Micro Lab Midterm

Brightfield microscope

Uses light to light up the background while the microorganisms appear dark

Compound microscope

Uses two lenses to magnify object observed

How are each of the objective lenses different?

Different resolutions and magnification

Scanning objective lens

4x

Low power objective lens

10x

High dry objective lens

40x

Oil immersion objective lens

100x

How can you calculate the total magnification?

magnification of ocular lens x magnification of objective lens

Procedure when putting microscope away

-lower stage

-remove slides

-wipe off oil on oil immersion lens

-put 4x lens in position

-turn off microscope, unplug, store cord in back

-turn knob head so eyepiece is facing the back

Culture media

liquid, broth, solid, or semisolid

broth media are dispensed in

tubes or flasks

solid agar can be poured in

test tubes, slanted tubes, or sterile petri dishes

agar deep

Agar is poured in test tube

agar slant

agar is poured in slanted tubes

agar plate

agar is poured in petri dish

chemically defined media

organism’s exact nutritional needs and chemical composition are known

complex media

complex substances and exact composition is unknown

enrichment media

complex media with additional additives for fastidious organisms

pure culture

only one microorganism

mixed culture

more than one microorganism

aseptic transfer

subculturing to transfer by sterilizing instruments under sterile condition to prevent contamination

Why is it important to keep things sterile?

prevent contamination from outside sources

How do you separate a mixed culture of bacteria?

streak plate technique

streak plate technique

reduce number of organisms on surface of agar into individual colonies

cocci

spheres

bacilli

rods

vibrios

curved rods

spirilla/spirochetes

spirals

pleomorphic

irregular

diplo-

pairs

staphylo-

grape-like

strepto-

chains

tetrad

quartets

sarcina

cube (2 tetrads)

Heat fix

denature cellular proteins, allowing amino acid side chains to adhere to slide, preventing cells from washing off cell during staining

negative staining

sample does not need to be heat fixed

disadvantage of heat fixation

cells are killed, cannot be observed in natural state

reagents in Gram stain

crystal violet, iodine solution, ethanol, safranin

role of crystal violet in gram stain

primary stain

role of iodine in gram stain

fixes crystal violet to cell wall

role of ethanol in gram stain

decolorizer

role of safranin in gram stain

counter stain

gram-positive bacteria result

thick peptidoglycan layer stains purple

gram-negative bacteria result

thin peptidoglycan layer stains pink

gram stain procedure

-crystal violet stains cells purple

-iodine solution forms insoluble complex with crystal violet and reinforces attachment of crystal violet dye to negatively charged groups in cell wall, membrane, and cytoplasm of bacteria

-ethanol dehydrates cell wall, trapping crystal violet or rinsing it away

- safranin stains cells red that were decolorized from ethanol

What happens if there is too much decolorizer added in gram stain?

removes crystal violet from gram positive cells, allowing them to stain pink

What happens if there is not enough decolorizer added in gram stain?

gram-negative bacteria will not lose color and remain purple

What happens if the bacteria is too old in gram stain?

gram-positive bacteria is unable to hold crystal violets and will appear pink

gram-positive bacteria

S. epidermidis, S. aureus, S. pneumoniae, S. pyogenes

gram-negative bacteria

E. coli, K. pneumoniae, Y. enterocolitica, S. enterica

Selective media

selects and isolates specific bacteria containing chemical substance that allows for specific growth of a particular organism, while inhibiting growth of another

examples of selective media

EMB, MacConkey

What type of bacteria do EMB and MacConkey media select for?

gram-negative

Differential media

contains chemical compounds that allow bacteria to distinguish between each other based on reactions to compounds in the media

examples of differential media

blood agar, EMB for lactose and sucrose fermentation

Selective/differential media

selective and differential media in one, inhibit and select for specific bacteria and help differentiate between bacteria that grows

examples of selective/differential media

MSA, EMB, MacConkey

Hemolytic activity

break down of red blood cells

nonhemolytic

produce no lysis of red blood cells, no change in surrounding medium, avirulent

alpha hemolytic

incomplete lysis of red blood cells, green halo around organisms, green color change in media

beta hemolysis

complete lysis of red blood cells, clear halo around colonies

catalase test

differentiate between staphylococcus and streptococcus

catalase

enzyme that converts hydrogen peroxide into water and oxygen

process of catalase test

drop of hydrogen peroxide on slide

positive catalase result

bubbles, oxygen is released

negative catalase result

no bubbles, no oxygen released

MSA (Mannitol Salt Agar)

selective/differential with high salt concentration and pH indicator

In MSA, organisms must tolerate what to grow?

high salt concentration