LABORATORY MANUAL

PHARMACEUTICAL BIOTECHNOLOGY

R13
III B.Pharm –II SEMESTER
 CONTENTS

S. No NAME OF THE EXPERIMENT

1 Test for sterility for moist heat sterilized product
2 Test for sterility for dry heat sterilized product
3 Test for sterility for gaseous sterilized product
4 Test for sterility of surgical dressing
5 Immobilization by entrapment technique
6 Isolation of antibiotic producing organisms from soil
7 Determination of minimum inhibitory concentration of an antibiotic

8 Microbiological assay of Amikacin by cup-plate method-

determination of unknown concentration

9 Microbiological assay of Amikacin by disc-plate method-

determination of unknown concentration

Experiment 1

TEST FOR STERILITY FOR MOIST HEAT STERILIZED PRODUCT

Aim: To perform test for sterility of a moist heat sterilized sample.

Requirements: Test tubes, Inoculation needles, Autoclave, Distilled water, Nutrient broth, 24
hours fresh culture.

Principle: Sterilization is a process in which the destruction or elimination of all living

organisms of all types including spores is brought about test for sterility are intended for
detecting the presence of viable microorganisms in all pharmacopeial preparations. The test for
sterility must be carried out under conditions to design to avoid accidental contamination of
products during testing. Precautions taken in this procedure must not adversely affect the
microorganisms that must be revealed in this test. Two types of controls are used in sterility
testing for interpretation of the results i.e. positive control and negative control.

Positive control: It is intended to ensure that sterility test system will support the growth of
microorganisms and is therefore likely to detect all viable microbial contamination.

Negative control: It ensures the sterility of all materials, medium, apparatus used in the test.

Autoclave is the best example for moist heat sterilization. Which is a double jacketed steam
chamber made up of stainless steel. It is operated at 15lb/inch2, at 121oC for 15 -20 min. At these
conditions the autoclave produces saturated steam and inactivates the contaminating organisms
by hydration and finally results in coagulation of proteins.

Procedure: The first step in experiment is the preparation of culture medium.

1. The nutrient broth is prepared using the following composition

Meat extract -0.3%

Peptone -0.1%

Sodium chloride -0.5%

Distilled water -100 mL

pH -7.2± 0.2

2. All the above ingredients should be dissolved in distilled water, made upto 100 mL and filter.
Then adjust the pH 7.2 by adding either NaOH or HCl solution. Then transfer the nutrient broth
into test tubes with volume of 5 mL.
 3. Transfer 2 mL of distilled water in separate test tubes.
4. All these test tubes are sterilized by autoclave at standard conditions.
5. After sterilization the positive control must be prepared by inoculating loop full of
microbial suspension into nutrient broth.
6. Negative control is prepared by adding fresh culture of microorganisms into sterilized
nutrient broth.
7. Transfer not less than 1 mL of moist heat sterilized water sample into the nutrient broth
under strict aseptic conditions using sterile syringe.
8. Now all the inoculated test tubes along with positive and negative control tubes are
incubated at 37oC for 24 hours.
9. After incubation the results are interpreted by comparing the sample tubes with positive
and negative controls.

Observation:

Positive control:

Negative control:

Sample tube:

Report:
Experiment 2

TEST FOR STERILITY FOR DRY HEAT STERILIZED PRODUCT

Aim: To perform test for sterility of a dry heat sterilized sample.

Requirements: Test tubes, Inoculation needles, Hot air oven, Kaolin, Nutrient broth, 24 hours
fresh culture.

Principle: Sterilization is a process in which the destruction or elimination of all living

organisms of all types including spores is brought about test for sterility are intended for
detecting the presence of viable microorganisms in all pharmacopeial preparations. The test for
sterility must be carried out under conditions to design to avoid accidental contamination of
products during testing. Precautions taken in this procedure must not adversely affect the
microorganisms that must be revealed in this test. Two types of controls are used in sterility
testing for interpretation of the results i.e. positive control and negative control.

Positive control: It is intended to ensure that sterility test system will support the growth of
microorganisms and is therefore likely to detect all viable microbial contamination.

Negative control: It ensures the sterility of all materials, medium, apparatus used in the test.

Hot air oven is recommended when the steam under pressure will make direct contact with the
materials to be sterilized may be degraded or destroyed. This is true for certain items of
glassware such as petridish, pipettes, oils, powders and some similar substances. The standard
conditions for dry heat sterilization using hot air oven is 160oC for 1-2 hours. Dry heat
sterilization kills the microorganisms by oxidation of the vital cell components.

Procedure: The first step in experiment is the preparation of culture medium.

1. The nutrient broth is prepared using the following composition

Meat extract -0.3%

Peptone -0.1%

Sodium chloride -0.5%

Distilled water -100 mL

pH -7.2± 0.2

2.All the above ingredients should be dissolved in distilled water, made upto 100 mL and filter.
Then adjust the pH 7.2 by adding either NaOH or HCl solution. Then transfer the nutrient broth
into test tubes with volume of 5 mL.
 3. All these test tubes are sterilized by autoclave at standard conditions.
4. Kaolin or talc is taken into petridish and sterilized by using hot air oven under standard
conditions.

5. After sterilization the positive control must be prepared by inoculating loop full of
microbial suspension into nutrient broth.

6. Negative control is prepared by adding fresh culture of microorganisms into sterilized

nutrient broth.

7. Take a pinch of kaolin or talc, which is sterilized previously by hot air oven and inoculate
into sterile nutrient broth tubes under aseptic conditions.

8. Now all the inoculated test tubes along with positive and negative control tubes are
incubated at 37oC for 24 hours.

9. After incubation the results are interpreted by comparing the sample tubes with positive
and negative controls.

Observation:

Positive control:

Negative control:

Sample tube:

Report:
Experiment 3

TEST FOR STERILITY FOR GASEOUS STERILIZED PRODUCT

Aim: To perform test for sterility of a gaseous sterilized sample.

Requirements: Test tubes, Inoculation needle, Forceps, Formaldehyde, Cotton, Nutrient broth,
24 hours fresh culture.

Principle: Sterilization is a process in which the destruction or elimination of all living

organisms of all types including spores is brought about test for sterility are intended for
detecting the presence of viable microorganisms in all pharmacopeial preparations. The test for
sterility must be carried out under conditions to design to avoid accidental contamination of
products during testing. Precautions taken in this procedure must not adversely affect the
microorganisms that must be revealed in this test. Two types of controls are used in sterility
testing for interpretation of the results i.e. positive control and negative control.

Positive control: It is intended to ensure that sterility test system will support the growth of
microorganisms and is therefore likely to detect all viable microbial contamination.

Negative control: It ensures the sterility of all materials, medium, apparatus used in the test.

Sterilization by formaldehyde is gaseous sterilization and is a chemical method. Formaldehyde is

stable only at high concentrations and elevated temperatures.

Formaldehyde solution is useful for sterilization of certain instruments. Formaldehyde in gaseous

form which can be used for disinfection and sterilization of closed areas. Humidity and
temperature have a prolonged effect on microbial action of formaldehyde. In order to sterilize
with formaldehyde the temperature must be above room temperature and the moisture between
60-80%. Formaldehyde kills the microbes by denaturation of the vital cell components

Procedure: The first step in experiment is the preparation of culture medium.

1.The nutrient broth is prepared using the following composition

Meat extract -0.3%

Peptone -0.1%

Sodium chloride -0.5%

Distilled water -100 mL

pH -7.2± 0.2
 2. All the above ingredients should be dissolved in distilled water, made upto 100 mL and
filter. Then adjust the pH 7.2 by adding either NaOH or HCl solution. Then transfer the
nutrient broth into test tubes with volume of 5 mL.
3. All these test tubes are sterilized by autoclave at standard conditions.

4. Small cotton balls are prepared and sterilized by gaseous sterilization. Place the
unsterilized cotton in upper chamber of the desiccator and place formaldehyde at lower
chamber.

5. Allow the formaldehyde vapours to release in closed conditions and the cotton get
sterilized.

6. After sterilization the positive control must be prepared by inoculating loop full of
microbial suspension into nutrient broth.

7. Negative control is prepared by adding fresh culture of microorganisms into sterilized

nutrient broth.

8. Take a small cotton ball with the help of sterile forceps, which is sterilized previously by
formaldehyde and inoculate into sterile nutrient broth tubes under aseptic conditions.

9. Now all the inoculated test tubes along with positive and negative control tubes are
incubated at 37oC for 24 hours.

10. After incubation the results are interpreted by comparing the sample tubes with positive
and negative controls.

Observation:

Positive control:

Negative control:

Sample tube:

Report:
Experiment 4

TEST FOR STERILITY OF SURGICAL DRESSING

Aim: To perform test for sterility of a surgical dressing.

Requirements: Test tubes, Inoculation needle, Forceps, Autoclave, Pieces of surgical dressing,
Nutrient broth, 24 hours fresh culture.

Principle: Sterilization is a process in which the destruction or elimination of all living

organisms of all types including spores is brought about test for sterility are intended for
detecting the presence of viable microorganisms in all pharmacopeial preparations. The test for
sterility must be carried out under conditions to design to avoid accidental contamination of
products during testing. Precautions taken in this procedure must not adversely affect the
microorganisms that must be revealed in this test. Two types of controls are used in sterility
testing for interpretation of the results i.e. positive control and negative control.

Positive control: It is intended to ensure that sterility test system will support the growth of
microorganisms and is therefore likely to detect all viable microbial contamination.

Negative control: It ensures the sterility of all materials, medium, apparatus used in the test.

Surgical dressings are used in surgeries on wounds. They are directly in contact with the tissues
and so, they must be sterilized and free from contaminating organisms.

Procedure: The first step in experiment is the preparation of culture medium.

1. The nutrient broth is prepared using the following composition

Meat extract -0.3%

Peptone -0.1%

Sodium chloride -0.5%

Distilled water -100 mL

pH -7.2± 0.2

2. All the above ingredients should be dissolved in distilled water, made upto 100 mL and filter.
Then adjust the pH 7.2 by adding either NaOH or HCl solution. Then transfer the nutrient broth
into test tubes with volume of 5 mL.

3. All these test tubes are sterilized by autoclave at standard conditions.

4. Take a piece of surgical dressing, fold in a piece of paper and prepare a small packet. Sterilize
in an autoclave by moist heat sterilization by keeping at 121oC at 15lb/inch2 pressure for atleast
15-20 min.

5. After sterilization the positive control must be prepared by inoculating loop full of microbial
suspension into nutrient broth.

6. Negative control is prepared by adding fresh culture of microorganisms into sterilized nutrient
broth.

7. Take a small piece of surgical dressing with the help of sterile forceps, and transfer into sterile
nutrient broth tubes under aseptic conditions.

8. Now all the inoculated test tubes along with positive and negative control tubes are incubated
at 37oC for 24 hours.

9. After incubation the results are interpreted by comparing the sample tubes with positive and
negative controls.

Observation:

Positive control:

Negative control:

Sample tube:

Report:
Experiment 5

IMMOBILIZATION BY ENTRAPMENT TECHNIQUE

Aim: To perform immobilization by entrapment technique.

Requirements: Calcium chloride, Sodium alginate, 24 hours cell suspension, Syringe, Beakers.

Theory:

The immobilized whole cell system is an alternative to enzyme immobilization. Unlike enzyme
immobilization, where the enzyme is attached to a solid support (such as calcium alginate or
activated PVA or activated PEI), in immobilized whole cell systems, the target cell is
immobilized. Such methods may be implemented when the enzymes required are difficult or
expensive to extract, an example being intracellular enzymes.

Procedure:

Preparation of cell suspension:

The organism was grown in nutrient broth medium for 24 hours. After incubation the cells are
harvested by centrifugation. The pellets are washed with sterile water. Finally 1-2 mL of sterile
water is added to make suspension. Cell suspension is used for immobilization of various
materials.

Sodium alginate solution (3%):

It is prepared by dissolving 3g of sodium alginate in 100 mL of hot distilled water. The contents
were stirred vigorously for 10 min to obtain thick uniform slurry without any undissolved gum
and sterilized by autoclave.

Calcium chloride solution (0.2 M):

A solution of calcium chloride is prepared by dissolving calcium chloride and make upto 1000
mL by using distilled water. The solution is stored at room temperature.

Sodium alginate and calcium chloride are used to prepare alginate beads containing whole cells.
The cell suspension is added to sodium alginate solution and stirred for 10 min to get uniform
mixture. The slurry is taken into sterile syringe and added slowly drop wise into 0.2 M calcium
chloride solution from 5 cm height and kept for 1 hour at 4oC. Beads are washed with sterile
water for 3-4 times. When beads are not used then they are stored in normal saline solution in
refrigerator. Experiment is conducted under aseptic conditions using laminar air flow unit.

Report:

Fig: Cacium alginate beads entrapped with microbial cell suspension

Experiment 6

ISOLATION OF ANTIBIOTIC PRODUCING ORGANISMS FROM SOIL

Aim: To screen the microorganisms that can be able to produce antibiotics from different soil
samples.

Requirements: Sterile growth medium, anti-bacterial and anti-fungal agents, soil samples, test
tubes, conical flasks.

Composition of media for bacterial growth:

Nutrient agar medium:

Beef extract- 10g

Peptone – 10 g

Sodium chloride – 5g

Agar -20g

Distilled water -1000 mL

pH -7 -7.2

Composition of the media for fungal growth:

Saboraud’s media:

Glucose – 20 g

Peptone -10 g

Agar – 15 g

Distilled water -1000 mL

pH – 5.6

Theory:

Screening consists of primary and secondary screening. It may be defined as use of selective
procedure to allow detection and isolation of interest from large to medium population.

Crowded plate technique:

Crowded plate technique is used for primary screening. Natural microbial sources such as soil
diluted to provide 200 -300 colonies per plate and sprayed on the surface of the agar plates yield
colonies which are distinct from neighboring colonies there by making easy for selection and
sub-culturing. For isolating fungal organisms the specific media is used to provide the fungal
growth. Addition of antibiotic i.e. antifungal agent in appropriate concentration prevents growth
of fungal colonies used in bacterial isolation and addition of anti-bacterial agents prevent growth
of bacteria in fungal isolation process. After isolation of that specific organism the
morphological characteristics are studied and sub cultured for future purpose.

Procedure:

Different sources of soil samples are collected e.g. garden soil, compost etc., in sterile empty
tubes. 1 g of each sample is taken into conical flask containing about 100 mL of sterile water and
flask is kept on shaker for 30 min to get uniform suspension and considered as stock culture.

1mL of soil suspended liquid is taken and diluted with distilled water to obtain 10-1 to 10-5
concentration of original samples. 10-3, 10-4, 10-5 dilutions may be considered for plating.

1 mL of these dilutions are mixed with warm liquefied media of nutrient agar and Sabouraud’s
agar media poured in petriplates for bacterial and fungal growth.

The plates are kept in incubator at 37oC for one day and for fungal growth, the plates are kept at
room temperature i.e. 28oC for 2-3 days.

If any bacterial and fungal colonies used to produce antibiotic there is a formation of clear zone
of inhibition around these colonies. From these colonies the organisms is isolated and streaked
on to a slants of selected media.

Report:
Fig: Isolation of antibiotic producing organisms from soil by employing serial dilution technique
Experiment 7

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION OF ANTIBIOTIC

Aim: To determine minimum inhibitory concentration (MIC) of given antibiotic.

Requirements:

Cultures: Nutrient broth cultures of Staphylococcus aureus, Bacillus cereus.

Media: Nutrient broth or nutrient agar.

Antibiotics: Amikacin

Apparatus: Test tubes, petriplates, pepettes, cork borer.

Equipments: Autoclave, hot air oven, refrigerator, incubator.

Principle:

Minimum inhibitory concentration is the minimum concentration of antibiotic microbial

component found to inhibit the growth of a particular test microorganism. It is applied to
disinfectant, anti-septic, preservatives and antibiotics. MIC values are usually expressed in terms
of µg/mL or units/mL. MIC of different antimicrobial is determined by broth dilution method or
solid dilution method. The advantages of solid dilution method are as follows

• Several microorganisms can be tested at the same time by using multi point inoculator.

• Contaminations are easily detected on solid media.

Procedure:

Broth dilution: Prepare nutrient broth in test tubes and label them. In first tube (UT), inoculums
is not added which is used for checking the sterility of the medium and as a negative control. In
all other test tubes 3-4 drops of inoculums is added to reach the concentration of microorganism
is 106 cells/mL. In all test tubes the test antimicrobial compound is added to get the final
concentration like 0.1 to 1µg/mL except uninoculated (negative control) and control (positive
control) tubes. The positive control is used to check the suitability of the medium for growth of
the test microorganism. A ll the test tubes are properly shaken and then incubated at 37oC for 2
days.

Solid dilution method: In this method the antimicrobial agent is mixed into molten agar and
then poured into petriplates. After solidification inoculums is spread on surface of agar medium.
All plates are incubated at 37oC for 2-3 days.
Observation and results:

S.No Tube number Vol. of medium Vol of test Presence of growth

(mL) antimicrobial
agent (mL)

Fig: Determination of minimum inhibitory concentration

Experiment 8

MICROBIOLOGICAL ASSAY OF AMIKACIN BY CUP-PLATE METHOD-

DETERMINATION OF UNKNOWN CONCENTRATION

Aim: To determine microbiological assay of Amikacin.

Apparatus: petridish, sterile nutrient agar medium, inoculating needle, test tubes, beakers,
volumetric flasks, sterile borer, 24 hours culture of Staphylococcus aureus.

Principle:

The microbiological assay is based upon the comparison of the inhibition of growth of
microorganism by measured concentration of the antibiotics to be examined with that produced
by known concentration of standard preparation of the antibiotic having a known capacity. The
potency of an antibiotic is expressed as the ratio of dose that inhibits growth of suitable
susceptible microorganism to the dose of an international biological standard, an international
biological reference standard of that antibiotic that produce similar inhibition. Properly validated
secondary reference material may also be utilized in the assay. To carry out assay a comparison
is made between inhibition of growth of microorganism produced by known concentration of
reference material and the inhibition produced by the measured dilutions of test substance. This
response can be measured by the diffusion method or in liquid medium by turbidimetric method.
The inhibition of microbial growth under standardized conditions may be utilized for
demonstrating the therapeutic efficacy of antibiotics.

The microbiological assays are very useful in resolving doubts regarding possible change in
potency of antibiotic and their preparations. Chemical methods cannot detect change in potency
of antibiotic and their products. So, one can rely on microbiological assay.

Two general methods are usually employed.

• Cylindrical/ cup plate method

• Turbidometric method

Procedure:

Required amount of nutrient agar medium is prepared and sterilized in an autoclave

1200C, 15 lbs pressure for 15 minutes. Microorganism is inoculated in the nutrient agar and is
transferred into sterile Petri plate under aseptic conditions and allowed to solidify. The cups are
made in the solidified media by using sterile metallic borer. Different concentrations of
Amikacin are 2, 4, 6, 8, 10, 12, 16 µg/mL are prepared and transferred into the Petridishes
containing cups by using micropipette. All the operations should be done in aseptic area. After
the inoculation of antibiotic the Petridishes are kept aside for the diffusion of antibiotic and
incubated at 370 C for 24 hours.

Observation and results:

As the concentration of antibiotic increases the zone of inhibition also increases

S.No Concentration Diameter of Average Average

(µg) zone of diameter diameter
inhibition (cm) (mm)

1. 2

2. 4

3. 6

4. 8

5. 10

6. 12

7. Unknown

Fig: Antibiotic zones produced by microorganism in cup plate method

Experiment 9

MICROBIOLOGICAL ASSAY OF AMIKACIN BY DISC-PLATE METHOD-

DETERMINATION OF UNKNOWN CONCENTRATION

Aim: To determine microbiological assay of Amikacin.

Apparatus: petridish, sterile nutrient agar medium, inoculating needle, test tubes, beakers,
volumetric flasks, sterile borer, 24 hours culture of Staphylococcus aureus.

Principle:

The microbiological assay is based upon the comparison of the inhibition of growth of
microorganism by measured concentration of the antibiotics to be examined with that produced
by known concentration of standard preparation of the antibiotic having a known capacity. The
potency of an antibiotic is expressed as the ratio of dose that inhibits growth of suitable
susceptible microorganism to the dose of an international biological standard, an international
biological reference standard of that antibiotic that produce similar inhibition. Properly validated
secondary reference material may also be utilized in the assay. To carry out assay a comparison
is made between inhibition of growth of microorganism produced by known concentration of
reference material and the inhibition produced by the measured dilutions of test substance. This
response can be measured by the diffusion method or in liquid medium by turbidimetric method.
The inhibition of microbial growth under standardized conditions may be utilized for
demonstrating the therapeutic efficacy of antibiotics.

The microbiological assays are very useful in resolving doubts regarding possible change in
potency of antibiotic and their preparations. Chemical methods cannot detect change in potency
of antibiotic and their products. So, one can rely on microbiological assay.

Two general methods are usually employed.

• Cylindrical/ cup plate method

• Turbidometric method

Procedure:

The standard amikacin antibiotic is taken and stock solution is prepared. From stock various
working standards are made. The growth inhibition measured by performing disc plate method
using molten agar medium. Standard loops of test microorganism is inoculated into molten
nutrient agar medium, poured into petriplate and then allowed for solidification. On solidified
seeded agar medium discs are placed which are already wetted with antibiotic solution and then
air dried.These discs are placed on agar medium at equal distance for about 4-5 discs per each
plate. All the operations should be done in aseptic area. After the inoculation of antibiotic the
Petridishes are kept aside for the diffusion of antibiotic and incubated at 370 C for 24 hours.

Observation and results:

As the concentration of antibiotic increases the zone of inhibition also increases

(Fig.24.1)

S.No Concentration Diameter of Average Average

(µg) zone of diameter diameter
inhibition (cm) (mm)
1. 2
2. 4
3. 6
4. 8
5. 10
6. 12
7. Unknown

Fig: Antibiotic zones produced by microorganism in disc plate method