(M) Lab: Biochemical Identification of Bacteria (Part 3) Flashcards

BIOCHEMICAL IDENTIFICATION FOR GRAM-NEGATIVE BACILLI

1
Q
  • It is less toxic
  • If you see G(-) bacilli under the microscope, you just get a test tube rack that contains a panel for the G(-) bacilli biochem tests
  • Routine has 7: TSI, LIA, Indole, Methyl red, Vogues-Proskauer, Citrate, Urease
  • From the MAC growth, sub-culture them into the seven tubes
  • Incubate
  • After 24 hours, interpret based on the pattern of reaction. Provided that you did the correct procedures.
A

Biochemical Identification for gram-negative bacilli

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2
Q
  • Uses a TSI medium: butted slant media
  • It contains: glucose, lactose, sucrose (separated in carbohydrate fermentation)
  • Detects the acid production, H2S, and gas production
A

Triple Sugar Iron (TSI)

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3
Q

Interpret/Report the following for TSI.

Slant: Yellow
Butt: Yellow

A

Report: A/A
Interpret: 2 to 3 sugars fermented

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4
Q

Interpret/Report the following for TSI.

Slant: Red
Butt: Yellow

A

Report: K/A
Interpret: 1 sugar fermented

Fermentation only occurs at the butt.

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5
Q

Interpret/Report the following for TSI.

Slant: Red
Butt: Red

A

Report: K/K
Interpret: No acid production/sugar fermented/Non-fermenter

Automatically no H2S nor gas

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6
Q

If only the butt is yellow, it indicates?

A

Fermentation

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7
Q

T or F: If bacteria can ferment >1 sugar, it can ferment the sugars one by one.

A

T

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8
Q

T or F: The first fermentation always happens at the slant portion wherein there is an anaerobic condition.

A

F (butt)

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9
Q

T or F: 2nd-3rd fermentation happens at the aerobic site → slant

A

T

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10
Q
  • Characteristic of Salmonella typhimurium, a large H2S producer
  • Only presumptive, not confirmatory
A

Blackening that almost occupies the entire medium, up to the slant

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11
Q
  • Uses Lysie agar plate (LAP), a butt-and-slant medium → recorded as a slant/butt basis
  • Also contains glucose
  • Also contains an H2S indicator, which may demonstrate blackening (+H2S) (ferric ammonium citrate)
  • Targets lysine
A

Lysine Iron Agar

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12
Q
  • (+) Decarboxylates lysine through the release of decarboxylase (observed at butt portion, anaerobically)
  • Deaminates due to the release of deaminase *(slant portion, aerobic)
A

Lysine Iron Agar

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13
Q

Report/Interpret based on LIA.

Slant: Purple
Butt: Purple

A

Report: K/K
Interpret: The organism is (+) for decarboxylation

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14
Q

Report/Interpret based on LIA.

Slant: Purple
Butt: Yellow

A

Report: K/A
Interpret:
(-) for decarboxylation

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15
Q

Report/Interpret based on LIA.

Slant: Red
Butt: Yellow

A

Report: R/A
Interpet: (+) deamination

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16
Q
  • Media is originally purple
  • The glucose of the media fermented a sugar at the butt portion resulting in acid production, turning the purple medium yellow
  • When the carboxylase is the released, the lysine from the medium produces cadaverine which is a basic substance, that reverts the yellow medium back to purple
17
Q
  • Yellow butt due to glucose fermentation, no decarboxylation, retaining the yellow color of the medium
18
Q
  • Changes occur in the slant (demonstrates deamination)
  • Deaminase changes medium from purple to red
19
Q
  • Performed in a semi-solid sulfide indole motility (SIM) medium that demonstrates sulfide production, indole test, and motility of the bacteria
  • If tryptophanase enzyme is released by bacteria, the enzyme utilizes the substrate in SIM: tryptophan, resulting to indole production
  • A common mistake in the lab is forgetting to add the reagent, resulting in no patterns observed, leading to false negatives
  • Another one is the wrong incubation for SIM medium *(It only requires room temperature)
    *
A

Indole Test

20
Q

Reagent for Indole Test?

A

Erlich’s Reagent → PDAB or p-dimethylaminobenzaldehyde

21
Q

Indole Test

Positive test?

A

Red ring on top of medium

22
Q

Indole Test

Negative results?

A

No red ring

23
Q
  • Uses the Methyl Red Vogues Proskauer (MRVP)
  • For observing which metabolic fermentative pathway is utuilized by the bacteria use
A

Methyl Red

24
Q

Methyl Red

  • Bacterium releases various acids creating a low pH in the medium (4.0-4.6)
  • Detected by methyl red
A

Mixed Acid Pathway

25
# Methyl Red Positive results?
Red solution
26
# Methyl Red Negative results?
Yellow *(enhanced original color of the medium or no change)*
27
* Also for determining which pathway is utilized, specifically: * Detects butylene glycol pathway * Enterobacteriaceae only one fermentation pathway: either mixed acid or butylene glycol, but never both
Vogues-Proskauer
28
# Vogues-Proskauer Butylene glycol pathway uses?
Alpha-naphthol and KOH
29
# Vogues-Proskauer Positive results?
Cherry red solution
30
# Vogues-Proskauer Negative results?
Chocolate brown
31
* Determines whether bacterium uses citrate as its own or its sole carbon source that releases ammonia * **Simmon’s citrate medium** incorporated with bromothymol blue as the indicator * Basic ammonia is detected by the medium turning from green to blue
Citrate Test
32
# Citrate Test Positive results?
Blue
33
# Citrate Test Negative results?
Green
34
* Checks if bacteria releases urease enzyme * In the urea agar incorporated with phenol red
Urease Test
35
# Urease Test ____ hydrolyzes urease to become basic ammonia turning the red medium to a deep pink or fuschia
Phenol red
36
# Urease Test Positive Results?
Deep or fuschia pink