Lecture 3 Flashcards
what does the test media for carb utilization test media contain
fermentation - O2 not required - high acid end products
-Oxidation where O2 is required - less acid products
Asaccharolytic - dont use CHO but use other organic molecules - no acid
-protein source pH indication
-Protein source, pH indicator & 1% of the carbohydrate being tested
fermenter: that produce alot of acid
- indicator like phenol red change to red in acidic conditions under pH 6
Oxidizer - produce less acid
- bromothymol blue
if glucose and lactose are both present then bacteria will use glucose first then lactose (monosac THEN disacc)
lac operon gene needs to be expressed to use lactose
no enzyme no lactose
what are some common indicators used in fermentation tests
Phenol Red
Andrades acid fuschin
Bromothymol blue
o/f test use and interpretation
- determines if orgs can use carbs aerobically (oxidative) or anaerobically (fermentative)
-To differentiate non-fermenting strict aerobic GNB from other bacteria
composed of 1% glucose , peptone , bromo blue, semi solid agar , Un-inoculated media starts off green
uses 2 tubes
-open tube for testing in aerobic conditions NO OIL AEROBIC
-closed tube for anaerobic conditions after adding sterile mineral oil
fermenter - both tubes yellow
oxidizer
-open - green with a little yellow on top
closed - all green with the oil on top
non fermenter/non oxidizer
-open is all green
-closed is all green with the oil on top
how does lactose fermentation work
lactose is formed from glucose and galactose
-B galactoside permease allows the lactose to enter the bacterial cell wall and once inside B galactosidase breaks the lactose down
if bacteria has both B galactosidase and the permease - LF
if bacteria has permease but no b galactosidase - NLF
if it has b galactosidase but not the permease it might take a while to produce the permease (through mutation or otherwise)- LLF - late lactose fermenter
what is the purpose ONPG
nitrophenyl
-differentiates from LLF from NLF because it detects permease negative and B galactosidase positive orgs
-ONPG is similar in structure to lactose except the glucose replaced with o-nitro phenyl
-ONPG is small enough to passively enter the bacterial cell wall without the permease enzyme
-Bacteria with the enzyme β galactosidase will degrade ONPG (colourless) to ortho nitro phenol (yellow) and galactose
Any shade of yellow is considered positive
-ANY SHADE OF YELLOW IS POSITIVE
what is the principle and ingredients in the TSI media (OXOID)
and how is it read
-detects fermentation glucose, lactose, and/or sucrose
-detects gas production
-detects H2S production
-used to get a presumptive for GN bacilli and are good for intestinal enteric pathogens
Ingredients
peptone
0.1% glucose, 1%lactose, 1%sucrose
ph indicator - phenol red
H2S indicators - ferric ammonium sulfate /ferric ammonium citrate and sodium thiosulfate
SLANT first then BUTT
A/A - YELLOW SLANT/ YELLOW BUTT - glucose, lactose and sucrose fermented
alkaline/akaline NA/NA- red/red - no cho fermented, aerobic organism
alkaline/acid NA/A - red/yellow butt - only glucose fermented
Gas is read:
CO2 and H2 are the gases that are produced in the breakdown on carbs
-a positive reaction is done by BUBBLES, CRACKS IN AGAR LIFTING OF AGAR
H2S is read
-na thiosulfate provides the sulfur
-the H2S reacts with ferrous sulfate to form a black precipitate
-black on the bottom
what is the principle behind TSI if the if the organism can only use 0.1% glucose
-glucose is used up first since it is a monosaccharide producing acid turning the whole medium yellow
-peptones are broken down in the presence of O2 on the slant and produces ammonia
-this leads to an reversion to alkaline pH in the SLANT = pink
the butt of the tube is still ana and more acidic staying yellow
what is the principle behind TSI if the if the organism can only use 0.1% glucose AND lactose 1% AND sucrose 1%
-lots of acid formed
-so many carbs that cant be used up in 18-24 hours so need to use peptones or reversion of PH
-BOTH SLANT AND BUTT will be yellow
what does the MRVP test help for
helps to determine whether an organism uses the mixed acid pathway =METHYL RED (ecoli)
or utilizes the2,3 BUTANEDIOL PATHWAY - VP vogues proskauer (entrobac)
after organ metabolizes glucose to pyruvate in glycolysis these are the different types of pathways
what is the principle of MR methyl red
-bacteria that ferments pyruvate via a mixed acid fermentation
-production of lactic acid and acetic acid which causes a low pH less than 4.2 that overcomes the buffering capacity of the broth causing a red color when the indicator is added
red=mixed acids = positive
yellow - negative
what is the principle of VP
-is when bacteria ferments pyruvate through the butanediol fermentation
-glucose breaks down the butanediol to acetoin under aerobic conditions and adds napthol which produces diacetyl causing a red color like a ring (POS)
CLEAR - uninoculated
pinkish/yellow - neg
LYSINE & ORNITHINE
DECARBOXYLASE what is the purpose and principle and what is used
-it detects the organisms ability to produce the correct decarboxylase enzyme to break down a specific amino acid
-decarb is when a carboxyl group is removed from an amino acid with the formation of an anime or diamine + CO2 (for carbon and energy)
-used for identification of gram negative bacilli
always need 3 tubes
-Decarboxylase control
-Decarboxylase lysine
-decarb ornithine
-the control tube contains glucose only so the bromocresol should turn it yellow because the organism eats glucose producing acid
ingredients in the media are glucose, an amino acid, bromocresol purple
-if the organism can ferment the glucose in the media - ACIDS are produced
- this is important because the decarboxylase org is only stimulated in an acid environment
First reaction is glucose fermentation creating an acid environment =yellow color
Acid condition is needed to stimulate the production of decarboxylase enzyme
The enzyme breaks down the amino acid to amines + CO2
These products shift the pH to the alkaline range = a change from yellow to purple
If the organism does not produce the enzyme, the media will remain in an acid state -stay yellow
DEAMINATION OF AMINO ACIDS
and which tests used this method
-when you remove an anime group from amino acid using enzymes it is called deaminases
- the production of deaminase enzymes is how some bacteria break down amino acids in order to get CO2 (carbon source) and energy ends with alkaline products
Arginine dihydrolase test
Phenylalanine deaminase test
what is the principle and purpose of Arginine dihydrolase test
-test the org ability to make arginine dehydrolase and citrulline ureidase to break down carbon and energy -GRAM NEG BACILLI
media contains arginine, glucose, bromocresol purple
-bacteria must produce arginine dihydrolase & citrulline ureidase
-the test media contains glucose and aa
-the fermentation creates a acid environment = YELLOW
-acid and ANO2 conditions are required to stimulate production of dihydrolase and ureidase enzymes.
-production of ammonia causes as alkaline change from yellow to purple
a control tube is also used to ensure fermentation and aid environment were achieved
what is the PHENYLALININE DEAMINASE TEST
(PPD/PPA) and what is its purpose and media components
-tests ability of orgs to make enzyme phenylalanine deaminase
-GN BACILLI
INGREDIENTS IN MEDIA:
* L-Phenylalanine
* NaCl
* Agar
-acid environment not needed for the production of phenylalanine deaminase that is why the broth does not have glucose
-if the bacteria produce the Phen Diamina the PHEN is broken down into phenyl pyruvic acid +ammonia and water using PHEN deaminase
-by adding 10% ferric chloride to phel pyruvic acid you can see a green color as a change
If phenylalanine deaminase not present = yellow color - neg
the green color is a positive
