Chapter-4 Isolation and Characterization of Escherichia coli 4.1 Sample source and collection of samples: Escherichia coli is known to be a colon bacteria which shows ubiquitous presence in many ecological niches. Aerobacter aerogenes which is a non faecal coliform is usually soil borne. Since the most common source of Escherichia coli is the sewage therefore sewage sample was collected from Nag-Nallah, Nagpur. 100 ml of sewage was collected in sterile 250 ml earlinmayer flask by tilting the flask at 45 0 to the direction of the flow. The flask was taken to the laboratory and the processing was done within an hour of collection. 10 ml of the sewage was filtered through Whatman No.1 filter paper to remove the residual solid matter. The filtrate was used as a raw source of Escherichia coli. 4.2 Isolation of Escherichia coli strain: 0.1 ml of the filtered sewage was spread over sterile plates of Nutrient Agar, McConkey s Agar and Eosin Methylene Blue (EMB) agar aseptically in triplicate. The plates were incubated at 37 0 C ± 2 0 C for 24 hours. The colonies showing typical characteristics of green metallic sheen on EMB agar and typical pink colour on McConkey s agar were identified as presumptive Escherichia coli colonies. These colonies were aseptically picked and purified by four way streaking method on sterile nutrient agar. These plates were incubated at 37 0 C ± 2 0 C for 24 hours and purification process was continued till pure axenic cultures were obtained. The colonies were denominated as EC1 to EC12 as 12 isolates showed similar results. The colony characteristics of the isolates are shown in Table 4.1A, B, C and the plates are shown in Photograph 4.1 4.3 Confirmation of Escherichia coli 4.3.1 Gram Reaction: All the twelve isolates namely EC1 to EC12 were subjected to gram reaction as per the method describe in chapter 3.2 the results are shown in Table 4.2 29
4.3.2 Motility: The twelve isolates grown in sterile nutrient broth, at 37 0 C ± 2 0 C for 24 hours. Without shaking the tube a loop full culture was taken out for hanging drop preparation these slides were observed under 10x and 45x for confirming the motility. The results are shown in Table 4.2 4.3.3 Scanning Electron Microscopy: All the twelve isolates were freshly grown in nutrient broth til they reach the exponential phase determine by first developing the growth curve of each isolate. The cells from the exponential phase were harvested by centrifuging at 1000g for 30 min. the bacterial pellets were then subjected to processing for SEM as per Kazunobu A and Akiko U. The Scanning Electron Micrographs is shown in Photomicrograph 4.2 4.4 Sugar Fermentation: Sugar medium containing BTB as indicator and 1% sugar were prepared along with Durham s tubes for confirmation of acid and gas productions. The carbohydrates and their derivatives in the present study included D-Glucose, L-Arabinose, Lactose, Maltose, D- Mannose, D-Mannitol, D-Sorbitol and D-Xylose. The tubes were incubated at 37 0 C ±2 0 C for 24 hours and acid and gas production are observed. Appearance of Yellow colour was considered as acid production and appearance of large air bubbles in the Durham s tube was considered as gas production. The results are shown in Table 4.3. 4.5 IMViC Characterization: All the twelve isolates were grown in tryptone water medium and incubated at 37 0 C ± 2 0 C for 24 hours. 5 ml of aliquot from the culture vessel was aseptically transfer to a fresh test tube and it was treated with kovacs reagent for confirming indol production. Similarly the isolates were grown in Voges proskauer medium for confirming methyl red and Voges proskauer test. Citrate utilization by the isolates was studied using the medium as describe in Chapter 3. The IMViC results of twelve isolates are shown in Table 4.4. 30
4.6 Enzyme studies of the isolates: Since Escherichia coli belongs to Enterobacteriaceae group therefore certain specific enzyme including OF test were conducted for final confirmation. The test include H 2 S Production, urea hydrolysis, Phenyl Alanin deaminase, Lysine decarboxylase, Lipase, ONPG, and OF Test. All these test were performed as per the standard procedure described in Collee et. al., and the composition discussed in chapter 3. The results are shown in Table 4.5 4.7 Reference strain for the study: Since our aim was to study the effect of exposure to transition metal on naturally occurring Escherichia coli. Therefore the initial work started with the isolation and characterization for Escherichia coli from sewage source. However for a complete picture of the response study it is always better to compare the results of experiments with standard reference strain. Hence a standard reference strain of Escherichia coli culture was procured from National Chemical Laboratory (NCL), Pune with Code No.NCIM 2065 which was ATCC culture hereafter referred to as Escherichia coli ATCC 8739. This culture was maintained after repeated sub-culture at 4 0 C. The culture was confirmed by Gram staining, Motility, Growth on Nutrient Agar, Eosin Methylene Blue (EMB) agar and McConkey s agar, IMViC, and Lactose fermentation. The results are shown in Table 4.6 4.8 Discussion: All the isolates were thoroughly examined with more than 80% characters of the family Enterobacteriaceae. Except for certain minor differences in the size and shape of colonies or size and shape of the organism, all the isolates were matching more than 99% hence it seems that all of them are technically belonging to Escherichia coli. In our further studies we selected only one isolate namely EC7 and hereafter referred to as the test strain of Escherichia coli or EC-Test. Similarly for comparison to reference strain the reference strain shall be referred to as EC- Reference ATCC 8739. 31
Table 4.1A Colony characteristics of the isolates on Mc Conkey s Agar Sr. No. Colour Margin Elevation Opacity Size Shape EC-1 Pink Entire Raised Opaque 0.5 mm Nucleated EC-2 Pink Entire Raised Opaque 0.4 mm Nucleated EC-3 Pink Entire Raised Opaque 0.6 mm Nucleated EC-4 Pink Entire Raised Opaque 0.8 mm Nucleated EC-5 Pink Entire Raised Opaque 0.7 mm Nucleated EC-6 Pink Entire Raised Opaque 0.6 mm Nucleated EC-7 Pink Entire Raised Opaque 0.5 mm Nucleated EC-8 Pink Entire Raised Opaque 0.8 mm Nucleated EC-9 Pink Entire Raised Opaque 0.4 mm Nucleated EC-10 Pink Entire Raised Opaque 0.6 mm Nucleated EC-11 Pink Entire Raised Opaque 0.7 mm Nucleated EC-12 Pink Entire Raised Opaque 0.5 mm Nucleated 32
Table 4.1 B Colony characteristics of the isolates on EMB Agar Sr. No. Colour Margin Elevation Opacity Size Shape EC-1 Green metallic sheen Entire Raised Opaque 0.7 mm Nucleated EC-2 Green metallic sheen Entire Raised Opaque 0.8 mm Nucleated EC-3 Green metallic sheen Entire Raised Opaque 0.7 mm Nucleated EC-4 Green metallic sheen Entire Raised Opaque 0.9 mm Nucleated EC-5 Green metallic sheen Entire Raised Opaque 0.7 mm Nucleated EC-6 Green metallic sheen Entire Raised Opaque 0.8 mm Nucleated EC-7 Green metallic sheen Entire Raised Opaque 0.7 mm Nucleated EC-8 Green metallic sheen Entire Raised Opaque 0.9 mm Nucleated EC-9 Green metallic sheen Entire Raised Opaque 0.8 mm Nucleated EC-10 Green metallic sheen Entire Raised Opaque 0.7 mm Nucleated EC-11 Green metallic sheen Entire Raised Opaque 0.8 mm Nucleated EC-12 Green metallic sheen Entire Raised Opaque 0.7 mm Nucleated 33
Table 4.1.C Colony characteristics of the isolates on Nutrient Agar Sr. no. Colour Margin Elevation Opacity Size Shape EC1 Pale Entire Raised Opaque Too small Pin headed EC2 Pale Entire Raised Opaque Too small Pin headed EC3 Pale Entire Raised Opaque Too small Pin headed EC4 Pale Entire Raised Opaque Too small Pin headed EC5 Pale Entire Raised Opaque Too small Pin headed EC6 Pale Entire Raised Opaque Too small Pin headed EC7 Pale Entire Raised Opaque Too small Pin headed EC8 Pale Entire Raised Opaque Too small Pin headed EC9 Pale Entire Raised Opaque Too small Pin headed EC10 Pale Entire Raised Opaque Too small Pin headed EC11 Pale Entire Raised Opaque Too small Pin headed EC12 Pale Entire Raised Opaque Too small Pin headed 34
Photograph 4.1A Growth of Escherichia coli on Eosin Methylene Blue (EMB) Agar culture media showing dark pink colour colonies with green metallic sheen Photograph 4.1B Growth of Escherichia coli on McConkey s Agar culture media showing pink colour colonies Photograph 4.1C Growth of Escherichia coli on Nutrient Agar culture media showing pin headed colonies 35
Microphotograph 4.2 Scanning Electron Micrograph EC-Test EC1: X-3500 EC2: X- 3500 EC3: - X-5000 EC4: X-7500 EC5: X-3500 EC6: X-5000 36
Microphotograph 4.2 continued EC7: X-5000 EC8: X-7500 EC9: X-5000 EC10: X-7500 EC11:X-5000 EC12:X-5000 37
Table 4.2 Morphological characters of isolates EC1 to EC12 Sr. No. Gram Reaction Shape of organism Size Motility EC1 Gram negative Rod or coccobacillary 1.2 µm x 2.2µm Sluggishly motile EC2 Gram negative Rod or coccobacillary 1.4 µm x 2.3µm Sluggishly motile EC3 Gram negative Rod or coccobacillary 1.6 µm x 2.2µm Sluggishly motile EC4 Gram negative Rod or coccobacillary 1.4 µm x 2.3µm Sluggishly motile EC5 Gram negative Rod or coccobacillary 1.2 µm x 2.6µm Sluggishly motile EC6 Gram negative Rod or coccobacillary 1.5 µm x 2.4µm Sluggishly motile EC7 Gram negative Rod or coccobacillary 1.3 µm x 2.4µm Sluggishly motile EC8 Gram negative Rod or coccobacillary 1.5 µm x 2.6µm Sluggishly motile EC9 Gram negative Rod or coccobacillary 1.4 µm x 2.3µm Sluggishly motile EC10 Gram negative Rod or coccobacillary 1.5 µm x 2.4µm Sluggishly motile EC11 Gram negative Rod or coccobacillary 1.2 µm x 2.6µm Sluggishly motile EC12 Gram negative Rod or coccobacillary 1.3 µm x 2.4µm Sluggishly motile 38
Table 4.3 Sugar fermentation by Isolates Sr. No. Glucose Arabinose Lactose Maltose Mannose Manitol Sorbitol Xylose EC1 A/G A A/G A A A/G A A EC2 A/G A A/G A A A/G A A EC3 A/G A A/G A A A/G A A EC4 A/G A A/G A A A/G A A EC5 A/G A A/G A A A/G A A EC6 A/G A A/G A A A/G A A EC7 A/G A A/G A A A/G A A EC8 A/G A A/G A A A/G A A EC9 A/G A A/G A A A/G A A EC10 A/G A A/G A A A/G A A EC11 A/G A A/G A A A/G A A EC12 A/G A A/G A A A/G A A N.B.: A = Acid production G = Gas production 39
Table 4.4 IMViC Characteristics of the isolates Sr. No. Indol MR VP Citrate EC1 + + - - EC2 + + - - EC3 + + - - EC4 + + - - EC5 + + - - EC6 + + - - EC7 + + - - EC8 + + - - EC9 + + - - EC10 + + - - EC11 + + - - EC12 + + - - N.B. : + = Indol production and acid Production - = Acetyl Methyl carbinol, non utilization of citrate. 40
Table 4.5 Biochemical characteristics of the isolates Sr. No. H 2 S Production Urea hydrolysis Phenyl Alanin deaminase Lysine decarboxylase Lipase ONPG OF Test EC1 - - - + - + F EC2 - - - + - + F EC3 - - - + - + F EC4 - - - + - + F EC5 - - - + - + F EC6 - - - + - + F EC7 - - - + - + F EC8 - - - + - + F EC9 - - - + - + F EC10 - - - + - + F EC11 - - - + - + F EC12 - - - + - + F F= Fermentative. 41
Table 4.6 Confirmation of reference strain Test Escherichia coli ATCC 8739 Gram Reaction Motility EMB Maccoknkye Agar IMViC Lactose Fermentation Gram Negative Small Rods Showed Brownian movement Colonies with profuse green metallic sheen Pink colour colonies I+ve, MR+ve, VP-ve, Citrate-ve Acid and gas within 48 Hours. 42