Secondary Stain? How to read results? Typical Microbes for each result?
Acid-Fast Stain
Differentiates based on cell wall content - Acid fast microbes have high mycolic wax contentn in walls (requires use of heat-fix)
Primary - Carbol Fuschin/ Mordant - “heat”/ Decolorizer - Acid-Alcohol/ Secondary - Methylene Blue
Acid fast (+) = cerise (pink-violet)/ Acid fast (-) = Blue
Acid fast (+) = Mycobacterium; Nocardia/ Acid fast (-) = all other microbial genera
Slide created using 2 different stains causing cells to stain differently based on structural differences.
What type of stain? Stain differentiates between what structures? Primary stain/ Mordant/ Decolorizer/ Secondary stain? How to read results? Typical microbes for each result?
Spore Stain
Heat driven dye is retained by spores (Spores are heat & chemical resistanct structures)
Primary = Melachite green / Mordant = “heat” / Decolorizer = Water/ Secondary = Safranin
Endospores = green center within pink microbe/ Free spores = small green oval bodies w/o surrounding pink microbe/ Vegetative (non-spore structures) = Pink
Bacillus (aerobic, gm + rod); Clostridium (anaerobic gm + rod; sporsarcinae (coccie)
What type of Plate is this? Selective/Differential? Results indicate what?
Phenylethyl Alcoholl Agar (PEA
Selective - for growth of gm (+) microbes
– Phenylethanol inhibits gm (-)
Growth indicicates gm (+)/ no growth indicates gm (-)
gm (+) = Staphylcoccus; Streptococcus; Bacillus; Mycobacterium
gm (-) = E. coli; Enterobacter aerogenes (E. aerogenes); Shigella; Salmonella
What type of plate is this? Selective/Differential? Results?
Blood Agar (BA) - 5% Sheep RBCs
Differentiates based on hemolytic ability of microbe
Beta Hemolysis - complete blood hemolysis; complete clearing around colong/ Alpha Hemolytic - particial blood hemolysis; partial clearing around colony (green-like appearance sometimes) / Gamma hemolytic - no blood hemolysis; no zone clearing around colony
Beta - Streptococcus pyogenesis, Staphylcoccus aureus
What type of plate is this? Sective/Differential? Results? Examples?
Eosin Methylene Blue Agar (EBM)
Slective for Gm (-)/ Differentiates lactose fermentation (+)/(-)
- EMB further differentiates pathway of lactose (+)…
- …(Mixed Acid fermentors) produce more acid & Methylene blue is absorbed
- …(2,3-Butanediol fermentors) produce less acid & Eosin is absorbed
Growth indicates gm (-)/ no growth indicates gm (+)
Lactose (+) show color change/ Lactose (-) show no color change
Dark blue-black colony indicates Mixed-acid/ Pale Pink or Lavender center indicates 2,3-butanediol
E. coli - strong mixed-acid fermentator; results in metallic-green sheen
E. aerogenes - 2,3-butanediol fermentator; results in Pale pink-lavender centers
P. aeruginosa - 2,3-butanediol fermentator; results in Pale pink-lavender centers
What type of plate is this? Selective/Differential? Results? Examples?
Desoxycholate Agar (DES)
Selective for Gm (-)/ Differentiates Lactose Fermentors (+)/(-)
- Desoxycholate agar inhibits growth of gm(+)
- Lactose fermentors produce acid which precipitates bile salts in media & absorbing neutral red dye/ non-Lactose fermentors do not absorb dye
Growth indicates gm (-)/ no growth indicates gm (+)
Lactose (+) appear red/ Lactose (-) do not absorb red dye
Lactose (+) - Eschericia (E. coli); Enterobacter (E. aerogenes); Pseudomonas (P. aeruginosa)
…E.coli (strong Lactose +) appears very red bc more acid produce
What type of plate are these? Selective/Differenetial? Results? Examples?
Starch Agar…
(2nd plate) Iodine has been added
Differential - detects Amylase enzyme
- Amylase hydrolyses starch to simple sugars
- Iodone appears blue/black when interacting with starch
Amylase (+) = absence of blue/black color around colony
Amylase (-) = presence of blue/black color
Bacillus is only Amylase (+)
What type of plate are these? Selective/Differential? Results?
Lipase Agar
Differential - detects Lipase
- Lipase hydrolyzes lipids to fatty acids & glycerol
- fatty acids lower pH just enough to produce dark blue precepitate around colonies
Lipase (+) = blue dye intensifies
Lipase (-) = no blue intensification
Lipase (+) - S. aureus (Staph)
What type of plate are these? Selective/Differential? Results?
Milk Agar
Differential - detects Caseinase
- Caseinase hydrolyzes casein (main Milk-protein) into amino acid products
- Casein give milk its white color…break down of casein causes milk to lose white-color
Caseinase (+) clearing of white-color around colony
Caseinase (-) no clearing of white-color
P. aeruginosa appears greenish on Milk agar
What type of Broth are these? Selective/Differential? Results? Examples?
Nitrate Broth
Differential - detects nitrate reductase (or Nitrase)
- Nitrase converts nitrate to nitrite
- if Nitrite present (after addition of reagents Nitrate I & Nitrate II) then broth turns red – indicating nitrase (+)
- if no color change then Zinc is added to tube…
…Zinc catalyzes reduction of nitrate to nitrite
…if broth turns red after addition of Zinc then said to be Nitrase (-)
…if broth does NOT turn red then said to be Nitrase (+) – (microbe reduced nitrate into product other than nitrite)
Nitrase (+) = either broth turns red OR broth does not turn red after addition of zinc
Nitrase (-) = broth turns red after addition of Zinc
P. aeruginosa = Nitrase (+) – reduces nitrate to products other than nitrite – broth colorless after addition of Reagents & Zinc
B. cereus = Nitrase (+) – reduces nitrate to nitrite – broth turns red after additino of reagents but before Zinc
Lactobacillus lactis = Nitrase (-) – broth turns red after addition of reagents & zinc
What type of test are these? Selective/Differential? Results?
Litmus Milk Broth
Differential - Detect moderate/heavy lactose (+), Detect Caseinase, Detects Reductase
…Litmus milk contains Lactose, Litmus, & Casein..
Pink Liquid = 2-3, Butanediol (lactose) fermentation (Moderate acid concentration)
Pink (hard or soft) curds = Mixed-acid (lactose) fermentation & coagulation of casein (by high acid conc)
Blue Liquid = alkaline rxn (caused from metabolism of Casein into amino acids which are deaminated & release NH3)
Reductino of LItmus = litmus reduced (reductase) & tube appears milk-opaque color (from casein)
Peptonization/Proteolysis = clearing of milk-opaque color; caseinase (+)
What type of test are these? Selective/Differential? Results?
Urea Broth
Differential - detects precense of Urease
Urea broth contains Urea & pH indicator Phenol Red
- Urease converts Urea to Ammonia & CO2
- when NH3 is release pH increases & turn phenol red indicator to cerise (more basic) to red (more neutral) color
Urease (+) = red to cerise color
- P. (Proteus) vulgaris
Urease (-) = yellow(more acidic) to orange
- S. (Staphylcoccus) aureus
what type of test are these? Selective/differential? REsults?
Simmons Citrate Slant
Differential - detects ability of microbe to utilize citrate as sole carbon source
- Bromothymol blue used as indicator of citrate (+) BUT NOT PRIMARY INDICATOR…
- Growth is primary indicator
Citrate (+) = GROWTH on slant &/or prussian blue color
- E. (Enterobacter) aerogenes
Citrate (-) = no Growth on slant & medium remains green
- E. coli
What type of test are each of these? (Left?/Middle/Right?) Slective/differential? Results?
Phenol Red (PR) Sugar Fermentation tests
Differential - detects ability of microbe to ferment selected sugar; detects production of gas (Durham tube)
- if able to ferment selected sugar then will lower pH of solution
- Phenol red used as indicator - turns solution red in basic conditions
Sugar Fermentation (+) = yellow
- E. coli
Sugar fermentation (-) = red (more neutral) to cerise (more basic)
- P. aerogenosa
(Left = Glucose/ Middle = Lactose/ Right = Mannitol)
What type of test is this? Selective/differential? REsults?
Oxidase test
Differential - presence of cytochrome oxidase
- Oxidase transfers electrons to oxygen (or in this test’s case to the reagent - Dimethyl-p-phenylenediamine hydrochloride)
- when oxidase transfers electrons to reagent, it turns dark blue-black color
Oxidase (+) = dark blue-black color
- P. aeroginoas
Oxidase (-) = no color change
Type of test? Selective/Differential? Results?
Phenylalanine Agar Slant (PPA)
Differential - detect phenylalanase
- Phenylalanase converts Phenylalanine to Phenylpyruvic acid (PPA) & NH3
- Ferric Chloride used as indicator
- Ferric chloride in presence of PPA appears deep green color
Phenylalanase (+) = green color
- P (Proteus) vulgaris
Phenylalanase (-) = no color change; yellow remains
Type of Test? selective/differential? results?
Tryptone Broth (Indole)
Differential - detects presence of tryptophanase
- Tyrptophanase converts tryptophane to indole, Pyruvic acid & NH3
- Indole easily tested for by adding Kovac’s reagent which appears red in presence of indole
Tryptphanase (+) = red-colored top
Tyrptophanase (-) = yellow colored top
Types of tests? Differential/Selective? REsults?
(left) Methyl Red MR & (right) Voges-Proskauer VP
Differential - MR detects Mixed acid ferm; VP detects 2-3, butanediol ferm
- Mixed acid ferm produces high concentration of acid - in presence of indicator methyl red, culture remains red
- 2,3 Butanediol ferm produces a less concentration of acid (compared to mixed acid) - in presence of Barrit’s reagents (VP I & VP II), brick-red color is produced
…Glucose –> Acetyl Methyl Carbinol (acetoin)–> 2,3 Butanediol - Aceotoin is actual substance being tested for in VP test
Mixed acid (+) = reagent remains red Mixed acid (-) = reagent loses red color
2,3 Butanediol (+) = produces brick red color
2,3 butanediol (-) = no brick red color
- Mixed acid - E. coli
- 2,3 - Butanediol - E. aerogenes
Type of Test(s)? Differential/Selective? Results?
SIM tests (Sulfur/Indole/Motility)
Differentiates for each of 3 tests: Sulfur (H2S production), Indole (Tryptophone), & Motility
Sulfur (+) = Black precipitate forms
Indole (Tyrptophone) (+) = turns cerise color after addition of Kovac’s reagents (reacts with Indole)
Motility (+) = growth away from inoculation line (cloudiness)
