industrialization and human activities have totally turned our environment to dumping sites for wast

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

http://en.wikipedia.org/wiki/File:Antibiotic_resistance.svg

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 β-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 μg), were purchased from Becton Dickinson and Company, USA. Ciprofloxacin (5 μg),

cotrimoxazole (25 μg), tetracycline (30 μg) and gentamicin (120 μg), nitrofurantoin (30μg)

were from Oxoid Ltd. UK. Nalidixic acid (30 μg). Erythromycin were from Mast

Diagnostics, UK.

3.1 Materials

Table-I Antibiotic discs used in the study:

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 μl) 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.

Sl. No. Antibiotic Letter Code Quantity Source

1 Amoxicillin A 30 μg Becton Dickinson, USA

2 Ciprofloxacillin CIP 5 μg Oxoid Ltd., UK

3 Cotrimoxazole SXT 25 μg Oxoid Ltd., UK

4 Gentamicin CN 120 μg Oxoid Ltd., UK

5 Imipenem IMI 10 μg Mast Diagnostics, UK

6 Nalidixic acid NA 30 μg Mast Diagnostics, UK

7 Nitrofurantoin NI 300 μg Oxoid Ltd., UK

8 Tetracycline TE 30 μg Oxoid Ltd., UK

9 Cephalexin CFX 30 μg Oxoid Ltd., UK

10 Penicillin G PG 10 unit Mast Diagnostics, UK

13 Erythromycin E 15 Mast Diagnostics, UK

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 thermometer.

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 Testa) 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) Testa) 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 Testa) 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-1Source DO BOD COD PH TemperatureWaste 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-2Specimen 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 Supplementedphenol

Nacl-10.gmTrepton-10.gmPhenol-10gmDistilled 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, E9H3, H4, H7

Table No-4.Media Compositio

n 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, E9H3, 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-5Isolates Code Gram Reaction Call

MorphologyA1 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-6Isolates Presence of sporeA1 Non sporeA3 Non sporeA7 Non sporeE2 Non sporeE5 Non sporeE6 Non sporeE9 Non sporeH3 Non sporeH4 Non sporeH7 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


Raised colonies

Spread growth

No growth

H3 Abundant, opaque golden growth


No growth No growth Yellow shiny colonies

4.1.7 Biochemical characteristics.

Sample

Isolate no.

Name of Biochemical Tests

Isolation no:

Lactose

Dextrose

Sucrose

TSI MIO MR

VP

Indole

Citrate

Urease

NitrateReduction

Cata la sa

Oxida se

PresumptiveOrganism

Name of biochemical tests

IsolationNo:

Lactose

Dextrose

Sucrose

TSI

MIO

MR

VP

Indole

Citrate

Urease

NitrateReduction

Cata la sa

Oxida se

PresumptiveOrganism

A1 AG

AG

AG

A.S/ A.B H2S-

M+

O+ - + - + - + + - Enterobacter spp

A3 - A AA.S/ A.BH2S-

M-

O+ - + - - - + + - Bacillus spp

A7 - AG

AG

A.S/ A.BH2S+

M+

O+ + - + - + + + - Proteus spp

E2 AG

AG A

A.S/A.BH2S-

M-

O+ + - + - - + + - Escherichia coli

E5 - - -No Change

M+

O+ - - - + - + + - Pseudomonas spp.

E6 - A AA.S/A.BH2S-

M-

O+ - + - - - + + - Bacillus spp

E9 AG

AG

AG

A.S/A.BH2S-

M-

O+ - - - + + + + - Klebsiella spp

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 organismsstudied 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 codeA1 H4Sensitive(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 25Cefalexin R S 22Tetracycline I 13 S 18Gentamycin R S 20Cotrimoxazole R S 30Ciprofloxacin R S 25Erythromycin R RNalidixic acid R I 13Nitrofurantoin

Antibiotic sensitivity test for Bacillus spp

Name of Antibiotic

Isolation code Isolation codeA3 E6Sensitive(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 RCefalexin R RTetracycline R RGentamycin RCotrimoxazole R RCiprofloxacin R RErythromycin R RNalidixic acid R RNitrofurantoin S 20 R

Antibiotic sensitivity test for Proteus spp

Name of Antibiotic

Isolation codeA7Sensitive(S)

Intermediate(I)

Resistant(R)

(S) Zone size (mm) (I) Zone size

(mm) (R)

Amoxicillin RCefalexin RTetracycline R

Gentamycin S 15Cotrimoxazole RCiprofloxacin RErythromycin RNalidixic acid RNitrofurantoin S 20

Antibiotic sensitivity test for Escherichia coli

Name of Antibiotic

Isolation codeE2Sensitive(S)

Intermediate(I)

Resistant(R)


(mm) (R)

Amoxicillin RCefalexin RTetracycline RGentamycin S 17Cotrimoxazole RCiprofloxacin RErythromycin RNalidixic acid RNitrofurantoin R

Antibiotic test for Klebsiella spp

Name of Antibiotic

Isolation codeE9Sensitive(S)

Intermediate(I)

Resistant(R)


(mm) (R)

Amoxicillin S 23Cefalexin S 25Tetracycline S 20Gentamycin S 18Cotrimoxazole RCiprofloxacin RErythromycin S 23Nalidixic acid RNitrofurantoin I 14 R

Antibiotic sensitivity test for Pseudomonas spp

Name of Antibiotic

Isolation code Isolation codeE5 H7Sensitive(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 RCefalexin S 30 RTetracycline S 20 RGentamycin S 21 RCotrimoxazole S 20 RCiprofloxacin S 27 RErythromycin S 25 RNalidixic acid R RNitrofurantoin S R

Antibiotic sensitivity test for Staphylococcus aureus

Name of Antibiotic

Isolation codeH3Sensitive(S)

Intermediate(I)

Resistant(R)

(S) Zone size (I) Zone size (R)

(mm) (mm)Amoxicillin RCefalexin RTetracycline RGentamycin RCotrimoxazole RCiprofloxacin I 12Erythromycin RNalidixic acid I 14Nitrofurantoin 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 M-O+

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 worldAs 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.In my present studies, I took sample from ACME Laboratories the sample is taken from both raw west water and ETP.

A total number of 39 bacterial strains were isolated from the waste water reservoir The samples were collected from surface layer, deep layer and sediment. with cell morphology, staining, biochemical tests were performed for identification of the isolates at genus level and 10 isolates were identified. A number of pathogenic organisms were detected from the raw waste water they are Enterobacter spp, Bacillus spp, Proteus spp, Escherichia coli, Pseudomonas spp, Klebsiella spp, Staphylococcus spp,.

When the isolated organisms were tested their reaction to certain antibiotics a tremendous picture was focused. Pseudomonas and bacillus were totally resistant to normal antibiotics. Staphylococcus aureus were moderately resistant, Escherichia coli was getting multi drug resistance, proteus was superbug and emerging resistance was found in klebsiella.All of the isolates are dangerous not only for human but also for other living creatures.Proteus infections of seven days can lead for death. It is responsible for kidney failure and infection.Staphylococcus aureus cause weaken immunity to the human body.Escherichia coli is generally opportunistic and very severe. This infection rise when body’s defense mechanism faces many other strong infections.Bacillus and klebsiella spp cause various types of respiratory problems not only in human beings but also in other birds and fishes.We can easily imagine how dangerious the matter could be if they persist in our ecosystems, because if one component of ecosystem is in problem the whole system will suffer as each component is crucially dependent on the other components.

Then second sample from ETP when tested gave satisfactory result. it contain a very low level of microorganisms which is very normal. it includes a little amount of coliforms which is safe for environment. Simple analysis result was also satisfactory. the pH, temperature, turbidity, was of enough standard. The values were ph- 7.3, temperature 24.80 C, BOD 23.85mg/L, COD 19.22mg/L, DO 6.61mg/L.So we can say that if the treated water is released in nature it will not be harmful to environment.This comment was done on the basis of microbiological tests of the treated water.The same picture has been confirmed when the treated water reservoir was visited the natural scenario was excellent. There were phytoplanktons in the upper layer of the pond. The water was enough transparent and also live fishes were seen in the pond.So it is easily understood that if the water is released to outside river or land the water will not do any harm to environment.As ACME Laboratories is one of the most reputed ,largest and leading pharmaceuticals of Bangladesh, expectations are high to do their duties and responsibilities to the society and country.It is a matter of great satisfaction that they are performing their social responsibilities very sincerely.

Conclusion:

In this era of industrialization it is the demand of time to implement ETP in full force . it will be a matter of joke if we ignore this. But it is a matter of sorrow that most industries do not have ETP. The waste from the industries are directly thrown away to nature and the waste pollutes water source i.e. rivers, lakes, agricultural lands. The fishes are contaminated from the polluted water source. It causes various lethal disease to human .It creates an unbearable damage to the environment .This mistreatment is one of the most responsible factors of increasing global warming. This is a punishable crime. Again untreated waste cause sterility to the agriculture land slowly.So we can say thanks to the authority of ACME Laboratories as they have taken the right decision to save the nature. Other should take this important decision within time in hand. Only we i.e. the humans have the awareness and should take steps can save the environment from destruction.Our government should take more necessary steps to check whether ETP is running within the industry or not and what is the efficiency level of ETPs.

The above study was performed in a small scale of time with just two samples from an industry. A more and deep study is suggested with more samples from different industry.

References

Atlas RM. Principles of Microbiology. Missouri, St. Louis, USA: CV Mosby Company, 491 (1995).

ASM COLLOQUIUM REPORT (1999) Antimicrobial Resistance, An Ecological Perspective. American Society for Microbiology, Washington DC, pp. 1–14.

Ahmad, S. & Yadava, J.N.S. 1979 Rapid detection of b-lactam antibiotic resistance among clinical isolates of Escherichia coli. India Veterinary Medical Journal 3, 256–259.

Ajayi, S.O. & Osibanjo, O. 1981 Pollution studies on Nigerian Rivers. II: water quality of some Nigerian rivers. Environmental Pollution. (Series B) 2, 87–95.

American Public Health Association. 1985 Standard methods for the examination of water and waste water, 16th edn. 1268 pp. Washington DC: APHA Inc. ISBN 0-87553131-8.

Bakare, A.A. & Oyedeji, S.I. 2001 Genotoxicity of leachates from a rural waste dump using two bioassays. Journal of Nigerian Society for Experimental Biology 1 11–21.

Bakare, A.A., Lateef, A., Amuda, O.S. & Afolabi, R.O. 2003 The Aquatic toxicity and characterization of chemical and microbiological constituents of water samples from Oba River, Odo-oba, Nigeria. Asian Journal of Microbiology, Biotechnology and Environmental Sciences 5, 11–17.

Bauer, A.W., Kirby, W.M.M., Sherris, J.C. & Turck, M. 1966 Antibiotic susceptibility testing by a standard single disc diffusion method. American Journal of Clinical Pathology 45, 493–496.

Buchanan, R.M. & Gibbons, N.E. (eds). 1974 Bergey’s manual of determinative bacteriology. 8th edn. Baltimore: The Williams and Wilkins Company. ISBN 0-68301117-0.

Cappuccino, Sherman; Microbiology, A Laboratory Manual, Seventh edition

Chukwura, E.I. & Okpokwasili, G.C. 1997 Impact of Brewery Wastewater on Recipient Aquatic Environments. In Biotechnology for Development in Africa: Proceedings of an International conference organized by Foundation for African Development through International Biotechnology (FADIB) held at Enugu, Nigeria, 9–13 Feb, 1997, eds. Okafor, N., Okereke, G., Miambi, E. & Odunfa, S. pp. 225–233. Enugu: Ochumba Press Ltd. ISBN 978 2791 19-9.

"Drug Resistant Infections: Riding Piggyback". The Economist. November 29, 2007. http://www.economist.com/displaystory.cfm?story_id=10205187&fsrc=RSS.

E Jawetz, JL Melnick, EA Adelberg (1987) Enteric Gram negative rods in review of Medical Microbiology 17th Edition-Appleton and Lange Norwalk, Connecticut/ LosAltos,California USA.Pp 233-235.

The Journal of Antibiotics 63, 423-430 (August 2010) | doi:10.1038/ja.2010.62

Goossens H, Ferech M, Vander Stichele R, Elseviers M (2005). "Outpatient antibiotic use in Europe and association with resistance: a cross-national database study". Lancet 365 (9459): 579–87. doi:10.1016/S0140-6736(05)17907-0. PMID 15708101.

Golam Mohammed 2002, Removal of organic wastes from effluent of pharmaceuticals industries by photo chemical degradation on TiO2 and also by adsorption, filteration on hydrate ferric oxide (HFO) and manganese hydroxide coated porous and Mesoporous silicate and aluminates surface in Environmental Research Laboratory Department of chemistry Jahangirnagar University, Saver, Dhaka,Bangladesh.Pp 46, 75.

Gerad J. Tortora, Berdell R. Funke, Chemisty L. case (1998) cell wall and the Gram stain mechanism in Microbiology an introduction 6th edition-Addision Wesly Longman, Inc.California, USA. Pp 234-235.

McARTHUR JV and TUCKFIELD RC (2000) Spatial Patterns in Antibiotic Resistance among stream bacteria: Effects of industrial pollution. Appl. Environ.Microbiol. 66 (9) 3722–3726. Michel J.Pelczer, Jr, E.C.S Chan and Noel R.Kriez (1998) Aquatic Microbiology, in Microbiology, 5th edition, McGraw-Hill company, Toyo, Paris, Pp 569-576.

M Alamgir Hossin, ANM Fakhruddin, Sirajul Islam Khan. BGMS. Volume 13, Number 01, March 2007. Article of the public Health at Stake by the Water pollution of peripheral river System of Dhaka. Bangladesh Journal of Medical science.Pp 44-46.

N Nahar, ANM Fakhruddin and H Rashid.Volume 21,Number 2, December 2004, Bangladesh Journal of Medical science. Aromatic Hydrocarbon-Degrading Bacteria in Industrial Chemical Water Contaminated Sites Near Dhaka City. Pp68-71.

http://en.wikipedia.org/wiki/PubMed_Identifier

http://en.wikipedia.org/wiki/Digital_object_identifier

James G.Cappuccion, Natalie Sherman (1996) MR-VP test in Microbiology A Laboratory Manual, 7th Edition, Addition-wesley, Tokyo, Japan, Pp 163-164.

R.C. Dubey, D.K. Maheshwari (2002 Practical Microbiology,1st edition.India.

Susan Sudavary, Maryadele J. O Neil, Ann Smith, Patricia E. Heckelman, An Encyclopedia of chemicals, Drugs, And Biological, in the Merck Index, 11th edition. Merck and CO, Inc. Rahway, Newjersey, USA.Pp 91, 303,820,1193, 1521, 954, 593, 11, 387, in Serial Nos of 58,610, 1971,2414,3713,3716,5095,5096,5942, 7482,9581.

WHO(January 2002). "Use of antimicrobials outside human medicine and resultant antimicrobial resistance in humans ". World Health Organization. http://www.who.int/mediacentre/factsheets/fs268/en/index.html

WHO in standard operating procedure, Guidelines for Drinking water quality 2nd edition Volume 1,2,3. World Health Organization, Geneva Pp 51-72.

Appendix-1

Composition of the media used:

Nutrient Agar Grams/Liter

Peptone 5.0 Bacto beef extract 3.0 NaCl 5.0 Agar 15.0 Distilled water 1000 mlPH 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Nutrient Broth Grams/Liter

Peptone 5.0NaCl 5.0Beef Extract 1.5Yeast Extract 1.5PH 7.4Distilled water 1000 ml


Mac Conkey agar Grams/Liter

Bacto Peptone 17.0Proteas Peptone 3.0 Lactose 10.0Bile Salt 1.5Agar 15.0Neutral red 0.03Crystal violet 0.001Distilled water 1ooomlP H

7.1 Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Eosine methylene blue (EMB) agar Gram/Liter

Peptone 10.0Lactose 10.0K2HpO4 2.0Eosin 0.4Methylene blue 0.065Agar 20.0Distilled water 1000mlPH 6.8Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Blood Agar Gram/Liter

Tryptone 14.0Peptone 4.5Yeast extract 4.5NaCl 5.0Agar 15.0Distilled water 1000mlPH 7.3


KF Streptococcal (KFSA) Agar Gram/Liter

Protease Peptone 10.0Yeast Extract 10.0NaCl 5.0Sodium Glycerophosphate 10.0Maltose 20.0Lactose 1.0Sodium Azide 0.4Brom cresol purple 0.015Agar 20.0PH 7.2


Trepton- Sodium (T1-N1) Broth Gram/Liter

Trypticase 10.0NaCl 10.0Distilled water 1000mlPH 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes

Mueller Hinton Agar Gram/Liter

Beef infusion 2.0Bacto casamino acid (technical) 17.5Starch 1.5Bacto agar 17.5Distilled water 1000mlPH 7.3


Mueller Hinton Broth Gram/Liter

Beef, infusion from 300Casein Hydrolysate (Acid) 17.5Soluble Starch 1.5PH 7.4


Sodium Chloride Broth Gram/Liter

Beef extract 0.1NaCl 0.375Distilled water 1000ml

Sterilized at 1210C under 151b/in2 pressure for 15 minutes. Endo Agar Gram/Liter

Peptone 10.0Lactose 10.0Dipotasssum phosphate 3.5Sodium sulphat 2.5Basic Fuchsin 0.5Agar 15.5PH 7.5


Mannitol Salt Agar Gram/Liter

Proteas peptone 10.0Beef extract 1.0D-Mannitol 10.0NaCl 75.0Phenol red 0.025Agar 20Distilled water 1000ml


Normal Saline Gram/Liter

NaCl 0.85Distilled water 1000ml

Autoclaved at 1210C for 15 minutes.

APPENDIX-II

Composition of the media used in biochemical test

MR-VP broth Gram/LiterPeptone 7.0Dextrose 5.0Dipotassium phosphate 5.0Distilled water 1000mlPH 6.9


Triple Sugar Iron (TSI) Agar Gram /Liter

Peptone 10.0Tryptone 10.0Yeast Extract 3.0Lactose 10.0Saccharose 10.0Dextrose 1.0Ferrous Sulphate 0.2Sodium Chloride 5.0Sodium Thiosulphate 0.3Phenol Red 0.024Agar 12.0

PH 7.4Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Simmons citrate Agar Gram/Liter

Magnesium sulphate 0.2Manoammonium phosphate 1.0Dipotassium phosphate 1.0Sodium citrate 2.0Sodium Chloride 5.0Agar 15.0Brom-Thymol Blue 0.08PH 6.8


Urea broth medium Gram/Liter

Urea 20.0Yeast extract 0.1KH2PO4 9.0K2HPO4 9.5Phenol red 0.01Distilled water 1000mlPH 6.8Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Indol tryptopon broth medium Gram/Liter

Tryptone 10.0Distilled water 1000ml


Carbohydrate fermentation media Gram/Liter

Mannitol l5.0Beef extract 1.0Peptone 10.0NaCl 5.0Bromocresol purple 0.015Distilled water 1000PH 6.8 Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Nitrate broth Gram/Liter

Peptone 5.0

Beef extract 3.0NaCl 5.0Potassium nitrate 1.0Agar 1.0Distilled water 1000mlPH 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes. APPENDIX-III

Composition of chemicals and reagents

Crystal violet

Solution-ACrystal violet (90% dye content) 2.0gEthyl alcohol (95%) 20.0mlSolution-BAmmonium oxalate 0.8Distilled water 80.0 ml

Note-Mix the solution A and B

Gram’s iodineIodine 1.0gPotassium iodide 2.0gDistilled water 300.0ml

Ethyl alcohol (95%)

Ethyl alcohol (100%) 95.5mlDistilled water 5.0ml

Safranin

Safranin O 0.25mlEthyl alcohol (95%) 10.0mlDistilled water 100.0ml

Methyl red solution

Methyl red .04gEthanol 40gDistilled water 100mlMethyl red dissolved in ethanol and diluted water.

Malachite green

Malachite green 5.0gDistilled water r 100ml

Kovac’s reagent (for detection of indole)

P-Dimethylaminobenzaldehyde 5.0gAmyl alcohol 75.0mlHydrochloric acid (concentrated) 25.0ml

concentrated P-Dimethylaminobenzaldehyde was dissolved in the amyl alcohol and HCl was added slowly.

Barrit’s reagentSolution-Aα-naptho l5.0gEthanol(Absolute) 95.0g

α-naptho was dissolved in ethanol with constant stirring.

Solution-B

KOH 40.0gCreatine 0.3gDistilled water 100ml

Hydrogen peroxide

3% aqueous solution of H2O2 was prepared from the H2O2 absolute solution.