Microbial Limit Test & Effectiveness of Preservatives

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Microbial limit test and effectiveness of antimicrobial Preservatives : 

Microbial limit test and effectiveness of antimicrobial Preservatives Prepared by: Ankit Parmar M.Pharm , sem I Q.A. department Guided by : Mrs. Parula a. patel HOD, Q.A. Department, S. J. Thakkar Pharmacy College, Rajkot. 1

Index : 

Index 1. Introduction to microbial limit test 2. Why to need microbial limit test?? 3.What to do for MLT ? 4. Media for organisms 5. Methods 6. Interpretation of test 7.Test specification by product 8. What is antimicrobial effectiveness preservatives? 9. Microbial limit specifications 10. applications 2

1. Introduction to microbial limit test : 

1. Introduction to microbial limit test This test are designed to perform qualitative quantitative estimation of the no of viable aerobic micro-organisms present or detecting the presence of designated microbial species in pharmaceutical product. The most care must be taken while performing microbial test so that contamination from outside can be avoided. Microbial limit testing of raw material as well as finished pharmaceutical products can help to determine whether the product complies with the requirements of the BP, IP or USP. materials . 3

2. Why to need microbial limit test??: 

2. Why to need microbial limit test?? 4

3. What to do for MLT ? Preliminary testing:: 

3. What to do for MLT ? Preliminary testing: The method given herein are invalid unless it is demonstrated that the test specimen to which they are applied do not themselves inhibit the multiplication of under the test condition of micro-organism that can be present. Therefore, inoculate diluted specimen of substance being examined with separate viable culture of (1) E.coli (2) S.aures (3) S.typhi (4) Psudomonas aeruginosa 5

If organism fails to grow in medium the procedure should be modified by: : 

If organism fails to grow in medium the procedure should be modified by: a) incresing the volume diluents with quantity of diluents remain same, or b) incresing a sufficient quantity of inactivating agent in diluents ,or c) combining aforementioned modification so as to permit growth of organisms in media. If inhibitory subtances are present in sample 0.5 soyalecithin & 4%of polysorbate 20 may be added to the culture medium. Repeat the same procedure using fluid casin digest - soys lecithin -polysorbate20 medium to demonstrate neutralization of preservative or other antimicrobial agent in test material. 6


Conti.. If inspite of incorporation of suitable inactivating agent &a substantial increase in volume of diluents it is still not possible to recover the viable culture described above & where article is not suitable for applying the membrane filtration method it can be assumed that the failure to isolate the inoculated organism may be due to the bactericidal activity of product. This may be indicated that the article not likely to be contaminated with the given species of organism. Monitoring should be continued to establish the spectrum of inhibition & bactericidal activity of product. 7

4. Media:: 

4. Media: culture media may be as per procedure & they have similar ingredient &/or yield media comparable to those obtained from the formula. Where agar specified in formula, use agar that has moisture content nmt15 %. Where water is mention in formula, use purified water. Media should be sterilized by heating in autoclave at 115°c for 30 min. In preparing media as per formula, dissolve the soluble solid in water, using the heat if necessary to effect complete solution & add HCL&NAOH in sufficient quantities to yield require pH in medium. SAMPLING: USE 10ML OR 10G SPECIMEN FOR EACH OF TEST. 8

Medium for growth of organisms : 

Medium for growth of organisms (i) Soybean-casein digest agar medium (for bacteria ) Casein peptone 15.0 g Soybean peptone 5.0 g Sodium chloride 5.0 g Agar 15.0 g Water 1,000 ml Mix all the ingredients autoclave for 15-20 minutes at 121º. Its pH becomes 7.1 to 7.3. after autoclaving. (ii ) Sabouraud glucose agar medium (for Fungi ) Peptone (derived from meat and casein) 10.0 g Glucose 40.0 g Agar 15.0 g Water 1,000 ml Mix all the ingredients and autoclave for15-20 minutes at 121°. Its pH becomes 5.4-5.8. 9



(I) Total aerobic microbial count : 

(I) Total aerobic microbial count PREPARATION OF TEST FLUID: Water soluble product : Dissolve 10g or 10ml of the sample in buffer or fluid medium & adjust volume to 100ml. Product insoluble in water(non fatty): Take 10g of sample, grind to fine powder & suspend it in buffer or fluid medium & adjust the volume to 100ml. - A suitable surface-active agent such as 0.1%w/v of polysorbate 80 may be added to assist the suspension of poorly wettable substance. Fatty product : Homogenise 10g or 10ml of sample with 5g ofpolysorbate20 or polysorbate80. -If necessary ,heat to nmt 40`c for 30min. -Add 85ml of buffer or fluid medium. -Adjust the PH to about 7. 11

(A) Membrane filtration: : 

(A) Membrane filtration: Use membrane filter 50mm in diameter & having nominal pore size ngt 0.45um or less. Sterilized the filters,filteration apparatus,media & other apparatus used. Transfer 10ml or quantity of each dilution contain 1g of preparation being examined to each of two membrane filter & filter immediately. If necessary dilute the pretreated preparation so that 10-100 colony count may be expectd . 12

Filtration conti..: 

Filtration conti .. After filtration wash the each filter three or more time with appropriate fluid such as phosphate buffer,sodium chloride-peptone buffer or fluid medium. For fatty susbtance add polysorbate20 or polysorbate80 to washing. Transfer one of the membrane filter, intended for enumeration of bacteria to surface of plate of casein soyabean digest agar & intend for enumeration of fungi to surface if sabouraud dextrose agar with antibiotics. Incubate the plate for 5 days, unless more reliable count is obtanied in shorter time,at 30 to 35°c in test for bacteria & 20 to 25°c in test for fungi. Count the number of colonies that are formed. Calculate the no of organism per g or ml of preparation being examined 13

(B) Plate count method : a. Pour plate method : 

(B) Plate count method : a. Pour plate method FOR BACTERIA: Use Petri dish 9 to 10 cm diameter, add to each dish a mixture of 1ml of the pretreated preparation & about 15ml of liquefied casein soyabean digest agar at nmt 45°c If necessary dilute the preparation as described above so that colony count nmt 300may be expected. Incubate the plate at 30 to 35 °c for 5 days unless more reliable count is obtained in shorter time. Calculate the result using plate with greatest no. of colonies but taking 300 colonies per plate as maximum consistent with good evaluation. FOR FUNGI: Use saboraud dextrose agar with antibiotics & incubate the plate at 20 to 25 °c for 5 days. Calculate the result using plate with nmt 100 colonies. 14

b. Spread plate method : 

b. Spread plate method Place 0.05-.2 ml of test fluid on solidified dried surface of agar medium spread it uniformly using spreader. Proceed under same condition as for the pour plate method. 15


(C)MULTIPLE TUBE METHOD Use 12 test tubes : 9 containing 9 ml of soybean-casein digest medium each and 3 containing 10 ml of the same medium each for control. Prepare dilutions using the 9 tubes. First, add 1 ml of the test fluid to each of three test tubes and mix to make 10- times dilutions.(100ul) Second, add 1 ml of each of the 10-times dilutions to each of another three test tubes and mix to make 100-times dilutions.(10ul) Third, add 1 ml of each of the 100-times dilutions to each of the remaining three test tubes and mix to make 1,000- times dilutions (1ul) Incubate all 12 test tubes for at least 5 days at 30 - 35°c. No microbial growth should be observed for the control test tubes. If the determination ofthe result is difficult or if the result is not reliable, take a 0.1ml fluid from each of the9 test tubes and place it to an agar medium or fluid medium, incubate all media for 24-72 hours at 30-35°c, and check them for the absence or presence of microbial growth. Calculate the most probable number of microorganisms per ml or gram of the sample. 16

6.Interpretation of test :: 

6.Interpretation of test : Mainly three methods are used : 1. total colony count 2. turbidity seen in sample 3. zone of inhibition 17

7. Test specification by product type : 

7. T est specification by product type 18

Where ,TAMC =total aerobic microbial count and TYMC = total yeast micribial count: 

Where ,TAMC =total aerobic microbial count and TYMC = total yeast micribial count 19


8 . WHAT IS THE ANTIMICROBIAL EFFECTIVENESS TEST? The Antimicrobial Effectiveness Test demonstrates the effectiveness of the preservative system in a product . A product is inoculated with a controlled quantity of specific microorganisms. Then compares the level of microorganisms found on a control sample versus the test sample over a period . Precautions: Challenge tests should be conducted under conditions that prevent accidental contamination of the product during the test . 20


TEST FOR EFFECTIVNESS OF ANTIMICROBIAL PRESERVATIVE Antimicrobial preservative are substance added to nonsterile dosage form to protect them from microbial growth or from micro-organism that are introduced inadvertently during or subsequent manufacturing process. All antimicrobial agent are toxic substances for maximum protection of patient, concentration of preservative shown to effective in final packged product should be below level that may be toxic to human beings. . 21


PRESERVATIVE IDEALS: Broad spectrum of activity. Effective over wide pH range. Stable to light & elevated temp for expected shelf of product. Soluble in formulation at the required concentration. No effect over colour , odour ,rheological property of formulation. It should be non volatile. Compatible with formulation component & packaging. Non toxic at in used concentration. It must not too oil soluble. It must be cost effective. 22

widely used preservative : 

widely used preservative Acids: benzoic acid, paraben , sorbic acid. Alcohols: ethyl, isopropyl, chlorbutol , bronopol . Biguanids : chlorhexidine , polyhexamethylene biguanide . Halogen: hypochlorite, povidone -iodine, chloroform. Organic mercurials : mercury, silver, thiomersal Aldehyde : formaldehyde, glutardehyde . Phenolics : Cresols, chlorcresol , bisphenol . Qutarnary ammonium -compound: cetrimide , benzalkonium chloride. 23

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Preparation Preservative Concentration % w.v Injections Phenol Cresol Chlorocresol Phenylmrcuric nitrate Benzyl alcohol 0.5 0.3 0.1 0.001 1.0 Eye drops Phenylmercuric nitrate or acetate Chlorhexidine acetate Benzalkonium chloride 0.002 0.01 0.01 Mixtures Chloroform Benzoic acid Methyl paraben Alcohol Sulphur dioxide 0.25 0.1 0.1 12-20 400 parts/10 6 Creams Parabens Chlorocresol Dichlorobenzyl alcohol Cetyltrimethyl ammonium bromide Phenylmercuric nitrate 0.1-0.2 0.1 0.05-0.2 0.01-0.1 0.001 Tablets Methylparaben 0.1 Preservatives used in pharmaceutical preparations The following Table gives a list of usual preservatives used in pharmaceutical preparations. 24


PRESERVATIVE INTERACTION: Tragacanth , acaccia , & agar are aninonic in nature & will adsorb cationic preservative. Starch can bind substituted phenols but this can be over come by increase conc of phenols. Protein can interact both with cationic & anionic preservative. Cationic & anionic surfactant contraindicated with anionic & cationic preservative respectively. PEG interact particularly with phenols. 25

Which organism should be used to challenge the preservative? : 

W hich organism should be used to challenge the preservative? Test organisms : The following test organisms are used in the test Candida albicans , ATCC 10231 Aspergillus niger , ATCC 16404 Escherichia coli , ATCC 8739 Pseudomonas aeruginosa , ATCC 9027 Staphylococus aureus , ATCC 6538 In order to prevent any phenotypic changes in the strains used, the organisms used in the test should not be more than 5 passages made from the original culture. Media: All the media used in the tests should be tested for growth promotion. 26


PREPARATION OF INOCULUM : Challenges microbes can be grown on agar surface or they may grown in broth culture. To harvest bacterial & c.albican culture,use sterile saline TS ,washing surface growth ,collecting it in suitable vessel, & add sufficient sterile solution TS to obtain microbial count of about 10 8 cfu per ml. To harvest the cell of A.niger use sterile solution TS to contain polysorbate 80 & add sterile solution TS to obtain count of about 10 8 cfu per ml. Bacterial & yeast suspension are to be use within 24hr of harvest,but the fungal preparation may be stored under refrigeration for up to 7 days. 27


PRODUCT CATEGORIES: For testing purposes, the USP has divided test articles into four separate categories Category 1 – Injections, other parenterals including emulsions, otic , sterile nasal products made with aqueous bases or vehicle. Category 2 – Topically used products made with aqueous bases or vehicles, non-sterile nasal products, and emulsions including those applied to mucous membranes. Category 3 – Oral products other than antacids made with aqueous bases or vehicles. Category 4 – Antacids made with an aqueous base . 28


PROCEDURE: The test can be conducted either in five original container, Inoculate each container with one of prepared & standardized inoculum & mix. The volume of suspension inoculum used is between is 0.5% to1% of product. The concentration of test organism that is to added to product category( 1,2& 3) are such that final concentration of test preparation is after preparation is between 10 5 to 10 6 cfu per ml of product. For category 4 product the final concentration of test preparation is between 10 3 to 10 4 cfu per ml of product. The concentration of viable micro-organism in each test preparation is estimated by plate count method. Incubate the inoculated containers at 22.5°c temperature & Determine the viable count by plate-count method at 7, 14, and 28 days subsequent to the inoculation. The calculated concentration of cfu per ml present at the start of the test. calculate the percentage of reduction in cfu per ml for each organism at the stated test intervals and express the changes in terms of percentage of initial concentration. 29


CRITERIA FOR ANTIMICROBIAL PRESERVATIVE For category 1 product: Bacteria: Not less than 1.0 log reduction from initial calculated count at 7 days, Not less than 3.0 log reduction from initial count at 14 days , No increase from 14`days count at 28 days Yeast &Mould: No increase from initial calculated count at 7,14 & 28 days. For category 2 product 30 Bacteria: Not less than 2.0 log reduction from initial count at 14days,& no increase from 14day`count at 28 days. Yeast &Mould: No increase from initial calculated count at 14 & 28 days.

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For category 3 product Bacteria: Not less than 1.0 log reduction from initial count at 14 day& no increase 14 days` count at 24 day . Yeast &Mould: No increase from initial calculated count at 14 & 28 days For category 4 product Bacteria, yeast & mould: No increase from initial calculated count at 14 & 28 days 31

9.Microbial limit specification: 

9.Microbial limit specification ! The ICH Harmonized Tripartite Guideline Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances Q6A Step 4 October 6 1999- Decision Tree #8: Microbiological Attributes of Non-Sterile Drug Products 32

10. Applications :: 

10. Applications : Microbiological attributes of non sterile pharmaceutical products Microbiological evaluation of clean room & other controls of environment Microbiological controll for dietary supplements. 33

References :: 

References : 1) “ Indian Pharmacopoeia 2007”, Government Of India, Ministry Of Health And Family Welfare, Volume-1, Controller Of Publication, Delhi Appendix-2.2.9, page no.35 -44. 2) “U.S.Pharmacopoeia-24”,(61) , page no,1814-1817. 3) “Guide to microbiological control in pharmaceuticals and medical devices”,Edited by Stephen P. Denyer,Rosamund M. Baird,CRC press, second edition page no.53. 4) www.atcc.org 34

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