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