Lab 3

Question 1

Camplobacter blood agar

Answer 1

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

Question 2

Eosin Methylene blue agar

Answer 2

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

Question 3

EMB vigourus fermentation

Answer 3

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)

Question 4

EMB slow fermentation

Answer 4

colonies apper pink instead of dark purple

Question 5

EMB non-fermenters

Answer 5

colonies are the same colour of the agar

Question 6

MacConkey agar

Answer 6

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)

Question 7

Red MacAgar =

Answer 7

lactose fermentaiton

indicator of coliforms like E. coli

Question 8

Clear MacAgar=

Answer 8

non=fermenting coliforms such as Shigella (causes dysentery) and Salmonella (which causes typhoid)

may also be red if they do not deaminate the peptone

Question 9

Pseudomonas F agar

Answer 9

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

Question 10

Citrate utilization test

Answer 10

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

Question 11

IMViC Tests

Answer 11

commonly used to differentiate Gram - bacilli, particularly members ofthe family enterobacteriaceae which includes that coliforms

Indole

Methyl Red

Voges-Proskauer

Citrate

Question 12

Indole Test

Answer 12

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

Question 13

Methyl red test

Answer 13

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

Question 14

Metabolic aspects of the methyl red test

Answer 14

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

Question 15

Nitrate reduction test theory

Answer 15

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

Question 16

Nitrate reduction test media

Answer 16

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

Question 17

single step reduction of nitrate

Answer 17

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

Question 18

No colour change in the nitrate reduciton test after the additon of reagent a and b

Answer 18

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

Question 19

Triple sugar iron agar media constituents

Answer 19

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

Question 20

Glucose only fermenter TSI

Answer 20

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

Question 21

Sucrose/ lactose fermentation TSI

Answer 21

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

Question 22

non-lactose/ sucrose/ glucose fermentation TSI

Answer 22

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)

Question 23

Why is timing of reading TSI fermentation tests important

Answer 23

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

Question 24

TSI sulfur reduction

Answer 24

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

Question 25

Urease test

Answer 25

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

Question 26

Sulfur Reduction Test (SIM agar)

Answer 26

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