Auburn Microbiology Lab Final!

BSL 1

microorganisms not known to cause disease in healthy adults; protective equipment not required

BSL 2

indigenous microorganisms that can lead to diseases of varying severity in healthy adults; protective equipment required

BSL 3

indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission; immunizations may be required

BSL 4

microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections; rarely are there treatments or vaccines; diseases may be fatal

Brightfield microscope

simplest form of microscopy where light is either passed through, or reflected off, a specimen. Illumination not altered by devices that change properties of light; requires use of stains to visualize cells

Phase contrast microscope

converts the differences in optical density of cells into shades of brightness; allows visualization of morphology, external structures, and some internal structures; stain not required

Fluorescent Microscope

uses high intensity illumination to excite fluorescent molecules which absorb photons, are excited to a higher level, as they relax back to ground-state vibrational energy is lost and emission spectrum shifted to longer wavelengths; fluorescence emirates from sample

Dark field Microscope

Contrast is created by a bright specimen on a dark background. It is ideal for revealing morphology and external structures, but does not provide a great deal of information about internal structure. Stains are not required.

base/arm

frame of microscope

light source

LED

stage

horizontal area supporting the slide; has stage adjustment knobs

lens system

oculars, objectives, condenser

oculars

10X magnification

objectives

attached to nosepiece; magnification of 10X, 40X ( high dry), 100X (oil immersion)

total magnification

ocular power x objective power

condenser

collects and directs light from the light source to the slide; located under the stage

diaphragm

controls the amount of light that reaches the slide

focusing knobs

coarse focus knob (outer) & fine focus knob (inner)

fine focus knobs

used after reaching 40X and 100X

coarse focus knobs

only used when using 10X

immersion oil

same refractive index as glass; used with 100X objective only; fills gap between objective and slide to form a continuos lens path which increases image resolution by reducing light refraction

parfocal

ability of a microscope to remain relatively in focus when changing from lower to higher power objective

numerical aperture

mathematical expression that describes how the condenser lens concentrates and focuses the light rays from light source

resolving power

ability of a lens to show two closely spaced objects as distinct and separate

working distance

distance between the bottom of the objective lens and the slide

ubiquity of microorganisms

microorganisms are everywhere

simple stain

staining with a single stain; allows visualization of cell morphology, cell arrangement, and internal storage materials

methylene blue

stain used in simple staining

types of cell morphology

cocci, rods, spiral/curved

cocci

spherical morphology; singly, pairs, tetrads, chains, clusters

rods (bacilli)

rod shaped morphology; singly or chains

spiral/curved

corkscrew rods morphology

Hans Christian Gram

responsible for Gram stain

Gram positive

have thick peptidoglycan layer in cell wall, retain crystal violet, purple

Gram negative

thin layer of peptidoglycan in cell wall; do not retain crystal violet; pinkish (safranin)

Gram stain steps

crystal violet, Gram's iodine, ethanol, safranin

crystal violet

primary stain (60 sec)

Gram's iodine

mordant (60 sec)

ethanol

decolorizer (10 sec)

safranin

counter stain (60 sec)

endospore

allow microorganisms to survive environmental conditions that are not favorable to growth

endospore staining

spores- malachite green (green)

vegetative cells- safranin (red)

standard plate count (SPC)

most common method of determining the number of live bacteria in a sample

spread plate technique

used with serial dilutions, one of the most common standard plate count methods

colony forming units (CFU)

SPC reports; between 30-300 considered statistically valid

serial dilutions

lab procedure in which an amount of one substance is added to a sterile solvent to reduce the concentration of original substance

#cells counted x #dilutions x reciprocal of dilution factor

= # cells in original sample

complex medium

exact composition and amounts of amino acids, vitamins, and growth factors are not exactly known in this medium

defined medium

specific chemical composition is know and the individual components are weighed out exactly to make up the medium

selective medium

allow certain bacteria to grow but will inhibit others from growing

differential medium

cause some bacteria to take on an appearance that distinguishes them from other bacteria

Agar

complex polysaccharide isolated from seaweed

autoclave

heating media to 121C for at least 15 min at 15 psi of steam pressure

fastidious

slow growers requiring specific nutrients and growth conditions

E coli

found in human intestine

anaerobic

can grow without oxygen

aerobic

requires oxygen to grow

pyscrophiles

optimal growth between -5C and 20C; in icy waters

mesophiles

optimal growth between 20C and 50C; most bacteria

thermophiles

optimal growth between 50C and 80C; in soils

hyperthermophiles

optimal growth is above 80C; deep in ocean floor

psychrotrophs

bacteria that can grow at temperatures higher or lower than their optima

Proteus, Pseudomonas, Campylobacter, Leuconostoc

psychrotrophs that can grow at refrigerator temps and cause food spoilage

prodigiosin

red pigment antibiotic tested for treatment of pancreatic cancer; in Serratia marsecens

neutrophiles

grow at or near neutral pH (7)

acidophiles

grow at acidic pH values (<7)

alkaliphiles

grow at alkaline pH values (>7)

UV light

non ionizing short wavelength radiation that falls between 4nm and 400nm in visible spectrum; shorter wavelength=more damaging

obligate (strict) aerobe

must grow in oxygen

MOST Pseudomonas species

microaerophiles

aerobic bacteria that prefers 2-10% oxygen

facultative anaerobes

grows well in aerobic conditions but can also grow anaerobically when oxygen isn't available

E. coli

aerotolerant anaerobes

tolerate oxygen and grow in its presence but do not require oxygen for energy production

Streptococcus pyogenes

obligate (strict) anaerobes

are harmed or killed by oxygen

Clostridium

toxic forms of oxygen

hydrogen peroxide & superoxide

Bacitracin disk (0.02-0.05 IU)

Purpose: Differentiates microorganisms based on susceptibility to bacitracin.

Procedure: Use a swab to make a lawn streak on a TSA or SBA plate. Apply a Bacitracin disk to the center of the lawn streak plate. Gently tap the disk to secure it onto the media.

Incubate for 24 hours at 37°C then read the zone of inhibition.

Interpretation:

Susceptible: Any size zone of inhibition around the disk

Resistant: No zone of inhibition (growth up to the edge of the disk)

BHI with 6.5% NaCI

Purpose: Differentiates microorganisms based on their ability to grow in 6.5% NaCl.

Procedure: Inoculate a BHI broth containing 6.5% NaCl with a small amount of organism. Incubate for 24-48 hours at 37°C with a loose cap. Too heavy of an inoculum will give a false positive result.

Interpretation:

Positive: Growth (turbidity)

Negative: No growth (clear)

Bile esculin hydrolysis

Purpose: Differentiates microorganisms based on their ability to hydrolyze esculin to esculetin and dextrose. The esculetin reacts with ferric citrate in the medium to form a dark brown-black complex.

Procedure: Using a loop, inoculate the media by streaking in a zig-zag pattern up the slant. Incubate for 24 hours at 37°C with a loose cap.

Interpretation:

Positive: Blackening of the entire slant

Negative: No color change or less than 1⁄2 of the slant is black

catalase

degrades H2O2 to oxygen and water

EMB (eosin methylene blue agar)

Purpose: Selects microorganisms based on their ability to grow in presence of eosin Y and methylene blue. Differentiates microorganisms by their ability to vigorously ferment lactose and produce acid.

Procedure: Using a sterile loop, inoculate an EMB plate and streak for isolation. Incubate the plate at 37°C for 24 hours.

Interpretation:

Positive (metallic green sheen): Growth + / Acid ++. Vigorous lactose fermenter with excessive acid formation (primarily, but not only, E. coli)

Positive (purple colonies): Growth + / Acid +. Moderate to slow lactose fermenter with moderate acid formation (non-E. coli coliforms)

Negative: Growth + / Acid -. Pink or colorless colonies (no lactose fermentation) or no growth

Growth at 5°C (walk-in cooler)

Purpose: Differentiates microorganisms based on their ability to grow at 5°C.

Procedure: Using a sterile loop, inoculate a TSA plate and streak for isolation. Incubate the plate at 5°C for 3-5 days.

Interpretation:

Positive: Growth in quadrants 3 or 4 of plate

Negative: No growth in quadrants 3 or 4 of plate (clear)

Growth at 41°C

Purpose: Differentiates microorganisms based on their ability to grow at 41°C.

Procedure: Using a sterile loop, inoculate a TSA plate and streak for isolation. Incubate the plate at 41°C for ONLY 24 hours.

Interpretation:

Positive: Growth in quadrants 3 or 4 of plate

Negative: No growth in quadrants 3 or 4 of plate (clear)

Growth at 45°C

Purpose: Differentiates microorganisms based on their ability to grow at 45°C.

Procedure: Using a sterile loop, inoculate a TSA or SBA plate and streak for isolation. Incubate the plate at 45°C for ONLY 24 hours.

Interpretation:

Positive: Growth on plate

Negative: No growth (clear)

superoxides

converted to oxygen and H2O2 by superoxide dismutase

beta hemolysis

complete lysis of red blood cells around a colony; clear zone around colonies

alpha hemolysis

breakdown of red blood cells producing a greenish discoloration around colonies

gamma hemolysis

do not exhibit any hemolysis of blood and have no effect on red blood cells in a blood agar plate

starch hydrolysis

detected by adding gram's iodine to a starch agar plate; media turns black/brown; if starch degraded, media adjacent to growth will be clear after adding iodine

Bile esculin agar

if organism can hydrolyze esculin, media will turn dark brown or black; positive only if over half media turns dark

coagulase

insulating small tube of rabbit blood plasma with loopful of organism; clotting=positive

Positive: Coagulation of the plasma (clot formation)

Negative: No coagulation/clot formation

MSA (mannitol salt agar)

ability to grow at 7.5% NaCl and ferment mannitol; Positive: Growth+/Acid+(yellow colonies)

Negative: Growth+/Acid- or Growth-/Acid- (colorless, no yellow, no growth)

phenol red carbohydrate tubes

arabinose, lactose, trehalose, mannitol, sucrose, xylose, etc;

positive: yellow (gas produced & carb fermented)

negative: red (stays red)

KOH test

confirms gram stain on whether 3% KOH can lyse bacterial cell wall and release DNA

strings present: gram negative

no strings: gram positive

citrate

diff. based on their ability to utilize citrate as a sole carbon source and ammonium salts as sole nitrogen source

positive: growth w/ intense blue color

negative: no growth/no color change (stays green)

methyl red

diff based on ability to produce and maintain stable acid end products from glucose fermentation

positive: red color (immediate)

negative: yellow color/no color

Nitrate reduction

diff based on ability to reduce nitrate to nitrite or ammonia or nitrogenous gas

positive: red color develops

negative: no red color develops; red develops after zinc is added

Novobiocin

Purpose: Differentiates microorganisms based on susceptibility to novobiocin

Procedure: Use a swab make a lawn streak on a TSA or SBA plate. Apply a Novobiocin disk to the center of the lawn streak plate. Gently tap the disk to secure it onto the media. Incubate for 24 hours at 37°C then read the zone of inhibition.

Interpretation:

Susceptible: Zone of inhibition >=17mm

Resistant: Zone of inhibition <= 16mm

O/F glucose (oxidation/fermentation)

oxidative or fermentative metabolism of carb in tube

oxidative: yellow color in open tube only

fermentative: yellow color in both open tube and closed tube

neither: green or blue color in both open and closed tubes

oxidase

presence of enzyme cytochrome C oxidase in bacteria

positive: blue or purple in 10-30 sec

negative: no color change or blue color after 30 sec

phenylalanine

to produce phenylalanine deaminase- enzyme removes amine group from amino acid and releases as free ammonia; produces phenylpryuvic acid

positive: green

negative: yellow

Production of Red Pigment at 25°C

Purpose: Differentiates microorganisms by their ability to produce a red pigment when incubated at 25°C. Procedure: Inoculate a TSA plate and incubate at 25°C for 24 hours. Observe for red pigment (prodigiosin) production.

Positive: Red pigment

Negative: No pigment production