What are the three types of media for isolating bacteria?

selective, differential and enrichment

3 common dilution techniques

pour plate
spread plate
streak plate

True or false. Diluting may exclude some bacteria that are low in numbers.

true
only predominant bacteria may be observed
special media may be used to prevent this

Enrichment media is used to

enrich the growth of microbes of in low numbers or that are outcompeted by other bacteria that grow faster.
Nutrients are added to enhance specific microbes growth- desired microbes increase in #; others not affected

Differential media

different bacteria will look different on special media plates

Selective media

apply substances that inhibit undesired microbes or for bacteria that we want to flourish

Enrichment Media characteristics:

Typically broths
contain chemicals that ENHANCE the growth of desired bacteria
other bacteria grow also

Selective Media characteristics:

contain chemicals that prevent the growth of unwanted bacteria or microbes without inhibiting growth of desired bacteria or microbes

Differential media characteristics:

media used to distinguish one bacterium from another
visual differences as microbes grow

Nutrient Agar is

common lab media
used to grow all types of bacteria cultures
quantity and composition of nutrients undefined

Nutrient agar contains:

peptone- partially digested protein
salt less than 1%
agar- solidifying agent from algae/ powdered form
distilled or deionized water
sometimes sugar is added

Mannitol Salt Agar characteristics

selective and differential
NaCl selective for Staphylococcus
-few other can tolerate the high salt concentration (E-coli can do this sometimes)
Differential:
-mannitol fermenters ---> turns yellow due to acid production
-mannitol non-fermenters ---> no color change of agar

Mannitol contains-

mannitol
NaCl (7.5%)
peptone
phenol red
agar

MSA is selective for?

salt

MSA is differential for?

to differentiate between pathogenic and non-pathogenic staph

Which staph is pathogenic?

Staphylococcus aureus

The MSA plate is selective against

all bacteria except staph

What is phenol red?

pH indicator
yellow pH < 6.8 turns yellow-acidic
Stays red pH 7.4-8.4 stays same
Pink > 8.4

If the mannitol is yellow

the staph aureus has metabolized the mannitol creating an acid

MSA plate:
poor growth or no growth

organism is inhibited by NaCl and is not Staphylococcus

MSA plate:
good growth no color change

Organism is not inhibited by NaCl and is non-pathogenic staph

MSA plate:
Yellow growth or halo

organisms produces acid from mannitol fermentation
and is possible Staphylococcus aureus

MSA plate:
Red growth (no halo)-growing well

organism does not ferment mannitol
nonpathogenic mannitol

True or false. Any yellow color is from the metabolizing of the mannitol and creating an acid.

true

MSA is a conclusive test or presumptive?

presumptive

Mannitol salt fermentation

Organism metabolizes the mannitol during cellular respiration causes fermentation of the mannitol creating an acid causing a yellowing or halo- converted to acid and Staph aureus break down

Eosin is a

negative stain- only stains the background

Methylene blue is a

simple stain

Eosin Methylene Blue (EMB) is

selective and differential

EMB is used for the

isolation of fecal coliforms and differentiate between different coliform bacteria

EMB is selective for:

selective against gram (+) - only see gram (-) growing on these plates

EMB is differential for:

in sugars coliforms can ferment lactose and are gram (-)they might be other gram (-) on plate but they can't ferment the lactose.
if they ferment lactose it turns pink
do not ferment lactose - no color change

Coliforms that ferment lactose on EMB turns it

pink

True or false. All coliforms are gram (-).

true

Which organism ferments lactose aggressively and doesn't turn pink due to the aggressive fermentation but metallic green or black.

E-coli

Most coliforms are found in the?

intestinal tract of humans and animals

Coliforms are gram (-) or gram (+)

(-)

Coliforms belong to the ____________ family.

Enterobacteriaceae

True or false coliforms ferment lactose?

true

They can survive with or without O2 so they are?

aerobic or facultative anaerobes

Coliforms are endospore formers or nonendospore formers?

nonendospore formers

Coliforms produce ____ and _____ within 48 hours at 37*C

gas and acid

True or false several members are pathogens that cause diarrhea.

true

Positive for coliform could be potential_____.

fecal contamination

EMB has no ______ ________.

pH indicator

EMB plate:
poor growth or no growth

organism is inhibited by eosin and MB
gram positive

EMB plate:
Good growth

organism not inhibited by eosin or MB
gram negative

EMB plate:
growth is pink

organism ferments lactose with little acid production
possible coliform

EMB plate:
growth is dark with or without metallic sheen

organism ferments lactose and/or sucrose with much acid production
probable coliform

EMB plate:
growth is colorless (no pink, no dark or metallic sheen)

organism does not ferment lactose or sucrose
noncoliform

EMB plate is selective against ________ and differential for _______ and _________.

gram (+)
diff. between coliform and non-coliform

MacConkey agar is

selective and differential

MC agar is selective

bile salts and crystal violet against gram (+)
inhibits gram (+)

MC agar is differential

differentiate between coliform (lactose fermenters) and non-coliforms (non-lactose fermenters)

The pH indicator for MCagar is

neutral red

In MC agar if there is a drop in pH it turns

red-pink in color

MC agar:
poor or no growth

organism is inhibited by crystal violet/ and or bile salts
gram (+)

MC agar:
good growth

organism is not inhibited by bile salts and/or crystal violet
gram negative

MC agar:
pink to red growth with or without bile precipitate

organism produces acid from lactose fermentation
probably coliform

MC agar:
Growth is colorless not red or pink

organism does not ferment lactose
non-coliform

The procedure for exercise 12:

4 different agar plates for the known bacteria: Ps. aeruginosa, staph aureus, staph epidermidis, E-coli
EMB
MC agar
Nutrient
MSA

4 different plates for the unknown bacteria: mixed culture
Section each plate off in 1/4s and streak each one with a different microbe (for each diff. agar)
For the other 4 plates streak the plate with a different microbe

What is the purpose of peptone in the agar?

To provide nutrients and agar to the bacteria- it is their source of carbon and nitrogen and to facilitate the culture's growth

What is the purpose of agar in the media?

To solidify the bacteria in the media

What ingredient makes mannitol salt selective?

salt (NaCl)

Growth on EMB:
(+)
metallic green
colorless

E-coli gram
Ps. aeruginosa

Growth on EMB/MSA
(-)
yellow
white

Staph aureus
Staph epidermidis

Nutrient agar that contains vancomycin is

Agar containing vancomycin will only allow vancomycin-resistant organisms to grow.
selective

EMB agar that contains sorbitol is

differential to differentiate ecoli 0157