1.0 OBJECTIVE:
To determine the bioload ofbacteria ,yeast and molds and specific pathogens in raw material and finished products.
2.0 SCOPE
This SOP is applicable to Quality Control Department (Microbiology Lab).
3.0 RESPONSIBILITY :
Sr. Executive.
4.0 ACCOUNTABILITY :
Department Head
5.0 PROCEDURE:
5.1 SAMPLE PREPARATION:
5.1.1 Total Aerobic Microbial Count :
Use the plate method for substance that are sufficiently soluble or translucent to permit the use of method; otherwise, use the multiple tube method.
5.1.2 For Water Soluble Products :
Dissolve 10 gms or 10 mL of substance in case of Raw material and 5gm or 5ml of finished product being examined in 100 mL, of soyabean casein digest medium and fluid lactose broth.
A suitable surface active agents such as 0.1% w/v of polysorbate, may be added to assist the suspension of poorly wettable substances.
5.1.3 For Fatty Products :
Homogenize 10gm or 10 mL of the preparation being examined with 5 gms of polysorbate 20, or polysorbate80. If necessary, heat to not more than 40°C. Mix carefully. Add 85mL of buffered sodium chloride – peptone solution pH7.0 or any other suitable medium shown to have no antimicrobial activity like fluid soyabean casein digest medium and make up the volume 100ml. Temperature should not exceed more than 40°C. If necessary maintain this temperature for the shortest time for necessary formation of an emulsion and is any case for not more than 30 minutes. If necessary, adjust the pH around 7.0
6.0 Determination of total microbial count in raw materials :
PROCEDURE :
6.1 a) General procedure for total microbial count : (Bacteria and Fungi)
1.0 Transfer 1 mL of dilution of substance being examined aseptically into two sterile pertiplates having 9 to 10 cm in diameter, labeled on plate as ‘Bacteria’ and on other plate as ‘Fungi’.
Add 15 ml of freshly prepared, sterilized soyabean casein digest agar into one pertiplate labeled as ‘Bacteria’ and add 15 mL of sterile sabouraud dextrose agar into second pertiplate labeled as ‘Fungi’ and allow the plates to solidify at room temperatures. Prepare at least two such plates for bacteria and two for fungi. Incubate the bacterial plates at 37°C for 3 days and incubate the fungi plates at 20° to 25° C for 5 days. Count the colonies and calculate the results.
6.2 b) Membrane filtration :
6.2.1 Use membrane filter 50 mm in diameter and having a nominal pore size not greater than 0.45 µm the effectiveness of which in retaining bacterial has been established for the type of preparation being examined. Sterilized and assemble the filtration the filtration apparatus.
6.2.2 Transfer 10 mL or quantity of each dilution containing 1 gms of the preparation being examined to each of two membrane filters and filter immediately. If necessary, dilute the pretreated preparation so that a colony count of 10 to 100 may be expected. Wash each of about 100 ml, of a sterile 1% peptone solution, pH 7.0. for fatty substances add to the liquid polysorbate 20 or polysorbate 80. Transfer one of the membrane filters, intend for the enumeration of bacterial , to the surface of the plate of casein soyabean digest agar and the other, intended for the enumeration of fungi, to the surface of the plate of sabouraud dextrose agar.
6.2.3 Incubate the plates for 5 days, unless a more reliable count is obtained in shorter time, at 37°C in the test for bacterial and 20° to 25°C in the test for fungi. Count the number of colonies that are formed, calculate the number of micro-organisms per gms or per ml, of the preparation being examined.
7.0 Determination of pathogenic organism for raw materials and finished products :
Dissolve 10 gms / 10mL ( Incase of viscous material 5 gm/5ml) , refer current Standard Testing Procedure for aerobic microbial count and for pathogens.
Pseudomonas aeruginosa
To determine the bioload ofbacteria ,yeast and molds and specific pathogens in raw material and finished products.
2.0 SCOPE
This SOP is applicable to Quality Control Department (Microbiology Lab).
3.0 RESPONSIBILITY :
Sr. Executive.
4.0 ACCOUNTABILITY :
Department Head
5.0 PROCEDURE:
5.1 SAMPLE PREPARATION:
5.1.1 Total Aerobic Microbial Count :
Use the plate method for substance that are sufficiently soluble or translucent to permit the use of method; otherwise, use the multiple tube method.
5.1.2 For Water Soluble Products :
Dissolve 10 gms or 10 mL of substance in case of Raw material and 5gm or 5ml of finished product being examined in 100 mL, of soyabean casein digest medium and fluid lactose broth.
A suitable surface active agents such as 0.1% w/v of polysorbate, may be added to assist the suspension of poorly wettable substances.
5.1.3 For Fatty Products :
Homogenize 10gm or 10 mL of the preparation being examined with 5 gms of polysorbate 20, or polysorbate80. If necessary, heat to not more than 40°C. Mix carefully. Add 85mL of buffered sodium chloride – peptone solution pH7.0 or any other suitable medium shown to have no antimicrobial activity like fluid soyabean casein digest medium and make up the volume 100ml. Temperature should not exceed more than 40°C. If necessary maintain this temperature for the shortest time for necessary formation of an emulsion and is any case for not more than 30 minutes. If necessary, adjust the pH around 7.0
6.0 Determination of total microbial count in raw materials :
PROCEDURE :
6.1 a) General procedure for total microbial count : (Bacteria and Fungi)
1.0 Transfer 1 mL of dilution of substance being examined aseptically into two sterile pertiplates having 9 to 10 cm in diameter, labeled on plate as ‘Bacteria’ and on other plate as ‘Fungi’.
Add 15 ml of freshly prepared, sterilized soyabean casein digest agar into one pertiplate labeled as ‘Bacteria’ and add 15 mL of sterile sabouraud dextrose agar into second pertiplate labeled as ‘Fungi’ and allow the plates to solidify at room temperatures. Prepare at least two such plates for bacteria and two for fungi. Incubate the bacterial plates at 37°C for 3 days and incubate the fungi plates at 20° to 25° C for 5 days. Count the colonies and calculate the results.
6.2 b) Membrane filtration :
6.2.1 Use membrane filter 50 mm in diameter and having a nominal pore size not greater than 0.45 µm the effectiveness of which in retaining bacterial has been established for the type of preparation being examined. Sterilized and assemble the filtration the filtration apparatus.
6.2.2 Transfer 10 mL or quantity of each dilution containing 1 gms of the preparation being examined to each of two membrane filters and filter immediately. If necessary, dilute the pretreated preparation so that a colony count of 10 to 100 may be expected. Wash each of about 100 ml, of a sterile 1% peptone solution, pH 7.0. for fatty substances add to the liquid polysorbate 20 or polysorbate 80. Transfer one of the membrane filters, intend for the enumeration of bacterial , to the surface of the plate of casein soyabean digest agar and the other, intended for the enumeration of fungi, to the surface of the plate of sabouraud dextrose agar.
6.2.3 Incubate the plates for 5 days, unless a more reliable count is obtained in shorter time, at 37°C in the test for bacterial and 20° to 25°C in the test for fungi. Count the number of colonies that are formed, calculate the number of micro-organisms per gms or per ml, of the preparation being examined.
7.0 Determination of pathogenic organism for raw materials and finished products :
Dissolve 10 gms / 10mL ( Incase of viscous material 5 gm/5ml) , refer current Standard Testing Procedure for aerobic microbial count and for pathogens.
Pseudomonas aeruginosa
Escherichia
Salmonellae
Staphylococcus
ACCEPTANCE CRITERIA :
*-Total microbial count including bacteria and yeast and moulds.
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