Identification of an Unknown Organism

#22
Nathan Cain
Microbiology 201
Wed. 11 o’clock Lab
Aim:

To identify an unknown organism based of morphological characteristics as well as metabolic reaction to

various media

Principle:

Take an unknown organism and perform staining, differential media tests, and colony cultivation.

 Gram staining
- Identifies between Gram positive and Gram negative bacteria
- Allows for identification of cell type and arrangement under bright field microscopy
 Metabolic Testing
- Used to establish the metabolic activity based of different nutrient utilization
- Can distinguish between similar enteric organisms
- Establish a biochemical profile
 Levine EMB Agar
- Dilute the sample by streaking and establish visually different colonies
- The media used is selective to enteric bacteria
- Media used allows for differential analysis of metabolic waste product

Material:

 General Materials
- Organism #22 Suspension
- Inoculating loop
- Bunsen Burner
 Gram Staining
- Clothespin
- Slide
- Bibulous Paper
- Microscope
- Immersion Oil
- Crystal Violet
- Gram’s Iodine
- Decolorizer (95%) Ethanol
- Safranin
 Metabolic Tests
- Test tubes containing liquid media:
Phenol Red Glucose – GLU
Phenol Red Lactose – LAC
Phenol Red Sucrose – SUC
MR Broth – MR
Voges-Proskauer (VP) broth – VP
Urea Broth – U
- Test Tubes Containing Solid Media:
SIM Agar – SIM
Citrate Agar - CIT
 Additional Materials
- Indole (Kovac’s) reagent
- (VP) reagent A
- (VP) reagent B
- Methyl Red solution
- Levine EMB agar plate
Gram Staining:

1) Sterilize Inoculating Loop and allow to cool

2) Dip Inoculating loop into Organism Suspension
3) Deposit culture to slide surface
4) Allow slide to dry on slide warmer
5) Fix culture to slide by passing quickly through flame sample side down
6) Flood sample side with crystal violet and allow culture to remain submerged for one minute; flush
with water
7) Flood slide with Gram’s Iodine for 1 minute and rinse with water.
8) Rinse sample location with 3-4 drops of decolorizer; immediately rinse with water(Keep slide
tilted during entire step so that Decolorizer does not sit on sample area)
9) Counter stain culture with Safranin for 1 minute then rinse with water
10) Dry on Bibulous Paper and view culture under 100x Objective with immersion oil
11) Record Results

Metabolic Activity Tests:

1) Sterilize Inoculation Loop in flame and allow to cool

2) Collect a loop of culture and transfer into test tube GLU
3) Repeat steps 1 and 2 for test tubes LAC, SUC, MR, VP, U
4) Sterilize Innoculation loop and allow to cool
5) Collect loop of culture and plunge into tube containing SIM
6) Repeat step 4; collect loop of culture and deposit on the surface of SIM media
7) Repeat step 4; collect loop of culture and deposit on the surface of CIM
8) Incubate VP and U tubes at 37º C for 48 hours
9) Incubate remaining tubes at 37º C for 24 hours

Levine EMB Agar Plate Inoculation:

1) Sterilize Inoculation loop and allow to cool

2) Collect loop of culture and deposit on agar surface for point A(Marking location aids in quick
identification)
3) Sterilize loop and allow to cool; beginning at point A slide loop across the edge of the dish on the
surface of Agar to make Streak B
4) Sterilize loop and allow to cool; rotate dish 90º(away from point A) and beginning at end of Streak
B slide the loop along the edge of the dish making several streaks across the agar surface(Streak
C)
5) Sterilize loop and allow to cool; rotate dish 90º(in same direction as previously) and beginning at
end of Streak C slide the loop along the edge of the dish making several streaks across the Agar
surface(Streak D)
6) Repeat Step 5 to create Streak E
7) Sterilize loop and allow to cool, now make multiple streaks in center of dish originating from
Streak E, making no contact with locations A-D
8) Cover and label dish; Incubate at 37° C for 24 hours.

After Incubation:

1) To the VP tube; add 3 drops of VP reagent B and flick tube, add 3 drops of VP Reagent A and
flick tube. Set to the side, if positive results should appear in 30 minutes.
2) To the SIM tube; add 3 drops of Indole ( Kovac’s) reagent. Set aside, if positive results should
appear in 5 minutes.
3) To the MR tube; add 4 drops of methyl red and shake tube, results should appear.
4) Record results from all 8 tubes.
5) Record and interpret results from colonies on Levine EMB agar plate.
Results:

Gram-Staining indicated Gram ¯ single celled bacillus organisms. There appeared to be a

contamination of Gram+ paired cocci.

Sugar Fermentation Tests:

Metabolism of sugars leads to metabolic waste(acids) being produced which changes the color of
the pH indicator(Phenol Red) within the from Red(sugar not utilized, negative reaction) to
Yellow(Sugar metabolized, positive reaction)

- Glucose -> positive

- Lactose -> positive
- Sucrose -> positive

SIM agar indole test:

SIM contains the amino acid tryptophan. A bacterium producing the enzyme tryptophanase breaks down
tryptophan to indole:

- Tryptophan+tryptophanase –––> Indole + Pyruvic acid + NH3.

Indole reacts with the indole (Kovac’s) reagent to produce a red colored ring at the agar surface.

- Indole prduction -> negative

SIM agar H2S test:

H2S is produced from the metabolism of Na2S2O3. H2S reacts with Fe2+ salts to form FeS which is black in
color.

- H2S production - > negative

MR Test:

Some bacteria metabolize glucose to produce small amounts of acid, with other non-acidic
metabolites like ethanol and 2, 3-butanediol. The addition of methyl red (a pH indicator) reveals
the amount of acid produced. Red indicates a large amount of acid produced, orange or yellow is
indicative of slight acid production
- Acid production -> negative

VP Test:

The 2,3 butanediol produced from sugar metabolism cannot be detected directly. It’s metabolic
pre-cursor acetylmethylcarbinol is detected with the VP reagent.

- Butanediol production -> positive

Citrate test:

Citrate is present in the medium as sodium citrate. When the organism metabolized citrate through the
Krebs cycle to CO2, sodium is left behind as sodium hydroxide. The medium becomes alkaline, and the pH
indicator turns blue.

- Citrate Utilization -> positive

 Urea Test:

Organisms producing the enzyme urease during growth break down the urea in the medium to CO2 and
ammonia. The ammonia being alkaline, the pH indicator turns purple.
- Urease production -> negative

Levine EMB agar test:

Levine EMB agar contains the sugar lactose, and dyes eosin and methylene blue. If the organism
metabolizes lactose and produces large quantities of acid, the colonies appear dark purple in transmitted
light. In reflected light, a metallic green sheen may be seen.

Colony Characteristics:
Size: 3mm, moderate size
Form: Circular
Elevation: Raised
Pigmentation: Light pink with slight lightening of tint towards the center
Margin: Entire
No additional color or sheen was observed therefore acid was not produced

Table:

Organism GLU LAC SUC SIM SIM MR VP CIT U EMB

(indole) (H2S)
Unknown + + + – – – + + – –
organism #
22
Enterobacter + + + – – – + + – –
aerogenes

Citrobacter + + + – + + – + – –

freundii

Escherichia + + – + – + – – – +

coli

Klebsiella + + + – – + + + + –

pheumoniae

Conclusions:
Enterobacter aerogenes is a Gram negative single cell bacillus. It is normally found within the
human gastrointestinal tract and is non-pathogenic in healthy individuals. The organism has been found
to cause many socomial infections especially in terms of urinary tract infections, wound and soft tissue
infection, and lower respiratory tract infections. Due to the hospital environment some strains have
developed antibiotic resistance though many infections do respond to common treatment. Most
commonly the organism is transmitted during extended stays within the hospital especially the ICU.
Patients involving invasive style treatment are the most susceptible and infection is rarely community
based. Proper identification is important to determine effective methods.