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Questions and Answers
What is the primary purpose of the Gram stain technique?
Which of the following bacteria stain positive for the acid-fast stain?
What does a positive result in the carbohydrate fermentation test using phenol red indicate?
In coliform testing, what is the significance of performing a definitive test after a presumed test?
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Which of these is NOT a method used to test for starch hydrolysis?
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What is the purpose of the Kirby-Bauer method?
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What does SIM stand for in microbiological testing?
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What does a positive result indicate in the catalase test?
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Which of the following bacteria can be identified through the endospore stain?
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In the context of carbohydrate fermentation tests, a positive result for lactose fermentation is indicated by what color change in phenol red?
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What does a positive result in the gelatin hydrolysis test indicate about the bacteria?
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Which chemical is typically used in the Gram stain procedure to develop the color of the cells?
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What is the function of the immersion oil in microscopy?
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What type of results would you expect from a positive citrate test?
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In the context of coliform testing, what does the appearance of green shiny colonies on EMB media signify?
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What is the purpose of the decarboxylase test in microbiological studies?
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Study Notes
Lab Safety
- Follow all lab safety rules
- Dispose of materials properly
Microscope
- Parts, magnification, measuring (unit size will be given), function of parts, focusing, use of immersion oil
Bacteria Morphology
- Bacillus cereus: Rod shaped
- Staphylococcus aureus: Round shaped, clusters
- Escherichia coli: Rod shaped
Common Lab Techniques
- Fixing bacteria to a slide: Heat fixation is used to kill bacteria and attach them to the slide.
- Aseptic technique: Prevents contamination of cultures and the environment.
- Streak plate method: Used to isolate individual colonies of bacteria.
Gram Stain
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Steps:
- Primary stain (Crystal violet)
- Mordant (Gram's Iodine)
- Decolorizer (Ethanol or acetone)
- Counter stain (Safranin)
- Chemicals Used: Crystal violet, Gram's iodine, ethanol or acetone, safranin
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Results:
- Gram-positive: Purple
- Gram-negative: Pink/red
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Testing: Differentiates bacteria based on their cell wall structure.
- Bacillus cereus: Gram-positive
- Staphylococcus aureus: Gram-positive
- Escherichia coli: Gram-negative
Acid Fast Stain
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Steps:
- Primary stain (Carbolfuchsin)
- Mordant (Heat)
- Decolorizer (Acid alcohol)
- Counter stain (Methylene blue)
- Chemicals Used: Carbolfuchsin, acid alcohol, methylene blue
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Possible Results:
- Acid-fast: Red/pink
- Non-acid-fast: Blue
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Testing: Detects the presence of mycolic acid in the cell wall of bacteria.
- Mycobacterium tuberculosis: Acid-fast positive
Endospore Stain
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Steps:
- Primary stain (Malachite green)
- Mordant (Heat)
- Decolorizer (Water)
- Counter stain (Safranin)
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Possible Results:
- Spores: Green
- Vegetative cells: Red/pink
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Testing: Detects the presence of endospores, a resistant form of bacteria.
- Bacillus cereus: Endospore positive
Coliform Testing
- Presumed test: Detects the presence of coliform bacteria using lactose fermentation.
- Definitive test: Confirms the presence of coliform bacteria through specific biochemical tests.
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Results for definitive tests:
- Positive: Indicates coliform bacteria are present.
- EMB media: Coliforms produce a metallic sheen on EMB media.
- Testing: Presence of coliform bacteria, which are indicators of fecal contamination.
Carbohydrate Fermentation
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Phenol red tests:
- Glucose: Ferments glucose, producing acid, which turns the medium yellow.
- Sucrose: Ferments sucrose, producing acid, which turns the medium yellow.
- Lactose: Ferments lactose, producing acid, which turns the medium yellow.
- Positive results: Yellow color, positive carbohydrate fermentation.
- Negative results: Red color, no carbohydrate fermentation.
- Testing: Determines which sugars a bacterium can ferment.
Catalase Test
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Possible results:
- Positive: Bubbles indicate the presence of catalase, which breaks down hydrogen peroxide.
- Negative: No bubbles.
- Testing: Detects the production of catalase.
Decarboxylase Test
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Possible results:
- Positive: Yellow color indicates the production of decarboxylase, which breaks down amino acids.
- Negative: Purple color.
- Testing: Detects the production of decarboxylase, which breaks down amino acids, producing an alkaline byproduct.
Starch Hydrolysis Test
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Possible results:
- Positive: Formation of a clear zone around the bacterial growth indicates the production of amylase, which breaks down starch.
- Negative: No clear zone.
- Testing: Detects the production of amylase, which breaks down starch.
Gelatin Hydrolysis Test
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Possible results:
- Positive: Liquid indicates gelatin hydrolysis by bacterial gelatinase.
- Negative: Solid.
- Testing: Detects the production of gelatinase, which breaks down gelatin.
Citrate Test
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Possible results:
- Positive: Blue coloration indicates the use of citrate as a carbon source.
- Negative: Green color.
- Testing: Detects the use of citrate as a carbon source.
Spirit Blue Test
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Possible results:
- Positive: Formation of a blue halo around the growth indicates the presence of lipase, which breaks down fats releasing fatty acids.
- Negative: No blue halo.
- Testing: Detects the production of lipase, which breaks down fats.
SIM Test
- What it stands for: Sulfide-Indole-Motility agar
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Possible results:
- Sulfide production: Black precipitate.
- Indole production: Red ring after adding Kovac's reagent.
- Motility: Diffuse growth throughout the medium.
- Testing: Detects the production of hydrogen sulfide, indole, and motility.
MR-VP Test
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Possible results:
- Methyl red (MR) positive: Red color.
- VP (Voges-Proskauer) positive: Pink to red color after adding VP reagents.
- Testing: Detects the production of mixed acids (MR test) or acetoin (VP test) as end products of glucose fermentation.
Kirby-Bauer Method
- Testing: Identifies antibiotic susceptibility patterns of bacteria.
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Enzymes produced by bacteria in tests:
- Amylase: Breaks down starch
- Catalase: Breaks down hydrogen peroxide
- Decarboxylase: Breaks down amino acids
- Gelatinase: Breaks down gelatin
- Lipase: Breaks down fats
- Protease: Breaks down proteins
Lab Safety
- Follow all lab safety rules and regulations.
- Dispose of materials and chemicals properly.
Microscope
- Parts: Identify components, including ocular lens, objective lenses, condenser, diaphragm, stage, and fine/coarse focus knobs.
- Magnification: Calculate total magnification using objective lens and ocular lens magnifications.
- Measuring: Use a calibrated ocular micrometer to estimate size of objects.
- Focusing: Use coarse adjustment knob for initial focus, then fine adjustment for precise focus.
- Immersion Oil: Use immersion oil with the highest magnification objective (100x) to improve clarity.
Bacteria Morphology
- Bacillus cereus: Rod-shaped bacteria.
- Staphylococcus aureus: Spherical bacteria arranged in clusters.
- Escherichia coli: Rod-shaped bacteria.
Common Lab Techniques
- Fixing Bacteria to a Slide: Heat fixation kills bacteria and attaches them to the slide.
- Aseptic Technique: Techniques to prevent contamination of cultures & prevent the spread of microbes.
- Streak Plate Method: Method to isolate pure cultures of bacteria by spreading a sample across a petri dish.
Gram Stain
- Steps: 1. Crystal violet staining. 2. Iodine treatment. 3. Decolorization with alcohol. 4. Safranin counterstain.
- Chemicals: Crystal violet, iodine, alcohol, safranin.
- Results: Gram-positive bacteria appear purple, gram-negative bacteria appear pink.
- Testing: Distinguishes bacterial cell wall types.
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Organism Results:
- Bacillus cereus: Gram-Positive.
- Staphylococcus aureus: Gram-Positive.
- Escherichia coli: Gram-Negative.
Acid-fast Stain
- Steps: 1. Carbol fuchsin staining (heated). 2. Acid-alcohol decolorization. 3. Methylene blue counterstain.
- Chemicals: Carbol fuchsin, acid-alcohol, methylene blue.
- Results: Acid-fast bacteria appear red, non-acid-fast bacteria appear blue.
- Testing: Detects presence of mycolic acid in cell walls.
- Positive Bacteria: Mycobacteria.
Endospore Stain
- Steps: 1. Malachite green staining (heated). 2. Water decolorization. 3. Safranin counterstain.
- Results: Spores appear green, vegetative cells appear pink.
- Testing: Detects presence of endospores, resistant structures formed by some bacteria.
- Positive Bacteria: Bacillus, Clostridium.
Coliform Testing
- Presumed Test: Detects presence of coliform bacteria (e.g., Escherichia coli) in water samples by utilizing media that allows coliforms to ferment lactose with gas production.
- Definitive Test: Confirms the presence of coliform bacteria by identifying specific biochemical characteristics.
- Results: Positive results indicate presence of coliforms.
- Coliforms on EMB Media: Coliforms produce metallic green sheen on EMB agar.
- Testing: Assess the safety of water sources.
Carbohydrate Fermentation
- Phenol Red Broth: Contains a sugar (glucose, sucrose, or lactose) and indicator dye, phenol red.
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Results:
- Positive: Yellow color indicates acidic fermentation.
- Negative: Red color indicates no sugar fermentation.
- Testing: Detects ability of bacteria to ferment specific sugars.
Catalase Test
- Steps: Place a drop of hydrogen peroxide on a slide, add bacteria.
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Results:
- Positive: Production of bubbles indicates catalase enzyme is present.
- Negative: No bubbles indicate catalase is absent.
- Testing: Detects presence of catalase enzyme, which breaks down hydrogen peroxide to water and oxygen.
Decarboxylase Test
- Media: Contains amino acid, indicator dye, and base.
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Results:
- Positive: Yellow color indicates decarboxylation of amino acid.
- Negative: Purple color indicates no decarboxylation.
- Testing: Detects presence of decarboxylase enzyme, which removes carboxyl group from amino acids.
Starch Hydrolysis Test
- Media: Contains starch.
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Results:
- Positive: Formation of a clear halo around bacterial growth indicates starch hydrolysis.
- Negative: No halo indicates starch is not hydrolyzed.
- Testing: Detects presence of amylase enzyme, which breaks down starch to simpler sugars.
Gelatin Hydrolysis Test
- Media: Contains gelatin.
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Results:
- Positive: Liquid medium after incubation indicates gelatin hydrolysis.
- Negative: Gelatin remains solid after incubation.
- Testing: Detects presence of gelatinase enzyme, which breaks down gelatin.
Citrate Test
- Media: Contains citrate as the only source of carbon.
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Results:
- Positive: Blue color indicates citrate utilization.
- Negative: Green color indicates no citrate utilization.
- Testing: Detects ability of bacteria to utilize citrate as a carbon source.
Spirit Blue Test
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Results:
- Positive: Blue color indicates presence of lipase enzyme.
- Negative: No blue color indicates absence of lipase.
- Testing: Detects presence of lipase enzyme, which breaks down lipids.
SIM Test
- SIM: stands for sulfur indole motility.
- Media: Contains cysteine, tryptophan, ferrous sulfate, and a semisolid agar.
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Results:
- Black precipitate: Production of hydrogen sulfide (H2S)
- Red ring: Production of indole from tryptophan.
- Diffuse growth: Motility detected.
- Testing: Detects production of H2S, indole, and motility.
MR-VP Test
- Media: Contains glucose.
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Results:
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Methyl Red (MR) Test:
- Positive: Red color indicates production of acids.
- Negative: Yellow color indicates no acid production.
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Voges-Proskauer (VP) Test:
- Positive: Red color indicates production of acetoin.
- Negative: No color change.
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Methyl Red (MR) Test:
- Testing: Tests for different metabolic pathways: MR detects mixed acid fermentation. VP detects butanediol fermentation.
Kirby-Bauer Method
- Method: Antibiotic sensitivity testing that uses antibiotic discs on a petri dish inoculated with bacteria.
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Results: Zones of inhibition around antibiotic discs indicate sensitivity to the antibiotic.
- Larger zones of inhibition mean greater sensitivity.
- Testing: Determine antibiotic susceptibility of bacteria.
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Description
Test your knowledge on lab safety rules, microscope usage, and bacteria morphology in this quiz. You'll also cover common techniques like heat fixation and Gram staining. Prepare to demonstrate your understanding of essential lab practices and bacterial identification.
