1. OBJECTIVE
1.1. To define a procedure and steps to be carried out for Potable water testing
2. PROCEDURE:
2.1. Total Microbial Count: Used petri dish 9 cm. in diameter, add to each dish 1 ml of the sample, add 15-20 ml. liquefied Soybean casein digest agar medium for bacteria and for fungi take 15-20 ml. liquefied Sabouraud dextrose agar with antibiotics at not more than 450C. Prepare for each medium at least to petri dishes for each label of dilution. Incubate the plates at 300C - 350C for bacteria and 200C -250C for fungi for four days. Take the arithmetic average of the counts and calculate the no. of CFU/ml.
2.2. Limit: Not more than 500 CFU/ml.
2.3. Identification of Pathogenic Bacterial species:
2.3.1.1. Escherichia coli: Transfer 100 ml. sample to the membrane filter (0.45 micron) and filter the sample. After filtration transfer the membrane on the surface of the Mac-conkey agar and incubate 350C-370C for 18-72 hrs. Growth of red, non-mucoid colonies of gram negative rods indicates the possible presence of E-coli. This confirmed by the indole production. The sample passes the test if such colonies are not seen or if the confirmatory test are negative.
2.3.1.2. Salmonella: Filter the 100 ml. sample through the membrane filter (0.45 micron) and transfer the membrane in 100 ml. soybean casein digest broth homogenize and incubate 300C-370C for 18-24 hrs. Transfer 1 ml. of the enrichment culture to 10 ml. of broth tetrathionate bile brilliant green. Incubate the tube at 350C- 370C for 18-24 hrs.
2.3.1.2.1.1. Subculture on at least two different agar chosen from the table given as under. Incubate the plates at 350C- 370C for 18-72 hrs. The probable presence of Salmonellae is indicated by the growth of the cultures on the media plates given as per the table.
|
Medium |
Description of colony |
|
Brilliant green agar |
Small, transparent and colorless,
or opaque, pinkish or white(
frequently surrounded by a pink or red zone) |
|
Desoxycholate Citrate
agar |
Colorless and opaque, with or without black centers. |
|
Bismuth Sulphite agar |
Black or green |
|
Xylose - lysine
-desoxycholate agar |
Red with or without black center. |
2.3.1.2.1.2. Transfer separately a few of the suspect colonies to triple sugar iron agar in tubes, using surface and deep inoculation and the same time inoculate a tube of urea broth. Incubate at 360C-380C for 18-24 hrs. The presence of Salmonellae is provisionally confirmed if in the deep inoculation but not in the surface culture, there is a change of color from red to yellow and usually a formation gas with or without production of hydrogen sulphide in the agar and with the absence of red color in urea broth. If acid but gas is produced in stab culture, the identity of the organisms should be confirmed by agglutination test.
2.3.1.3. Pseudomonas aeruginosa: Take 100 ml. sample filter by membrane filter (0.45 micron), transfer the membrane filter to 100 ml. soybean casein digest broth, homogenize and incubate at 350C- 370C for 24-48 hrs. Examine the medium for growth and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of Cetrimide agar, incubate at 350C- 370C for 18-24 hrs. If upon examination none of the plate contains colonies heaving the characteristics as per the table given below.
|
S.N. |
Medium |
Characteristics
colonial morphology |
Fluorescence
in UV light |
Oxidase
test |
Gram
Stain |
|
1. |
Cetrimide agar |
Generally greenish |
Greenish |
Positive |
Negative rods |
|
2. |
Pseudomonas agar medium for detection of
fluorescein |
Generally colorless to yellowish |
Yellowish |
Positive |
Negative rods |
|
3. |
Pseudomonas agar medium for detection of
pyocyanin |
Generally greenish |
Blue |
Positive |
Negative rods |
2.3.1.3.1.1. The test specimen meets the requirements for freedom from Pseudomonas aeruginosa. If any colony confirming to the description as per table, carry out the oxidase test and pigment test.
2.3.1.3.1.2. Streak the representative suspect colonies from the agar surfaces to Pseudomonas agar medium for detection of fluorescein and Pseudomonas agar medium for detection of pyocyanin contained in petri dishes. Cover and invert the inoculated media and incubate at 330C- 370C for not less than three days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies confirming to the description in the table given as above. If growth of suspect colonies occurs, place 2 or 3 drops of a freshly prepared 1 % w/v solution of N, N, N1, N1, Tetramethyl-4-phenylenediamine dihydrochloride on filter paper and smear with the colony; if there is no development of a pink color changing to purple, the sample meets the requirements of the tests for the absence of Pseudomonas aeruginosa.
2.3.1.4. Staphylococcus aureus: Take 100 ml. sample filter by membrane filter (0.45 micron), transfer the membrane filter to 100 ml. Soya bean casein digest broth, homogenize and incubate at 350C- 370C for 24-48 hrs. Examine the medium for growth and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of Vogel Johnson agar or Baird Parker agar or Mannitol Salt agar incubate at 350C- 370C for 18-24 hrs.
2.3.1.4.1.1. If upon examination of the incubated plates, none of them contains colonies having the characteristics as per above table for the media, the sample meets the requirements for the absence of S. aureus. It growth occurs, carry out the coagulase test. Transfer suspect colonies from the agar surface of any of the media listed in above table to individual tube, each containing -0.5 ml. mammalian plasma with or without additives, incubate in water bath at 370 C. Examining the tubes at 3 hrs and subsequently at suitable intervals up to 24 hrs. If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of S.aureus.
2.3.1.4.1.2. Limit for all Pathogens: Absent per 100/ml.
END OF THR DOCUMENT
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