Lab 3 Flashcards
Camplobacter blood agar
selective blood agar medium containing vanomycin, polumixin B, trimethoprim and cephalothin
inhibit the growth of most bacteria (3x antibiotics) including those of the gastrointestinal tract
good media to attempt and isolate Campylobacter spp. like C. jejuni.
can however, also isolate other Gram + that are not normally found in the gastrointestinal tract
Eosin Methylene blue agar
selects for Gram - bacteria by containing eosin Y and methylene blue.
Also helps to differentiate gram negative organisms on their ability to ferment lactose into lactic acid
EMB vigourus fermentation
acidic conditions that are produced by vigorus fermentation make the agar a dark purple colour or black/green matallic sheen.
=thermotolerant coliforms (bacilli in faecal contamination)
EMB slow fermentation
colonies apper pink instead of dark purple
EMB non-fermenters
colonies are the same colour of the agar
MacConkey agar
selects for G- organisms and differentates them based on lactose fermentation
G+/- are inhibited by crystal violet and bile salts
eneteric bacteria typically grow best on EMB agar
pH indicator = neutral red (red @ 6.8 and below, above 6.8 it is colourless)
Red MacAgar =
lactose fermentaiton
indicator of coliforms like E. coli
Clear MacAgar=
non=fermenting coliforms such as Shigella (causes dysentery) and Salmonella (which causes typhoid)
may also be red if they do not deaminate the peptone
Pseudomonas F agar
non-selective agar that isused to differentiate between Pseudomonas species and other bacteria
can also differentiate between P. aeruginosa ans P. fluorescens
contians lower levels of iron than typicla media which causes organisms to produce siderophores; which are iron-scavenging molecutes
P. fluorescens both prodcue a siderophore = pyocerdin aka fluorescein = green under uv
P. aeruginosa produces a siderophore pyocyanin = blue under uv
Citrate utilization test
determines if the organism can use citrate as it’s sole carbon source
Looks for the enzyme citrate permease, uptake of citrate on conversion to pyruvate
simmons citrate agar, citrate only carbon, and ammonium phosphate as teh only source of nitrogen
pH indicator bromothymol blue turns green at 6.9, and blue at 7.6
citrate utalization will end up in ammonium phosphate conversion to ammonia (NH3) which increases the pH of the agar turning it blue
part of the IMViC tests that are commonly used to differentiate Gram - bacilli, particularly members ofthe family enterobacteriaceae which includes that coliforms
IMViC Tests
commonly used to differentiate Gram - bacilli, particularly members ofthe family enterobacteriaceae which includes that coliforms
Indole
Methyl Red
Voges-Proskauer
Citrate
Indole Test
distinguishes bacteria that produce indole by hydrolyzing the aa trp using tryptophanase.
part of the IMViC tests that are commonly used to differentiate Gram - bacilli, particularly members ofthe family enterobacteriaceae which includes that coliforms
Performed on SIM agar, a semi-solid medium made of casein and animal tissue for aa and iron-containin sodium thiosulfate for sulfur (can however be performed as a spot test)
indole produciton is detected via the addtion of kovacs reagent (DMABA in hydrochloric acid) the clear agent turing red is a potitive and results from the DMABA reacting with any indole
Methyl red test
detects teh ability to produce acidic end products from mixed-acid fermentation of glucose
part of the IMViC tests that are commonly used to differentiate Gram - bacilli, particularly members ofthe family enterobacteriaceae which includes that coliforms
MRVP broth which is used for both Methyl red and the Vogues-Proskauer tests.
Phosphate buffer
Methyl red uses the ph indicator methyl red post incubation instead of barrits reagent in teh VP test
Mixed acid ferm is able to overe come the buffer and turns the media red insead of yellow which is a negative result
Metabolic aspects of the methyl red test
the acids that are produced by mixed-acid fermenting bacteria such as lactic acid, acetic acid and formic acid are stable and remain at pH 4 in the tube
Methyl red negative organisms convert these acids into products like 2,3-butanediol and acetoin which raises the pH (less acid) to 6
red +, yellow -, orange = inconclusive
Nitrate reduction test theory
Nitrate (NO3-) can be reduced by the enzyme nitrate reducate to produce nitrite (NO2-) and water.
some bacter have the ability to perform multi-step denitrification and convert ammonia to molecular nitrogen
Tests for the ability of aerobes and facultative anerobes to reduce nitrate (TEA) in the absence of Oxygen
Nitrate reduction test media
Nitrate reduction test mediasemisolid media, with peptone and potassium nitrate.
0.1% agar reduces the diffusion of oxygen
no colour indicator is in the broth
post incubation, sulfanilic acid (reagent a) and a-naphthylamine (reagent b) are added
single step reduction of nitrate
if nitrite is present (NO2-) it will for nitrous acid (HN02) wit the reagents to make a red pigment
this means single step reduction of nitrate to nitrite has occured
No colour change in the nitrate reduciton test after the additon of reagent a and b
either no reduction of nitrate, or, it was rapidly reduced beyond nitrite to ammonia or nitrogen
to determine what has happened, zinc is added and if act as a catalyst to reduce nitrate to nitrite
red after zinc means nothing has happened during incubation
nothing after zinc means that there was rapid reduction of nitrate to ammonia or molecular nitrogen
Triple sugar iron agar media constituents
is a differential test based on fermentation of glucose, lactose and sucrose and sulfur reduction
Agar contains 1% lactose, 1% sucrose and 0.1% glucose (dextrose).
also contains beef extract and peptone, and sodium thiosulfate as a reducible sulfur compound
pH indicator = phenol red
media is on a slant but with more agar to provide an anaerobic environment
Glucose only fermenter TSI
produce acid, lower the media pH turning the medium yellow within a few hours
recall only 0.1% glucose, so it is quickly consumed and then the organisms in the aerobic slant will deaminate the peptone and aa in the media turning the slant red
red slant, yellow butt is positive for glucose fermentation and negative for lactose fermentation and sucrose fermentation
Sucrose/ lactose fermentation TSI
turn the medium full yellow
does not specify which of the two has been fermented, and if the organism is capable of fermenting glucose as well
non-lactose/ sucrose/ glucose fermentation TSI
tube has red alkaline slant and a orange butt (for organisms that can only catabolize peptone aerobically)
Tube has a red alkaline butt red slant (aerobic and anaerobic itilization of peptone)
Why is timing of reading TSI fermentation tests important
an early reading might result in glucose fermentation falsely looking like lactose or sucrose fermentation (before the glucose has been used up)
However, reading too late, i.e. after the lactose and sucrose are depleted would show a yellow butt and red slant falsely suggesting the organism only ferments glucose
TSI sulfur reduction
Bacteria can anaerobically reduce sulfur to hydrogen sulfide (H2S) by reducing thiosulfate ot hydrolysis of the aa cysteine which is present in TSI peptone into pyruvate.
either way, colourless H2S reacts with iron ions in the medium to for ferrous sulfide (FeS) which is a black ppt.
acidic conditions are required for this reaction to occur
black ppt = sulfur reduction and fermentation
Urease test
Urea is produced form decarboxylation of some AA
urea can be broken down into ammonia and CO2 by bacteria which contain the enzyme urease
many bacteria can metabolize urea, but few can do it fast enough to give a positive result
pH indicator = phenol red
media also has a buffer (potassium phosphate)
rapid hydrolysis of urea to ammonia will overcome the buffer and lead to an increase of pH turning the media form orange to pink
negative would be yellow
Sulfur Reduction Test (SIM agar)
SIM agar is a multifunction medium that can be used to detect sulfur reduction, indole production ondmotility
same mechanism for dectecting sulfur reduction as in TSI
Sulfur (thiosulfate in media) reduction to hydrogen sulfide (H2S)
bacteria can anaerobically reduce sulfur from thiosulfate or the amino acid cystiene from peptone into pyruvate
either reaction produces hydrogen sulfide whihc reacts with the iron ions in the media to form ferrous sulfide a visible black ppt
