BACTERIOLOGY LABORATORY

EXERCISE NO. 6- BSMT 3A

GRAM-NEGATIVE BACILLI: Enterobacteriaceae

MATERIALS AND INSTRUMENTS

Tube media- TSI, SIM, SCA, LIA, MRVP broth, Urea Agar
Plated Media - BAP, MAC, EMB
Kovac's reagent (para-dimethylaminobenzaldehyde)
MRVP reagents
Inoculating loop and needle
Alcohol lamp
Specimen
18-to 24-hour Nutrient Broth/Nutrient Agar of E. coli, Salmonella spp. and Citrobacter spp.

METHODS:
Triple Sugar Iron Agar (TSIA) Test
Principle: Gram-negative bacteria which can ferment sugar such as glucose, lactose,
and/or sucrose are identified using the triple sugar iron agar through the production of
acidic end products detected by the pH indicator after overnight incubation, while gas and
hydrogen sulfide formation is exhibited by the presence of bubbles and black precipitate
in the medium, respectively.

Sulfide Indole Motility (SIM) Test

Principle: The sulfide indole motility medium contains a small amount of agar which after
18 to 24 hours of incubation allows motile bacteria to spread out from the line of
inoculation. The addition of the Kovac's reagent detects organisms that can hydrolyze the
tryptophan in the medium forming pink or red color ring of indole and those that produce
black precipitate are positive for hydrogen sulfide production.

Citrate Utilization Test

Principle: Bacteria that can utilize the sodium citrate in the Simmons citrate agar as the
sole carbon source convert the ammonium phosphate component in the medium to
ammonia and further to an alkaline product ammonium hydroxide causing the
bromothymol blue pH indicator to change the color of the agar slant from green to blue.

Lysine Iron Agar (LIA) Test

Principle: The amino acid lysine present in the lysine iron agar is either decarboxylated
anaerobically in the butt producing a purple color alkaline product known as cadaverine
or deaminated aerobically creating a burgundy- colored slant. The absence of both
enzymatic reactions maintains the purple color of the slant and forms a yellow color of the
butt due to glucose fermentation, while the sodium thiosulfate in the agar detects
hydrogen sulfide production.
Urease (Christensen’s Urea Agar) Test
Principle: Hydrolysis of urea by urease, a constitutively expressed enzyme, produces
ammonia and CO2. The formation of ammonia alkalizes the medium and the change in
pH is detected by the change in color from phenol red from light orange at pH 6.8 to
magenta (pink) at pH 8.1.

General Procedure:
1. Perform correctly the Gram staining.
2. From a pure culture plate, inoculate E. coli and Citrobacter separately on BAP and
MAC. Follow the order of inoculation.
3. Incubate all the agar plates at 35°C for 18 to 24 hours.
4. Interpret the results on MAC.
5. Keep the pure culture in MAC for the biochemical tests.
6. Record your observations.
7.Inoculation of organisms into TSIA, SIM, SCA, LIA and Urea Agar should all be
performed consecutively without reheating the inoculating needle in-between streaking
and stabbing techniques until the last tube medium.
Growth on Mac Conkey (MAC) Agar
Lactose Fermenters: Pink colonies
Non-lactose fermenters: Colorless colonies

PROCEDURE ON THE BIOCHEMICAL TEST- TSIA, SIM, SCA, LIA and Urea Agar
1. Using a sterile inoculating needle, touch the top of a well-isolated colony from MAC
and:
• Hold the TSIA with the other hand and aseptically stab through the center
of the butt within half-inch of the bottom of the agar.
• Pull the needle and streak the surface of the slant moving the needle
upward.
• Aseptically plug the cotton of the TSIA. Set aside.
• Do not reheat the needle.
• Hold the SIM tube and inoculate aseptically by stabbing the agar halfway.
• Aseptically plug the cotton of the SIM. Set aside.
• Do not reheat the needle.
• Hold the SCA tube and inoculate aseptically by streaking the slant starting
from the bottom of the medium.
• Aseptically plug the cotton of the SCA. Set aside.
• Do not reheat the needle.
• Hold the LIA tube and aseptically stab through the center of the butt. Pull
the needle and streak the surface of the slant moving the needle upward.
• Aseptically plug the cotton of the LIA. Set aside.
• Hold the Urea Agar tube and aseptically stab through the center of the butt.
Pull the needle and streak the surface of the slant moving the needle
upward.
• Aseptically plug the cotton of the Urea Agar. Set aside.
2. Sterilize the inoculating needle.
3.Label properly all the tubes.
4. Incubate all the tubes for 18 to 24 hours at 35°C. Do not over incubate.
5. After incubation, add 1 to 3 drops Kovac's reagent in the SIM tube.
6. Interpret the results. Record your observations.

TSIA Test Results

Lactose Fermenters: Acid slant/ Acid butt (A/A)- Yellow/Yellow
Nonlactose fermenters: Alkaline slant/ Acid butt (K/A) - Red/Yellow
Positive Gas production: Presence of bubbles or splitting of the media
Positive H&S production: Presence of black precipitate in the butt
Quality Control:
A/A, +Gas, -H2S: Escherichia coli (ATCC culture)
K/A, +/- Gas, +H2S: Salmonella typhimurium (ATCC Culture)
K/A, + Gas, + H2S: Proteus mirabilis (ATCC Culture)
K/A, -Gas, -HS: Shigella flexneri (ATCC Culture)
K/K: Pseudomonas aeruginosa (ATCC Culture)

SIM Positive Results

Sulfide: Black precipitate in the medium
Indole: Pink-to wine-colored ring after the addition of the Kovac's reagent
Motility: Spreading out from the line of inoculation
Quality Control
Motility and Indole Test Positive: Escherichia coli (ATCC Culture)
Motility and Indole Test Negative: Klebsiella pneumoniae (ATCC Culture)

Citrate Utilization/SCA Test Result

Positive: Blue colored slant
Negative Green colored slant (no change in the color of the slant)
Quality Control
Positive Blue Color: Enterobacter aerogenes (ATCC Culture)
Negative/No color change: Escherichia coli (ATCC Culture)

LIA Test Result

Alkaline slant/Alkaline butt (K/K):(-) Lysine deamination
(+) Lysine decarboxylation without glucose fermentation

Alkaline slant/Acid butt (K/A): (-) Lysine deamination

(-) Lysine decarboxylation with glucose fermentation

Red slant/Acid butt (R/A): (+) Lysine deamination

(-) lysine decarboxylation with glucose fermentation
Positive H2S production: Presence of black precipitate
Quality Control
Alkaline slant/Alkaline butt: Escherichia coli (ATCC Culture)
Alkaline slant/Alkaline butt with H2S production: Salmonella typhimurium (ATCC Culture)
Citrobacter freundii (ATCC Culture)
Red slant/Acid butt: Proteus mirabilis (ATCC Culture)

Urease (Christensen’s Urea Agar) Test Result

Positive: Magenta or Pink in the agar slant or butt.
Negative: No color change in the agar.
Quality Control:
Rapid Positive Urease: Proteus mirabilis (ATCC Culture)
Late Positive Urease: Klebsiella pneumoniae (ATCC Culture)
Negative Urease: Escherichia coli (ATCC Culture)

METHOD: Methyl Red and Voges-Proskauer (MRVP) Test

Principle: Bacteria which can maintain stable mixed acid end products from glucose
fermentation creates a bright red color after adding the methyl red reagent at pH 4.4 into
the MRVP Broth. The alpha-naphthol and potassium hydroxide reagents confirm the
generation of substances such as acetoin that can neutralize the products of fermentation
through the formation of a red color.

PROCEDURE ON MRVP TEST

1. Inoculate aseptically the MRVP broth with a drop of bacterial suspension from a 24-
hour old brain-heart infusion (BHI) culture.
2. Incubate the broth at 35°C for a minimum of 48 hours,
3. Divide the broth into two (2) aliquots for MR test and VP test
4. For the MR test, add 5 to 6 drops of MR reagent per 5 mL of broth. Read the reaction
immediately.
5. For VP test, add 6 drops of alpha-naphthol and 2 drops of KOH to 1 mL of broth. Shake
well after adding each reagent. Observe after 5 minutes.

Note: MRVP broth should be incubated for complete 48 hours because the acid end
products have higher detectable capacity if allowed to maintain the same environment
over time.

MRVP Test Result

MR Positive: Bright red color (mixed acid fermentation)
MR Negative: Yellow color
MR Inconclusive: Shades of orange (negative result is sometimes orange color)
VP Positive: Red color (acetoin production)
VP Negative: Yellow color
Quality Control
MR Positive/VP Negative: Escherichia coli (ATCC Culture)
MR Negative/VP Positive: Enterobacter aerogenes (ATCC Culture)
 EXERCISE NO. 6- BSMT 3A
GRAM NEGATIVE BACILLI: ENTEROBACTERIACEAE

NAME AND GROUP NO.: DATE:

POST LAB ACTIVITY

A. DATA AND RESULTS

Table 1. Biochemical Reactions

Organism TSI Sulfide Indole Motility Citrate LIA Urease MR
VP
MR

VP

Table 2. Colonial Characteristics

Organism Description of the Colony

#1 BAP

MAC

B. QUESTIONS (Maximum of 4 sentences)- 20 pts

1. Do we need a separate inoculum for every tube in the routine biochemical test?
Justify your answer.

2. Which culture medium is preferred as the source of the inoculum for all the
biochemical tests under Enterobacteriaceae? Justify your answer.
3. What makes Mac Conkey agar both a differential and a selective medium?

4. If the TSI medium will be incubated for more than 24 hours, would there be any
changes in the reaction? Justify your answer.