CONTENT Definition Objective Preliminary testing Different media Sampling Methods Total aerobic microbial count

Membrane filtration Plate count Serial dilution

Tests for specified micro-organism Escherichia coli Salmonella species Pseudomonas aeruginosa Staphylococcus aureus

Application References

DEFINITION

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.

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OBJECTIVE[1]

Microbial limit tests are designed to estimate the number of viable aerobic organisms present in pharmaceutical products and raw materials.

The microbial limit testing of raw material as well as finished pharmaceutical products can help to determine whether the product complies with requirement of IP.

The most care must be taken while performing microbial test so that contamination from outside can be avoided.

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Preliminary Testing[7]

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The method given herein are invalid unless it is demonstrated that the 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

  a

1 ml of NLT 10-3 dilutions of 24 hr broth culture

+

buffer solution pH 7.2, fluid soyabean-casein digest medium or fluid lactose medium

+

test material

inoculate incubate

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Continue . . .

If the organisms fail to grow in the relevant medium the procedure should be modified by

(a) increasing the volume of diluents with the quantity of test material remaining the same, or

(b) incorporating a sufficient quantity of a suitable inactivating agent in the diluents, or

(c) combining the above modifications so as to permit growth of the organisms in the media.

If inhibitory substances are present in the sample, 0.5% of soya lecithin and 4% of polysorbate 20 may be added to the culture medium.

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Continue . . .

Alternatively, repeat the test as described using fluid casein digest-soya lecithin- polysorbate 20 medium to demonstrate neutralization of preservatives

OR

other antimicrobial agents in the test material.

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Media[1]

Baird – Parker Agar Medium Bismuth Sulphite Agar Medium Brilliant Green Agar Medium Buffered Sodium Chloride-Peptone Solution pH 7.0 Casein Soyabean Digest Agar Medium Cetrimide Agar Medium Desoxycholate-Citrate Agar Medium Fluid Casein Digest-Soya Lecithin-Polysorbate 20

Medium

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Continue: Fluid Lactose Medium Lactose broth Medium Levin Eosin-Methylene Blue Agar Medium MacConkey Agar Medium (culture of enterobacteria) MacConkey Broth Medium Mannitol Salt Agar Medium Nutrient Broth Medium Nutrient Agar Medium Pseudomonas Agar Medium for Detection of

Flourescein

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Continue: Pseudomonas Agar Medium for Detection of

Pyocyanin Sabouraud Dextrose Agar Medium Sabouraud Dextrose Agar Medium with Antibiotics Selenite F Broth Fluid Soyabean-Casein Digest Medium Tetrathionate-Bile-Brilliant Green Broth Medium Triple Sugar-Iorn Agar Medium Urea Broth Medium Vogel-Johnson Agar Medium Xylose-Lysine-Desoxycholate Agar Medium

(selective media for salmonella)

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Notes[1]

Where agar is specified in a formula, use agar that has a moisture content of not more than 15%.

Where water is called for in a formula, use purified water.

The media should be sterilized by heating in an autoclave at 115°c for 30 minutes.

In preparing media dissolve the soluble solids in the water, using heat if necessary, to effect complete solution an add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the required pH in the medium when it is ready for use. Determine the pH at 25°c ± 2°c

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Some common ingredients and its use[8]

Agar: A solidifying agent which is a complex polysaccharide

derived from marine algae. It has no nutritional value in media. It is bacteriological inert. It is stable at different temperature used for incubation.

Peptones: Protein is large, relatively insoluble molecules that a

minority of organism can utilized directly, but a partial digestion by acid or enzyme reduces protein to shorter chain of amino acids called peptone. These small, soluble fragments can be digested by most bacteria.

It should be stored in a tightly closed container as it is hygroscopic in nature.

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Continue:

Meat extract: It is prepared from fresh meat by hot water extraction. It contains water soluble constituents of animal tissue

that is carbohydrates, organic nitrogen compound, water soluble vitamins and mineral salts.

Yeast extract: It is particularly rich in vitamin B. It also contains carbohydrates, amino acids, inorganic

salts, growth factors.

 

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TERMS[8]

Culture medium: A nutrient material prepared for growth of micro-organism in a laboratory is called culture medium.

Culture: The microbes can grow and multiply in or on a culture medium are referred to as a culture.

Nutrient broth: if the complex media is in liquid form, it is called nutrient broth.

Nutrient agar: when agar is added to media, it is called nutrient agar.

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TYPES OF CULTURE MEDIA[8]

Selective: suppression of unwanted microbes and encouraging desired microbes.

Differential: differentiation of colonies of desired microbes from others.

Enrichment: similar to selective but designed to increase numbers of desired microbes to detectable levels.

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SAMPLING[1]:

Use 10 ml or 10 g specimens for each of the tests specified in the individual monograph.

PRECAUTION: The microbial limit tests should be carried out under The microbial limit tests should be carried out under

conditions designed to conditions designed to avoid accidental contamination avoid accidental contamination during the test. during the test.

The precautions taken to avoid contamination must be The precautions taken to avoid contamination must be such that, they such that, they do not adversely effect any micro do not adversely effect any micro organism organism that should be revealed in the test.that should be revealed in the test.

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METHODS[1]:

 1. TOTAL AEROBIC MICROBIAL COUNT:

Unit: cfu/ml or gm[3]

colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal numbers. Unlike direct microscopic counts where all cells, dead and living, are counted, CFU measures viable cells. For convenience the results are given as CFU/ml (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids.

This technique allows the determination of the number of CFU per ml in the sample, and thus the degree of contamination in samples of water, vegetables, soil or fruits, and in industrial products and equipment.

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Pre-treatment of sample:- To dissolve or dilute the sample use

Phosphate buffer (pH 7.2) Sodium chloridepeptone buffer solution Fluid medium used for the test.

If not specified:- Use 10 g or 10 ml of the sample Adjust the test fluid to pH 6.8 Use the test fluid within one hour after

preparation.

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Water soluble products:

10 g or 10 ml of the sample + buffer or fluid medium

mix

make up to 100 ml.

If necessary, adjust the pH to about 7.

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Products insoluble in water (non-fatty) :

Take 10 g or 10 ml of the sample

grind

fine powder + buffer or fluid medium

suspend(use blender or surfactant to disperse )

Make it up to 100 ml

 

A suitable surface-active agent such as 0.1% w/v of

polysorbate 80 may be added to assist the suspension of

poorly wettable substances.

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Fatty products :

10 g or 10 ml of the sample + buffer or fluid medium

+surfactant (polysorbate 20 or polysorbate 80)

Prepare emulsion and make up to 100 ml.

If necessary, warm at a temperature not exceeding 40˚c to emulsify the sample.

Avoid warming for more than 30 minutes.

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A. Membrane filtration method

10 ml or dilution containing 1 gm sample membrane filter(50 mm in diameter,

pore size NGT 0.45 µm)

Residue

Wash it with buffered sodium chloride-peptone solution

pH 7.0 [For fatty substances add to the liquid polysorbate 20 or polysorbate 80.]

Transfer the filter on media for enumeration

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Membrane filtration assembly and A sterile screw-capped container[4][6]

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•Count the number of colonies that are formed. Calculate the number of micro-organisms per gm or per ml of the preparation being examined.

Laminar air flow unit[6]

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B. Plate count method[1]

a)Pour -plate method

b)Surface -spread method

a)Pour plate method :-

Take Petri dishes 9 to 10 cm in diameter

1 ml of the pretreated preparation + 15ml (15-20 ml as per U.S.P) of liquified media at NMT 45°c.

If necessary, dilute the pretreated preparation .

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b) Surface-spread method:-

Spread the pretreated preparation on the surface of the solidified media in a Petri dish of the same diameter .

Prepare at least two such Petri dishes using the same dilution and incubate.

If necessary dilute the pretreated preparation

For bacteria :- Count the 300 colonies per plate as the maximum consistent with good evaluation.

For fungi :- Calculate the results using plates with not more than 100 colonies.

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Plate showing the colony[11] colony counter[12]

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C. Serial Dilution Method (Multiple tube method)[9]

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. 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.

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.

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Continue: 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 the 9 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

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2. TESTS FOR SPECIFIED MICRO ORGANISMS

As per IP Escherichia coli Salmonella Pseudomonas aeruginosa Staphylococcus aureus

Preparetion of test fluid:- Proceed as described under the test for total aerobic

microbial count . Using lactose broth or medium which have no

antimicrobial activity in place of buffered sodium chloride-peptone solution pH 7.0.

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Escherichia coli[3][6] is a Gram negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2 and by preventing the establishment of pathogenic bacteria within the intestine.

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As per IPEscherichia coli[1]

Take sterile screw-capped container

add sample+50 ml of nutrient broth

shake

allow to stand for 1 hour (4 hours for gelatin)

Shake it again

Loosen the cap

Incubate at 36°c to 38°c for 18 to 24 hours

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Continue: Primary test – 1.0 ml culture to a 5 ml of MacConkey broth. Incubate

at 36°c to 38°c for 48 hours. If the contents of the tube shows acid and gas, carry out

secondary test.

Secondary test –

Add 0.1 ml of the contents to

(a) 5 ml of MacConkey broth, and

(b) 5 ml of peptone water

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Incubate in a water-bath at 43.5°c to 44.5°c for 24 hours and examine tube

(a) for acid and gas

(b) for indole For indole add 0.5 ml of Kovac’s reagent, shake well. If a red colour is produced in the reagent layer indole

is present. The presence of acid, gas and indole in the secondary

test indicates the presence of Escherichia coli.

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Continue: Carry out a control test by repeating the primary and

secondary tests adding 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli organisms, prepared from a 24 hour culture in nutrient broth, to 5 ml of MacConkey broth.

The test is not valid unless the results indicate that control contains Escherichia coli.

Kovac's reagent[10]: A reagent used to detect the presence of indole which is used in identification of bacteria.

Indole test[10] :The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to split indole from the amino acid tryptophan.

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Salmonella[3][6] is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria. Flagella which project in all directions (i.e. peritrichous). They cause illnesses like typhoid fever, paratyphoid fever, and the food borne illness.

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Salmonella[1]

Take sterile screw-capped container

add

sample+100 ml of nutrient broth

shake

allow to stand for 4 hour

Shake it again

Loosen the cap

incubate at 35°c to 37°c for 24 hours

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Primary Test - Add 1.0 ml of the culture to each of the two tubes containing

(a) 10 ml of selenite F broth

(b) Tetrathionate bile-brilliant green broth

Incubate at 36ºc to 38ºc for 48 hours. From each of these two cultures, subculture on at least

two of the following four agar media: bismuth sulphite agar, brillinat green agar, desoxycholate-citrate agar and xylosetysine desoxycholate agar.

Incubate the plates at 36ºc to 38ºc for 18 to 24 hours

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Test for salmonella:

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Continue: Upon examination, if none of the colonies conforms to

the description given in Table, the sample meets the requirements of the test for the absence for the genus Salmonella.

If any colonies conforming to the description in Table are produced, carry out the secondary test.

Secondary test: Subculture any colonies showing the characteristics

given in Table in triple sugar- iron agar by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle, and at the same time inoculate a tube of urea broth.

Incubate at 36ºc to 38ºc for 18 to 24 hours.

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Continue: The formation of acid and gas in the stab culture. The absence of acidity from the surface growth. The absence of a red colour in the urea broth.

Indicates the presence of salmonella Carry out the control test by repeating the primary and

secondary test using 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 salmonella organisms, prepared from a 24-hour culture in nutrient broth.

The test is not valid unless the results indicate that the control contains Salmonella.

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Pseudomonas aeruginosa[3]

It is a gram negative bacterium which can cause disease in humans and non-human animals. It is found in soil, water, skin flora, and most man-made environments throughout the world.

It uses a wide range of organic material for food; in animals, the versatility enables the organism to infect damaged tissues or people with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonization occur in critical body organs such as the lungs, the urinary tract, and kidneys, the results can be fatal.

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Continue[3][6]

P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Pyocyanin refers to "blue pus", which is a characteristic of infections caused by Pseudomonas aeruginosa.

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1 ml or 1 gm containg sample + 100 ml of fluid soyabean-casein digest medium

Mix Incubate at 35ºc to 37ºc for 24 to 48 hours.

If growth is present, streak a portion of the medium on

the surface of petri dishes of cetrimide agar medium. Cover and incubate at 35ºc to 37ºc for 18 to 24 hours. If upon examination, none of the plates contains colonies

having the characteristics listed in Table for the media used, the sample meets the requirement for freedom from Pseudomonas aeruginosa.

If any colonies conforming to the description in Table are produced, carry out the oxidase and pigment tests.

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Tests for Pseudomonas aeruginosa

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Oxidase and pigment tests:

Streak representative suspect colonies from agar surface of cetrimide agar on the surfaces of pseudomonas agar medium for detection of fluorescein and for detection of pyocyanin contained in Petri dishes.

Cover and invert the inoculated media and incubate at 33º to 37º for not less than 3 days. Examine the streaked surfaces under ultra-violet light. Examine the plates to determine whether colonies conforming to the description in previous table are present.

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Continue:

If 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 colony; if there is no development of a pink color, changing to purple, the sample meets the requirements of the test for the absence of Pseudomonas aeruginosa.

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Staphylococcus aureus [3][6]

S. aureus are gram positive cocci (in clusters) which can cause a range of illnesses from minor skin infections, such as pimples, scalded skin syndrome, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, chest pain, bacteremia, and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections.

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Proceed as described under Pseudomonas aeruginosa. If, upon examination of the incubated plates, none of them contains colonies having the characteristics listed in Table for the media used, the sample meets the requirements for the absence of Staphylococcus aureus.

If growth occurs, carry out the coagulase test.

Staphylococcus aureus[1]

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Continue:

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Continue: Coagulase test: Transfer representative suspect colonies from the agar

surface of any of the media listed in Table to individual tubes, each containing 0.5 ml of mammalian- preferably rabbit or horse-plasma with or without additives.

Incubate in water-bath at 37º examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours.

If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of Staphylococcus aureus.

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APPLICATION MICROBIOLOGICAL ASSAY OF ANTIBIOTIC

DRUGS DISINFECTION EFFICACY TEST OF

DISINFECTANTS AND ANTISEPTICS STERILITY TEST OF STERILISED

PHARMACEUTICALS TESTS FOR MICROBIAL LIMITS FOR NON-

STERILE PHARMACEUTICAL AND BIOLOGICAL PRODUCTS

TESTING OF WATER

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REFERENCES1. The Indian pharmacopoeia, IP-1996, volume-2, Indian

pharmacopoeia commission, Ghaziabad, India, 1996. Appendix-9.4,A-110:A-117

2. The United States Pharmacopoeia,USP-25 NF-20, 2002, p 1873-1878

3. http://en.wikipedia.org

4. http://productimage.tradeindia.com/00530916/s/1/Membrane-Filtration-Assembly-PC-Single-Unit-.jpg

5. http://en.wikipedia.org/wiki/Candida_albicans

6. http://www.google.co.in/image

7. http://www.usp.org/pdf/EN/meetings/asMeetingIndia2009/session2Track2Tirumalai_1.pdf

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Continue:8. Gerard J. Tortora, Berdell R. Funke, Christine L. Case,

Microbiology An Introduction, Eighth Edition, Pearson Education, 2005, p 193-199

9. http://ffcr.or.jp/zaidan/FFCRHOME.nsf

10. http://en.wikipedia.org/wiki/Indole_test

11. http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Yeast_Plate_Count/07_yeast_0.2mL_plate_P7201181.jpg

12. http://chemicoscientific.com/images/Digital_Colony_Counter.jpg

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