Environmental biotechnology laboratory manual

Environmental
Biotechnology
Laboratory Manual

Prof. Ismail Saadoun


ISLAMIC UNIVERSITY OF GAZA
DEPARTMENT OF BIOTECHNOLOGY

ENVIRONMENTAL BIOTECHNOLOGY
LABORATORY MANUAL

Prof. Dr. Ismail Saadoun
Department of Applied Biological Sciences, Jordan University of
Science and Technology, P.O. Box 3030, Irbid- 22110, Jordan.
Phone: +962-2-7201000-Ext. 23460; Fax: +962-2-7201071. E-mail
address:

i


Copyright 2008.
All rights reserved. No part of this publication may be reproduced, stored in a retrieval
system, or transmitted, in any form or by any means, electronic, mechanical, photocopying,
recording, or otherwise, without prior written permission of the author.

Prof. Dr. Ismail Saadoun
Department of Applied Biological Sciences, Jordan University of Science and Technology,
P.O. Box 3030, Irbid- 22110, Jordan. Phone: +962-2-7201000-Ext. 23460; Fax: +962-27201071. E-mail address:

ii


PREFACE
This manual has been designed for an undergraduate level laboratory sessions in
environmental biotechnology. The manual is divided into experiments that belong to a
particular category. An experiment will be carried out each week and some times may be
continued in the week after.
It should be noted that the first exercise in this manual require a repetition of basic techniques,
and most results call for observations and tabulations.
Prior to each lab session, careful orders and preparations are required which can be found in
the procedure or the appendix sections. Each experiment contains the following basic
sections:
Introduction
Background and principles behind the assays performed.
Procedure
A detailed description of the materials, equipment needed to conduct the experiment and the
method to be followed. Detailed listing of laboratory media, cultures, and special chemicals
are also included.
Results
The experimental analysis data are lay out as tables and figures. Reports of the field visits are
also included as instructed.
References and further readings
A listing of useful articles and books is also provided.
Appendix
Media, buffers and solutions used in each experiment are provided. Their composition and
companies which supply them are also included.

Prof. Dr. Ismail Saadoun
Dept. of Biotechnology and Genetic Engineering

Dept. of Applied Biological Sciences
Jordan University of Science and Technology
Irbid-22110, JORDAN
Tel: (Work) 962-2-7201000, Ext. 23460
Fax: 962-2-7201071
E-mail: ;
iii


ISLAMIC UNIVERSITY OF GAZA
Dept. of Biotechnology
Environmental Biotechnology Lab

Contents
Introduction and Orientation/ Review of Microbial Techniques
Isolation and Characterization of Bacteria from Crude Petroleum Oil
Contaminated Soil
Growth Response of Bacteria on Petroleum Fuel (Diesel)
Enrichment for Uric Acid Utilizing Bacteria
Environmental Detection of Streptomycin-Producing Streptomyces spp.
by Using strb1 and 16S rDNA-Targeted PCR
Field Trip (Main Wastewater Treatment Plant in Gaza)
Molecular Detection of Fecal Coliforms (E. coli) in Water by PCR
Field Trip (Main Landfill Site in Gaza)
How the community deals with domestic solid waste?
(Collection, disposal and treatment)
Interaction of Plant Seeds with Diesel for Potential Use in the
Remediation of Diesel fuel Contaminated Soils
Detection of Alkylbenzenesulfonate-Degrading Microorganisms
Risks of Genetically Modified Organisms (GMOs)

Appendices

iv

Pages
6-12
13-15
16-21
22-24
25-28
29
30-35
36-37
38-46
47-49
50-55
56-58


ISLAMIC UNIVERSITY OF GAZA
Dept. of Biotechnology
Environmental Biotechnology Lab
Lab Schedule
The aim of this lab course is to provide an understanding of the metabolic capability of
microorganisms to reverse and prevent environmental problems. Topics will cover: Sewage
treatment, control of domestic, agricultural and industrial wastes, biocontrol of pests and
molecular detection of microorganisms in the environment. Scientific visits are hopefully to be
worked on with proper arrangements.
Week
Exercise

Introduction and Orientation/ Review of Microbial Techniques
1
Isolation and Characterization of Bacteria from Crude Petroleum Oil
2
Contaminated Soil
3
Continue
Growth Response of Bacteria on Petroleum Fuel (Diesel)
4
Enrichment for Uric Acid Utilizing Bacteria
5
Environmental Detection of Streptomycin-Producing Streptomyces spp.
6
by Using strb1 and 16S rDNA-Targeted PCR
7
Mid Term Exam
Field Trip (Main Wastewater Treatment Plant in Gaza)
8
Molecular Detection of Fecal Coliforms (E. coli) in Water by PCR
9
Continue
10
Field Trip (Main Landfill Site in Gaza)
How the community deals with domestic solid waste?
(Collection, disposal and treatment)
Interaction of Plant Seeds with Diesel for Potential Use in the
13
Remediation of Diesel fuel Contaminated Soils
Detection of Alkylbenzenesulfonate-Degrading Microorganisms
14

Risks of Genetically Modified Organisms (GMOs)
15
16
Final Exam

v

Pages
6-12
13-15
16-21
22-24
25-28
29
30-35
36-37
38-46
47-49
50-55
-


Review of Microbial Techniques
CULTURAL TRANSFER
The procedure for transferring a microbial sample from a broth or solid medium or from a
food sample is basically the same. The sample is collected with a sterile utensil and
transferred aseptically to a sterile vessel. Two implements commonly used for collecting and
transferring inoculum are the cotton swab and the platinum needle or loop. The swab is used
in instances where its soft nature and its fibrous qualities are desired such as in taking a throat
mucus sample or in sampling the skin of an apple. A platinum needle or loop is used in those

instances where a more concentrated microbial sample is available, such as in a contaminated
water sample. A typical culture transfer proceeds as follows:
1. In one hand hold the wire loop as you would a pencil;
2. Heat the wire loop until red;
3. Allow it to cool for a moment (this prevents burning or boiling of the medium when it
contacts the loop);
4. Holding the culture container in the other hand, remove the cover by grasping it between
the small finger and the palm of the loop holding hand;
5. Flame the container by passing the vessel top through the flame slowly (2 to 3 sec) in order
to sterilize the rim;
6. Insert the wire loop and take the sample;
7. Reflame the container top and replace the lid;
8. Open and flame the top of the receiving vessel as you did with the sample vessel;
9. Inoculate the sample into the vessel;
10.Reflame and cap the receiving vessel-;
11.Flame the loop to resterilize it.
All vessels used need to be clearly labeled for identification. The date and name of the person
using the vessel should be included along with the other pertinent information, (e.g, medium
type, control, concentration, etc.). All swabs, medium tubes, culture plates, and other items
contaminated with microbes should be autoclaved before washing or disposal.
PLATING
Isolation of individual microbial types may be obtained by dilution methods. The dilution, a
reduction of microbial cell concentration, may be achieved by spreading a small amount of
culture across a wide medium surface. This technique is called streaking. Bacterial cell
dilution may also be carried out using a series dilution scheme, a small amount of initially
concentrated culture is introduced into a volume of medium or physiological saline and then
homogeneously dispersed into that volume. Physiological saline (0.85% NaCl) is used to
protect cells from sudden osmotic shock thus preventing cell rupture, a sample of the new
volume may be redispersed in yet another dilution volume to achieve further cell number
reduction, by transferring known volumes of sample culture to known volumes of dilution

media, one can calculate the reduction in cell concentration achieved, for example, if one
introduced 1 ml of a sample into 9 ml of medium, one would have reduced the initial
concentration by a factor of ten.

1


Please refer to a dilution scheme for practice in making dilutions. In dilution schemes one
must maintain aseptic technique. All transferring items must be microbe free. All new media
or dilution media must be sterile.
A pipet is used to transfer volumes of liquid. The pipette should be clean and sterile. It should
be equipped with a pipette bulb or pro-pipette so that oral contact and the potential danger of
inhaling the microbial sample is avoided. Always place pipettes in germicidal washing
solution immediately after use.
Dilution of cultures made by volume dilution may be plated out in Petri dishes and then
incubated to allow the microbes time to grow. A typical plating procedure would be as
follows:
1. Pipette 1 or 0.1 ml of a known dilution of a sample into the bottom section
(smaller plate) of a sterile Petri dish;
2.

Within 20 min add 12-15 ml of warm (46-48°C) fluid medium to this Petri dish;

3. Cover the dish;
4.
Swirl it gently to disperse the sample throughout the medium, (a figure eight pattern
holding the dish flat on the table is the recommended swirl pattern: care should be taken to
prevent splashing of the medium onto the lid of the dish);
5.
6.

Allow the plate to stand, cool, and solidify;
Invert the Petri dish (medium surface pointing down) and incubate in this position.

Petri dishes are incubated upside down to prevent water from condensation from standing on
the medium surface during incubation. Pools of surface water would result in the loss of
individual surface colonies since bacterial cells forming in the colonies could use the water
pools as vehicles to reach the medium. After a period of incubation microbial growth may be
observed. If sufficient dilution has been achieved, individual colonies of microbes may be
clearly seen. It is assumed that colonies arises from single microbial cells, thus an individual
colony represents only one microbial type. This assumes that the microbes in the original
culture were not clustered and that a true homogenous dispersion was achieved. (Shaking the
solution with glass beads helps to break up cells clusters.) by picking out individual colonies
and transferring them to a new sterile medium, microbial isolation can be achieved.
Isolation is also achieved using the streaking technique. This involves the aseptic
transfer of a small quantity of culture to a sterile Petri dish containing medium. The most
common implement for streaking is the wire loop.
Streaks should be performed by initially introducing an inoculum of the culture onto a
small area of the medium plate surface. This is called ‘the well’. After inoculating the well,
the transfer loop is re-flamed, allowed to cool, and then touched on a remote corner of the
plate to remove any heat remaining. Beginning with the sterile loop in the well a streak is
made across a corner of the medium surface. (This spreads a bit of the culture out over the
2


medium—dispersing or diluting the culture.) the loop is re-flamed, cooled, and the streaking
continued until all the available medium surface is utilized. On a typical plate 3-5 streaks can
be made.

Remember: the streaking loop must be re-flamed after each streak.

Both processes, streaking and volume dilution, reduce and disperse the cell
concentration onto the medium. Upon incubation both dilution procedures should produce
isolated colonies of a single strain. The dilution technique has added use, in that upon
sufficient dilution, all the colonies from the dilution can be seen as separate individual spots
when plated. By counting these spots and multiplying that number by the dilution factor for
the plate, one can arrive at an estimate of the number of organisms in the original culture
solution.
As a rule of thumb only those incubated plates which have between 30 and 300
colonies are used to determine organism concentration in the original culture. Thirty is taken
as the lower limit since statistically this many individual colonies are required for accuracy in
calculation. Three hundred is taken to be the upper limit] because difficulty is encountered in
counting more than this number of colonies accurately.
Motility Testing
Many microbes are motile. Motility can be checked by inoculating a culture sample into a
semisolid medium. This is done with an inoculating needle which is stabbed straight down
and pulled straight out of the tube. Upon incubation, a non-motile colony will produce a
single line of growth along the needle jab line, while a motile colony will give a wider band
of growth.
The hanging drop mount is used to check motility. It is prepared by placing a ring of
lubricating grease around the rim of the recession in the hanging drop slide. A drop of culture
medium or a water suspension of a culture is then placed on a coverslip. The coverslip is
inverted so that the drop is clinging to the lower side, and the coverslip is laid to rest on the
slide—being supported by the ring of grease. This mount has the advantages that motility of
live, motile microbes can be observed.

3


Staining
A method of biochemical differentiation is staining. Staining operates on the principal that

different types of microbes have different chemical constituents making up their cellular
components. For example, the Gram stain operates on the principle that some cells retain a
crystal violet-iodine complex after leaching with an alcohol solvent, these cells generally have
complex membranes which result in retention of the blue complex and are thus called gram
positive. Other microbials with less complex membranes are not affected by the mordant,
iodine. The dye in these cells is washed out and replaced by a safranin counter-stain (red).
These cells are said to be gram negative. There are many other types of cellular dyes. There
are basic dyes specific for nuclear material, other cellular elements, and spores.
Objectives:
This exercise will review the technical skills required to successfully function in an analytical
microbiology laboratory. This exercise will enable you to:
1. Transfer cultures, streak plates and inoculate slants;
2. Carry out dilution schemes to obtain microbial counts;
3. Determine microbial motility by two methods;
4. Carry out gram and spore stains;
Materials:
Broth and slant of: Escherichia coli, Bacillus subtilis, Staphylococcus aureus
Broth mix of: Staphylococcus aureus and Escherichia coli
Tryptone glucose extract agar (TGEA)
1 ml pipettes
Petri plates
99 ml dilution blanks
Gram and spore stains
Semi-solid agar tubes
Procedure:
A. Microbial Isolation
1. Flask of agar medium are kept in a 48°c oven to maintain their fluidity, label _____
plates of TGEA and pour 1—15 ml of the medium into these plates and allow them to
cool and solidify for streaking and spread plating.
2. The instructors have prepared 4 different types of broth cultures. You will dilute out each

of these 4 different cultures, 2 by spread plating techniques and 2 by pour plating methods.
Your instructor will explain these procedures, as well as designate which of the cultures are
to be spread or pour plated and to what dilution. Dilution schemes should be worked out

4


first on paper to avoid confusion. (Note —examples of dilution schemes are given at the
end of this exercise)
3. If the TGEA plates prepared in step 1 have solidified, proceed to streaking so that isolated
colonies may be observed. Streak out samples from all 4 broth cultures.
Which of the cultures are to be spread or pour plated and to what dilution? Dilution
schemes should be worked out first on paper to avoid confusion. (Note —examples of
dilution schemes are given at the end of this exercise)
4. When all plates have cooled and solidified, invert and incubate at 37 c for 48 hr. Count
the plates from the dilution(s) yielding between 25 and 250 colonies. Calculate the
bacterial cell concentration in the original culture. Observe the streak plates.
Exchange class data.
B. Microbial Motility
1. Obtain 3 tubes of semi-solid agar and inoculate each tube with one of the 3 culture types
using an inoculating needle. Omit the mixed culture sure to label each tube, incubate tubes at
37c for 48 hr.
C. Staining
Use the broth cultures provided
sources for microorganisms to stain

and

the

plates

streaked

for

isolation

as

1. Make gram stains of the E. coli, S. aureus, B. subtilis and the mixed culture according to
the procedure described by your instructor. Observe these stains under the microscope
using the oil immersion magnification.
2. Make a spore stain of the cultures assigned to you. Observe it under the microscope using
the oil immersion objective. Can you observe distinct spore bodies? If so, are they
terminal, subterminal, or central? Are cells swollen at the spore location?

Dilution Calculations
Dilution factor = initial dilution x subsequent dilutions x amount plated Count per ml (or g) =
reciprocal of dilution factor x colonies counted
Example:
A sample was diluted initially 1:100 (1 ml of in 99 ml sterile diluent). A subsequent 1:10
dilution (1 ml of the initial dilution into 9 ml sterile water) was prepared. Finally, 0.2 ml of
the final dilution plated and 64 colonies were counted on the plate.
Initial dilution x subsequent dilutions x

amount plated = dilution factor

1/100 x 1/10x0.2 =0.0002 or 10- 2 x 1 0 - 1 x 2 x l 0 - 1 = 2 x 1 0 - 4

5


reciprocal of dilution factor x colonies counted = count per ml
5000 (or 5 x 103) x 64 = 320,000 (or 3.2 x 105)

A. Plate count results should be reported to two significant figures only.
Example:
If the dilution factor used was 106 and 212 colonies were counted, the count/ml would be
calculated thus,
Reciprocal of dilution factor x colonies counted count/ml = count/ml
106 x 212 = 2 . 1 2 x 1 0 8
Then, the answer should be rounded off to 2.1 x 108 colony forming units (CFU) per ml.
B. Only those plates with between 25 and 250 colonies should be used to calculate plate
counts.
Counting Colonies on Plates and Recording Results
Refer to the prepared handout for details.
References:
American Public Health Association. 1985. Standard methods for the examination of dairy
products. 15th edition (APHA: N.Y.). Chapter 5, standard plate count method.

6


7


Isolation and Characterization of Bacteria from Crude Petroleum Oil
Contaminated Soil
Introduction

Petroleum fuel spills as a result of pipeline raptures, tank failures and various other
production storage and transportation accidents is considered as the most frequent organic
pollutants of soil and ground water (BOSSERT et al. 1984; MARGESIN and SCHINNUR,
1997) and classified as hazardous waste (BARTHA and BOSSERT, 1984).
DAGLEY (1975) suggested that indigenous oil utilizing microorganisms, which have
the ability to degrade organic compounds, have an important role in the disappearance of oil
from soil. This microbiological decontamination (bioremediation) of the oil-polluted soils is
claimed to be an efficient, economic and versatile alternative to physiochemical treatments
(ATLAS, 1991; BARTHA, 1986).
In this experiment, enumeration of bacteria and assessment of microbial diversity will be
conducted for soils polluted by petroleum fuel spills. Also, the ability of different bacterial
cultures to transform diesel fuel using a simple and rapid test will be investigated.
PROCEDURE
Collection of samples:
-Collect soil samples of 1 kg from different gas stations contaminated with petroleum fuel
spills. They can collected down to 10 cm depth, after removing approximately 3 cm of the soil
surface.
Sample processing:
-Crush each soil sample, thoroughly mixe and sieved through a 2 mm pore size siever
(Retsch, Germany) to get rid of large debris. The sieved soil will then be used for the isolation
purposes.
-Place the samples in polyethylene bags, close tightly and store at 4±1 °C.
Isolation of bacteria:
-Suspend samples of 1g in 100 ml of sterile distilled water, agitate on a water-bath shaker
(100 rpm, 30 min), serially dilute up to 10-6.
-Spread aliquots of 0.1 ml from each dilution over the surface of nutrient agar plates.
Bacterial identification:
-The morphological characterization of each isolate will be first performed, noticing color,
size, and colony characteristics (form, margin, and elevation).
-Perform Gram stain test for each isolate.

8


-Grow the isolates at 42 °C.
Biochemical tests:
The following biochemical tests will be used in the identification studies: gelatin liquefaction;
citrate utilization; oxidase; catalase; growth at 6.5% sodium chloride; fluorescent pigment
production; indole formation; glucose fermentation and nitrate reduction (CAPPUCCINO and
SHERMAN 1996).
-Place the isolates in phenol red glucose broth to determine glucose fermentation as well as
gas production.

Results
Table 1. Total bacterial count and diversity in soils (at 10 cm depth) polluted with petroleum
fuel.
Sample
No.

Locality

Time of Exposure (Year) to
Petroleum Oil Spill

Colour

CFU x
105/gm

Colony
Types

1
2
3

Table 2. Morphological and physiological properties of the different bacterial isolates.
Species

Biochemical and cultural criteria
Oxidase

Citrate

Mr/VP

Indole

TSI
Gelatin

Sp. 1
Sp. 2
Sp. 3
Sp. 4
Sp. 5
Gram reaction for the isolates:

9

Nitrate

Reduction

Growth
at 42 ºC

Motility

Species
Identified


References
ATLAS, R.M., 1991. Microbial hydrocarbon degradation-bioremediation of oil spills. J.
Chem. Technol. Biotechnol. 52, 149-156.
BARTHA, R., 1986. Biotechnology of petroleum pollutant biodegradation. Microbial
Ecology 12: 155-172.
BARTHA, R. and BOSSERT, I., 1984. The treatment and disposal of petroleum refinery
wastes. In Petroleum Microbiology, ed. ATLAS, R.M. New York: Macmillan Publishing Co.
pp. 1-61.
BARTHA, R. and BOSSERT, I., 1984. The treatment and disposal of petroleum wastes. In
Petroleum Microbiology, ed. Atlas, R.M.. New York: Macmillan Publishing Co. pp. 553-578.
BOSSERT, I.D., KACHEL, W.M., and BARTHA, R., 1984. Fate of hydrocarbons during oily
sludge disposal in soil. Appl. Environ. Microbiol., 47, 763-767.
BOSSERT, I.D., and COMPEAU, G.C. 1995. Cleanup of petroleum hydrocarbon
contamination in soil. In Microbial Transformation and Degradation of Toxic Organic
Chemicals, ed. YOUNG, L.Y., and CERNIGLIA, C.E. New York: Wiley-Liss, Inc., pp. 77126.
BOSSERT, I.D., and BARTHA, R., 1984. The fate of petroleum in soil ecosystems. In
Petroleum Microbiology, ed. ATLAS, R.M. New York: Macmillan Publishing Co. pp. 435474.
Cappuccino, J. G. & Sherman, N., 1996. Microbiology: A Laboratory Manual. The
Benjamin/Cummings Publishing Company, Inc. New York, pp. 129–182. ISBN 0-8053-67461

DAGLEY, S., 1975. A biochemical approach to some problems of environmental pollution.
Essays Biochem., . 11: 81-138.
MARGESIN, R. and SCHINNUR, F., 1997 Efficiency of indogenous and inoculated coldadapted soil microorganism for biodegradation of diesel oil in Alpine Soils. Appl. Environ.
Microbiol., 63, 2660-2664.
Further Readings
1-Saadoun, I. 2002. Isolation and characterization of bacteria from crude petroleum oil contaminated soil and
their potential to degrade diesel. J. Basic Microbiol. 42 (6): 420-428.
2-Saadoun, I. 2004. Recovery of Pseudomonas spp. from chronocillay fuel-oil polluted soils in Jordan and the
study of their capability to degrade short chain alkanes. World J. Microbiol. Biotech. 20 (1): 43-46.
3-Saadoun, I. 2005. Production of 2-methylisoborneol by Streptomyces violaceusniger and its transformation by
selected species of Pseudomonas. J. Basic Microbiol. 45 (3): 236-242.
4-Ziad Al-Ghazawi, I. Saadoun and A. Al-Shak’ah. 2005. Selection of bacteria and plant seeds to grow on diesel
fuel to be used in remediation of diesel contaminated soils. J. Basic Microbiol. 45 (5): 251-256.
5. Saadoun, I., M. Alawawdeh, Z. Jaradat and Q. Ababneh. 2008. Growth of hydrocarbon-polluted soil
Streptomyces spp. on diesel and their analysis for the presence of alkane hydroxylase gene (alkB) by PCR.
World Journal of Microbiology and Biotechnology. DOI 10.1007/s 11274-0089729-z.

10


Growth Response of Bacteria on Petroleum Fuel
Introduction
Information on hydrocarbon (HC) degradation is required to determine the feasibility
to use microorganisms such as bacteria in the removal of petroleum-based pollutants from the
environment. Degradation of these pollutants by microorganisms has been assessed by a
variety of strategies. Early efforts at petroleum prospecting were based on the detection and
enumeration of HC-degrading bacteria that were associated with soils overlaying petroleumbearing formation (BRISBANE and LADD, 1965; DAVIS, 1967). Others included the
seeding of the environment with cocktails of oil-utilizing bacteria (Dave et al. 1994).
The straight-chained alkanes are usually the easiest hydrocarbons to be degraded
which are usually converted to alcohol via a mixed function oxygenase activity and through a

chemical pathway resulting finally in the formation of fatty acids (Sanger and Finnarty 1984).
Since alkanes are one of the main components of diesel fuel, thus the detection of
alcohol production as a result of alkane oxidation would be an applicable approach for
detecting the activity of microorganisms on diesel.
Simple Alkane
Monooxygenase ↓ O2/ NADH+H+
Alcohol + NAD++H2O

Alcohol Dehydrogenase ↓ NAD +
Aldehyde + NADH+H+
Aldehyde Dehydrogenase ↓ NAD+
Fatty Acid + NADH+H+

↓
β-Oxidation
Jacobs et al. (1983) reported that the detection of alcohol formation is a simple, rapid
and suitable method for the primary, semiquantitative screening of organisms capable of
ethanol production. Saadoun and E-Magdadi (1998) adopted this method to screen organisms
capable of degrading geosmin. In this experiment, the method of Jacobs et al. (1983) has been
modified in order to determine the ability of different bacterial strains in degrading diesel fuel
by transforming the diesel fuel to alcohol. The test is based on the following reactions:
Ethanol + nicotinamide adenine dinucleotide (NAD+)
↑↓ alcohol dehydrogenase
acetaldehyde + NAD+ + H+
NAD+ + H+ + 2,6-dichlorophenolindophenol (DCPIP) [oxidized, blue]
↑↓ 5-methyl-phenazinium methylsulphate (MPMS)
NAD+ + 2,6-DCPIP [reduced, yellow]
11

12


PROCEDURE
Microbial culture:
The different bacterial species recovered from different soils contaminated with petroleum
fuel spills (previous experiment) will be used to study their ability to degrade diesel fuel.
Growth on diesel:
-Incoculate colonies of different bacteria (previous experiment) into 50 ml mineral salts
medium (MSM) of Leadbetter and Foster (1958) (Per liter: FeSO4 1 mg, MgSO4.7H2O 200
mg, Na2HPO4 210 mg, NaH2PO4 90 mg, CuSO4.5H2O 5 µg, H3Bo3 10 µg, MnSO4.5H2O 10
µg, ZnSO4.7H2O 70 µg, MoO3 10 µg, CoSO4 10 µg, KCl 40 mg, CaCl2 15 mg, NH4Cl 500 mg
and NaNO3 2 mg) supplemented with 0.05% (v/v) diesel sterilized by filtration through 0.45
unit membranes (Millipore Corp. MA, USA).
-Incubate at 28°C and 200 rpm for 21 days.
-The growth response of each of the above isolated bacteria on diesel can initially determined
at 7 days intervals by physical appearance (turbidity) and measuring the optical density (O.D.)
at 540 nm using Bausch and Lomb Spectronic colorimeter 20 (Bausch and Lomb Inc.,
Rochester, NY).
-Determine the dry weight of cells/ml of the cell suspension by placing 2ml volume of the
final cell suspension in pre-weighed aluminum tares and dry at 65°C for over night before
weighing.
-Determine the growth on diesel by the ‘hole-plate diffusion method’ as follows:
-Pour 20 ml of mineral salts agar medium (MSM) into Petri dishes.
-Inoculate plates with the above test organisms using a sterile swab.
-Remove cores of 6 mm diameter from the agar. Fill up the holes with 50 µl of filter sterilized
diesel. The control hole will be filled with sterile distilled water only.
-Incubate the agar plates with the bacterial isolates overnight at 28 °C.
-Record the results after 48 hrs by physical appearance of growth surrounding the holes.
Growth conditions:

-Inoculate 6 slants of yeast extract-dextrose (YD) agar [per liter: 10 g dextrose, 10 g yeast
extract (YE), 0.5% (v/v) glycerin, pH 7.5] with the different bacteria then incubate at 28 ºC
for 48h.
Adaptation of bacteria on diesel:

13


-Inoculate cells into 100 ml broth of yeast extract (YE) (0.1%)-peptone (0.1%) plus 0.1%
(v/v) diesel, then incubate at 28 °C with shaking at 100 rev/min for 12 h.
-Centrifuge the whole mixture of each flask for 5 min at 4000 rev/min then suspend the pellet
in the same medium and incubate under the same conditions. The last step will be repeated
three times, then wash the cells three times with 0.1 mol/L phosphate buffer, pH 7.5.
-Suspend the pellets in a small volume (5 ml) of the same phosphate buffer.
Assay for diesel degradation:
The test of JACOBS et al. (1983) will be conducted to detect the biodegradation of diesel,
hoping that the bacterial strains used the monoxygenase pathway in the biodegradation
process.
-Perform the test in duplicate at 28 ºC in a small test tube containing the following: 20 µl 2,6dichlorophenolindophenol (DCPIP) (Acros organic, NJ, USA), 0.05 mol/L; 30 µl 5-methylphenazinium methylsulphate (5-MPMS) (Acros organic, NJ, USA), 0.05 mol/L; 25 µl of
0.1% (v/v) diesel, 5 µl of 0.15 M NAD solution and 25 µl of washed cells.
-Compare the change in the colour with four controls. The first control contains no diesel
(substrate), the second contains no NAD+ and the third contains no cells. A fourth control
consists of heating the cells for 10 min at 90 ºC.
-Follow the reaction at 1 hour, 2 hour, 6 hr and 12 hr.

Results
Table 1. Growth response of different bacterial isolates on diesel as measured by turbidity
and dry weight
Growth Measurements/Time (days)
Bacteria

O.D. (540 nm)
Dry Weight (mg/ml)
7
14
21
7
14
21

Readings at zero time = 0.0

14


Table 2. Action of different bacterial species on diesel as indicated by colour change
Colour change from dark blue to other colours at different time intervals by each bacterial
species
Reaction
condition
+NAD+/29˚C

Time

Sp. 1

Sp. 2

Sp. 3

1h.

2h.
6h.
12h.

Result
-NAD+/29˚C

1h.
2h.
6h.
12h.

Result
+NAD+/90˚C

1h.
2h.
6h.
12h.

Result

15

Sp. 4

Sp. 5

Sp. 5

16


References
BRISBANE, P.G., and LADD, J.N., 1965. The role of microorganisms in petroleum
exploration. Annu. Rev. Microbiol., 19, 351-364.
DAVIS, J.B., 1967. Petroleum Microbiology. Elsevier Publishing Co., New York.
JACOBS, C.J., PRIOR, B.A. and DEKOCK, M.J. 1983 A rapid screening method to detect
ethanol production by microorganisms. J. Microbiol. Methods 1, 339-342.
LEADBETTER, E.R. and FOSTER, J.W. 1958. Studies of some methane utilizing bacteria.
Arch. Microbiol. 30, 91-118.
SAADOUN, I. and EL-MIGDADI, F., 1998. Degradation of geosmin-like compounds by
selected species of Gram–positive bacteria. Lett. Appl. Microbiol, 26, 98-100.
SANGER, M. and FINNARTY, W. 1984. Microbial metabolism of straight-chain and
branched alkanes. In Petroleum Microbiology ed. ATLAS, R.M.New York: Macmillan. pp.
1-61.

Further Readings
1-Saadoun, I. and F. Al-Meqdadi. 1998. Degradation of geosmin like compounds by selected species of Grampositive bacteria. Lett. Appl. Mibrobiol. 26: 98-100.
2-Ziad Al-Ghazawi, I. Saadoun and A. Al-Shak’ah. 2005. Selection of bacteria and plant seeds to grow on diesel
fuel to be used in remediation of diesel contaminated soils. J. Basic Microbiol. 45 (5): 251-256.
3. Saadoun, I., M. Alawawdeh, Z. Jaradat and Q. Ababneh. 2008. Growth of hydrocarbon-polluted soil
Streptomyces spp. on diesel and their analysis for the presence of alkane hydroxylase gene (alkB) by PCR.
World Journal of Microbiology and Biotechnology. DOI 10.1007/s 11274-0089729-z.

17


Enrichment for Uric Acid Utilizing Bacteria

Introduction
Enrichment or selective culture techniques were first used by Winogradsky and Beijernick in
their extensive studies of soil microorganisms. It is based upon the diversity of
microorganisms which exist in nature. Microbiologists use this technique to create an in vitro
environment in the laboratory which favors the isolation of a particular microorganism. This
is achieved in two ways:
1-Optimal conditions for growth are selected
2-The most rapid growth rate for the desired organism is selected
Generally enrichment culture is done in liquid-batch culture where medium
composition and physical parameters such as temperature can be controled and or varied,
selective inhibitors can also be added to the medium to control or inhibit unwanted organisms.
For example, cyclohexamide added to the medium will inhibit the growth of fungi which
might otherwise overgrow the desired bacterial species. Of course, also important is the
source of the inoculum for the enrichment.
Enrichment for the bacterium Bacillus fastidiosus that is able to grow on uric acid or
allantoin was first described by den Doceren de Jung in 1929. Only uric acid or allantoin can
be metabolized by the bacterium. Uric acid and allantoin are breakdown products of purine
(Fig. 1).
In this experiment, you will enrich for B. fastidiosus which can grow on uric acid.
Media
1-Uric acid broth tubes
0.5% uric acid + mineral salt (MS) base in tap water
MS base: NH4Cl 1.0 g; Na2HPO4.2H2O 2.14 g; KH2PO4 1.04 g; MgSO4.7H2O 0.2 g; Trace
salt solution 10 ml; water 1000 ml, pH 7.0.
Trace salt:
FeSO4.7H2O

300 mg

MnCl2.4H2O

180 mg

Co(NO3)2.6H2O

130 mg

ZnSO4.7H2O

40 mg

H2MoO4 (Molybdic acid) or molybdenum
trioxide

20 mg

CuSO4.5H2O

1 mg

CaCl2

1000 mg

HCl (0.1 N)

1000 ml

2-Uric acid (UA) agar plates: MS base + 0.5% UA + 1.5% agar
3-UA-yeast extract (YE) broth and agar: same as 1 and 2 + 0.5% YE

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4-Glucose MS agar: Glucose 0.5%, MS + 1.5% agar. The glucose is sterilized separately and
added to the sterile MS agar base.
5-Glucose-Casein hydrolysate agar: glucose 0.2%, casein hydrolysate 0.5%, 1.5% agar.
6-Malate-YE slants: 0.2% malate, 0.5% YE, 1.5% agar
7-YE-NA: 1.5% NA, 0.2% YE, 1% agar in tap water (water agar)
Procedure
1-Inoculate each of 2 tubes of uric acid broth with about 0.2 gm of soil. It is preferable to get
soil sample that is contaminated with chicken manure. Why?
2-Pasteurize one broth tube by heating in a water bath at 80-85 ºC/5 min.
3-Incubate the tubes at 37 ºC / 3 days.
4-Prepare wet mounts and Gram stains of your culture.
5-Repasteurize the culture which you pasteurized in the first lab period and streak this culture
and the unpasteurized culture on separate plates of uric acid agar. Be sure to mark your plates
as pasteurized and unpasteurized.
6-Incubate at 37 ºC /3 days.
7-Look for white rhizoid spreading colonies which adhere tenaciously to the agar surface.
Prepare Gram stains of one of the colonies.
8-Transfere a single colony to a sterile test tube containing 0.5 ml of water. Mix thoroughly to
make a turbid suspension.
9-Inoculate slants of UA agar, YE-NA, glucose-MS agar, glucose-casein hydrolysate agar and
sodium malate-YE agar. Incubate at 37 ºC for 5-7 days.
11-Record your results. Did growth occur on any of the media other than the uric acid agar? If
so, prepare Gam stains and characterize the organism which grew on these media.
Bacillus fastidiosus:
Large rods, 1.5-2.5 µm x 3-6 µm; stain uniformly; often in chains, motile, with lateral
flagella. Gram positive in the early stages of growth. Endospores oval to cylindrical, 1.4 -1.7

µm x 1.8-3 µm; occupy most of the interior of the shorter sporangia; terminal or subterminal
in longer rods; may lie obliquely to the axis of closely septate filaments; produce little or no
swelling of the sporangium; have a stainable surface after release.

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