Microbio Lab Midterm 1

Aspectic technique

combination of skills that is necessary for work safety and prevent contamination

Example of Aseptic Techniques

• passing loop in flame

• flaming the mouth of tubes/flasks

• not placing lid/cap on the table

culture

media that contain microbes

contamination

unwanted microorganism in the culture, sample, or sterile enivronments

Sterile

completely free from bacteria

Pure culture

contain one type of microorganism

Mixed culture

contain two or more types of microorganism

contaminated culture

contain unwanted organism that supposed to be pure culture

turbid

cloudy appearance in liquid culture

colony

a visible cluster of microorganism that grow on solid nutrient medium

inoculate

process of introducing microorganism in a suitable environment for growth and study

independent variable

what you change in the experiment

dependent variable

what effect that change

liquid medium

broth

solid medium

agar (visible mass)

3 types of solid medium

1. plates

2. slants

3. semi-solid agar

defined media

the exact formula of the chemicals is known

complex media

at least one ingredient in the media whose exact chemical makeup is unknown

general purpose media

contain general nutrients for most organisms to grow

enriched media

contain additional nutrients to allow certain organisms to grow

selective media

prevent growth of some organisms while promoting others to grow

differential media

distinguish between types of microorganisms based on their ability to carry out biochemical reaction

to obtain fresh, active growing culture

broth

to get isolated colonies

petri plate

to preserve a culture for later use

agar slant

to see a characteristic (ex: motility)

semi solid deep

How do you obtain a pure culture?

streak plate isolation method by spreading microorganism on the surface of solid growth medium and thinning it out

light microscopes

use light in order to see image

brightfield

producing a darker image using a light background

darkfield

see a light image on a dark background

electron microscopes

use electron beams to visualize image

transmission electron microscope

tend to be more ideal for visualizing internal structures

scanning electron microscope

visualizing the 3D surface

40x

4500 um

100x

1800 um

400x

450 um

1000x

180 um 

staining

applied color to see certain feature of a specimen before seeing under a microorganism

uncolored ion

counterion

positive charged ion

basic dye

negatively charge

acidic dye

positive stain

a dye that absorbed by the cells/organisms

• positive charge of stain is attracted to negative charge on cell surface

• use heat fixed

negative stain

dye the background (producing an outline of the organisms)

• negative charge of stain is repelled from negative charge of cell surfaces

bacilli

rod shaped

cocci

spherical cell

spiral

spiral, vibrio (comma shape)

diplo

two

strepto

chain

staphylo

clusters

gram staining

help distinguish the bacteria based on their cell wall

capsule staining

combine simple and negative stain that allow us to see bacteria capsule

vegetative cells

sensitive to extreme temp and radiation 

endospores

resistant to harsh environment

obligate aerobes

need oxygen (20-25%)

obligate anaerobes

kill by oxygen

facultative anaerobes

need or no oxygen

aerotolerant

no oxygen

microaerophillic bacteria

low oxygen

obligate aerobes (pattern)

top only

obligate anaerobe (pattern)

bottom only

facultative anaerobes (pattern)

mostly top, some bottom

aerotolerant (pattern)

even growth

microaerophillic bacteria (pattern)

band below surface

Phenlethyl Alcohol Agar (PEA)

• inhibits Gram-negative

• Allow Gram-positive to grow

• inhibitor: phenyl ethyl alcohol

Bile Esculine Agar (BEA)

Selective / Differential Media

• bile (inhibitor)

• inhibit most Gram-positive

• allow organisms that can grow in bile

What is BEA trying to observe?

bacteria that have enzyme esculinase

What is the substrate for BEA?

esculin

What is the indicator for BEA?

Bile

BEA postive test

turn black

BEA negative test

nothing

Blood Agar

Differential Media

• try to observe hemolysin

Blood Agar substrate

RBC

Indicator Blood Agar

RBC

positive test blood agar

use all RBC

negative test blood agar

doesn’t use all RBC

Mannitol Salt Agar (MSA)

selective and differential

inhibitor: 7.5% concentration NaCl

• inhibit most bacteria (non-halophilic)

• grow halophilic bacteria (salt tolerant)

what is MSA trying to observe?

mannitol

substrate MSA

mannitol

inhibitor (differential - MSA)

phenol red - if a bacteria can ferment mannitol, it will produce acid byproduct turning the phenol red to yellow

positive test MSA

yellow

negative test MSA

remain red/pink

Catalase Test

Differential

• detect the enzyme catalase

• SOD + Catalase break down hydrogen peroxide to produce water and O2

• during aerobic metabolism, the toxic oxygen damage DNA so SOD and catalase help detoxifies

positive test catalase

bubble

catalase negative

no bubble

capsule primary stain

negative stain (congo red)

capsule secondary stain

crystal violet

gram primary stain

crystal violet

gram stain mordant

gram’s iodine (form crystal violent complex: fixing primary color)

gram stain decolorizer

ethanol - turn gram negative colorless

gram stain secondary

safranin - get the gram negative

acid fast primary stain

carbol fushsin

acid fast mordant

heat (get the color in the cell)a

acid fast decolorizer

acid alcohol - get rid stain off vegetative cellsa

acid fast secondary color

methylene blue - stain vegetative cells

endospore primary stain

malachite green

endospore mordant

heat (steam) - open up pore to get stain into the cellse

endospores decolorizer

water - remove stain off vegetative cells

endospore secondary

safranin - stain vegetative cells