exp 5: determinatio of coliform in water by MPN Flashcards
what is the potability of water determinedby
the KINDS of microorgsnims not the number
as long as pathogens are not present
What does the public health service drinking eater standards state baout water
it should be entirely free of pathogens
how are pathogens introduce
fecal origins, seweage system
coliform bacteria used as an indicator why
bc sewages have alot of them, associated with fecal matter
-can be detected easier and quicker than pathogens
-presence indicates presence of fecal matter, not of harmful microorganisms as coliform are non-pathogenic
Sequence of most probably number method (MPN test)
1) Presumptive test
-innoculate known amount of water into lauryl tryptose tubes, if it contains gas after 48h, then it is PRESUMED to have fecal matter and unsafe to drink
2) Confirmed Test
-sometimes pos result of presumed may be due to the presence of other bacteria such as clostridium or bacillus so this test is done to ensure that it is coliform
-loopful from laural tryptose added to green lactose bile broth (more selective for coligorm)
-if gas, then pos
-OR can make a eosin methylene blue (EMB) or ENDO streak plate of laural broth (pos if GREEN METALLIC SHEEN typical coliform colonies or PINK colonies with dark center (E.aerogenes) is present
3)Completed Test
-innoculate the colonies from the confirmed test into Lauryl tryptose broth, checks to amke sure that acid/gas is produced)
-streak a nutrient agar slant using the same colony (slant used to do a gram test to see if it is gram-neg, nonsporeforming bacilli : COLIFORM)
what does coliform look like on endo or EMB plate (select for gram neg)
-GREEN METALLIC SHEEN (ecoli)
-PINK WITH DARK CENTER (e.aerogenes)
METHOD
PRESUMPTIVE
-3 double strength samples LAURYL
-6 single strength LAURL
-added creek water to it
-observe for gas
CONFIRMED TEST
add three loopfuls of lauryl to green lactose bile broth
-streak plate the highest diluted (one of the 0.1 ml single strength) on the plate
COMPLTETD
innoculate one laural tube and an agar slant
on slant make a gram stain and look to see if it is gram neg rods with no spores
What is the MOST PROBABLE NUMBER
-techniqie to coint the # of cells
-the most probable number of coliform present in the sample
-use the MPN index to determine
-essentailly just look at how many of the double strength adn how many of the single strength got pos results and match to it
Why can EMB be used for confirmed test
because it inhibits the growth of gram pos due to methylene blue
-gram neg LACTOSE FERMENTERS produce dark center colonies
-coliform, ecoli and enterbacter can be dofferenctiated on it based on how they look. ecoli have grreen shee n e aero does not
-select broth of the highest dilution (so one of the 0.1 ml ones) for test
why is ecoli a better indicator of sewage presence than enterbacter aerogenes
ecoli not normally found in soil whereas enterobacter is
what does ecoli look like on emb agar compared to e aerogenes
ECOLI: metallic green sheen, flat
E.Aerogenes: non metallic sheen, gummy, mucoid, larger
what was seen on the gram stain
PINK RODS
what were the results
MPN
presumptive: 4.6 coliform/ml
confirmed: 4.6 coligorm/ml
