Microbial limit tests I.P by Dr.P.srinivas
- 1. Microbial limit tests I.P
- 2. The microbiological quality of non-sterile pharmaceutical or cosmetic material can be controlled by using two methods
- 3. Pathogencity of specific Microorganisms I.P E.coli: Enterotoxins/Diarrhoel diseases. Hence, exclude from pharmaceutical materials. Salmonella species: Initiate infections by ingestion/ Excluded from pharm. materials because they represent major infection. S.aureus: Originate on skin /Limit tests for S.aureus are most likely to be applied to topical products. P.aeruginosa: Pathogen infects vulnerable sites eg: eyes opportunist/immunity/topical products/resistant to preservatives
- 4. Preliminary testing/Antimicrobial activity of test sample The methods given here are invalid if test sample show antimicrobial activity. Hence, check inhibition effect of sample. Test procedure: 1. 1ml of 10-3 dil of 24hrs broth culture of Mo’s in 1st dilution of test material (in buffer pH 7.2 Medium.( Soya bean casein digest agar medium) .If Mo’s fail to grow modified by Increase the volume of diluents with test material Incorporate inactivating agent Combine above for so as to permit the growth of Mo’s.
- 5. 0.5% of soya lectin and 4% of polysorbate are added to sample to inactivate inhibitory substances Repeat test as above by using fluid casein digest soya lecithin- polysorbate 20 medium to neutralize preservatives other antimicrobial agents If inhibitory substances are present/Later soluble the total Aerobic microbial count may be used If all tests fail to inactivate the activity of test sample/ article not contaminated with test sample/ article not contaminated with microbial species
- 6. A. Total aerobic microbial count Total viable Mo’s / unit wt OR volumes of sample is common in Mo’s Pretreatment of sample as follows • Water soluble products: Dissolve 10gms or 10 ml of test sample as monograph/ buf. Nacl. Peptone solution(pH 7.0) any other medium/ to 100ml medium. •Non- fatty water insoluble products: same as A + 0.1w/v of polysorbate 80 may be added to assist the suspension of poorly wettable substances. •Fatty products: 10gms or 10ml(as monograph + 5gms polysorbate 20 or 80, if necessary heat/40 deg in water bath or oven. Maintain 40 deg/ formation of emulsion of emulsion total aerobic microbial count present in test substances determined by follows
- 7. 1. Transfer 10ml of diluted sample though membrane filter of 50mm diameter of 0.45µm if necessary dil. for require colony count. 2. Wash mem.fil. With successive quantities of 100ml of buffered Nacl. peptone broth soln./for fatty prods. Add polysorbate 80 &20. 3. Transfer one of mem.filter to SDA plate with antibiotics. 4. Incubate 30 -40oC /48hrs for bacteria and 20-25oC /5 days for fungi. 5. Calculate No. of Mo’s/ml.
- 8. (ii).Total plate count 1. Mix 1ml of preheated sample +15ml of liquefied SCD agar pour into plate at 45oC or spread on surface. 2. Incubate plates at 37oC and 24 -25oC/ 2-3 days for fungi. 3. Enumerate no. Mo’s/ml.
- 9. (iii) Most probable number (MPN) 1. Take 14 test tubes of similar size place 9 ml of sterile fluid soyabean casein digest medium. 2. Arrange twelve of the tubes in four sets of 3 tubes each. One among serves as control. 3. Prepare stock solution of sample and dispense 1ml into one set each of three tubes of conc. named 100µg/ µl and into tube A and from it to tube B. 4. Now ,where the concentrations of tube B and second set is 10 µg/ µl and set three is 1µg/ µl. 5. Close well and incubate all tubes and examine the growth in each test tubes. The 3 tubes remain clear. Interpret with reference table indicate MPN of MO’s per gm/ml of test sample
- 10. Most probable number of multiple tube or serial dilution method
- 11. Prescribed quantity of test sample + 50ml of nutrient broth Shake and incubate at 370C/24 hrs 1ml enrichment culture + 5ml of Mackonkey broth Perform primary test Shake and incubate at 370C for 48 hours If tube shows acid and gas formation then perform secondary test B.(a). Detection of E.coli 0.1ml enrichment culture + 5ml Mac Conkey broth 0.1ml enrichment culture + 5ml peptone water incubate at 370C/24 hrs Presence of acid and gas incubate at 370C/24 hrs 0.5ml of Kovacs regt. Shake and detect presence of red colour Indole (+ve) Indicates the presence of E.coli
- 12. (b) .Detection of Salmonella 1gm or 1ml of the test sample+100 of NAB Perform primary test Shake &incubate at 37C/24hrs 1ml enrichment culture +10ml selenite F- broth 1ml of enrichment culture +10ml of tetrathionate bile-brilliant green broth Incubate at 370C /48 hrs Bismuth sulphite agar (BSA) Sub culture each of two cultures, on at least two of the following 4 agar media Deoxcholate citrate agar (DCA) Brilliant green agar BGA Xylose lysine deoxycholate agar (XLDCA) Incubate all plates at 37C/24hrs and observe the colony characteristics Small, tranparent and colourless or apaque,pinking or white Black or green Colourless and opaque with or without black centers Red with or without black centers If none of the colonies confirm to the characteristics on the different media, the sample meets the requirements of the absence of the salmonella. If colonies are formed confirming on the basis discription, carrry out the secondary test. Subculture any colonies showing the positive characteristics Triple sugar iron agar Slant TSIA (stab) Urea broth Incubate at 370C /24hrs Absence of acidity Formation of acid and gas Absence of red colour Indicates presence of Salmonella Perform secondary test
- 13. Pre-heat &inoculate 100ml of fluid soyabean casein digest medium with a quantity of the 1gm(or as specified in monograph) test sample Mix& incubate 37C0/48hrs Examine the medium for growth, if growth is present, streak on the surface of cetrimide agar medium incubate at 370C for 24 hours (c). Detection of P.aeruginosa Greenish colonies Streak representative colonies on the surfaces of Pseudomonas agar medium for detection of fluorescein and pyocyanin Examine the plates to confirming the colonies as yellowish (flurescein) and blue (pyocyanin) colour Incubate at 370C for not less than 3 days and examine under U.V light If colony confirms for colour and growth, add growth, add 2-3 drops of 1%w/v solution of N,N,N1, N2 tetramethyl-4-phenylenediamine di-hydrochloride on filter paper and smear with the colony. No greenish colonies (Sample free from p.aeruginosa) Permorm oxidase test If there is no development of pink colour (changing to purple) Absence of pseudomonas aeruginosa
- 14. Pre-heat &inoculate 100ml of fluid soyabean casein digest medium with a quantity of the 1gm(or as specified in monograph) test sample Mix& incubate 37C0/48hrs Examine the medium for growth, if growth is present, streak on the surface of following medium (d). Detection of S.aureus Vogel-Johnson agar incubate at 37C0/24hrs If none of colonies have the characteristics given as above for the media used that indicates absence of S.aureus. If growth occurs and colony shows the above specific charecteristics, carry out coagulase test. Black colonies surrounded by yellow zones Transfer representative colonies from the agar surface into a test tube containing 0.5 ml of mammalian, preferably rabbit or horse plasma with or with out additives. Baired-Parker agar Incubate in water broth at 37C0 and examining the tubes at 3 hrs and subsequently at suitable intervals up to 24 hrs. If no coagulation is observed that indicates absence of S. aureus Mannitol-salt agar Yellow colonies with yellow zones Black shiny colonies surrounded by clear zones