Lab Exam 1 Flashcards
(28 cards)
Eosin Methylene Blue Agar - 15
Purpose: to differentiate and select Gram - microbes (coliforms). Ex. E.coli
Dyes/pH Indicators/Reagents: eosin methylene blue dyes (acidic)
Fermentation: lactose
Pos. Results: green metallic colonies (when acids produced by lactose fermentation react with eosin methylene blue dyes)
Neg. Results: colorless or pink colonies
Water Coliform Test (DSPRLB tubes & EMB plate) - 15
Purpose: isolation of fecal coliforms
Dyes/pH Indicators/Reagents: double-strength Phenol red lactose broth (acidic)
Phenol Red Both Results: contains a Durham tube for trapping gas produced during bacterial growth
- Yellow broth, bubble in tube = lactose fermentation with acid and gas end products
- Yellow broth, no bubble in tube = lactose fermentation with acid and products, no has produced
- Red broth, no bubble in tube = no fermentation
- Pink brother, no bubble in tube = degradation of peptone; alkaline end product
EMB Results: select a tube with gas to streak a EMB plate
- Poor growth or no growth = inhibited by eosin methylene blue (gram-positive)
- Good growth = not inhibited by eosin methylene blue (gram-negative)
- Growth is pink and mucoid = ferments lactose with little acid production (possible coliform)
- Growth is pink (purple, with or without green metallic sheen) - ferments lactose and/or sucrose with much acid production (probable coliform)
- Growth is colorless - no fermentation of lactose or sucrose (non-coliform)
Starch Hydrolysis - 111
Purpose: to determine if an organism can breakdown starch into glucose which requires the enzyme amylase
Dyes/pH Indicators/Reagents: uses Lugol’s Iodine (acidic)
Fermentation: utilizes starch as an energy source after it has been broken down into glucose
Pos. Results: clear zone around organisms due to the presence of amylase (breaks down starch into glucose). Lugol’s iodine will not react with glucose
Neg. Results: dark zone around organism due to the reaction of starch and Lugol’s iodine (organism did not produce amylase)
Gelatin Hydrolysis Test - 89
Purpose: gelatin is a protein derived from collagen. The gelatin test is used to determine a microbe’s ability to produce gelatinases.
Dyes/pH Indicators/Reagents:
- Nutrient gelatin differs from other solid media because the solidifying agent (gelatin) is also the substrate for enzymatic activity
Pos. Results:
- Gelatin is liquid = gelatinase is present
Neg. Results:
- Gelatin is solid = no gelatinase is present
Catalase
Purpose: identify organisms that produce enzyme catalase
Dyes/pH Indicators/Reagents: uses hydrogen peroxide (acidic)
Pos. Results: bubbles produced when oxygen is released. Hydrogen peroxide breaks down catalase
Neg. Results: no bubbles
Simmons Citrate - 80
Purpose: determines the ability of an organism to use citrate as its sole carbon source
Dyes/pH Indicators/Reagents: Bromthymol blue (acidic)
Fermentation: utilizes citrate as a carbon source
Pos. Results: agar slant turned from green to blue due to sodium carbonate reaction with bromthymol blue. In conjunction, you will see bacterial growth on slant
- Blue; small amount of growth = citrate is utilized
- No color change; growth = citrate is utilized
Neg. Results: agar slant remains green and no bacterial growth
- No color change; no growth = citrate is not utilized
Carbohydrate Metabolism - 86
Purpose: determine the ability of the organism to ferment various carbohydrates
- an organic molecule acts as an electron donor and one or more of its organic products acts as the final electron acceptor
Dyes/pH Indicators/Reagents: Phenol red broth (acidic) + durham tube
Fermentation: sucrose, lactose, glucose
Results:
- Yellow, bubble in tube = fermentation with acid and gas end products
- Yellow, no bubble in tube = fermentation with acid and end products; no gas produced
- Orange red, no bubble in tube = no fermentation
- Pink, no bubble in tube = degradation of peptone; alkaline end products
Whats a coliform?
Gram-negative, rod (bacillus) shaped, facultative anaerobe (can grow with or without oxygen)
Fermentation: lactose with the production of acid and gas
Often used to test sanitary of quality of water, food, contamination of fecal matter, etc.
Present in digestive tract of animals and found in their waste products, also found in plant and soil material
SIM test - 113
Purpose: tests for sulfur reduction, motility and indole production which means the organism was to metabolize tryptophan
Dyes/pH Indicators/Reagents: Kovac’s Reagent
Fermentation: Sulfur
Pos. Results:
- Black in medium = sulfur reduction (Hydrogen Sulfide is produced)
- Red/Magenta in medium = presence of indole (due to kovac’s reagent). Tryptophan is broken down into indole and pyruvic acid
- Growth radiating outward from the stab line = motility
Neg. Results: no black color, no magenta color (didn’t metabolize with tryptophan when Kovac’s was added), no turbidity (clear)
Methyl Red-Voges-Proskauer (MR-VP) - 119
Purpose: MR indicates an organism’s ability to produce large amounts of acid from glucose. The VP test is used to detect the presence of acetoin.
Dyes/pH Indicators/Reagents: Methyl red dye (acidic) & V-P uses potassium hydroxide and α-naphthol
Fermentation: glucose
Methyl Red test Results:
Pos. Results:
- Red = produced large amounts of acid (mixed acid fermentation)
Neg. Results:
- No color change = no production of large amounts of acid (no mixed acid fermentation)
VP Test Results:
Pos Results:
- Red = acetoin produced (2,3-butanediol fermentation)
Neg. Results:
- No color change = acetoin is not produced
Gram Stain (Differential Stain) - 57
Purpose: distinguishes between bacteria with thick peptidoglycan cell walls (gram +) and those with thinner cell walls and an outer membrane (gram -)
Dyes/pH Indicators/Reagents:
- Primary Stain: crystal violet (basic) (60 seconds)
- Mordant: Gram’s iodine (60 seconds)
- Decolorizer: Acetone Alcohol (until clear but no longer than 30 seconds)
- Counterstain: Safranin (basic) (60 seconds)
Pos. Results: retain the primary stain, crystal violet so it appears purple/blue
Neg. Results: loses the primary stain during decolorization so it appears safranin due to the counterstain, which is pink/red
Acid-Fast Stain (Differential Stain) - 63
Purpose: whether or not a cell has an extra lipid layer with mycolic acid (most bacteria cells do not have an extra layer). Extra layer is used for protection against phagocytosis
- Used to identify bacteria in the genus Mycobacterium
Dyes/pH Indicators/Reagents:
- Primary stain: Carbolfuschin (basic) (60 seconds)
- Mordant: Heat (5 min)
- Decolorizer: Acid alcohol (until clear)
- Counterstain: Methylene Blue (basic) (60 seconds)
Pos. Results: maintains primary stain of carbolfuschin, stays red
Neg. Results: loses primary stain and appears blue from the counterstain
Endospore Stain (Special Stain) - 66
Purpose: detect the presence and location of endospores in bacterial cells
- Keratin is the chemical structure in the spore coat that also resists staining
Dyes/pH Indicators/Reagents:
- Primary: Malachite green (basic) (60 seconds)
- Mordant: Heat (5 min)
- Decolorizar: Water
- Counterstain: Safranin (basic) (60 seconds)
Fermentation: vegetative cells undergo spore formation
Pos. Results: endospores appearing green
Neg. Results: vegetative cells appearing pink/red
Negative Stains - 50
Purpose: stains the background of a smear, leaving the bacterial cells colors. Allows for visualization of their morphology (shape and size) and arrangement
Dyes/pH Indicators/Reagents: India Ink (acidic)
+ Results: cells are negatively charged. The India Ink is also negatively charged, so the cell will repel it. The background will appear black but not the cell.
Capsule Stain - 65
Purpose: differential stain used to detect cells capable of producing an extracellular glycocalyx (capsule)
- increases virulence making them less vulnerable to phagocytosis
Dyes/pH Indicators/Reagents:
- Spreader - place one drop and slide across
- Air dry
- Crystal Violet (basic) (2 min)
- Copper Sulfate (10 drops)
Pos. Results: the capsule remains unstained and appears as a white halo between the cells ad the colored background
Neg. Results: no capsule
Flagella Stain - 69
Purpose: to visualize and identify bacterial flagella (helps with cell motility–movement)
Dyes/pH Indicators/Reagents: wet mount of preparation of a spirillum & viewed with phase contrast
- A mordant assists with coating
Results: the flagella are stained and reveals whether a bacteria has a flagella or not. Eukaryotic microbes tend to have 1 flagella, while prokaryotic microbes tend to have multiple
How does UV light inhibit bacterial growth?
Damages DNA through the formation of pyrimidine dimers, which disrupts the DNA replication and transcription
- Take off the lid of the plate so the UV light doesn’t get absorbed into the lid
How does salt inhibit bacterial growth?
Reduces water activity of the environment –> osmosis: dehydrates the bacteria. Creates a hypertonic environment since bacteria will have a higher water concentration inside their cells.
How does heat inhibit bacterial growth?
Denatures proteins and damages cell structures, including the membrane and nucleic acids.
High temperatures can damage DNA and RNA, hindering the ability to replicate and function
What is a zone of inhibition? How can you determine if a bacterium is resistant or susceptible to an antibiotic using the zone of inhibition?
The clear area that forms around an antibiotic disk on an agar plate where bacterial growth has been prevented.
- A larger zone = bacterium is likely susceptible (effective)
- Small or no zone = the bacterium is likely resistant (not effective)
Disk Diffusion Test
Purpose: examines the effects of various chemical disinfectants on microbial growth
Results:
Size of zone of inhibitions
Antibiotic Disk Diffusion Test (Kirby-Bauer Method) - 291
Purpose: used to measure the effectiveness of antibiotics and other chemotherapeutic agents on pathogenic microorganisms. In many cases, it is an essential tool in prescribing the appropriate treatment for a patient.
Dyes/pH Indicators/Reagents:
- Muellar-Hinton agar: pH between 7.2 and 7.4, poured into a depth of 4 mm.
- The depth is important because it effects the diffusion.
- Thick agar slows lateral diffusion and this produces smaller zones
Results:
- As the drug moves through the agar, it established a concentration gradient. If the organism is susceptible to it, a clear zone (zone of inhibition) will appear around the disk where the concentration is high enough to stop the growth.
Lipid Hydrolysis (Lipase test)/Spirit Blue Agar Test (Plate) - 93
Purpose: to identify if the bacteria produce the enzyme lipase (hydrolizes triglycerides (fats) into glycerol and fatty acids) – breakdown lipids
Prep for Spirit Blue Agar Test: emulsion with tributyrin oil, yeast extract, spirit blue dye
Fermentation: lipase
Pos. Results: a clear or light-colored halo zone around the bacterial colonies where lipase activity is present = lipase present
Neg. Results: no halo or zone = lipase absent
Decarboxylase Test - 83
Purpose: determines if the bacteria can produce enzymes that remove carboxyl groups from specific amino acids
Dyes/pH Indicators/Reagents:
Lysine decarboxylase tubes & decarboxylase control tubes
+ mineral oil
pH = bromocresol purple (acidic)
Fermentation: alkaline byproducts that raise the pH of the medium
Pos. Results:
- Purple: decarboxylation (accumulation of alkaline)
Neg. Results:
- Yellow: glucose fermentation, no decarboxylation
- No color change: does not ferment glucose, no decarboxylation
