“SCREENING FOR ANTIBIOTIC PRODUCERS IN SOIL FROM THE BANKS OF SEWER CANALS, AND TESTING THE EFFICACY OF ANTIMICROBIAL COMPOUNDS OBTAINED, AGAINST COLIFORMS”

1. “SCREENING FOR ANTIBIOTIC PRODUCERS IN SOIL FROM THE BANKS OF SEWER CANALS, AND
TESTING THE EFFICACY OF ANTIMICROBIAL COMPOUNDS OBTAINED, AGAINST COLIFORMS”
(DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD
OF THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY)
By
JAMMALA VAMSIKRISHNA
Regd. No: 27016062010
Submitted to
DEPARTMENT OF MICROBIOLOGY
SRI VENKATESWARA UNIVERSITY, TIRUPATHI.
Under the supervision of
Sri. P. VIJAYA KUMAR, M.Sc , (Ph.D)
Department of Microbiology,
Government Degree College, Naidupet.
(Affiliated to Vikrama Simhapuri University)

2. INTRODUCTION
 Among microbial flora, Antagonism is a very common phenomenon. Microbes exhibit antagonism at every
niche.
 antimicrobial compounds inhibit the growth of other microbes in the same or other microenvironment.
Ex - Penicillin, Cephalosporin, Streptomycin etc.,
 Present study antimicrobial compounds produced in soil from the banks of sewer canals against coliforms like
E. coli and salmonella.
 E.coli is a is a gram-negative , facultative anaerobes , rod-shaped, most commonly found in the
lower intestine of warm-blooded organisms .
 Salmonella is a rod-shaped , gram-negative bacteria of the Enterobacteriaceae family. The two species
of Salmonella are Salmonella enterica and Salmonella bongori are most commonly found.
 interaction of the sewage contaminants with the indigenous microflora of soil at the sewer canal-banks, could
invariably lead to antagonism, which may result in production of new or modified antimicrobial compounds
against the contaminants which is the basic principle of present study.

3. E.coli Salmonella
 coliforms When cultured on EMB agar medium E.coli produce a green metallic sheen through
fermenting lactose and producing strong acids and salmonella produce a colour less colonies.
 Coliforms when cultured on MacConkey agar medium E. coli developed fish-eye like colonies with pink
periphery. This further the identification of the E.coli. And Salmonella spp. developed colour less
colonies.
 “Crowded plate" method used and detected for antibiotic producers in soil.
 The produced antimicrobial compounds are extracted by using solvent – solvent extraction method .
 This extracted antimicrobial compounds are tested against coliforms by using disc diffusion method
(Kirby-Bauer method)

4.  “To isolate, identify and grow E. coli and Salmonella species as pure cultures”
 “To screen for antibiotic producers from soil samples at the banks of sewer
canals”
 “To produce and extract antibiotics from the isolated antimicrobial cultures”
 “To test for antimicrobial activity of the antimicrobial compounds obtained
against the isolated cultures of E. coli and Salmonella spp.”
OBJECTIVES
To address the research problem, the work is divided into four major objectives as described below.

5. Isolation and identification of E. coli and Salmonella spp.
Materials and Methods
 Sewage samples were collected from different sewer canals, using thread-tied screw-cap tubes at Naidupeta,
Nellore District.
 These samples were pooled, and serial dilution of the sample was made up to 10-8 dilution with autoclave-sterilized
distilled water.
 1000 mL each of EMB agar, MacConkey agar media were prepared respectively. The media were sterilized in an
autoclave at 121ºC temperature and 15 lbs pressure for 20 minutes. 20 mL aliquots of media were poured into
sterile petri-plates and the media were allowed to sit for solidification.
 100 mL of nutrient agar medium (NAM) was prepared. 10 mL aliquots of the NAM were poured into sterile test
tubes, using sterile glass pipettes, the tubes were labeled and cotton-plugged. Cotton-plugged tubes with medium
were kept in a slant position, until solidification so as to obtain NAM-slants for sub-culturing.

6.  0.1 mL of serially-diluted sewage samples from each dilution were spread on the EMB agar medium-plates by using
sterilized L-shaped bent-glass rod. The plates were labeled appropriately.
 These plates were incubated at 37ºC for 24-48 h in incubator which has a thermostat.
 Upon incubation the plates were observed for the growth of desired colonies. EMB agar-plates were screened for the
colonies that produce green-metallic sheen around them, which is a characteristic feature of E. coli which ferment
lactose and produce acid rapidly which is responsible for green metallic sheen. Colorless or light purple colonies were
identified to be lactose non-fermenters.
 These colonies were picked separately by using an inoculation loop and streaked on to MacConkey agar medium-
plates, by quadrant-streaking method of streak-plate culturing; and the plates were labeled appropriately.
 These plates were incubated at 37ºC for 24-48 hrs in incubator which has a thermostat.
 Upon incubation the plates were observed for the growth of desired colonies. Fish-eye colonies with pink periphery
were identified as E. coli colonies, colorless colonies were deemed to be Salmonella spp.
 Identified colonies were picked separately, using inoculation loop and streaked on NAM-slants, which were prepared
earlier. Labeling was done appropriately. Sub-cultures were incubated at 37ºC for 24-48 h. Once growth occurred, the
sub-cultures were transferred to refrigerator (4ºC) for storage until further use.

7. Primary screening for antibiotic producing microbes in soil samples obtained from the banks of
various sewer canals by crowded plate technique
 100 mL of nutrient agar medium was prepared as per the composition . The media were sterilized in an autoclave at
121ºC temperature and 15 lbs pressure for 20 minutes. 20 mL aliquots of media were poured into sterile petri-plates
and the media were allowed to sit for solidification.
 Upon solidification, a pinch of soil inoculum was sprinkled uniformly on sterile nutrient agar plates. The plates were
incubated at 37ºC for 72 h or more in incubator which has a thermostat.
 During incubation, the plates were observed at different intervals, for colonies which had clear zone of inhibition
surrounding them i.e. no growth would be seen in their vicinity. In other words, compounds secreted by these
colonies do suppress the growth of other microbes around those colonies.
 Such colonies were picked-up, and sub-cultured.

8. Scaling-up was done by the following procedure
 250 mL each of nutrient broth, and czpek-dox broth media were prepared respectively. The media were sterilized in an
autoclave at 121ºC temperature and 15 lbs pressure for 20 minutes.
 Upon cooling, the conical flasks with media were transferred to sterile laminar air-flow chamber, where, plenty of the
microbial cultures that produce antimicrobial substances were added separately to the flasks, and labelled appropriately.
Cultures that possess fungal morphology were inoculated into czapek-dox broth.
 The flasks were incubated at 37ºC for 24-72 h in an electric shaker-incubator which has a thermostat. Besides optimum
temperature, continuous agitation was provided in shaker-incubator.
Production of antimicrobial substances

9. Production of antimicrobial substances was achieved by the following procedure
 1000 mL each of nutrient broth, and czapek-dox broth media were prepared respectively, as production media.
The production-media were sterilized in an autoclave at 121ºC temperature and 15 lbs pressure for 20 minutes.
 Upon cooling to room temperature, the conical flasks with media were transferred to sterile laminar air-flow
chamber, where, scaled-up cultures were added to the production media.
 The flasks were then incubated at 37ºC for 72 h or more for mycelial cultures in an electric shaker-incubator
which has a thermostat. At the end of the incubation, antimicrobial compounds were deemed to be produced and
diffused into the broth media, as per the principle.

10. Extraction of antibiotics from broth media by solvent extraction method
 Equal volumes of extraction media and methanol were taken in a separating funnel and the mixture was shaken
vigorously for 5 min.
 An equal volume of n-hexane was added to the mixture followed by shaking the contents vigorously for 5 min.
 The contents were then allowed to settle for 30 min. The lower aqueous layer is collected into sterile conical flask
and the upper hexane-extract is collected into another conical flask.
 The first three steps were repeated twice, to extract any remaining compounds from the aqueous phase, and the
hexane-extract thus obtained was pooled.
 n-hexane extract was transferred to a wide beaker, covered with filter paper, and dried gradually, by evaporating
n-hexane.
 The extract from fungal sample was referred to as ‘extract-1’ and that from bacterial culture was referred to as
extract-2
 The dried extract was stored in a refrigerator until further use.

11. Assay of the antimicrobial activity of the extracted antimicrobial substances - disc diffusion method
(Kirby-Bauer method)
 Filter paper discs were cut using a punching machine and 100 mL of nutrient agar medium was prepared a. The paper-discs and
the media were sterilized in an autoclave at 121ºC temperature and 15 lbs pressure for 20 minutes. 20 mL aliquots of media
were poured into sterile petri-plates and the media were allowed to sit for solidification.
 Antibiotic extract was taken from refrigerator and cooled to room temperature. The extract was re-dissolved in about 0.5 mL of
n-hexane. Upon sterilization, the paper-discs were placed in this antibiotic-solution and dried in laminar air-flow chamber.
 Separate inoculum of each test organism (E. coli and salmonella spp) was prepared by dispersing the test cultures in a few mL
of sterile distilled water taken in a test tube.
 0.1 mL of this inoculum was added to the surface of the NAM plates and was spread by using sterile L-shaped bent-glass rod.
Immediately, antibiotic discs were placed on the surface of the medium with the help of a sterile forceps, by positioning them
distant enough from each other and the rim of the plate. Each plate was also placed with kanamycin-discs as control.
 The plates were incubated at 37ºC for 24-48 h in an electric incubator which has a thermostat.
 Upon incubation, the plates were observed for zone of inhibition around the antibiotic-discs. The area of the zone of inhibition
was calculated using the formula “Πr2”. In which, r denotes the radius of the zone of inhibition, and the results were tabulated.

12. RESULTS
Isolation and identification of E. coli and Salmonella spp.
• Colonies with green metallic-sheen periphery were observed on EMB agar. This is a characteristic feature of E.
coli. Therefore, the colonies were identified as that of E. coli. and colour less colonies is identified as
salmonella spp.
• ‘Fish-eye’ like colonies with pink periphery were observed on MacConkey agar. This is a characteristic feature
of E. coli. Therefore, the colonies were identified as that of E. coli.

13. Primary screening for antibiotic producing microbes in soil samples obtained from the banks of various
sewer canals.
 One microbial colony was found to be antagonistic, forming a clear zone of growth inhibition around it. However,
this colony has fungus-like mycelial morphology. Therefore this organism was assumed to be a fungus.
 Another colony with a clear zone of growth inhibition around it was also observed in the crowded plate, which was
having the morphological characteristics of a bacterial colony.
Crowded plate technique

14. Production of antimicrobial compounds
 The microbial strain that was assumed to be a fungus was well grown in, the fungal medium that was used for
scale-up process, confirming the assumption. Therefore this culture was continued to grow in czapek-dox broth for
production of antibiotics.
 Another microbial strain was assumed to be a bacterium; it has grown well in nutrient broth medium, giving rise to
enormous growth in nutrient broth. Hence, this culture was grown in the same medium for production of
antibiotics.
Nutrient Broth Czapek-Dox Broth

15. Extraction of antibiotics from broth media by solvent extraction method
 Two separate extracts one each from the unknown fungal culture and the unknown bacterial culture were made.
The final dried extract was highly viscous, indicating the positive outcome of the solvent extraction procedure.

16. Assay of the antimicrobial activity of the extracted antimicrobial substances
 Extract-1 had showed highest antimicrobial activity against both the coliforms.
 When compared the antibacterial efficacy against the isolated strain of E. coli, extract-1 had 1.45 times higher efficacy
than that exhibited by kanamycin, an antibiotic that is known to be effective against gram negative bacteria like E. coli.
 When compared the antibacterial efficacy against the isolated strain of Salmonella spp., the antimicrobial potential of
extract-1 was approximately 16 times greater than that of the kanamycin.
Effect of antimicrobial compounds on growth of coliforms.
Test organism
Area of the inhibitory zone (cm2)
Blank Extract-1 Extract-2 Kanamycin
E. Coli 0.0 18.1 3.8 12.5
Salmonella 0.0 16.6 2.0 1.1
Data are Mean values of duplicate assays, with negligible standard deviation. Formula used to calculate the area of the
circle was “Πr2”. Three to four values of diameter were taken using cm scale and average diameter was divided by two to
get the radius (r)

17. Figure: Assay of antimicrobial activity by disc diffusion test
against Salmonella spp. culture. E1 - extract-1; E2 - extract-2;
the other unlabeled disc was blank.
Figure :Assay of antimicrobial activity by disc diffusion
test against E. coli culture. E1 - extract-1; E2 - extract-2;
B – blank disc; K – kanamycin disc.

18. Figure 11: Graphical representation of the assay of the
antimicrobial activity of the antibiotic extract on
Salmonella spp.
Figure 10 Graphical representation of the assay of the
antimicrobial activity of the antibiotic extract on
Salmonella spp.

19. CONCLUSION
 Highest antimicrobial activity was shown by extract-1 against both the isolated coliform strains, which is
promising, as the compound inhibited the growth of the test organisms to for greater extent than did
kanamycin
 Although the extract-2 was twofold effective than kanamycin, against Salmonella spp. Further research on
the compound is lesser promising than the effect of extract-1. However, identification of this bacterial
strain, strain improvement, and optimization of the production process could be of some research interest
and future purpose