Microbio Lab Practical

total magnification

ocular x objective

parfocal

remains in focus when changing magnification

working distance

distance between objective lens and slide

Field of view

area viewed through ocular

most appropriate magnification for bacteria

oil

most appropriate magnification for fungi

10X or 40X

ubiquitous

they are everywhere

brightfield microscopy

low contrast

dead, stained cells

phase-contrast microscopy

high contrast

living, unstained cells

increasing the contrast between the cells and their background without staining them

telescopic eyepiece

used to align the phase rings of the microscope

streaking for isolation purpose

place inoculum, then decrease concentration over a set of streaks

coccus

circle

bacillus

rod-shaped

spirillum

spiral

coccus

single-celled

diplococci

pairs

tetrad

group of 4 cocci

streptococci

chain-like morphology

staphylococci

grape-like cluster

Brownian motion

bombarded with water molecules

Streaming

follow convection currents, nutrients

True motility

any direction

flagella

organelle that drives bacterial motility

monotrichous

one flagella at the end

lophotrichous

multiple flagella at one end

petrichous

multiple flagella at all ends

amphitrichous

multiple flagella at two poles

hanging drop slide

• small amount of Vaseline placed near each corner of the cover glass with a toothpic

• two loopfuls of organisms are placed in the cover glass

• depression slide is pressed against Vaseline on cover glass and quickly inverted

• completed preparation can be examined under oil immersion

semisolid media

0.4% agar

agar slants

1.5% agar

motility is visualized by

Tetrazolium dye

reacts with living organisms to turn red

simple staining

use of a single strain to color a bacterial cell

Gram stain is an example of a

differential stain

differential stain

use of multiple stains or dyes to differentiate cells or cellular structure

bacterial smear preps

• adhere cells to the microscope slides so they aren’t washed off during staining and washing steps

• ensure shrinkage doesn’t occur so cell morphology does not differ

What cellular structures absorb crystal violet dye?

peptidoglycan and lipids

iodine is a

mordant: fixes the dye to the peptidoglycan

alcohol does what to the cell

decolorizes the cell by dissolving lipids

Gram positive

thick peptidoglycan layer, only one cell membrane

Gram negative

two membranes (outer and inner), thin peptidoglycan layer

Gram staining common mistake

• if you wash your cells with too much alcohol they might mistakenly appear Gram negative

• rod shaped cells may look round (like cocci) if heated too much

Gram staining steps

1. crystal violet

2. Gram’s iodine

3. ethanol

4. Safranin

capsule stain

stains around the gelatinous capsule lauer

spore stain

stains the durable endospores

acid-fast stain

low pH dissolves the waxy outer layer, allowing the cells to absorb stain

what conditions limit bacterial growth?

• temperature

• desiccation

• oxygen

• radiation

• acidity

• pressure

• chemicals

• nutrients

obligate aerobes

requires O2 for growth/survival

cellular respiration

microaerophiles

prefer low concentrations of O2

2-10% O2

facultative anaerobes

can grow with or without O2

cellular respiration and fermentation

obligate anaerobes

cannot tolerate O2

aerotolerant anaerobes

tolerate oxygen but don’t perform cellular respiration

anaerobic jar

• for culturing obligate anaerobes

• oxygen is removed from chamber by combining with hydrogen to form water

  • this reaction is catalyzed by palladium pellets

psychrophiles

< 15 C

psychotrophs

15-20 C

often associated with food spoilage

mesophiles

20-45 C

most human pathogens

thermophiles

65-79 C

hypothermophiles

> 80 C

thermodurics

survive but do not grow at high temperature

What is the pigment that makes Serratia marcescens red?

prodigiosin

temperature regulated

osmosis

the movement of water across a membrane due to solute concentration

osmotic pressure

the force required to prevent water from moving across a membrane

hypotonic

solute concentration higher inside the cell, water moves into the cell

isotonic

solute concentration same inside and outside the cell, water moves out from the cell

halophiles

10% NaCl or higher

halotolerant

can handle 5-10% NaCl

osmophiles

high sugar concentrations

plasmolysis

when cytoplasm of a hypertonic cell recedes from the cell wall

acidophiles

pH below 7

neutrophiles

neutral pH, around 7

alkaliphiles

pH above 7

defined media

you know exactly everything that is in it

complex media

general compounds put into the media, but don’t know what all components are down to the molecular level

allows the growth of many types of organism

selective media

contains a growth inhibitor for a specific group of organisms

usually a dye, specific chemical, NaCl, pH or an antibiotic

organisms resistant to the inhibitor will grow

differential media

usually contains a pH indicator in some way differentiates between physiological types of organisms

enriched media

compounds are added to further supplement the nutrients in complex media for fastidious organisms

autoclave

huge pressure cooker

most media

baking

oven, dry heat

glassware

filtration

filter

heat labile solutions

chemical methods

phenolics, alcohols, halogens, quaternary ammonium compounds, aldehydes, ethylene oxide gas

radiaion

UV at 260 nm

plasticware, antibiotics, vitamins, food

volumetric

defined volume

serological

measured to the end of the tip

blow out

measuring

not measured to the tip

do not blow out

micropipettes

used for transferring smaller volumes

standard plate count

serial dilution

numbers between 30 - 300 colony-forming units are considered statistically valid

CFU/mL =

# of colonies * DF / inoculum

spectrophotometer measures

the absorbance/optical density of a culture

turbidity of a culture is

measured and relates to growth

spread plate

• inoculate onto agar surface

• spread with glass hockey stick

• surface colonies only

pour plate

• inoculate onto bottom of empty petri plate

• inoculum into plate before getting melted agar tube

• wipe water off outside tube

• pour melted agar over inoculum swirl gently in figure 8 to mix well

• pour plate - both large surface colonies and small eliliptical subsurface colonies count booth

EMB is an example of ____ and ____ mediaa

selective and differential

two dyes in EMB are

eosin and methylene blue

EMB is selective because

methylene blue kills gram-positive bacteria

so you are selecting for Gram-negative

EMB is differential because

eosin changes color (dark purple) under acidic conditions → lactose fermentation produces acid, lowers pH

• Gram-negative bacteria that ferment will look dark

• E. coli will look metallic green

• non-fermenters

Endo agar is slightly ____ and ______ media

selective and differential

endo agar has a

fuchsin sulfite indicator

• fermenters will have red/pink colonies, non-fermenters will be colorless

endo agar is

slightly selective because of the sodium sulfite and basic fuchsin in the media inhibits growth of gram-positives

presumptive test

MPN value determination

MPN value determination

identifies the presence of lactose fermenting coliforms like E. coli

endospores of the Bacillus and Clostridium genera are resistant to

high temperatures