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Microbial Assays

This document provides guidance on microbiological assays for measuring compounds like vitamins and antibiotics using microorganisms. It describes two common assay methods - the cylinder-plate method which measures inhibition zones, and the turbidimetric tube method which measures inhibition of microbial growth in solution. Details are given on apparatus, temperature control, media preparation, standard and sample solutions, organisms and inoculation, assay designs including controls, and procedures for each method. The goal is to establish assays that precisely measure the potency of test samples as compared to a reference standard.

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504 views

Microbial Assays

This document provides guidance on microbiological assays for measuring compounds like vitamins and antibiotics using microorganisms. It describes two common assay methods - the cylinder-plate method which measures inhibition zones, and the turbidimetric tube method which measures inhibition of microbial growth in solution. Details are given on apparatus, temperature control, media preparation, standard and sample solutions, organisms and inoculation, assay designs including controls, and procedures for each method. The goal is to establish assays that precisely measure the potency of test samples as compared to a reference standard.

Uploaded by

gopi018
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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M IC R O B IA L A SSAYS

Microbiological assay Method of

measuring compounds such as vitamins


and amino acids, using microorganisms
A biological assay is defined as a

practical procedure whereby the potency


of material of unknown potency is
estimated by comparison of its effect in a
biological system with that of a reference
standard of known or defined potency

M IC R O B IA L A SSAYS O F
A N TIB IO TIC S
The activity (potency) of antibiotics may be demonstrated under

suitable conditions by their inhibitory effect on microorganisms.


Two general methods are employed,
1. The cylinder-plate or plate assay
2. The turbidimetric or tube assay
. The first depends upon diffusion of the antibiotic from a vertical

cylinder through a solidified agar layer in a petri dish or plate to


an extent such that growth of the added microorganism is
prevented entirely in a circular area or zone around the
cylinder containing a solution of the antibiotic.
. The turbidimetric method depends upon the inhibition of growth
of a microbial culture in a uniform solution of the antibiotic in a
fluid medium that is favorable to its rapid growth in the absence
of the antibiotic.

Apparatus
All equipment is to be thoroughly cleaned before and after each
use. Glassware for holding and transferring test organisms is
sterilized by dry heat or by steam.
Temperature Control
Thermostatic control is required in several stages of a microbial
assay, when culturing a microorganism and preparing its
inoculum, and during incubation in plate and tube assays.
Maintain the temperature of assay plates at 0.5 of the
temperature selected. Closer control of the temperature (0.1
of the selected temperature) is imperative during incubation in
a tube assay, and may be achieved in either circulated air or
water, the greater heat capacity of water lending it some
advantage over circulating air.

Media and Diluents

Dissolve the ingredients in water to make 1 L, and adjust the solutions


with either 1 N sodium hydroxide or 1 N hydrochloric acid as required,
so that after steam sterilization the pH is as specified

Ingredient
Pepton
Pancreatic digest
of casein
Yeast extract
Beef extract
Dextrose
Papaic digest of
soyabean
Agar

A
6.0
4.0

B
6.0
-

C
5.0
-

D
6.0
4.0

E
6.0
-

F
6.0
-

G
9.4
-

H
17.0

I
10.0
-

J
15.0

3.0
1.5
1.0
-

3.0
1.5
-

1.5
1.5
1.0
-

3.0
1.5
1.0
-

3.0
1.5
-

3.0
1.5
-

4.7
2.4
10.0
-

2.5
3.0

10.0
-

5.0

15.0

15.0

15.0

15.0

15.0

23.5

12.0

17.0

15.0

Glycerin
Polysorbate 80
Sodium chloride
Dipotassium
Hydrogen
Phosphate

3.5
3.68

10.0
-

10.0
5.0
2.5

10.0
3.0
-

5.0
-

Potassium
dihydrogen
phosphate

1.32

Final pH (after
sterilisation)

6.56.6

6.56.6

6.95
7.05

7.8 8.0

7.88.0

5.86.0

6.0 6.2

7.1
7.3

6.9 7.1

7.2 7.4

Preparation of the Standard

To prepare a stock solution, dissolve a quantity of a given antibiotic,


accurately weighed, or the entire contents of a vial of Reference
Standard, where appropriate, in the solvent, and then dilute to the
required concentration as indicated. Store in a refrigerator, and use
within the period indicated.

Standard Stock Solution


Antibiotic
Method
Amikacin
Amphotericin B
Bacitracin
Bleomycin
Carbenicillin
Doxycycline
Erythromycin
Framycetin
Gentamicin
Kanamycin
Kanamycin B
Neomycin
Novobiocin
Nystatin
Polymyxin B

B
A
A
A
A
B
A
A
A
A
A
A
A
A
A

Final stock
concentration
per /ml
1 mg
1 mg
100 units
2 units
1 mg
1 mg
1 mg
1 mg
1 mg
800 units
1000 units
1 mg
1 mg
1000 units
10,000 units

Rifampicin

1 mg

Test Dilution
Use before(no
Final
of days)
diluent

Median dose g
or units per ml

Incubation
temp. (oC)

14
Same day
Same day
14
14
5
14
14
30
30
30
14
5
Same day
14

Water
B5
B1
B6
B6
Water
B2
B2
B2
B2
B2
B2
B4
B4
B4

10 g
1.0 g
1.0 unit
0.04 units
20 g
0.1 g
1.0 g
1.0 g
0.1 g
0.8 units
1.0 unit
1.0 g
0.5 g
20 units
10 units

32 35
29 31
32 35
32 35
36 - 37.5
35 37
35 37
30 35
36 - 37.5
37 39
32 35
36 - 37.5
32 35
29-31
35 - 39

B1

5.0 g

29 31

On the day of the assay, prepare from the stock solution five or more test
dilutions, the successive solutions increasing stepwise in concentration,
usually in the ratio of 1:1.25 for a cylinder-plate assay or smaller for a
turbidimetric assay

Preparation of the Sample

From the information available for the preparation to be assayed (the


Unknown), assign to it an assumed potency per unit weight or
volume, and on this assumption prepare on the day of the assay a
stock solution and test dilution as specified for each antibiotic but with
the same final diluent as used for Reference Standard. The assay with
five levels of the Standard requires only one level of the Unknown at a
concentration assumed equal to the median level of the Standard.

Organisms and Inoculum


Antibiotic
Amikacin
Amphotericin B
Bacitracin
Bleomycin
Carbenicillin
Doxycycline
Erythromycin
Framycetin
Gentamicin
Kanamycin sulphate

Kanamycin B
Neomycin
Novobiocin
Nystatin
Oxytetracycline
Polymyxin B
Rifampicin
Streptomycin
Tetracycline

Test Organism
Staphylococcus aureus
Saccharomyces cerevisiae
Micrococcus luteus
Mycobacterium smegmatis
Pseudomonas aeruginosa
Staphylococcus aureus
Micrococcus luteus
Bacillus pumilus
Bacillus subtilis
Staphylococcus epidermidis
Bacillus pumilus

Bacillus subtilis
Staphylococcus epidermidis
Staphylococcus epidermidis
Saccharomyces Cerevisiae
Bacillus cereus var, mycoides
Staphylococcus aureus
Bordetella bronchiseptica
Bacillus subtilis
Bacillus subtilis
Klebsiella pnumoniae
Bacillus Cereus
Staphylococcus aureus

P reparation of Inoculum
Test organism

Temp.
(C)

Time

Medium

Bacillus Cereus
var. mycoides
Bacillus
pumilus

32-35

5 days

Amount
(ml per
100 ml)
As required

Antibiotics
assayed.

32-3

5 days

As required

Bacillus
subtilis
Bordetella
bronchiseptica
Klebsiella
Pneumoniae
Micrococcus
luteus (9341)
Micrococcus
luteus (10240)
Mycobacterium
smegmatis
Pseudomonas
aeruginosa2
Saccharomyces
cerevisiae
(9763)
Saccharomyces
cerevisiae
(2601)
Staphylococcus
aureus

32-35

5 days

As required

Oxytetracycline
Tetracycline
Framycetin
Kanamycin
sulphate
Framycetin

32-35

24 hr

0.1

Polymyxin B

36-37

24 hr

0.1

Streptomycin

32-35

24 hours

1.5

Erythromycin

32-35

24 hr

0.3

Bacitracin

36-37.5

48 hr

1.0

Bleomycin

36-37.5

24 hr

0.5

Carbenicillin

29-31

48 hr

1.0

Amphotericin B

29/31

48 hr

1.0

Nystatin

32-35

24 hr

0.1

Amikacin
Doxycycline
Oxytetracycline

A ssay D esigns
Two alternative designs are recommended in IP
3-level(or 2-level) factorial assay
1-level assay with a standard curve

For a factorial assay, solutions of 3 or 2 corresponding

test dilutions for both the standard and the unknowns


are prepared on the day of the assay, For a 1-level
assay with a standard curve, prepare instead solutions
of five test dilutions of the standard and a solution of a
single median test level of the unknown as described in
the same sections

M ethods
The m icrobiologicalassay ofantibiotics m ay be carried outby
M ethod A or
M ethod B.

CYLINDER-PLATE OR CUP-PLATE

METHOD

a previously liquified medium appropriate to the assay is


Inoculated with the requisite quantity of suspension of the
micro organism, The suspension is added to the medium at a
temperature between 40 and 50C and inoculated medium
is immediately poured into the petri dishes or large
rectangular plates to give a depth of 3 to 4 mm.

The prepared dishes or plates must be stored in a manner so


as to ensure that no significant growth or death of the test
organism occurs before the dishes or plates are used and
that the surface of the agar layer is dry at the time of use.

C YLIN D ER -P LATE O R C U PP LATE M ETH O D


Solutions of known concentrations of the standard

preparation are prepared by using buffer solution and


solutions of the corresponding assumed of the antibiotic
to be examined. These solutions are applied to the
surface of the solid medium in sterile cylinders or in
cavities prepared in the agar
These dishes or plates are left standing for 1 to 4 hours

at room temperature or at 4c, as appropriate, as a


period of pre-incubation diffusion to minimize the
effects of variation in time between the applications of
the different solutions. Then they are incubated for
about 18 hours at 3 70c and accurately measured the
diameters or areas of the circular inhibition zones.

TU R B ID IM ETR IC O R TU B E A SSAY
The
methodO
has
M ETH
Dthe advantage of a shorter incubation period for the growth

of the test organism (usually 3 to 4 hours) but the presence of solvent


residues or other inhibitory substances affects this assay more than the
cylinder plates assay and care should be taken to ensure freedom from such
substances in the final test solutions. This method is not recommended for
cloudy or turbid preparations.

Five different concentrations of the standard solution are prepared for


preparing the standard curve by diluting the stock solution of the Standard
Preparation of the antibiotic and increased stepwise in the ration 4:5.

1 ml of each concentration of the standard solution and of the sample


solution is placed in each of the tubes in duplicate. To each tube 9 ml. of
nutrient medium previously seeded with the appropriate test organism are
added

Three control tubes are prepared, one containing the inoculated culture
medium (culture control), another identical with it but treated immediately
with 0.5 ml of dilute formaldehyde solution (blank) and a third containing
uninoculated culture medium.

All the tubes are placed randomly distributed or in a randomized block


arrangement, in an incubator or water-bath and maintain them at the
specified temperature for 3 to 4 hours. After incubation 0.5 ml of dilute
formaldehyde solution is added to each tube. The growth of the test
organism measured by determining the absorbance at about 530nm of each
of the solutions in the tubes against the blank,

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