BIOL240L exam

What is the purpose of a simple stain?

determine cell morphology

what stains were used for a simple stain?

methylene blue, basic fuchsin, crystal violet

What is the purpose of the Gram stain?

differentiate between gram-positive and gram,-negative bacteria

what colour are gram-positive organisms?

purple

why are gram positive organisms purple?

gram positive organisms are not easily decolourized, so they retain the purple stain

what colour are gram negative organisms?

red or pink

why are gram negative organisms red or pink?

they are easily decolourized, so they only retain the safranin

what are some reasons that a gram-positive organism may appear gram-negative or gram-variable?

culture is old

in an acid medium

T/F only fresh culture should be used for the gram stain

True

can iodine be added before primary stain in a gram stain?

No. Iodine causes crystal violet to bond to the peptidoglycan in the cell walls, preventing the crystal violet from being washed off.

what is a mordant?

substance which fixes primary stain in bacterial cells

what does acid-fast stains identify

tuberculosis bacillus, mycobacterium tuberculosis, mycobacterium leprae

why is an acid-fast stain used over a simple stain in some situations?

some bacteria do not stain with simple and gram stain, mycobacterium

are acid-fast bacteria gram-positive or gram-negative?

gram-positive

example of an acid-fast negative bacteria

staph aureus

example of an acid-fast positive bacteria

mycobacterium phlei

why would mycobacteria walls be “waxy” in nature?

high levels of mycelia acids

why is a bacteria called “acid-fast”?

organisms ability to retain a primary stain when treated with acid

which method was used in class?

kinyoun method or cold stain

what colour will a gram-positive organism be after decolorization in an acid-fast stain?

purplish-blue colour

what is the purpose of the secondary stain?

The main aim objective of this stain is to differentiate bacteria into acid fast group and non-acid fast groups

what colour was staph aureus?

blue, negative

what colour was mycobacterium phlei?

pink/purple, positive

what is the purpose of the spore stain?

identify size and presence of spores

what is done differently with heat fixing for the spore stain?

heat fix 20 times instead of usual 3

does the negative stain require heating?

no

after endospore staining, the colour of the vegetative cells will appear _____?

red

why are we using the cold method instead of the hot method?

can endospores be stained by conventional methods, such as a simple stain?

no

when is a negative stain ineffective?

large bacterial populations(smear), bacterial species excrete high concentration of exogenous neg. charged products.

what is a benefit of the negative stain?

safe for heat fragile bacteria, does not cause heat distortion

what bacteria is tested with the negative stain?

bacillus brevis

is nigrosin an example of a cationic or anionic dye?

anionic

what colour is the bacteria in an negative stain

clear

what is the purpose of the capsule stain?

reveal the presence of the bacterial capsule

what does the capsule consist of?

polysaccharides, polypeptides and/or carbs

What is the reason behind adding the lysis solution after resuspending the cells?

This is what allows the plasmid DNA to be separated from the bacterial chromosome.

Why do you want to avoid transferring the white precipitate to the spin column? What \n would you suggest we do in order to avoid disrupting the white precipitate?

If too much precipitate is included in the transferred liquid, the column will get clogged and the purification will fail

Why is it important that we do not skip the wash solution step?

What is the purpose of the elution buffer?

what is selective media?

contains specific chemicals which do not affect the growth of the organism you wish to isolate, but discourage the growth of other microorganisms

differential media

contains dyes/chemicals which allow the observer to distinguish between types of bacterial colonies that have developed after incubation

what is enriched media?

some microorganisms require specific nutrients such as vitamins and other growth promoting substances

what is the appearance of Ea on EMB?

lots of growth on outside, looks like bubbles

what is the appearance of Ef on KF strep?

slight growth, red in colour

what does EMB agar inhibit the growth of?

gram positive, staph e

what is the purpose of the streak plate method?

obtain isolated colonies from an innoculum by creating areas of increasing dilution on a single plate.

what did the streak plate colonies of staph e look like?

circular/punctiform

what did the streak plate colonies of e coli look like?

circular/punctiform

what did the streak plate colonies of the mixed bacteria look like?

punctiform

what is the morphology of staph e?

cocci

what is the morphology of e coli?

rod-shaped

what would a gram stain of a mixed population (staph a & e coli) look like?

gram variable, staph a is gram positive and e coli is gram negative

what is the importance of pure culture?

allow for the study of one species without the worry of contamination from other organisms.

what is the purpose of the pour plate method?

This technique is used to isolate microbial colonies by serial dilution and then counting the colony forming units

how to calculate CFU

number of colonies/ 100 x dilution factor+ 2

why are capsules difficult to stain>

Because most capsules are so tightly packed, they are difficult to stain because most standard stains cannot penetrate the capsule.

what does sterile mean?

free of all life, including viruses

what are signs that a broth is not sterile?

murky/cloudy, opaque

what are signs a broth is sterile?

clear, transparent

what are the two types of sterilization?

physical and chemical

examples of physical sterilization?

heat, radiation, filtration

what is the standard plate count method?

counts the number of viable organisms present in a sample

What are some advantages to SPC?

counts only viable organisms, accurate for samples with low bacterial loads

What are some disadvantages to SPC?

some organisms die from contact with molten agar, colonies may have developed from a single cell or cluster of cells, it is impossible to tell the difference

what do motile bacteria possess?

flagella

what is Brownian motion?

bacteria that look like they are moving but are not

A sign of true motility?

zig-zag pattern

why is the hanging drop method used to look for motility?

the pressure of a normal slide would stop movement

why do we not stain bacteria when testing for motility?

it would kill the bacteria

what is the purpose of the bacterial flagella experiment?

determine the mode of flagellar insertion of any flagellated bacteria

what is peritrichous flagella

flagella that is all around the cell

what is polar flagella

flagella that gathers at one side or opposite sides of the cell

What is the benefit to using a DNA ladder when conducting gel electrophoresis?

Without using a DNA ladder, built by DNA fragments of known sizes, we would be \n unable to estimate the size of our DNA running through the gel. The ladder acts as a \n reference point that allows us to determine what the size of our DNA might be

You are cautioned, when pouring the agarose into the casting, to avoid bubbles. Why \n do you think that bubbles in the gel would interfere with the electrophoresis?

The basis of the gel is that the DNA you’ve extracted and run go through the pores of \n the agarose gel; the migration of the DNA fragments will be impeded by the presence of \n bubbles in the gel

what is the purpose of the nutrition of microorganisms experiment?

determine minimal growth requirements for certain microorganisms.

what does L planetarium need for growth?

nitrogen

what bacteria had the ability to grow in just agar?

b subtitles and p fluorescens

what does e coli require for growth?

nitrogen and carbs

fermentation of carbs:

some bacteria can ferment simple carbs, producing acidic, alcoholic or gaseous end products in the process

production of indole:

indole is a by-product of the metabolic breakdown of the amino acid tryptophan, if indole is present it combines with the reagent to produce a red colour

activity of urease:

some bacteria can split the urea molecule releasing carbon dioxide and ammonia. This can be detected as the pH becomes alkaline and the indicator becomes dark pink

production of hydrogen sulfide:

this is produced when amino acids containing sulfur are metabolized by microorganisms. if the medium contains metallic ions, the hydrogen sulfide formed during growth combines with metallic ions to form a metal sulfide that blackens the medium.

what bacteria produces hydrogen sulfide?

p vulgaris

in carb fermentation, what does a purple result mean?

neutral

in carb fermentation, what does a yellow result mean?

acid production

in carb fermentation, what does a dark blue result mean?

alkaline production

what bacteria produces a positive indole result?

e coli

what colour indicates indole production?

red/pink ring

what colour indicates urease activity?

pink

what bacteria has urease activity?

p vulgaris

what does catalase positive mean?

production of oxygen, bubbles have formed

what is the purpose of temperature on microbial growth experiment?

observe the effects of temps on levels of microbial growth and pigment production of different bacteria

at what temperature did an grow best

20-25 degrees

what bacteria grew best at 55 degrees

bacillus stear

what does hypotonic mean?

low solute content

what does hypertonic mean?

high solute content

what was the purpose of the pH experiment

find the optimum concentration of hydrogen ions in which it grows best

what bacteria grew best in high sodium concentration?

halosalinarium

what bacteria grew best in low sodium concentrations?

e coli