Term
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Definition
1) Non-selective - most organisms can grow (general purpose media)
2) Selective - Inhibits growth of some groups, but allows for growth of others.
3) Differential - Allows for identification of certain species or groups of organisms.
4) Enrichment - Used to isolate organisms from complex samples. |
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Term
| Solidifying agent used in our lab |
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Definition
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Term
| Difference between broth and solid media |
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Definition
| Broth does not contain agar; solid media does |
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Definition
| Inanimate object that can harbor pathogens |
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Term
| Ubiquity of microorganisms |
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Definition
| Microorganisms are EVERYWHERE |
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Definition
| Methodology used to reduce contamination of cultures, people, and workspace |
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Definition
| To introduce a microorganism into |
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Term
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Definition
| Pellicle, Turbidity, Sediment, and Flocculent |
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Term
| Benefits of a digital pipette |
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Definition
| Allows the experimenter to transfer precise volumes of liquid from one container to another. |
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Term
| Number of cells a colony arises from |
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Definition
One
(i.e. 1 colony = 1 cell) |
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Term
| Understand the microscope and total magnification |
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Definition
| See PowerPoint from Lab 2 |
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Term
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Definition
Serves to:
1) Isolate pure colonies of bacteria from a mixed culture of bacterial species
2) Observe morphology of the isolated colonies for identification and classification |
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Term
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Definition
Used to evenly distribute bacterial cells over entire plate surface (2 possible results):
1) Spread concentrated samples to create an even lawn of bacterial growth
2) Spread diluted sample of bacteria so individual colonies can be seen
*100μL is the standard amount used for spread plates* |
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Term
| What is used with 100X on our microscopes |
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Definition
| Immersion oil (or Ooyle, if you're from AL) ;) |
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Term
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Definition
Draw a circle on the back of a clean slide with marker
Flame loop
If making smear from an agar plate:
-Apply a couple of drops of water to slide and smear a small amount of bacteria in the water
-Flame loop
If making smear from a broth culture:
-Take a loopful of bacteria and smear on the slide (No Water)
-Flame loop
Make sure to spread bacteria evenly over most of the slide surface to create a thin smear
Air dry smear completely
Apply enough methanol to cover and fix the smear, let sit for 5-10 seconds
Rinse with water |
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Term
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Definition
Fixing serves 2 purposes:
1)Adheres the cells to the slide
2) Kills the organism |
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Term
| Difference between simple and Gram staining |
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Definition
| Simple uses only ONE stain, while Gram uses a primary and a secondary stain. |
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Term
| Gram stain steps and components |
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Definition
Step 1: Flood slide with Crystal Violet for 30 seconds - 1 minute
Wash with H2O
-Acts as Primary Stain
Step 2: Flood slide with Gram’s Iodine for 30 seconds - 1 minute
Wash with H2O
-Acts as Mordant
Step 3: Decolorize with Acetone alcohol for a few seconds
Wash with H2O
-Acts as Decolorizer
Step 4: Flood slide with Safranin for 30 seconds - 1 min
Wash with H2O
-Acts as Secondary Stain |
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Term
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Definition
| Differentiate between Gram-positive and Gram-negative bacteria. Also allows determination of (1) cell morphology, (2) size, and (3) arrangement. |
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Term
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Definition
+ = Blue (purple)
- = Pink |
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Definition
| Allows bacterium to survive in poor environmental conditions |
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Term
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Definition
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Term
| What characteristics can be determined from simple stain |
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Definition
Cellular morphology
Cellular arrangement |
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Term
| Cell wall component that dictates Gram reaction |
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Definition
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Term
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Definition
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Term
| Endospore staining procedure (Schaeffer-Fulton method) |
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Definition
Step 1: Flood slide with Malachite Green
-Acts as Primary Stain
Step 2: Heat over steam bath for 5 min
-Acts as Mordant
Step 3: Rinse with Water for a few seconds
-Acts as Decolorizer
Step 4: Flood slide with Safranin for 2 min
-Acts as Secondary Stain |
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Term
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Definition
Used primarily to stain & identify members of the genera Mycobacterium and Nocardia
-A differential stain used to detect the presence of mycolic acids in the cell wall
-Cells typically don’t stain well with Gram Stain method
-Acid-fast organisms stain pink and non-acid fast organisms stain light blue with these methods |
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Term
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Definition
Used to visualize gelatinous coating that surrounds some bacteria
SMEAR IS NOT HEATED or the capsule melts!
Background is stained by one of the following stains:
(a) Nigrosin
(b) India Ink
(c) Congo Red
Counterstain with a basic stain to stain cells
*Important to use older cultures so capsules have time to form* |
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Term
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Definition
Many bacteria are motile due to production of flagella
Flagella are very fine, hair-like structures that are difficult to stain due to their small size and delicate nature
Requires the addition of mordant and stain to increase the diameter of flagella for microscopic observation |
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Term
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Definition
Monotrichous (single polar)
Lophotrichous (clump of tuft at one or both poles)
Amphitrichous (single flagella at each pole)
Peritrichous (covered) |
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Term
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Definition
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Term
| Spore-producing famililes |
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Definition
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Term
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Definition
Selective Agent: NaCl (Salt)
-Selects for Staphyloccus spp.
Differential Agent: Mannitol
-Differentiates pathogenic from non pathogenic Staphylococcus spp.
pH Indicator: Phenol Red
-Indicates acid production due to mannitol fermentation |
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Term
| Phenylethyl Alcohol Agar (PEA) |
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Definition
Selects for Gram positives
-Growth of Gram negative organisms should be reduced or inhibited
Selective Agent: Phenylethyl Alcohol
Not Differential |
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Term
| Eosin Methylene Blue Agar (EMB) |
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Definition
Selects for Gram negatives
-Growth of Gram positive organisms should be reduced or inhibited
Selective Agents: Eosin Y & Methylene Blue
Differential Agent: Lactose
-Differentiates lactose fermenters from lactose non fermenters
Indicator: Eosin Y
-Indicates acid production due to lactose fermentation
E. coli is easily identified by the production of green, metallic sheen |
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Term
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Definition
Selective for Gram negatives
-Growth of Gram positive organisms should be reduced or inhibited
Selective Agents: Crystal Violet & Bile Salts
Differential Agent: Lactose
-Differentiates lactose fermenters from lactose non fermenters
pH Indicator: Neutral Red
-Indicates acid production due to lactose fermentation |
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Term
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Definition
Selective agent: None
Differential agent: Blood |
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Term
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Definition
β (beta) hemolysis - complete lysis of RBCs
α (alpha) hemolysis - partial lysis of RBCs
γ (gamma) hemolysis - no lysis of RBCs |
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Term
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Definition
Identifies organisms that produce the enzyme catalase
Differentiates families of Gram positive cocci
-Catalase (+) Micrococcaceae from
-Catalase (-) Streptococcaceae
Substrate: hydrogen peroxide
(+) test: bubbles form immediately
(-) test: no bubbles |
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Term
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Definition
Identifies bacteria that produce the respiratory enzyme cytochrome c oxidase
Differentiates families of Gram negative rods
-Oxidase (-) Enterobacteriaceae from
-Oxidase (+) Pseudomonadaceae
Reagent: Oxidase reagent
(+) test: blue color appears within seconds
(-) test: no color change |
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Term
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Definition
Differentiates S. aureus from other Gram-positive cocci
Substrate: rabbit plasma
(+) test: formation of white clumps
(-) test: milky white liquid (no clumps) |
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Term
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Definition
| Media: Starch Agar Plate (SA) Reagent: (Lugol's) Iodine (I2KI - Iodine Potassium Iodide)
After incubation, plate is flooded with iodine:
(-) test: Iodine binds with remaining starch in the plate to produce a dark brown/black color
(+) test: As a result of starch breakdown, a clear area or halo will be visible around the colony that produces amylase |
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Term
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Definition
Differentiates organisms based on their ability to hydrolyze urea with urease
Helpful in detecting rapid urease positive organisms such as Proteus sp. from other Enterobacteriaceae
Media: Urea agar slant
Positive reaction overcomes the buffer in the medium and changes the color of the slant
(+) test: Hot pink slant
(-) test: Peach or yellow slant
pH Indicator: Phenol Red |
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Term
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Definition
| The ability or inability of an organism to grow in the presence of molecular oxygen (O2) |
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Term
| Classificaitons of aerotolerance |
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Definition
Obligate aerobe - requires O2 for growth
Falcultative anaerobe - can grow in the presence or absence of O2
Obligate anaerobe - cannot grow in the presence of O2
Microaerophile - requires low levels of O2
Capnophile - requires elevated levels of CO2 |
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Term
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Definition
A liquid medium formulated to promote growth of a fastidious anaerobes without an anaerobe jar
-Creates anaerobic conditions by reducing oxygen to water
Oxygen Indicator: Resazurin
If oxygen present = Pink/Red
If no oxygen present = Colorless |
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Term
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Definition
Provides an anaerobic environment as a result of an added anaerobic gas pack
Oxygen indicator: Methylene Blue
If oxygen present = blue
If no oxygen present = colorless (white) |
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Term
| Most prevalent mutation caused by UV |
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Definition
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Term
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Definition
Short wave length
Mutagenic because it excites nucleic acid bases in DNA
Penetrates glass and plastic poorly
Prolonged exposure is lethal to cells |
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Term
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Definition
Used to detect and directly count fecal coliforms in water
Normally use a 0.45 µm filter (0.22-0.45)
It is very important to know the size of your target organism or compound to choose the correct filter
E. coli ~ 1 μm X 2 μm
Coliform counts are calculated per 100 mL of water
Media: Endo broth
Membrane filter is transferred to sterile Petri dish with sterile pad saturated with Endo broth |
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Term
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Definition
Coliforms & Fecal Streptococci (including Enterococci) are used as possible indicator organisms for sewage contamination.
-Coliforms are comprised of many organisms including the fecal coliform Escherichia coli.
-E. coli is a normal intestinal colonizer of humans and other warm-bloodied animals .
-The EPA recommends Total Coliform counts for drinking water, E. coli counts for recreational fresh water & Enterococci for recreational salt water. |
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Term
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Definition
Selects for Gram(-) organisms
Selective Agents: Sodium Sulfite & Basic Fuchsin
Differential Agent: Lactose
Indicators: Sodium Sulfite & Basic Fuchsin
Lactose fermenters = metallic sheen OR dark pink
Nonlactose fermenters = light pink |
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Term
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Definition
Bacterial populations can be enumerated in two ways:
(1) Direct - actual number of cells
(2) Indirect - relative amounts compared to beginning of experiment |
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Term
| Purpose of performing viable plate count |
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Definition
| To obtain actual numbers of viable cells |
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Term
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Definition
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Term
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Definition
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Term
| Countable plate considerations |
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Definition
| 30-300 CFU. Below or above this number is considered statistically unreliable |
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Term
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Definition
-Systematically reduces cell density, producing at least one dilution that will yield a countable plate
-Provides a reference to link the unknown (original cell density) with the known number of colonies on the plate
-In a serial dilution, each transfer reduces the amount of original broth in the solution
-Each dilution is given a dilution factor
-The dilution factor of each new solution is compounded by the dilution factor of the previous tube |
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Term
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Definition
| After incubation the colony forming units (CFU) are counted -The term CFU is used since more than one cell could form one colony -Only a plate with a colony count between 30-300 CFU can be used to determine OCD -Plates below or above this range are considered statistically unreliable -OCD = CFU/FDF (combination of volume plated and dilution factor = final dilution factor) -Ex: 47 CFU growing on 10-6 plate OCD = 47/10-6 OCD = 47 x 106 = 4.7 x 107 (correct scientific notation) |
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Term
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Definition
| Temperature at which growth rate is maximum (optimal) |
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Term
| Temperature classifictions |
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Definition
Psychrophiles: -10 to 20°C
Psychrotrophs: 0°C to 30°C
-Their optimum temperature falls in the mesophilic range, however, they can grow at lower temperatures at slower rates
-These are typically food spoilage organisms
Mesophiles: 10°C to 50°C
Thermophiles: 40°C to 75°C
Hyperthermophiles: >75°C |
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Term
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Definition
Series of tests used to differentiate Enterobacteriaceae
-Includes:
Indole
Methyl Red
Voges-Proskauer
Citrate |
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Term
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Definition
Media: Tryptone broth containing tryptophan
Purpose: Determine bacteria’s ability to produce indole from tryptophan
Reagent: Kovac’s Reagent (after incubation)
5 drops of reagent
(+) reaction = red ring
(-) reaction = yellow or colorless ring |
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Term
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Definition
Media: MRVP broth
Bacteria are inoculated into MRVP broth, divided after incubation
Purpose: Detects organisms capable of producing stable acid end products from mixed acid fermentation of glucose
Reagent: Methyl Red Reagent
Usually combined with VP test
(+) reaction = media turns red
(-) reaction = no color change |
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Term
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Definition
Media: MRVP broth
Bacteria are inoculated into MRVP broth, divided after incubation
Purpose: Detects production of acetoin & 2,3-butanediol
Reagents:
α-naphthol is added FIRST (mix well)
40% potassium hydroxide added SECOND (mix well)
Usually combined with Methyl Red test
(+) reaction = red color change
(-) reaction = no color change |
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Term
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Definition
Media: Simmon’s Citrate agar
Purpose: Determine bacteria’s ability to utilize citrate as sole carbon source
pH indicator: Bromthymol blue
Increase in pH = color change to blue
(+) reaction = blue agar
(-) reaction = green agar |
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