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MICR 203 > lecture 5 - biochem tests > Flashcards

lecture 5 - biochem tests Flashcards

1
Q

biochem tests are used to differentiate

A

enterobacteriaceae or now refered to as order:
Enterobacterales

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2
Q

why do we do biochem tests?

A
  • detect what substrate it uses to produce energy
  • different pathwats
  • acidic or basic byproducts
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3
Q

acid fushin (andrades)

A

acid - pink
basic - pale yellow

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4
Q

bromcrescol green

A

acid: yellow
basic: blue

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5
Q

bromcresol purple

A

acid: yellow
basic: purple

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6
Q

bromthymol blue

A

acid: yellow
basic: blue

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7
Q

methyl red

A

acid: red
basic: yellow

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8
Q

neutral red

A

acid: red
basic: yellow

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9
Q

phenol red

A

acid: yellow
basic: red

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10
Q

cresol red

A

acid: yellow
basic: red

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11
Q

the main sources of energy for bacterial growth are:

A

carbohydrates + peptones

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12
Q

what are peptones?

A

smaller denatured proteins that are easier to use because small and readily available

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13
Q

carbohydrates produce:

A

acid in anaerobic, CO2, or aerobic conditions

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14
Q

peptones produce:

A

alkaline in aerobic only

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15
Q

hugh & leifsons O/F test

A
  • differentiates glucose oxidizer (carb - acid) or fermenter (peptone - alkaline)
  • lots of glucose
  • little peptone
  • bromthymol blue indicator
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16
Q

H&L O/F Test pos/neg

A

F: Enterobacterales
O: Neiseseria, Pseudomonas, Stentrophomonas, Burkholderia

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17
Q

Carbohydrate Fermentation Test:

A
  • determines ability to ferment diff. sugars to form acids
  • basic media, 1% of each sugar. phenol red
  • also has durham tube for gas production
  • if pos, turns yellow (acid formed)
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18
Q

glucose is ______ for all enterobactererales

A

yellow!

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19
Q

glucose –> pyruvic acid –>

A

mixed acid path - acidic byproduct

OR

neutral products

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20
Q

methyl red test

A
  • inoculate MRVP
  • add methyl red
  • red = mixed acid
  • no change = ?
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21
Q

Voges-Proskauer Test

A
  • inoculate MRVP broth
  • add a-naphthol & 40% KOH, shake gently, observe for 10-15min
  • red = acetoin (neutral path)
  • no change = no acetoin produced
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22
Q

MRVP results

A

most members of Enterobacterales give opposite rxns
- E.coli = MR (+) & VP (-)
- Klebsiella, Serratia, Enterobacter = MR(-) & VP (-)
- Proteus mirabilis = MR(+) & VP (+)

23
Q

TSI Agar

A
  • gives info about:
  1. Fermentation of glucose (0.1%), lactose (1%), sucrose (1%)
  2. Production of CO2 gas during ferm. (CO2 - aerogenic, none - anaerogenic)
  3. Production of H2S gas –> black
24
Q

why is glucose in such a small percent?

A

looking at organisms that all ferment glucose so you do not need very much

25
TSI Media
- peptone (basic growth media) - glucose, lactose, sucrose - phenol red - sodium thiosulphate and ferrous sulfate for H2S
26
how do you read a TSI agar test?
top to bottom
27
producton of H2S gas
some microbes can liberate colourless H2S gas from: - sulphur containing amino acids (cystein/methionine) sodium thiosulphate --> ferrous sulphate + colourless H2S gas --> ferrous sulphide (black ppt)
28
if you see black ppt (H2S), do we call it Alk or Ac underneath?
always acid
29
other H2S indicators include:
- ferrous citrate (most common) - ferric ammonium citrate - lead acetate
30
why is incubation time critical for TSI?
- do not want all of the glucose consumed and then have the organism turn to using peptone
31
why do caps need to be loose for TSI test?
allow CO2 to escape to maintain aerobic bc no peptone use with w/o O2
32
Indole Test
- organism produce tryptophanase, hydrolize tryptophan to produce INDOLE, pyruvic acid, NH3 - can be detected w/ Kovacs, p-aminobenzaldehyde or cinnamaldehyde reagent - red in alcohol layer = pos for indole
33
Indole Test - pos organisms
- E.coli - Klebsiella oxytoca - Proteus spp. (except mirabilis & penneri)
34
Urease Test
- ability to split urea --> CO2 and NH3 (alkaline byprod.)
35
Urea Medium
- peptone - glucose - urea - phenol red
36
Urease Test Results
Rapid Pos - Proteus mirabilis Delayed Pos - Klebsiella pneumoniae Neg - Eschericia coli
37
Decarboxylase Test
- some organisms can decarboxylate AA to produce amine and CO2 - yellow = AA was not decarboxylated - purple/turbid = AA was decarboxylated
38
Decarboxylase Media
- Moeller's Medium - basic medium - glucose - amino acid - pH indicator (bromcresol purple and cresol red) - acid overlay * very important*
39
what amino acids get decarboxylated during the decarboxylase test?
Lysine --> Cadaverine Ornithine --> Putrescine Arginine --> Citrulline all + CO2
40
phenylalanine deaminase (PPA/PDA) & tryptophasn deaminase (TDA) tests + reactions
- some organisms can remove amino group from phenylalanine or tryptophan Phenyl --> phenylpyruvic acid + NH3 / Tryptophan --> indolepyruvic acid add indicator (ferric chloride) --> dark green or brown
41
PDA/TDA is useful for differentiating:
urease positive organisms - proteus, providencia & morganella
42
citrate test
- some org. use sodium citrate as sole source of carbon for growth - citrate + ammonium phosphate --> ammonia + ammonium hydroxide - alkaline --> blue
43
Citrate Test Media/Conditions
- sodium citrate -ammonium phosphate - bromthymol blue - O2 required - light inoculum (not too much or false pos bc feed off each other) - some org. dont produce colour change but have growth, still negative
44
Citrate Test Results
Pos: Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia & Providencia species Neg: Escherichia, Shigella, Morganella, Yersinia Variable: Proteus species
45
Nitrate Reductase Test
- if org produces nitrate reducate, will reduce nitrate (NO3) to nitrite (NO2) - then reacts w/ added sulfanilic acid + a-napthylamine to form red dye (rxn 1) - some reduce nitrite to nitrogen gas (N2) (rxn 2), wont react w reagents from rxn 1 - add zinc to determine if nitrate was not reduce, so zinc will reduce nitrate to nitrite + reagents = red OR - nitrate was reduced to N2, no colour will develop - can detect nitrogen gas w/ durham tube
46
ONPG test
- o-nitrophenyl-B-D-galactopyranoside --> galactose + O-nitrophenol (ignores permease) - ANY yellow = pos
47
Gelatinase Test
- uses gelatin w/gelatinase, cover charcoal - if organism can break down gelatin, it is pos bc charcoal released
48
Gelatinase Test Results
pos: proteus & serratia species AND Pseudomonas fluorescens Neg: Pseudomonas putida
49
Motility Test
- stab, if cloudy then motive, can have pH indicator
50
a quicker way to witness bacterial motility is
wet preparation
51
what is a notable non-motile bacteria?
bacillus anthracis
52
Motility Results
Non-motile Enterobacterales - Klebsiella & Shigella Yersinia enterocolitica - motile at 25C but not 35C Listeria spp. (GPB) - umbrella motility in tube - tumbling in wet mount Bacillus anthracis (GPB) - NON MOTILE!
53
MUG Test
- for E.coli O157:H7 stool pathogen - produces toxin and read under UV light - pos - glows - neg - no glow (E.coli O157:H7) - 4-methylumbelliferyl-B-D-glucoronide --> (B-glucoronidase) --> 4-methylumbelliferone

MICR 203 (2 decks)

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