Micro Lab

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Endo Agar

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- Selects for gram –
- Diferential for lactose, lactose + = red colonies and surrounding medium
- Coliforms produce a golden metallic golden sheen

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M-staphylococcus broth

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Enriched media containing 10% NaCl, which selects for Staphylococcus since Staphylococcus prefer the higher salt concentration, which inhibits most other organisms

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DNase

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- Tests for exoenzyme DNase which is able to hydrolyze DNA
- Zones of clear around streaks either before or after addition of 0.1N HCl is a positive result for the presence of DNase

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Staphylococcus 110 Medium

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- Contains Mannitol and 7.5% NaCl, but lacks Phenol Red as in MSA plate
- Selects for Staphylococcus and allows for development of natural colony pigment formation unlike in MSA

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Phenol Red Manitol Salt Agar (MSA)

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- Selects for Staphylococcus due to high salt concentration 7.5%
- Medium is red, but plate and colonies will turn yellow if organisms are mannitol positive

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Coagulase

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-Incubate bacteria in small tube of plasma overnight
-If plasma becomes clumpy and or solidifies, then bacteria are coagulase positive
-Test is only valid on gram + staphylococcus like bacteria since gram negative bacteria are able to provide false positive reactions from a non coagulase like mechanism.

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Desoxycholate Citrate

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- Selects for gram -, lactose – microorganisms
- Some Lactose + colonies do grow but they will appear Red

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SS Agar

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- On this medium Salmonella usually produces a black colony, Shigella a colorless colony & all lactose positive colonies appear red

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Brilliant Green Agar (BGA)

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Differential for lactose/sucrose fermentation
- Lactose/ sucrose fermenting organisms produce yellow/ green or yellow colonies and turn the surrounding media yellow/green
- Non-lactose/ sucrose fermenting organisms produce opaque red/ pink/ white colonies and turn the surrounding media red

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Bismuth Sulfide Agar (BSA)

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- A dull green color; Salmonella typhi produces a black or very dark brown color

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Tetrazolium chloride (Motility)

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Contains Tetrazolium chloride, a growth indicator which turn red in the presence of growing bacteria. Therefore Red color away from the inoculation line is an indicator of growth.
Red color is only a growth indicator and indicates that tetrazolium chloride is reduced , the red color does not mean motility. Where the red color appears is an indication of motility, not the appearance of red.

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IMViC
(Indol, Methyl Red, Voges-Proskauer, and Citrate)

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IMVC
E. coli ++--
E. aerogenes --++

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OF Glucose
(OXIDATOR, FERMENTOR)

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Used to determine if a bacteria can use glucose in an oxidative (aerobic) or fermentative (anerobic) condition.
Two tubes are inoculated, with one of the tubes covered in mineral oil to prevent air from reaching the media. Media contains pH indicator (Brom Thymol Blue) which turns yellow if the glucose is utilized and acids are produced.

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OF Glucose
(OXIDATOR, FERMENTOR)
Results

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Open Tube: Yellow, Closed Tube: Green = Oxidation (O)
Open Tube: Yellow, Closed Tube: Yellow = Fermentation (F)
Open Tube & Closed Tube: Green, No Reaction to Glucose
Open Tube: Green, Closed Tube: Yellow = Ambiguous
GREEN - DYE COLOR
YELLOW - acidic pH

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Kliger’s Iron Agar (KIA)

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Tests for ability to ferment glucose and or lactose, tests for H2S production, gas production

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Kliger’s Iron Agar (KIA) mechanism

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- phenol red pH idicator
- lower pH = yellow color
- Glucose producers = small amount of acid
- Lac producers = large amount of acid
- Deamination = Red Slant
- Lac + = acid overneutralizes deamination so yellow
- Lac - = little acid so cannot diffuse whole medium so R/Y
- if bac. contain enzyme cystein disulfurase H2S produced
- if cracks or empty space gas prodution (CO2)

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Kliger’s Iron Agar (KIA) - reading

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R\R = Glucose - , Lac -
Pseudomonas, Alkalingenes
R\Y = Glucose +, Lac -
Proteus, Shigella, Salmonella
Y\Y = Glucose +, Lac +
Ecoli, Enterobacter
R\B = Glucose +,Lac -,H2S+
Salmonella, Proteus
Y\B = Glucose +,Lact+,H2S+

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Litmus Milk

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Lactose fermentation, reduction of litmus, presence of caseinase, and the deamination of amino acids to produce NH3

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Litmus Milk - Acid Reaction

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Pink Liquid due to drop in pH from the fermentation of lactose
Lac +, Cas -

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Litmus Milk - Acid Curd Reaction

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Pink Solid due to acid production and coagulation of casein causing the solid formation
Lac +, Casinase +

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Litmus Milk - Reduction

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Litmus is reduced and is caused to be colorless and the tube appears white since only the Milk remains.

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Litmus Milk - Alkaline Reaction

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Blue liquid = protein breakdown produces amino acids that are deaminated and release ammonia (pH increase)

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Litmus Milk - petonization/Proteolysis

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Clearing of medium (may be brown or amber) caused by enzyme caseinase which breaks down the white protein casein in milk to individual amino acids.

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Phenylalanine (PPA)

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Phenylalanine -> Phenylpyruvic Acid (PPA) + NH3
Tests for the presence of the enzyme phenylalanase which converts Phenylalanine to PPA and NH3. Ferric Chloride in the presence of PPA will appear a deep green color.
Green Color = + for Phenylanalase enzyme

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Simmons Citrate

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-Tests for the ability of a microorganism to utilize citrate as the sole carbon source.
- If + organism can grow in citrate and media will turn Prussian Blue
- Blue medium = citrate + microorganism
-

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SIM

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- Tests for Sulfur (H2S production), Indole, and Motility
H2s positive = black precipitate
Indole positive = Kovacs Reagent turns red after addition
Motility positive = growth away from inoculation line (appears as cloudiness in tube)

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Hydrogen Sulfide Production (H2S)

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Cystein -> H2S + Amino Acrylic Acid -> Imino Acid -> Pyruvic Acid + NH3
Tests for the enzyme cystein desulfurase which removes the sulfur side chain from cystein to produce H2S, which when in the presence of iron salts (contained in Klinger’s Iron Agar and SIM medium) forms a black precipitate. This Black Precipitate is a positive test for cystein desulfurase
Proteus = H2S positive

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Urea Broth

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Urea -> 2NH3 + CO2
Tests for the enzyme Urease which converts urea to ammonia and CO2. Urea broth contains the substrate urea and the pH indicator phenol red. When ammonia is released the pH of the solution increases and once the pH is above 8.1 the phenol red indicator will appear Red. The red color indicates a positive test for Urease.
Proteus = Urinase +

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Tryptophan (Indole)

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Tryptophan -> Pyruvic Acid + Indole + NH3
Tests for the enzyme tryptophanase which converts trytophane to indole and pyruvic acid. Indole can easily be tested for by adding Kovac’s Reagent (p-dimethylaminobenzaldehyde, amyl butyl alcohol, and HCl) which will appear red in the presence of the product indole.

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Nitrate Test

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- NO3- + 2e +2H+ -> NO2 + H2O
- NO2 → →→ N2 + NH3 other products
Tests for the ability of microorganisms to reduce Nitrate
Organisms are grown in Nitrate broth which contains nitrates (NO3-), Reagents Nitrate I (Sulfanilic Acid) and Nitrate II (dimethyl-alpha-naphthylamine) are added to the broth, if NO2-, a product of nitrate reduction, is present the broth will appear red. If the broth is red at this point the microorganism is said to be Nitrate pos. If the broth is not red, Zinc is added to the tube. Zinc is a catalyst which will convert NO3- to NO2-. Since the nitrate reagents are still in the tube, if NO3- was still present the broth will turn red. If the broth turns red after addition of zinc, the microorganisms are said to be Nitrate neg. If the broth does not turn red, the NO3- of the Nitrate broth was reduced by the microorganism to products other than NO2-, and is therefore said to be Nitrate pos.

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Nitrate Test - Reagents

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Nitrate I (Sulfanilic Acid) Nitrate II (dimethyl-alpha-naphthylamine)

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Kovac’s Reagent

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p-dimethylaminobenzaldehyde, amyl alcohol, and HCl)

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Oxidase Test

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Oxidase is an enzyme which can oxidize aromatic amines to form colored products. Indicator
dimethyl-p-phenylenediamine hydrochloride in the presence of oxidase will turn a dark blue black color.

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Oxidase Test - Reagents

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dimethyl-p-phenylenediamine hydrochloride

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Catalase

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2H2O2 -> 2H2O + O2
H2O2 produced during oxygen utilization is toxic. Catalase is an enzyme which converts hydrogen peroxide to water and oxygen,
Addition of H2O2 to the culture of question and production of oxygen bubbles = catalase +

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Voges-Proskauer (VP)

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HCOOH → acetyl methyl carbinol →2,3 butanediol
Tests for 2,3 butanediol fermentor
2,3 butanediol fermentors produce less acid and more neutral products than Mixed Acid Fermentors.
- AMC (acetoin) is easier to detect than 2,3butendiol
- AMC will produce red brick color when in presence of oxygen and VP1 and VP2.
-VP2 used to itensify color
- Red Brick = 2,3butandiol fermentor

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Voges-Proskauer (VP) Reagents

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Barrit’s reagents, also known as VPI (alpha-naphthol) and VPII (KOH)

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Methyl Red (MR)

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- HCOOH→CO2 + H2
-Tests for a Mixed Acid Fermentor
- Mixed Acid Fermentors produce lots of acid from the fermentation of sugars. This acid ultimately results in the lowing of the pH below 5.1, so when the indicator methyl red is added to the culture the methyl red remains red.
Escherichia = MR +

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Sugar Fermentation Tubes

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-Contain sugar of interest, phenol red and Durham tube.
- Fermentor of sugar = production of acid = decrease in pH = yellow color change
- Production of gas during fermentation = gas bubble in Durham tubes
- Use of peptone = increase in pH = dark red color

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Lipase Plate

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Tests for the presence of the enzyme lipase which hydrolyzes fat to form glycerol and fatty acids. The production of the fatty acids lowers the pH just enough to produce a dark blue precipitate when a microorganism is Lipase positive.

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Milk Agar

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Tests for the presence of the enzyme Caseinase, which hydrolyzes casein into amino acid products. Casein gives milk its white color so a breakdown in casein causes the milk plate to lose its white color and become clear around the Caseinase positive colonies.

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Starch Agar

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Tests for the presence of Amylase, which hydrolyses starch to simple sugars. Iodine is added to starch plate and appears blue/black when interacting with starch. If amylase is present starch will be hydrolyzed and the blue/black color will not be observed around the amylase positive colonies.