1.0 OBJECTIVE : To laid down procedure for microbial limit test in for the estimation of the number of viable aerobic micro-organisms present and for detecting the presence of designated microbial species in the Raw materials and finished products.
2.0 SCOPE : This procedure is applicable for microbial limit test in raw materials, finish products for estimation of number of viable aerobic micro-organisms present and for detecting the presence of designated microbial species.
3.0 RESPONSIBILITY : Microbiologist, Q.C. In charge.
4.0 REQUIREMENTS: 70% I.P.A, 0.2µ micron filter, Petri plates, Culture media, Inoculums loop, Buffered sodium chloride-peptone solution pH 7.0, Autoclave, incubator, Kovac’s reagent
5.0 PROCEDURE :
5.1 PRECAUTIONS:
5.1.1 Mix the specified culture in the appropriate media at specific temperature.
5.1.2 Carry out all the inoculation and transfer procedure ascetically.
5.1.3 During mixing and pouring of media take care no air bubble should be formed.
5.1.4 Incubate the plates at specified time period and temperature in inverted position.
5.1.5 Keep LAF switched on at least 15 minutes before working.
5.2 DETERMINATION OF BACTERIAL COUNT:
5.2.1 After receiving upon the raw material or finish product for microbial limit test take the sample entry into the respective register of microbial limit test.
5.2.2 Prepare the specified media plates for total microbial count.
5.2.3 Pre-incubate these sterile prepared plates for 24 to 48 hours before using to check for contamination.
5.2.4 Bring all the autoclaved accessories to be used; in microbiological analysis area.
5.2.5 Disinfect hands with 0.2µ filtered 70% I.P.A. and wear sterilized hand glove.
5.2.6 Take an autoclaved Petri plate and weigh the 10 to 15g of sample in it (for solid sample) and keep it in clean polyethylene bag. Transfer it to microbial lab.
5.2.7 Take the weighed sample or 10ml of liquid sample in sterile buffered sodium chloride-peptone solution pH 7.0 and mix well. If necessary adjust the pH of solution to 7.0.
5.2.8 If inhibitory substances are present in the sample, then add 0.5% of soya lecithin and 4% of polysorbate 20 to the culture medium.
5.2.9 In case of water insoluble substances or fatty substances use polysorbate –80 to prepare solution.
5.2.10 Take 1ml of prepared sample solution in duplicate Petri plates and Pour about 15ml of liquefied sterile Soyabean Casein digest agar media cooled at about not more than 45° C.
5.2.11 Slowly rotate to mix the content of Petri plates and allow to solidify the media at room temperature for about 30minutes.
5.2.12 Prepare Positive control by Streaking one Soyabean Casein digest agar plate with Bacillus subtilis.
5.2.13 Prepare Negative control by mixing 1ml of sterile buffered sodium chloride-peptone solution pH 7.0 and 15ml of Soyabean Casein digest agar media as it is.
5.2.14 Keep all above prepared plates in an incubator at 30°C to 35°C for 2.-3 days in inverted position for Bacterial count.
5.2.15 After 2.-3 days incubation; count the number of colonies formed, and calculate the results using plates with the greatest number of colonies but taking 300 colonies per plate as the maximum consistent with good evaluation.
Calculation: Average cfu of bacteria x dilution factor.
5.3 DETERMINATION OF FUNGAL COUNT :
5.3.1 Proceed similarly as mentioned in bacterial count and take 1ml of prepared sample solution in duplicate Petri plates and Pour about 15ml of liquefied sterile sabouraud dextrose agar media cooled at about not more than 45° C.
5.3.2 Slowly rotate the plates to mix the content and allow solidifying the media at room temperature for 15-20 min.
5.3.3 Prepare Positive control by Streaking one sabouraud dextrose agar media plate with C. albicans ATCC 10231 and proceed as above.
5.3.4 Prepare Negative control by mixing 1ml of sterile buffered sodium chloride-peptone solution pH 7.0 and 15ml of sabouraud dextrose agar media as it is.
5.3.5 Keep all these prepared plates in an incubator at 20°C to 25°C for 5 days in inverted position for fungal count.
5.3.6 After 5 days incubation; count the number of colonies formed, and calculate the results using plates with the greatest number of colonies but taking 300 colonies per plate as the maximum consistent with good evaluation.
Calculation: Average cfu of fungus x dilution factor.
5.3.7 Record the result into the protocol for Microbial Limit Test.(Annex .).
5.4 TEST FOR ABSENCE OF PATHOGENS:
5.4.1 Tests for E. coli:
5.4.1.1 Prepare the sample dilution as described in ‘Determination of bacterial count’ but using lactose broth and incubate this tubes at 35°C to 37°C for 18 to 24 hours.
5.4.1.2 Shake the tube and transfer 1ml of this solution in 100ml of sterile Mac Conkey broth medium, and incubate at 36° to 38° C for 48 hours.
5.4.1.3 After incubation shake the tube and transfer loop full portion of Mac Conkeys broth on the surface of sterile Mac Conkey agar Petri plate.
5.3.1.4 For positive control proceeding same as above using E. coli organism instead of sample and streak a portion of on the surface of sterile Mac Conkey agar Petri plate
5.3.1.5 For negative control proceeding same as above except without sample or organism streak a portion of incubated sterile Mac Conkey broth medium on the surface of sterile Mac Conkey agar Petri plate.
5.3.1.6 Incubate these plates at 35° to 37°C for 18 to 72 hours in invert position.
5.3.1.7 After completion of incubation observe the plates the plates, Growth of red, non-mucoid colonies of gram negative rods, indicates the possible presence of E. coli.
5.3.1.7 This is confirmed by Indole test by Taking suspect colonies in 10ml of pre-sterilized peptone water, keep it at 37°C for 4 to 5 hours. Add 4 to 5 drops of Kovac’s reagent. Production of red coloured ring indicates positive test for E. coli.
5.3.1.8 If the above colony characters are not observed it indicates the absence of E. coli.
5.3.2 Tests for Salmonella
5.3.2.1 Proceed same as step-1 as described in E-Coli and transfer 1.0ml of the enrichment culture to each of two tubes containing 10ml of sterile Selenite F broth and sterile tetrathionate brilliant green bile broth.
5.3.2.2 Incubate these tubes at 36°C to 38°C for 48 hours.
5.3.2.3 From each of these two cultures subculture on at least two of the following four sterile agar media plates; bismuth sulphite agar, brilliant green agar, desoxycholate-citrate agar and xylose-tysine-desoxycholate agar.
5.3.2.4 Incubate these plates at 36º to 38º C for 18 to 24 hours.
5.3.2.5 After incubation examination the plates; if none of the colonies conforms to the description given in Table given below, the sample meets the requirements of the test for the absence for the genus Salmonella.

 
5.3.2.6 During observation, if the characteristic of the colony identifies as given in the table then perform the secondary test using triple sugar iron agar & Urea Broth.
5.3.2.7 In triple sugar- iron agar by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle, and at the same time inoculate a tube of urea broth.
5.3.2.8 Prepare the positive control by proceeding with above test using 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Salmonella abony (NCTC 6017) organisms, prepared form a 24-hour culture in nutrient broth, for the inoculation of the tubes sterile Selenite F broth and sterile tetrathionate brilliant green bile broth.
5.3.2.9 Incubate all these plates at 36º to 38º C for 18 to 24 hours.
5.3.2.10 The formation of acid and gas in the stab culture (with or without concomitant blackening) and the absence of acidity from the surface growth in the triple sugar iron agar, together with the absence of a red colour in the urea broth, indicate the presence of salmonellae.
5.3.2.11 If acid but no gas is produced in the stab culture, the identity of the organism should be confirmed by agglutination test.
5.3.3 Test for pseudomonas aeruginosa
5.3.3.1 Proceed same as step-1 as described in E-Coli and transfer 1.0ml of the enrichment culture to 100 ml of fluid soyabean-casein digest medium and mix well.
5.3.3.2 Incubate these tubes at 35°C to 37°C for 24 hours. Examine the medium form growth is present.
5.3.3.3 Streak a portion of the medium on the surface of Sterile Petri plates of cetrimide agar medium.
5.3.3.4 For Positive control proceed same as above method except in case of sample using Pseudomonas organism.
5.3.3.5 For Negative Control, keep one plate of cetrimide agar as it is proceeding with same method except without inoculation of sample and organism.
5.3.3.6 Incubate all these plates at 35º to 37º for 18 to 24 hours.
5.3.3.7 If upon examination, none of the plates contains colonies having the characteristics listed in Table given below for the media used, the sample meets the requirement for freedom from Pseudomonas aeruginosa.
5.3.3.8 If any colonies conforming to the description in Table given below are produced, carry out the oxidase and pigment tests.
5.3.3.9 For Oxidase Test Streak representative suspect colonies from agar surface of cetrimide agar on the surfaces of sterile plates of pseudomonas agar medium for detection of fluorescein and pseudomonas agar medium for detection of pyocyanin contained.
5.3.3.10 Over and invert the inoculated media and incubate at 33º to 37º for not less than 3 days.
5.3.3.11 Examine the streaked surfaces under ultra-violet light, to determine whether colonies
conforming to the description in Table are present.
5.5.12 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 colony.
5.5.13 If there is no development of a pink colour, changing to purple, the sample meets the requirements of the test for the absence of Pseudomonas aeruginosa.

5.3.4 For Staphylococcus aureus
5.3.4.1 Proceed as described under pseudomonas, Place the prescribed quantity in a sterile screw capped container, add 100ml. Soyabean casein digest Broth, shake and allow it to stand for 1 hour. Loosen the cap and incubate at 37°C for 24-48 hours.
5.3.4.2 Examine the medium for growth and if growth is observed, streak a portion of the enriched culture on sterile agar media Petri plates of vogel Johnson agar, Mannitol salt agar, Baird parker agar plates.
5.3.4.3 For Positive control proceed same as above method except instead of sample using Staphylococcus aureus.
5.3.4.4 For Negative control keep one plate each of vogel Johnson agar, Mannitol salt agar, and Baird parker agar plates as it is proceeding with same method except without inoculation of sample and organism.
5.3.4.5 Incubate all these plates at 35°C –37°C for 18 to 24 hours.
5.3.4.6 Identify the characteristic of the colony as given in table below. Sample meets the requirements for the absence of Staphylococcus aureus.
5.3.4.7 If growth occurs as described in table given below, then carry out the coagulase test.
5.3.4.8 Transfer representative suspect colonies from the agar surface of any of the media listed in Table given below to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without additives.
5.3.4.9 Incubate in water-bath at 37º C and examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours.
5.3.4.10 If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of Staphylococcus aureus.
S.No MEDIUM Characteristic Colony Morphology
1. Vogel Johnson agar Black surrounded by yellow zones.
2. Mannitol salt agar Yellow colonies with yellow zones.
3. Baird parker agar Black, shiny, surrounded by clear zones of 2 to 5 mm
5.3.4.11 Record the result into the protocol for Microbial Limit Test For pathogens. (Annex)
 
6.0 ABBREVATION:
FTGM : Fluid thioglycolate medium
SCDM : Soyabean casein digest medium
IPA : Iso propyl alcohol
LAF : Laminar air flow
SOP : Standard operating procedure
QC : Quality Control