QUALITY ANALYSIS OF NON – STERILE THERAPEUTIC PRODUCTS -MICROBIAL LIMIT TEST

 QUALITY ANALYSIS OF NON – STERILE THERAPEUTIC PRODUCTS -MICROBIAL LIMIT TEST 

AIM: 

To the qualitative and quantitative estimations of specific viable microorganisms present in samples. To test the microbial quality of non -sterile pharmaceutical product 

INTRODUCTION: 

The microbial limit test (MLT) is performed to assess how many and which of certain viable microorganisms are present in non-sterile pharmaceutical, healthcare or cosmetics manufacturing samples that range from raw materials to finished products. The test provides information about the safety of the tested product by determining if quantitative limits for certain microorganisms are exceeded. It covers testing for bile tolerant gram-negative bacteria as well as for Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, clostridia and Candida albicans. The samples are prepared by mixing multiple portions taken randomly from the ingredient or product to be tested. If the samples contain antimicrobial substances, these have to be eliminated beforehand by dilution, neutralization, filtration, inactivation or other appropriate means. Great care must be taken to prevent microbial contamination from the outside. 

PRINCIPLE: 

This test is based on the principle that the microbiological quality of non-sterile pharmaceutical materials can be controlled by the adoption of both the standards.

 1. The first is a limit on the total viable count. 

 2. The second is the exclusion of specific pathogens.

MATERIALS REQUIRED:

• Sterilized glass wares- Glass plates, test tubes, pippets flask etc. 
• Incubators- BOD and Bacteriological for 20-25°C, 30- 35°C & 40-45°C.
•  Laminar Air Flow- Horizontal LAF. 
• Dry Heat Sterilizer- About 200°C.
•  Filter Paper Disc- It is made up of cellulose nitrate and have 0.45um pore size. 
• Other Materials- Sterilized media, weight machine, filtration unit etc. 
• Culture Media 
• BRILLIANT GREEN AGAR
•  BISMUTH SULPHITE AGAR 
• CETRIMIDE AGAR 
• EOSINE METHYLENE BLUE AGAR 
• MACCONKEY AGAR 
• MANNITOL SALT AGAR 
• PSEUDOMONAS AGAR 
• SABOURAUD CHLORAMPHENICOL AGAR
• SOYABEAN CASEIN DIGEST AGAR 
• TRIPLE SUGAR IRON AGAR 
• BUFFERED PEPTONE WATER
•  FLUID SELENITE CYSTEINE BROTH
•  MACCONKEY BROTH 
• PEPTONE WATER 
• SOYABEAN CASEIN DIGEST MEDIUM
• TETRATHIONATE BRILLINT GREEN BILE BROTH

METHODS: 

There are two types of method of microbial limit test

1. Direct inoculation- In this method sample is directly inoculated into the media and that are incubated.

 2. Membrane filtration method- In this method sample solution is filter with filter membrane and then this kifilter membrane is transfer to the freshly prepared sterilized media. Above methods are performed for

1.  Total Bacterial Count- Quantitative method
2.  Total Fungal Count -Quantitative method
3.  Pathogen testing -Qualitative method

QUANTITATIVE METHOD: 

TOTAL BACTERIAL COUNT(TBC) / TOTAL FUNGAL COUNT(TFC); 

Four Method are employed for this test:

1.  Filtration method 
2.  Pour pate method 
3. Spread plate method 
4.  Serial Dilution Method 

Membrane Filtration Method; (Filtration method) 

Introduction: 

This method is applied to the sample which contains antimicrobial substances. Use membrane filters of an appropriate material with a pore size of 0.45 um or less. 

Requirements: 

Sterilized filter membrane disc (0.45um), membrane filtration unit, 0.1% bacteriological peptone water without tween 80 or 20 (800ml), 0.1%peptone water with tween 20 (3 X 100ml), soyabean casein digest media (SA) and saboured dextrose agar (SD) medium, glass wares etc. 

Pretreatment of sample: 

• Water soluble product: 10 gm./10 ml sample +70 ml buffered normal saline solution+ make up volume up to 100 
• insoluble product: 10 gm./ /10 ml sample +70 ml buffered normal saline solution + make up volume up to 100+ 0.1% w/v polysorbate 80 (surface active agent) 
• Fatty product: 10 gm./10 ml sample +60 ml buffered normal saline solution+ 5 gm. polysorbate 80 or polysorbate 20+ gentle heat + make up volume up to 100.  

PROCEDURE: 

1 .Firstly arrange the all requirements and take all precaution before starting work. 

2.Now take 1.0 gm of sample and mix into 800 ml of 0.1% of bacteriological peptone water without tween 80.

 3 Then filter it with the help of filter membrane disc. 

4. Now given them washing with 0.1% of peptone water which contain1.0% tween 20, in 3 times of 100ml.

 5.After filtration cut the filter membrane disc in two half pieces and transfer on freshly prepared SA and SD plate. 

6. Now incubates the plates. SA plates for 5 days at 35-37 °C. and SD plates for 5 days at 20-25°C. 

7.After the completion of the incubation period observe plate & count the No. of colonies.

2.SERIAL DILUTION:

• Procedure: 

1. Take the pre-treated sample. 
2. Take the sixtest tube each containing 9 ml saline solution. 
3. Mark them 101 to 10-6. 
4. Open the cotton plug of the flask with your right hand and procure 1 ml of sample form the flask with the help of micropipette and then plug the flask again. Adequately unplug the test tube and pour the sample in 10 saline tube containing 9 ml saline and then close the led of plate. 
5. Vortex the tube 10¹ for 2minute, unplug the tube and take 1 ml solution in micropipette.
6. Unplug 103 saline tube and pour the solution of micropipette inside it. 
7. Follow the previous step to attain the 10-6 dilution. 
8. Follow the pour plate method to pour 103-106 saline tube in petri plate with 3replica.
9. Incubate all the petri plate in inverted position for 72 hrs. at 22- 25°C and later 24 hrs. at 30-35°C.
10.  Observe the petri plate at regular interval of time. After incubation complete then count the colonies with digital colony counter under SOP. 
11. Record the colony count and documented in report of product.

3.Pour plate method:

Procedure: 

1. Take the treated sample. 
2. Remove cotton plug with your right hand and last two finger, take up the flask and take 1 ml sample by micropipette with your right hand too. After taking sample plug the flask again and adequately open the lid of petri plate with your left hand& finger and toe and then pour the sample in the plate. 
3.  Now Remove the cotton plug of media containing flask as mention before and pour approx. 12 ml of media inside the plate cavity and lid the plate. 
4. Rotate the plate anti-clockwise and clockwise gently so the media mixed well with 1 ml of sample. Be precautious that media should not touch the lid of petriplate. 
5. Let the plate solidify. After solidification invert all the petri plate. o Incubate all the petri plate in inverted position for 72 hrs. at 22- 25°C and later 24 hrs. at 30-35°C.
6.  Observe the petri plate at regular interval of time. After incubation complete then count the colonies with digital colony counter under SOP. Record the colony count and document in report of product.

4.SPREAD PLATE METHOD 

Procedure: 

1. Take the pre-treated sample. 
2. Take the solidify media containing plate. 
3. Open the cotton plug of the flask with your right hand and procure 1 ml of sample form the flask with the help of micropipette and then plug the flask again. Adequately open the led of media containing plate and pour the sample and then close the led of plate. 
4. Put down the micropipette and pick up the spreader and put the plate at rotating plate, open it with left hand and apply gently the spreader on the surface of plate with right hand. Remain it touch with surface until all sample spread well.
5.  Let the plate dry from surface for 1 min. 
6. After dry invert all the petri plate. 
7. Incubate all the petri plate in inverted position for 72 hrs. at 22- 25°C and later 24 hrs. at 30-35°C. 
8. Observe the petri plate at regular interval of time. After incubation complete then count the colonies with digital colony counter under SOP. 
9. Record the colony count and documented in report of product. 

2.Qualitative estimation (Pathogen detection) 

In qualitative estimation the four more pathogenic bacteria are detecting under this test, which are following: 

1.Escherichia coli

2.Pseudomonas aeruginosa 

3.Staphylococcus aureus 

4.Salmonella 

In qualitative estimation the two more pathogenic fungi are detecting under this test, which are following:

1. Candida albicans 
2. Aspergillus niger 

1. Escherichia coli 

• These include identification of E. coli, bacteria pathogenic to human body and causes infection of stomach. It is detected by using specific, differential media which support growth of only E. coli. 

• Primary test: Pipette 1 ml of enrichment culture into tubes containing 5 ml Mac Conkey & broth and incubate at 36-38 °C for 48 hrs. If the content shows acid and gas carry out the secondary test. 
• Secondary test: After incubation, if the tube shows presence of acid and gas, transfer 0.1 ml from tube to each of two tubes containing, 5 ml Mac Conkey and broth and other containing 5 ml peptone water and Incubate broth tubes in a water bath at 43.5°C to 44.5°C for 24 hrs. After incubation examine tube (a) for acid and gas (b) for indole. If the tubes shows turbidity then one loop full culture is streaked over EMB and MCA and incubate them for 24 hrs. at 43.5°C to 44.5°C.
• Tertiary test: To test for indole production add 0.5 ml of kovac's reagent, shake well and allow to stand for 1 min., if a red color is observed in the reagent layer, indole is present, The presence of acid and gas and of indole indicates presence of Escherichia coli.

2. Staphylococcus aureus:

Identification of Staphylococcus aureus is the detection of pathogenic bacteria which causes infection in human body. It can be identified by using specific, differentiation media which supports growth of only Staphylococcus aureus. 

Primary test: 

• Place the prescribed quantity in a sterile screw-capped jar containing 100 ml of soybean casein digest medium and incubate at 32-37°C for 24-48 hrs

• Subculture on a plate containing a layer of mannitol salt agar medium or Vogel Johnson agar medium and incubate at 32-37°C for 18-24 hrs. 

• Examine the resulting growth by Gram & stain and apply the coagulase test. Gram positive cocci (in cluster) in yellow colonies (on mannitol salt agar medium) and in colonies, black surrounded by yellow zones (on Vogel Johnson agar medium) and giving a positive coagulase test indicates the presence of Staphylococcus aureus.

 Confirmatory test: (coagulase test) 

• Transfer representative suspect colonies from the agar surface of mannitol salt agar medium or Vogel Johnson agar medium to individual tubes, each containing 0.5 ml of 28 mammalian, preferably rabbit or horse plasma with or without additives. Incubate in water bath at 37°C examining the tubes after 24 hrs. 

• If coagulation in any degree is observed, the test is positive

 

3. Pseudomonas aeruginosa. 

The identification or detection of pathogenic bacteria which causes infections to the human body can be identified by using specific differential media which support only the growth of Pseudomonas aeruginosa. 

• Primary test: Pre-treat the preparation as described above. 
• Place the prescribed quantity in a sterile screw caped jar containing 100 ml of soybean casein digest medium and incubate at 35-37°C for 24 to 48 hrs. Observe the medium for growth.
• If any growth is observed, subculture a portion of medium on a plate containing a layer ofCetrimide Agar and incubate 35-37°C for 18 to 24 hrs. 
• If none of the plate contains colonies having characteristics given in the table, carry out  the confirmatory test. 
• Confirmatory test: (oxidase and pigment test) Streak representative suspect colonies from the agar surface of cetrimide agar medium on the agar surface of 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 35+2°C for not less than three days.
•  Examine the streaked surface under UV light. 
• Examine the plate to determine the whether colonies having the characteristics, given in table, are present. Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of oxidase test. 
• Upon the colonial growth place or transfer colonies to strip or disks of filter papers that previously has been impregnated with N, N, N, N, -tetra-methyl, 4 phenyl adenine; if there is no development of pink color, changing to purple, the specimen meets the requirement of the test for the absence of Pseudomonas aeruginosa. 
• The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and bio-chemical test, if necessary.

4. Salmonella sp: 

Add 1 ml of enrichment culture to 10 ml of Rappaport vassiliadis salmonella enrichment broth and incubate at 30-35°C for 18 to 24 hrs. Subculture Salmonella on xylose lysine Deoxychollate agar media with inoculating loop.

 

OBSERVATION &RESULT: 

In Microbial limit test, microorganisms are count and microorganisms are compared with the ATCC/MTCC culture for detection of pathogenic bacteria. 

The bacterial culture of ATCC/NCTC/MTCC used, are given below:

• Escherichia coli- ATCC No. 8739 
• Pseudomonas aeruginosa -ATCC No. 9027 
• Staphylococcus aureus-ATCC No. 6539 
• Salmonella- NCTC No. 6017 
• Candida albicans -ATCC No. 10231 
• Aspergillus niger-ATCC No. 16404

1.QUANTITATIVE METHOD: 

2.QUALITATIVE METHOD: 

• No colony on selective media shows the absence of pathogenic microorganisms in the sample. 

• Hence it is compliance with the IP, BP, USP specification