MLT: Microbial Limit Test

1.0 Equipment Required

LAF
Filtration Assembly
Sterile 0.45 micron membrane filter
Sterile Pipettes – 1 ml and 10 ml

2.0 Material Required

Sterile 0.1% w/v peptone 3 X 100 ml
70% IPA solution
Sample for testing
SCDA and SCDA plates
SDA and SDA plates
SCDM 3 X 100 ml

3.0 Procedure

3.1 Total Aerobic Microbial Count

3.1.1 Collect the sample to be tested for the microbial limit test as per sampling plan.
3.1.2 Use specified quantity of sample for each of the test specified in the individual monograph and pre-treat the sample as the following method-
3.1.3 Water Soluble Product: Dissolve 10 gm or dilute 10 ml of sample, unless otherwise specified, in sterile 90 ml peptone water or buffered sodium chloride-peptone solution pH 7.0
3.1.4 Product Insoluble In Water: Suspend 10 gm or 10 ml sample, unless otherwise specified, in sterile 90 ml peptone water with 0.1% polysorbate 80 or buffered sodium chloride-peptone solution pH 7.0
3.1.5 Fatty Products: Homogenize 10 gm or 10 ml of the sample, unless otherwise specified, with 5 g of polysorbate 80. If necessary heat to not more than 40°C. Mix carefully while maintaining temperature on the water bath. Add 85 ml of sterile peptone water or buffered sodium chloride-peptone solution pH 7.0

3.2 Examination of sample by Membrane Filtration Method

3.2.1 Aseptically connect the rubber tube of sterile manifold to receiver tank and receiver tank rubber pipe to vacuum pump.
3.2.2 Using sterile smooth tip forceps, place a 47 mm diameter 0.45m sterile membrane filter on the center of the filter support screen. Without disturbing the filter, place the funnel on top of the filter holder base.
3.2.3 Separately transfer 10 ml of pretreated sample from step no. 3.1.3 to each two 90 ml of sterile 0.1% peptone water. Mix well and transfer the whole quantity of dilution to each of two membrane filters and filter immediately.
3.2.4 Wash both the membrane filter with each 3 x 100 ml of sterile 0.1% peptone water into the filtration funnel and filter under partial vacuum
3.2.5 After completion of filtration process, shut off the vacuum with the help of vacuum control key.
3.2.6 Transfer one of the membrane filters, intended for the enumeration of bacteria, to the surface of the plate containing SCDA and other, intended for the enumeration of fungi, to the surface of the plate of SDA.
3.2.7 For positive control carry out the same procedure in duplicate except for sample use 100 ml fluid A inoculated with 100 bacterial cells and another 100 ml fluid A is inoculated with c.albicans intended for identification of total aerobic and fungal count respectively.
3.2.8 For negative control carry out the same procedure except for sample use 100 ml sterile 0.1% peptone water.
5.3.2.9 Incubate the plates for 5 days unless a more reliable count is obtained in shorter time, at 30 to 35°C in the test of bacteria and 20 to 25°C in the test for fungi.
3.2.10 Count the number of colonies that are formed. Calculate the number of cfu per gram or per ml of the sample being examined.

3.3 Examination of sample Plate Count Method

3.3.1 Use this method for fatty products and product insoluble in water.
3.3.2 Use 90 mm sterile Petri plate. Take a four-Petri plate and label two plates for bacteria and remaining two for fungi count. Transfer 1 ml quantity of each pretreated dilution sample solution to each of four Petri plates.
3.3.3 Add 15 ml of sterile liquefied SCDA at not more than 45°C, into two plates labeled for bacterial count.
3.3.4 Then add 15 ml of sterile liquefied SDA at not more than 45°C, into two plates labeled for the fungal count.
3.3.5 Allow to solidify the plates at room temperature, invert and incubate at 30 to 35°C for 5 days and 20 to 25°C for 5 days respectively.
3.3.6 Count the number of colonies that are formed. Calculate the number of cfu per gram or per ml of the sample being examined.

3.4 Test For Specified Microorganisms

3.5 For Membrane Filtration Method

3.5.1 Follow the same procedure described under 3.2.1 to 3.2.5, transfer one of the membrane filter, intended for enrichment of E. coli and Salmonella to a tube containing 100 ml of sterile nutrient broth, and other membrane intended for enrichment of Pseudomonas aeruginosa and Staphylococcus aureus, to a tube containing 100 ml of sterile Soybean casein digest medium.
3.5.2 For positive control carry out the same filtration procedure in duplicate except for sample use 100 of peptone water inoculated with approx 100 cells of E. coli or Salmonella and another 100 ml of peptone water inoculate with Staph. aureus or Ps. aeruginosa and transfer the membrane to 100 ml of sterile nutrient broth and soybean casein digest medium respectively.
3.5.3 For negative control carry out the same procedure except for sample use 100 ml of sterile peptone water for both the tubes.

3.6 For Plate count method (Direct Inoculation)

3.6.1 Use this method for Fatty products and Product insoluble in water.
3.6.2 Transfer separately 1 ml quantity of pretreated sample from step no. 3.1.4 and 3.1.5 to a tube containing 100 ml of sterile nutrient broth and soybean casein digest medium.
3.6.3 For positive control inoculate approx 10 to 100 cells of E. coli or salmonella into nutrient broth and Staph. aureus or Ps. aeruginosa in Soybean casein digest medium.
3.6.4 For Negative control inoculate 1 ml of sterile peptone water in both the medium.
3.6.5 Incubate all the tubes at 35 – 37°C for 18 to 24 hours.
3.6.6 Observe the tubes for growth, by means of turbidity. If the growth is present in the sample tube and positive control tube and absent in negative control tube, proceed for further identification of specific microorganisms i.e. E. coli, Salmonella, Ps aeruginosa and Staphylococcus aureus.
3.6.7 If growth is not observed in the sample tube and negative control tube and observed in positive control tube, need not proceed for further identification of specific microorganisms i.e. E. coli, Salmonella, Ps aeruginosa and Staphylococcus aureus.

3.7 Escherichia coli

3.7.1 By means of inoculating loop, streak a portion from enrichment culture (obtained from the nutrient broth of the previous test) on the surface of MacConkeys agar plate.
3.7.2 Simultaneously carry out the positive control by streaking a growth of E. coli on the surface of MacConkeys agar plate. For negative control incubate the plate as it is without inoculation.
3.7.3 Invert and incubate all the plates at 35 to 37°C for 24 hours.
3.7.4 Upon examination, if none of the colonies are brick red in color and have a surrounding zone of precipitated bile, the sample meets the requirements of the test for the absence of Escherichia coli.
3.7.5 If the colonies described above are found, transfer the suspect colonies individually to the surface of Levine eosin-methylene blue agar medium.
3.7.6 Simultaneously carry out the positive control by streaking a growth of E. coli on the surface of MacConkeys agar plate. For negative control incubate the plate as it is without inoculation
3.7.7 Invert and incubate all the plates at 35 to 37°C for 24 hours
3.7.8 Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light, the sample meets the requirements of the test for the absence of Escherichia coli

3.8 Salmonella

3.8.1 Primary Test: Aseptically add 1.0 ml of the enrichment culture (obtained from the nutrient broth of the previous test) to each of two tubes containing (a) 10 ml of sterile Selenite F broth and (b) tetrathionate-bile-brilliant green broth and incubate at 35 to 37°C for 24 to 48 hours.
3.8.2 From each of these two cultures subculture on at least two of the following four agar media: Bismuth sulfite agar, Brilliant green agar, Deoxycholate-citrate agar and Xylose-lysine-deoxycholate agar.
3.8.3 Simultaneously carry out the positive control by streaking a loop full growth of Salmonella on the surface of one of the above media, which is used for testing. For negative control incubate the agar plate without streaking or inoculation.
3.8.4 Invert and incubate all the plates at 35 to 37°C for 18 to 24 hours.
3.8.5 Upon examination, if none of the colonies confirms to the description given in Table-1, the sample meets the requirements of the test for the absence of the genus Salmonella.
3.8.6 If any colonies confirming to the description in Table-1, carry out the secondary test.
Table-1

Sr. No	Medium	Description of colony
1	Bismuth sulphite agar	Black or green
2	Brilliant Green Agar	Small, transparent and colorless, or opaque, pinkish or white (frequently surrounded by a pink or red zone)
3	Deoxycholate-citrate agar	Colorless, and opaque, with or without black center.
4	Xylose-lysine-deoxycholate agar	Red with or without black centers.

3.8.7 Secondary Test: Subculture any colonies showing the characteristics given in Table –1, in triple sugar-iron agar by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle.
3.8.8 At the same time inoculate a tube of urea broth. Incubate at 36 to 38°C for 18 to 24 hours.
3.8.9 Upon examination, no evidence of tubes having alkaline (red) slant and acid (yellow) butt (with or without concomitant blackening of the butt from hydrogen sulfide production), the sample meets the requirements of the test for absence of genus salmonella.

3.9 Pseudomonas aeruginosa

3.9.1 Streak a portion of the medium from soybean casein digest medium (obtained from the nutrient broth of previous test) on the surface of cetrimide agar medium,
3.9.2 Simultaneously carry out the positive control by streaking a loop full growth of Ps. aeruginosa on the surface of cetrimide agar. For negative control incubate the cetrimide agar plate without inoculation. Invert and incubate all the plates at 35 to 37°C for 18 to 24 hours.
3.9.3 If, upon examination, none of the plates contains colonies having the characteristics listed in Table-2 for the media used, the sample meets the requirements for freedom from Ps. aeruginosa.
3.9.4 If any colonies confirming to the description in the table – 3 are produced, carry out the Oxidase and pigment test.
Table-2

Medium	Colony characteristic	Fluorescence in UV light	Oxidase	Gram stain
Cetrimide Agar	Generally greenish	Greenish	Positive	Negative rods
Pseudomonas agar for detection fluorescein	Generally colorless to yellowish	Yellowish	Positive	Negative rods
Pseudomonas agar for detection Pyocyanin	Generally greenish	Blue	Positive	Negative rods.

3.9.5 Pigment Test: Streak representative suspect colonies from the agar surface of cetrimide agar on the surface of pseudomonas agar medium for detection of fluorescein and pseudomonas agar medium for detection of Pyocyanin.

3.9.6 Cover and invert the inoculated plates and incubate at 33 to 37°C for not less than 3 days.
3.9.7 Examine the streaked surface area under UV light and determine whether colonies confirming to the description in Table-2.
3.9.8 Oxidase Test: If the 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 suspected colony. If there is no development of a pink color, changing to purple, the sample meets the requirements of the test for absence of Pseudomonas aeruginosa.

3.10 Staphylococcus aureus

3.10.1 Streak a portion of the medium from soybean casein digest medium (obtained from the nutrient broth of the previous test) on the surface of one of the agar medium listed in Table-3
3.10.2 Simultaneously carry out the positive control by streaking a loop full growth of Staphylococcus aureus on the surface of agar medium. For negative control incubate the agar plate without inoculation.
3.10.3 Invert and incubate all the plates at 35 to 37°C for 18 to 24 hours
3.10.4 If, upon examination, none of the plates contains colonies having the characteristics listed in Table-3 for the media used, the sample meets the requirements for freedom from Staphylococcus aureus.
3.10.5 If any colonies confirming to the description in the table – 3 are produced, carry out the coagulase test.
Table – 3

Sr. No	Selective Medium	Colony characteristic	Gram Stain
1	Vogel-Johnson agar	Black surrounded by yellow zones	Positive cocci in clusters
2	Mannitol-salt agar	Yellow colonies with yellow zones	Positive cocci in clusters
3	Baird-Parker agar	Black, shiny, surrounded by clear zone of 2 to 5 mm	Positive cocci in clusters

3.10.6 Coagulase Test: Transfer representative suspect colonies from the agar surface or any of the media listed in Table-3 to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse plasma with or without additives.
3.10.7 Incubate at 37°C and examine the tubes at 3 hours and subsequently at suitable intervals up to 24 hours.
3.10.8 If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of Staphylococcus aureus.

4.0 Precaution

4.1 Keep the hands clean and used strictly IPA rinsed hand gloves throughout the operations.
4.2 Run positive and negative control along with each test.
4.3 The microbial limit test must be carried out under LAF.
4.4 For pour plate method, if necessary dilute the sample in the sample solution to obtain 100 to 300 cfu.

5.0 Frequencies

Batch wise

6.0 Abbreviation

SOP: Standard Operating Procedure
IPA: Isopropyl Alcohol
LAF: Laminar Air Flow
cfu: Colony forming unit
SCDA: Soybean casein digest Agar
SCDM: Soybean casein digest medium
SDA: Sabouraud Dextrose Agar