• 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 term ‘growth’ is used to designate the presence & presumed proliferation of viable micro-organism.• The most care must be taken while performing microbial test so that contamination from outside can be avoided.
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
• 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 –soyslecithin,-polysorbate20 medium to demonstrate neutralization of preservative or other antimicrobial agent in test material.• Where inhibitory substance are contained in product & latter is soluble, the Membrane filtration method may be used.
• 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.
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.
( 1) TOTAL AEROBIC MICROBIALCOUNTPREPARATION 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. Membrane filtration:• Use membrane filter 50mm in diameter & having nominal pore size NGT 0.45 um orless.
• 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.• 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.
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 NMT300 may 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. 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.
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 of the 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.
• Test for specified micro organism• - E.COLI: Place the prescribed quantity in sterile screw-capped container, add 50ml of nutrient broth, shake allow to stand for 1hr &incubate at 37° for 18 to 24hr.• Primary test: Add 1ml of enrichment culture to tube contain 5ml MacConkey broth&incubate in water bath at 36 to 38° for 48hr.
• If content show acid &gas carry out secondary test. • Secondary test: Add 0.1ml of content of tube containing (a)5 ml of MacConkey broth (b)5ml of peptone water incubate in a water bath at 43.5 to 44.5° for 24hr &examine tube for (a) acid &gas (b) indole• For indole: Add 0.5 ml of kovac’s reagent, if red colour is produced ,indole is present.
• That indicates presence of e.coli.• For control: Repeat primary &secondary test adding 1.0ml of enrichment culture &volume of broth containing 10 to 50 e.coli organism,prepared from 24hr culture in nutient broth,to 5ml MacConkey broth. The test is not valid unless the result indicate that the control contain e.coli.
• OTHER TEST: streak a portion from enrichment culture on surface of MacConkey agar medium.cover the dish &incubate.• -If none of the colonies are brick-red in colour, sample meet the requirement of test for absence of e.coli.• If colony described above are found, transfer the suspect colony to surface of Levine eosin ethylene blue agar medium. cover &incubate.
• Upon examination, none of colony exhibit both metallic sheen under reflected light & blue-black under transmitted light ,sample meet requirement test for absence of E.coli. • SALMONELLA: Transfer a quantity of pretreaed prepration being examined containing 1g or 1ml of product to 100ml of nutrient broth in sterile screw capped jar ,shake & incubate at 35 to 37 for 24hr.
• Preliminary test; Add 1.0 ml of enrichment culture to each of two tubes containing(A)10ml of selenite F broth & (B)tetrathionet -bile- briliant green broth &incubate at 36 to38° for 48 hr.• From each of this two cultures subculture on following four agar medium & incubate at 36 to 38°for24 hr• if none of colonies conform to description given in table, sample meet requirment for absence of salmonella.
Characteristic Colonial MorphologySelective Medium Brilliant Green Small, transparent, colorless or pink to white Agar Medium opaque (frequently surrounded by pink to red zone) Xylose-Lysine- Red, with or without black centers Desoxycholate Agar Medium Bismuth Sulfite Black or green Agar Medium
• Secondary test: subculture any colonies showing characteristics given in table in triple sugar-iron agar by first inoculating the surface of slope & at the same time inoculate a tube of urea broth &incubate both at 36 to 38 for 18 to 24 hr. • The absence of acidity from the surface growth in triple sugar iron agar & together with absence of red colour in urea broth, indicates the presence of salmonella.
• FOR CONTORL: Repeat the primary &secondary test using 1.0ml of enrichment culture& volume of broth containing 10 to 50 salmonella organism, prepare from 24hr broth culture in nutrient broth, for inoculation of tubes (a) &(b).• The result is not valid unless the result indicate that the control contains salmonella.
• PSUDOMONAS AERUGINOSA: • Inoculate 100ml of fluid soyabean casein digest medium with quantity of solution ,suspension or emulsion thus obtained containing 1g or 1ml of preparation being examined &incubate at 35 to 37for 24 to 48hr
Characteristic Fluorescence Selective Colonial in Medium Morphology UV Light Oxidase Test Gram Stain Centrimide Generally Greenish Positive Negative rodsAgar Medium greenishPseudomonas Generally Yellowish Positive Negative rodsAgar Medium colorless to for yellowishDetection of FluorescinPseudomonas Generally Blue Positive Negative rodsAgar Medium greenish forDetection of Pyocyanin
• If upon examination none of colonies having characteristics listed in table for media used, sample meet requirement for absence of micro- organisms.• If colony conform to description in table ,carry out oxidase & pigment test.• OXIDASE &PIGMENT TEST:• Streak representative suspect colony from cetrimide agar medium on surface of pseudomonas agar medium for
detection of florescein &pseudomonas agar medium for detection of pyocyanin contained in Petri dish & incubate at 33 to 37 °for NLT 3 days.• Examine the streak surface under u.v light.• Examine plate to determine whether colonies conforming to description in table are present.
• If, growth of suspect colonies occur ,place 2 or 3 drops freshly prepared 1%w/v solution of N,N- tetramethyl-4-phenylenediamine dihydrochloride on filter paper &smear with colony.• If there is no development of pink colour changing to purple, the sample meet requirements of test for absence of pseudomonas aeruginosa.
• STAPHAYLOCOCCUS AURES:• Proceed as described under psudomonas aeruginosa ,if upon examination of incubate plate. none of them contain colony having characteristic listed table sample meet requirment for absence of S.aures.• If, growth occurs ,carry out COAGULASE test. Transfer repsentative suspect colony from agar surface to individual tubes, each contain 0.5ml of mammalian,
• preferably horse or rabbit plasma with or without additive.• Incubate in water bath at 37 °examine tubes at 3hr & subsequently at suitable interval up to 24hr.• If no coaggulation is observed sample meet requirment of test for absence of S.aures
CharacteristicSelective Medium Colonial Morphology Gram Stain Vogel-Johnson Black Surrounded by yellow Positive cocci Agar Medium zone (in clusters) Mannital-Salt Yellow colonies with yellow Positive cocci Agar Medium zones (in clusters) Baird-Parker Black, shiny, surrounded by Positive coccid Agar Medium clear zones 2 to 5 mm (in clusters)
• REFERENCES:• (1)INDIAN PHARMACOPIEA• (2)U .S. P• (3) BY: JAMES SWARBRICK ENCYCLOPEDIA OF PHARMACEUTICAL• TECHNOLOGY,THIRD EDITION ,VOL-1• (4) BY: GILBERT S. BANKER• MARTIN M . RIGER• PHARMACEUTICAL DOSAGE FORM DISPERSE SYSTEM, VOL-2