Lab 4 Flashcards
Oxidase test
the last enzyme of the ETC = Complex IV aka cytochrome c oxidase make sthe final electron transfer from cytochrome c to the TEA, oxygen
this test specifically detects cytochrome c oxidase, which can oxidise cytochrome c and catalyze the reduction of cytochrome c by tetra-methyl-p-phenylenediamine dihydrochloride (TMPD)
positicve = purple on the filter paper within 30s
do not transfer cultures from agar with purple dyes like EMB or Mac agar
note that some species can us e oxygen as a tea but do not have cyto c oxidase so they will apper -ve
Indole test
detects bacteria that produce indole via the hydrolysis of aa tryptophan using tryptophanase
part of the IMViC taht are commonly used to differentiate gram negative bacilli particularly members of the family enterobacteriaceae
done on sim agar
works because the bacteria may have tryptophanase to hydrolyse the tryptophan in the casein and animal protien into pyruvate, ammonia and indole
indole is detected via kovacs reagent
postive result = red
Why is it impractical to test each potential pathogen taht can contaminate water individually
many are difficult to culture or are present in such low concentrations that they are difficult to isolate using confentional micobiological tecniques
What is an indicator species
an indicator organism is a species that is realitively easy to isolate, culture and identigy that are frequently found associated with pathogens
sewage often often contains such pathogens, and rather than testing for those individuals, if a if we identify a coliform and or faecal streptococci, with the assumption that they are associated with pathogens, the water can be declared unsafe to drink
coliform bacteria
gram negative bacilli that are non-sporulating, can produce acids and gas from the fermentation of lactose.
they may be found in the water, soil and vegetation but are a signficant portion of the bacteria found in the faeces of warm-blooded animals
criteria of porable water
potable = safe for human consumption
means that their is less thatn 1 coliform CFU per 100mL
TCC
total coliform count
performed on m-Endo agar, incubated at 37c
selects for gram - organisms and differentiates then based on lactose fermentation
contians sodium lauryl sulfite, sodium deoxycholate and basic fuchsin to inhibit gram positives
lactose non fermenters produce a faint, pink colony (lactose is the only carb )
with fermenters, sodium sulfite reacts with acetaldehyde to produce dark rosy colonies
vigourous fermentation leads to metallic gold sheen as seen in Escherichia coli and Klebsiella pneumoniae (from the acidic conditions)
m-endo is commonly used for total coliform counts
FCC
faecal coliform counts
done on m-FC agar
incubated at 44.5c
sodium deoxycholate (bile salts) inhibits the growth of gram positives. Rosolic acid inhibits the non-faecal coliforms. Analine blue indicates lactose fermentation.
note: not all coliforms that can grow at 44.5c and produce acid from lactose are faecal in origin, so the term thermotolerant coliforms is becoming more popular. However, most of the organisms taht grow on mFC agar are found in faeces and can be used to indicate faecal contaminaiton
FSC
Faecal streptococcal counts
are done on kenner fecal (KF) agar and is incubated at 37c
aodium azide suppresses gram-0negative organsims
maltose and lasctose are the fermentable carbs, and can be fermented by most faecal streptococci
fermentaion is detected by bromo9cresol purple, whihc will change from purple to yellow around fermenting organisms
origin of pollutiohn
the ratio of faecal coliforms to faecal streptococci can be interprented to suggest the origin of the pollution
faecal soliforms are indicative of human pollution and faecal streptococci are indicative of animal pollution
not as reliable as it was once thought to be because faecal streptococci tent to persist in the enviroment longer than coliforms so, the furhter the sampling gets from the source, the less accurate the data becomes
FCC/FSC < 0.7
poultry and livestock pollution
2 < FCC/FSC < 4
Human and animal pollution
FCC/FSC > 4
human pollution
Quantitative analysis of water with membrane filtration
counts CFUs in “clean” liquids
for example, if a sample has less than 1 CFU/mL, plating is unlikely to reveal any visible growth, (cant really put one 1mL on an agar plate without flooding it)
bacteria can still cause disease at this dilution tho!
highly reproducable which is swag
How does membrane filtration work
a water sample is passed through a porous membrane that traps microorgansism or other debris larger than 0.45 um. (sometime is hooked up to a vacuum pump)
once a measured volume passes through, the filter is placed on an agar plate
