1. A novel cost-effective cover slip holder was designed using microscope slides, a glass cutter, and silicone adhesive to resemble the shape of the roman numeral "II". 2. A modified cover slip culture technique was developed where actinomycete cultures were grown sandwiched between two coverslips to allow for in situ microscopy without disturbing morphological structures. 3. The cover slip culture was placed in the custom holder and observed under the microscope to examine the substrate mycelium, aerial hyphae, and spore chains of the actinomycetes.

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A NOVEL, COST –EFFECTIVE COVER SLIP HOLDER FOR
IN SITU MICROSCOPY OF COVER SLIP CULTURES OF
ACTINOMYCETES
RAVINDRAGOUDA PATIL1*
, G. JEYASEKARAN2
AND
S. A. SHANMUGAM2
1
CURRENT AFFILIATION: FISHERIES RESEARCH AND
INFORMATION CENTER, KVAFSU, HESSARAGHATTA,
BENGALURU- 560089, KARNATAKA STATE, INDIA.
2
FISHERIES COLLEGE AND RESEARCH INSTITUTE,
TNFU, THOOTHUKKUDI, TAMIL NADU, INDIA.
Corresponding author’s e-mail: ravi.patil30@gmail.com
ABSTRACT:
A novel, cost effective cover slip holder was designed and used. A
modified cover slip culture method was developed and used. The routine
smear preparation technique disturbs the morphological details of the aerial
hyphae and spore chains of the actinomycete culture and makes it difficult
for identification. The two coverslips with the inoculated medium
sandwiched was incubated in a petri plate humid chamber. The cover slip
culture was placed within the cover slip holder device and observed for the
nature of substrate, aerial hyphae and the nature of spore chains. A novel,
cost-effective cover-slip holder was designed in the laboratory using micro
slides, glass cutter and silicone adhesive. The cover slip holder after
completion, resembled the shape of roman numeral “II”. The cover slip
culture was placed within the cover slip holder device and observed for the
nature of substrate, aerial hyphae and the nature of spore chains.
KEY WORD: Cover slip culture, Cover slip holder, Actinomycete, In situ
microscopy, Mycelium, Spor.
Universal Impact
Factor0.9285:2012;
1.2210:2013
Index Copernicus
ICV 2011: 5.09
ICV 2012: 6.42
ICV 2013: 15.8
ICV 2014:89.16
NAAS Rating
2012 : 1.3;
2013-2014-2015:2.69
SJIF 2012: 3.947,
2013: 4.802
INFOBASE INDEX
2015:4.56
COSMOS IMPACT
FACTOR
2015: 4.366
Received on:
23rd June 2016
Revised on:
18th August 2016
Accepted on:
18th August 2016
Published on:
1st September 2016
Volume No.
Online & Print
79 (2016)
Page No.
60 to 64
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INTRODUCTION:
The routine smear preparation technique, leads to disruption of the aerial hyphae, disruption of the
spore chains of the actinomycete culture and makes it very difficult to get the in situ details, thereby
proper identification becomes a difficult task. The nature of substrate mycelium, aerial mycelium and
nature of spore chains form one of the important characteristics used in the actinomycete taxonomy
(IMTECH, 1998). Hence, cover –slip culture technique is one of the important microbial culture
techniques which facilitates in situ microscopy investigations of the actinomycete cultures without
distorting the mycelial and spore chain structures (Kawato and Shinolue, 1959). When the cover slip
culture is used for microscopic observation, there is a need for a device/tool which helps in moving
the cover slip culture on the stage for changing the field since the normal clamps are designed for
holding and moving the standard micro-slides with the samples on the stage. Hence, in the present
study, efforts were made to design a novel, cost-effective cover-slip holder device for the effective in
situ microscopy studies of the cover slip culture of actinomycete isolates.
MATERIAL AND METHODS:
Modified cover-slip culture technique using SCA medium: A drop of sterile and molten SCA
medium (Fig 1, Table 1) was placed on a sterile cover slip. Another sterile cover slip was placed
on the medium with the help of sterile forceps and gently pressed upon, so that the medium
spread uniformly over the cover slip.
Figure 1. Cover Slip with a drop of molten SCA medium
TABLE 1: Composition of Starch Casein Agar (SCA) (g/l)
Soluble starch 10.0
Vitamin free casamino acids 0.3
Calcium Carbonate CaCO3 0.02
Fe3SO4.7H2O 0.01
KNO3 2.0
MgSO4.7H2O 0.05
NaCl 5.0
Agar 18.0
Aged Seawater Make up to 1L
pH 7.1±0.1

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When the medium was still in semi-solid condition, the upper cover slip was removed gently with the
help of forceps by sliding horizontally. Care was taken to see to it that the margins of the medium were
quite inside the outer margins of the lower cover slip. Inoculation of the medium on the cover slip was
done at three spots along each of the edges of the medium with the highly inhibitory marine
actinomycete isolates (Patil et al., 2001). Another sterile cover slip was placed on this with the help of
a sterile forceps (Fig 2). The coverslips with the inoculated medium were placed on a bent glass stand
placed on sterile cotton soaked with sterile distilled water in a sterile Petri plate which served as a
humid chamber (Fig 3). The culture was incubated at room temperature for 7 days and the culture was
killed by exposure to chloroform vapours for 15-20 minutes (Patil, 1999). The cover slip culture was
placed within the cover slip holder device and observed for the nature of substrate, aerial hyphae and
the nature of spore chains.
Figure 2. Cover slip overlaid on first cover slip with the SCA medium and the inoculum
Figure 3. Cover slip culture of actinomycete placed in the petri plate humid chamber on the
bent glass rod placed over the soaked cotton
Cover slip Holder Device: A novel, cost-effective cover-slip holder was designed in the laboratory
using micro slides, glass cutter and silicone adhesive. Two standard micro-slides, each with a
dimension of 75 mm x 25 mm, were scored with glass cutter at the center of the slide vertically and
cut into halves. Then, the two halves were placed facing each other with a gap equal to 24 mm (width
of the standard square cover slip of 20mm + 2 mm gap on either side). Then the other two halves
were cut for a length equal to 24 mm (length of the standard square cover slip of 20mm + 2 mm gap
on either side). These two halves were placed between the two major halves resembling the shape of

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roman numeral “II” (Fig 4). The two minor halves were glued to the two major halves using the
silicone adhesive. The distance between the two minor halves was equal to the length of the cover
slip in addition to 2 mm gap on either side. These slight gaps between the cover slip outer edges and
the inner edges of the holder were filled with narrow rectangular card board strips (Fig 5).
Figure 4. The novel, cost-effective Cover slip holder
Figure 5. Cover slip culture of actinomycete placed in the cover slip holder with the card board
strips
RESULTS AND DISCUSSION:
The routine cover slip culture methods involve inoculation of the plates with the actinomycete with
swabs and then insertion of 3-4 sterile square cover slips at an angle 450
followed by incubation. Later
the cover slips with over growths of actinomycete were carefully removed and subjected to
microscopic studies using wet mounts. The use of such cover slip culture methods were reported by
many workers (Sathiyaseelan and Stella, 2011; Kalyani et al., 2012; Kandasamy et al., 2012). The
slide culture method for the wet mount microscopy studies for the nature of aerial hyphae and the
nature of spore chains were reported by some other workers (Kavitha and Vijayalakshmi, 2007). This
is the first ever report of modified cover slip culture method allowing the actinomycete to grow in
between two cover slips as well as the designing and usage of the cover slip culture holder.

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CONCLUSION:
This modified cover slip culture method followed in this study allows the use of high power
magnification microscopy studies. The novel, cost-effective cover slip culture holder device was
designed, fabricated and successfully used in the present study for the in situ microscopic studies of
the actinomycete cultures within the coverslips.
ACKNOWLEDGEMENTS:
The authors express their sincere thanks to the Dean, Fisheries College and Research Institute,
Thoothukkudi, erstwhile, TANUVAS, Tamil Nadu and Indian Council of Agricultural Research
(ICAR), New Delhi for providing the laboratory facilities and financial assistance for carrying out
this research work.
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