Journal of Research in Biology - Volume 4 Issue 1

Journal of Research in Biology An International Scientific Research Journal
Original Research
Diversity of freshwater diatoms from few silica rich habitats of Assam, India
Authors: ABSTRACT:
Journal of Research in Biology
Dharitri Borgohain and Diatoms are a ubiquitous class of phytoplankton of extreme importance for
Bhaben Tanti*. the biogeochemical cycling of minerals such as silica. Few places of Nagaon district of
Assam, India viz., Jiajuri, Borhola, Thanajuri and Chapanala have been recognized as
the highest silica zones by Geological Survey of India. No any research has been
conducted to explore the diatom diversity at this important silica rich habitat. In the
present investigation, the morphology and diversity of freshwater diatom species
were investigated during May 2012 to April 2013. The samples were subjected to acid
Institution:
Department of Botany, wash treatment followed by microscopic observations. Altogether 103 species of
Gauhati University, diatoms belonging to 20 genera were recorded. Occurrence of diatom varied in all the
Guwahati - 781014, Assam, four different study sites. The dominant genera includes: Stauroneis, Kobayasiella,
India. Eunotia, Pinnularia, Nitzschia, Gomphonema, Frustulia, Surirella, Achnanthes,
Rhopalodia, Navicula, Synendra, Encyonema, Achnanthidium, Cymbella, Hippodonta,
Tabularia, Actinella, Encyonopsis and Luticola. Notably, all the diatom species
belonged to pennate type.
Corresponding author: Keywords:

Bhaben Tanti. Freshwater diatoms, silica rich soil, diatom diversity, Geological Survey of
India.
Email Id: Article Citation:

Dharitri Borgohain and Bhaben Tanti.
Diversity of freshwater diatoms from few silica rich habitats of Assam, India.
Journal of Research in Biology (2014) 4(1): 1162-1173
Web Address:
http://jresearchbiology.com/ Dates:
documents/RA0410.pdf. Received: 07 Jan 2014 Accepted: 29 Jan 2014 Published: 15 Feb 2014
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1162-1173 | JRB | 2014 | Vol 4 | No 1

An International
Scientific Research Journal www.jresearchbiology.com
Borgohain and Tanti, 2014
INTRODUCTION (262010 N latitude and 925130 E longitude) deposits

Diatoms belonging to the class Bacillariophyceae occur friable quartzite covering an area of 0.373 km 2 and
are the major group of single-celled photosynthetic possible reserve is 3.5 million tones. Thanajuri hill
eukaryotic algae which can be found in almost all (2612 35 to 261310 N latitude 924840 to 925035
aqueous and humid environments. Diatoms are an E longitude and) is situated in the northern part of Karbi-
important component of phytoplankton in freshwaters. Anglong plateau and southern part of Nagaon district.
There are over 250 genera of diatoms with more than The possible reserves of glass sand is about 1.788
100,000 species (Gurung et al., 2012, Van Den Hoek million tones. Friable quartzite occurs in Borhola (2626
et al., 1997), which includes both marine and the 15 N latitude and 925645 E longitude) covering an
freshwater environments. These microscopic autotrophic area of 0.595 km2 and the possible reserve of glass sand
microalgae possess highly ornamented cell wall is about 1.25 million tones. Till date, there is no any
composed of glass silica (SiO2) called frustules which extensive work on the detailed investigation of diatom
provide a variety of shapes from nano to micro-scale diversity in these silica rich regions of Assam. Set in this
structures. Diatoms can occur in large amounts, either backdrop, the present investigation is assessed for the
solitary or in colony and is cosmopolitan in distribution. exploration of diatom, having the genetic ability to
A major constituent of the plankton family, diatoms are deposit natural silica over their cell surface in
free floating, planktonic or attached to a substrate and characteristics nanoporous forms.
benthic forms (Werner, 1977). Diatoms are important
from the point of the biogeochemical cycling of silica. MATERIALS AND METHODS
Diatoms play a very significant ecological role by fixing Sample collection and growth conditions
about 25% carbon globally. The diatoms of North East Samples were collected from aquatic and semi-
region of India are still largely unexplored and aquatic habitats of the four study sites- Jiajuri, Borhola,
unexploited. Friable quartzites belonging to the Shillong Thanajuri and Chapanala from May 2012 to April 2013
groups of rocks occur sporadically along eastern most (Fig.1). The freshly collected samples were immediately
part of the Nagaon district. Borhola, Chapanala, Jiajuri transferred to Diatom Medium (DM) proposed by
and Thanajuri are some of the important places where Beakes et al., (1988) which was standardized with slight
friable quartzites are found abundantly. About 75% of modifications and the composition of stock (per 200ml)
the glass sand may be recovered from this friable includes- Ca(NO3)2. 4H2O 4g, KH2PO4 2.48 g,
quartzite by using different methods of beneficiation MgSO4.7H2O - 5 g, NaHCO3- 3.18 g, EDTAFeNa-
(Goswami, 2006). 0.45g, EDTANa2 0.45g, H3BO3 0.496g, MnCl2.
The Geological Survey of India (GSI) has found 4H2O 0.278g, (NH4) 6Mo7O24.4H2O 0.20g,
significant reserves of silica deposits in the Jiajuri region Cyanocobalamine - 0.008g, Thiamine HCl 0.008g,
between the district of Nagaon and Karbi Anglong in Biotin 0.008g and Na2SiO3.9H2O 22.8g.
Assam (Borpuzari, 2012). The area is located about One ml of each stock solution was added to make
30kms South-East from Nagaon and is adjacent to Jiajuri the final volume of 1L with distilled water, and adjusted

Tea Estate. The deposit is bounded by latitude 26 18 0 to pH 6.8. For solid medium, 1.5% agar was added. The
to 2619 0 N and longitude 9252 55 to 9254 15 E. cultures were allowed to grow at 3K light at 18-20C for
2
Jiajuri hill covers an area of 2.9 km and the possible 20-22 days. Repeated sub-cultures were done on the
friable quartzite is about 7.4 million tones. Chapanala solid medium to obtain pure cultures of diatom species.
1163 Journal of Research in Biology (2014) 4(1): 1162-1173
Figure 1: Map showing the four study areas (source: www.mapsofindia.com).
Cleaning diatom frustules by acid wash method for until the cell suspension become less acidic. To confirm
microscopic analysis the complete removal of organic matters, a drop of
In order to analyze the diatom frustules for cleaned samples was observed under the microscope.
microscopic studies, a cleaning procedure was needed For light microscopy (LM) observation, the
that removed the external organic matrix covering the slides were prepared by evaporating drops of the cleaned
frustules. Plankton samples were subjected to acid wash diatoms suspended in distilled water onto cover-slips and
method according to the protocol of Hasle and Fryxell the mounting was done by using Naphrax (a specific
(1970) before light microscopic observations. About diatom mountant with refractive index 1.74). The slides
20ml of liquid cultures were transferred into a beaker and were examined carefully under 1000x magnification and
treated with equal quantity of concentrated H2SO4 and the diatom images were documented in Nikon ECLIPSE
agitated gently. Freshly prepared KMnO4 was added to E200 with photo micrographic attachment.
the sample until the sample had a purple tint. Then Identification of diatoms
freshly prepared oxalic acid (COOH)2 was added to The diatoms obtained through laboratory pure
obtain clear solution. The sample was centrifuged at cultures were identified by consulting various literatures
2500 rpm for 15 min and then rinsed with distilled water and monographs (Gandhi, 1955; Husted, 1959; Hendey,
Journal of Research in Biology (2014) 4(1): 1162-1173 1164

1964; Patrick and Reimer 1966; Prescott, 1975; culture were enumerated.
Desikachary, 1989; Round et al., 1990; Nautiyal et al., Out of 103 diatoms species obtained in pure
1996; Anand, 1998; Gurung et al., 2013). cultures, 25 diatoms were found to be of different species
of Nitzschia representing 24.3% of the total diatom flora.
RESULTS AND DISCUSSION Further, there were 17 different species of Gomphonema,
During the present investigation, a total of 103 15 different species of Navicula, 14 different species of
species of freshwater diatoms belonging to 20 genera of Pinnularia and 5 different species of Eunotia
class Bacillariophyceae were reported from the silica rich representing 16.5%, 14.6%, 13.6% and 4.9%
soils of Nagaon district of Assam i.e. Jiajuri, Borhola, respectively. There were four different species of
Thanajuri and Chapanala. The prominent genera in Stauroneis, Cymbella (3.9% each), followed by Frustulia
terms of its abundance and frequency were Nitzschia and Synendra (2.9% each) and Achnanthes and
(25), Gomphonema (17), Navicula (15), Pinnularia (14), Achnanthidium (1.9% each). The remaining diatoms viz.
Eunotia (5), Stauroneis (4), Cymbella (4), Frustulia (3), Surirella, Tabularia, Encyonema, Actinella,
Synendra (3), Achnanthes (2), Achnanthidium (2) and Encyonopsis, Rhopalodia, Luticola, Hippodonta and
single species of the following diatoms: Actinella, Kobayasiella were represented by only one species
Luticola, Encyonema, Hippodonta, Surirella, Tabularia, showing 8.7% out of the total diatoms identified in pure
Encyonopsis, Kobayasiella and Rhopalodia. Pure cultures (Fig. 2).
cultures of diatoms obtained in this study were identified Taxonomic account:
upto their genus level (Fig. 3-9). Morphological Taxonomic description of the 20 pennate
descriptions of the diatom isolates obtained in pure freshwater diatom genera obtained in the four silica rich
Diversity of diatom flora
Figure 2: Representation of diatom flora diversity.
1165 Journal of Research in Biology (2014) 4(1):1162-1173

sites during the study period are described below: coarse, 2-4 middle striae short and thick, radiate in the
Class: Bacillariophyceae middle, convergent towards apices.
Order: Bacillariales Class: Bacillariophyceae
Family: Naviculaceae Order: Cymbellales
Genus: Navicula Bory 1822, Cleve 1894 Family: Gomphonemataceae
Navicula sp. (Fig. 3 A-O) Genus: Gomphonema C.A. Agardh 1824
Valves 36 m long, 14 m broad, broadly Gomphonema sp. (Fig. 5 A-L, 6 M-Q)
elliptical with convex margins; ends slightly produced, Valves 45 m long and 8 m broad, clavate with
slightly capitate rounded; raphe thin, straight; central capitate head pole and slightly capitate foot pole; axial
nodules distinct; axial area narrow, linear; central area area linear, narrow, and widening into a small circular
somewhat obliquely rectangular; striae 23 in 10 m, very central area with an isolated pore on the primary side of
fine. the central nodule; raphe straight with distinct central
Class: Bacillariophyceae nodules; striae 10-11 in 10 m, punctate and slightly
Order: Naviculales radiate, wider at the centre of the valve.
Family: Pinnulariaceae Class: Bacillariophyceae
Genus: Pinnularia Ehrenberg 1843 Order: Naviculales
Pinnularia sp. (Fig. 4 A-N) Family: Amphipleuraceae
Valves 53 m long, 11 m broad, linear, more or Genus: Frustulia Lange-Bertalot
less parallel margins with slightly tapering, broadly Frustulia sp. (Fig. 6 A-C)
rounded ends; raphe thick, straight, placed on one side Valves 71-160 m long and 15.3-30.2 m
with distinct, unilaterally curved central nodules and broad, rhombic-lanceolate, narrowing sharply to the
curved terminal fissures; axial area distinct, linear; rounded apices. Axial and central areas narrow but
central area large reaching the sides; striae 7 in 10 m, distinct. Transverse striae perpendicular to the raphe at
A C D E
B
H
G
F
M N
I
J
K L
Figure 3(A-O): Navicula.

Figure 4: (A-N) Pinnularia
the center of the valve, sometimes becoming slightly Family: Diadesmidaceae

convergent towards the ends of the valve, but radiate at Genus: Luticola (Ehrenberg) D. G. Mann, 1990
the apices, striae 20-30 in 10 m. Luticola sp. (Fig. 6 E)
Class: Bacillariophyceae Valves 12-24 m long and 7-9 m broad, linear
Order: Cymbellales to linear-elliptical. Transapical striae radiate throughout,
Family: Cymbellaceae composed of two to four rounded areolae. Largest
Genus: Encyonema (Berkeley) Kutzing areolae near the valve margins. One isolated, circular
Encyonema sp. (Fig. 6 D) stigma present, striae 18-20 in 10 m.
Valves 37-91 m long and 15-30 m broad, Class: Bacillariophyceae
robust and broadly dorsiventral and symmetrical to the Order: Cymbellales
transapical axis. Dorsal margin normally arched, ventral Family: Cymbellaceae
margin biarcuate to convex. Valve apices bluntly Genus: Encyonopsis (Grunow) Krammer, 1997
rounded. Raphe straight with central endings deflected Encyonopsis sp. (Fig. 6 F)
dorsally and apical ends deflected ventrally, striae coarse Valves 21-25 m long and 5.1-6.3 m broad,
and 8-21 in 10 m. cymbelloid with dorsal margin strongly curved and
Class: Bacillariophyceae straight ventral margin. Axial area narrow, straight and
Order: Naviculales without a central area. Small central nodule. A stigmoid
1167 Journal of Research in Biology (2014) 4(1):1162-1173
A B C
G
H
D
E
F
I J
K L
Figure 5 (A-L):Gomphonema.
Presented near the dorsal central striae, striae 14.2-16 in Class: Bacillariophyceae
10 m. Order: Eunotiales
Class: Bacillariophyceae Family: Eunotiaceae
Order: Rhopalodiales Genus: Actinella Lewis, 1864
Family: Rhopalodiaceae Actinella sp. (Fig. 7 C)
Genus: Rhopalodia Otto Muller, 1895: 57 Valves 76-140 m long and 5.7-8 m broad,
Rhopalodia sp. (Fig. 7 A) arcuate, asymmetrical to both the apical and transapical
Valves 21-30 m long and 6-9 m broad, axes. External distal raphe ends extending slightly to the
isopolar and dorsiventral, lanceolate-elliptical in shape, valve face on both ends. Striae parallel, striae 13-19 in
acute apices. The dorsal margin curved and straight at 10 m.
the ventral margin. Striae composed of a single row of Class: Bacillariophyceae
puncta composes. Fibulae radiate, striae 14-20 in 10 m. Order: Achnanthales
Class: Bacillariophyceae Family: Achnanthaceae
Order: Naviculales Genus: Achnanthidium Kutzing, 1844
Family: Naviculaceae Achnanthidium sp. (Fig. 7 D and E)
Genus: Kobayasiella Lange-Bertalot, 1999 Valves 6.2-14 m long and 2-3.7 m broad,
Kobayasiella sp. (Fig. 7 B) linear-elliptic, slightly or more elongated near the end,
Valves 22-26 m long and 5-7 m broad, linear- and with bluntly rounded poles. Striae slightly radiate
lanceolate with convex sides and short, capitate apices. and often a shortened striae near the small central area,
The axial area is narrow and nearly linear. The central axial area narrow, striae 19-21 in 10 m.
area is small and elliptical and bordered by alternately Class: Bacillariophyceae
long and short striae, striae 35-40 in 10 m. Order: Bacillariales

Figure 6: (A-C) Frustulia, D Encyonema, E-Luticola, F-Encyonopsis, (M-Q) Gomphonema.
Family: Eunotiaceae and distantly placed, striae 13 in 10 m.

Genus: Eunotia Ehrenberg 1837 Class: Bacillariophyceae
Eunotia sp. (Fig.7 F-J) Order: Bacillariales
Valves 68m long, 12 m broad, slightly arched, Family: Bacillariaceae
dorsal margin convex with two wavy ridges at the Genus: Nitzschia Hassall, 1845: 435
middle, gradually narrowing towards the ends, ventral Nitzschia sp. (Fig. 8 A-Y)
margin concave; ends slightly constricted on the dorsal Valves 27-30 m long and 5.2-6.7 m broad,
side, slightly produced, rounded; raphe thin; polar linear with concave sides and wedge shaped, constricted
nodules distinct, on the ventral side near the apices; produced ends, striae very fine, almost indistinct, striae
striae 13 in 10m, coarse, lineate, parallel, somewhat 31-35 in 10 m.
radiate and closely placed near apices. Class: Bacillariophyceae
Class: Bacillariophyceae Order: Naviculales
Order: Fragilariales Family: Naviculaceae
Family: Fragilariaceae Genus: Hippodonta (Ehrenberg)
Genus: Synendra Ehrenberg 1832: 87 Hippodonta sp. (Fig. 9 A)
Synendra sp. (Fig. 7 K-M) Valves 20.2-29 m long and 5.5-8 m broad,
Valves 44 m long and 3.2- 3.8 m broad, linear elliptic-lanceolate, ends subcapitate to capitate. Raphe
with narrow and capitate ends. The central area reaches straight, filiform, central pores fairly close. Striae
the margins. Pseudo raphe linear and broad. Striae strong

Figure 7 A: Rhopalodia, B- Kobayasiella, C- Actinella, D and E- Achnanthidium,

(F-J) Eunotia, (K-M) Synendra.
noticeably broad, radiate in the middle, convergent at the Class: Bacillariophyceae

ends, striae 9-11 in 10 m. Order: Fragilariales
Class: Bacillariophyceae Family: Fragilariaceae
Order: Surirellales Genus: Tabularia (C. Agardh) D.M. Williams and
Family: Surirellaceae Round
Genus: Surirella Turpin 1828 Tabularia sp. (Fig. 9 E)
Surirella sp. (Fig. 9 B) Valves 21-400 m long and 3.1-5.3 m broad,
Valves 55-65 m long and 30-34 m broad, elliptic or elongate and variable in outline, from narrowly
heteropolar, ovate with broad rounded ends. Middle line linear to linear- lanceolate or lanceolate valves with
absent. Middle field linear-lanceolate. Striae very thick, rounded or capitate ends, striae 7.4-25 in 10 m.
widening towards the middle, set at unequal distances, Class: Bacillariophyceae
Striae 11-16 in 10 m. Order: Cymbellales
Class: Bacillariophyceae Family: Cymbellaceae
Order: Achnanthales Genus: Cymbella, C.A. Agardh 1830
Family: Achnanthaceae Cymbella sp. (Fig. 9 F-I)
Genus: Achnanthes C.A. Agardh (1824) Valves 118 m long, 24 m broad, ventricose,
Achnanthes sp. (Fig. 9 C & D) curved, asymmetric, dorsal side convex, ventral side
Valves 12.5-16 m long and 5-7 m broad, slightly concave with middle inflation; ends slightly
rectangular-elliptical to almost quadrate in the middle constricted, produced rounded; raphe thick, arcuate,
portion, constricted at the ends which are rostrate. Axial excentric with ventrally curved central nodules; axial
area narrow and central area linear reaching the margins. area not narrow; central area elliptical with 3-4 isolated

Figure 8(A-R):Nitzschia
Figure 8(S-Y):Nitzschia
stigmata at the ends of the middle ventral striae; striae central pores and curved terminal fissures. Axial area
8-10 in 10 m, punctate, radiate. moderate, linear or slightly widened between the middle
Class: Bacillariophyceae and ends: Striae radial, striae 20-22 in 10 m.
Order: Naviculales It is interesting to note that all the diatom taxa
Family: Stauroneidaceae belonged to pennate type. No centric forms of diatom
Genus: Stauroneis Ehrenberg, 1843 were found in all the four sampling sites. Majority of the
Stauroneis sp. (Fig. 9 J-M) forms were solitary and colonial forms were absent. The
Valves 62-66 m long and 15-18 m broad, dominant genera includes- Gomphonema, Nitzschia,
lanceolate with abruptly constricted, somewhat produced Stauroneis, Navicula, Frustulia, Eunotia and Pinnularia
capitate ends. Raphe thick with slightly unilaterally bent which were common in all the sampling sites in all the

Figure 9. A- Hippodonta, B- Surirella, C and D- Achnanthes, E- Tabularia, (F-I) Cymbella, (J-M) Stauroneis.
seasons throughout the year. Kobayasiella, Cymbella, ACKNOWLEDGEMENT

Synendra, Achnanthidium and Tabularia were abundant The author would like to acknowledge UGC-
only in Chapanala while Luticola, Encyonema occurred SAP (Special Assistance Programme) for providing
in Borhola. Pennate diatoms like Achnanthes, Basic Scientific Research (BSR) fellowship in carrying
Encyonopsis, Hippodonta, Actinella and Rhopalodia out the work.
were found only in Jiajuri. Only pennate diatom
Surirella was found in Thanajuri. REFERENCES
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CONCLUSION Singh Mahendra Pal Singh Publication, Dehradun, India.
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Chapanala of Nagaon district of Assam harbours rich
Beakes GW, Canter HM and Jaworski GHM. 1988.
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Zoospore ultrastructure of Zygorhizidium affluens and Z.
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investigations on the diatom flora of North- East India is
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Affordable Charges
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Original Research
Detection of biofilm formation in urinary isolates: need of the hour
Authors: ABSTRACT:
Saha R1*, Arora S1, Das S1,

Gupta C1, Maroof KA2, The purpose of the study was to estimate biofilm (BF) formation in urinary
Singh NP1 and Kaur IR1. catheterized patients, by comparing three methods i.e. Tissue culture plate method
(TCP), Congo Red Agar method (CRM) and Tube method (TM) and to study the
antimicrobial resistance pattern in BF producing and non BF producing isolates. A total
Institution:
of 130 urinary catheterized patients were taken as the study group. From one milli
1. Department of
Microbiology, University litre of urine sample isolates > 102 colony forming units per milli litre were screened
College of Medical Sciences for the detection of BF by TCP, TM and CRM. Antibiotic sensitivity test for both BF
and Guru Teg Bahadur producing and non BF producing bacterial and fungal isolates were done as per CLSI
Hospital, Dilshad Garden, guidelines. From 130 urine samples in our study group, 55 samples grew
Delhi 110095, India. microorganisms of significance, of which 11 samples were poly-microbial in nature. Of
these biofilm production was seen in 49 microorganisms (89.09%) by any of the three
2. Department of methods used. TCP method picked up 69% of biofilm producers as compared to TM
Community Medicine, and CRM which picked up only 36% and 27% biofilm producers respectively. Our study
University College of reveals TCP method as the more dependable one as compared to TM and CRA
Medical Sciences and Guru methods for the quantitative biofilm detection, so it can be recommended as a
Teg Bahadur Hospital, screening method in laboratories.
Dilshad Garden,
Delhi 110095, India.
Keywords:
Biofilm, biofilm detection, Congo Red Agar.
Corresponding author: Abbreviations

Rumpa Saha. BF - Biofilms; TCP - Tissue Culture Plate; CRM - Congo Red Method; TM - Tube
Method; CLSI - Clinical Laboratory Standard Institute; CAUTI - Catheter associated
Urinary Tract Infection; CLED - Cysteine Lactose Electrolyte Deficient; BHIB - Brain
Heart Infusion Broth; TSB - Trypticase soy broth; ELISA - Enzyme linked
immunosorbent assay; MHA - Muller Hinton Agar; MIC -Minimum Inhibitory
Concentration; ATCC - American type culture collection; GPC -Gram positive cocci;
GNB - Gram negative bacilli.
Web Address: Article Citation:

http://jresearchbiology.com/ Saha R, Arora S, Das S, Gupta C, Maroof KA, Singh NP and Kaur IR.
documents/RA0395.pdf. Detection of biofilm formation in urinary isolates: need of the hour.
Dates:
Received: 01 Dec 2013 Accepted: 08 Feb 2014 Published: 17 Feb 2014

1174-1181 | JRB | 2014 | Vol 4 | No 1

An International
Saha et al., 2014
INTRODUCTION patient to become incontinent, thus leading to failure of

Indwelling urinary catheters play an essential medical device.
part in the management of disorders of the urinary tract, There are different methods for the estimation of
especially in the elderly and disabled patients. These biofilm formation including Tissue culture plate method,
urinary catheters serve as a portal of entry for Tube method, Congo Red agar method, bioluminescent
microorganisms leading to Catheter Associated Urinary assay, light or fluorescence microscopic examination,
Tract Infections (CAUTI). Many of these microbes confocal laser scanning microscope and piezoelectric
colonize and adhere to the artificial surface of the sensor (Mathur et al., 2006).
indwelling catheters, which then forms biofilms. There is paucity of data in Indian literature
Biofilms are communities of microorganisms which are regarding biofilm formation in urinary catheterized
embedded within a matrix of extracellular polymeric patients. This study was undertaken with the aim to
material and display an altered phenotype. Based on the estimate biofilm formation in urinary catheterized
type and length of the stay of a gadget, composition of patients, to compare three methods i.e. Tissue culture
microorganism in a biofilm may vary from one to plate method (TCP), Congo Red method (CRM) and
numerous. The same is true for urinary catheter biofilms Tube method (TM) for biofilm production and to study
where number of organisms is directly proportional to antimicrobial resistance pattern in biofilm producing
length of exposure. isolates.
Microorganisms commonly isolated from
indwelling urinary catheters are Staphylococcus MATERIALS AND METHODS
epidermidis, Escherichia coli, Klebsiella pneumoniae, The study was done over a period of one year
Enterococcus faecalis, Proteus mirabilis and Candida sp from April 2008 March 2009 at department of
(Donlan, 2001). Microbiology, of our tertiary care hospital after obtaining
Biofilms carry important clinical repercussions clearance from Institutional Ethical Committee. A total
as they provide a niche for survival of microbes, by of 130 urinary catheterized patients were taken as study
conferring protection to microbes from drying, group who gave informed consent to the work. One ml of
mechanical damage and other influences from external urine samples were collected from catheter with aseptic
environment, human immune system and antimicrobial precautions and the samples were immediately sent to
agents (Costerton et al., 1995; Mah and Toole, 2001). the Microbiology laboratory. The samples were plated on
High antimicrobial concentrations are required to Cysteine Lactose Electrolyte Deficient (CLED) medium.
inactivate organisms growing in biofilms and resistance The age, sex, days of catheterization of the patients were
may often increases thousand folds. (Stewart and noted. Isolates were identified by standard
Costerton, 2001) microbiological procedures. The presence of > 102 c.f.u./
Moreover biofilms act as a persistent source of ml in aseptically collected urine was taken as significant
infection or may provide reservoir for new infections. bacteriuria (Winn et al., 2006). The cultures were
The biofilms often leads to crystalline material blocking maintained on nutrient agar slopes, Enterococci were
the catheters and induce complications like painful maintained on brain heart infusion slopes and Candida
distension of the bladder, urolithiasis, reflux of infected species were maintained on Sabourauds Dextrose Agar
urine resulting in pyelonephritis and sometimes urinary (SDA) slopes. Control strains used for biofilm
leakage around the outside of the catheter causing the production in the study were: S. epidermidis ATCC
Saha et al., 2014
35984 (strong biofilm producer), S. epidermidis ATCC of bacteria adhering to surface and forming biofilms.
35983 (moderate biofilm producer) and S. epidermidis Experiments were performed in triplicate. Interpretation
ATCC 12228 (non biofilm producer), Acinetobacter of biofilm production was done according to the criteria
baumannii ATCC 19606 and Candida albicans ATCC of Stepanovie et al., (2007). (Table 1)
90028. Tube method:
Biofilm formation was detected by the following A quantitative method was used as described by
three methods:- Christensen et al., (1982). Ten milli litre of BHI broth
Tissue culture plate method (Christensen et al., 1995): with 1% w/v glucose was taken in test tubes and was
Isolates from freshly subcultured plates were inoculated with loop full of microorganism from
inoculated in trypticase soy broth (TSB) with 1% w/v overnight culture plates and incubated at 37C for 24 hrs.
glucose and incubated for 18 hours at 37C in stationary The tubes were washed with PBS (pH 7.3) after
conditions and then diluted to 1:100 with fresh TSB. decanting the culture. The dried tubes were then stained
Individual wells of sterile polystyrene 96 well flat with crystal violet (0.1% w/v) for 30 minutes after fixing
bottom microtitre plates were filled with 200l aliquots with sodium acetate (2% w/v) for 10 minutes. Through
of diluted culture. Un-inoculated TSB served as a control washing was again done with de-ionized water to remove
to check sterility and non specific binding of media. excess stain. Tubes were then kept in inverted position
Control strains were also inoculated in triplicate. The for complete drying. Biofilm formation was detected by
microtitre plate was incubated for 24 hrs at 37C. After the presence of visible film on the wall and bottom of the
incubation contents of each well was removed by tapping tube. Ring formation at the liquid culture interface was
the plates. After washing the wells for four times with taken as negative. The amount of biofilm formation was
200l of phosphate buffer saline (PBS pH 7.2), the scored according to the results of control strains and
floating planktonic bacteria were removed. The biofilms graded as 0, 1, 2 and 3 denoting absent, weak, moderate
thus formed in plates were fixed using 2% w/v sodium and strong biofilm formation respectively. Experiments
acetate for 10 minutes and tainted with 0.1% w/v crystal were performed in triplicate.
violet for 30 minutes. After washing thoroughly with de- Congo red agar method (Freeman et al., 1989):
ionized water to remove any excess stain, the plates were Congo red media was prepared as a concentrated
dried. Micro-ELISA auto-reader at the wavelength of aqueous solution of 0.8 g/l of Congo red and autoclaved
540 nm was used to measure the Optical Density (OD) of separately from other medium constituents [brain heart
the stained adherent micro-organisms. The OD540 value infusion broth (37 g/l), sucrose (50 g/l), agar (10 g/l)];
of sterile medium, fixative and dye were averaged and then added when agar gets cooled to 55C. The required
subtracted from all test values. The mean OD540 value microbial strains were inoculated on the prepared media
from a control well was deducted from all test OD540 and incubated aerobically for 24 hrs at 37C. Growth of
values. These OD540 values were considered as an index black colonies with a dry crystalline consistency was
taken as positive biofilm production; pink colonies with
Table 1. Interpretation of biofilm production
occasional darkening at the centre of the colonies were
Average OD value Biofilm production
non biofilm producers. Black colonies without dry
OD540C/ OD540C < ~ 2x OD540C Non/weak
crystalline colonial morphology indicated indefinite
2x OD540C < ~ 4x OD540C Moderate
results. The experiment was performed in triplicate and
> 4x OD540C Strong
repeated for three times.

Saha et al., 2014
Table 2. Comparison of biofilm production by three methods TCP, TM and CRM
Isolate TCP (%) TM (%) CRM (%) No BF producer (%)

Gram positive organism n-12 11(91.66) 2(16.66) 2 (16.66) 1 (8.33)
Staphylococcus aureus n = 8 7 1 2 1
Enterococcus sp n=4 4 1 0 0
Gram negative organism n-37 24(64.86) 17(45.94) 12 (32.43) 4 (10.81)
Escherichia coli n = 20 13 11 5 3
Klebsiella sp n=7 4 3 3 1
Citobacter sp n=2 1 0 1 0
Proteus sp n=2 1 1 2 0
Acinetobacter sp n=2 2 1 0 0
Pseudomonas sp n=4 3 1 1 0
Candida sp n-6 3 (50) 1(16.66) 1 (16.66) 1 (16.66)
Candida albicans n=2 1 0 0 1
Candida tropicalis n=4 2 1 1 0
Total n = 55 38(69.09) 20(36.36) 15 (27.27) 6 (10.90)
Antimicrobial susceptibility testing was done RESULTS

on Muller-Hinton agar (MHA) for both biofilm Among 130 urine samples from our study group,
producing and non biofilm producing bacterial isolates 55 samples grew microorganisms of significance of
by Kirby Bauer disk diffusion method as per Clinical and which 11 samples were polymicrobial in nature. Of these
Laboratory Standards Institute guidelines (CLSI, 2006). biofilm production was seen in 49 microorganisms
The antibiotic panels used were 25g Cotrimoxazole, (89.09%) by any of the three methods used. All sets of
30g Cefotaxime, 30g Vancomycin, 300 units polymicrobial organisms were biofilm producers. All
Nitrofurantoin, 10g Norfloxacin, 120g High level comparisons were done keeping TCP as gold standard.
gentamicin, 30g Tetracycline, 30g Amikacin, 10g The different organism isolated and their biofilm
Gentamicin, 10g Imipenam, 100g Piperacillin; 10g producing capacity is compared in Table 2.
Tazobactam and 300 units Polymyxin B . Antibiotics TCP method picked up 69% (38) of biofilm
discs were procured from HiMedia Laboratories Pvt. Ltd, producers as compared to TM and CRM which picked up
India. only 36% (20) and 27% (15) of biofilm producers
Antifungal susceptibility profile of BF forming respectively. This difference was found to be highly
and non biofilms forming Candida isolates was done by significant (x2 = 17.55, P < 0.001). Table 3 shows
determining MIC for Amphotericin B, Itraconazole and sensitivity and specificity of TM and CRM. By TCP
Fluconazole by microdilution method as described by method, the number of strong biofilm producers were 20
CLSI guidelines (CLSI, 2008). Candida albicans ATCC
Table 3. Diagnostic parameters TM and CRM for
90028 were used as control.
Biofilm detection
Statistical Analysis:
Parameters TM CRM
Data entered in MS Excel and SSPS 17.0 were
Sensitivity 34.21% 21.05%
used for data analysis. Chi square test was used to
Specificity 58.82% 58.82%
compare proportions between various groups.
Positive Predictive Value 65.00% 53.33%
Sensitivity, Specificity and predictive values were
Negative Predictive Value 28.57% 25.00%
calculated using the standard formulae.
Saha et al., 2014
Table 4. Comparison of antimicrobial resistance pattern of BF producer with

non BFproducers
Antimicrobial agents BF producer (%) Non BF producer (%)
Staphylococcus aureus n =8 n= 7 n=1
Cotrimoxazole 6(85.71) 1 (100)
Cefotaxime 5(71.42) 1 (100)
Vancomycin 0 0
Nitrofurantoin 3(42.86) 0
Norfloxacin 6(85.71) 0
Enterococcus n-4 n=4 n=0
Vancomycin 1 (25) -
High level Gentamicin 4(100) -
Nitrofurantoin 2 (50) -
Norfloxacin 4(100) -
Tetracycline 4(100) -
Gram negative organism n=33 n= 21 n = 12
Amikacin 15 (71.43) 6 (50)
Gentamicin 15 (71.43) 6 (50)
Cotrimoxazole 18 (85.71) 6 (50)
Imipenam 7 (33.33) 1 (8.33)
Piperacillin-Tazobactam 15 (71.43) 5 (41.67)
Norfloxacin 17 (80.95) 8 (66.67)
Nitrofurantoin 13 (61.90) 4 (33.33)
Pseudomonas n = 5 n=4 n=1
Amikacin 3 (75) 1 (100)
Gentamicin 3 (75) 1 (100)
Imipenam 3 (75) 0
Piperacillin-Tazobactam 2 (50) 0
Polymyxin B 0 0
Norfloxacin 3 (75) 0
Candida spp n = 6 n=3 n=3
Fluconazole 2 (66.67) 1 (33.33)
Itraconazole 3 (100) 2 (66.67)
Amphotericin B 0 0
and the same by TM and CRM was 3 and 14 respectively however it was not significant except for Cotrimoxazole
and this difference was found to be highly significant (x2 = 4.911, P = 0.0266).
(x2 = 21.4, P < 0.001, d.f = 2). (Figure1). When degree Biofilm production has also increased
of biofilm production was compared, TM showed similar significantly with the days of catheterization (x2 = 16.88,
detection rate with TCP for moderate biofilm producers, P < 0.001) (Figure 3).
but the same is not true for strong biofilm producers.
This difference was also highly significant.(x2 = 21.06, DISCUSSION
P < 0.001, d.f = 1). Figure 2 shows colonies of biofilm More than 40% of all healthcare associated
and non biofilm producers on Congo Red medium. infections are due to CAUTI. Eradication of biofilm
The antimicrobial resistance pattern of the based catheter related infection is often challenging
biofilm producing isolates is given in Table 4. Among because they exhibit increased resistance to antimicrobial
the gram negative organism, the resistance was more for therapies by various mechanisms (Douglas, 2003).
biofilm producers as compared to non biofilm producers

Saha et al., 2014
Figure 1 Degree of biofilm formation by TCP, TM and CRM
In this study we evaluated 55 isolates by three CRM picked up greater number of biofilm producers
different screening methods for their ability to form among the Gram negative bacilli (GNB). This difference
biofilms. In our study we have found that TCP method was however not significant (x2 = 197, P = 0.1226,
detected biofilm formation in 69% of isolates. We have d.f = 2).
used 1% sucrose in BHI for growing biofilms in TM detected 36% of isolates as biofilm
microtitre plate. Addition of sugar increases the biofilm producers while 63% isolates were identified as non
production; as reported by other authors (Mathur biofilm producers. TM is only 34.21% sensitive, 58.82%
et al.,2006; Bose et al., 2009 ; Hassan et al., 2011). specific for biofilm detection. This is not consistent with
Overall TCP method detected maximum biofilm the findings of Mathur et al., 2006; Bose et al., 2009
producers. The ability to detect biofilm production of from India, who reported higher sensitivity and
Gram Positive Cocci (GPC) was less for TM and CRM specificity for Tube method. In our study, this method
method as compared to TCP method whereas TM and correlated well with TCP for identifying moderate
biofilm producers (30.90% i.e. 17 / 55), but detection
rate for high biofilm producer was very low (5.45% i.e.
3/55). This difference may be due to the inter-observer
variability in the reading of results, resulting in low
sensitivity and specificity in our study.
Only 27% isolates were identified as biofilm
producers by CRM similar to Ruzicka et al., 2004 who
detected 43.5% of biofilm producers by this method.
This was higher in comparison to the 3-6% detection rate
by other workers from India and Pakistan (Mathur et al.,
2006; Bose et al., 2009; Hassan et al., 2011). The
sensitivity and specificity, however, remained low
Figure 2. Colonies of biofilm and non biofilm (21.05% and 58.82% respectively). Surprisingly, in this
producers on Congo Red agar medium
study CRM outscores TM in the detection of high
Saha et al., 2014
CONCLUSIONS
The ability of microorganisms to form biofilms
on the medical devices is a challenge for the clinicians
because biofilm associated microorganisms are much
more resistant to antimicrobial agents, which may result
in treatment failure. Therefore effective treatment
strategies should be explored to deal such infections. Our
Days of catheterization findings indicate that TCP is a suitable and reproducible
method for the screening of biofilm producers in health
Figure 3. Relationship of Biofilm production with
duration of catheterization. care setups.
biofilm producers. CRM detected 25.45% (14/55) while REFERENCES:

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Affordable Charges
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Original Research
Foraging and pollination behavior of Apis mellifera adansonii Latreille

(Hymenoptera: Apidae) on Glycine max L. (Fabaceae) flowers at Maroua
Authors: ABSTRACT:
Fernand-Nestor To assess the impact of Apis mellifera adansonii on pod and seed yields of
Tchuenguem Fohouo1 and Glycine max, its foraging and pollinating activities were studied in Maroua, during the
Dounia1-2*. two season seasons (August-September 2010 and 2011). Observations were made on
51 to 17866 flowers per treatment. Treatment 1 represented by free flowers;
treatment 2 bagged flowers and treatment 3 flowers visited only by A. m.
adansonii. In addition, all flower visitors were recorded. The abundance of
bee, duration of visits, impact of activity of A. m. adansonii on fruiting
percentage, the influence of this bee on formation of pods, number of seeds in
Institution: each pods and average of normal seeds (well developed) were recorded.
1. Laboratory of Zoology, Individuals from 28 species of insects were recorded on the flowers of G. max, after
Faculty of Science, two years of observations. Apis mellifera adansonii with 23.18% of 954 visits was the
University of Ngaoundr, most frequent, followed by Polyrachis sp. 1 (14.77%), Macronomia vulpina (14.22%),
Ngaoundr, Cameroon. Lipotriches collaris (11.07%). This honey bee intensely and exclusively foraged for
nectar. The mean foraging speed was 12.56 5.79 flowers per minute. Flowers
2. Laboratory of Zoology,
Higher Teacher Training visited by insects had higher fruiting rate compared with the others while
College, University of those bagged had the lowest. Apis mellifera adansonii foraging resulted to a
Yaound I, Yaound, significant increment in fruiting rate by 14.14 and 11.98%, as well as the
Cameroon. number of seeds per pod by 36.95 and 35.65%, and the percentage of normal
seeds by 32.61 and 29.26% respectively in 2010 and 2011. The installation of A.
m. adansonii colonies in G. max plantations is recommended to improve pod and
seeds production of this species.

Dounia. Apis mellifera adansonii, Glycine max, flower, visit, nectar, pollination.

Fernand-Nestor Tchuenguem Fohouo and Dounia.
Foraging and pollination behavior of Apis mellifera adansonii Latreille (Hymenoptera:
Apidae) on Glycine max L. (Fabaceae) flowers at Maroua.
Web Address: Dates:

http://jresearchbiology.com/ Received: 15 Jan 2014 Accepted: 04 Feb 2014 Published: 11 April 2014
documents/RA0415.pdf.
1209-1219 | JRB | 2014 | Vol 4 | No 1

An International
Fohouo and Dounia, 2014
INTRODUCTION MATERIALS AND METHODS

Glycine max is an annual plant originated from Study site, experimental plot and biological material
Northern and Central regions of China (Hymowitz, 1970). The experimental is carried, from June to October,
The plant is an annual, herbaceous, erect, and can reach a in 2010 and 2011 at Ma ye l - I bb (Latitude 10 62' N,
height of 1.5m; there are cultivars of soybean Longitude 1433' E and altitude 400 m), Maroua, Far
indeterminate, determinate and semi-determinate growth North Region of Cameroon. This Region belongs to the
(Gallais and Bannerot, 1992). The first leaves are Savanna zone, with unimodal rainfall (Letouzey, 1985).
simple, opposite and swallowed, while the following are It has a Sahel-Sudanian climate type, characterized by
trifoliate and alternate; the pod is straight or slightly two seasons: a more extended dry season (November to
curved, with a length of two to seven cm; the seed is May) and a brief rainy season (June to October) (Kuete
generally oval, but may vary depending on the cultivar, et al., 1993). The maximum rainfall and
almost spherical, elongated and flattened (Hymowitz and temperature are 1100mm and 38C respectively
Harlan, 1983). Flowers are grouped by two to eight on a (Kuete et al., 1993). The experimental plot was 28m x
short racemes inserted on the stem axile sheets and are 5m. The biological material was represented by Apis
purple or white (Boyeldieu, 1991). Each flower has a mellifera adansonii Latreille (Hymenoptera: Apidae),
tubular calyx of five sepals, a corolla of five petals, a and others insects present in the environment. Seed of
single carpel and ten stamens, nine of which being G. max was provided by the Institute of Agricultural
welded and the tenth is free (Hymowitz and Harlan, Research for Development (IARD).
1983). Each flowers Produce nectar and pollen which Sowing and weeding
attract insects (Milfont et al., 2013). The reproduction On t h e June 12, 2010 and June 15, 2011, the
system is autogam/allogam (Ibarra-Perez et al., 1999). experimental plot was cleaned and divided into 24
Soybean is grown primarily for its seeds, which have subplots, each measuring 1m 1m. Sowing and weeding
many uses in the food and industrial sectors (USDA, was done as described by Douka and Tchuenguem
2002). It is a major edible oil and vegetable sources of (2013).
protein (38-40%) for the feed of men and other animals Determination of the reproduction system of Glycine
(Boyeldieu, 1991; Tien et al., 2002; USDA, 2002). max
Currently the production of G. max in Cameroon is low On July 22, 2010, eight subplots carrying 106
whereas the demand for seeds is high (MINADER, plants with 34395 flowers at the bud stage were labeled.
2010). Therefore, it is important to investigate on the Four subplots carrying 80 plants with 17187 flowers
possibilities of increasing the production of this plant were left to be open pollinated (treatment 1) (figure 1)
in the country. This can be done if flowering insects of and four subplots carrying 17208 flowers were
G. max in each region are well known and exploited protected with gauze mesh prevent to insect or other
(Milfont et al., 2013). Unfortunately no research has pollinating animals vi s i t s (treatment 2) (figure 2).
been reported on the relationships between G. max and On July 28, 2011, the experiment was repeated, for
its anthophilous insects in Cameroon. In Maroua A. m. treatment 1 four subplots carrying 80 plants with 17866
adansonii visit flowers of G. Max (unpublished data), and flowers and four treatment 2 four subplots carrying 80
this study is carried out to assess the effects of foraging plants with 15875 flowers.
activities of A. m. adansonii on yields of G. max. Twenty days after shading of the last flower,
the number of pods was assessed in each treatment.
Figure 1. Glycine max plot showing unprotected Figure 2. Glycine max plot showing isolated
plants in bloom. plants in bloom.
The podding index (Pi) was then calculated as described (Tchuenguem, 2005).
by Tchuenguem et al., (2004): Pi = F2/F1, where F2 is During the same time that A. m. adansonii
the number of pods formed and F1 the number of encountered on flowers were registered, the types of
viable flowers initially set. floral products collected by this bee were noted. This
The allogamy rate (Alr) from which derives the parameter was measured to determine if A. m. adansonii
autogamy rate (Atr) was expressed as the difference in is strictly a pollenivore, nectarivore or pollenivore and
podding indexes between treatment 1 (unprotected nectarivore. This could give an idea on its implication as
flowers) and treatment 2 ( bagged flowers) as follows a cross pollinator of G. max.
(Demarly, 1977): In the morning of each day, the number of
Alr = [(Pi1 - Pi2) / Pi1] 100, Where Pi1 and Pi2 opened flowers was counted. The determination of
are respectively the podding average indexes of frequency of visits, the duration of A. m. adansonii on
treatments I and II. Atr = 100 Alr. the flower of G. max was recorded according to
Study of the foraging activity of Apis mellifera Tchuenguem (2005). The number of pollinated visits,
adansonii on Glycine max flowers the abundance of foragers, the number of flowers
The frequency of A. m. adansonii in the flowers visited by A. m. adansonii per minute was recording
of G. max was determined based observations on every day of observation. The method of observation was
flowers of treatments 1 in 2010 and 2011. Experience followed as given by Tchuenguem et al., (2004).
were made on 17187 individual opened pollinated The foraging speed was calculated according to
flowers (treatment 1) each day, from July 26 to August Jacob Renacle (1989) by this formula: Vb = (Fi/di) x
20, 2010 and from August 2, to August 2 4 , 2011 at 60 where di is the time (s) given by a stopwatch and Fi is
7 8 h, 9 10 h, 11 12 h, 13 14 h, 15 16 h and 17 the number of flowers visited during di. The
18 h. Capture and determination of insects that visited int era cti on bet ween A. m. adansonii and the
G. max flowers was realize as described by Borror and competitors and the attractiveness exerted by the flower
White (1991). of other plant species around the experimental plot on
The determination of the relative frequency of all A. m. adansonii were recorded (Tchuenguem et al.,
insects visit the G. max flowers was calculated 2004). The climatic factor (temperature and humidity)

was registered as described by Douka and Tchuenguem seeds (well developed) was then calculated for each
(2013). treatment 3.
Evaluation of the impact of Apis mellifera adansonii Data analysis
and other insects on Glycine max yields Data were analyzed using descriptive statistics,
This evaluation was based on the impact of students t-test for the comparison of means of the two
visiting flowers on pollination, the impact of pollination samples, correlation coefficient (r) for the study of the
on fructification of G. max, and the comparison of association between two variables, chi-square (2) for
yields [fruiting rate, mean number of seeds per pod and the comparison of two percentages using SPSS statistical
percentage of normal (well developed) seeds] of software and Microsoft Excel.
treatments 1 and 2. The fruiting rate due to the activity
of insects (Fri ) was calculated as follows by Tchuenguem RESULTS
et al., (2004): Fri = {[(Fr1 Fr2) / Fr1] 100} Reproduction system of Glycine max
Where Fr1 and Fr2 are the fruiting rate in treatments According to table 2 : the allogamy rate was
1 and 2. 6.59% and 5.38% respectively in 2010 and 2011 and
The fruiting rate (Fr) is: Fr = [(F2/F1) 100] autogamy rate was 93.41% and 94.62% respectively in
Where F2 is the number of pods formed and F1 the 2010 and 2011. Glycine max (used in our experiments)
number of flowers initially set. has a mixed reproduction system autogamous -
At maturity, pods were harvested from each allogamous, with the predominance of autogamy.
treatment. The mean number of seeds per pod and the Frequency of A. m. adansonii in the floral entomofauna
percentage of normal seeds were then calculated for of Glycine max
each treatment. Among the 532 and 422 visits of 24 and 24
Evaluation of the pollination efficiency of insect species counted on G. max flower in 2010 and
Apis mellifera adansonii on Glycine max 2011, respectively, A. m. adansonii was the most
In 2010, along with the development of r e p r e s e n t e d insect with 132 visits (24.81 %) and 91
treatment 1 and 2, 11 plants belonging to four subplots visits (21.56 %), in 2010 and 2011, respectively. The
and carrying 47 flowers were protected using gauze mesh difference between these two percentages is not
(treatment 3). In 2011 the same experience was repeated significant (2 = 1.39 df = 1 p > 0.05) (Table 1). In
but on 16 plants carrying 51 flowers. Between 7 and 2010, the highest mean number of A. m. adansonii
9am, of each observation date, the evaluation or the simultaneously in activity was one per flower (n = 50; s
efficiency pollination of A. m. adansonii on G. max = 0) and 2.88 per 1000 flowers (n = 60; s = 3.53; maxi
was realized as according of Douka and Tchuenguem = 19). In 2011, the corresponding figures were one per
(2013). The impact (Frx) of A. m. adansonii to fruiting flower (n = 50; s = 0) and 1.97 p er 10 0 0 fl ow er s (n
rate was calculated as follows by Tchuenguem et al., = 60; s = 2.59; maxi = 12). The difference between the
(2004) the formula: mean number of foragers per 1000 flowers in 2010 and
Frx = {[(Fr3 Fr2) / Fr3] x 100} 2011 was highly significant (t = 9.19; df = 118, p <
Where Fr3 and Fr2 are the fruiting rates in treatment 0.001).
3 (protected flowers visited exclusively by Activity of Apis mellifera adansonii on Glycine max
A. m. adansonii) and treatment 2 (protected flowers). Floral reward harvested
The number of seeds per pod, the percentage of normal During each of the two flowering periods, A. m.
respectively. The difference between the duration of the

visit in 2010 and 2011 is higher significant (t = 22.25;
df = 221, p < 0.001). For the two cumulated years the
mean duration of a flower visit were 2.55 sec.
Foraging speed of Apis mellifera adansonii on Glycine
max flowers
On the pot of G. max, A. m. adansonii visited
between 4 and 24 flowers/min in 2010 and between five
and 25 flowers/min in 2011. The mean foraging speed
was 11.65 flowers/min (n = 50; s = 5.77) in 2010 and
13.48 flowers/min (n = 50; s = 5.82) in 2011. The
Figure 3. Apis mellifera adansonii collecting nectar in difference between these means is highly significant (t =
a flower of Glycine max
- 7.95; df = 98, p < 0.001). For the two cumulated years
adansonii was found to collect nectar intensively and the mean foraging speed was 12.56 flowers /min.
exclusively (Figure 3). Effect of climate on foraging activity of Apis mellifera
Relationship between visits and flowering stages adansonii on Glycine max flowers
Visits were most numerous when the number of Climatic condition seem not to influence the
open flowers was highest (Figure 4) Furthermore a activity of A. m. adansonii. T he correlation was
positive and significant correlation was found between negative and not significant (r2010 = - 0.34; df = 11; p
the number of G. max opened flowers and the number > 0.05 and r2011 = 0.28; df = 11; p > 0.05) between the
of A. m. adansonii visits in 2010, as well as 2011 number of A. m. adansonii visits on G. max flowers
(r2010 = 0.90; df = 8; p < 0.05; r2011 = 0.85; df = 8; p < and the temperature. It was positive and not significant
0.05). Apis mellifera adansonii foraged on G. max (r 2010 = 0.48; df = 11; p > 0.05 and r2011 = 0.07; df = 11;
flowers throughout the blooming period, with a peak of p > 0.05) between the number of A. m. adansonii visits
activity situated between 10 and 11am (Figure 5). and relative humidity (Figure 6).
Duration of visits per flower Impact of anthophilous insects on pod formation and
In 2010 and 2011, the mean duration of A. m. seed yields of Glycine max
adansonii visit is 2.50 sec (n = 132; s = 1.34; maxi = 6 During nectar harvest on G. max, foraging
sec) and 2.61 sec (n = 91; s = 1.40; maxi = 6 sec) insects always shook flowers and are regularly in contact
Figure 4: Variation of number of flowers and Figure 5. Variation of number of flowers and visits of
number of visits of Apis mellifera adansonii on the Apis mellifera adansonii on the flowers of Glycine
flowers of Glycine max in 2010 and 2011. max according to daily time in 2010, 2011.

with the anthers and stigma (Figure 3), increasing cross normal seeds in opened flowers was higher than that of
pollination possibility of G. max fruiting rate, number of protected flowers in 2010 and 2011. The percentage of
seeds per pod and percentage of normal seeds in different the normal seeds due to the action of insects was 24.81%
treatments (Table 2). in 2010 and 20.90% in 2011. For all the flowers studied,
a - The difference observed was highly the percentage of the normal seeds due to flowering
significant between fruiting rate of free opened flowers insects was 22.85%.
(treatment 1) and that of bagged flowers (treatment 2), Pollination efficiency of Apis mellifera adansonii on
the first year (2 = 248.73, df = 1, p < 0.001) and the Glycine max
second year (2 = 299.84, df = 1, p < 0.001). T he Apis mellifera adansonii foragers were always
fruiting rate of t r e a t m e n t 1 ( unprotected flowers) in contact with the stigma and the anthers of G. max
was higher than treatment 2 (protected flowers) in 2010 (contacts with anthers and stigma was 100% for all
and in 2011. The fruiting rate due to the action of insects visits). C on sequ en t l y t his bee increased
was 5.92 and 5. 81% in 2010 and 2011 respectively. possibilities of the pollination of G. max flowers.
For the two cumulated years, the fructification rate due to a - the difference observed between the fruiting
the influence of insects was 5.86%. rate of treatments 2 and that of treatment 3 was highly
b - For the mean number of seeds per pod, significant in 2010 (2 = 7.73; df = 1; p < 0.001) as
there was a highly significant difference between well as 2011 (2 = 6.93; df = 1; p < 0.001). T he
treatments 1 and 2 (t2010 = 4315.78; df = 30462; p < fruiting rate of flowers exclusively visited by A. m.
0.001; t2011 = 5958.33; df = 30670; p < 0.001). adansonii (treatment 3) was higher than those of bagged
Consequently, a high mean number of seeds per pod in flowers (treatment 2). The fruiting rate due to A. m.
treatment 1 (opened flowers) were noticed compared to adansonii activity was 14.14% and 11.98% respectively
treatments 2 (bagged flowers). The number of seeds per in 2010 and 2011. The percentage of the fruiting rate
pod attributed to the activity of insects was 26.11% in due to A. m. adansonii activity was 13.06 %
2010 and 36.47% in 2011, giving an overall mean of b - There was a highly significant difference
31.29%. between treatments 2 and 3 (t = 64.76; df = 14821; p <
c - There was a highly significant difference 0.001) the first year and the second year (t = 49.28; df =
between the percentage of normal seed of treatment 1 14023; p < 0.001). High mean number of seeds per pod
and that of treatment 2 in the first year (2 = 4329.98; df of flowers of treatment 3 was noticed compared to
= 1; p < 0.0001) as well as the second year (2 = flowers of treatment 2. The augmentation of the number
6094.38; df = 1; p <0.0001). Thus, the percentage of of seeds per pod due to A. m. adansonii was 36.95% and
Figure 6. Daily distribution of A. m. adansonii visits on 17187 and 17866 G. max flowers over 10 days in 2010
(A) and 10 days in 2011 (B) respectively, mean temperature and mean humidity of the study site.

Table 1. Diversity of floral insects on Glycine max in 2010 and 2011, number and
percentage of visits of different insects
Insects 2010 2011
Order Family Genus, species, sub-species n1 p1% n2 p2%

n
Hymenoptera Apidae Apis mellifera adansonii 132 24.81 91 21.56
Amegilla sp. 1 n 4 0.75 0 0
Xylocopa sp. 1 n 3 0.56 1 0.24
Halictidae Macronomia vulpina n 87 16.35 51 12.09
Lipotriches collaris n 56 10.53 49 11.61
Megachilidae Chalicodoma sp.1 n 13 2.44 2 0.47
Megachile sp. 1 n 3 0.56 1 0.24
Megachile sp. 2 n 0 0 4 0.95
Formicidae Polyrachis sp. 1 sh 79 14.85 62 14,69
Vespidae Synagris cornuta n 11 2.07 4 0.95
(1 sp.) n 1 0.19 0 0
Sphecidae Philanthus triangulum pr 6 1.13 2 0.47
(1 sp.) pr 1 0.19 0 0
Lepidoptera Pieridae Catopsilia florella n 28 5.26 29 6.87
(sp. 1) n 17 3.20 8 1.90
(sp. 2) n 12 2.26 3 0.71
Nymphalidae (1 sp.) n 19 3.57 23 5.45
Acraeidae Acraea acerata n 13 2.44 17 4.03
Diptera Muscidae Musca domestica n 26 4.89 49 11.61
Drosophilidae Drosophila sp. 1 n 12 2.26 8 1.90
Syrphidae (1 sp.) n 2 0.38 3 0.71
Calliphoridae (1.sp.) n 3 0.56 0 0
Hemiptera Coreidae Anoplocnemis curvipes n 1 0.19 1 0.24
Pyrrhocoridae Dysdercus voelkeri n 1 0.19 2 0.47
Orthroptera (sp.1) lv 0 0 5 1.18
(sp.2) lv 0 0 2 0.47
Nevroptera (sp.1) pr 2 0.38 1 0.24
(sp.2) pr 0 0 4 0.95
Total 28 species 532 100 422 100
Comparison of percentages of Apis mellifera adansonii visits for two years: 2 = 1.39 ([df = 1; P > 0.05]).
n1: number of visits on 17187 flowers in 10 days.
n2: number of visits on 17866 flowers in 10 days.
p1 and p2: percentages of visits.
p1 = (n1 / 532) x 100.
p2= (n2 / 422) x 100.
n: Visitor collected nectar.
lv: Visitor eating leaves.
sh: visitor shelter
pr: Predation.
sp.: Undetermined species.

35.65% respectively in 2010 in 2011. The percentage of attributed to the variation of the number of colonies of
the mean number of seeds per pod attributed to the this honey bee around the experimental site. The peak of
activity of A. m. adansonii was 36.30%. activity of A. m. adansonii on G. max flowers was at
c - There was highly significant difference between 10 and 11am, which correlated to the period
between the percentage of normal seed of treatment 3 of highest availability of nectar on G. max flowers. The
and that of treatment 2 in first year (2 = 67.76; df = 1; positive and highly significant correlation between the
p < 0.001) as well as the second year (2 = 58.58; df number of G. max flowers and the number of A. m.
= 1; p < 0.001). The percentage of normal seeds in adansonii visits indicates the attractiveness of G. max
treatment 3 was higher than in treatment 2. The nectar with respect to this bee. The significant
percentage of the normal seeds due to A. m. adansonii difference observed between the duration of visits in
was 32.61% in 2010 and 29.26% in 2011. T he 2010 and 2011 could be attributed to the availability of
percentage of the number of seeds per pod attributed to nectar, the floral morphology of this crop or the variation
the activity of A. m. adansonii was 30.93%. in the diversity of flowering insects from one year to
another. At Maroua in 2010 and 2011 (in the rainy
DISCUSSION season), A. m. adansonii intensely and regularly
Honey bee was the main floral visitor of harvested nectar on the flowers of G. max during
G. max during the observation period. This bee has flowering periods. This could be attributed to the needs
been reported as the main floral visitor of this Fabaceae of colonies during the flowering period. During our
in USA (Rortais et al., 2005) and Brazil (Milfont et al., investigations, the interruption of visits by other insects
2013). Apis mellifera adansonii was also shown to be the or the same honey bee reduced the duration of A. m.
most abundant floral visitors of other Fabaceae members adansonii visits. Similar results were found in
such as Phaseolus coccineus in Yaound, Cameroon Cameroun by Tchuenguem et al., (2009b) and Douka
(Pando et al., 2011a), and Phaseolus vulgaris in and Tchuenguem (2013) on flowers of Vigna
Ngaoundr, Cameroon (Kingha et al., 2012) and in unguiculata (L.) (Fabaceae) and Phaseolus vulgaris
Maroua by Douka and Tchuenguem (2013). The (Fabaceae) respectively. It indicates that
significant difference between the percentages of A. m. A. m. adansonii can increased the possibility of
adansonii visits for the two studied years could be pollination of G. max flowers. During the collection of
Table 2. Glycine max yields under pollination treatments.
Seeds / Pod Total Normal % normal

Treatment Year Flowers Pods Fruiting rate
seeds seed seed
Mean sd
Unlimited visits 2010 17187 15688 91.28% 3.14 1.42 48853 42609 87.22
Protected plot 2010 17208 14776 85.87% 2.32 1.01 34162 22415 65.61
Protected plot 2011 17866 16697 93.46% 3.92 2.06 66020 57137 86.54
Bagged flowers 2011 15875 13974 88.03% 2.49 1.52 63176 43250 68.46
A. m. adansonii 2010 47 47 100.00% 3.68 1.84 152 148 97.37
A. m. adansonii 2011 51 51 100.00% 3.87 1.88 217 201 92.63

nectar, A. m. adansonii foragers regularly come into differ between plant varieties and /or region.
contact with the stigma and carry the pollen to the anthers
for stigma. The weight of A. m. adansonii sh oot t h e CONCLUSION
fl ower s of G. max dur in g nectar collection and this This study reveals that t h e v a r i e t y o f G. max
movement played a positive role in liberation of pollen st udi ed is a nectariferous bee plant that obtained
by anthers for the optimal occupation of the stigma. benefits from the pollination by insects among which A.
This phenomenon was also reported by Ahrent and m. adansonii is the must important. The comparison of
Caviness (1994) and Rortais et al., (2005) on G. max. pods and seeds set of unprotected flowers with that of
Thus in addition to their direct pollination role, flowers visited exclusively by A. m. adansonii
A. m. adansonii foragers also indirectly effected self- underscores the value of this bee in increasing pods and
pollination and cross-pollination of G. max flowers. The seed yields as well as seed quality. The installation of
positive and significant contribution of A. m. adansonii A. m. adansonii c o l o n i e s to G. max fi el d s should
in pods, seed yields and percentage of normal seeds of be recommended for the increase of pod and seeds
G. max is justified by the action of this bee on yields of this valuable crop.
pollination. The similar have been obtain in Britain
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Original Research
Determining the Natural Gypsophila L. (Coven) Taxa Growing

in Tunceli (Turkey)
Authors: ABSTRACT:
Mustafa Korkmaz1* and 56 species belonging to 60 taxa (out of 126 species in the World) of
Hasan Ozelik2. Caryophyllaceae family grows naturally in Turkey with Gypsophila sps L. as the third
largest genus. The endemism ratio of the genus is 60% in Turkey. Because Turkey is
the gene center of Gypsophila and economically very valuable; determining the
geographic distribution and biological characteristics of the taxa is very necessary.
They have well-developed roots, that prevent soil erosion. Because of containing
saponin (10-25 %) in their root, its extract is used as fire extinguisher, gold polisher,
Institution: cleaner and softener of delicate fabrics and crispness giving substance for halva. It is
1. Erzincan niversity, also used for making liqueur, herbal cheese, ice cream and some other foods. Some
Science and Arts Faculty, taxa are boron hyper acumulators and vegetative mining can be conducted by hyper
Department of Biology, accumulation. They are also thought to be the cleaning tools for toxid areas by
Erzincan-Turkey. fitoremediation.
In this study, 12 records from eight Gypsophila taxa were collected around
2. Sleyman Demirel
Tunceli. These are G. aucheri Boiss. (1), G. elegans Bieb. (1), G. pallida Stapf. (2),
niversity, Science and Arts
Faculty, Department of G. perfoliata L. var. perfoliata (1), G. ruscifolia Boiss. (3), G. sphaerocephala Fenzl ex
Biology, Isparta-Turkey. Tchihat var. cappadocica Boiss. (1), G. venusta Fenzl (1) and G. viscosa Murray (2).
With addition of G. briquetiana Schischk. and G. hispida Boiss. the total number is
reaching to 10 and it shows that the city is an important diversity center of the genus.
G. aucheri, G. briquetiana and G. sphaerocephala var. cappadocica are endemic to
Turkey and G. pallida, G. perfoliata L. var. perfoliata, G. venusta and G. viscosa are
determined to be new records for Tunceli.
Keywords:
Corresponding author:
Coven, Gypsophila, Habitat, Biodiversity, Tunceli, Turkey.
Mustafa Korkmaz.

Mustafa Korkmaz and Hasan Ozelik.
Determining the Natural Gypsophila L. (Coven) Taxa Growing in Tunceli (Turkey).
Web Address: Dates:
http://jresearchbiology.com/ Received: 04 Feb 2014 Accepted: 05 Mar 2014 Published: 16 April 2014
1220-1227 | JRB | 2014 | Vol 4 No 1

An International
Korkmaz and Ozelik, 2014
INTRODUCTION 2008; Korkmaz et al., 2010; Korkmaz and zelik,

Caryophyllaceae family distributes mostly in 2011a).
Mediterranean region of southern hemisphere. It has a Turkish Covens are commonly obtained from
large diversity with over 2000 species. Gypsophila L. Gypsophila graminifolia Bark. G. arrostii Guss.var.
genus, which has 126 species on the World, has natural nebulosa (Boiss. and Heldr.) Bark., G. eriocalyx Boiss.,
distribution in the Irano-Turanian and Mediterranean G. bicolor (Freyn&Sint.) Grossh., G. perfoliata L.,
phytogeographic regions (Williams, 1989; Sumaira et al., G. venusta Fenzl subsp. venusta and Ankyropetalum
2008). There are about 500 species of Caryophyllaceae gypsophiloides Fenzl. (nan, 2006; Kl, et al., 2008).
family in Turkey. More than half of totally 126 G. ruscifolia Boiss. and G. bitlisensis Bark. are the least
Gypsophila species in the world are found in Caucasian, preferred species. The most preferred species are
the North Iraq and the North Iran regions. There are G. bicolor, G. arrostii and A. Gypsophiloides (Baytop,
about 56 Gypsophila species found in Turkey. Many of 1984; zelik, and, zgke, 1999; Korkmaz and
them are known from the type collection. G. heteropoda zelik, 2011a).
Freyn & Sint. subsp. minutiflora Bark. is a rare endemic Saponin chemical was first produced from the
taxon peculiar to Cappadocica sub region in Inner roots of Saponaria officinalis (Baytop, 1984). The
Anatolia of Turkey and an endangered taxa on global amount of saponin in the roots of Gypsophila taxa differs
scale (Ekim et al., 2000; Ozhatay et al., 2005). from 4 % to 25 % (Sezik, 1982). Gypsophila bicolor
Gypsophila L. is the third biggest genus of (Van veni), G. arrostii var. Nebulosa (Beyehir,
Caryophyllaceae family after Silene L. and Dianthus L. Isparta veni), G. perfoliata (Nide veni),
(Davis, 1967; Davis et al., 1988; Gner et al., 2000; G. venusta subsp. Venusta and G. eriocalyx
elik et al., 2008; Korkmaz and zelik, 2011b).The (orum-Yozgat veni) are most preferred taxa for
most important factor for the distribution of this genus is obtaining coven extract in Turkey (Heroldand Henry,
the soil structure which contains gypsum, lime and 2001; Battal, 2002).
calcium; these are important for these plants to grow. Soap root extract is composed of sugar, resin and
There are gypsum habitats around Sivas, ankr, saponin. It protects the plant from germ and fungal
orum, Ankara, Eskiehir, Nide and Erzincan. Because infection, increases the nutritive value and facilitates the
of that, Gypsophila taxa are rich in these areas. digestion. The production phases of the extract starts
Soap root has been exported from Anatolia for a with cutting the roots in the form of chips and continuous
long time. The collection of coven from natural habitats with boiling them for two times. After second boiling
and extraction have been increasing rapidly especially in stage the extract can be obtained. (Korkmaz et al., 2010;
the Eastern and South-east Anatolia for nearly 40 years Korkmaz and zelik, 2011a).
(Kl, et al., 2008). In Turkey Gypsophila taxa are The main areas of the use of them are in the food
generally known by the name ven Otu and they are industry, the chemistry, in hygiene industry, in
mostly used by the public for different purposes. The horticulture, in mining, in whitening gold and in fire
word Soaproot or Soapworth terms are used for extinguishers. They have antimicrobial effect and used in
Gypsophila species; in Europe the members of the genus medicines. Every year the average export of soap root
are widely known as Babys Breath. In Turkey the from Turkey is about 90 tones by gaining approximately
plants are also called Dii ven, Tarla veni, Helva 66 000 US Dollars (Baytop, 1984; Korkmaz and zelik,
veni, ark veni by the local people (Kl, et al., 2011a; zelik and zgke, 1996).
This study was aimed to determine the Gypsophila taxa herbarium specimen. Economic importance of the taxa is
naturally distribute in the province of Tunceli city of given according to our early papers (zelik and
Turkey. zgke, 1999; Korkmaz et al., 2010; Korkmaz and
zelik, 2011a,b).
MATERIALS AND METHODS As it is given in the Table-2, endemic taxa and
Material of this study contains Gypsophila taxa the risk categories, phytogeographic regions, altitudes,
growing around Tunceli. With regard to this aim we have life forms and new records have been determined.
collected eight taxa of the genus from 13 different Turkish names of Gypsophila taxa grows around Tunceli
localities in the area. Collection date, record number, have been determined from Trkiye Bitkileri Listesi
habitat types and some other properties of the identified (Gner et al., 2012) as they were given in Table 2.
taxa were determined (and given in Table 1). For the Endemic taxa of the genus and their threat categories
identification of taxa Flora of Turkey and the East have been determined from Ekim et al. (2000) and given
Aegean Islands (Davis, 1967) has been used extensively. in the same table.
Identifications were done with the help of stereo-zoom
microscope. Identified samples were converted to
Table 1. Locality and habitat informationof Gypsophila taxa collected around Tunceli
Record
No Taxon Date Locality Habitat
number
1 G. aucheri Boiss. K: 1769 03.07.2009 Tunceli: Tunceli-Pertek, 10 km Rocky places
to Pertek
2 G. elegans Bieb. K: 1741 02.07.2009 Tunceli: Erzincan- Plmr, Rocky places
near to Plmr
K: 1740 02.07.2009 Tunceli: Erzincan- Plmr, Rocky places
near to Plmr
3 G. pallida Stapf.
K: 1748 02.07.2009 Tunceli: Tunceli- Ovack, 40 Inclined slopes
km to Ovack
4 G. perfoliata L. var. K: 1745 02.07.2009 Tunceli: Plmr-Tunceli, near Rocky slopes
perfoliata to Plmr
K: 1746 02.07.2009 Tunceli: Plmr-Tunceli, 30 Rocky slopes
km to Tunceli
K: 1760 02.07.2009 Tunceli: Tunceli-Ovack, 10 km Flowing slopes
5 G. ruscifolia Boiss. to Ovack
K: 1761 02.07.2009 Tunceli: Ovack, Munzur Rocky places
ay Gzeleri
6 G. sphaerocephala K: 2588 12.06.2011 Tunceli-Erzincan, Munzur Rocky slopes
Fenzl ex Tchihat var. Mountain
cappadocica Boiss. K: 2638 11.07.2011 Tunceli-Erzincan Munzur Slopes
Mountain
7 G. venusta Fenzl K: 1749 02.07.2009 Tunceli: Tunceli- Ovack, 25 Rocky slopes
km to Ovack
8 G. viscosa Murray K: 1750 02.07.2009 Tunceli: Tunceli Ovackaras, Rocky slopes
25 km to Ovack
K: 1752 02.07.2009 Tunceli: Tunceli-Ovack, 10 km Rocky places
to Ovack
K: Korkmaz

Table 2. Taxonomic information of Gypsophila taxa growing around Tunceli
Taxon name P.G. Altitude Life New record or

No Endemic Fl.
(Turkish name) region (m) form recorded before
1 G. aucheri Boiss. Endemic 6-7 Ir.-Tur. 1200-1600 P Tunceli, Pertek
(Ta veni) (VU)
2 *G. briquetiana Schischk. Endemic 7-8 Ir.-Tur. 1700-2500 P Tunceli, Ovack,

(Gl evgeni) (LR) Munzur Mountain
3 G. elegans Bieb. - 6-7 Ir.-Tur. 650-2600 A New record to

(Ho ven) Tunceli
4 *G. hispida Boiss. - 6-7 Ir.-Tur. 1100-2150 P Tunceli, between

(Kll ven) Tunceli and Ovack
5 G. pallida Stapf. - 6-8 Ir.-Tur. 850-2000 P New record to

(ark veni) Tunceli
6 G. perfoliata L. var. - 6-8 - 1000-1500 P New record to

Perfoliata (Helvac veni) Tunceli
7 G. ruscifolia Boiss. - 6-7 Ir.-Tur. 300-1800 P Tunceli, Ovack

(Acem veni)
8 G. sphaerocephala Fenzl ex Endemic 7-8 Ir.-Tur. 800-1900 P Tunceli, Munzur

Tchihat var. cappadocica Boiss. (LR) Mountain
9 G. venusta Fenzl - 5-7 Ir.-Tur. 300-1600 P New record to

(Kara ven) Tunceli
10 G. viscosa Murray - 4-6 Ir.-Tur. 350-1400 A New record to

(Sadrl ven) Tunceli
* :Gypsophila taxa not available in the area, P: Perennial, A: Annual, P.G.: Phyto-geographic, Fl.: Flowering period
RESULTS AND DISCUSSION G. aucheri, G. briquetianaand G. sphaerocephala var.

The results of the study are summarized in Table cappadocica are endemic taxa available in the vicinity.
-1 and Table-2. As seen in Table-1, 8 Gypsophila taxa Threat (risk) category of G. aucheri is Vulnerable (VU)
were collected from the area in 2009 and 2011. All of the and the other two taxa is at the category of Low Risk
plant samples were collected from Plmr, Tunceli, (LR). Flowering periods of the taxa changes from April
Ovack and Munzur Mountains. Generally, the collected to August. All of the determined taxa are Irano-Turanian
plants are naturally grown in rocky and slopy places. phytogeographic region elements and distributes from
Photograph of all collections were taken during the field 800 to 2500 m altitudes in the area. G. elegans
work. Totally 8 Gypsophila taxa were collected from 13 and G. viscose are annual life forms and the others are
different localities. As seen in Table-2 there are 10 perennial life forms. G. aucheri, G. briquetiana,
Gypsophila taxa determined in the flora of Tunceli. G. elegans, G. hispida, G. ruscifolia and
G. sphaerocephala var. cappadocica are early recorded as forming a natural border between Erzincan and
in Tunceli but, G. pallida, G. perfoliata var. perfoliata, Tunceli. The width of the mountain is 25-30 km and the
G. venusta and G. viscose (4 taxa) are new records. length of it is 100-130 km. Altitude of the area changes
Habitat types of Gypsophila taxa growing naturally in from nearly 850 to 3462 m. The climate of the area is hot
the province are rocky places, in clined or flowing slopes and dry summers and long and snowy winters.
and slopes of mountains. Their flowering period starts in According to the study there are 1407 vascular plant
July. The general vegetation type of the plants are arid or species. The number of endemic species is 275 and some
semiarid steppes. of them were described as new to science. In this study
Soap roots have economic value in medicine, G. briquetiana Schischk., G. sphaerocephala,
food, decoration and cleaning and chemistry to produce G. ruscifolia, G. elegans Bieb, G. bitlisensis Bark. and
saponin. It is used as fire extinguisher, gold polisher, G. hispida Boiss. are given in the list of the plants.
fabric, cleaner and for purification of contaminated soil Munzur Dalar is one of the most important BA
such as by removing the boron. In addition, it is possible (nemli Bitki Alan) of Turkey with its very rich floristic
to perform vegetative mining by boron diversity. Munzur Valley is also an important national
hyper-acumulation from soil to the upper parts of the park of the country. There are 43 plant species peculiarto
plant (Babaolu et al., 2004; Korkmaz and zelik, Munzur Dalar. In addition to the study of Yldrml
2011a). Turkish soaproot is mostly obtained from (1995) zhatay et al. (2005), this is another important
G. graminifolia, G. bicolor, G. arrostii var. nebulosa, study on biological diversity of the mountains.
G. eriocalyx, G. perfoliata var. anatolica, G. venusta and Gypsophila briquetiana Schischk., Gypsophila elegans
Ankyropetalum gypsophiloides species and the gene Bieb. and Gypsophila ruscifolia Boiss. are three species
center of both of the species is Turkey (Korkmaz and of the genus growing in the area of Munzur mountains
zelik, 2011a,b). The harvest time of these plants is (Koyuncu and Arslan, 2009). Polat et al. (2012)
from March to June. Because the roots of these plants are evaluated ethno botanical studies performed in the
generally used, the plants dont produce seeds for the Eastern Anatolian region including Tunceli. According
next years. So, the plants are increasingly disappearing to this study there are only five ethnobotanical study
from the nature and under the threat of extinction. This (Tuzlac ve Doan, 2010; Yldrml, 1985; 1991; 1994
problem becomes more important when the plants are a;b) conducted in Tunceli. Also in another study
rare or endemic. Because of unemployment soap roots performed by Karlda in (2009) related with both of
have been collected for a long time in the rural parts of Elaz and Tunceli, they determined local names and
the country. For preservation of Gypsophila species they medicinal uses of 53 plants.The least studied cities in
should not only be collected from nature but also its East Anatolian region are Ar, Ardahan, Bingl, Bitlis,
cultivation should be planned and other soap root Erzincan, Kars, Mu, Hakkari and Tunceli. So, it is
yielding plant species should be identified. necessary to record and prevent ethnobotanical culture in
The most important floristic study related with these cities by conducting news tudies (Polat et al.,
Tunceli in the area is Flora of Munzur Dalar 2012).
(Yldrml, 1995). The mountains are situated between
Erzincan and Tunceli in B7 grid square and in CONCLUSION:
Irano-Turanian phytogeographic region. It starts from There are 60 naturally growing Gypsophila taxa
Kemaliye and reach to Plmr at the west-east direction in the Turkey. Many species of the genus are highly

potential to be used in economy. G. sphaerocephala and the support provided by the institution
G. perfoliata are known as boron hyper accumulators
and they are very important for boron mining. Because REFERENCES
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Original Research
Distribution pattern of birds in Banni Grassland of

Kachchh district, Gujarat, India
Authors: ABSTRACT:
Mukesh H. Koladiya1,
ArunKumar Roy Mahato2, Birds are interesting group of animals which are distributed in all major types
Nikunj B. Gajera3 and habitat. Banni is one of the large grassland of India invaded by Prosopis juliflora, an
Yatin S. Patel4. alien plant species. Invasion of this species and some other natural and anthropogenic
factor leads the grassland converted into a mixture of heterogeneous habitats. A study
was attempted to understand the distribution of birds in this heterogeneous
grassland. The habitats were identified based on dominant species of plants. The
population estimates of birds were surveyed using line transects method and point
Institution: count census method.
1,2,3. Gujarat Institute of A total of 91 species were recorded during the survey in the various habitats
Desert Ecology, Bhuj, of this grassland. Among the seven habitats, sparse Prosopis was the most diverse
Kachchh-370001, Gujarat. habitat for bird species whereas Prosopis-Capparis was the least diverse habitat for
bird species. The highest mean population density of birds were recorded in Prosopis-
4. Samarth Organization
Capparis (15.9 individuals/km2), while lowest recorded in sparse Prosopis habitat (9
Trust, Vijapur, Mehsana-
individuals/km2). It was found that, Prosopis-Salvadora (23.109.47) was the most
382870, Gujarat.
dense and Prosopis-Capperis (8.845.26) was the least dense habitat for common
birds of Banni grassland. In conclusion, bird species diversity and their population
density estimates were varied among the various heterogeneous habitats of Banni
grassland both in time and space gradients.
Mukesh H. Koladiya. Bird, distribution, density, habitat, Banni grassland, Kachchh

Mukesh H. Koladiya, ArunKumar Roy Mahato, Nikunj B. Gajera and Yatin S. Patel.
Distribution pattern of birds in Banni Grassland of Kachchh district, Gujarat, India.
Journal of Research in Biology (2014) 4(1):1228-1239
Web Address: Dates:

http://jresearchbiology.com/ Received: 10 Feb 2014 Accepted: 24 Feb 2014 Published: 16 April 2014
1228-1239 | JRB | 2014 | Vol 4 | No 1

An International
Koladiya et al., 2014
INTRODUCTION: understanding on the distribution pattern and habitat

Various group of animals varied from survival preference of bird communities over heterogeneous
strategies in a landscape which are evolved in long environment is very much essential for conservation and
course of evolution. The distribution patterns of animals management of birds in regional as well as in local
in various habitats are preferred in response to various environment (Kattan and Franco, 2004).
uses and selective processes (Clark and Shutler, 1999). Banni grassland is one of the rich areas of birds
The distributions of life forms are not typically random due to its varied micro-habitat and act as a seasonal
in the habitat and it is generally assumed that non- wetland. The distribution pattern of birds across the
random distribution of life forms is due to natural grassland is very less understood due to the lack of study
selection (Southwood, 1977). The distribution range in the area. Therefore, the present study was conducted
across a heterogeneous landscape may depend on the to understand the pattern of distribution of birds in time
habitat selected by the species, and animal which favors and space gradient in the grassland for their conservation
their distribution in a greater proportion of the habitat and management.
(Veech et al., 2011).
Banni grassland is one of the largest remnant MATERIALS AND METHODS:
grassland of India. The landscape of this grassland is flat Study Area:
and most part of it is filled with water during monsoon Banni, the largest remnant grassland in India,
which makes the grassland as a wetland. The soil situated on the south-west portion of the Kachchh
salinity is normally high in most of the part due to its Biosphere Reserve (KBR) and attached to the fringes of
connection with Great Rann of Kachchh (GRK), a salt greater Runn of Kachchh (2319' to 2352' N latitude and
inflated marshy land. To protect the grassland from salt 6856' to 7032' E longitude), encompassing an area of
intrusion from GRK, Prosopis juliflora was introduced over 2,600 km2 is taken into consideration for our study
in fringe areas of GRK to check desertification in Banni (Fig-1). A large tract of the southern part of Banni
grasslands. In present, P. juliflora is proved to be an grassland is marshy land and salty waste remains a
invasive species for the grassland and now major part of wetland in the monsoon season, known as Little Rann of
the grassland is invaded by the species. Banni, which separates the Banni grassland from the
Birds are very important animal for this mainland of Kachchh district (Shah and Somusundaram,
ecosystem as they are good indicators of biodiversity. 2010). The climate of the Banni is arid and semi-arid
Birds are one of the typical groups of animal distributed type therefore, the temperature is high during most of the
in large landscape and even some species prefer to live in time and it reaches a maximum of 48-49C during May-
heterogeneous environment distributed over continents. June and low during winter season (8-10C) in the
To understand the processes of habitat selection and month of January and February. The average yearly
preference by birds is dependent on an accurate rainfall of this grassland is 317 mm with scanty rainfall
representation of the patterns of habitat occupancy and droughts are the common phenomenon of this area.
(Wiens et al., 1987). Birds generally colonize in an area The grassland is situated in the semi-arid bio-
having presence of suitable habitat for their survival climatic zone of India. The major part of grassland is
needs (Veech et al., 2011). The distribution pattern of now invaded by Prosopis juliflora, an invasive alien
birds might also influence by distribution patterns of bird species. The grassland has varied types of habitat patches
species richness (Shiu and Lee, 2003). The above that attract large number of birds. Further, the seasonal
Figure 1. A map of Banni grassland, and its location in the Kachchh district of Gujarat.
water bodies (locally known as Dhandh) inside the Banni Habitat classification:
region serve as the wintering ground for many migratory Banni was earlier divided by 10 habitat types by
species of birds. Koladiya et al. (2012). In the present study, the Banni
grassland was divided into 7 major habitat types based
METHODOLOGY: on the dominant plant species. It includes; Dense
A preliminary survey was made to whole of the Prosopis, Moderate Prosopis (medium Prosopis
Banni grassland for identifying transect location and density), Sparse Prosopis, Prosopis-Capparis Mixed,
number of transect location required for the survey. Prosopis-Suaeda-Calotropis Mixed, Prosopis-Salvadora
Based on this survey various micro-habitats were Mixed and Suaeda Dominant. The vegetation of the
identified. A total of 60 km distance was covered by study area was also recorded by making quadrate on the
walking through various transects. The field data were line transect and calculated the density of vegetation by
collected by two observers during the whole study period using Misra (1968).
between the months of June 2009 to May 2011. The Avi-faunal Survey:
birds were identified using the field guide produced by The population and distribution of birds were
Ali (1996) and survey was conducted by using standard recorded using line transect method and point count
data sheet, GPS-Garmin, binocular (8X40) and camera. census method (Bibby et al., 1992; Bhupathy, 1991). A
total of 51 transects were laid down in the whole

Table 1. Major plant species density and birds population density in various
micro-habitats of Banni grassland
Vegetation Mean individuals of bird/Km2

Habitat class
Dominant species Density/ Ha Winter Summer Monsoon
Dense Prosopis (DP) Prosopis juliflora 1200.00 12.4 4.50 20.5
Moderate Prosopis (MP) Prosopis juliflora 833.33 12.3 4.30 17.4
Sparse Prosopis (SP) Prosopis juliflora 483.33 8.9 2.80 15.3
Prosopis juliflora 733.33

Prosopis-Capparis mixed (PC) 15.5 3.00 29.1
Capparis decidua 1400.00
Prosopis juliflora. 1050.00

Prosopis-Suaeda-Calotropis
Suaeda sps. 2133.30 7.8 4.40 16.6
mixed (PSC)
Calotropis sps. 8933.30
Prosopis juliflora 433.33

Prosopis-Salvadora mixed (PS) 21.4 5.70 17.2
Salvadora sps. 366.67
Suaeda dominant (SD) Suaeda sps. 10000.00 13.0 4.20 20.4
MeanSD - - 13.14.50 4.120.98 19.54.64
surveyed area. The presence of individual and group of and found in all habitats except Suaeda dominant habitat.
birds within 25 m radius of circular plot was made in The flag ship and dominant species of plants in the seven
every 200 m distance along the line transect. The species identified habitat were Prosopis juliflora, Capparis
of bird was identified using binoculars and with the help decidua, Suaeda spp., Calotropis spp. and Salvadora
of Ali and Ripley (1983) and Grimmett et al.(2006). spp. The density of major plant species calculated in
Generally, the surveys were made during the morning each habitat type is given in table-1.
(7.30 am to 11.30 am) and afternoon (4.00 pm to 6.30 Species Richness and diversity:
pm) hours of each season during 2009 and 2011. A total of 91 Species of avi-fauna belonging to
The data recorded during the study was used to 62 genera under 35 families and 11 orders were observed
calculate vegetation density, birds population density during the whole study period (given in Annexure-I).
(Gaston, 1973; Burnham et al., 1980) and tested by Among the total observed bird species, 59 were resident
ANOVA between micro-habitat using Microsoft Excel and 32 were migratory in nature. The number of bird
2007. species recorded in Banni grassland based on their
feeding guilds included; granivorous (32 species),
RESULTS AND DISCUSSION: insectivorous (30 species), frutivorous (12 species),
Habitat category & Vegetation density: piscivorous (10 species) and others (7 species). Based
Among the seven identified habitats of Banni on the transect survey in various seasons, the maximum
grassland Prosopis juliflora is the most dominant species bird species recorded during monsoon (83 species), next
Figure 2. Seasonal Avian species richness in various habitat of Banni grassland
to that in winter (67 species) and minimum during lowest in Prosopis-Capparis mixed (H= 0.91) habitat
summer (32 species). (fig-2). The above result highlighted that avian species
The total number of avian species was recorded diversity was also lower in comparison to the species
lower than number of species (163) recorded by Gajera diversity recorded by Gajera et al. (2012, 2013a, 2013b)
et al. (2012, 2013a, 2013b) in wetland, arid grasslands in wetland, grassland and mining areas distributed in
and mining areas respectively distributed in western part western parts of Kachchh district.
of Kachchh district. It is also noted that 56 species of Distribution of birds in various micro-habitat:
birds recorded alone from the Pena thattah, a seasonal Out of the total species recorded during the
wetland located in the western part of Banni grassland by whole study period, the number of bird species recorded
Koladiya et al. (2013). in 7 identified habitats were as follows; dense Prosopis
The species diversity (Shannon_H) was recorded (45 species), moderate Prosopis means Prosopis density
to found highest in Sparse Prosopis (H=2.20) habitat and between more than 500 and less than 1000 individuals/
Figure. 3. Bird species diversity in various habitats of Banni grassland, Kachchh

Figure 4. Seasonal abundance (%) of birds in Banni grassland of Kachchh, Gujarat

ha. (56 species), sparse Prosopis (60 species), Prosopis- season; moderate Prosopis and Prosopis-Suaeda-
Capparis mixed (28 species), Prosopis-Suaeda- Calotropis are the most preferred habitat during the
Calotropis mixed (50 species), Prosopis-Salvadora month of summer (Fig-3). The percent of species
mixed (30 species) and Suaeda dominant (40 species) recorded in each type of habitat in seasonal basis is
respectively. The above result highlighted that sparse shown in Figure-4.
Prosopis was the rich habitat for bird species diversity We found that the mean population density
and Prosopis-Capparis mixed was the least supportive (Mean SD) of birds was highest during monsoon
habitat for bird species diversity in Banni grassland. The season (19.54.64) and least density during summer
number of species diversity between three season season (4.120.98). The seasonal population density of
(summer, monsoon and winter) was significantly varied birds in various habitats of Banni grassland is given in
(F=14.40, df=2, p<0.001) while species diversity table-1. It was found that the highest population density
between various habitat were significantly not varied. of birds was found in Prosopis-Capparis mixed habitat
On analysis of seasonal distribution of bird (29.1 individuals/km2) during monsoon and least density
species in 7 identified habitats of Banni grassland, it was was recorded in sparse Prosopis habitat (2.8 individuals/
found that sparse Prosopis, Prosopis-Suaeda-Calotropis km2) during summer season. The mean population
and dense Prosopis were the preferred habitat during density of birds recorded in various habitats of Banni
monsoon season; moderate Prosopis, dense Prosopis and grassland is shown in fig-5. Among the various habitat,
Suaeda dominant are the preferred habitat during winter the highest mean population density of birds were
Figure 5. Population density of birds in various habitats of Banni grassland, Kachchh
recorded in Prosopis-Capparis (15.9 individuals/km2) the most dense habitat and Prosopis-Capparis
2
and Prosopis-Salvadora habitats (14.8 individuals/km ) (8.845.26) was the least dense habitat for the common
while lowest mean population density was recorded in birds of Banni grassland.
2
sparse Prosopis habitat (9 individuals/km ). The result
revealed that the density of birds in Banni grassland was CONCLUSION:
higher in relation to the density of birds recorded by In conclusion, the diversity of birds in banni
Gajera et. al (2013b) in western part of Kachchh. grassland is rich with sparse Prosopis is the richest
Distribution pattern of common birds: habitat compare to other habitat in relation to species
We analyse the population density estimates of diversity. Prosopis juliflora, an invasive alien species of
commonnly occuring 10 species of birds in identified plant in the grassland is playing major role in the
seven habitat types of Banni grassland (Table-1). It was distribution of avi-fauna in this region. Prosopis juliflora
found that, Prosopis-Salvadora was the most dense is the dominant species of plant of this grassland which
habitat of six common species of birds viz. house crow, provide habitat for nesting of birds and greater visibility
lark, babblar, dove, bee eater and bul bul; sparse of birds for preying. Based on the results of the study, it
Prosopis was the most dense habitat of pegion and was found that monsoon season attracts more number of
drongo; dense Prosopis for sand groose and Prosopis- species of birds in the grassland because large portion of
Suaeda-Capparis was the most dense habitat for the grassland is converted into seasonal wetland during
francolin. Similarly, Suaeda dominent was the least the season. However, habitats with dominance of mixed
dense habitat of four species viz. babblar, dove, bee eater vegetation are the dense in habitat for birds compared to
and bul bul; Prosopis-Capparis and Prosopis-Suaeda- other habitats of the grassland.
Capparis were the least dense habitat for three species of
common birds viz. house crow, francolin, dansgroose ACKNOWLEDGEMENTS:
and lark, pigeon, drongo respectively. On estimating the We would like to thank Dr. R. V. Asari, Director,
overall mean density (MeanSD) of common birds, it Gujarat Institute of Desert Ecology (GUIDE) for
was found that, Prosopis-Salvadora (23.109.47) was providing logistic supports and his encouragement. We

are thankful to Mr. Yatin Patel for his help in Plant data (3):166-170.
analysis for the manuscript. We are also thankful to all Gaston AJ. 1973. Methods for estimating bird
scientist and scholars of Terrestrial Ecology Division of populations. J Bomb Nat Hist Soc., 72(2): 271283.
GUIDE for their help and valuable comments. We are
Grimmett R, Inskipp C and Inskipp T. 2006. Pocket
grateful to State Forest Department, Gujarat for
Guide to the birds of the Indian sub-continent. Oxford
providing funds for conducting this study.
University Press, New Delhi. 384 p.
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Ali S and Ripley SD. 1983. A Pictorial Guide to the elevational gradients in the Andes of Colombia: area and
birds of the Indian subcontinent. Bombay Natural mass effects. Global Ecol Biog. 13(5): 451-458.
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Koladiya MH, Gajera NB and Vijay Kumar V. 2013.
Ali S. 1996. The book of Indian birds, Bombay Natural Status, diversity and distribution of avifauna in Banni
History Society. 345 p. Grassland of Kachchh district. 3(1): 43-47.
Bhupathy S. 1991. Population and resource utilization Koladiya MH, Mahato AK Roy, Shah JP and
of waterfowl in Keoladeo National Park, Bharatpur. Vijaykumar V. 2013. Avifauna of Pena Thathh: A
Ph.D. Thesis. Rajasthan University, Jaipur. Lesser known Wetland in Banni Grassland of Kachchh
district, Gujarat, India. Intern J Res BioSci. 2(1): 59-65.
Bibby CJ and Burgess ND. Hill DA. 1992. Bird census
Techniques. Academic press, U.K. London. Misra R. 1968. Ecology Work Book, Oxford and IBH
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Burnham KP, Anderson DR and Laake JL. 1980.
Estimation of Density from Line Transect: Sampling of Shah J and Somusundaram S. 2010. Preliminary GIS
Biological populations. Wildlife Monograph. 72: 202 p. and Remote sensing analysis on Banni grasslands,
Kachchh. PRAJ J. Pure App. Sci. 18: 15 - 17.
Clark RG, Shutler D. 1999. Avian habitat selection:
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(1): 272-287. Species Richness along Elevational Gradients in Taiwan.
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Gajera NB, Mahato AK Roy and Vijay Kumar V.
2012. Wetland birds of arid region-a study on their Southwood TRE. 2011. Habitat, the Templet for
diversity and distribution pattern in Kachchh. Columban Ecological Strategies? J Ani Ecol., 46(2):337-365.
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Gajera NB, Mahato AK Roy and Vijay Kumar V. of habitat on the range expansion of a native and an
2013a. Status, Distribution and Diversity of Birds in introduced bird species. J Biog. 38(1): 6977.
mining environment of Kachchh, Gujarat. Intern J
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2013b. Status, diversity and conservation of grassland 147.
birds in arid region of Kachchh. Intern J Rec Sci Res., 4

Annexure I
List of bird species recorded in various habitat of Banni grassland
S. No Family Scientific Name Common Name MS Habitat

1 Phasianidae Francolinus pondicerianus Grey Francolin R DP, MP, SP, PSC,SD
2 Upupidae Upupa epops Common Hoopoe R MP, SP, SD
3 Coraciidae Coracias garrulus European Roller RM MP, SP, SD
4 Coracias benghalensis Indian Roller R SP, PC
5 Meropidae Merops orientalis Green Bee-eater R DP, MP, SP, PC, PS
6 Merops leschenaulti Chestnut-Headed R DP, MP, SP, PC, PS

Bee-Eater
7 Cuculidae Eudynamys scolopacea Asian Koel R SP, PC, PS
8 Centropodidae Centropus sinensis Greater Coucal R MP, SP, SD
9 Psittacidae Psittacula krameri Rose-Ringed Parakeet R PC, PS, SD

10 Apodidae Apus affinis House Swift R MP, SP, SD
11 Strigidae Bubo bubo Eurasian Eagle-Owl R DP, MP, SP
12 Columbidae Columba livia Blue Rock Pigeon R DP, MP, SP, PC

13 Streptopelia decaocto Eurasian Collared Dove R DP, MP, SP, PC
14 Streptopelia tranquebarica Red Collared Dove R DP, MP, SP, PC
15 Streptopelia chinensis Spotted Dove R DP, MP, SP, PC
16 Streptopelia senegalensis Little Brown Dove R DP, MP, SP
17 Pteroclididae Pterocles exustus Chestnut-bellied R DP, MP, SP, PC

Sandgrouse
18 Pterocles indicus Painted Sandgrouse R DP, MP, SP, PC

19 Accipitridae Circus pygargus Montagu's Harrier RM MP, PSC
20 Circus aeruginosus Eurasian Marsh Harrier WV DP, MP, PSC
21 Circus cyaneus Hen Harrier WV DP, MP, PSC
22 Circus macrourus Pallid Harrier R DP, MP, PSC
23 Accipiter badius Shikra R MP, SP, PSC
24 Elanus caerulus Black-Shouldered Kite R MP, SP, PSC
25 Milvus migrans Black Kite R MP, SP

26 Pandion haliaetus Osprey RM SP, SD
27 Aquila pomarina Lesser Spotted Eagle R DP, MP, PSC
28 Aquila nipalensis Steppe Eagle WV DP, MP, PSC
29 Falconidae Falco tinnunculus Common Kestrel WV DP, MP, PSC
30 Lanidae Lanius excubitor Grey Shrike RM DP, MP, PSC, PS

31 Lanius cristatus Brown Shrike M DP, MP, PS, SD

32 Lanius vittatus Bay-backed Shrike R DP, MP, PS, SD
33 Lanius schach Rufous-tailed Shrike R DP, MP, PS, SD
34 Lanius meridionalis Southern Grey Shrike RM DP, MP, PS, SD

35 Corvidae Corvus splendens House Crow R DP, MP, SP, SD
36 Corvus macrorhynchos Jungle Crow R DP, MP, SP, SD
37 Dicrurus macrocerus Black Drongo R DP, MP, PS, SD

38 Muscicapidae Saxicola jerdoni Jerdon's Bushchat R MP, SP, PS, SD
39 Saxicola caprata Pied Bush Chat R MP, SP, PS, SD
40 Oenanthe deserti Desert Wheatear RM MP, SP, PSC, SD
41 Oenanthe picata Variable Wheatear M SP, PSC, SD
42 Oenanthe isabellina Isabelline Wheatear M SP, PSC, SD
43 Copsychus saularis Oriental Magpie Robin R DP, MP, SP, PC, SD
44 Saxicoloides fulicata Indian Robin R DP, MP, SP, PC, SD
45 Sturnidae Sternus roseus Rosy Starling WV DP, MP, PS
46 Acridotheres tristis Common Myna R DP, MP, PS
47 Acridotheres ginginias Bank Myna R DP, MP, PS
48 Paridae Parus nuchalis Pied Tit R MP, SP, SD

49 Hirundinidae Hirundo rustica Barn Swallow WV SP, SD
50 Hirundo smithii Wire-tailed Swallow R SP, SD
51 Hirundo daurica Red-Rumped Swallow R SP, SD

52 Delichon urbica Northern House-Martin RM SP, SD
53 Pycnonotidae Pycnonotus cafer Red-Vented Bulbul R DP, MP, PC, PSC,PS,SD
54 Pycnonotus leucotis White-eared Bulbul R DP, MP, PC, PSC,PS,SD
55 Cisticolidae Prinia buchanani Rufous-fronted Prinia R DP, SP, PSC, PS
56 Prinia inornata Plain Prinia R DP, SP, PSC, PS
57 Prinia sylvatica Jungle Prinia R DP, SP, PSC, PS
58 Prinia socialis Ashy Prinia R DP, SP, PSC, PS
59 Sylvidae Orthotomus sutorius Common Tailorbird R DP, MP, PC, PSC, PS
60 Hippolais caligata Booted Warbler R DP, SP, PC, PSC, PS
61 Turdoides caudatus Common Babbler R DP, MP, PC, PSC, PS
62 Turdoides malcolmi Large Grey Babbler R DP, MP, PC, PSC, PS
63 Turdoides striatus Jungle Babbler R DP, MP, PC, PSC, PS
64 Alaudidae Galerida cristata Crested Lark R SP, PC, PSC
65 Eremopterix grisea Ashy-crowned, Sparrow-Lark R SP, PC, PSC

66 Mirafra erythroptera Indian Bushlark R DP, MP, PC, PSC
67 Mirafra cantillans Singing Bushlark R MP, SP, PC, PSC
68 Calandrella raytal Short-toed lark M MP, SP
69 Galerida deva Sykes's Crested Lark R MP, SP, PSC
70 Nectarinidae Nectarinia asiatica Purple Sunbird R DP, SP, PC, PSC, PS
71 Passeridae Passer domesticus House Sparrow R SP, PSC, PS
72 Anthus rufulus Paddyfield Pipit RM DP, PSC, PS
73 Lonchura malabarica Indian Silverbill R DP, PC, PSC, PS
74 Motacilla alba White Wagtail WV SP, PSC
75 Motacilla flava Yellow Wagtail WV SP, PSC
76 Motacilla cinerea Grey Wagtail WV SP, PSC
77 Ploceus philippinus Baya Weaver R SP, PC
78 Alcedinidae Alcedo atthis Common Kingfisher R MP, PSC
79 Dacelonidae Halcyon smyrnensis White-breasted Kingfisher R SP, PSC
80 Cerylidae Ceryle rudis Pied Kingfisher R SP,PC, SD
81 Gruidae Grus grus Common Crane WV SP, PSC, SD
82 Grus virgo Demoiselle Crane WV SP, PSC, SD
83 Charadridae Vanellus indicus Red-Wattled Lapwing R MP, PSC, SD
84 Anhingidae Anhinga melanogaster Darter R PSC, SD

85 Ardeidae Bubulcus ibis Cattle Egret R MP, PSC, SD
86 Casmerodius albus Great Egret R SP, PSC, SD
87 Egretta garzetta Little Egret R SP, PSC, SD
88 Mesophoyx intermedia Intermediate Egret R SP, PSC, SD
89 Threskiornithidae Pseudibis papillosa Black Ibis R MP, PC, SD
90 Platalea leucorodia Eurasian Spoonbill R SP, PC, SD
91 Ciconidae Mycteria leucocephala Painted Stork R SP, PC, SD
MS: Migratory Status, R: Resident, RM: Resident Migratory, WV: Winter visitor, DP: Dense Prosopis, MP:
Moderate Prosopis, SP: Sparse Prosopis, PC: Prosopis-Capparis mixed PSC: Prosopis-Suaeda-Calotropis mixed,
PS: Prosopis-Salvadora mixed, SD: Suaeda dominant

Annexure II. Photographs showing Banni grassland and a few birds sited
Banni grassland Galerida deva
Accipiter badius Grus grus
Aquila nipalensis Upupa epops

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Original Research
Determination of age and growth by scale of a population of common trout

(Salmo trutta macrostigma, Dumeril, 1858) at the level of Sidi Rachid River
(Ifrane. Morocco)
Authors: ABSTRACT:
Abba H 1, Belghity D1,

Benabid M2 and Chillasse L3.
Institution:
1. Biology and Health The determination of age and growth from the scales of trout river (Salmo
Laboratory. Environmental trutta macrostigma, Dumeril, 1858) at Sidi Rachid River; was employed out of 438
and Parasitology Team / specimens used the size varies between 6.3 cm and 37.5 cm, the relation linking the
UFR Doctoral Parasitology growth in length of the fish and the growth of the scale. Varied according to the
compared: Medical and equation Log Lt = 0.8674 Log Rt + 0.5349, with a coefficient of correlation( r) =
Veterinary Applications." 0.86592138. The period of the end of growth to this population of trout is between
Sciences Faculty. Ibn Tofail December and January, this period is characterized in the middle of the atlas by
University. Knitra B.P. important reductions in temperature on one hand, the decrease of the network
133, 14000. Morocco.
trophique on the other hand which gets coincided with the period of reproduction of
2. National Center of the trout. The resumption of the growth is made in a important way from March. The
Hydrobiology and age of the trout's determined by scales varies between 0 + to 5 +. The retro measures
Pisciculture (NCHP) Azrou are lower than those observed and the equation of theoretical growth of Van
Morocco. Bertalanffy is of the following type: Lt = 34, 96 (1-exp-0,309 (t-0, 27)).
3. Laboratory of biodiversity
and wet zones .Uni My
Ismail. Faculty of Science.
Meknes.
Abba H River trout, age, growth, scales, Sidi Rachid River. Morocco
Article Citation:
Email Id: Abba H, Belghity D, Benabid M and Chillasse L.
Determination of age and growth by scale of a population of common trout
(Salmo trutta macrostigma, Dumeril, 1858) at the level of Sidi Rachid River (Ifrane. Morocco)
Dates:
Web Address: Received: 19 Dec 2013 Accepted: 15 Jan 2014 Published: 16 April 2014
http://jresearchbiology.com/
1240-1246 | JRB | 2014 | Vol 4 | No 1

An International
Abba et al., 2014
INTRODUCTION seasonal rhythm with fast growth at the spring and

The fishing of salmonids constitutes one of the summer and a stops its growth during winter period. This
main concerns of the members of fishing associations in annual growth rate is marked on the various osseous
the nation. Both the common trout (Salmo trutta structures among which scales are present. The study of
macrostigma, Dumeril, 1858) and the rainbow trout these osseous structures will allow determining the
(Oncorhynchus mykiss) are appreciated in the fishing period of the stop of growth and consequently the age
sport. This activity plays an important role in the and its relation with the size of the specimens of the
socioeconomic development of the region. To alleviate population of trout in the Sidi Rachid River.
the disappearance of the endemic common trout, the
administrators in Morocco resort to the repopulation of MATERIALS AND METHODS
rivers with vesicle alevins stemming from artificial Presentation of the environment of study
reproduction which is carried out at the salmon farming The environment of study (Figure-1) is Sidi
station of Ras El Ma. Rachid River, present in the geographical coordinates of
For a long time, numerous studies were 59'N N and 3328'W W. It is at a height of 1620m and
conducted in the determination and knowledge of the belongs to the rural district of Ait Ali Ouikoub (province
lines of fish the populations of in various aquatic circles. of Ifrane). The brook is fed by the sources of Sidi Rachid
Besides the parameters size and mass, we also quote the of which it takes its name with a maximum debit of 172
age of the fish. These various biological lines once L/S (Abba, 2011) for a main source as well as the waters
determined, can be exploited in the perspectives of from the station of salmon farming of Ras El Ma (Abba
management of the various types of peaches et al., 2011). From the morphomtric point view, the
professionally. The estimation of the age of a fish is of a River presents a low width which can vary from 2m to 6
big importance to understand the dynamics of a m, and a depth which does not exceed 1m generally.
population. This determination of the age can be made Biological material
either in a direct way, or in an indirect way. In this study, Sampling of fishes
we limited ourselves to one of the direct methods by The method used in our case is the electric
means of the osseous structures (Spillmann, 1961; fishing realized by technicians' team of the National
Bagliniere et Maisse, 1990). Although the use of scales Center of Hydrobiology and Fish farming of Azrou. The
for certain species are questioned (Pikitch et Demory, number of fish every month varies between 30 and 50
1988), the scales are used for a majority of families with specimens. For every sinned fish, we have proceed to the
species dulaquicoles and amphihalines temperate or measure of its total length (Lt (cm)) with an ichtyometer,
cold regions to be known, almonds, cyprinids and and before putting it back in the housing environment,
precedes (Bagliniere et Lelouarn, 1987; Meunier, 1987; scales in number from 6 to 20 were removed in the zone
Bouhbouh, 2002). During this study, method used for the recommended for salmonids according to Ombredane
determination of age and growth of brown trout (Salmo and Richard(1990). Scales are then tidied up in
trutta macrostigma, Duerile 1858) is by the number, size envelopes and numbered for further study in the
and pattern of scales. Indeed, the growth of the laboratory with a microfiche (40).
structures mineralized as the scales is proportional to the Determination of the structure of the population
length of the fish (Lea, 1910; Hattour et al., 2005). In The determination of the number of classes of
temperate zones, the growth of the fish presents a size of the population of trout at the level of the Sidi
Abba et al., 2014
Figure 1: Situation of area of study (Extracted from the map of Azrou. E: 1/50. OOO;
division of the map, 1974)
Rachid River during the period of study was made by extension (AM). The latter is used to determining of the
applying the ruler of Sturge. Number of class = 1 + (3, 3 period of stop of growth. The front of the scale generally
log N), were N is the sample Size. held to salmonids (Bagliniere et al., 1991) is used for the
Preparation and reading of scales determination of the total shelf R and other shelves r
The preserved scales dried on the referenced corresponding to the various annuli, r1, r2, r3 to rn. The
envelopes were taken and rubbed between fingers and measure was made by means of a graduated ruler on a
cleaned by the water to eliminate any sorts of residues device microfiche for the same swelling (42). To work
(Jearld, 1983). The examination of scales can be made by always in the same condition, the measure of the beam
several ways. The reading chosen in this work as the was made on the main line, which corresponds to the
determination of the age of the fish was made by a reader previous field of the scale. The Extension Margin (EM)
of microfiche. The criteria used for the determination of was calculated according to, Benabid (1990).
rings for the stop of growth vary according to the Determination of the retro calculation on growth
species. For the salmon kind, the criteria are generally as The relation binding the size of the fish and the
follows: shelf of its scale is linear and is determined by the
Contraction of several circuli in the form of a following formula (Bryuzgin, 1970): L = b Ra (or Log L
concentric band making the tour (ballot) of the scale = a Log R + Log b), with, , L:: length of the fish (cm)
(Bagliniere and Lelouarn , 1987); in the capture, R: the previous shelf of the scale of the
Discontinuity of circuli or absence of discontinuity fish (cm) ie., distance between the center of the scale and
of the circuli in which the thickness decreases; its outside edge according to a direction strictly constant,
Stepping of the circuli of the annulus on those a: and , b: are constants.
previously trained in the side fields either The formula of Le Cren (1947) and Philippart,
Measures made on scales (1975) allows then the retro calculation of the size of the
The rings of ruling of growth allow making fish every age. Log Ln = Log L + a (Log Rn - Log R).
measurements on the scale to calculate the marginal With, Ln length calculated at the time of the training of
the nme ring of the stop of growth in mm; L: length

Abba et al., 2014
observed by some fish in mm; , R:: length observed by 44 % (Ombredane and Richard, 1990).
the previous beam of the scale in mm; , Rn: length of The determination of the period of appearance of
the previous beam of the scale up to the nme ring in the rings to the stop of growth was made by monthly
mm; and , a: constant. The theoretical model of growth analysis of the variations of average Marginal Extension
used is the one of Von Bertalanffy (1938): (Lt = L [1- (AM) on 387 trout's which presents normal scales.
exp (-K (t-t0))]). (Benabid, 1990; Bouhbouh, 2002). During this study, some scales do not present rings on
With K (years- 1): growth rate; L (cm): cut that the fish the stop of growth; it is about scales of truitelles
in time infinite should have; t0 (years): the age in the stemming from on-the-spot cross-posted or born alevins
worthless length. from March, 2007. The (figure-3) shows the results
obtained for all the scales of fishes representing stops of
RESULTS AND DISCUSSION growths.
The histogram of the structure of population of The analysis of variations of the results showed
the trout (Figure-2) shows a good representation of the that Marginal Extension presents the minimum only one
individuals and the size of which is between 14 (the marked well for December and January. This minimum
Middle = 13.8) and 17 cm (the Middle of 16.8).This type translates not only shows the ring of wintry stop of
of structure is a characteristic of young populations. This growth but also it corresponds to the period of
structure is explained by the fact that the adults are heavyweight at the river trout. Indeed this stop of growth
generally fished by farmers in the station of fish farming is not only due to the period of reproduction which slows
as a source of gametes during the period of artificial down the growth of the fish but also on the severe
reproduction which comes true in the station of Ras El conditions which exist during this period of year as the
Ma. important decrease of temperature and trophiques
Among 438 individuals sampled during the resources (Pourriot and Meybeck, 1995) which are
period of study, the number of river trout presenting generally due to the snow coverage which knows in this
scales of regeneration is 50 specimens, this constitutes a region. The resumption of the growth begins gradually
number raised with regard the size of the sample; it is 11, from February and reaches its maximum during August.
Figure 2: Representation schedules of various classes of common trout and their staff
at the level of the Sidi Rachid River during the period of study

Abba et al., 2014
Figure 3: Monthly evolution of Average Marginal Extension

(AME) of the river trout
This important growth is due to the favorable conditions The introduction of the coefficient of regression
of the housing environment as the temperature and the of the relation length (Lt) and length (R) of the scale
abundance of the food reserves, on 438 scales examined gives the following equation: Log Ln = Log L + 0.8674
(51, scales of regeneration), the age is between 0 + and (Log Rn - Log R) (Le Cren, 1947; Benabid, 1990;
4+ for sizes going from 6.3 cm to 37.5 cm. The Bouhbouh, 2002). The total retro measure lengths from
determination of the size of the trout's at the various the equation above are listed in the table -1.
moments of their life is based on the principle of The results obtained for the total retromeasures
proportionality of the growth of the scale with that of its lengthes are used for the determination of the annual
body. For this end, the equation connecting the previous average linear increase ( C ) as well as the specific speed
beam R of the scale and the total length (Lt) used in this of growth noted VSC established by Ricker ( 1958 ):
study was determined as continuation. Log Lt = 0, 8674 C = Ln-Ln-1. (Ln and Ln-1: annual lengthes retro
Log Rt + 0, 5349. The relation between the total length measures in time n and n-1 expressed in years. VCS = Ln
of the body of the trout (Lt) and the length of the -Ln-1 100/Ln-1. The obtained results showed that, the
previous shelf of its scale (R) (Figure-4) can be calculated total retro measures lengths are quite lower
allometrique (Giles and Giguere, 1992). than the observed annual average lengths. This
Figure 4: Relation between the length of the fish and the previous shelf of
its scale at the common trout of the Sidi Rachid River.

Abba et al., 2014
Table 1: Linear retro measures at the Growth of common trout (combined Sexes)
Age Age Observed average Length averages retro measures (mm)
group group length (mm) I II III IV V
2008 I 134.60 91.20
2007 II 152.40 103.27 141.25
2006 III 190.09 104.71 147.90 177.07 - -
2005 IV 263.35 112.20 165.95 213.79 245.47 -
2004 V 318.11 128.82 190.54 234.42 275.42 309.02
Number of fish retro measures 358.00 285.00 194.00 83.00 9.00
Annual average length retro measures 108.63 161.41 208.42 260.44 309.03
Standard deviation 13.84 20.04 29.04 21.17 -
Increase in annual average length (mm) 91.20 37.98 29.17 31.68 33.61
Specific speed of growth 44.39 28.33 14.85 12.20
difference of length can give some explanation by the of the age. The use of reliable software can give even
fact that the observed average lengths correspond to the more reliable results for this equation because the sizes
lengths of fish at various moments of the year or the sinned in other circles sometimes exceed 40cm.
growth is made. On the other hand the total retro
measures lengths correspond to the lengths of fish at the CONCLUSION
time of the training of annuli stag of stop of growth The use of scales and other osseous structures
during December generally. The average lengths allow determining particularly the aspects of age and the
observed to both sexes and individuals of the indefinite analysis of dynamics of a fish population growth. With
sex do not present notable difference for age groups I (1 salmonids, the most recommended method is the scale,
+) and II (2 +). Beyond this age, we notice a variation in despite some disadvantages such as the difficulty of
favour of females (age groups III (3 +) and IV (4 +)), to scales reading or the high number of scales of
become slightly raised to the males of age group V (5 +). regeneration that we obtain. Similarly, the use of another
These variations can be due to the sexual maturity which method can be very beneficial and will allow having
influences the growth and which is premature in a more information.
general way at males. Also, the retro measure averages
are slightly superior at the females than at the males of ACKNOWLEDGEMENTS
the same age group. As for the specific speed of the I thank the persons in charge of the station of fish
growth, it is very important for the class II (2 +) and it farming of Ras El Ma/ Azrou/ Morocco.
exceeded 40 % (combined sexes and various sexes). The
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Corresponding author: Keywords:

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This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

1162-1173 | JRB | 2014 | Vol 4 | No 1

INTRODUCTION (262010 N latitude and 925130 E longitude) deposits

Figure 1: Map showing the four study areas (source: www.mapsofindia.com).

Journal of Research in Biology (2014) 4(1): 1162-1173 1164

Diversity of diatom flora

Figure 2: Representation of diatom flora diversity.

1165 Journal of Research in Biology (2014) 4(1):1162-1173

Figure 3(A-O): Navicula.

Journal of Research in Biology (2014) 4(1): 1162-1173 1166

Figure 4: (A-N) Pinnularia

the center of the valve, sometimes becoming slightly Family: Diadesmidaceae

Journal of Research in Biology (2014) 4(1): 1162-1173 1168

Figure 6: (A-C) Frustulia, D Encyonema, E-Luticola, F-Encyonopsis, (M-Q) Gomphonema.

Family: Eunotiaceae and distantly placed, striae 13 in 10 m.

1169 Journal of Research in Biology (2014) 4(1): 1162-1173

Figure 7 A: Rhopalodia, B- Kobayasiella, C- Actinella, D and E- Achnanthidium,

noticeably broad, radiate in the middle, convergent at the Class: Bacillariophyceae

Journal of Research in Biology (2014) 4(1): 1162-1173 1170

1171 Journal of Research in Biology (2014) 4(1): 1162-1173

seasons throughout the year. Kobayasiella, Cymbella, ACKNOWLEDGEMENT

Journal of Research in Biology (2014) 4(1): 1162-1173 1172

Gurung L, Tanti B, Buragohain AK and Borah SP.

Gurung L, Buragohain AK, Borah SP and Tanti B.

Hasle GR and Fryxell GA. 1970. Diatoms: cleaning

Hendey NI. 1964. An introductory account of the

Husted F. 1959. Die Kieselalgen Deutschlands,

Nautiyal R, Nautiyal P and Singh HR. 1996. Pennate

Patrick R and Reimer CW. 1966. The diatoms d

(1): 668-672. Advantages

1173 Journal of Research in Biology (2014) 4(1): 1162-1173

Detection of biofilm formation in urinary isolates: need of the hour

Saha R1*, Arora S1, Das S1,

Corresponding author: Abbreviations

Web Address: Article Citation:

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

1174-1181 | JRB | 2014 | Vol 4 | No 1

INTRODUCTION patient to become incontinent, thus leading to failure of

Journal of Research in Biology (2014) 4(1): 1174-1181 1176

Table 2. Comparison of biofilm production by three methods TCP, TM and CRM

Isolate TCP (%) TM (%) CRM (%) No BF producer (%)

Antimicrobial susceptibility testing was done RESULTS

Table 4. Comparison of antimicrobial resistance pattern of BF producer with

Journal of Research in Biology (2014) 4(1): 1174-1181 1178

Figure 1 Degree of biofilm formation by TCP, TM and CRM

biofilm producers. CRM detected 25.45% (14/55) while REFERENCES:

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1181 Journal of Research in Biology (2014) 4(1): 1174-1181

Foraging and pollination behavior of Apis mellifera adansonii Latreille

Corresponding author: Keywords:

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Web Address: Dates:

1209-1219 | JRB | 2014 | Vol 4 | No 1

INTRODUCTION MATERIALS AND METHODS

Journal of Research in Biology (2014) 4(1): 1209-1219 1211