LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
Laboratory #10
Non-Fermentative Gram-Negative Bacilli
Skills= 7 points
Objectives:
At the completion of this lab, the student should be able to:
1. Select the appropriate culture media to optimize the laboratory recovery of nonfermentative gram- negative bacilli.
2. Demonstrate the following reactions of Pseudomonas aeruginosa and Escherichia coli:
a. Growth characteristics on BAP, MacConkey and CNA agar
b. Growth in TSI
c. Oxidase reaction
d. Pigment production
e. Oxidative vs. fermentative capabilities
Materials:
Miscellaneous media (TSA,CNA, MacConkey) cultures of:
Escherichia coli
Pseudomonas aeruginosa
2 Triple Sugar iron (TSI) slants
Oxidase test materials
4 O-F glucose tubes
Mineral oil
References:
1. Mahon and Manuselis, Textbook of Diagnostic Microbiology, Third Edition, Chapter 22
Overview:
The gram-negative, nonfermentative bacilli differ from the Enterobacteriaceae, Vibrio,
Aeromonas, and other aerobic, facultative gram-negative rods by not fermenting
carbohydrates, and thereby producing no change on triple sugar iron (TSI) slants. Most nonfermentative bacilli (NFB) are opportunistic pathogens that primarily attack the debilitated
host. Although these bacteria account for approximately 15% of the isolates seen in the
clinical lab- by far, the most common NFB is Pseudomonas aeruginosa. Pseudomonas
aeruginosa has been implicated in wound and hospital-acquired infections, cystic fibrosis
patients, septicemia, and osteomyelitis.
Procedures:
1. Determine amount of growth, colony characteristics, and hemolysis for Escherichia coli
and Pseudomonas aeruginosa on BAP, MacConkey, and CNA agars and record results
on report form.
MLAB 2434 – Laboratory 10 – Page 1
LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
2. Inoculate one TSI with Pseudomonas aeruginosa and one TSI with Escherichia coli and
incubate at 35° C for 24 hours. Read and record results according to established protocol.
3. Perform an oxidase test on Escherichia coli and Pseudomonas aeruginosa and record
results on report form, according to established protocol.
4. Pigment Production
Discussion: Three types of pigment, pyocyanin, pyoverdin (also called fluorescein),
and pyorubin, may be produced by various strains of Pseudomonas. Pyocyanin is bluishgreen, non-fluorescing, water and chloroform soluble pigment which is a phenazone
derivative. It constitutes a reversible oxidation-reduction system and acts as a respiratory
catalyst, enabling the organism to use oxygen after the cytochrome system has been
poisoned by cyanide. Pyoverdin (fluorescin) is a greenish-yellow, fluorescent, water
soluble, chloroform-insoluble pigment which is formed in the presence of phosphate.
Pyorubin is a pink soluble pigment.
Procedure:
A. Observe any pigment produced by Pseudomonas aeruginosa and Escherichia coli on
any of the agars and record results by giving the color AND the name of the pigment.
Pseudomonas
species
P. aeruginosa
P. fluorescens
P. putida
P. cepacia
P. stutzeri
P. maltophilia
P. pseudomallei
Pyocyanin
Pyoverdin
(fluorescin)
Pyorubin
+
-
+
+
+
-
-(+)
-
5. Oxidation-Fermentation (OF) Test
Discussion: Bacteria produce acid from carbohydrates by two methods. One is an
anaerobic process called fermentation; the second, designated oxidation, is an aerobic
process. Since gram negative bacteria which attack carbohydrates usually do so
exclusively by either fermentation or oxidation, the determination of the type of
carbohydrate metabolism carried out by an organism is of taxonomic significance.
Enteric fermentation broth carbohydrate media will detect acid production by
fermentative organisms. Oxidative organisms do not usually produce a detectable
amount of acid in enteric fermentation broth media since the acidity produced by
oxidation is less than by fermentation and because sufficient alkaline products are
frequently produced from the peptone in the medium to neutralize the acid produced by
oxidative metabolism. Triple sugar iron agar (TSI) can be used to differentiate
fermentative from oxidative bacteria. A TSI reaction of acid butt with or without an acid
slant is indicative of a fermenter. No change of the butt and slant of TSI or slight
MLAB 2434 – Laboratory 10 – Page 2
LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
alkalinization of the slant is indicative of an oxidizer or non-utilizer of sugars.
Occasionally, an oxidizer will show slight acidity on the slant of TSI. In cases where the
TSI reaction is doubtful, an oxidation-fermentation test can be carried out using OF
medium with 1% glucose (dextrose) as described.
An OF medium should be used to determine the pattern of carbohydrate metabolism of
oxidative bacteria. OF base medium contains less peptone than do the fermentation
bases and contains 0.3% agar. The agar prevents convection currents and allows the acid
produced to remain concentrated in the medium adjacent to where it is produced and not
be diluted by mixing throughout the medium.
Procedure:
1. Two tubes are required for the OF test. Inoculate two, each with P. aeruginosa and E.
coli by using a straight inoculating needle. Transfer a generous amount of the test
organism to the OF medium by stabbing four (4) times approximately 1/3 the depth
of the medium.
2.
Overlay one inoculated tube from each pair with 1 mL of mineral oil.
(The tubes
overlayed with oil are called "closed" tubes). This overlay prevents the diffusion of
oxygen into the medium and creates an anaerobic condition in the tube.
3. Incubate with caps loose at 35° C for 24 hours.
4. Following incubation, interpret the results based on the interpretive table below.
5. On the report form, record as Oxidative (O), fermentative (F), or negative (N).
Negative indicates no fermentative or oxidative metabolism of the carbohydrate in
the media.
Reading and Interpretation:
Open Tube
Closed Tube
Method of Glucose
Utilization
Yellow (A)
Yellow (A)
Fermentative
Yellow (A)
Blue-Green (-)
Oxidative
Blue-Green (-)
Blue-Green (-)
Non-utilizer or
possible no growth
MLAB 2434 – Laboratory 10 – Page 3
LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
MLAB 2434 – Laboratory 10 – Page 4
LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
NAME ___________________________________
DATE __________________________________
Laboratory #10: Non-Fermentative Gram-Negative Bacilli
Report Form
Points: 7
Organism
BAP *
CNA *
Mac *
TSI
(A/K/G/H2S)
Oxidase
(P/N)
Pigment
Oxidation/Fermentation
( F/ O/ N)
E. coli
P. aeruginosa
* Include amount of growth, colony morphology, and hemolysis. If no growth present, notate it.
MLAB 2434 – Laboratory 10 – Page 1
LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
Name:_______________
Date:_______________
Lab #10: Non-Fermentative Gram-Negative Bacilli
Study Questions
Points= 12
1.
Define “oxidation”. (1 pt.)
2.
Explain how oxidation differs from fermentation. (1 pt.)
3.
Explain the use of OF media in differentiating between oxidative and fermentative
organisms. (1 pt.)
4.
Why is it necessary to keep the caps loose on OF media after inoculation? (1 pt.)
5.
How do the non-fermentative gram-negative bacilli different from the Enterobacteriaceae
with respect to oxidase activity? (1 pt.)
6.
Which non-fermentative gram-negative bacillus is most often implicated in infections? List
at least three (3) common sites of infection for this organism. (2 pts.)
7.
List as least four (4) characteristics of Pseudomonas aeruginosa. (2 pts.)
MLAB 2434 – Laboratory 10 – Page 1
LABORATORY #10
Non-Fermentative Gram-Negative Bacilli
8.
Nonfermenting gram-negative rods will typically produce the following reactions on KIA or
TSI: (1 pt.)
a. Acid/Acid (A/A)
b. Acid/alkaline(A/Alk)
c. Alkaline/acid (Alk/A)
d. Alkaline/alkaline (Alk/Alk)
9.
An organism producing a yellow color in the open tube and a blue-green color in the closed
tube of O-F glucose medium is said to: (1 pt.)
a. Ferment glucose
b. Oxidize glucose
c. Not utilize the carbohydrate
d. Be nonviable
10. Quality control on O-F glucose tubes with ATCC strains of organisms gave the following
reactions: (1 pt.)
E. coli
Ps. aeruginosa
Open tube
Yellow
Yellow
Closed tube
Yellow
Yellow
The results are:
a. Acceptable
b. Unacceptable; they are consistent with mineral oil layered onto the open and
closed tubes
c. Unacceptable; they are consistent with mineral oil not layered onto the
closed tubes
d. Unacceptable; they are consistent with mineral oil layered onto the open
MLAB 2434 – Laboratory 10 – Page 2