BIO 204 Laboratory Quiz 2: Microbial Tests and Characteristics

extremophiles

any organism living in an ecological niche outside of normal conditions

what are the 5 basic environmental growth parameters of microbial growth?

temperature, pH, osmolarity, pressure, and oxygen

normal environmental conditions

68-104 degrees F (20-40 degrees C), pH 6-8, 1.0 atm pressure, 0.9% salt, ample nutrients

can microbes regulate their own temperature?

no, optimal growth depends on finding the correct environment

psychrophiles

14-68 degrees F, prefer cold temperatures; enzymes that require less energy, flexible membrane

mesophiles

68-104 degrees F, normal temperatures; most human pathogens

thermophiles/hyperthermophiles

105-250 degrees F, prefer hot temperatures; structurally stronger enzymes, chaperone proteins, compact membranes with stronger bonds, higher GC content in genome (more stable DNA)

pH

concentration of hydrogen ions/protons (H+) in solution

acidophiles

pH 0-5; membranes with decreased proton permeability (ex. helicobacter pylori)

neutralophiles

pH 6-8; most human pathogens

alkaliphiles

pH 8-11; sequester enzymes near core of cell (away from harsh extracellular pH)

what is vital to preventing disease?

controlling microbial growth

cleaning products to kill microbes

antiseptics and disinfectants

antiseptics

kill microbes on living material (skin); alcohol, iodine, hydrogen peroxide

disinfectants

kill microbes on non living surfaces; bleach, lysol, chlorine, detergents

UV irradiation

short wavelength light, 10-400 nm; damages microbial DNA by inducing pyrimidine dimers; commonly used in labs to sterilize euqipment

what cells are resistant to UV irradiation?

cells that repair DNA damage

antibiotics

chemical compounds that can selectively kill bacterial cells; purified from fungi or bacteria, or synthesized chemically

spectrum of activity

range of species that a given antibiotic works against; depends on target of the antibiotic

broad spectrum

works against both gram-positive and gram-negative

narrow spectrum

works against either gram-negative or gram-positive, but not both (more specific to which bacteria they kill)

selective toxicity

antibiotics need to be specific for a bacterial target that is not found in host cells

kirby-bauer antibiotic sensitivity test

uses mueller-hinton agar; non-selective, non-differential; round filter paper disks soaked with different antibiotics; antibiotics diffuse into agar during incubation

what is measured to determine antibiotic sensitivity?

the zone of inhibition, area of no growth

sensitive

killed by antibiotic

intermediate

some survival

resistant

survived antibiotic

different microbes can use different types of metabolism by?

consuming different nutrients, utilizing different enzymes, generating different metabolic byproducts

what can help identify microbial species?

identification of unique sets of enzymes using biochemical enzyme tests

catalase test

enzyme tested for catalase; breaks down hydrogen peroxide (toxic ROS) into water and oxygen; helps microbes live in environment with high oxygen concentration

how does the catalase test work?

expose cells to hydrogen peroxide, look for the production of oxygen (bubbling)

oxidase test

enzyme tested for cytochrome oxidase; transfers electrons in ETS from donor molecules to oxygen (O2); a type of oxidoreductase

how does the oxidase test work?

streak bacterial cells across pre-coated surface on a dryslide; presence indicates the microbe is highly aerobic

dryslide

surface is coated with an oxidase reagent that takes the place of O2; reagent changes color when reduced

oxidase test results

positive is a purple color and negative is no color change

starch hydrolysis test

enzyme tested for amylase; breaks down complex starch (amylose) polysaccharides into individual glucose membranes

how does the starch hydrolysis test work?

streak microbe across starch agar and expose to iodine (starch agar has starch as one of the nutrient sources)

starch hydrolysis test results

positive for amylase creates a halo and negative will have starch right up to bacteria

urease test

enzyme tested for urease; breaks down urea into ammonia via hydrolysis; production of ammonia increases pH to make environment less acidic

how does the urease test work?

urea broth is inoculated with microbe and incubated; urea broth includes a pH indicator (phenol red) that changes color when pH changes

urease test results

positive for urease is a color change to pink and negative is a color change to yellow

fermentation

electrons of NADH are added back onto pyruvate, forming organic waste products (acids, alcohols, gasses); no O2 is used

carbohydrate (sugar) fermentation tests

metabolism tested for fermentation of sugars (glucose, lactose, and sucrose)

what are the main sugar fermentation products?

acid and gas (depends on enzymes found in the microbial species)

how do carbohydrate fermentation tests work?

grow microbe in a broth of the specific sugar, look for production of acids (color change/phenol red is pH indicator) and gasses (bubbles)

durham tube

inverted tube in broth that collects gas bubbles for visualization

carbohydrate fermentation test results

positive for fermentation is yellow and has air bubbles and negative is red/orange and no air bubbles

methyl red test

metabolism tested for fermentation of glucose; produce multiple acids: acetic acid, formic, acid, other organic acids; more fermentation enzymes = more types of products

how does the methyl red test work?

grow microbe in glucose broth, then add pH indicator (methyl red) to look for production of several mixed acids (color change)

methyl red test results

positive for mixed acid fermentation will be red and negative will be yellow

voges-proskauer test

metabolism tested for acetoin (precursor to 2,3-butanediol)

2,3-butanediol

an organic liquid used to make plastics and pesticides

how does the voges-proskauer test work?

grow microbes in glucose broth, then add reagents to detect production of 2,3-butanediol precursors (acetoin, 2,3-butanediol can't be detected directly)

what 2 reagents are added to detect acetoin?

naphthol and potassium hydroxide (KOH); oxidize acetoin to form colorful product (diacetyl)

voges-proskauer test results

positive for 2,3-butanediol fermentation will be a dark red color and negative will have no color

methyl red and voges-proskauer tests

can be combined using MR-VP broth; inoculate together, add test reagents separately

citrase test

enzyme tested for citrase permease; breaks down citrate to eventually produce pyruvate and carbon dioxide; allows bacteria to use citrate as their carbon source

how does the citrase test work?

grow microbes on agar with citrate as sole carbon source; uses simmons citrate agar slants; degradation of citrate in media increases the pH; pH indicator is bromothymol blue

citrase test results

positive for citrase is blue and negative is green

staphylococcus

gram-positive, clusters of round cells; part of the human microbiome (skin); facultative anaerobes, halotolerant

streptococcus

gram-positive, chains of round cells; part of the human microbiome (oral, gut, skin); aerotolerant but use fermentation

grouping staphylococcus and streptococcus is based on?

hemolysis of RBCs; uses blood agar plates (5% sheeps blood), a differential media

alpha hemolysis

partial lysis of RBCs produces greenish halo on agar

beta hemolysis

full lysis of RBCs produces clear halo on agar; contains most pathogens

gamma hemolysis

no lysis of RBCs means agar stays dark red

tests to distinguish staph from strep

catalase test: ability to breakdown toxic hydrogen peroxide into water and O2; positive = bubbles and negative = no bubbles

mannitol salt agar (MSA) test

identifies staph species; trait tested for manitol fermentation; produces acids when fermented

how does the MSA test work?

grow microbe on mannitol salt agar, look for growth and production of acids (color change); phenol red is pH indicator

mannitol salt agar (MSA)

selective because of high salt (7.5%) --> only staph species are halotolerant; strep will not grow

differential because of mannitol (1%) fermentation

MSA test results

positive for mannitol fermentation is yellow and negative is red

optochin sensitivity test

allows us to distinguish between alpha-hemolytic strep species; uses blood agar plates

optochin

antibiotic that interferes with microbial ATP production

how does the optochin antibiotic sensitivity test work?

streak out lawn of bacteria, add optochin-soaked disk, measure zone of inhibition after growth

optochin sensitivity test results

sensitive is 14 mm or greater and resistant is less than 14 mm

bacitracin sensitivity test

allows us to distinguish between beta-hemolytic strep species; uses blood agar plates

bacitracin

antibiotic that interferes with peptidoglycan synthesis

how does the bacitracin sensitivity test work?

streak out lawn of bacteria, add bacitracin-soaked disk, measure zone of inhibition after growth

bacitracin sensitivity test results

sensitive is any zone of inhibition and resistant is no zone of inhibition