Micro lab 1

microbiota

bacteria found in the body that play role in development and homeostasis

resident bacteria

microorganisms that colonize an area from months to years (immune system)

transient bacteria

microbes that are temporarily present (direct contact and don’t multiply)

Bacteriostatic

stops the growth of certain microbes (handwashing)

bactericidal

kills microbes (handwashing)

Discard in sharps only

microscope slides, cover slips, glass pipettes, other sharp

autoclavve bags in white bucket

petri dish and disposable tubes

glass tubes

remove labels and place in racks

agar deep tubes

make pour plates and rinse; place in racks with other tubes

dilution bottles

autoclave tubes andd plates

sterile

absent of microorganisms

sterilize bottles and test tubes

flame mouth

sterilize loop and needle

hold in flame until bright red

sterilize plates

use lid as shield

subculture

agar slant

used for bacterial storage

agar plate

isolate, calculate, study bacteria characterisistics

tsb broth

used for fresh bacteria or for large numbers

pure culture

single type of organism growing in absence of other organism

mixed colony

contains two or more bacteria species growing in same environment

bacterial colony

visible mass of bacteria seen in agar

Quadrant streak

used when bacteria has a high cell concentration

Zig zag streak

sample suspected of having low sample concentration

spread plate method of isolation purpose

isolate bacteria species and quantify

how to do spread plate method of isolation

serial dilution then pour plate method (pipette + sterile medium added; should be even)

ubiquitous

found everywhere

4 necessary macromolecules

lipids, sugars, nucleic acids, and protein

fastidious

depend on environment to obtain organic material; picky

non fastidious

less dependent on environment for organic material

heterotrophs

eat other organisms to obtain energy

autotrophs

make their own energy

how to distinguish fastidious vs. non fastidious

if growth on undefined complex media, probably fastidious compared to a chemically defined media

subculture

transfer of bacteria from one media to another

example of complex media

Nutrient Agar TSA/TSB

complex media

exact composition unknown

defined media

chemical amount and identity known

resolution

the ability of a microscope to distinguish details of a specimen

resolving power

limit of resolution on LM 0.2 um

microscope is called a 3-lens system

eyepiece lens, condenser lens, and objective lens

calculate magnificatino

(ocular lens magnification)(magnification of objective lens)

oil immersion lens

used for 100x; clean microscope with lens paper; oil minimize light loss

as magnification increases

working distance decreases

smear

thin layer of bacteria added to microscope slide

purpose of heat fixation

adhere and kill and coagulate proteins

basic stain

positively charged; stains bacterial cells with negative membrane (crystal violet, methylene blue, safranin)

acidic stain

negatively charged; stains background (nigrosin, india ink, congo red)

why is gram stain called a differential stain

uses more than one dye to differentiate between gram-negative and positive bacteria

gram positive

more teichoic acid (negative charge) and peptidoglycan (resist decolorization) trap crystal violet

gram negative

more lipid dissolved with a thin peptidoglycan layer (less resistant to decolorization)

primary dye of gram stain

crystal violet

gram stain mordant

gram iodine; enhance CRYSTAL-violet

decolorization of gram stain

95% ethanol or ethanol/acetone solution; most important step as gram negative becomes colorless

gram stain counter stain

safranin; gram-negative turn pink

factors that affect gram stain

too much/little exposure to decolorizer', smear preparation, age of bacteria (24 hrs. max.)

Acid-fast stain purpose

differentiate bacteria from mycobacterium, nocardia and diagnose tuberculosis

Acid fast positive

red rods; contains mycolic acid (waxy layer)

acid fast negative

blue bacilli (no waxy layer)

Zhiel Nelson

heat (steam) as mordant

Kinyoun method

cold method (lipid soluble stain)

first step of acid-fast

prepare heat-fixed smear and put filter paper + Ziehl’s carbolfuschin stain over steam (mordant) for 5 minutes then rinse

second step of acid-fast

rinse with acid alcohol (decolorize)

third step of acid-fast

add methylene blue (counterstain) for one minute and blot dry

purpose of capsule stain

differentiate between bacteria that have capsules

capsule

polysaccharide structure with neutral charge

no heat fixation in capsule stain

avoid damage to capsule, cell is already adhered to slide with serum

positive results for capsule stain

use acidic stain in capsule stain

to stain background

use basic stain

to stain bacteria (not capsule)

sheep serum

adhere bacteria to slide in capsule stain

stains in capsule stain

congo red (negative stain) and Maneval’s stain (positive)

purpose of endospore stain

differential stain for organisms that produce endospores

endospore

dormant version of bacterial cell (unsuitable growth conditions); highly resistant; green

vegetative cell

active form of bacterial cell; red pink

how are endospores made

when vegetative cell is placed in unfavorable conditions they sporulate as a survival mechanism and become endospores and can germinate back into a vegetative cell

primary dye of endospore stain

malachite green

mordant of endospore stain

steam

counterstain of endospore stain

safranin

psychrophiles

below 20 degrees celsius

psychotrophs

fluctuate between 0 and 30 degrees celsius

mesophiles

20 to 40 degrees celsius

thermophiles

above 40 degrees celsius

extreme thermophiles

65 to 110 degrees celsius

acidophile

below pH 5.5

neutrophile

range pH 5.5-8.5

alkaliphile

over pH 8.5

changes in pH in acidophiles

acid tolerance response if pH drops below 5.5 protein synthesis pumps (H+) out; if pH drops below 4.5 acid-shock and heat-shock proteins synthesized

changes in pH in neutrophiles

exchange potassium (antiport)

changes in pH in alkiphiles

exchange internal sodium ion for external protons

osmosis

movement of water from an area of low solute to high solute concentration

halophiles

grow in salt concentration of 3% or higher

extreme halophiles

Require a salt concentration from 15 up to 25%

osmotolerant

Grow in a wide range of salt concentration typically 3% or less

halotolerant

non-halophilic microorganisms, able to grow at high salt concentrations

antiseptic

antimicrobial substance that can be applied to living tissue

disinfectant

kills microorganisms on nonliving skin

percent reduction

((i-f)/i) x 100

zone of inhibition

area surrounding that has no bacterial growth (mm)

what UV wave can kill organisms?

UV-C (alters DNA)

Light Repair (photoreactivation)

DNA photolyase monomorizes dimer (reverse reaction)

Excision Repair (Dark repair)

UvrABC detects UV light damage, hellicase removes DNA, DNA Poly. 1 adds missing nucleotides, DNA ligase glues gap (phosphate bonds)