Enrichment & Isolation of

Purple Nonsulfur Bacteria

Dominado, K., Esberto, I., Manuel, K., Molao, Y.
ABSTRACT
This study was carried out to isolate and microscopically observed the purple
non-sulfur bacteria in a pond sample. It was first grown in a glass bottle for 2-3 weeks in
a BIM media, until change in color was noticed. It was then streaked into a BIM agar for
isolation. It was incubated in an improvised anaerobic chamber using bell jar in a close
room with a minimal light. The colonies after inoculation was colorless or rusty which
gives a negative result in this experiment. Microscopy was done and the cells were
green pigmented and rod shaped. According to Uffen & Wolfe (1970), PNSB colonies
remained colorless after incubating in a dark condition. Therefore, the colonies turned to
be colorless or rusty due to lack of light during incubation. The initial identification of
isolated PNSB following evaluation of identification key stated that they belong to the
genus Rhodobacter.
carbon, nitrogen and sulfur (Kondo et al.,

INTRODUCTION

2010).

The major free-living, nitrogen-fixing

microbial

systems

the

preeminent photoheterotrophs, are capable

inhabit

of photoautotrophy, and possess diverse

These

capacities for dark metabolism and growth

through

(Madigan & Jung, 2009). Some purple

bacteriochlorophyll and carotenoid which

bacteria are called purple nonsulfur bacteria

are divided into two principal classes which

because it was originally thought that they

include the green bacteria and the purple

were unable to use H2S as an electron

bacteria (Poretsky, 2003).

donor for the reduction of CO2 to cell

photosynthetic
floodwater
organisms

The

include

Purple nonsulfur bacteria, natures

bacteria

and

that

surface

absorb

purple

soil.

light

nonsulfur

bacteria

material. In fact, H2S can be used by most

(PNSB) are one of the most diverse

species in this group, although the levels

photosynthetic bacteria. These bacteria are

ideal for purple sulfur bacteria are typically

called nonsulfur because it was originally

toxic to most purple on sulfur bacteria.

thought that they were unable to use sulfide

Some purple nonsulfur bacteria can

as an electrondonor for the reduction of

also grow anaerobically in the dark using

CO2 to cell material. The PNSB are known

fermentative

to play an important role in the circulation of

metabolism, and most can grow aerobically

or

anaerobic

respiratory

in darkness by respiration. The anaerobic

1 | Page

environment is generated microbiologically-

Media used for isolation was the BIM

aerobes use glycerol until the oxygen is

and was only added by a 7.5 g of agar, with

depleted, and then are unable to ferment it

a pH of 7.0.

and so stop growing. The tubes turn dark

Enrichment

brown to bright red, or sometimes green,

The

because of the organisms photopigments.

Enrichment and isolation of purple
nonsulfur bacteria is easy using a mineral
salts medium supplemented with an organic
acid

as

carbon

source.

Such

media,

inoculated with a mud, lake water, or

sewage

sample

and

incubated

anaerobically in the light, invariably select

for purple nonsulfur bacteria (Madigan et al.,

pond

water

sample

was

aseptically transferred to sterile empty glass

bottle, containing BIM, to the top of the neck
of the tube until no air was left. A syringe
needle was put on the tip of the bottle cover,
and a paper was wrapped over the base of
the needle to absorb the liquid displaced
from the tube by the cover. This was
incubated for 2-3 weeks in the lighted area
inside the laboratory.
Growth of purple non-sulfur bacteria

2012).

is indicated by red, purple, or brown color.

MATERIALS AND METHODS

Growth of sulfur reducing organisms is

indicated by an opaque black color (metal

Source of Organisms
The organism was isolated from the
pond water sampled from BFAR, Kabacan,
Cotabato and was inoculated in a Basic
Isolation Media.

sulfides).
After 3 weeks of incubation and the
bottle was brightly colored, 2 drops was
transferred to a fresh BIM bottle. It was
followed by the same procedure mentioned
above.

Media
Media used for enrichment was

Isolation

Basic Isolation Media (BIM) composed of

After another 3 weeks of incubation,

(NH4)SO4, 0.5 g; K2HPO4, 2.5 g; MgSO4, 0.1

and the bottle was brightly colored again. A

g; NaCl, 1 g; NaHCO 3, 2.5 g; Yeast extract,

sample of the enrichment from the tube by

0.75 g and Glycerol, 0.75 g per 500 mL of

the syringe was drawn, and inoculated in a

distilled water. Transparent empty glass

BIM plate. It was incubated for a week

bottles with cover were used instead of

inside an improvised anaerobic bell jar.

Balch tubes.

2 | Page

After a week of incubation, another

fresh plate was prepared and streaked with
a well- isolated colony.
Microscopy
Cultures were wet mounted in the
slide for morphological characterization.

A
.

Experiment Guide for Observations

The purple non-sulfur bacteria can
be

classified

by

gross

microscopic

morphology into 5 groups:

B
.

Fig. 1. Setup for the enrichment of purple

nonsulfur bacteria in a BIM inoculated with

Rhodomicrobium -often in chains

pond water during 3 weeks of incubation. (a)

Rhodobacter - very large short rods or

cocci
Rhodopseudomonas -straight or bent rods

Pond only - Negative Control. (b) Pond water

and BIM.

Rhodospirillum -large spirals

Rhodocyclus - which look like a lockwasher.

RESULTS AND DISCUSSION

Purple

nonsulfur

bacteria

were

isolated in Basic Isolation Media (BIM)

bottle and pond water from BFAR, Kabacan.
Positive result indicates turning the color

Fig. 2. Colorless PNSB colonies.

into red, purple or brown, but in this case its

Purple nonsulfur bacteria should

red. Inoculation of the water sample on the

growth of

have a well- pigmented colony. But in this

colorless colonies of bacteria after a week

case it is colorless. In the study of Uffen &

of incubation (Figures 1 and 2). Microscopy

Wolfe (1970), entitled Anaerobic Growth of

was done and green pigmented bacterial

Purple

cells were observed (Figure 3).

Conditions, after their 10 day incubation

BIM

agar

yielded

optimum

Nonsulfur

Bacteria

under

Dark

that bacterial growth is apparent but the

colonies remained colorless. We concluded
that due to the incubation of our anaerobic
3 | Page

bell jar in a close room with minimal light,

conditions, so that this experiment will be

the colonies become colorless. Therefore,

validated.

light is necessary during incubation which

turned out to be an error in our experiment.
REFERENCES
Kondo, K., Nakata, N., & Nishihara, E.
(2010). Effect of the purple nonsulfur
bacterium
(Rhodobacter
sphaeroides) on the Brix, titrable
acidity, ascorbic acid, lycopene and

- carotene in tomato fruit.

Fig. 3 Microscopic observation showing

green pigmented cells.

Cocci

and

rod-cell

shape

were

observed under the microscope. Through its

morphological characteristics we concluded
that it is Rhodobacter species.

CONCLUSION
Purple

nonsulfur

bacteria

were

isolated from pond water sample using

Basic

Isolation

Medium

enrichment.

Colonies were colorless due to lack of light.

Microscopic

observation

revealed

the

bacterial isolate belongs to the genus

Rhodobacter.
RECOMMENDATION
Conduct another study on purple
nonsulfur bacteria comparing light and dark

4 | Page

International
journal
of
agriculture and environment,
743-746.carotene in tomato
International
journal
of
agriculture and environment,
743-746.

food,
8(2),
fruit.
food,
8(2),

Madigan, M.T. & Jung, D.O (2009). An

Overview
of
Purple
Bacteria:
Systematics,
Physiology,
and
Habitats
Madigan, M.T., Martinko,J.M., Stahl,D.A.,
Clark, D.P. (2012) Brock Biology of
Microorganisms. Pearson Education,
Inc.,
publishing
as
Benjamin
th
Cummings. 12 Edition.
Poretsky, R. S. (2003). Finding a niche: The
habits and habitats of purple nonsulfur bacteria. Retrieved from
http://www.mbl.edu.
/microbialdiversity/files/2012/08/200
3_poretsky.pdf
Uffen, R.T and & Wolfe R.S (1970).
Anaerobic
Growth
of
Purple
Nonsulfur Bacteria Under Dark
Conditions. American Society for
Microbiology. Vol 104, No. 1