Industrialization and human activities have totally turned our environment to dumping sites for waste materials

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Industrialization and human activities have totally turned our environment to dumping sites for waste materials

1. Introduction

Industrialization and human activities have totally turned our environment to dumping sites for waste materials. As a result, many water resources have been rendered unwholesome and hazardous to man and other living systems (Bakare et al. 2003). The toxic substances discharged into water bodies are not only accumulated through the food chain (Odiete 1999), but may also either limit the number of species or produce dense populations of microorganisms (Okafor 1985). Pollution of the surface and underground water wastes is widespread, thereby rendering them unsuitable for man’s use (Ajayi & Osibanjo 1981; Kakulu & Osibanjo 1992; Odiete 1999; Bakare et al. 2000; Bakare & Oyedeji 2001; Bakare et al. 2003). In addition, since many industries lack effluent treatment plants, the untreated wastes are either deposited on the ground or discharged into nearby natural water bodies (Chukwura & Okpokwasili 1997; Odiete 1999).

Industrial effluents are wastes generated by the industry during the process of drugs manufacturing. Here we identify and isolate microorganisms from effluent of the industry and also measure the quality of effluent treatment plant of ACME Laboratory.

ACME Laboratories produces almost 400 products and The ACME Agrovet & Beverage Ltd, savar, manufacture fruit juice, mineral water and many more. Discharges waste material in reservoirs situated within the area of the industry.

ACME Laboratory an industry producing (amoxicillin, ampicillin, cephalexin, cloxacillin, flucloxacillin). The waste disposal of this industry contains of a range of chemical substances including methylene chloride, isopropyl alcohol, pivalic acid, triethylamine, small amount of amoxicillin and its degradation products, ethanol acetone

Cephalexin and its degradation products, ethyl acetate, methyl ketone and small amount of cloxacillin its degradation products. (Susan et al 1989, Golam Mahammed, 2005).

In this study, we undertake the microbiological assessment of the effluent of a pharmaceutical company name ACME Laboratory whose effluent is discharged through an effluent treatment plant. After treatment the treated water is reserved into a man-made lake. When it rains, the water is washed down the terrain into a stream, where as in the dry season a substantial portion of the water sinks into the ground.

Microorganisms of polluted environment bear resistant property to that pollutant. Like other normal microbes they are identified based on morphology, nutritional requirement and growth characteristic. Comparing the viability of microorganisms of polluted environment to that of natural habitat,. A chemical industry without waste treatment facility always discharges materials in natural habitat harmful to living world comprising human, animal, plants, and the important microbial community.

We assess the effect of polluting waste water and treated water from an effluent treatment plant (ETP) on microbes from a reputed industry namely ACME Laboratories Ltd.

1.1 General information:

Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. Bacterial cell components have their own defense mechanism against their lethal components.

Fig: Bacterial components of cell responsible for resistance against antibiotics ( natural )

Antibiotic resistance evolves via natural selection acting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by conjugation, transduction, or transformation. Many antibiotic resistance genes reside on plasmids, facilitating their transfer. If a bacterium carries several resistance genes, it is called multi resistant or, informally, a superbug.

Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place. The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.

Most of the microorganisms get mutated to antibiotic resistance under untreated condition due to their long time presence in that condition to survive

Antibiotic resistance of organisms are now creating worse situation day by day. The rate of resistance is increasing regularly. Therefore it is now creating a very critical condition to check. The increasing antibiotic resistance is a matter of concern not only in treating diseases but also in treating environment that is the waste management. There are a number of factors that are responsible for getting antibiotic resistance among the microorganisms . some are as follows:

· The wide spread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria.

· They are often used in animals but also in other industries which at least in the case of agricultural use lead to the spread of resistant strains to human populations.

· In some countries antibiotics are sold over the counter without a prescription which compounds the problem. In human medicine the major problem of the emergence of resistant bacteria is due to misuse and overuse of antibiotics by doctors as well as patients.

· Other practices contributing towards resistance include the addition of antibiotics to the feed of livestock.

· Household use of antibacterial in soaps and other products, although not clearly contributing to resistance, is also discouraged (as not being effective at infection control).

· Unsound practices in the pharmaceutical manufacturing industry can contribute towards the likelihood of creating antibiotic resistant strains.

· Certain antibiotic classes are highly associated with colonization with superbugs compared to other antibiotic classes. The risk for colonization increases if there is a lack of sensitivity (resistance) of the superbugs to the antibiotic used and high tissue penetration as well as broad spectrum activity against “good bacteria”.

1.2 Mechanisms

Bacteria use different mechanisms to protect themselves from natural, synthetic and semi synthetic compounds that are

The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:

1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of ?-lactamases.

2. Alteration of target site: e.g. alteration of PBB—the binding target site of penicillin’s—in MRSA and other penicillin-resistant bacteria.

3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.

4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface. Some bacterial spp use it. But it is now getting attention. They posses efflux transporters of very broad substrate specificity. Some bacteria are able to withstand synthetic and semi synthetic agents by pumping out.

2. Objective:

(a) To isolate microorganisms from chemically polluted water source.

(b) To isolate microorganisms from ETP to check its efficiency.

(c) To characterize the isolates microbes with resistance pattern to antimicrobial drugs.

(d) To indicate the spreading of drug resistant microbial strains to community endangering people’s health.

(e)To focus on efficiency level of ETP of ACME Laboratories.

2.1 Outline of the study

1. Objective Selection

2. Sample collection

3.simple analysis like alkality, ph, temperature, etc

4.BOD

5.COD

6. Microbiological analysis of sample

a. Microscopic examination

b. Isolation of microbes by culturing the sample

c. Cultural characterization of the isolates

d. Colony characters on solid media

e. Morphological and staining properties

f. Biochemical properties

g. Antibiotic sensitivity testing of the isolates

h. Survival study of the isolates with polluted water

3. Materials and Methods

The duration of the study was from May 2010 to October 2010 at Gonoshasthaya Vaccine Research and Diagnostic Laboratory (GVRL), Department of Microbiology, Gono Bishwabidyalay. MacConkey agar was purchased from Oxoid Ltd., England and Tropic soy agar for blood agar plates was from Becton Dickinson and Company, USA. Mueller Hinton agar was purchased from Sanofi Diagnostics, Pasteur, France. Antibiotic discs amoxicillin (30 ?g), were purchased from Becton Dickinson and Company, USA. Ciprofloxacin (5 ?g), cotrimoxazole (25 ?g), tetracycline (30 ?g) and gentamicin (120 ?g), nitrofurantoin (30?g) were from Oxoid Ltd. UK. Nalidixic acid (30 ?g). Erythromycin were from Mast Diagnostics, UK.

3.1 Materials

Table-I Antibiotic discs used in the study:

Sl. No.
Antibiotic
Letter Code
Quantity Source
1 Amoxicillin A 30 ?g Becton Dickinson, USA
2 Ciprofloxacillin CIP 5 ?g Oxoid Ltd., UK
3 Cotrimoxazole SXT 25 ?g Oxoid Ltd., UK
4 Gentamicin CN 120 ?g Oxoid Ltd., UK
5 Imipenem IMI 10 ?g Mast Diagnostics, UK
6 Nalidixic acid NA 30 ?g Mast Diagnostics, UK
7 Nitrofurantoin NI 300 ?g Oxoid Ltd., UK
8 Tetracycline TE 30 ?g Oxoid Ltd., UK
9 Cephalexin CFX 30 ?g Oxoid Ltd., UK
10 Penicillin G PG 10 unit Mast Diagnostics, UK
13 Erythromycin E 15 Mast Diagnostics, UK

Sheep blood was collected from animal house of GVRL. Other ingredients were of appropriate analytical and commercial standards.

Both qualitative and quantitative microbiological analyses were performed. For quantitative routine examination, the samples were mixed thoroughly before plating. Inoculation on media was done with pre-calibrated platinum loop to deliver a measured quantity (1.0 ?l) of sample.

For culturing MacConkey-agar and 5% sheep blood-agar plates were used that can support the growth of most Gram-negative bacilli and staphylococci (Baron and Finegold, 1990). The inoculated plates were incubated at 37ºC overnight. Plates with tiny colonies or no growth were extended to further incubation up to 48 hour.

For qualitative analysis, identification of organisms was done by conventional methods through culturing of samples followed by biochemical tests (Monica, 1984). Antimicrobial sensitivity was performed by disc diffusion method on Mueller-Hinton agar plates for organisms other than streptococci which needed blood agar plates.

Glass wares and other materials:

The different types of sterilized glass ware and materials are used.

1. Experimental test tube.

2. Durham` s fermentation tube.

3. Stopper of test tube.

4. Petri dish.

5. Conical flask.

6. Pipette.

7. Slide.

8. Microscope

9. Immersion oil.

10. Cotton swab

11. Thermometer.

12. Beaker

13. Jar, Cylinder.

14. Electric Balance.

15. Toothpick

16. Spirit lamp and

17. Bacteriological loop

18. Bacteriological straight wire etc.

3.2 Methods

3.2.1 Sample collection:

Sample was collected from the untreated water reservoir of ACME Laboratories and from the pond where treated water was discharged. Waste water samples were collected from surface, deep layer and sediment of the reservoir and tasted for the presence of microorganisms.

3.2.2 Sample collation site

Samples were collected from ACME Laboratories.

Production plant waste water reservoir (sampling point-1)

ETP (sampling point-2)

Treated water reservoir

Fig: overall process of ACME Laboratories

3.2.3 study site

This study was carried out in the Department of Microbiology, Gono Bishwabidyalay (University ).

3.3 Chemical parameters of water

3.3.1 COD of water

(a) Poured 50 ml of water sample in a conical flask (100 ml capacity).

(b) Similarly, took 50 ml distilled water in a flask.

(c) Poured 5 ml K2Cr2O7 solution separately in both flasks.

(d) Incubated the flasks at 1000 C for one hour keeping in a water bath.

(e) Thereafter, removed the flasks to cool for 10 minutes.

(f) Mixed 5 ml KI solution, and 10 ml of H2SO4 solution in each flask.

(g) Transferred 0.1 M sodium thiosulfate solution in burette fitted in titration assembly, and titrated with both the samples in flasks till pale yellow color disappears. In each case noted the amount of sodium thiosulfate solution used.

(h) Added 1 ml of starch solution to both the flasks. Color turned blue.

(i) Again titrated with sodium thiosulfate as above till complete disappearance of blue color.

3.3.2 DO of water

(a) Collected water sample in a glass bottle (250 ml) in such a way that water bubble should not come out.

(b)Pipette separately 2 ml of manganese sulfate and 2 ml of alkaline iodine-azide solutions.

(c)Added these solution in succession at the bottom of bottle and placed the stopper of bottle

(d)Shook the bottle upside down for about 6-8 times. There developed brown precipitate.

(e)Left the bottle for a few minutes, the precipitate settled down.

(f)Added 2 ml of concentrated H2SO4 in the bottle. Shook properly so that brown precipitate might dissolve.

(g)Took a clean flask and pour 50 ml of this water sample. Titrated it against 0.025 N sodium thiosulfate solution taking in a burette until pale straw color develops.

(h)Add 2 drops of starch solution on the flask. Color of contents changed from pale to blue.

(i)Again titrated against thiosulfate solution until the blue color disappeared.

3.4.1 Alkalinity of water

(a) Took 50 ml of water sampler in a conical flask (100 ml capacity).

(b) Added a few drops of phenolphthalein indicator.( If color of the water does not change, it means that phenolphthalein alkalinity is nil due to absence of carbonates in the water sample. Moreover, if there develops pink color, determine phenolphthalein Alkalinity).

(c) Poured 0.1N Hcl solution in burette and titrated with water sample. Noted the end point of when pink color become colorless.

(d) Took another 50 ml of water in flask and added 2-3 drops of methyl orange in to it. Color turned to orange.

(e) Transferred 0.1N Hcl solution in to burette in titration assembly and titrated with the water sample. Methyl orange added until yellow color change to pink. Noted the end point.

3.4.2 PH of Water

The PH of water sample was determined correctly by PH meter.

3.4.3 Temperature of water

The temperature of water sample was determined correctly by thermo- meter.

3.5 Isolation of viable organisms.

Each specimen was streaked on nutrient agar plate and incubated over night. Next day, the types of colonies were observed and recorded.

3.6 Screening for phenol tolerant organism.

Two sets of test tubes were prepared with phenol containing media. One set had T1N1 media with 1% phenol. Another set had distilled water with 1% phenol. Both sets were inoculated with the isolates and incubated at 370 C for 3 days, the tubs were observed for growth.

3.7 Gram Staining.

(a) Obtained clean glass slides

(b) Using sterile technique, prepared a smear of each of the organisms. Did this by placing a drop of water on the slide, and then transferring each organism separately to the drop water with a sterile, cooled loop. Mixed and speeded organism by means of a circular motion of the inoculating loop.

(c) Allowed smears to air-dry and then heat fixed in the usual manner.

(d) Gently flooded smears with crystal violet and let stood for 1 minute. Gently washed with tap water.

(e) Gently flooded smears with Gram’s iodine mordant and let stood for 1 minute. Gently washed with tap water.

(f) Decolorized with 95% ethyl alcohol. Gently washed with tap water.

(g) Counterstain with safranin for 45 sec.

(h) Gently washed with tap water.

(i) Examined under oil immersion.

3.8 Spore Staining. (Schaeffer-Fulton Method )

(a) Obtained one cleaned glass slide.

(b) Made smears in the usual manner using sterile technique.

(c) Allowed smear to air dry, and heat fixed in the usual manner.

(d) Flooded smear with malachite green and placed on top of a beaker of water sitting on a warm hot plate. Allowing the preparation to steam for 2 to 3 minutes. Prevent the stain from boiling by adjusting the hit plate temperature.

(e) Removed slide from hot plate, cool, and wash under running tap water.

(f) Counterstain with safranin for 30 sec.

(g) Washed with tap water.

(h) Examined under oil immersion.

3.9 Biochemical test

3.9.1 .Catalase Test

a) A colony of the bacteria from a plate was picked up and transferred on a glass slide in a drop of water.

b) A few drops of 3% H202 was placed (dilute 30% commercial solution 1: 10) over the slide.

c) Production of gas bubbles (released oxygen) indicated appositive reaction.

3.9.2 Oxidase Test

a) A portion of the test organism was picked up from agar plate by means of a sterile wooden-pick.

b) Streaking on to the filter paper soaked with the oxidase reagent.

c) Formation of a dark purple color developed within 5-10 seconds indicated positive for oxidase

3.9. 3. Triple Sugar Iron (TSI) agar Test

a) A loop of bacteria was spread across the surface of the agar.

b) A needle of bacteria was inserted (stabbed) into the bottom (butt) of the tube.

c) The tubes were kept at 37° C for 24 hours for incubation.

d) The tubes ere examined.

3.9.4 Motility Iodole and Ornithine decarboxylate ( MIO) Test

a) Inoculated with 2-3 similar colonies from an over night growth, by stabbing with a straight needle to over half the depth.

b) The tube was inoculated at 37° C for 24 hours with loosen caps.

c) Examined by naked eyes for motility and ornithine decarboxylate.

3.9.5 Methyl red (MR) Test

(a) Sterile MR-VP broth was inoculated with the test organism and following incubation at 37° C for 24 hours.

b) Few drops of methyl red solution were added.

c) A distinct red color indicated MR positive test while yellow or orange color indicated a negative result.

3.9.6 Voges proskaur (VP) Test

a) Sterile MR-VP broth was inoculated with the test organism and following incubation at 37° C for 24 hours.

b) After incubation, 5 rops of naphtha solution and 5 drops of KHO solution were added.

c) The development of a bright red or pink-red color was recorded as a positive result.

3. 9.7 Indole Test.

a) Tryptophan containing broths were inoculated with bacteria.

b) The tubes were incubated at 37° C for 24 hours.

c) Added 0.5 ml of the Kovac’s reagent after the bacterial growth. If indole positive, after 2 minutes a red color ring appeared at the junction of medium and reagent in the tube

3.9.8 Citrate Utilization Test.

a) A loop of bacteria was spread across the surface of the agar.

b) A needle of bacteria was inserted (stabbed) into the bottom (butt) of the tube.

c) Then the tubes were kept at 37° C for 24 hours for incubation.

d) Finally the tubes were examined.

3 .9 .9 Urea Test.

a) The organisms were inoculated in tubes containing urea slant.

b) The tubes were incubated at 37° C for 24 hours.

c) Bright red color indicated positive result.

3.9.10 Carbohydrate Fermentation.(sucrose, lactose, dextrose)

(a) Phenol red broth medium containing inverted Durham’s tube was inoculated with the test organism.

(b) Incubated at 370 C for 24 hours.

(c) Chang in color indicated acid production while formation of bubble in the Durham’s tube indicated gas production.

3.9.11 Nitrate Reduction Test

a) Sterile nitrate broth was inoculated with the test organism.

b) Incubated for 24 hours at 37° C.

c) Following incubation one drop of sulfanilic acid and one drop of napthalamine were added.

d) Formation of a red color indicated reduction of nitrate. If no color developed, zinc dust was added and absence of any color again indicated a positive result.

3.9.12 Mannitol Fermentation Test.

a) one loop of test organism was inoculated in the mannitol broth.

b) Incubated the tube at 37° C for 24 hours.

c) The tubes were examined, in case of producing acid, the color of the medium would be changed from purple to grayish yellow. The medium remaining purple color indicated that no acid was produced.

3.9.13 6.5% Nacl test

a) The organisms were in inoculated in 6.5 % Nacl broth.

b) The tubes were incubated at 37° C for 24 hours.

c) The results were observed.

3.10 Antibiotic Sensitivity Assay Of Bacterial Isolates.

Bacterial susceptibility to anti microbial agent was determined in vitro by using the standardized agar disc-diffusion method known as the Kirby Bauer ( Barry and Thom berry, 1985 ). Antibiotic and the disc potencies used were following

Imipenem (10 µg), Azithromycin (15µg), Amoxicilin (30 µg), Tetracyclin (30 µg), Nalidixic Acid (30 µg), Erythromycin (15 µg), Vancomycin (30 µg), Cephalexin (30 µg), Amikacin (30 µg), Penicillin-G (10 units).

(a) Labeled the covers of each of the agar plates with name of the test organisms was inoculated.

(b) Using sterile technique, inoculated all agar plates with their respective test organisms as follow:

1) Dipped a sterile cotton swab into a well mixed saline test culture and removed excess inoculums by pressing the saturated swab against the inner wall of the culture tube.

2) Using the swab, streaked the entire agar surface horizontally, vertically, and around the outer edge of the plate to ensure a heavy growth over the entire surface.

(c) Allowed all culture plates to dry for about 5 minutes.

(d) Distributed the individual antibiotic discs at equal distance with forceps dipped in alcohol and flamed.

(e) Gently pressed each disc down with the wooden end of the cotton swab or sterile forceps to ensure that the discs adhered to the surface of the agar.

(f) The plates were then inverted and incubated at 370 C for 24 hours.

(g) After incubation, the plates were examined and the diameter of the zones of complete inhibition were measured in mm.

(h) The zone diameter for individual anti-microbial agents were used to determine susceptible, intermediate, and resistant categories by referring to an interpreting table ( Barry and Thom berry, 1985 ).

3.11 Observation of cultural characteristic.

3.11.1 Blood Agar.

Each isolate was streaked on blood agar and incubated at 370C for over night. Next day demonstrated the patterns of haemolysis produced by organisms.

3.11.2 Eosin Methylene Blue (EMB) Agar.

Each isolate was streaked on EMB agar plate and incubated at 370C for over night. Next day demonstrated morphological characteristics of the bacterial colonies.

3.11.3 MSA

Each isolate was streaked on MSA plate and incubated at 370C for over night. Next day demonstrated morphological characteristics of the bacterial colonies.

3.12 Oxygen Utilization Test.

(a) Using sterile technique, inoculated each experimental organism by introducing two drops of the culture from a sterile pipette into the appropriately labeled tubes of molten agar.

(b) Vigorously rotated the freshly inoculated molten infusion agar between the palms of the hands to distribute the organisms.

(c) Placed inoculated test tubes in an upright position in the ice water bath to solidify the medium rapidly.

(d) Incubated the tubes at 370C for 24 hours.

4.1.1 Result of chemical parameter of waste water and treated water.

DO, COD, Alkalinity, PH, and temperature of the water was determined .The result is recorded in below table.

Table No-1

Source DO BOD COD PH Temperature
Waste water 2.5mg/L 750mg/L 1190.0mg/L 6.5 35.00 C
Treated water 6.61mg/L 23.85 19.22 mg/L 7.3 24.80 C

4.1.2 Result of isolation of viable organisms.

Each water sample was streaked on NA plate and types of colonies were observed and record. The result is recorded in below table.

Table No-2

Specimen Nutrient Agar Plate
Surface water Different colonies of 17 morphological types were found code numbered A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11,A12,A13,A14,A15,A16,A17.
Deep water Different colonies of 12 morphological types were found code numbered E1,E2,E3,E4,E5,E6,E7,E8,E9,E10,E11,E12.
Sediment water Different colonies of 10 morphological types were found code numbered H1,H2,H3,H4,H5,H6,H7,H8,H9,H10.

[All colony types were repeatedly sub cultured in nutrient agar to get isolated pure culture of each type of colony ] .

4.1.3 Screening.

All colony types were inoculated in T1N1 broth, T1N1 supplemented with phenol and diluted phenol solution and there growth was observed for 3 days. The result is recorded in below table.

Table No-3.

Media composition Isolates Code Growth after 3 days.
T1N1

Supplemented

phenol

Nacl-10.gm

Trepton-10.gm

Phenol-10gm

Distilled

water-1000ml

A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11,A12,A13,A14,A15,A16,A17.

E1,E2,E3,E4,E5,E6,E7,E8,E9,E10,E11,E12.

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10.

A1, A3, A7

E2, E5, E6, E9

H3, H4, H7

Table No-4.

Media Composition Isolates Code Growth after 3 days.
Dilute phenol solution 0.5gm phenol crystal in 5ml water. (100µg/ml) A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11, A12,A13,A14,A15,A16,A17.

E1,E2,E3,E4,E5,E6,E7,E8,E9,E10,E11,E12.

H1, H2, H3, H4, H5, H6, H7, H8, H9, H10.

A1, A3, A7

E2, E5, E6, E9

H3, H4, H7

4.1.4 Result of Gram staining.

Physical, Staining, and cultural properties of the bacterial strains isolated from industrially polluted waste water are recorded in below table.

Table No-5

Isolates Code Gram Reaction Call Morphology
A1 Gram (-) Ve Rod
A3 Gram (+) Ve Rod
A7 Gram (-) Ve Rod
E2 Gram (-) Ve Rod
E5 Gram (-) Ve Rod
E6 Gram (+) ve Rod
E9 Gram (-) ve Rod
H3 Gram (+) ve Cocci
H4 Gram (-) Ve Rod
H7 Gram (-) Ve Rod

4.1.5 Result of Spore staining.

Table No-6

Isolates Presence of spore
A1 Non spore
A3 Non spore
A7 Non spore
E2 Non spore
E5 Non spore
E6 Non spore
E9 Non spore
H3 Non spore
H4 Non spore
H7 Non spore

4.1.6 Colony character of the isolated organisms in different media:

Table No-7

Isolate no. Nutrient agar Blood agar MacConkey agar EMB MSA
A1, H4 Thick, white, glestining growth Alpha haemolytic Thick, mucoid, pink colonies No growth
A3, E6 Slightly elevated, Beta -haemolytic No growth
A7 Blue grey spreading colonies Beta haemolytic colonies Circular spread colonies Reddish type growth No growth
E2 White, moist, glistening growth White, dry type colonies Pink, umbunate colonies Green metallic sheen No growth
E9 White, translucent, raised growth Alpha haemolytic colonies Pink, moist colonies Growth found No growth
E5, H7 Moist, greenish, spreaded colonies Beta haemolytic colonies Raised colonies Spread growth No growth
H3 Abundant, opaque golden growth Beta haemolytic colonies No growth No growth Yellow shiny colonies

4.1.7 Biochemical characteristics.

Name of biochemical tests
Isolation

No:

Lactose Dextrose Sucrose T

S

I

M

I

O

MR VP Indole Citrate Urease Nitrate

Reduction

Ca

ta

la

sa

Oxi

da

se

Presumptive

Organism

A1 AG AG AG A.S/

A.B

H2S

M+

O+

+ + + + Enterobacter spp
A3 A A A.S/ A.B

H2S

M

O+

+ + + Bacillus spp
A7 AG AG A.S/ A.B

H2S+

M+

O+

+ + + + + Proteus spp
E2 AG AG A A.S/A.B

H2S

M

O+

+ + + + Escherichia coli
E5 No Change M+

O+

+ + + Pseudomonas spp.
E6 A A A.S/

A.B

H2S

M

O+

+ + + Bacillus spp
E9 AG AG AG A.S

/A.B

H2S

M

O+

+ + + + Klebsiella spp
Sample Isolate no. Name of Biochemical Tests
Isolation no: Lactose Dextrose Sucrose TSI MIO MR VP Indole Citrate Urease Nitrate

Reduction

Ca

ta

la

sa

Oxi

da

se

Presumptive

Organism

H3 A A A A.S/

A.B

H2S

M+

O+

+ + + Staphylococcus aureus
H4 AG AG AG No Change M+

O+

+ + + + Enterobacter spp.
H7 No Change M+

O+

+ + + Pseudomonas spp.

4.1.8 Identification of isolated organisms

studied for the isolates from the polluted waste water for identification at isolate at The staining properties, colonial morphology, and biochemical properties were genera level.

Isolate code Presumptive organisms
A1 Enterobacter spp
A3 Bacillus spp
A7 Proteus spp
E2 Escherichia coli
E5 Pseudomonas spp.
E6 Bacillus spp
E9 Klebsiella spp
H3 Staphylococcus aureus
H4 Enterobacter spp.
H7 Pseudomonas spp.

4.1.9 Result of sensitivity test

Sensitivity of the isolated bacterial strains was done on Mueller Hinton agar plate medium with the standard antibiotic discs from commercial source. Zone of inhibition was determined by referring to an interpreting table ( Barry and Thornsberry, 1985 ).

Antibiotic sensitivity test for Enterobacter spp

Name of Antibiotic Isolation code Isolation code
A1 H4
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R) (S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin R S 25
Cefalexin R S 22
Tetracycline I 13 S 18
Gentamycin R S 20
Cotrimoxazole R S 30
Ciprofloxacin R S 25
Erythromycin R R
Nalidixic acid R I 13
Nitrofurantoin

Antibiotic sensitivity test for Bacillus spp

Name of Antibiotic Isolation code Isolation code
A3 E6
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R) (S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin R R
Cefalexin R R
Tetracycline R R
Gentamycin R
Cotrimoxazole R R
Ciprofloxacin R R
Erythromycin R R
Nalidixic acid R R
Nitrofurantoin S 20 R

Antibiotic sensitivity test for Proteus spp

Name of Antibiotic Isolation code
A7
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin R
Cefalexin R
Tetracycline R
Gentamycin S 15
Cotrimoxazole R
Ciprofloxacin R
Erythromycin R
Nalidixic acid R
Nitrofurantoin S 20

Antibiotic sensitivity test for Escherichia coli

Name of Antibiotic Isolation code
E2
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin R
Cefalexin R
Tetracycline R
Gentamycin S 17
Cotrimoxazole R
Ciprofloxacin R
Erythromycin R
Nalidixic acid R
Nitrofurantoin R

Antibiotic test for Klebsiella spp

Name of Antibiotic Isolation code
E9
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin S 23
Cefalexin S 25
Tetracycline S 20
Gentamycin S 18
Cotrimoxazole R
Ciprofloxacin R
Erythromycin S 23
Nalidixic acid R
Nitrofurantoin I 14 R

Antibiotic sensitivity test for Pseudomonas spp

Name of Antibiotic Isolation code Isolation code
E5 H7
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R) (S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin S 25 R
Cefalexin S 30 R
Tetracycline S 20 R
Gentamycin S 21 R
Cotrimoxazole S 20 R
Ciprofloxacin S 27 R
Erythromycin S 25 R
Nalidixic acid R R
Nitrofurantoin S R

Antibiotic sensitivity test for Staphylococcus aureus

Name of Antibiotic Isolation code
H3
Sensitive

(S)

Intermediate

(I)

Resistant

(R)

(S) Zone size (mm) (I) Zone size (mm) (R)
Amoxicillin R
Cefalexin R
Tetracycline R
Gentamycin R
Cotrimoxazole R
Ciprofloxacin I 12
Erythromycin R
Nalidixic acid I 14
Nitrofurantoin R

Fig. 1: Growth of E. coli in EMB agar, confirmed with green metallic sheen

Fig. 2: Growth of E. coli on MacConkey Agar

Fig.3: Growth of Klebsiella on MacConkey

Fig. 4: Swarning Proteus on Blood agar

Fig. 5: Pseudomonas showing Haemolysis on Blood Agar

Fig. 6: Steaphylococcus on MSA

Fig. 7: Catalase test ( right shows positive and left is negative )

Fig. 8: Oxidase Test ( right shows positive and left is negative )

Fig.9: TSI Test ( Left shows color change of butt and showing H2S production

Middle is showing acid slant/ acid butt with gas production, right is no change)

Fig. 10: MIO Test ( left is control, second tube is M+O+, third tube is MO+

and the fourth is negative)

Fig. 11: MR- VP Test ( Left is control, Middle is MR positive

And right is VP negative)

Fig. 12: Indole Test ( Left is control, middle is positive and right is negative )

Fig. 13: Citrate utilization ( left is negative and right is positive )

Fig. 14: 6.5% NaCl Test ( left is negative and right is positive )

Fig. 15: Mannitol fermentation test ( left is control, middle is negative

and right is positive )

Fig. 16: Nitrate reduction test (left is control, second and third is positive and right is negative)

Fig: 17. Urea test (right is control middle is negative and left is positive )

Fig.18: multi drug resistant pattern of an isolates

Fig. 19: moderate sensitivity of an isolate

Fig. 20: gram negative bacilli

Fig. 21: gram positive cocci

Discussion:

Emerging antibiotics resistance of microorganisms is now a burning questions. The increasing resistance rate to various antibiotics is now creating problems.

In industrial site it is a problem to manage the waste material that is very harmful for the environment as well as for all living creatures and for our dearest world

As we are the best creature of God we should try to save our environment for our existence. For this reason waste management is very important.

It is expected from the industrial plants that they must have industrial plant(ETP). So that their treated material(waste) which is released to the environment do not cause any harm to the environment components.

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