REVIEW OF LITERATURE A good indicator of fecal wastes exists in higher numbers in the human intestine and feces. It should not be very threatened to humans, detectable in environmental water human fecal pollution is to be separated from animal pollution. . Industrial effluents are needs to be in check through different water quality control quality assurances on the water is very essential for their establishment.
The several water industries are undergoing the good check control for water practices. Waste water are effluents of degradation of water practices at several set up industries. Primary water treatment is very beneficial for out coming effluents of waste water that is relevance so to secondary water treatments that is considered as BOD are known as biological oxygen demand and DO that is considered as dissolved oxygen concentration Dissolved oxygen is the good amount of the total oxygen level present in the water sample. Several procedures are applied for calibration of both the DO and BOD.
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These are the good water practices for increasing the quality of water control and waste water properties check under standard practices. Waste water degrading from the industries are on the quality check control by several practices under consideration of analysis of waste water treatment. Industrial effluents are good sources of pollution to several aspects preventing the measures for their goos quality control is beneficialEXPERIMENT NO.1BASIC MICROBIOLOGICAL TECHNIQUES-:MEDIA PREPARATION1-PREPARATION OF NUTRIENT AGAR MEDIUMComposition of N.A. medium for 100 ml:Agar = 1.5 gNacl =0.
5gPeptone =0.5gBeef extract = 0.3 gD/W = 100 mlProcedure-:For 100 ml of nutrient agar media Firstly 100 ml of distilled water was poured in a flask.Weigh down the amount of peptone=0.
5g, beef extract=0.3g, Nacl 0.5g and agar =1.5g then added to 100 ml distilled water.It is made sure that all the above components dissolved properly.Adjust down the pH of medium to 7.0 by using the ph meter by adding acid HCL.
The medium were sterilized inside the autoclave at 121 C temperature and 15 lbs pressure in Pascal second for period of 45 minutes properly Autoclave was allowed to cool the prepared N.A. medium is placed inside the laminar air flow. PREPARATION OF SABARAUD DEXTROSE AGAR CompositionMycological peptoneAgarDextroseSteps involved in the preparation of SDA medium-:Firstly weighed down the correct composition of the SDA of 3.0 was weighed in the 100 ml of D/W.Then medium were added in the conical flask and dissolved in the required ml of distilled water.The solution was well mixed and required volume was made by adding distilled water.
The medium were sterilize by autoclaving at 15 psi , 121 C temperature for 15-20 minutes.PREPARATION OF POTATO DEXTROSE AGAR MEDIUMPotato Dextrose Agar-:Preparation of 100 ml Synthetic PDA medium-:3.9 g of PDA powder is used for making 100ml PDA medium as recommended.100 ml distilled water requires 3.9 g PDA.1000 ml distilled water requires 39g PDA.
100 ml distilled water = 39/1000ml *100 ml =3.9 g PDA in 100 ml.Steps involved in the preparation of 100 ml PDA medium-:3.9 g PDA powder was weighed under weighing balance. PDA was added to conical flask and dissolved in 100 ml distilled water.
The solution was boiled or heated on the heating plate between40-60 C.The medium was sterilized by autoclaving at 15 lbs psi pressure121 C temperature for 15-20 minutes.EXPERIMENT NO.2Aim-: To perform Gram staining of given bacteria and differentiate both Gram –positive and Gram negative bacteriaMaterials Required-:Single bacterial specimen of isolated E.
coli strainGram Crystal Violet with large transfer pipetteGram iodine with large transfer pipetteGram stain Decolourizer with large transfer pipetteSafraninGlass slide with cover slipProcedure-:A single glass slide is gently wash with distilled water and dry inside hot air oven for few minutes.A small drop of Specimen was placed on the clean glass slide.The sample was spread down over a large surface with loop forming thin filmThe suspension was just allowed to complete Air drySmear was heat fixed onto glass slideEntire area of bacteria was covered with Gram crystal violet and left at room temperature for 1 minute the slides was rinsed for 5 seconds under slow running water.The bacteria was covered with the Gram iodine and left at room temperature for 1 minute the slides was rinsed for 5 seconds under slow running waterThe Gram stain Decolourizer Solution was added drop wise until the blue –violet color no longer visible on the sampleEXPERIMENT NO.
3Aim- Lactophenol cotton blue staining /mounting of fungiRequirements-Fungal culture old 5-7 daysLacto phenol cotton blue in a dropper bottleCover slipBunsen burner 02 mounted needles70% ethanol MicroscopeProcedure-A drop of lacto phenol cotton blue was placed on a clean glass slideA small spore of fungus was transferred preferably and spore bearing structures into the drop using a flamed cooled needleThe materials was teased down very gently by using the 02 mounted needleThe stain was then mixed gently with the mould structureA cover slip was placed over the preparation taking care to avoid trapping air bubbles in the stainExamine specimen under the microscope of 40 X.EXPERIMENT NO. 4TO PERFORM STREAK –PLATE TECHNIQUE-Materials –24 to 48 hours nutrient broth cultures of bacterial strainSterile Nutrient agar platesInoculating loopBunsen burnerProcedure-: Single bacterial culture of isolated E. coli was picked for streaking.The streak plate labeled on the bottom with the name of specified organism to be incubated with a markerThe loop was sterilized by the right hand and introduced into the isolated culture plate Inoculums was placed on agar surface at the edge further and inoculums streaked from side to side in parallel lines across the surface of area.The rest of the agar surfaces are now used to complete the streakingThe streak plate was incubated at 37 C in an inverted position for 48 hrs of bacterial growthEXPERIMENT NO.5Aim-: Microbial count for RO Water plant Sample through Serial Dilution MethodCollection of sample :The waste water sample was collected from R.O unit manufacturing plant located at Nawabgunj, Kanpur city.
Materials Required-:Water SampleTest tubesPetri dishesSterile tips and micropipette70% ethanol0.5% saline solutionSCDM & PDA mediaProcedure-;Firstly prepare the 5 blank test tubes with 9ml 0.5% saline each tubes.
100 ml SCDM agar and 100ml PDA agar media was prepared according to the composition.These medium were sterilized in an autoclave at 121C, 15 psi pressure for 15- 20 minutes.6 Sterile Petri plates were taken and SoyabeanCasein Digest Agar Medium was poured in them under the aseptic condition inside laminar air flow 100ml SCDM media and 100 ml PDA media was also poured in sterile Petri plates. The plates were kept for solidifying then 5 test tubes were taken which were filled by the 9ml sterile distilled water and marked them as 10-1 .10-2 ,10-3 ,10-4 ,10-5 dilutions.1ml of the sample was mixed properly in 1st test tube micropipette from the beaker and was mixed to the test tube which was been marked as 10-2.containing the 9ml sterile waterEXPERIMENT N0.6AIM- To determine total coliform test of waste water sampleMaterials -:MacConkey Broth of 50 mlOne single sample of RO water sample100 ml flaskPREPARATION OF MACCONKEY BROTH OF 50 ML50 ml of MacConkey broth was prepared accordingly for one water sample in 100 ml flask.
Sterilized in an autoclavePROCEDURE-:After autoclaving broth was kept inside the Laminar air flow.Now the single water sample of 5 ml inoculated in Macconkey broth with the sterilized pipette and tip.After inoculation put down the conical flask inside the BOD incubator shaker for 2 days at 37 CObserved for growth after 2 days of incubationOBSERVATIONS AND RESULTS 1 .
PREPRATION OF DIFFERENT TYPES OF MEDIA Nutrient Agar, Potato Dextrose Broth and MacConkey Broth – Nutrient Agar 100ml prepared in 100 ml Distilled Water and PDA Agar is prepared in 100 ml D/W water.3068320182880-622935127000POTATO DEXTROSE AGAR MACCONKEY AGAR (100ML) (100ML)2. GRAM STAININGStain shows pink color indicating that it was gram negative1714560960RESULT – NEGATIVE3. FUNGAL STAINING LACTOPHENOL COTTON BLUE STAINING1714530480RESULT- Fungal Stain Examined at 40X4. RESULTS FOR STREAK PLATING ON SPECIMEN OF E. coli7048501968505.
Isolated bacterial colonies on SCDM and PDA plates from RO Water Plant Sample25946103054354933958255RESULT-: Separate Bacterial colonies was observed in 10-4 dilution CFU = No. Of colonies /Amount of plate*Dilution rateNo. of Bacterial Colonies= 6*10-4/0.1=6*10-5 CFU/g/ml.
6. Result for the presence of Coliform bacteria in RO Water plant Sample625475345440Sample 1- RO Water plantRESULT- No typical colonies appeared on MacConkey agar media. The water sample is Coliform negative.