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

Question 1

• 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.

Answer 1

Biochemical Identification for gram-negative bacilli

Question 2

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

Answer 2

Triple Sugar Iron (TSI)

Question 3

Interpret/Report the following for TSI.

Slant: Yellow
Butt: Yellow

Answer 3

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

Question 4

Interpret/Report the following for TSI.

Slant: Red
Butt: Yellow

Answer 4

Report: K/A
Interpret: 1 sugar fermented

Fermentation only occurs at the butt.

Question 5

Interpret/Report the following for TSI.

Slant: Red
Butt: Red

Answer 5

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

Automatically no H2S nor gas

Question 6

If only the butt is yellow, it indicates?

Answer 6

Fermentation

Question 7

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

Answer 7

T

Question 8

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

Answer 8

F (butt)

Question 9

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

Answer 9

T

Question 10

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

Answer 10

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

Question 11

• 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

Answer 11

Lysine Iron Agar

Question 12

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

Answer 12

Lysine Iron Agar

Question 13

Report/Interpret based on LIA.

Slant: Purple
Butt: Purple

Answer 13

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

Question 14

Report/Interpret based on LIA.

Slant: Purple
Butt: Yellow

Answer 14

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

Question 15

Report/Interpret based on LIA.

Slant: Red
Butt: Yellow

Answer 15

Report: R/A
Interpet: (+) deamination

Question 16

• 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

Answer 16

K/K

Question 17

• Yellow butt due to glucose fermentation, no decarboxylation, retaining the yellow color of the medium

Answer 17

K/A

Question 18

• Changes occur in the slant (demonstrates deamination)
• Deaminase changes medium from purple to red

Answer 18

R/A

Question 19

• 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)
  *

Answer 19

Indole Test

Question 20

Reagent for Indole Test?

Answer 20

Erlich’s Reagent → PDAB or p-dimethylaminobenzaldehyde

Question 21

Indole Test

Positive test?

Answer 21

Red ring on top of medium

Question 22

Indole Test

Negative results?

Answer 22

No red ring

Question 23

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

Answer 23

Methyl Red

Question 24

Methyl Red

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

Answer 24

Mixed Acid Pathway

Question 25

Methyl Red

Positive results?

Answer 25

Red solution

Question 26

Methyl Red

Negative results?

Answer 26

Yellow (enhanced original color of the medium or no change)

Question 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

Answer 27

Vogues-Proskauer

Question 28

Vogues-Proskauer

Butylene glycol pathway uses?

Answer 28

Alpha-naphthol and KOH

Question 29

Vogues-Proskauer

Positive results?

Answer 29

Cherry red solution

Question 30

Vogues-Proskauer

Negative results?

Answer 30

Chocolate brown

Question 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

Answer 31

Citrate Test

Question 32

Citrate Test

Positive results?

Answer 32

Blue

Question 33

Citrate Test

Negative results?

Answer 33

Green

Question 34

• Checks if bacteria releases urease enzyme
• In the urea agar incorporated with phenol red

Answer 34

Urease Test

Question 35

Urease Test

____ hydrolyzes urease to become basic ammonia turning the red medium to a deep pink or fuschia

Answer 35

Phenol red

Question 36

Urease Test

Positive Results?

Answer 36

Deep or fuschia pink