CONTENTS

PAGE NO.
SL.NO TOPIC

1 INTRODUCTION 1-12

2 AIM AND OBJECTIVE 13

3 REVIEW OF LITERATURE 14-25

4 METERIALS AND METHODS 26-34

5 RESULT 35-57

6 GRAPHS 58-63

7 DISCUSSION 64-65

8 SUMMARY 66-67

9 REFERENCE 68-71

10 PHOTOS 72-73

1
 INTRODUCTION

Microbiological assay is a technique where the potency (or)

concentration of a chemical substance may be determined by it's
effect on the growth on Microorganism, that may be to promote the
growth of microorganism by substances such as certain vitamins (or)
amino acids (or) to inhibit the growth in the case of antibiotics and
other substances having similar properties.

A refinement of the assay is seen over a period of time.

Despite the efforts and some success of chemistry in replacing
microbiological assays with chemical (or) physical method of assay,
microbiological assay remains today the only practical method of
assay for some antibiotic assays.

Estimation of the drug

Physical analysis in which the physical properties of the
formulation are taken into consideration. This method includes
spectrophotometer and chromatography.
Chemical analysis is based on the chemical nature and
recovery of the drug in question. It includes titrimetrv and colour
reactions: Instrumental analysis is carried out using various
instruments like spectrophotometer, GLC, etc.
Biological analysis depends on the response of the organism
for the drug. Biological methods estimate the biologically active
potency of the drug in a living system. So these methods consider
more sensitive and appropriate in terms of activity in a living
system.
Biological assay can be performed using animals, animal

2
preparations microbial cells.
In assay performed using animals, maintenance of sterile
animals is very difficult and the immune system of the animal may
interfere with the assay process (or) can influence the assay result.
Preparations and maintenance of animal preparation and isolated
living tissues without contamination is difficult. Comparatively it is
very easy to maintain and handle the microbial cells, aseptically
without contamination.
Measurement of the response of the assay can be carried out in many
ways.

 Turbidomentry (16 to 24 hours incubation)

 Gravimetry (Fungal growth measurement)
 Direct cell count in haemocytometer.
 Packed cells.
 Determination of pH (after 18 hrs of lactic acid bacteria)
 Measuring diffusion zone.

By whatever way growth is determined, a standard curve is

prepared by plotting growth (turbidometry, weight of mycelium,
acid produced. etc.,) Vs concentration of sample to be analyzed.
From the standard curve the amount of substance in question in
various levels of test solution is determined by interpolation

Basic technique of microbiological assay

There are two major practical techniques, the agar diffusion
assay and Tube assay. Which are applicable to both growth
promoting and growth inhibiting substances.

3
 In agar diffusion assay a nutrient agar gel is inoculated
uniformly with a suitable sensitive test organism. Active substances
are allowed to diffuse from an aqueous reservoir into the agar. Upon
incubation a zone is formed. The is one of inhibition in the case of
growth inhibitory substances and of exhibition in case of growth
prom oting substance. The width· of the zone depends on the
concentration of the active substance and provides the quantitative
basis for the assay.
In the tube assay the extent of growth of an organism in a liquid
nutrient medium is dependent on the quantity of active substance that is
added to and mixed uniformly with liquid medium. The techniques for
growth promoting and growth inhibiting substance differ in several
aspects. The growth or organism is measured interns of turbidity.
Table-1 Method for assay of antibiotics and microorganisims
METHOD
S. ATCC NCIM
ANTIBIOTIC CUP/TUBE ORGANISM
No No. No.
ASSAY
Staphylococcus
1 Amikacin Tube assay 29737 7447
aureus
Saccharomyces
2 Amphotercin - B Plate assay 9763 10716
cerevesiae
3 Bacitricin Plate assay Micrococcus 10240 7743
lute us
Candicidin
4 Tube assay Saccharomyces 9763 10706
Pseudomonas
5 Carbenici Hin Plate assay 25619 -
aeruf!.inosa
Staphylococcus
6 Chlorotetracycline Tube assay 29737 7447
aureus
Staphylococcus
7 Cloxacillin Plate assay 29737 7447
aureus
Staphylococcus
8 Demeclocyclin Tube assay 29737 7447
aureus
9 Dihydrostreptomycin Plate assay Bacillus subtilis 6633 8236
Staphylococcus
10 Doxycycline Tube assay 29737 7447
aureus

4
 Staphylococca
11 Gentamycin Plate assay 12228 8853
epidermids
Staphylococcus
12 Neomycin Plate assay 12228 8853
epiderm ids

13 Erythromycin Plate assay MicrOccus lute us 9341 8553

Saccharomyces
14 Nystatin Plate assay 9763 10716
cerevesiae
Bacillus
15 Oxytetracyclin Tube assay cereus 11778 10320
var. Mycoides
Staphylococcus
16 Penicillin - G Plate assay 29737 7447
aureus
17 Streptomycin Tube assay Bacillus subtilis 6633 8236
Staphylococcus
18 Tetracycline Tube assay 29737 7447
aureus
Borditella
19 Polymyxin - B Plate assay 4617 -
bronchoiseptica
8236
20 Vancomycin Plate assay Bacillus subtilis 6633
.
21 CWoramphenical Tube assay E.coil 10536 -

Table-2 Method for assay of vitamins and micro-organisms

SL NO VITAMIN MICRO ORGANISMS ATCC NO
USED FOR ASSAY

1 Vitamin B1 Lactobacillus fermenti 9338

2 Vitamin B2 (L.helveticus) 7469
3 Vitamin B5
a) Pantothenic acid Lactobacillus arobinoses 8041
(L.Plantarum)
b) Calcium 8041
pantothenate L.Plantarum
c) -
Pantothenol Leuconostoc mesentiroides

5
 Nicotinic acid
4 L.arabinosus
(niacin)
a) 8041
Nicotinic acid & Leuconostoc mesentiroides
b) -
Nicitinamide

Saccharomyces 9080
carisburgenesis
8043
5 Vitamin B6
Streptococcus faecails
7469
Lactobacillus caesai

6
Folic acid (free) 8043
a) Streptococcus faecails
Lactobacillus casei
7469
b) L.arabinosus
Total folic acid 8014
7 L. casei
Biotin 7469
S. carisburgenesis
9080

Lactobacillus leichaminnii
8 Vitamin B12 7830
a)
Ochromonas
True -
menhamensis(Protozoan)
b) Vitamin B12
-
Euglena gracilis(Algae)
Para-
9 aminobenzoic Neurospora crossa -
acid
S. carisburgenesis
10 Inosital 9084

Estimation of Calcium panthothenate

Calcium panthothenate content in the pharmaceutical
preparation can be estimated in Physics, Chemical and Biological
methods. In biological methods the estimation in done by
microbiological assay calcium pantothenate can be assayed
microbiologically with sensitivity using Lactobacillus plantarum
ATCC -08014 known as Lactobacillus arabinosus.

6
 Chemical methods are based on the fact that platothenic acid, its
salts and panthothenal undergo acid or alkaline hydrolysis. In alkaline
hydrolysis pantethonic acid and pantothenal yield -alanine and -alanol,
respectively which on acid hydrolysis are determined by suitable color
reactions.

Calcium panthothenate content in the pharmaceutical

preparation can be estimated in Physics, Chemical and Biological
methods. In biological methods the estimation in done by
microbiological assay calcium pantothenate can be assayed
microbiologically with sensitivity using Lactobacillus plantarum ATCC
-08014 known as Lactobacillus arabinosus.

Estimation of vitamin B 12
Vitamin Bl2 content in the pharmaceutical preparation can be
estimated in physical and biological methods. In biological methods,
the estimation is done by microbiological assay.
Vitamin Bl2 can be assayed with sensitivity using
lactobacillus liechmannii, Lactobacillus lactis and phagocyte
protozoan Ochromonas melhamensis.All these organisms can only
respond to true vitamin Bl2 but not to the pseudo types. The algae
Euglena gracilis is also used, but the incubation period is 4-7 years
which is disadvantageous to analyze specially lactobacillus species,
is a commonly available bacteria having gram positive rod shape.
Lactobacillus lactis is the important bacteria easily found in milk,
which is a good liquid diet for men. So it well be easy to analyze
vitamin Bl2 through this bacteria.

7
 Inspite of many methods development in pharmaceutical
analysis microbiological method of estimation of vitamin Bl2 is one of
the proffered methods. When the quantity of Bl2 is to less in a particular
formulation, the option left is microbiological assay due to its
sensitivity. Another advantage with microbiological assay is that it
represents a true effect on a living cell Cyanocobalamine is (5,6 -
dimethy ] benzimadazolyl Cyanocobalamide) .

Perhaps the discovery of no other vitamin has caused so much

worldwide intent and attention as the Government in 1948 of a red
crystalline compound, designated as vitamin B 12 which relieved the
symptoms of pernicious anemia, when given in minute dosage.

Vitamin Bl2 or the ant pernicious anemia factor is dark red

crystalline odorless, tasteless powder Cyanocobalamine is highly
hygroscope in a hydrous condition. Cyanocobalamine is soluble in 80
parts of water. Sparingly soluble in alcohol, in soluble in chloroform,
acetone and ether. Description of vitamin BI2 occurs on heating at
above pH 9. Cyanobalamine is heat stable in presence of dilute at pH 4-
7 at 100°C

In Cyanocobalamine - N group is co-ordinates to the cobalt

atom, but other forms also exists with -OH (Hydroxo Cobalamine_
-N02 (Nitrito -cobalmine) and HS04 (sulfito - cobamine) vitamin BI2
functions as cobmide coenzyme involved in many biochemical process
in biological systems.

Deficiency of Vitamin B 12 in humans results in loss of appetite,

failure of growth, macrocytic and magaloblastic anemia, pernicious
anemia and deranged metabolism in nervous tissue. Vitamin B 12 is
required around 1-2 micro grams per day for adults. The vitamin B 12 is
absorbed through gastro intestinal tract after binding to the intresic
factor. In blood it is found attached to transcobalamins.

8
 Vitamin BI2 is stored in liver and can diffuse across placenta.
The deficiency of BI2 occurs mostly due to the absence of intrensic
factor. Vitamin BI2 is abscenest in plant materials. Strptomyces group
of organisms produces it. Animal tissues like liver, kidney etc., are
good sources of this vitamin. Egg yolk and milk contains adequate of
this vitamin.

Doxycycline
Doxycycline hydroxide is(4s,4ar,5s,5ar,6r,12as)-4-dimethyl
amino1,4,4a,5,5a,6,11,12apenahydrox6methyl11,11dioxonapthacene
,2corboxamide,hydrochloride,hemiethanolate,hemihydrate,hemihydr
ate.an antimicrobial substance obtained from oxytetracycline or
metacycline.
It is a hygroscopic crystalline powder soluble in 3 parts of
water in the 4 parts methyl alcohol sparingly in alcohol particularly
in soluble in solutions of alkali hydroxides and carbonates.
Doxycycline hydrocloride should be kept in a tightly closed
container protected from light because even in he absence of light it
is gradually degraded on exposure to humid atmosphere the
decomposition being faster at higher temparature.

Antimicrobial action
Doxycycline hydrochloride is mainly bacteriostatic
with a broad spectrum of antimicrobial activity they interfere with
bacterial protein synthesis by reversibly binding 1030 s ribosome.
They are preventing the binding of aminoacyltransfer RNA to the m
RNA ribosome complex. they are usually active against
mycoplasmas rickettsias and spirochaetas and also against maney
aerobic and anaerobic gram positive and gram negative pathogenic

9
bacteria and some protozoa it is more effective then tetracyclines
against many species.

Absorption

Doxycycline hydrochloride is readily absorbed from the

gastrointestinal track. The absorption is not significantly affected by
the presence of food in the stomach or deodenum. It is biological
half life varies from about 15-22 hours. It is more lipid soluble from
tetracycline. It is readily absorbed and excreted more slowly than
most other tetracyclines and effective blood concentrations are
maintained for longer periods. So that, dosage once daily is crucially
adequate

Adverse effects

 It has a lower affinity for binding with calcium and it may cause
less tooth discoloration than other tetracycline’s
 Gastrointestinal effects including nousea, vomiting and diarrhea,
irritation of the mucosa may occur.
 It interfere with most defence mechanisms including depression
of chemoloxis,phagocytosis and intracellular metabolismof
polymorph nuclear leucocytes.
 Intravenous Doxycycline hydrochloride causes allergic reaction.
 It cause photosensitivity -reaction.!t is a frequent cause.
 It also impairs the material learned during the night (USP 23, 19
as)

Erythromycin

The macrolides are hydroxylated macrocyclic lactones

containing 12 - 20 carbon atoms in the primary ring. There are 37
known members of this class but only one, erythromycin and ifs
derivatives is of clinical importance. Macrolides binds to the 50 s
subunit of the bacterial ribosome. The main effect on grampositive
organisms is the grater permeation of the cell membrane. Macrolides
do not bind to mammalian ribosomes.

Erythromycin is produced by S. Erythreus. Erythromycin

estolate is white, crystalline power, odourless and tasteless. It is

10
insoluble in water, freely soluble in ethanol (96%), methanol and
chloroform.

About 20 % erythromycin estolate is absorbed as the base and 80 % as

ester, the later undergoes hydrolysis. Further hydrolysis at bacterial cell
level contributes to the activity of the drug.

Erythromycin is narrow spectrum antibiotic and most effective

against gram positive cocci, mycoplasma and the agent of legionnaires
disease are inhibited any low concentrations of the drug. It is active
against bacteria that have developed resistance to streptomycin. It
shows low activity against some of the important disease causing gram
negative organism group. There is no cross resistance between
erythromycin and other macrolides. It is the drug of choice in the
treatment of whooping cough, diphtheria, diarrhoea caused by
campylobactor and pneumonia from mycoplasma (or) legionella
infections. Erythromycin serves mainly as a backup drug when
penicillin allergy occurs. A large number of "Staphylococci" have
acquired resistance to the drug.

adverse effects

Adverse reactions attributable to erythromycin therapy are

uncommon and usually of little consequences. Nausea, vomiting and
occasionally diarrhea and stomatitis may occur with large doses.
Hepatic dysfunction with (or) without jaundice has occurred in
association with erythromycin estolate administration chiefly in adults.

Tetracycline
Tetracycline hydrochloride is a (4s, 4as, 5 as, 6 as, 12 as) - 4
dimethylamino - 1, 4, 4a, 5, 5a, 6, 11, I2a - actahydro - 3, 6, 10, 12a
pentahydroxy - 6 - methyl -I, 11- dioxnaphthalene - 2 - carboxamide
hydrochloride. It is an antibacterial antibiotic. It is obtained from
streptomyces species, cultured in an appropriate nutrient medium.

Tetracycline's contains variable quantity of water; it is a yellow,

odourless, crystalline powder. It is moderately hygroscopic, stable in
air, but exposure to strong sunlight, moist air causes it to darken,

11
potency affected in solutions of pH below 2 and is rapidly destroyed by
alkaline hydroxide solutions.
Since hydrochloride is most soluble form of tetracyclines, it is used for
parental administration and in solutions for tropical use.

Antimicrobial action

The tetracyclines are broad-spectrum antibiotics. They are

mainly bacteriostatic. They bind to the 30-s ribosomes and prevent
tRNA from combining with mRNA. Thus protein synthesis is inhibited.
The tetracyclins have activities against gram positive and gram
negative, mycobacteria, mycoplasma, treponema, leptospira,
actinomycetes, chlamydiae and plasmodia. The acid fast
mycobacterium tubercle is poorly susceptible. Various streptococci and
penumococci are resistant. The incidence of resistance among various
gram negative bacteria is high.

12
 AIM AND OBJECTIVES OF THE STUDY

By keeping these properties in view the present investigation

carried out with the following objectives.
 Microbiological assay of Cynocobalamine and Erythromycin by
using Cup plate assay method.
 Microbiological assay of pantothenic acid, doxycyclin,
Tetracycline by turbidometric assay

13
 REVIEW OF LITERATURE

We developed a kinetic, 96-well turbidimetric procedure that

is capable of testing the antimicrobial properties of six human
defensins concurrently on a single microplate. The defensins were
prepared by solidphase peptide synthesis and tested against gram-
positive bacteria (Staphylococcus aureus and Bacillus cereus) and
gram-negative bacteria (Enterobacter aerogenes and Escherichia
coli). Analysis of the growth curves provided virtual lethal doses
(vLDs) equivalent to conventional 50% lethal doses (LD50s),
LD90s, LD99s, and LD99.9s obtained from colony counts. On the
basis of their respective vLD90s and vLD99s, the relative potencies
of human myeloid defensins against S. aureus were HNP2 HNP1 >
HNP3 > HNP4. In contrast, their relative potencies against E. coli
and E. aerogenes were HNP4 HNP2 HNP1 HNP3. HD5 was as
effective as HNP2 against S. aureus and as effective as HNP4
against the gram-negative bacteria in our panel. HD6 showed little or
no activity against any of the bacteria in our panel, including B.
cereus, which was highly susceptible to the other five defensins. The
assay described provides a quantitative, precise, and economical way
to study the antimicrobial activities of host-defense peptides. Its use
has clarified the relative potencies of human defensins and raised
intriguing questions about the in vivo function(s) of HD6 (bryan
Ericksen et al.,2004).

The correlation and the level of agreement between the

standardized agar dilution and the agar disk diffusion methods for
antimicrobial susceptibility testing of Campylobacter were
investigated. A high-level agreement between the two methods was

14
evident for aminoglycosides and fluoroquinolones, while a low-level
agreement was observed for other antibiotics (Taradon
Luangtongkum et al.,2006).

Telavancin (TD-6424) is a novel lipoglycopeptide that

produces rapid and concentration-dependent killing of clinically
relevant gram-positive organisms in vitro. The present studies
evaluated the in vivo pharmacodynamics of telavancin in the mouse
neutropenic thigh (MNT) and mouse subcutaneous infection (MSI)
animal models. Pharmacokinetic-pharmacodynamic studies in the
MNT model demonstrated that the 24-h area under the
concentration-time curve (AUC)/MIC ratio was the best predictor of
efficacy. Telavancin produced dose-dependent reduction of thigh
titers of several organisms, including methicillin-susceptible
Staphylococcus aureus (MSSA) and methicillin-resistant
Staphylococcus aureus (MRSA), penicillin-susceptible and resistant
strains of Streptococcus pneumoniae, and vancomycin-resistant
Enterococcus faecalis. The 50% effective dose (ED50) estimates for
telavancin ranged from 0.5 to 6.6 mg/kg of body weight
(administered intravenously), and titers were reduced by up to 3
log10 CFU/g from pretreatment values. Against MRSA ATCC
33591, telavancin was 4 and 30-fold more potent (on an ED50 basis)
than vancomycin and linezolid, respectively. Against MSSA ATCC
13709, telavancin was 16 and 40-fold more potent than vancomycin
and nafcillin, respectively. Telavancin, vancomycin, and linezolid
were all efficacious and more potent against MRSA ATCC 33591 in
the MSI model compared to the MNT model. This deviation in
potency was, however, disproportionately greater for vancomycin
and linezolid than for telavancin, suggesting that activity of

15
telavancin is less affected by the immune status. The findings of
these studies collectively suggest that once-daily dosing of
telavancin may provide an effective approach for the treatment of
clinically relevant infections with gram-positive organisms (sharath
et al.,2004)

Macrolide antibiotics have an outstanding ability to

concentrate within host cells, particularly phagocytes. In the study
described in this paper five different macrolide antibiotics were
compared regarding the uptake and release kinetics in human
peripheral blood polymorphonuclear neutrophils (PMNs) and three
different cell lines, two phagocytic cell lines (RAW 264.7 and THP-
1) and an epithelial cell line (MDCK). Based on the results obtained,
the substances tested could be clustered into different groups.
Azithromycin constituted thefirst group, characterized by rapid and
nonsaturable uptake into phagocytic cells and a high degree of
retention in the preloaded cells. The second group included
erythromycin and clarithromycin. These two substances do not
exhibit cell specificity; consequently, they are taken up to a similar
extent and are released by all cell types studied. Ketolides
constituted the last group. Their uptake was saturable in cells of
monocytic lineage as well as in nondifferentiated cells of myeloid
lineage, and they were rapidly released from all the cell lines
studied.However, in PMNs, ketolide uptake was not saturable; and
unlike telithromycin, cethromycin rapidly egressed from the loaded
cells(Martina Bosnar et al.,2005). Successful application of growth
inhibition techniques for quantitative determination of bacteriocins
relies on the sensitivity of the applied indicator microorganism to the
bacteriocin to which is exposed. However, information on indicator

16
microorganisms' performance and comparisons in bacteriocin
determination with bioassays is almost non-existing in the literature.
The aim of the present work was to evaluate the parameter "indicator
microorganism" in bioassays carried out on solid –agar diffusion
assay- and liquid -turbidometric assay- substrates, applied in the
quantification of the most studied bacteriocin nisin (Maria pagianni
et al.,2006).
Various statistical classification methods, including
discriminant analysis, logistic regression, and cluster analysis, have
been used with antibiotic resistance analysis (ARA) data to construct
models for bacterial source tracking (BST). We applied the
statistical method known as classification trees to build a model for
BST for the Anacostia Watershed in Maryland. Classification trees
have more flexibility than other statistical classification approaches
based on standard statistical methods to accommodate complex
interactions among ARA variables. This article describes the use of
classification trees for BST and includes discussion of its principal
parameters and features. Anacostia Watershed ARA data are used to
illustrate the application of classification trees, and we report the
BST results for the watershed ( Bertram Price et al.,2006).

To study the antimicrobial susceptibility of six vaginal

probiotic lactobacilli. Methods. The disc diffusion method in
M¨ueller Hinton, LAPTg and MRS agars by the NCCLS (National
Committee for Clinical Laboratory Standards) procedure was
performed. Due to the absence of a Lactobacillus reference strains,
the results were compared to those of Staphylococcus aureus ATCC
29213.Mnimal Inhibitory Concentration (MIC) with 21 different

17
antibiotics in LAPTg agar and broth was also determined. Results.
LAPTg and MRS agars are suitable media to study antimicrobial
susceptibility of lactobacilli. However, the NCCLS procedure needs
to be standardized for this genus. The MICs have shown that all
Lactobacillus strains grew at concentrations above 10 μg/mL of
chloramphenicol, aztreonam, norfloxacin, ciprofloxacin,
ceftazidime, ceftriaxone, streptomycin and kanamycin. Four
lactobacilli were sensitive to 1 μg/mL vancomycin and all of them
were resistant to 1000 μg/mL of metronidazole. Sensitivity to other
antibiotics depended on each particular strain. Conclusions. The
NCCLS method needs to be standardized in an appropriate medium
to determine the antimicrobial susceptibility of Lactobacillus.
Vaginal probiotic lactobacilli do not display uniform susceptibility
to antibiotics. Resistance to high concentrations of metronidazole
suggests that lactobacilli could be simultaneously used with a
bacterial vaginosis treatment to restore the vaginal normal flora
(Virginia Oca na et al., 2006).

Studies on the synthesis of vitamins have led to no general

agreement as to the ability of specific micro6rganisms to produce
vitamin B or as to the extent of this synthesis among the lower plant
forms.This lack of agreement may be attributed in part to the failure
of investigators to keep clearly in mind the characteristics and
properties that define a vitamin and in part to the marked differences
in methods which have been employed. There is, on the one hand,
the group which has adhered rigidly to the conception of a vitamin
as defined by Drummond (1924), i.e., a substance of unknown
constitution neither fat, protein, carbohydrate, nor mineral salt,
necessary for the life and wellbeing of a suitable organism. On the

18
other hand, there is a group which has disregarded the requirements
of Drummond and has used as the criterion of the presence of
vitamin B, the ability of a substance to stimulate multiplication of a
specific microorganism. The yeast growth test of Williams (1919)
employs the multiplication of Saccharomyces cerevisiae
(Bacteriology Section et al.,1928).

DX-619 is a novel des-fluoro(6) quinolone with potent

activity against gram-positive pathogens. The in vivo activity of DX-
619 against Streptococcus pneumoniae was compared with those of
fluoro(6) quinolones, sitafloxacin, and ciprofloxacin in a mouse
model. Two strains of S. pneumoniae were used: a penicillin-
sensitive S. pneumoniae (PSSP) strain and a penicillin-resistant S.
pneumoniae (PRSP) strain. Furthermore, these strains showed
intermediate susceptibilities to ciprofloxacin. In murine lung
infections caused by PSSP, the 50% effective doses (ED50s) of DX-
619, sitafloxacin, and ciprofloxacin were 9.15, 11.1, and 127.6
mg/kg of body weight, respectively. Against PRSP-mediated
pneumonia in mice, the ED50s of DX-619, sitafloxacin, and
ciprofloxacin were 0.69, 4.84, and 38.75 mg/kg, respectively. The
mean _ standard error of the mean viable bacterial counts in murine
lungs infected with PSSP and treated with DX-619, sitafloxacin,
ciprofloxacin (10 mg/kg twice daily), and saline (twice daily) were
1.75 _ 0.06, 1.92 _ 0.23, 6.48 _ 0.28, and 7.57 _ 0.13 log10 CFU/ml,
respectively. Furthermore, the numbers of viable bacteria in lungs
infected with PRSP and treated with the three agents and not treated
(control) were 1.73 _ 0.04, 2.28 _ 0.17, 4.61 _ 0.59, and 5.54 _ 0.72
log10 CFU/ml, respectively. DX-619 and sitafloxacin significantly
decreased the numbers of viable bacteria in the lungs compared to

19
the numbers in the lungs of ciprofloxacin-treated and untreated mice.
The pharmacokinetic parameter of the area under the concentration-
time curve (AUC)/MIC ratio in the lungs for DX-619, sitafloxacin,
and ciprofloxacin were 171.0, 21.92, and 1.22, respectively. The
AUC/MIC ratio in the lungs was significantly higher for DX-619
than for sitafloxacin and ciprofloxacin. Our results suggest that DX-
619 and sitafloxacin are potent against both PSSP and PRSP in our
mouse pneumonia model(Yuichi Fukuda et al.,2005).

Bacterial genes defining intrinsic resistance to antibiotics

encode proteins that can be targeted by antibiotic potentiators. To
find such genes, a transposon insertion library of Acinetobacter
baylyi was screened with subinhibitory concentrations of various
antibiotics to find supersusceptible mutants. A DNA microarray
printer was used to replica plate 10,000 individual library clones to
select mutants unable to grow at 1/10 the MICs of 12 different
antibiotics. Transposon insertions in 11 genes were found to cause
an eightfold or higher hypersusceptibility to at least one antibiotic.
Most of the mutants identified exhibited hypersusceptibility to _-
lactam antibiotics. These included mutants with disruptions of genes
encoding proteins involved in efflux (acrB and oprM) as well as
genes pertaining to peptidoglycan synthesis and modification
(ampD, mpl, and pbpG). However, disruptions of genes encoding
proteins with seemingly unrelated functions (gph, argH, hisF, and
ACIAD0795) can also render cells hypersusceptible to β-lactam
antibiotics. A knockout of gshA, involved in glutathione
biosynthesis, enhanced the susceptibility to metronidazole, while a
knockout of recD, involved in recombination and repair, made the
bacteria hypersusceptible to ciprofloxacin. Disruption of acrB in

20
Escherichia coli rendered the cells hypersusceptible to several
antibiotics. However, knockout mutants of other homologous genes
in E. coli showed no significant changes in antibiotic MICs,
indicating that the intrinsic resistance genes are species specific
(Maria et al.,2006).

The pharmacodynamic properties governing the activities of

antibiotics against intracellular Staphylococcus aureus are still
largely undetermined. Sixteen antibiotics of seven different
pharmacological classes (azithromycin and telithromycin
[macrolides]; gentamicin [an aminoglycoside]; linezolid [an
oxazolidinone]; penicillin V, nafcillin, ampicillin, and oxacillin [_-
lactams]; teicoplanin, vancomycin, and oritavancin [glycopeptides];
rifampin [an ansamycin]; and ciprofloxacin, levofloxacin,
garenoxacin, and moxifloxacin [quinolones]) have been examined
for their activities against S. aureus (ATCC 25923) in human THP-1
macrophages (intracellular) versus that in culture medium
(extracellular) by using a 0- to 24-h exposure time and a wide range
of extracellular concentrations (including the range of the MIC to the
maximum concentration in serum [Cmax; total drug] of humans).
All molecules except the macrolides caused a net reduction in
bacterial counts that was time and concentration/MIC ratio
dependent (four molecules tested in detail [gentamicin, oxacillin,
moxifloxacin, and oritavancin] showed typical sigmoidal dose-
response curves at 24 h). Maximal intracellular activities remained
consistently lower than extracellular activities, irrespective of the
level of drug accumulation and of the pharmacological class.
Relative potencies (50% effective concentration or at a fixed
extracellular concentration/MIC ratio) were also decreased, but to

21
different extents. At an extracellular concentration corresponding to
their Cmaxs (total drug) in humans, only oxacillin, levofloxacin,
garenoxacin, moxifloxacin, and oritavancin had truly intracellular
bactericidal effects (2-log decrease or more, as defined by the
Clinical and Laboratory Standards Institute guidelines). The
intracellular activities of antibiotics against S. aureus (i) are
critically dependent upon their extracellular concentrations and the
duration of cell exposure (within the 0- to 24-h time frame) to
antibiotics and (ii) are always lower than those that can be observed
extracellularly. This model may help in rationalizing the choice of
antibiotic for the treatment of S. aureus intracellular
infections(Maritza Barcia-Macay et al.,2005).

The bactericidal pharmacodynamics of antibiotics against

Escherichia coli were analyzed by a concentrationkilling curve
(CKC) approach, and the novel parameters median bactericidal
concentration (BC50) and bactericidal intensity (r) for bactericidal
potency were proposed. By using the agar plate method, about 500
E. coli cells were inoculated onto Luria-Bertani plates containing a
series of antibiotic concentrations, and after 24 h of incubation at
37°C, all the viable colonies were enumerated. This resulted in a
sigmoidal CKC that could be perfectly fitted (R2 > 0.9) with the
function N _ N0/[1 _ er(x _ BC50)], where N is number of colonies
surviving on each plate with an x series of concentrations of an
antibiotic, and N0 represents the meaningful inoculum size.
Construction of the CKC method was based on the bactericidal
effect of each antibiotic against the bacterial strain versus the
concentration in two dimensions and may be a more valid, accurate,
and reproducible method for estimating the bactericidal effect than

22
the endpoint minimum bactericidal concentration (MBC) method.
Mathematically, the CKC approach was point symmetrical toward
its inflexion (BC50, N0/2); thus, 2BC50 could replace MBC. The
parameter BC1 can be defined as BC50 _ [ln(N0 1)/r], which is the
drug concentration at which only one colony survived and which is
the least critical value of MBC in the CKC. The variate r, which
determined the tangent slope on inflexion when N0 was limited,
could estimate the bactericidal intensity of an antibiotic. This
verified that the CKC approach may be useful in studies with other
classes of antibiotics and has considerable value as a tool for the
accurate and proper administration of antibiotics (Liu et al.,2004).

Antibiotics with novel mechanisms of action are becoming

increasingly important in the battle against bacterial resistance to all
currently used classes of antibiotics. Bacterial DNA gyrase and
topoisomerase IV (topoIV) are the familiar targets of
fluoroquinolone and coumarin antibiotics. Here we present the
characterization of two members of a new class of synthetic bacterial
topoII ATPase inhibitors: VRT-125853 and VRT-752586. These
aminobenzimidazole compounds were potent inhibitors of both
DNA gyrase and topoIV and had excellent antibacterial activities
against a wide spectrum of problematic pathogens responsible for
both nosocomial and community-acquired infections, including
staphylococci, streptococci, enterococci, and mycobacteria.
Consistent with the novelty of their structures and mechanisms of
action, antibacterial potency was unaffected by commonly
encountered resistance phenotypes, including fluoroquinolone
resistance. In time-kill assays, VRT-125853 and VRT-752586 were

23
bactericidal against Staphylococcus aureus, Streptococcus
pneumoniae, Enterococcus faecalis, and Haemophilus influenzae,
causing 3-log reductions in viable cells within 24 h. Finally, similar
to the fluoroquinolones, relatively low frequencies of spontaneous
resistance to VRT- 125853 and VRT-752586 were found, a property
consistent with their in vitro dual-targeting activities (Nagraj Mani
et al.,2006).

Resistance to β-lactam antibiotics in enteric Gram-negative

bacilli may be difficult to detect using standard methods of either
Kirby-Bauer disc diffusion (KBDD) or broth dilution for minimal
inhibitory concentration (MIC). This difficulty is due to genetic
differences in resistance determinants, differences in levels of gene
expression, and variation in spectra of enzymatic activity against the
substrate β-lactams used for susceptibility testing. We have
examined 95 clinical isolates reportedly susceptible to ceftazidime
and ceftriaxone, as originally determined by either KBDD or MIC
methods. The organisms studied here were isolated in 2002 from two
pediatric hospital centers (Seattle, USA and Shanghai, China). They
belong to the inducible β-lactamase producing Gram-negative
bacilli, such as Enterobacter spp., Citrobacter spp., Serratia spp.,
Morganella spp., Providencia spp., and Proteus vulgaris. A Kirby-
Bauer disc approximation (KBDA) method identified inducible
phenotypes of third-generation cephalosporin resistance in 76% of
isolates, which would otherwise be considered susceptible by
standard KBDD methods (Xuan Qin et al.,2004).

24
 The performances of three commercial broth microdilution
MIC assays adapted for use with fastidious organisms-the E test
(ET), Fastidious Antimicrobial Susceptibility panel (FAS), and FOX
Fastidious panel (FOX)-were compared with a MIC using Mueller-
Hinton broth with 5% lysed horse blood (MHLHB) to confirm
penicillin and cephalosporin resistance in clinical isolates of
Streptococcus pneumoniae. Of the isolates screened for penicillin
resistance, 5 (12.8%) were categorized as susceptible, 16 (41.0%)
were categorized as intermediate, and 18 (46.2%) were categorized
as resistant by MHLHB. Only the isolates exhibiting intermediate-
to-resistant MICs were included in the comparison. Agreement
within ±1 log2 dilution was found in 91, 21, and 76% of the ET,
FAS, and FOX MICs, respectively, compared with the MHLHB
MIC. No very major or major discrepancies occurred with the ET or
FOX; however, two very major interpretive errors occurred with the
FAS. Agreement between the ET and MHLHB for cefotaxime,
ceftriaxone, and cefuroxime was 88, 85, and 100%, respectively.
Less than 50% of cephalosporin MICs categorized as >0.5 ,ug/ml by
MHLHB were detected by FAS or FOX. Of the methods compared,
the ET was the most reliable alternative for susceptibility testing of
pneumococci (karen et al.,1994).

25
 MATERIALS AND METHODS

This present work was carried out at natco Pharma,during

December 2007 to February 2008. Microbiological assay is a
biological method of estimation of any pharmaceutical preparation
by their effects on growth of microorganisms. These effects may be
to promote the growth of that organism ex:- Vitamins, amino acids
etc. or to inhibit the growth of the organisms ex:-Antibiotics, heavy
metal salts .

Principle

The microbiological assay is based upon the comparison of

the growth of the bacteria by measured concentration of the sample
with that of the known concentration of the standard preparation of the
substance having known activity. For this generally two methods are
used.

1.Cup plate or cylinder plate method.

2.Tubidimetric or tube assay method.
In Cup or Cylinder plate assay, the potency of the sample is
estimated by measuring growth inhibition or zones.

In turbidimetric or tube assay, potency of the sample is

estimated by measuring turbidity of the test solutions (Dr. About-
emein).

26
Apparatus

Lab bench or suitable laminar air flow bench, pH meter, hot air
oven, autoclave, incubator, refrigerator, balance, zone reader, pipettes,
petridish containers, Bunsen burner, glass ware for holding and
transferring test organisms, volumetric flasks, conical flasks, separating
funnels, measuring

very equipment has to be cleaned and sterilized before and after

each use, cleaning of glassware was ensured by soaking to 24hours in
the cleaning solution (50grams potassium dichromate in 200ml water
+300ml concentrated sulfuric acid).

Basic requirement of culture media

Culture media must have sufficient moisture, adequate

nutrients, and the optimum pH range. A suitable oxidation -reduction
potential.

Water that has been used in culture media purified by

distillation, because during sterilization its calcium and magnesium
salts given heavy precipitate with the phosphates in peptones and meat
extract.

Nutrients are necessary in culture media for the growth of

organisms. These are nitrogen sources, carbohydrate sources, mineral
salts.

Peptones are used, as nitrogen and carbon sources these are

water soluble not coagulated by heat, because of they are amphoteric
nature. These are good buffers peptone from casein is rich in growth

27
factors and amino acids. They pass easily through the bacterial cell wall
and readily used for cell growth. Dextrose, glucose, lactose etc., is used
as carbohydrate sources. These are readily utilizable sources of energy.
Yeast extract used as nitrogen and carbon source. Which is
rich source of B-vitamins. It may be added as a growth supplement beef
Extract is a source of nucleotides amino acids peptides vitamins and
mineral salt solution is added to most culture media. These are together
with other constituents providing a suitable osmotic pressure.
The optimum pH range and suitable oxidation-reduction
potentials maintained in the media for the growth of the organisms.
Cultured media should be stored in a dark place. It is also important to
reduce loss of water to a minimum otherwise the may become too
concentrated for optimal bacterial growth. Evaporation is prevented by
screw caps or rubber seals and can be in plugged or capped tubes by
stored in a refrigerator.

Temperature control in Microbial Assay

Temperature maintenance plays a very important role in the

out come of microbiological assay as temperature influences the growth
of microorganisms. Any fluctuations in temperature may increase or
decrease the generation time of the test organism during incubation.
Normally the assay incubation is done at any constant temperatures
between 30-37oC. For this incubators with thermostat are used. These
incubators provide uniform temperature through circulating air, some
constant temperature maintenance water baths are also used for
incubation for more accurate temperature maintenance.

28
Cylinder- plate assay receptacles

Rectangular glass trays or glass or plastic petridishes

(approximately 20 10mm) having cover of suitable material and assay
cylinders made of glass, porcelain aluminum or stainless steel with
outside diameter (8mm) inside diameter (6mm) and length 10mm
instead of cylinders, holes 6 to 8mm in diameter may be bored in the
medium with a sterile borer or paper discs of suitable quality border or
paper may be used carefully clean the cylinders to remove all residues.
An occasional acid bath with above 2m nitric acid or with chromic
acid.

Turbidimetric assay receptacles

For assay tubes made of glass or plastic with 16mm 125mm

or 18 mm 150mm size reality uniform size are used (Nithyaanad).

Reference standards

The standard preparations for India are maintained at the

central drugs Laboratory, Calcutta. A unit refened to in the official
assay and tests is the specific activity contained in such an amount of
the respective standards preparation as is indicated by the ministry of
health &family welfare Government of India from time to time.

A standard preparation may be replaced by a working

standard prepared by any laboratory, which should be compared at
definite intervals under varying conditions with the standard. A full
record must be kept for each such working standard with the following
details.

29
 Working standard

As full a record as possible must be kept of each working

standard such as
 Name and batch! Identity number
 Date, potency was assigned
 Source
 Potency per mg
 Storage condition, expiry date
 Date of potency recheck

Standard solution

To prepare a stock solution, dissolve a quantity of the

standard preparation of a given sample accurately weighed and
previously dried and then dilutes to the required concentration. Store in
a refrigerator and use with in the period specified on the day of the
assay prepared from the stock solution five or more test dilutions, the
concentration usually in the ratio 1 : 1:25 for cup plate method or small
for tubidimetric method.

An experienced person should prepare a standard

solution. A new person preparation should be treated as sample and
evaluated where ever possible, the assay of the substance / solution
should be get assessed by other methods like UV, HPLC or other
techniques.

30
Assay design

Microbial assay gain markedly in precision by

segregation of relatively large sources of potential error and bias
thorough suitable experimental designs. In a cylinder plate assay the
essential comparisons are restricted to relationships between zone
diameter measurements with in their preparation and subsequent
handling. To count a turbid metric assay so that the difference in
observed turbidity will reflect in the environment created for the tubes
through close’s thermostatic control or the incubator and avoidance or
systematic bias by a random placement of replicate tubes in separate
tube racks. Each rack containing one set of treatments. The essential
comparisons are then restricted to relationship between the observed
turbidity with in racks. Within these

restrictions, two alternative designs are recommended

i.e. a three level or factorial assay and a one level with a standard curve.
For a factorial assay, solutions of a three or two corresponding set
dilutions were prepared for both standard and unknowns on the day of
the assay, as described under preparation of the standard and the
sample.

Precision of Microbiological Assay

The fiducially limits of error of the estimated potency

should be not less than 95%and not more than 105%of the estimated
potency unless otherwise stated in the individual monograph. This
degree of precision in the minimum acceptable for determining that
final product compiles with the official requirements and may be
inadequate for those deciding for example the potency which should be

31
stated on the label or used as the basis for calculating the quantity of
sample (vitamin)

To be incorporated in a preparation. In such

circumstances assays of grater precision may be desirable with, for
instance fiducially limits of error of the order of 98% to 102% with this
degree of precision, the lower fiducially limit lies close to the estimated
potency. By using this limit to assign potency to the sample either for
label ing or for calculating the quantity to be included in a preparation,
there is less likelihood of the final preparation subsequently failing to
comply with the official requirements for potency.

Turbidimetricmethod

The microbiological assay of calcium pantothenate carried

out by turbidimetric tube assay method (as per USP -23, 1995).

Stock Culture of Lactobacillus arabinosus

The organism L.arabinosus(ATCC No.8014) growth on

NCL media prepare stab culture in 3 or more the tubes, using a pure
culture of lactobacillus arabinosus incubating for 16-24 hours at any
selected temperature between 30-370C and store in a refrigerator.
Prepare a fresh stab of the stock culture every week.

32
Preparation of Inoculum

On one day before transfer cells from the stock culture of

L.arabinosus to a sterile tube containing 10ml of NCL liquid media
(NCL medium with out agar). Incubate this culture for 16-24 hours at
any selected temperature between 30-37oC. the cell suspension so
obtained is the Inoculum.

Preparation of Final Inoculum

After 24 hours incubation at 35 and 37°C of inoculated

tube centrifuged the complete Inoculums suspension aseptically and
decent. The supernatant liquid 10-ml of 0.85% Nacl solution is added
and shakes disperse the sediment and centrifuged the decent to the
supernatant. The collected cell suspension of deterent inoculated tubes
in one aseptic tube dilutes I ml of suspension to 100mi with steri Ie
0.85% Nacl solution.

One drop of final Inoculum added aseptically to each of

the test and sample assay tubes.

Preparation of Standard Stock Solution:

Accurate weighed 25mg of calcium pantothenate standard

powder dissolved in 500ml distilled• water (500mcg / ml). Stored in the
refrigerator.

Preparation of Standard Solution

25mg of standard drug is taken and it is dissolved in 500ml of
distil water from that 2ml is taken and dissolved in 100ml of distilled
water water from that 5ml is taken and dissolved in 250ml of distilled

33
 water. Ofter these serial dilutions the concentration of standared will
be0.02mcg/ml

Sample taken for analysis

The following pharmaceutical formulation is analyzed against the
standard solution. The composition of the formulation is as below.
25mg of sample drug is taken and it is dissolved in 500ml of distil
water from that 2ml is taken and dissolved in 100ml of distilled
water water from that 5ml is taken and dissolved in 250ml of
distilled water. Ofter these serial dilutions the concentration of
sampil will be0.02mcg/ml.

1.Fcnule - TR Capsules

Timed release capsules of ferrous sulfate with B- complex & Vitamin

-C Each Capsule Contains
(The sample fenule capsule has 205mg of pantothenic acid /
Capsule overage approximately added 100%, i.e., 5mg of pantothenic
acid per capsule)
Dried ferrous Sulfate BP 150 mg
Ascorbic acid BP 50 mg
Riboflavin BP 2 mg
Thiamin Mononitrate BP 2 mg
Nicotinamide BP 15 mg
Pyridoxine Hydrochloride BP l mg
Pantothenic Acid BP 2.5 mg
(As Calcium panothenate)

34
 RESULTS

Assay Of Vitamin B5

Test organism Lactobacillus plantrarum

Stock Standard

Soln. Cone. 50 mcg / ml

Standard Dilution

25mg of standard drug is taken and it is dissolved in 500ml of

distil water from that 2ml is taken and dissolved in 100ml of distilled
water water from that 5ml is taken and dissolved in 250ml of
distilled water. Ofter these serial dilutions the concentration of
standared will be0.02mcg/ml.

Sample Fenule Capsules

(NATCO PHARMA LIMITED)

Assay for Calcium pantothenate

Batch No. 9010329

Batch size 10,00,000

Mfg. Date 10-02-2006

Expiry date 12-02-2010

Sampling Date 15-01-2007

Average weight of capsule 422.61 mg

Labeled Amount 2.5 mg/ cap

Amount found 4.89mg/cap

35
 SAMPLE DILUTION

422.6mg of sample drug is taken and it is dissolved in 500ml of

distil water from that 2ml is taken and dissolved in 100ml of distilled
water water from that 5ml is taken and dissolved in 250ml of
distilled water. Ofter these serial dilutions the concentration of
sampil will be0.02mcg/ml.

Table-4 for assay of vit B5

Aliquot Transmission at 620nm Y 200- Observed Logml Log ml Differen

Ml from
Ml Std. (obser (observed)log Ce with
1 2 Total Graph ved) mI(real) of sign (x)
theassay
preparation
1.0 85.37 85.60 170.97 0.2903 1.02 0.0086 0.0086-0.000 0.0086
2.0 58.74 28.26 117.00 0.83 1.92 0.283 0.283-0.3010 -0.018
3.0 35.27 33.73 69.00 1.31 3.05 0.484 0.484-0.4771 - 0.0069
4.0 21.02 20.98 42.00 1.58 3.97 0.598 0.598-0.6021 - 0.0041

Total =0.037
Average (X) or M 1 the log relative potency with sign.

Anti log M = Antilog (Ml + log R), where R = mg of assumed

potency per unit dosage.

(Note: where average weight is taken into consideration)

mg per unit dosage = Antilog (0.037+ 1.3959)

Log R = 1.3959, M' + Log R = 1.3669,

R = mg of assumed potency X Wt. Taken I Av. Weight X No. of

Tablets
=25 x 0.5748/0.5774 x 1/1 = 24.887

Assay Of Vitamin B5

36
 Sampling Date 15-01-2007

Test organism Lactobacillus plantrarum

Stock Standard Soln. Cone. 50 mcg / ml

Standard Dilution

25mg of standard drug is taken and it is dissolved in 500ml

of distil water from that 2ml is taken and dissolved in 100ml of
distilled water water from that 5ml is taken and dissolved in 250ml
of distilled water. Ofter these serial dilutions the concentration of
standared will be0.02mcg/ml.

Sample Beplex forte tablets

(NATCO PHARMA LIMITED)

Assay for Calcium pantothenate

Batch No. 18199

Batch size 10,00,000

Mfg. Date 10-02-2007

Expiry date 12-02-2009

Average weight of capsule 577.04 mg

Labeled Amount 2.5 mg/ cap

Amount found 23.27mg/cap

SAMPLE DILUTION
577.4mg of sample drug is taken and it is dissolved in 500ml of
distil water from that 2ml is taken and dissolved in 100ml of distilled
water water from that 5ml is taken and dissolved in 250ml of distilled
water. Ofter these serial dilutions the concentration of sampil will
be0.02mcg/ml.

Table-5 for assay of vitB5

37
Aliquot Transmission at 620nm Y Observed Logml Log ml Differen
Ml from
Ml 200- Std. (obser (observed)log Ce with
1 2 Total Graph ved) mI(real) of sign (x)
theassay
preparation
1.0 85.37 85.60 170.97 0.29.03. 1.02 0.0086 0.0086-0.000 0.0086
2.0 52.55 53.15 106.00 0.09 2.1 0.322 0.322 - 0.3010 0.02
3.0 29.42 28.91 58.33 1.41 3.35 0.525 0.525 -0.4771 0.04
4.0 17.19 17.58 34.76 1.65 4.2 0.623 0.623 - 0.6021 0.02
Total 0.08

Average (X) or M 1 the log relative potency with sign.

Anti log M = Antilog (Ml + log R), where R = mg of assumed

potency per unit dosage.

Note: where average weight is taken into consideration)

mg per unit dosage =Antilog(0.08+1.3995)

Log R = 1.3995, M' + Log R = 1.4795, antilog(M + Log R) 30.164

R = mg of assumed potency X Wt. Taken I Av. Weight X No. of

Tables
R= 25 x 0.3278/0.3266 x 1/1 = 25.09

ASSAY OF VITAMIN B12

38
The protocol used for recording of the data and calculation of the

results are enclosed. This gives clear information about standard and

sample preparations treatment and method of evaluation.

Test Organism E-coli ATCCNo.11105

Amount of Inoculum used 10ml

Potency of Standard Substance 960.4 mcg/ cap

Standard Dilution

25mg of standard is dissolved in 500 ml of distilled water from that

take 2ml and dissolve in100ml of water from that take 10 ml and
dissolve in 50 ml it is used for standard high(S H) from that take 25
ml and dissolve in 50 ml of distilled water it is used for standard
low(SH)

SAMPLE -ZINFE - SR CAPSULES

39
 (NATCO PHARMA LIMITED)
Assay for Cyanocobalamine
Batch No. 0186
Batch Size 5,00,000
Mfg. Date 06.2006
Expiry Date 09.2009
Sampling Date 02.02.2007
Labeled Amount 15 mc/cap
AV. Weight / cap 464.95mg
Weight taken 3.605gms
Potency found 17.92mcg / cap
Amount found 140.34%
Sample Dilution
3.605mg of sample was taken and dissolved in100 ml distilled water
from that take 10 ml and dissolve in100 ml of water it is used for
sample high (UH). from that take 25 ml and dissolve in 50 ml of
distilled water it is used for sample low(UL).

Table-6 for assay of vitB12

Concentration Concentration Exhibition Zone Diameter Soms of

Level Level 1 2 3 4 Zone
SI 0.1 16.8 16.5 16.4 16.6 66.3
S2 0.05 15.8 14.60 15.02 15.20 60.6
Ul 0.1 17.4 17.2 17.62 17.82 70.04
U2 0.05 16.0 15.82 15.6 15.64 63.06

calculations

40
A)% Potency of the sample + Antilog (2.0 +a log I)
(in terms of standard)
Where "a" may have a+ve or -ve value and should be used algebraically.
(U1+U2) - (S1 + S2)
a= (U1-U2) - (Sl - S2)
Where Ul and U2 are the sums of the zones diameter with solution of the unknown of
high and low levels.
SI and S2 are the sums of the zone diameter with solution of the standard high and
low levels.
I = Radiation of dilution (SHG / SL)
Note: If the potency of the sample is lower than 60% or greater than 150% of
standard, the assay was invalid and was repeated using higher or lower dilution of the
sample solution.
(b) Potency of the sample was calculated from the formula
% Potency x assumed potency of the sample
100
(c) % Amount found: 140 : 34

Antilog + (13301-126.9) ( 0.3010)

6.98 + 5.7
Antilog2 + 6.2 x 0.3010
12.68

Antilog2 + 0.4889 x 0.3010

Antilog2.1472 = 140.34 of assumed

Actual potency = 140.34 x 3.0687 x 15

100 3.605

=17.92 mcg/ml
SAMPLE-GLOHEM-TR CAPSULES

(NATCO PHARMA LIMITED)

41
 Assay for Cyanocobalamine
Sampling Date 02.02.2006
Labeled Amount 10 mg/cap
AV. Weight / cap 469039mg/cap
Weight taken 4.6943gms
Potency found 14.46mcg / cap
Amount found 144.64%
Sample Dilution

4.6943 mg of sample was taken and dissolved in100 ml distilled water from that

take 10 ml and dissolve in100 ml of water it is used for sample high (U H). from

that take 25 ml and dissolve in 50 ml of distilled water it is used for sample

low(UL).

Table-7 for assay of vitB12

Concentration Concentration Exhibition Zone Diameter Soms of

Level Mcg/ml 1 2 3 4 Zone
SI 0.1 12.42 12.5 12.0 12.40 49.32

S2 0.05 11.0 11.0 15.24 11.24 44.48

Ul 0.1 12.8 12.8 12.62 12.62 50.84
U2 0.05 11.82 11.5 11.92 11.84 47.38

calculations
A)% Potency of the sample + Antilog (2.0 +a log I)

42
 (in terms of standard)
Where "a" may have a+ve or -ve value and should be used algebraically.
(U1+U2) - (S1 + S2)
a= (U1-U2) - (Sl - S2)
Where Ul and U2 are the sums of the zones diameter with solution of the unknown
of high and low levels.
SI and S2 are the sums of the zone diameter with solution of the standard high and
low levels.
I = Radiation of dilution (SHG / SL)
Note: If the potency of the sample is lower than 60% or greater than 150% of
standard, the assay was invalid and was repeated using higher or lower dilution of
the sample solution.

(b) Potency of the sample was calculated from the formula:

% potency x assumed p[otency of the sample

100

(c) % amount found: 144.64

Antilog 2+ [ 98.22-93.8] (0.3010)

3.46+4.84

Antilog 2+ 4.42 x (0.3010)

8.3

Antilog 2+ 0.16029

Antilog2.1472=144.64 of assumed

Actual potency =140.64 x 4.6939 x 10

43
 100 4.6943

= 14.46 mcg/ml

ASSAY DOXYCYCLIN
Assay of doxycyclin is den by tarbidomatric method .staphylococcus
aureus(ATCC 6538)is used for test organism .3 ml of test organism is
dissolved in 300ml of media tubes and tubes are incubated at 37 0c for 4-5
hrs.
Table-8 for assay of doxycyclin

Transmittance /ml /ml /ml /ml /ml

Sample
(530nm) 0.064 0.08 0.1 0.125 0.156
Potency 880
1 67.71 67.91 69.35 72.21 75.01 70.23
/mg
Weight
57mg 2 65.52 69.79 70.85 71.49 70.99 71.55
Dissolved 50ml Avg. 66.52 68.85 70.1 0 71.85 73.00 67.95
Stock 1000 Avg.69
solution µg/ml .91
..

44
concentrati
on

Standard curve = 0.098

Potency = 887.37fl/ml

Actual Potency = 934.07fl/ml

= 0.096 X 890

= 854.40. 0.1

Ethonal Free = 854.40 X 100/(100-1.55)

= 867.85mg

ASSAY OF TETRACYCLINE
Assay of tetracycline is den by tarbidomatric method .staphylococcus
aureus(ATCC 6538)is used for test organism .2 ml of test organism is
dissolved in 200ml of media tubes and tubes are incubated at 37 0c for 4-5
hrs.
Table-9 for assay of tetracyccline
Std B. No IP Ref /ml /ml /ml /ml /ml
Std 0.154 0.192 0.240 0.300 0.375
Potency 920mg
Weight 100 mg
Transmittance 1 64.96 70.41 73.47 78.05 83.18
Dissolved
530nm
in 100 ml 66.16 68.97 73.87 78.0 82.35

45
Stock 1000 2
Solution /ml 65.54 69.69 73.67 78.02 82.76
con Avg

SAMPLE OBSERVATION
Sample : Tetra Cycline Hcl IP
Batch No. : T 707379
Assumed potency : 950 /mg
Labeled potency : 100 mg/cap
Weight taken : 250.6 mg
Dissolved in : 250 ml
Transmission 1 : 75.03
At 530 nm 2 : 75.06
Avg : 75.06
Standard curve value (/ml) : 0.265
Potency (/mg) : 977.5 /mg
Standard Curve value on Graph
Potency = X Standard Potency
Median concentration

= 0.255 / 0.24 X 920

= 977.5 g/mg

46
ASSAY OF TETRACYCLINE
Assay of tetracycline is den by tarbidomatric method .staphylococcus
aureus(ATCC 6538)is used for test organism .2 ml of test organism is
dissolved in 200ml of media tubes and tubes are incubated at 37 0c for 4-5
hrs.
Table-10 for assay of tetracyccline
Std B. No PX2TCH /ml /ml /ml /ml /ml
660 0.154 0.192 0.240 0.300 0.375
Potency 1026.02 Transmittance
Weight 100.02
530nm 1 76.19 79.03 83.41 87.54 89.98
mg
Dissolved
in 100 ml 75.06 79.78 83.48 86.52 88.8

47
Stock 1000 2
Solution /ml Avg 75.62 79.41 83.44 87.03 89.15
con

SAMPLE OBSERVATION
Sample : Tetra Cycline Hcl IP
Batch No. : T 707379
Assumed potency : 950 /mg
Labeled potency : 100 mg/cap
Weight taken : 250.6 mg
Dissolved in : 250 ml
Transmission 1 : 82.5
At 530 nm 2 : 82.92
Avg : 82.705
Standard curve value (/ml) : 0.234
Potency (/mg) : 101.7 /mg

Standard Curve value on Graph

Potency = X Standard Potency
Median concentration

= 0.244 / 0.24 X 1026.62

= 101.7 g/mg

48
ASSAY OF TETRACYCLINE
Assay of tetracycline is den by tarbidomatric method .staphylococcus
aureus(ATCC 6538)is used for test organism .2 ml of test organism is
dissolved in 200ml of media tubes and tubes are incubated at 37 0c for 4-5
hrs.
Table-11 for assay of tetracyccline
Std B. No IP REF /ml /ml /ml /ml /ml
STD 0.154 0.192 0.240 0.300 0.375
Potency 920mg Transmittance
Weight 100mg
530nm 1 68.28 70.70 74.44 76.66 80.30
Dissolved
in 100 ml 69.24 72.43 75.53 76.08 80.50

49
Stock 1000
Solution /ml 2 68.76 71.56 73.98 76.42 80.43
con
Avg

SAMPLE OBSERVATION
Sample :Tetra Cycline Hcl IP 250mg capsule
Batch No. : 142
Assumed potency : 250 mg/cap
Labeled potency : 100 mg/cap
Weight taken : 251.3 mg
Dissolved in : 250 ml
Transmission 1 : 74.04
At 530 nm 2 : 75.11
Avg : 74.59
Standard curve value (/ml) : 0.240
Potency (/mg) :259.06 /mg

Standard Curve value on Graph

Potency = X Standard Potency
Median concentration

= 0.250/100x50/12x100/2x100/5x250/251.3x250
= 259.06mg(or)103.62mg

50
ASSAY OF ERYTHROMYCIN

Assay of erythromycin is den by cup plate method .micrococcus

luteus(ATCC 2103)is used for test organism .5ml of test organism is used.
Potency of standered substence is 661 mcg/mg.
Standard Dilution

The 1ml of erythromycin standard is taken and dissolved in 100 ml

of water From that 5 ml is take and dissolved in 25 ml of water, it is used
for Standard high (SH).from that 5ml is taken and dissolved in 25 ml of
distilled water it is used for Standard low(SL).

51
 Sample Dilution

184.7mg of erythromycin sample is taken and dissolved in

500 ml of water from that 1ml is taken and dissolved in 100 ml, from
that 5ml is taken and dissolved in 25ml of distilled water it si used
for the Sample high(UH).from that 5ml is taken and dissolved in 25
ml is taken it is used for Sample low(UL)

Sample : Erythromycin Estolate

B. No : UEE / 13
Wt. taken : 184.7
Potency found (mcg/mg) : 642.24 g/mg
Amount found (%) : 96.952 %

Table-12 for assay of erythromycin

Concentration Concentratio Inhibition Zone Diameter (in mm) = Sum of Zones for
Level n mcg/ml In petri Dishes each Level (mm)
1 2 3 4
S1 0.5 28.72 28.71 28.72 28.71 28.71
S2 0.1 20.71 21.71 21.42 21.28 21.28
U1 0.5 28.94 28.32 28.00 28.42 28.42
U2 0.1 21.42 21.47 21.00 21.29 21.29

Calculations
a) % potency of the sample = antilog (2.0 + a log 1)
(in terms of standard)

52
 (U1+U2) – (S1+S2)
Where a =
(U1-U2) + (S1-S2)

b) The potency of the sample was calculated from the formula

% potency x assumed potency of the sample

100

  49.71  49.99  
c) % Amount Found : Antilog 2   (0.69897) 
  7.13  7.43  

   0.28  
Antilog 2   (0.69897)
 14.56
  

Antilog (2-0.01344) = Antilog 1.98656

96.952 %
185.1
Actual potency = 96.952 x x661
184.7
642.24 g/mg

ASSAY OF ERYTHROMYCIN

Assay of erythromycin is den by cup plate method .micrococcus

luteus(ATCC 2103)is used for test organism .5ml of test organism is used.
Potency of standered substence is 671.78 mcg/mg.

Standard Dilution

The 1ml of erythromycin standard is taken and dissolved in 100 ml

of water From that 5 ml is take and dissolved in 25 ml of water, it is used
for Standard high (SH).from that 5ml is taken and dissolved in 25 ml of
distilled water it is used for Standard low(SL).
Sample Dilution

53
 184.7mg of erythromycin sample is taken and dissolved in
500 ml of water from that 1ml is taken and dissolved in 100 ml, from
that 5ml is taken and dissolved in 25ml of distilled water it si used
for the Sample high(UH).from that 5ml is taken and dissolved in 25
ml is taken it is used for Sample low(UL)

Sample : Erythromycin 250mg tablets

B. No : 021
Wt. taken : 254.72MG
Potency found (mcg/mg) : 252.32 MG
Amount found (%) : 101.00 %

Table-13 for assay of erythromycin

Concentration Concentratio Inhibition Zone Diameter (in mm) = Sum of Zones for
Level n mcg/ml In petri Dishes each Level (mm)
1 2 3 4
S1 0.5 27.56 28.42 27.72 27.64 117.34
S2 0.1 21.42 21.28 21.68 21.48 85.86
U1 0.5 27.76 27.74 27.82 28.34 111.66
U2 0.1 21.64 21.42 21.40 21.40 85.86

Calculations
a) % potency of the sample = antilog (2.0 + a log 1)
(in terms of standard)
(U1+U2) – (S1+S2)

54
 Where a =
(U1-U2) + (S1-S2)

b) The potency of the sample was calculated from the formula

% potency x assumed potency of the sample

100

  197.52  197.20  
c) % Amount Found : Antilog  2    (0.69897) 
  25.80  25.48  
  0.28  
Antilog  2    (0.69897) 
 14.56
  

Antilog (2+0.004361) = Antilog 2.004361

101.00
Actual potency = 101.98 x 254.54 x 250
100
= 255.32mg or100.92%

ASSAY OF ERYTHROMYCIN

Assay of erythromycin is den by cup plate method .micrococcus

luteus(ATCC 2103)is used for test organism .5ml of test organism is used.
Potency of standered substence is 661 mcg/mg.

Standard Dilution

The 1ml of erythromycin standard is taken and dissolved in 100 ml

of water From that 5 ml is take and dissolved in 25 ml of water, it is used
for Standard high (SH).from that 5ml is taken and dissolved in 25 ml of
distilled water it is used for Standard low(SL).
Sample Dilution

184.7mg of erythromycin sample is taken and dissolved in

500 ml of water from that 1ml is taken and dissolved in 100 ml, from

55
 that 5ml is taken and dissolved in 25ml of distilled water it si used
for the Sample high(UH).from that 5ml is taken and dissolved in 25
ml is taken it is used for Sample low(UL)

Sample :Erythromycin500mg tablets

B. No : 007
Wt. taken : 225.62mg
Potency found (mcg/mg) : 509.62mg
Amount found (%) : 101.98 %

Table-14 for assay of erythromycin

Concentration Concentratio Inhibition Zone Diameter (in mm) = Sum of Zones for
Level n mcg/ml In petri Dishes each Level (mm)
1 2 3 4
S1 0.5 28.64 28.79 28.24 27.74 113.41
S2 0.1 22.32 21.78 22.12 21.24 87.46
U1 0.5 2827 28.72 28.96 28.84 114.79
U2 0.1 22.00 21.24 21.74 21.76 86.74

Calculations
a) % potency of the sample = antilog (2.0 + a log 1)
(in terms of standard)
(U1+U2) – (S1+S2)
Where a =
(U1-U2) + (S1-S2)

b) The potency of the sample was calculated from the formula

56
 % potency x assumed potency of the sample

100

  201.53  200.87  
c) % Amount Found : Antilog  2    (0.69897) 
  28.05  25.95  

  0.66  
Antilog  2    (0.69897) 
  54  

Antilog (2+0.008542) = Antilog 2.008542

101.98 %
Actual potency = 101.98 x 225.50 x 500
100 225.62

509.62mg or 101.92%
ASSAY OF TETRACYCLINE HCl IP

Error! Not a valid link.

57
ASSAY OF TETRACYCLINE HCl IP

Error! Not a valid link.

58
ASSAY OF TETRACYCLINE HCl IP

Error! Not a valid link.

59
ASSAY OF DOXYCYCLINE

Error! Not a valid link.

60
ASSAY OF VIT. B5

Error! Not a valid link.

61
 ASSAY OF VIT. B5

4.5

4
Concentration of Standard

3.5

2.5

1.5

0.5

0
0 0.5 1 1.5 2

Standard OD values at 530 nm

DISCUSSION

62
 Potency test for vitamins and antibiotics were carried out in
the present study was based on the biological assay as prescribed in
IP.1996 and USP.1995.
Biological assays are primarily based on the use of micro
organisms wish act as indicator or test species for the particular
vitamins and antibiotics. They give a first hand information about
the invitro effect and use of vitamins and antibiotics.
In the present study microbiological assay of vitamins and
antibiotics detecting the potency of drug. The pantothenic acid and
cynacobalamine vitamins and doxycycline, tetracycline,
erytromicine antibiotics are detected by cup plate and turbidometrc
method by using micro organisms like L.plantarum, E.coli,
Staphylococcus aureus, Micrococcus luteus based on the activity of
drug the potency will be detected .the antibiotics inhibits the growth
of microorganisam, these are control the growth of gram positive
and gram negative bacteria. the vitamins are promoting the growth
based on the activity of drug .the both microbial assays physiological
state of medial of lagardamic phase.
The table-4&5 shows potency of pantothenic acid that is
24.887, 25.09 the table 6&7 shows potency of the cynacobalamine
that is17.92 mcg/ml, 14.46 mcg/ml, the table-8 shows potency of
doxycycline is 867.85mg,the table-9,10,11 shows potency of
tetracycline is 977.5 g/mg, 101.7 g/mg, 259.06mg,,the
table12,13,14 shows potency of erythromycin is 642.24 g/mg,
255.32mg, 509.62mg, these values detected from different tablets.s

63
 The results summarizes that the tests carried out in the
analytical laboratory for the estimation of potency of vitamin
pantothenic acid, cynacobalamine, antibiotics doxycyclin,
tetracycline, erythromycin, and their potency values were in the
limits of standard potency witch were specified in pharmacopoeia.
The day-to-day variation and the analyst-to-analyst variations
were also in the limits of the specified labeled amounts. Hence the
evolution was considered as realistic and validated method for
vitamins pantothenic acid, cynacobalamine, antibiotics doxycyclin,
tetracycline, erythromycin.

64
 SUMMARY
The total projection of this project work is on the Micro
Biological assay of Vitamins and Antibiotics. The project work
report containing two units. One is Vitamin assay and the other is
Anti Biotic assay. Vitamin Assay contains two parts. One is
Cyanocobalamine and the other is Calcium panthothenate.
Antibiotic. Assay contains three parts. i.e. Doxycycline Assay,
Erythromycin Assay, Tetracycline Assay.
The Assay Cyanocobalamine is carried out by cup or cylinder
plate method by using E.coli culture as inoculum. The cylinders are
loaded with dilute cyanocobalamine .. standard and test solutions. It
will slowly diffuses in to the agar bed and enhances the growth of
E.coli. after the incubation exhibition zones will be produced, from
which the potency can be calculated.
The assay of calcium panthothenate is carried out by turbidi
metric method by using Lactobacillus arabinosus. In this the sample
and standard is diluted and added I ml of sample to the media and
incubated it at 37°C after the incubation period. The samples was
measured by turbido metric method by using spectro photometer.
Depend on this measurements we will plot the graph and calculated
the value of calcium panthathenate in the given tablet.
The Assay Erythromycin is carried out by cup or cylinder
plate method by using M.luteus culture as inoculum. The cylinders
are loaded with dilute Erythromycin standard and test solutions. It
will slowly diffuses in to the agar bed and Inhibits the growth of
M.luteus. After the incubation inhibition zones will be produced,
from which the potency can be calculated.
The assay of doxycycline is carried out by turbidi metric
method by using S.aureus. In this the sample and standard is diluted

65
and added 1 ml of sample to the media and incubated it at 37°C after
the incubation period. The samples was measured by turbido metric
method by using spectro photometer. Depend on this measurements
we will plot the graph and calculated the value of doxycycline in the
given tablet.
The assay of Tetracycline is carried out by turbidi metric
method by using S.aureus. In this the sample and standard is diluted
and added Iml of sample to the media and incubated it at 37°C after
the incubation period .. The samples was measured by turbido metric
method by using spectro photometer. Depend on this measurements
we will plot the graph and calculated the value of Tetracycline in the
given tablet, As by using Micro Biology and Micro Organisms the
quantity of the samples in the given tablets can be measured through
assay methods in the pharmaceutical field.

66
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70
 PLATE-1
A)Spectrophotometer B) zone reader

C)Assay of cyncobalamine

D)Assay of erythromycin

71
 PLATE-2

E) Antibiotics F)Vitamins

72