BIO-ASSAY

Definition
Estimation of the concentration / potency of a
substance by measuring its biological response
in living systems
i.e.Observation of pharmacological effects on
[1] living tissues, or cells
[2] microorganisms
[3] animals
 Indications for Bioassay
 Active principle of drug is unknown

 Active pple cannot be isolated, e.g. insulin,

posterior pituitary extract etc.

 Chemical method is either

 not available
 if available, too complex,
 insensitive to low doses e.g. Histamine can be
bioassayed in microgram conc.
 Unknown Chemical composition, e.g. long
acting thyroid stimulator.

 Chemical composition of drug variable but

has same pharmacological action e.g.
cardiac glycosides isolated from diff
sources, catecholamines etc
 Principles of Bioassay
 Active
principle to be assayed should show the
same measured response in all animal species

 The degree of pharmacological response

produced should be reproducible under identical
conditions [Eg Adrenaline shows same rise in BP
in the same species under identical conditions: wt,
age, sex, strain / breed etc]
 The reference standard must owe its
activity to the principle for which the
sample is being bio assayed
 Activity assayed should be the activity of
interest
 Individual variations must be minimised /
accounted for
 Bioassay might measure a diff aspect of
the same substance compared to
chemical assay [Eg testosterone &
metabolites
 Factors affecting assays

 Concentration of cells
 Size of cells
 Opacity of cells
 Inoculum size
Factors affecting assays in general
 Media components
 Incubation Time
 Incubation temperature
 pH of medium
 Drugs
 Types of Assays
 Chemical Assays: Spectrophotometry,
Spectrofluorimetry, Chromatography

 Immunoassays

 Microbiological assays

 Animal assays.

 Plant assays ????

 Types of Bioassays
 [1]Quantal Assays [ Direct endpoint ]
 Elicits an ‘All or None’ response in
different animals
 Eg. Digitalis induced cardiac arrest in
guinea pigs
 hypoglycaemic convulsions in mice.
 Digitalis induced head drop in
rabbits
Calculation of LD50 in mice or rats
 Types of Bioassays 2
 [2] Graded Response Assays [mostly on tissues]
 Graded responses to varying doses
 Unknown dose response measured on same tissue
 Methods of Bioassay con1

Graded Response Assays [ Direct comparison

on same tissues]
 Interpolation: Conc. of unknown is read
from a standard plot of a log dose
response curve of at least 4 sub maximal
concentrations
 Methods of Bioassay 2.
 Matching / Bracketing: Const dose bracketed with varying

doses of standard till exact match is obtained

Used when test sample is too small

Inaccurate & margin of error difficult to estimate

Eg histamine on g. pig ileum, Posterior pituitary on

rat uterus
 Methods of Bioassay 3

 Multiple Point Assays

3 point assay [combines pples of

matching with interpolation]

4 point assay [combines pples of

matching with interpolation]

 3 point assay [2+1 dose assay]
 Fast & convenient
 Procedure [Eg Ach bioassay]
 Log dose response [LDR] curve plotted
with varying conc. of std Ach solutions
and given test solution
 Select two std doses s1& s2 [ in 1:2
dose ratio] from linear part of LDR [ Let
the corresponding response be S1, S2]
 Choose a test dose t with a response T
between S1 & S2
3 point assay [2+1 dose assay]
 Record 4 sets data [Latin square:
Randomisation reduces error] as follows
s1 s2 t
t s1 s2
s2 t s1
s1 s2 t
 Plot mean of S1, S2 and T against dose.
Calculate
 Log Potency ratio [ M ] = [ (T –S1) / (S2-S1) ] X
log d
[d = dose ratio]
 4 point assay [2 +2 dose assay]
 Procedure [Eg Ach bioassay]
 Log dose response [LDR] curve plotted
with varying conc. of std Ach solutions
and given test solution
 Select two std doses s1& s2 from linear
part of LDR [ Let the corresponding
response be S1, S2]
 Choose two test doses t1 & t2 with
response T1 &T2 between S1 & S2 ; Also
s2/s1 = t2/t1 = 2
4 point assay [2 +2 dose assay]
contd.
 Record 4 data sets [Latin square: Randomisation
reduces error]
s1 s2 t1 t2
s2 t1 t2 s1
t1 t2 s1 s2
t2 s1 s2 t1
 Plot mean of S1, S2 and T1, T2 against dose.
Calculate
 Log Potency ratio [M] = [ (T1 –S1 + T2 –S2) / (S2-S1
+ T2-T1) ] X log d [d = dose ratio]
 PRINCIPLES
1.THE LAW OF DIFFUSION
 Ficks Law
 Solutions for Linear Diffusion
 Solutions for Radial Diffusion
 Prediffusion
 Factors Influencing the Diffusion Coefficient
 Measurement of Diffusion Coefficient
2. THE LAW OF GROWTH AND MULTIPLICATION
 Critical Time
 Viable Count and Time
 Temperature and Growth Rate
 The Effect of the Inoculum
 Other Factors
3. THE LAW OF ABSORPTION,
PERMEABILITY, AND PARTITION

 General Principles
 The Relationship of Critical Population and
Critical Concentration
4. THE LAW OF SPECIFIC ANTIBIOTIC
ACTION
 Antibiotic Destruction or Deviation
 Specific Antibiotic Action
5.THE LAW OF STATISICS
 Biological Assay
 Antibiotic Assay
OTHER USES OF DIFFUSION METHOD
METHODS
 Surface Cultures
 Exhibition Zones

SENSITIVITY TESTS
 Assay methods for antifungal
compounds have followed the
patterns of antibacterial antibiotics,
measuring their effects on growth of
test organisms by
 Microscopy
 Turbidimetry
 Agar Dilution
 Agar Diffusion
 Respirometry.
 I . TURIDIMETRIC METHOD

Serial dilution assay method

Amphotericin B
Turbidimetric assay

o Most commonly used

technique for measuring growth
response of a microorganism.

o Turbidity reading in a liquid

medium is that of the growth of
microorganism in the medium.
Agar dilution method

o The antibiotic is incorporated into the unseeded

molten agar medium and the test microorganisms
are seeded on the surface.
o The advantage of this method is that more than one
type of organism can be tested directing comparing
the effect of one antifungal agent upon several
microorganisms.
ube dilution assay
oculate tubes containing sever
ncentrations of test compound
st organism and incubate.

TB
 bacteria
growth
10 1 .1 .01 .001
MIC
minimum inhibitory concentration
lowest concentration of a substa
inhibits growth of a test organism
TB
Diffusion technique

o Seeded agar is placed in test tubes

and layered with solution of antifungal
agent.
o This agent diffuses into the medium.
o The response was measured as the
distance from the liquid-agar interface
to the place in the tube where growth
first appeared.
gar diffusion assay
lawn of test bacteria
filter paper soaked
with test compoun

zones of inhibition
agar plate (no growth)

TB
Sensitivity of diffusion method can be influence
by:

1. Prediffusing the antibiotic in the agar by

holding plates for a period of time below the
optimal growth temperature of
microorganism

2. Decreasing thickness of agar layer

3. Reducing the number of the organism in the

seed
Sensitivity of diffusion method can be
influence by:

4. Varying the nutrient and salt

composition of the agar

5. Addition of subinhibitory level of the

antifungal agent to the agar prior to
pouring the plates.
 1. INTRODUCTION

 Assay method used for amphotericin B in

body fluids

 Activityof amphotericin B is influenced by

the presence of urine, serum, tissue
extracts, etc.
 2.TEST ORGANISM
Stock culture: grow Candida albicans for 1-2 days at 250 to 350 C on
sabouraud’s dextrose agar slants.

Inoculum: inoculate sabouraud’s dextrose broth from stock slant and

incubate 1-2 days at 250 C or 350 C.

Preparation of inoculated medium: add 100,000 units of penicillin and

100mg of streptomycin to 100ml of sterile sabouraud’s dextrose broth
(test medium).
- add 1ml of the inoculum to 100ml of medium and dispense (0.8ml)
the mixture into tubes, place in groups of four in racks.
- keep the racks at 50 C.
 3.PREPARATION OF STANDARDS AND
SAMPLES FOR ASSAY
Protect both standard and samples from light.
 Standards: solution of amphotericin B 100ug of
activity/ml of dimethyl sulfoxide.
- make further dilutions of the standard in normal
body fluid.
- make three final standard solutions (0.10,0.25 and
0.60ug/ml)
 Samples: depending on the expected potency,
make dilutions with appropriate normal body fluid.
 4.ASSAY DESIGN
Dosing, incubation and reading of assay:
- approximately 2ml each of sample and normal
body fluid are required for each test.
- to each of the 4 tubes add a quantity of sample
and compensating fluid.
- assay each of the three standard solutions as a
separate sample.
- incubate the racks of tubes overnight
- end point tube is the one containing the smallest
quantity of sample capable of inhibiting the test
organism .
Calculation: MIC = concentration of standard solution
dilution of the end point tube
II . PHOTOMETRIC ASSAY METHOD

Amphotericin B
 1. INTRODUCTION

This method is used for assay of

amphotericin B18.

2.TEST ORGANISM

Stock culture: grow Candida tropicalis for 1-2 days at

250 to 350 C on yeast beef agar slants.
2.TEST ORGANISM

 Inoculum:
- inoculate yeast from inoculum broth.
- incubate this first broth transfer overnight at 370
C on shaker.
- inoculate second flask with 25% from the
first broth transfer, incubate on shaker for 3 hrs and
chill rapidly.
3.PREPARATION OF STANDARDS AND
SAMPLES FOR ASSAY
Protect both standard and samples from
light
 Standards: amphotericin B under at -200C
to 300C
- solution of amphotericin B in dimethyl
sulfoxide at a concentration of about 1mg
of activity per millimeter.
- dilute this solution in DMW-1 to obtain
concentrations of 36.0, 0.9, 0.6, 0.4, and
0.267 ug/ml.
 Samples :
Solutions – 0.6 and 0.4 ug/ml DMW-1
Suspensions –sufficient amount of dimethyl
sulfoxide to be added
- shake for about ½ hr.
- dilute to an estimated 0.6
and 0.4 ug/ml in DMW-1.
Solids – add from 1 to 10 ml of dimethyl
sulfoxide for each mg of
amphotericin B.
-dilute the dimethyl sulfoxide extract
in DMW-1 to 0.6 and 0.4 ug/ml.
4. ASSAY DESIGN
Preparation of inoculated assay medium,
incubation, and reading:
- to the tubes already containing 0.06ml of
amphotericin B solution add 10ml of inoculated
assay medium
- incubate for 3 hrs at 370C on a shaker
- add 1ml of an aqueous 10% phenol solution to
each of the tube .
- read the per cent transmission of each tube
with spectrophotometer at a wavelength of 600-
660nm.
Calculation
 III . TURIDIMETRIC METHOD

Agar dilution assay method

Amphotericin B
 1.INTRODUCTION

• This method suited for testing the

susceptibility of fungi to anti-fungal
agents.
• Testing of clinical isolates
 2.TEST ORGANISM
Inoculum : Candida albicans
Preparation of assay plates : Dispense 300
ml aliquots in 500 ml flask and sterilize
Dilute amphotericin B solution in flask of
melted assay agar to the follation
concentaration 100, 50, 25, 12, 6, 3, 1.6,
0.8, 0.4 and 0.2ug/ml
Prepare one flask with out antibiotic for
control
 3.Preparation of Standards

 Weigh 25-30 mg of amphotericin B

reference standard and dissolve in
dimethyl sulfoxide to a concentration of 4
mg of activity per milliliter of the solvent
 4. Assay Design

 Filla 2 or a 5 ml pipette with the suspension of the

test organism and streak it across the surface of
one plate of each concentration
 Streak 3 to 10 different suspension on each plate
 Incubate for 3 to 4 days at room temperature
 Smallest concentration of amphotericin B that
causes inhibition is the MIC
 IV PHOTOMETRIC ASSAYING
PRINCIPLE
A Mixture of test substance and test
organism in nutrient medium is incubated,
and the growth response of the test
organism in measured.
Physical factors influencing turbidity.

1. Concentration of cells.
2. Size of cells.
3. Opacity of cells.
4. Arrangement
Calibration of photometers.

Preparation of Inoculum

Media Composition.

Incubation conditions.
 Response of bacteria.
A. Antibiotics

B. Growth Substances (amino acids &

vitamins)

Modes of Expressing Response

A. Antibiotic Dosage-Response Curve

B. Vitamins and Amino acids Curve

DESIGN AND VALIDITY

o ANTIBIOTICS
1.Validity
2.Design

o VITAMINS AND AMINO ACIDS

1.Nonlinear Assay Lines
2. Linear Assay Lines
SENSITIVITY

MEANING OF PHOTOMETRIC RESPONSE

VARIATIONS AND ERRORS

 Glassware Cleaning
 Volumetric Equipment
 Medium
 Organism
 Incubation
 Mechanical Errors
 Errors of Measurement
 Inherent Errors
MICROBIOLOGICAL
ASSAY
The main use of microbiological assay
is in determination of the potency of
growth –inhibiting substances –
especially antibiotics-and of growth
promoting substances-amino acids
and vitamins.
 Three main assay procedures

1.Agar diffusion assay (for growth-inhibiting & growth-

promoting substances)

o Devised by Biochemist Norman George Heatly,1940

for penicillin.
o Parallel-line assay.
2.Flask or Tube assay (for growth-promoting
substances)

o Was given by Snell and Strong in 1939.

o Slope ratio assays.
3.Tube assay (for growth-inhibiting
substances)

o Foster in 1942 described this assay for

penicillin.
 BASIS OF CALCULATION OF
POTENCY ESTIMATES
 There are two models for calculations:
1.The response (or a function of response) is
directly proportional to the logarithm of the dose of
active substance.

2.The response (or a function of response) is

directly proportional to the dose itself of active
substance.
These two models are the basis of parallel-line
assays and slope line assay respectively.
Microbiological Assay (MBA) is defined
here as the estimation of potency of a
growth-promoting (GPS) or growth-
inhibiting substances (GIS) by comparing
its quantitative effect on the growth of
specific microorganism with that of a
reference standard of defined potency.
 General guidance for conducting
Microbiological Assays

1.INOCULUM

 108/ml or 109/ml of cells

 May be vegetative cells or spore
suspension
 Exact size of inoculum is not critical as it
will not make a difference to the zone
diameter.
2. TEST SOLUTIONS

 Weighing
 Weighing or Measuring from a sample of the
unknown
 Dilution of the primary solution to test solution
level
 Problems with very dilute solutions
 Assay medium
Table 1
Permitted variation in capacity of volumetric bulb pipettes

Capacity Class A Tolerance Class B Tolerance

ml ±ml ±% ±ml ±%

1 0.007 0.70 0.150 1.50

2 0.010 0.50 0.020 1.00
5 0.015 0.30 0.030 0.60
10 0.020 0.20 0.040 0.40
25 0.030 0.12 0.060 0.24

Source :Extract adapted from BIS1583:1986.

Table 2
Permitted variation in capacity of volumetric flasks

Capacity Class A Tolerance Class B Tolerance

ml ±ml ±% ±ml ±%

5 0.02 0.40 0.04 0.80

10 0.02 0.02 0.04 0.4
25 0.03 0.12 0.06 0.24
50 0.05 0.10 0.10 0.20
100 0.08 0.08 0.15 0.15
250 0.15 0.06 0.30 0.12
500 0.25 0.05 0.50 0.10

Source :Extract adapted from BIS1792:1982.

3. SELECTION OF LATIN SQUARES AND THE
PLATING ROUTINE

 Was given by Fisher and Yates (1938)

 A Latin square design is an arrangement of numbers
1 to n in n rows and n columns in such a way that
each number appears once, and once only, in each
row and each column.
 In microbiological assays, n commonly has values
either 6 or 8 (6×6 and 8×8 latin squares respectively)
 4.ASEPTIC TECHNIQUES

• Test organism
• Inoculating the medium
• Assay plates (agar diffusion assay)
• Assay tubes (turbidimetric assay)
• Diluents
• Sample
• Test solution and effect of contamination
• Application of test solutions (agar diffusion
assay/ turbidimetric assay)
5.MEASURING RESPONSES

• AGAR DIFFUSION ASSAY

• TURBIDIMETRIC ASSAY