application of microbiology in pharma qc industry

1. APPLICATION OF MICROBIOLOGY
IN ORAL LIQUIDS
BY
RUDRA PRASAD CHAKRABORTY
(B.SC, B.TECH)

2. INTRODUCTION
 The microbiological quality of the pharmaceutical excipients used to
manufacture pharmaceutical & over the counter drug products may
significantly affect the outcome of individual processing steps &
microbiological attributes of the final drug products. Unlike active
pharmaceutical ingredients, excipients purchased from multiple suppliers &
in many cases are produced for the food, cosmetics, & not specifically for
the pharma industry so the microbiological quality control becomes
necessary for human health care purposes & company goodwill.

3. WHAT IS ORAL LIQUID
 Liquid preparations for oral use are usually solutions, emulsions or suspensions containing one or more active
ingredients in a suitable vehicle; they may in some cases consist simply of a liquid active ingredient used as such.
Liquid preparations for oral use are either supplied in the finished form or with the exception of Oral emulsions, may
also be prepared just before issue for use by dissolving or dispersing granules or powder in the vehicle stated on
the label. The vehicle for any liquid preparation for oral use is chosen having regard to the nature of the active
ingredient(s) and to provide organoleptic characteristics appropriate to the intended use of the preparation. Liquid
preparations for oral use may contain suitable antimicrobial preservatives, antioxidants and other excipients such
as dispersing, suspending, thickening, emulsifying, buffering, wetting, solubilizing, stabilizing, flavouring and
sweetening agents and authorized
 colouring matter. Liquid preparations for oral use may besupplied as multidose or as single-dose preparations.
Each dose from a multidose
 container is administered by means of a device suitable for measuring the prescribed volume. The device is usually
a spoon or a cup for volumes of 5 mL or multiples thereof, or an oral syringe for other volumes or, for Oral drops, a
suitable dropper. Additional information. Liquid preparations for
 oral use are often the dosage form of choice for paediatric use. Owing to the wide range of liquid preparations for
oral use and their long history of use, a variety of terms has been used to describe different members of this
category of preparation. These terms, which are not mutually exclusive and the definitions of which have changed
over time, include elixirs, linctuses, milks, mixtures and syrups. Such terms are still used within the titles of certain

4. CLASSIFICATION OF LIQUIDS
LIQUIDMonophasic
Oral use
Solution
Draught
Drops
Linctuses
Syrups
Elixirs
External use Parenteral Special use
Used in Oral
cavity
THROAT PAINTS
GLYCERITES
MOUTH WASHES
THROATSPRAYS
Used in other than
oral cavity
DOUCHES
ENEMAS
EYE DROPS
EYE LOTIONS
NASAL DROPS
INHALANTS
Biphasic
Liquid in liquid
Oral use
EMULSION
External
use
Liniments
Solids in liquid
Parenteral Oral
SUSPENSION
External
Lotion

5.  Advantages
 Immediately available for absorption.
 Administration convenient, particularly for infants, psychotic patients.
 Easy to color, flavor & sweeten.
 Liquids are easier to swallow than solids and are therefore particularly acceptable for
pediatric patient.
 A solution is an homogeneous system and therefore the drug will be uniformly
distributed throughout the preparation.
 Some drugs like aspirin, KCl can irritate gastric mucosa if used orally as a solid
dosage forms. But this effect can be reduce by solution system.
ADVANTAGES & DISADVANTAGES

6. ADVANTAGES & DISADVANTAGES
 Disadvantages
 Bulky than tablets or capsule, so difficult to carry transport.
 Less stable in aqueous system. Incompatibility is faster in solution than solid dosage
form.
 Patients have no accurate measuring device.
 Accident breakage of container results in complete loss.
 Solution often provide suitable media for the growth of microorganisms.
 The taste of a drug, which is often unpleasant, is always more pronounced when in
solution than in a solid form.

7. ORAL LIQUIDS
S.N. Liquid, External Preparation & Disinfectants
1 Trimethoprim & Sulphamethoxazole oral suspension IP
2 Paracetamol Syrup IP
3 Povidone–iodine Solution IP
4 Benzyl Benzoate Application IP
5 Piperazine Citrate Syrup IP
6 UVLON (Hospital Concentrate)
7 FLO-PHIL (THE PHENYL)
8 Nitrofurazone Ointment 0.2% w/w

8. EXAMPLE OF ORAL LIQUIDS

9. WHAT IS MICROBIOLOGY
 MICROBIOLOGY IS STUDY OF MICROBES SUCH AS
BACTERIA,VIRUS, PROTOZOA ETC
 THESE ORGANISMS CAN ONLY BE SEEN UNDER THE MICROSCOPE
 MICROBES HAVE EXSISTED ON EARTH FOR BILLION S OF YEARS
AGO & WERE HERE BEFORE ANIMAL & PLANT LIFE BEGINS
 MICROBES ACT AS GUARDIAN OF OUR PLANET ENSURING THAT
KEY MINERALS SUCH AS CARBON & NITROGEN ARE CONSTANTLY
RECYCLED
 THE STUDY OF MICROBIOLOGY STARTED AFTER 17th CENTURY
WHEN ROBERT HOOKE INVENTED MICROSCOPE &
MICROBIOLOGISTS LATER DISCOVERED THAT MICROBES ARE
FOUND JUST ABOUT ANYWHERE, LOUIS PASTUER IS CONSIDERED
AS THE FATHER OF MICROBIOLOGY.

10. Scope of a pharmaceutical
microbiology lab
 A pharmaceutical microbiology lab is involved in :
• Sterility Testing
• Detection, isolation and identification of microorganism
• Examining different samples with the use of microorganisms

11. ORAL MICROBIOLOGY
 The discipline of Oral Microbiology is a clinical specialty, undertaken by laboratory-based personnel,
which is concerned with the diagnosis and assessment of diseases of the oral and maxillofacial region.
It is a branch of Medical Microbiology and in common with Medical Microbiologists, Oral Microbiologists
provide reports and advice based on interpretation of microbiological samples.
 This document indicates the breadth and depth of the specialty of Oral Microbiology. The majority of
specialists are senior academics with honorary consultant status based in Dental Schools and the
majority of trainees will be competing for such posts. As such, trainees will be required, for their
academic advancement, to obtain higher academic degrees related to proficiency in research, as well
as specialist training in Oral

12. SAFETY CONCERNS &
REQUIREMENT OF QUALITY CONTROL UNIT
 SAFETY CONCERNS:-
 An important aspect of Good Manufacturing Practice for all pharmaceutical products is assuring the quality of all the starting
materials used. The need for analytical testing to check the identity and quality of starting materials is explained in detail in section
14 of the current WHO GMP guidelines 1 . Failure to ensure that starting materials are of the required quality can have very
serious consequences. Increasingly countries are dependent on the importation of starting materials for use in the production of
medicines. Starting materials often change hands many times before reaching the manufacturer of the final marketed product and
there are many opportunities for the material to undergo relabelling along the distribution and trade chain (see WHO Guideline on
Good Trade and Distribution Practices for Pharmaceutical Starting Materials1). As a result, starting materials required for
production of pharmaceutical products can become contaminated or materials may be supplied that no longer correspond to what
is stated on the label in terms of quality or identity, either accidentally or as a result of negligence and sometimes fraud. The most
documented incidents of contamination involve liquid preparations for oral use manufactured with excipients such as glycerol and
propylene glycol that have been contaminated, adulterated or mixed up with diethylene glycol. Such incidents have been
 responsible for hundreds of deaths throughout
 the world (see, for example, editorial in WHO
 Bulletin 2001, 79 (2)). Ingestion of diethylene
 glycol often leads to death through kidney

13. IMPORTANCE OF MICROBIOLOGY IN
PHARMACEUTICAL INDUSTRY
 MICROBIAL CONTAMINATION IN PHARMACEUTICAL PRODUCTS HAS
MASSIVE CONSEQUENCES & COMPANY SUFFERS ENORMOUS
DAMAGE WHEN A DRUG PRODUCT IS RECALLED & THE DIRECTHIT
WILL CAUSE AS LOSS OF PRODUCT SALES, DECREASED
CUSTOMER CONFIDENCE, DAMAGE TO THE LEGAL PROCEEDINGS.
 QUALITY CONTROL IS AN ESSENTIAL FUNCTION OF THE
PHARMACEUTICAL INDUSTRY.DRUG MANUFACTURERS MUST GO
THROUGH THE THOROUGH TEST OF RAW
MATERIALS,PROCESSES,EQUIPMENT,TECHNIQUES,ENVIROMENTS
& PERSONAL IN ORDER TO ENSURE THEIR FINISHED PRODUCTS
ARE CONSISTENT, SAFE, EFFECTIVE AND PREDICTABLE.

14. RECOMMENDED ACCEPTANCE
CRITERIA FOR ORAL PREPARATION
 acceptance criteria for oral dosage forms, other than herbal medicines, containing raw material of natural
origin for which antimicrobial pretreatment is not feasible and for which the competent authority accepts
TAMC of the raw material exceeding 103 CFU/g or CFU/mL (see Table 1), were exempted from the PDG
harmonization. The text is provided to give information and guidance and is not regarded as an analytical
requirement. The acceptance criteria do not apply to herbal medicines (i.e. herbs, herbal materials, herbal
preparations and finished herbal products). For such preparations reference should be made to Quality
control methods for herbal materials: Determination of microorganisms (World Health Organization, 2011),
and WHO guideline on assessing quality of herbal medicines with reference to contaminants and residues
(Determination of microbial contaminants; Annex 5 – Determination of microorganism) (World Health
Organization, 2007). The presence of certain microorganisms in non-sterile preparations may have the
potential to reduce or even inactivate the therapeutic activity of the product and has a potential to adversely
affect the health of the patient. Manufacturers have, therefore, to ensure a low bioburden of finished dosage
forms by implementing current guidelines on good manufacturing practice (GMP) during the manufacture,
storage and distribution of pharmaceutical preparations.Microbial examination of non-sterile products
microorganisms. Acceptance criteria for non-sterile pharmaceutical products based upon the total aerobic
microbial count (TAMC) and the total combined yeasts/moulds count

15. CONDITIONS FOR QC TESTS IN
PHARMACEUTICAL INDUSTRY
 THE PLANT FUNCTIONS AS PER WHO/SOP/GMP GUIDELINES
 DIFFERENT CONDITIONS AS PER STANDARD GUIDELINES FOR
AYURVEDIC & ALLOPATHY MEDICINES
 AYURVEDIC- TOTAL AEROBIC COUNT 100000 CFU/ml.
 TOTAL FUNGAL COUNT 1000 FUNGI/ml
 ALLOPATHY- TOTAL AEROBIC COUNT 1000 CFU/ml.
 TOTAL FUNGAL COUNT 100 FUNGI/ml.
 PREMISES SHOULD BE CAREFULLY MAINTAINED ENSURING THAT
REPAIR AND MAINTANANCE OPERATIONS DO NOT PRESENT ANY
HAZARD TO THE QUALITY OF PRODUCTS
 TEMPARATURE, HUMIDITY & VENTILATION SHOULD BE APPROPRIATE
SUCH AS THEY DO NOT ADVERSELY AFFECT DIRECTLY OR INDIRECTLY.
.

16. QUALITY CONTROL UNIT
microbiology
chemical
Packaging

17. LIST OF EQUIPMENTS,MATERIALS &
SAMPLES IN THE TESTS
 EQUIPMENTS- HOT AIR OVEN, AUTOCLAVE,INCUBATORS,LAMINAR
FLOW HOODS,MICROSCOPES,PIPETTES,,TIPS,TEST-TUBES,PETRI
DISHES,WEIGH MACHINE,SLIDES,INNOCULATION LOOPS &
MEDIUMS( BOTH SOLID & LIQUID)
 MATERIALS-BUFFER PEPTONE MEDIUM, FLUID LACTOSE MEDIUM,
MCCONKEY BROTH,NUTRIENT BROTH & AGAR,SALENITE F BROTH,
XYLOSE LYCIN CHOCOLATE AGAR, SOYABEAN KESIN DIGEST AGAR
MEDIUM, SABARATOSE DEXTROSE AGAR, BISMATH SULPHIDE
AGAR,TRYTOPHAN SUGAR IODINE AGAR.
 SAMPLES- BOTH AYURVEDIC & ALLOPATHY SUCH AS
CARMINOZYME,JVINE,LIVOSIN ETC.

18. IMAGES OF EUIPMENTS & MATERIALS

19. PROCEDURE
MICROBIOLOGICAL TESTING OF NON STERILE PRODUCTS:-
 "The significance of microorganisms in non-sterile pharmaceutical
products should Be evaluated in terms of the use of the product, the
nature of the product, and the potential hazard to the user." The USP
recommends that certain categories be routinely tested for total
counts and specified indicator microbial contaminants. For example
natural plant, animal and some mineral products for Salmonella, oral
liquids for E.Coli, topicals for P. aeruginosa and S. Aureus, and
articles intended for rectal, urethral, or vaginal administration for
yeasts and molds. A number of specific monographs also include
definitive microbial limits.Therefore, each company is expected to
develop microbial specifications for their non-sterile products.

20. PROCEDURE
 MEDIA:-
 Begin the inspection with a review of analyses being conducted and inspect the plates and tubes of
media being incubated (caution should be exercised not to inadvertently contaminate plates or tubes of
media on test). Be particularly alert for retests that have not been documented and "special projects" in
which investigations of contamination problems have beenidentified. This can be evaluated by
reviewing the ongoing analyses (product or environmental) for positive test results. Request to review
the previous day's plates and media, if available and compare your observations to the recorded entries
in the logs. Inspect the autoclaves used for the sterilization of media. Autoclaves may lack ability to
displace steam with sterile air. For sealed bottles of media, this would not present a problem. However,
for non-sealed bottles or flasks of media, non- sterile air has led to the contamination ofmedia. In
addition, autoclaving less than the required time will also allow media associated contaminants to grow
and causea false positive result. These problems may be more prevalent in laboratories with aheavy
workload. Check the temperature of the autoclave since overheating can denature and even char
necessary nutrients. This allows for a less than optimal recovery of already stressed microorganisms.
The obvious problem with potential false positives is the inability to differentiate between inadvertent
medium contamination and true contamination directly associated with the sample tested.

21. PROCEDURE
 STERILITY TESTING:-
 contaminated or potentially contaminated drug products made by aseptic processing
 and later recalled was also made available. Many of the investigations/inspections of the recalled
products started with a list of Initial sterility test failures. FDA review of the manufacturer's production,
controls, investigations and their inadequacies, coupled with the evidence of product failure (initial
sterility test failure) ultimately led to the action. The USP points out that the facilities used to conduct
sterility tests should be similar to those used for manufacturing product. The USP states, "The facility for
sterility testing should be such as to offer no greater a microbial challenge to the articles being tested
than that of an aseptic processing production facility". Proper design would, therefore, include a
gowning area and pass-through airlock. Environmental monitoring and gowning should be equivalent to
that used for manufacturing product.

22. PROCEDURE
METHODOLOGY & VALIDATION OF TEST:-
 During inspections, including pre-approval inspections, evaluate the
methodology for microbiological testing. For example, we expect test
methods to identify the presence of organisms such as Pseudomonas
cepacia or other Pseudomonas species that may be objectional or
present a hazard to the user. Where pre-approval inspections are being
conducted, compare the method being used against the one submitted in
the application. Also verify that the laboratory has the equipment
necessary to perform the tests and that the equipment was available and
in good operating condition on the dates of critical testing. The USP
states that an alternate method may be substituted for compendial tests,
provided it has been properly validated as giving equivalent or better
results.

23. LIST OF TESTS
E.COLI TEST
SALMONELLA TEST
PSEUDOMONAS AERUGONSA
TEST
STAPHYLOCOCCUS AURUES
TEST

24. E.COLI TEST
Place the prescribed quantity in a sterile screw capped
container, add 50 ml of nutrient broth, shake allow to stand
for 1 hour( 4 hours for gelatin) & shake again.loosen the
cap & incubate at 37C for 18-24 hours.
PRIMARY TEST- add 1.0 ml of the enrichment culture to a
tube containing 5ml of macconkey broth. Incubate in a
water bath at 36-38C for 48 hours. If the contents of the
tube show acid & gas carry out secondary test.

25. E.COLI TEST
SECONDARY TEST- Add 0.1 ml of the tubes containing(a) 5ml macconkey broth &(b) 5ml of peptone
water . incubate in a water bath at 43.5-45C for 24 hours & examine the tube(a) for acid and gas & (b) for
indole. To test for indole, add 0.5 ml of kovac’s reagent, shake well and allow for 1min, if a red color is
produced in the reagent layer indole is present. The presence of acid & gas & of indole in the secondary
test indicates the presence of E.coli.
Carry out a control test by repeating the primary & secondary tests adding 1.0ml of the enrichment culture
and volume of broth containing 10-50 E.coli organisms, prepared from a 24 hour culture in nutrient broth,
to 5ml of macconkey broth. The test is not valid unless the results indicate that the control contains E.coli.

26. SALMONELLA TEST
 Transfer a quantity of the pretreated preparation being examined containing 1g or 1ml of the product to
100ml of nutrient broth in a sterile screw capped jar ,shake & allow to stand for 4 hours & shake again,
loosen the cap and incubate at 35-37C for 24 hours .
 PRIMARY TEST- Add 1.0 ml of the enrichment culture to each of the two tubes containing (a) 10ml of
selenite broth & (b) tetrathionate bile brilliant green broth and incubate at 36-38C for 48 hours. From
each of these two cultures subculture atleast two of the following four agar media: bismuth sulphate agar,
brilliant green agar, deoxychocolatecitrate agar, xylose-lycin-deoxychocolate agar. Incubate the plates at
36-38C for 18-24 hours. Upon the examination if none of the colonies conforms to the description, thre
sample meets the requirements of the test for the absence of the genus salmonella.
 If any colonies conforming salmonella produced , carry out the secondary test.

27. SALMONELLA TEST
SECONDARY TEST- subculture any colonies showing the characteristics given
in triple sugar iron agar by first inoculating the surface of the slope and
then making stab culture with same inoculating needle, and at the
same time inoculate a tube of urea broth . Incubate at 37C for 18-24
hours. The formation of acid & gas in stab culture (with or without
concomitant blackening) and the absence of the acidity from the
surface growth in the triple sugar iron agar, together with the absence
of red color in the urea broth , indicate the presence of salmonella . If
acid but no gas is prodecd In the stab culture , the identity of the
organisms should be confirmed by agglutination test.

28. TEST FOR SALMONELLA
MEDIUM DESCRIPTION OF COLONY
Bismuth sulphate agar black or green
Brilliant green agar small, transparent/opaque
generally pinkish /white
Deoxy chocolate-citrate agar with/without blak centres
Xylose-lysine-deoxychocolate with or without red/ black centres
Agar

29. PSEUDOMONUS
AERUGINOSA

 Pretreat the preparation being examined as described & inoculate 100ml of fluid soyabean-casein
digest medium with a quantity of the solution , suspension or emulsion thus obtained containing 1g of
the preparation being examined. Mix & incubate at 35-37C for 24-48 hours. Examine the medium for
growth, steak a portion on the surface of cetrimide agar medium, each plated on petri dishes. Cover &
incubate at 35-37C for 18-24 hours.

 If upon examination , none of the plates contains colonies having the characteristics for the media used
the sample meets the requirement freedom from pseudomonas, if any colonies conforming the
description than carry out the oxidase & pigent test.


30. PSEUDOMONUS
AERUGINOSA
 Streak representative suspect colonies from the aar surface of cetrimide agar on the surface of
pseudomonas agar medium for detection of procyanin in petri dishes, cover & invert the inoculated
media & incubate at 33-37C for not less than 3days. Examine the streaked surfaces under ultra-violet
light. Examine the plates to determine whether colonies conforming to the description.
 If growth of suspect colonies occurs, place 2-3 drops of freshly prepared 1% w/v solution of N,N,N1,N1-
tetramethyl-4-phenylenediamene dihydrochloride on filter paper & smear with the colony; if there is no
development of pink color, changing to purple, the sample meets the requirements of the test for the
absence of pseudomonas aeruginosa.

31. TEST FOR PSEUDOMONAS
MEDIUM COLONIAL MORPHOLOGY FLUROSCENCE IN U-V OXIDASE TEST STAIN
Cetramide greenish greenish positive negative
Agar.
Pseudomonus yeelowish yellowish positive negative
Agar medium for
Detection of
Fluroscein.
Pseudomonus greenish blue positive negative
Agarmedium for
Detection of
procyanin

32. STAPHYLOCOCCUS AURUES
If upon the examination of the incubated plates, none of them contains colonies having the characteristics
for the media used the sample meets the requirements for the absence of staphylococcus aurues.
If the growth occurs carry out the coagulase test. Transfer representative suspect colonies from the agar
media of individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or
without additives. Incubate in water bath at 37C & examining the tubes for 3hours & subsequently for
suitable periods up to 24 hours . If no coagulation in any stage found the sample is free of
STAPHYLOCOCCUS AURUES.

33. Summary
 Using good practices in pharmaceutical microbiology labs will
ensure that all the tests are performed by experienced
professionals in a very hygienic and disinfected environment
using proper devices to get accurate results

34. REFERENCE
Jiraporn CHINGUNPITUK,
 Nanosuspension Technology for Drug Delivery,
 Walailak J Sci & Tech 2007; 4(2): 139-153.
V. B. Patravale, Abhijit A. Date and R. M. Kulkarni,
 Nanosuspensions: a promising drug delivery strategy
 JPP 2004, 56: 827–840
Rong Liu
 Water-Insoluble Drug Formulation
 Second Edition, page no. 122-123

35. THANK YOU