Lab - Stains & Biochemical Tests
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| What is the medium used in the Catalase Test? | Nutrient Agar
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| What are the ingredients in the Catalase Test? | Nutrient Agar: 1. Nothing major 2 seaweed
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| How do you inoculate and incubate the Catalase Test? | 1. Streak plate 2. Incubate @ 35 degrees C for 48 hours
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| How do you perform the Catalase Test? | Take bacteria, place on microscope slide, add hydrogen peroxide
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| How do you read the Catalase Test? | 1. Pos: Bubbles: 2. Neg: No Bubbles
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| What happens chemically in the Catalase Test? | Presence of catalase enzyme breaks down hydrogen peroxide, release H2O & O2
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| What do the results mean in the Catalase Test? | Catalase is used by bacteria to help them live in presence of oxygen and to detoxify hydrogen peroxide
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| What is the medium used in the Gelatin Hydrolysis / Liquefaction Test? | Nutrient Gelatin
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| What are the ingredients in the Gelatin Hydrolysis / Liquefaction Test? | 1. Gelatin 2. Peptone
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| How do you inoculate and incubate the Gelatin Hydrolysis / Liquefaction Test? | Stab Inoculate & 35°C for 48 hours
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| How do you perform the Gelatin Hydrolysis / Liquefaction Test? | After incubation, place tubes in fridge for 20 mins to solidify any negative tests; after cooling, check to see if Nutrient Gelatin is solid or liquid
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| How do you read the Gelatin Hydrolysis / Liquefaction Test? | 1. Pos: Gelatin is Liquid 2. Neg: Gelatin is Solid
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| What happens chemically in the Gelatin Hydrolysis / Liquefaction Test? | Presence of gelatinase enzyme breaks down the gelatin and liquefies it
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| What do the results mean in the Gelatin Hydrolysis / Liquefaction Test? | After breaking down the gelatin, the cell can take up the individual amino acids and use them for metabolic purposes.
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| What is the medium used in the Phenol Red Carb Fermentation Test? | Phenol Red Broth
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| What are the ingredients in the Phenol Red Carb Fermentation Test? | 1. 1 Carb: (glucose, lactose, or sucrose) 2. Peptone 3. Phenol red indicator 4. Durham tube (only in glucose tube)
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| How do you inoculate and incubate the Phenol Red Carb Fermentation Test? | 1. Loop inoculate broth 2. Incubate at 35°C for 48 hours.
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| How do you perform the Phenol Red Carb Fermentation Test? | After incubation period, observe the color change.
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| How do you read the Phenol Red Carb Fermentation Test? | 1. Pos: Yellow broth (pH = 6.8) 2. Neg (no glucose fermentation or peptone deamination): Red broth 6.9 < pH < 7.3; 3. Neg (no glucose fermentation):Pink broth (pH > 7.4) 4. Bubble: Gas end product
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| What happens chemically in the Phenol Red Carb Fermentation Test? | If bact can ferment carb/sugar, acid produced, pH lowered. If the bacteria can't ferment glucose, but deaminates peptone amino acid, medium becomes alkaline, raising pH
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| What do the results mean in the Phenol Red Carb Fermentation Test? | Metabolic process by which org. mol. acts as an e- donor (become oxidized in process) and 1+ of its organic prods act as final e- acceptor.
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| What does it mean when phenol red broth is red at top & yellow at bottom for Phenol Red Carb Fermentation test? | Carbs have run out, so bact uses amino acid & makes alkaline prods to change pH (reversion)
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| What is the medium used in the Methyl Red Test? | MR-VP Broth
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| What are the ingredients in the Methyl Red Test? | 1. Peptone 2. Glucose 3. Phosphate buffer
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| How do you inoculate and incubate the Methyl Red Test? | 1. Loop inoculate broth 2. 35°C for 48 hours
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| How do you perform the Methyl Red Test? | After incubation, transfer 1ml of broth into a new sterile tube; add 4-5 drops of Methyl Red and read result immediately.
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| How do you read the Methyl Red Test? | 1. Positive: Red (pH = 4.4) 2. Negative: Yellow (pH = 6.2).
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| What happens chemically in the Methyl Red Test? | If the bacteria can undergo mixed acid fermentation, the acid byproducts will overcome the phosphate buffer and lower the pH. If it cannot, the pH will remain the same.
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| What do the results mean in the Methyl Red Test? | Fermentation is a metabolic process by which an organic molecule acts as an electron donor (becoming oxidized in the process) and one or more of its organic products act as the final electron acceptor.
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| What is the medium used in the Voges-Proskauer Test? | MR-VP Broth
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| What are the ingredients in the Voges-Proskauer Test? | 1. Peptone 2. Glucose 3. Phosphate buffer
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| How do you inoculate and incubate the Voges-Proskauer Test? | 1. Loop inoculate broth 2. 35°C for 48 hours
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| How do you perform the Voges-Proskauer Test? | After incubation, xfer 1ml of broth into a new sterile tube; add 18 drops of Barritt’s A (a-naphthol) and 18 drops of Barritt’s B (potassium hydroxide) and mix frequently for 1.5 hours; observe color change.
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| How do you read the Voges-Proskauer Test? | 1. Positive: Red 2. Negative: Yellow
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| What happens chemically in the Voges-Proskauer Test? | If bact undergo 2,3-butanediol ferment., acid prods converted to acetoin & 2,3-but'l. Adding VP reagents oxidize acetoin to diacetyl, which reacts w/ guanidine nuclei fr. peptone to produce red color.
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| What do the results mean in the Voges-Proskauer Test? | Fermentation is a metabolic process by which an organic molecule acts as electron donor (becoming oxidized in the process) and one or more of its organic products act as the final electron acceptor.
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| What is the medium used in the Nitrate Reduction Test? | Nitrate Broth
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| What are the ingredients in the Nitrate Reduction Test? | 1. Peptone 2. Potassium nitrate
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| How do you inoculate and incubate the Nitrate Reduction Test? | 1. Loop inoculate broth
2. Incubate at 35°C for 48 hours.
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| How do you perform the Nitrate Reduction Test? | After incubation, add 8 drops each of Reagent A & B & mix; wait 10 mins & observe color change. If no color change, confirm add Reagent C to confirm negative; let sit for 10 mins & observe color change.
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| What is Reagent A in the Nitrate Reduction Test? | N,N-dimethyl-a-naphthylamine
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| What is Reagent B in the Nitrate Reduction Test? | Sulfanilic acid
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| What is Reagent C in the Nitrate Reduction Test? | Zinc dust
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| How do you read the Nitrate Reduction Test? | 1. Pos: Red color before adding Reagent C 2. Neg: No color change before adding Reagent C and a red color change after adding Reagent C.
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| What happens chemically in the Nitrate Reduction Test? | If the bacteria can reduce nitrate, nitrous acid will react with the added reagents to produce a red color.
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| What do the results mean in the Nitrate Reduction Test? | If bacteria has nitrate reductase, it reduces nitrate to nitrite to nitrous acid. If after adding Reagent C, still no color change, nitrate is reduced to ammonium, nitric oxide, and nitrous oxide.
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| What is the medium used in the Oxidase Test? | Trypticase Soy Agar Plate
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| What are the ingredients in the Oxidase Test? | 1. Amino Acids 2. Casein 3. Iron-containing compound 4. Sulfur (sodium thiosulfate)
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| How do you inoculate and incubate the Oxidase Test? | 1. Streak Plate 2. Incubate at 35°C for 48 hours
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| How do you perform the Oxidase Test? | Use sterile toothpick, get large amount of bacterial growth & place a small dot on the BBL DrySlide. After 20 seconds (the time it takes for Pseudomonas to give a positive result), observe the color on the slide
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| How do you read the Oxidase Test? | 1. Pos: Dark blue/purple color within 20 seconds
2. Neg: No color change to dark blue/purple within 20 seconds.
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| What happens chemically in the Oxidase Test? | If the test is positive, the reagent, which acts as an artificial electron donor, will oxidize cytochrome c
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| What do the results mean in the Oxidase Test? | A positive color indicates the presence of cytochrome c oxidase, which is a carrier molecule in the Electron Transport Chain. It transfers electrons to oxygen, thus reducing it to water
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| What is the medium used in the Indole (Tryptophan Hydrolysis) Test? | 1% Tryptone broth
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| What are the ingredients in the Indole (Tryptophan Hydrolysis) Test? | 1. Amino Acids 2. Casein 3. Iron-containing compound 4. Sulfur (sodium thiosulfate)
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| How do you inoculate and incubate the Indole (Tryptophan Hydrolysis) Test? | 1. Loop inoculate broth 2. Incubate at 35°C for 48 hours.
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| How do you perform the Indole (Tryptophan Hydrolysis) Test? | After incubation, add 6 drops of Kovac’s Reagent (para-dimethyl-aminobenzaldehyde) and mix; observe color change.
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| What is Kovac's Reagent called? | para-dimethyl-aminobenzaldehyde
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| How do you read the Indole (Tryptophan Hydrolysis) Test? | 1. Pos: Red in the alcohol layer of Kovac’s reagent 2. Neg: No color change.
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| What happens chemically in the Indole (Tryptophan Hydrolysis) Test? | Kovac’s Reagent reacts with indole present and produces a red reagent layer.
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| What do the results mean in the Indole (Tryptophan Hydrolysis) Test? | The presence of tryptophanase hydrolyzes tryptophan and water to produce indole, ammonia, and pyruvic acid.
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| What is the medium used in the Urease Test? | Urea Broth
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| What are the ingredients in the Urease Test? | 1. Urea 2. Phenol red indicator
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| How do you inoculate and incubate the Urease Test? | 1. Loop inoculate broth 2. Incubate at 35°C for 48 hours
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| How do you perform the Urease Test? | After incubation, observe color change.
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| How do you read the Urease Test? | 1. Pos: Pink (pH above 8.4) 2. Neg: Yellow / orange (pH < 8.4).
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| What happens chemically in the Urease Test? | When urea is broken down, it produces alkaline products, changing the pH > 8.4
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| What do the results mean in the Urease Test? | The presence of urease breaks down urea and water into carbon dioxide and ammonia.
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| What is the medium used in the Glucose/Lactose Fermentation and Hydrogen Sulfide Production Tests? | Kligler's Iron Agar
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| What are the ingredients in the Glucose/Lactose Fermentation and Hydrogen Sulfide Production Tests? | 1. 0.1% glucose 2. 1% lactose 3. Peptone 4. phenol red (indicator) 5. ferrous sulfate (indicator)
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| How do you inoculate and incubate the Glucose/Lactose Fermentation and Hydrogen Sulfide Production Tests? | 1. Stab butt and streak slant 2. Incubate at 35°C for MAX 18-24 hours
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| How do you perform the Glucose/Lactose Fermentation and Hydrogen Sulfide Production Tests? | After incubation, observe color change
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| How do you read the Glucose/Lactose Fermentation Tests? | Glucose/Lactose Fermentation: 1. Pos: Yellow butt/slant 2. Neg: Red butt/slant
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| How do you read the Hydrogen Sulfide Production Test? | 1. Pos: Black precipitate in agar 2. Neg: No black precipitate; Bubbles: Gas production
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| What happens chemically in the Gluc/Lac Fermentation Tests if the bacteria can ferment carbohydrates? | If the bacteria can ferment carbohydrates, it will start to use up the glucose, and then the lactose, producing acid byproducts and lowering the pH
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| What happens chemically in the Gluc/Lac Fermentation Tests if the bacteria can NOT ferment carbohydrates? | If the bacteria cannot ferment carbohydrates, it will use up the peptone, producing alkaline products, and raising the pH
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| What happens chemically in the Hydrogen Sulfide Production Test if bacteria can reduce sulfur? | If the bacteria can reduce sulfur, the ferrous sulfate will react with it to produce the black precipitate
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| What do the results mean in the Glu/Lac Ferm & Hydrogen Sulfide Prod Tests? | Fermentation is metabolic process by which org mol acts as e- donor (becoming oxidized in the process) and 1+ of its organic products act as final electron acceptor.
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| What is the medium used in the Citrate Test? | Simmons Citrate Agar
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| What are the ingredients in the Citrate Test? | 1. Sodium citrate (as sole carbon source) 2. ammonium phosphate 3. bromthymol blue dye (indicator).
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| How do you inoculate and incubate the Citrate Test? | 1. Loop inoculate or stab butt and streak slant 2. Incubate at 35°C for 48 hours
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| How do you perform the Citrate Test? | After incubation, observe color change
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| How do you read the Citrate Test? | 1. Pos: Blue (pH = 7.6) 2. Neg: No color change (pH = 6.9)
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| What happens chemically in the Citrate Test? | Bacteria that can utilize citrate convert ammonium phosphate to ammonia and ammonium hydroxide, which alkalinizes the agar, changing the medium color to blue
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| What do the results mean in the Citrate Test? | Citrate-permease can break down citrate and convert it to pyruvate, which can be converted to a variety of products. Citrate is also used in the Krebs Cycle.
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| What is the medium used in the Casein Hydrolysis Test? | Skim Milk Agar
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| What are the ingredients in the Casein Hydrolysis Test? | 1. Casein 2. Yeast extract 3. Dextrose 4. Powdered milk
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| How do you inoculate and incubate the Casein Hydrolysis Test? | Inoculate with an inch-long line down the center of the plate 2. Incubate at 35°C for 48 hours
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| How do you perform the Casein Hydrolysis Test? | After incubation, remove the plate and observe
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| How do you read the Casein Hydrolysis Test? | 1. Pos: Clearing in agar 2. Neg: No clearing in agar.
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| What happens chemically in the Casein Hydrolysis Test? | The bacteria uses casein as a source of amino acids for uptake
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| What do the results mean in the Casein Hydrolysis Test? | The presence of cesease/caseinase, which is released by the bacteria, breaks down the casein to produce a clear area around growth.
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| What is the medium used in the Starch/Amylose Hydrolysis Test? | Starch Agar
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| What are the ingredients in the Starch/Amylose Hydrolysis Test? | 1. beef extract 2. Soluble starch 3. Agar
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| How do you inoculate and incubate the Starch/Amylose Hydrolysis Test? | Inoculate with an inch-long line down the center of the plate 2. Incubate at 35°C for 48 hours
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| How do you perform the Starch/Amylose Hydrolysis Test? | After incubation, flood the plate with iodine and observe color change.
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| How do you read the Starch/Amylose Hydrolysis Test? | 1. Pos: Clearing around growth 2. Neg: No clearing around growth.
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| What happens chemically in the Starch/Amylose Hydrolysis Test? | The iodine reacts with the starch to produce a blue/black color, but if the starch is broken down by the bacteria, there will be a clear zone where there is no starch present
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| What do the results mean in the Starch/Amylose Hydrolysis Test? | If the bacteria has amalyase, it can break down starch for metabolic value since it is too large to pass through the bacterial cell membrane.
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| How is the gram stain done? | 1. heat fix slide 2. Add Crystal Violet for 1 minute 3. Add Iodine 4. Decolorize with 95% ethanol or ethanol/acetoneand 5. Add Safranin 6. Look under microscope
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| How do you read the results of the gram stain? | 1. Postive: Purple 2. Negative: Pink
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| What happens chemically in the gram stain test? | The Crystal Violet and Iodine stick to the peptidoglycan layer of gram positive cells, while the decolorizer washes away the stain from gram negative cells, which are counterstained with Safranin.
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| How is the Endospore stain test done? | 1. Heat fix slide 2. Cover the smear with bibulous paper and apply Malachite Green, steaming for 7-10 minutes (keep moist) 3. Counterstain with Safranin for 1 minute 4. Look under a microscope.
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| How do you read the results of the endospore stain test? | 1. Postive: Green 2. Negative: Pink
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| What happens chemically in the endospore stain test? | The steaming forces the Malachite Green to stain the spores, and the non-spores are decolorized with water.
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| How is acid fast stain test done? | 1. Heat fix 2. Cover smear w/bibulous paper & apply ZN carbolfuchsin stain, steaming for 5 min (keep moist) 3. Decolorize w/acid-alcohol for 20 sec 4. Counterstain with Methylene Blue for 1 min 5. Look under microscope.
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| How do you read the results for the acid-fast stain test? | 1. Pos: Reddish-Purple 2. Neg: Blue
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| What happens chemically in the acid-fast stain test? | The presence of mycolic acids in the cell walls absorbs the stain.
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| What is the medium used in the motility test? | S. Medium
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| What are the ingredients in the motility test? | Triphenyl tetrazolium chloride (TTC) as growth indicator.
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| How do you inoculate & incubate the motility test? | 1. Stab inoculate with bacterial growth 2. Incubate at 35°C for 48 hours.
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| How do you perform the motility test? | After incubation period, observe the color change.
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| How do you read the motility test? | 1. Pos: Red throughout tube 2. Neg: Red only in stab line
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| What happens chemically in the motility test? | As the bacteria move throughout the tube, the TTC indicates where they are.
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| What is the medium used in the O2 Requirement Test? | Fluid Thioglycollate Medium (FTM)
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| What are the ingredients in the O2 Requirement Test? | 1. Yeast extract 2. Casein 3. dextrose 4. sodium thioglycollate 5. L-cystine 6. Resazurin as an oxygen indicator
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| How do you inoculate & incubate the O2 Requirement Test? | 1. Loop inoculate broth 2. Incubate at 35°C for 48 hours.
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| How do you perform the O2 Requirement Test? | After incubation period, observe the growth pattern
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| How do you read the O2 Requirement Test? | 1. Aerobe: Growth at the top 2. Facultative Anaerobe: Growth at the top and throughout the tube 3. Anaerobe: Growth at the bottom of the tube.
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| What happens chemically in the O2 Requirement test? | The medium is a reducing agent that removes the oxygen from the bottom of the tube, allowing anaerobes to grow
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