(M) Lab: Biochemical Identification of Bacteria (Part 3) Flashcards
BIOCHEMICAL IDENTIFICATION FOR GRAM-NEGATIVE BACILLI
- 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.
Biochemical Identification for gram-negative bacilli
- Uses a TSI medium: butted slant media
- It contains: glucose, lactose, sucrose (separated in carbohydrate fermentation)
- Detects the acid production, H2S, and gas production
Triple Sugar Iron (TSI)
Interpret/Report the following for TSI.
Slant: Yellow
Butt: Yellow
Report: A/A
Interpret: 2 to 3 sugars fermented
Interpret/Report the following for TSI.
Slant: Red
Butt: Yellow
Report: K/A
Interpret: 1 sugar fermented
Fermentation only occurs at the butt.
Interpret/Report the following for TSI.
Slant: Red
Butt: Red
Report: K/K
Interpret: No acid production/sugar fermented/Non-fermenter
Automatically no H2S nor gas
If only the butt is yellow, it indicates?
Fermentation
T or F: If bacteria can ferment >1 sugar, it can ferment the sugars one by one.
T
T or F: The first fermentation always happens at the slant portion wherein there is an anaerobic condition.
F (butt)
T or F: 2nd-3rd fermentation happens at the aerobic site → slant
T
- Characteristic of Salmonella typhimurium, a large H2S producer
- Only presumptive, not confirmatory
Blackening that almost occupies the entire medium, up to the slant
- 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
Lysine Iron Agar
- (+) Decarboxylates lysine through the release of decarboxylase (observed at butt portion, anaerobically)
- Deaminates due to the release of deaminase *(slant portion, aerobic)
Lysine Iron Agar
Report/Interpret based on LIA.
Slant: Purple
Butt: Purple
Report: K/K
Interpret: The organism is (+) for decarboxylation
Report/Interpret based on LIA.
Slant: Purple
Butt: Yellow
Report: K/A
Interpret:
(-) for decarboxylation
Report/Interpret based on LIA.
Slant: Red
Butt: Yellow
Report: R/A
Interpet: (+) deamination
- 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
K/K
- Yellow butt due to glucose fermentation, no decarboxylation, retaining the yellow color of the medium
K/A
- Changes occur in the slant (demonstrates deamination)
- Deaminase changes medium from purple to red
R/A
- 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)
*
Indole Test
Reagent for Indole Test?
Erlich’s Reagent → PDAB or p-dimethylaminobenzaldehyde
Indole Test
Positive test?
Red ring on top of medium
Indole Test
Negative results?
No red ring
- Uses the Methyl Red Vogues Proskauer (MRVP)
- For observing which metabolic fermentative pathway is utuilized by the bacteria use
Methyl Red
Methyl Red
- Bacterium releases various acids creating a low pH in the medium (4.0-4.6)
- Detected by methyl red
Mixed Acid Pathway
Methyl Red
Positive results?
Red solution
Methyl Red
Negative results?
Yellow (enhanced original color of the medium or no change)
- 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
Vogues-Proskauer
Butylene glycol pathway uses?
Alpha-naphthol and KOH
Vogues-Proskauer
Positive results?
Cherry red solution
Vogues-Proskauer
Negative results?
Chocolate brown
- 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
Citrate Test
Positive results?
Blue
Citrate Test
Negative results?
Green
- Checks if bacteria releases urease enzyme
- In the urea agar incorporated with phenol red
Urease Test
Urease Test
____ hydrolyzes urease to become basic ammonia turning the red medium to a deep pink or fuschia
Phenol red
Urease Test
Positive Results?
Deep or fuschia pink
