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. 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 beneficial
BASIC MICROBIOLOGICAL TECHNIQUES-:
1-PREPARATION OF NUTRIENT AGAR MEDIUM
Composition of N.A. medium for 100 ml:
Agar = 1.5 g
Beef extract = 0.3 g
D/W = 100 ml
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
Steps 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 MEDIUM
Potato 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.
Aim-: To perform Gram staining of given bacteria and differentiate both Gram –positive and Gram negative bacteria
Single bacterial specimen of isolated E. coli strain
Gram Crystal Violet with large transfer pipette
Gram iodine with large transfer pipette
Gram stain Decolourizer with large transfer pipette
Glass slide with cover slip
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 film
The suspension was just allowed to complete Air dry
Smear was heat fixed onto glass slide
Entire 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 water
The Gram stain Decolourizer Solution was added drop wise until the blue –violet color no longer visible on the sample
EXPERIMENT NO. 3
Aim- Lactophenol cotton blue staining /mounting of fungi
Fungal culture old 5-7 days
Lacto phenol cotton blue in a dropper bottle
02 mounted needles
A drop of lacto phenol cotton blue was placed on a clean glass slide
A small spore of fungus was transferred preferably and spore bearing structures into the drop using a flamed cooled needle
The materials was teased down very gently by using the 02 mounted needle
The stain was then mixed gently with the mould structure
A cover slip was placed over the preparation taking care to avoid trapping air bubbles in the stain
Examine specimen under the microscope of 40 X.
EXPERIMENT NO. 4
TO PERFORM STREAK –PLATE TECHNIQUE-
24 to 48 hours nutrient broth cultures of bacterial strain
Sterile Nutrient agar plates
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 marker
The 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 streaking
The streak plate was incubated at 37 C in an inverted position for 48 hrs of bacterial growth
Aim-: Microbial count for RO Water plant Sample through Serial Dilution Method
Collection of sample :The waste water sample was collected from R.O unit manufacturing plant located at Nawabgunj, Kanpur city.
Sterile tips and micropipette
0.5% saline solution
SCDM & PDA media
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 water
AIM- To determine total coliform test of waste water sample
MacConkey Broth of 50 ml
One single sample of RO water sample
100 ml flask
PREPARATION OF MACCONKEY BROTH OF 50 ML
50 ml of MacConkey broth was prepared accordingly for one water sample in 100 ml flask.
Sterilized in an autoclave
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 C
Observed for growth after 2 days of incubation
OBSERVATIONS 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.
POTATO DEXTROSE AGAR MACCONKEY AGAR
2. GRAM STAINING
Stain shows pink color indicating that it was gram negative
RESULT – NEGATIVE
3. FUNGAL STAINING LACTOPHENOL COTTON BLUE STAINING
RESULT- Fungal Stain Examined at 40X
4. RESULTS FOR STREAK PLATING ON SPECIMEN OF E. coli
Isolated bacterial colonies on SCDM and PDA plates from RO Water Plant Sample
Separate Bacterial colonies was observed in 10-4 dilution
CFU = No. Of colonies /Amount of plate*Dilution rate
No. of Bacterial Colonies
6. Result for the presence of Coliform bacteria in RO Water plant Sample
625475345440Sample 1- RO Water plant
RESULT- No typical colonies appeared on MacConkey agar media. The water sample is Coliform negative.