TEST NAME SUBSTRATE ENZYME DETECTED WHAT LEADS TO COLOR CHANGE? WHAT DOES A POSI
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Question
TEST NAME
SUBSTRATE
ENZYME DETECTED
WHAT LEADS TO COLOR CHANGE?
WHAT DOES A POSITIVE TEST LOOK LIKE?
WHAT DOES A NEGATIVE TEST LOOK LIKE
HOW DO YOU INOCULATE THE MEDIA?
TIPS
Citrate Utilization: Used to characterize enteric bacteria, (IMViC tests)
Catalase Test: hydrogen peroxide is toxic to cells – catalase breaks it down
Carbohydrate Utilization:
Change in pH w/ fermentation of a carbohydrate
Glucose
Lactose
Sucrose
Gelatin Utilization:
Hydrogen Sulfide Production: (This data is obtained from the TSI test)
H2S is an end product when enzyme hydrolyzes amino acid cysteine
Indole Production:
Used to characterize enteric bacteria, (IMViC tests)
Methyl Red (pH indicator) Test:
To determine if glucose can be converted to acidic products (lactate, acetate, formate)
Used to characterize enteric bacteria, (IMViC tests)
Only inoculate 1 MRVP tube. This culture will be split after incubation. ½ of the culture will serve as the MR (methyl red part of the test while the other ½ will be used as the VP (Vogues Proskauer) part. For the MR test add methyl red.
Vogues-Proskauer Test:
To see if glucose can be converted to acetoin.
Used to characterize enteric bacteria, (IMViC tests)
This is run on ½ of the MRVP culture. For the VP test add KOH and alpha-napthol
Motility Test:
Presence of flagella (not a true biochemical test)
Not applicable
Not applicable
Not applicable
Oxidase Test
Urea Utilization:
Detection of urease, which breaks down urea into ammonia.
Triple Sugar Iron (TSI) Agar:
Determine both carbohydrate fermenatation and hydrogen sulfide H2S, production in enteric bacteria
glucose
sucrose
lactose
DIFFERENTIAL AND SELECTIVE AGARS:
Eosin-methylene blue (EMB) agar:
Selects for gram-negative bacteria. Differentiates lactose fermentors from nonfermentors. Pathogenic gram neg usually can not ferment lactose.
Not applicable
Not applicable
MacConkey Agar:
Selects for gram-negative bacteria. Differentiates lactose fermentors from nonfermentors. Pathogenic gram neg usually can not ferment lactose.
Not applicable
Not applicable
Mannitol Salt Agar:
Selects for organisms that tolerate high salt. Inhibitory to most bacteria because it contains 7.5% NaCl, but Staphylococci grow well.
Not applicable
Not applicable
Heavy inoculation of some non-halophiles will result in growth so don’t use too much culture!
TEST NAME
SUBSTRATE
ENZYME DETECTED
WHAT LEADS TO COLOR CHANGE?
WHAT DOES A POSITIVE TEST LOOK LIKE?
WHAT DOES A NEGATIVE TEST LOOK LIKE
HOW DO YOU INOCULATE THE MEDIA?
TIPS
Explanation / Answer
Citrate utilization:
Substrate: Citrate agar
enzyme detected: citrate permease
what leads to color change: ammonium salts that result from citrate metabolization, increases alkalinity
positive: blue
negative: green
how to inoculate media: take the center of a well isolated colony and put it in the media moving it back and forth. Incubate it for 4-6 days to see if there is a change in color.
tips: be careful to adjust the ph of the media at 6.9
Catalase test
Substrate: 3% to 15% of H2O2
enzyme detected: catalase
what leads to color change: no color change, instead, bubbles are formed because catalase breaks down H2O2 into oxigen and water
positive: bubbles are formed
negative: absence of bubbles
how to inoculate media: small inoculum is exposed to the H2O2 and the bubble are or aren´t instantly formed.
tips: helps identify streptococcus (negative) and staphylococcus (positive) which are morphologically identical.
Glucose utilization
Substrate: glucose
enzyme detected: any from the glucolisis procedure
what leads to color change: if glucose is metabolized the pH drops because of the presence of acids
positive: yellow
negative: green or blue
how to inoculate media: Use 1% of glucose. The agar is stabbed with the inoculum to put the bacteria at the bottom of the tube. Place it at 35-37°C for 24 hours.
tips: for anaerobic bacteria use a layer of sterile mineral for oxygen barrier.
Lactose utilization:
Substrate: lactose
enzyme detected: permease and beta galactosidase
what leads to color change: if lactose is fermented, the pH will drop
positive: yellow
negative: magenta to pink or red.
how to inoculate media: the inoculum is transfered to the tube with phenol red lactose broth and incubated at 35-37°C for 24 hours
tips: no other reagents needed.
Sucrose Utilization:
Substrate: sucrose
enzyme detected: sucrase
what leads to color change: if sucrose is fermented, the pH will drop
positive: yellow
negative: magenta, red or pink
how to inoculate media: the inoculum is transfered to the tube with phenol red sucrose broth and incubated at 35-37°C for 24 hours
tips: no other reagents needed.
Gelatin Utilization:
Substrate: nutrate gelatin medium
enzyme detected: gelatinase
what leads to color change: the presence of gelatin makes the media solidify. If there is a metabolism of gelatin by gelatinase, the media won´t solidify.
positive: after refrigeration the media won´t solidify.
negative: after refrigeration the media will solidify
how to inoculate media: take several colonies and stab the media 4 to 5 times separated by half inch. Incubate for 48 hrs at 37°C. Refrigerate for at least 30 minutes
tips: re-incubate negative tubes for up to two weeks to be absolutely sure they are negative.
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