Microbiology Lab exercise 12- Special Media for Isolating Bacteria
selective, differential and enrichment
What are the three types of media for isolating bacteria?
pour plate
spread plate
streak plate
3 common dilution techniques
true
only predominant bacteria may be observed
special media may be used to prevent this
True or false. Diluting may exclude some bacteria that are low in numbers.
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
Enrichment media is used to
different bacteria will look different on special media plates
Differential media
apply substances that inhibit undesired microbes or for bacteria that we want to flourish
Selective media
Typically broths
contain chemicals that ENHANCE the growth of desired bacteria
other bacteria grow also
Enrichment Media characteristics:
contain chemicals that prevent the growth of unwanted bacteria or microbes without inhibiting growth of desired bacteria or microbes
Selective Media characteristics:
media used to distinguish one bacterium from another
visual differences as microbes grow
Differential media characteristics:
common lab media
used to grow all types of bacteria cultures
quantity and composition of nutrients undefined
Nutrient Agar is
peptone- partially digested protein
salt less than 1%
agar- solidifying agent from algae/ powdered form
distilled or deionized water
sometimes sugar is added
Nutrient agar contains:
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 Salt Agar characteristics
mannitol
NaCl (7.5%)
peptone
phenol red
agar
Mannitol contains-
salt
MSA is selective for?
to differentiate between pathogenic and non-pathogenic staph
MSA is differential for?
Staphylococcus aureus
Which staph is pathogenic?
all bacteria except staph
The MSA plate is selective against
pH indicator
yellow pH < 6.8 turns yellow-acidic
Stays red pH 7.4-8.4 stays same
Pink > 8.4
What is phenol red?
the staph aureus has metabolized the mannitol creating an acid
If the mannitol is yellow
organism is inhibited by NaCl and is not Staphylococcus
MSA plate:
poor growth or no growth
Organism is not inhibited by NaCl and is non-pathogenic staph
MSA plate:
good growth no color change
organisms produces acid from mannitol fermentation
and is possible Staphylococcus aureus
MSA plate:
Yellow growth or halo
organism does not ferment mannitol
nonpathogenic mannitol
MSA plate:
Red growth (no halo)-growing well
true
True or false. Any yellow color is from the metabolizing of the mannitol and creating an acid.
presumptive
MSA is a conclusive test or presumptive?
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
Mannitol salt fermentation
negative stain- only stains the background
Eosin is a
simple stain
Methylene blue is a
selective and differential
Eosin Methylene Blue (EMB) is
isolation of fecal coliforms and differentiate between different coliform bacteria
EMB is used for the
selective against gram (+) - only see gram (-) growing on these plates
EMB is selective 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
EMB is differential for:
pink
Coliforms that ferment lactose on EMB turns it
true
True or false. All coliforms are gram (-).
E-coli
Which organism ferments lactose aggressively and doesn't turn pink due to the aggressive fermentation but metallic green or black.
intestinal tract of humans and animals
Most coliforms are found in the?
(-)
Coliforms are gram (-) or gram (+)
Enterobacteriaceae
Coliforms belong to the ____________ family.
true
True or false coliforms ferment lactose?
aerobic or facultative anaerobes
They can survive with or without O2 so they are?
nonendospore formers
Coliforms are endospore formers or nonendospore formers?
gas and acid
Coliforms produce ____ and _____ within 48 hours at 37*C
true
True or false several members are pathogens that cause diarrhea.
fecal contamination
Positive for coliform could be potential_____.
pH indicator
EMB has no ______ ________.
organism is inhibited by eosin and MB
gram positive
EMB plate:
poor growth or no growth
organism not inhibited by eosin or MB
gram negative
EMB plate:
Good growth
organism ferments lactose with little acid production
possible coliform
EMB plate:
growth is pink
organism ferments lactose and/or sucrose with much acid production
probable coliform
EMB plate:
growth is dark with or without metallic sheen
organism does not ferment lactose or sucrose
noncoliform
EMB plate:
growth is colorless (no pink, no dark or metallic sheen)
gram (+)
diff. between coliform and non-coliform
EMB plate is selective against ________ and differential for _______ and _________.
selective and differential
MacConkey agar is
bile salts and crystal violet against gram (+)
inhibits gram (+)
MC agar is selective
differentiate between coliform (lactose fermenters) and non-coliforms (non-lactose fermenters)
MC agar is differential
neutral red
The pH indicator for MCagar is
red-pink in color
In MC agar if there is a drop in pH it turns
organism is inhibited by crystal violet/ and or bile salts
gram (+)
MC agar:
poor or no growth
organism is not inhibited by bile salts and/or crystal violet
gram negative
MC agar:
good growth
organism produces acid from lactose fermentation
probably coliform
MC agar:
pink to red growth with or without bile precipitate
organism does not ferment lactose
non-coliform
MC agar:
Growth is colorless not red or pink
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
The procedure for exercise 12:
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 peptone in the agar?
To solidify the bacteria in the media
What is the purpose of agar in the media?
salt (NaCl)
What ingredient makes mannitol salt selective?
E-coli gram
Ps. aeruginosa
Growth on EMB:
(+)
metallic green
colorless
Staph aureus
Staph epidermidis
Growth on EMB/MSA
(-)
yellow
white
Agar containing vancomycin will only allow vancomycin-resistant organisms to grow.
selective
Nutrient agar that contains vancomycin is
differential to differentiate ecoli 0157
EMB agar that contains sorbitol is