1. OBJECTIVE
1.1. To define a procedure and steps to be carried out for the count of unsterile products.
2. PROCEDURE FOR ESCHERICHIA COLI AND SALMONELLA
2.1. Identification of Pathogenic Bacterial species:
2.1.1. Escherichia coli: Transfer 100 ml. sample to the membrane filter (0.45 microns) and filter the sample. After filtration transfer the membrane to the surface of the Mac-conkey agar and incubate 350C-370C for 18-72 hrs. The growth of red, non-mucoid colonies of gram-negative rods indicates the possible presence of E-coli. This is confirmed by the indole production. The sample passes the test if such colonies are not seen or if the confirmatory test is negative.
2.1.2. Salmonella: Filter the 100 ml. sample through the membrane filter (0.45 micron) and transfer the membrane in 100 ml. soybean casein digest broth homogenise 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.
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 in the table.
|
Medium |
Description of colony |
|
Brilliant green agar |
Small, transparent and
colourless, or opaque, pinkish or
white( frequently surrounded by a pink or red zone) |
|
Desoxycholate Citrate
agar |
Colourless and opaque, with or without black centres. |
|
Bismuth Sulphite agar |
Black or green |
|
Xylose - lysine
-desoxycholate agar |
Red with or without black
centre. |
Transfer separately a few of the suspect colonies to triple sugar iron agar in tubes, using surface and deep inoculation and at 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 colour 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 colour in urea broth. If acid but gas is produced in stab culture, the identity of the organisms should be confirmed by the agglutination test.
LIMIT : Absent / gm
2.1.3. PSEUDOMONAS: Take 1 gm/one ml in a sterile screw-capped jar containing 100 ml of cetrimide broth and incubate at 30 Deg. C - 32 Deg. C for 24 hours. Subculture on a plate containing a layer of cetrimide agar and incubate at 30 Deg. C - 32 Deg.C for 48 hours. apply oxidase test.
2.1.3.1. OXIDASE TEST: Place 2 or 3 drops of freshly prepared one per cent w/v solution of NNN'N' tetramethyl-p- phenylene diammonium dichloride on a piece of filter paper ( Whatman No.1) and smear with the suspect colony. If a purple colour is produced within 5 to 10 seconds, the test is positive.
Limit : Absent / gm
2.1.4 STAPHYLOCOCCUS AUREUS: Take 1 gm/1 ml of sample in a sterile screw-capped jar containing 100 ml of fluid casein digest medium and incubate 30 Deg. C - 32 Deg. C of 72 hours. Subculture on a plate containing a layer of mannitol salt agar medium and incubate at 30 Deg.C-32 Deg.C for 48 hours. Examine the resulting growth by gram's stain and apply the coagulase test. Gram-positive cocci( in clusters ), yellow colonies in colour and +ve coagulase test indicates the presence of Staphylococcus aureus.
2.1.4.1 COAGULASE TEST: Transfer suspect colonies from mannitol salt agar Surfaces in the tube containing 0.5 ml of mammalian, plasma, incubate in a water bath at 37 Deg.C, examine the tubes at three hours and subsequently at suitable intervals up to twenty-four hours. If coagulation in any degree is observed., the test is positive.
Limit : Absent / gm
END OF THE DOCUMENT
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