Pharmaceutical microbiology (practical)

Practical notes
on
Pharmaceutical Microbiology

(PM 502)

LEVEL THREE

Department of Microbiology & Immunology

Faculty of Pharmacy,
Delta University for Science and Technology

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 Pharmaceutical microbiology (practical)

Table of Content
no. Subject Page
1. Laboratory Safety 3

2. Pour Plate Technique 5

3. Techniques for Isolation of Pure Cultures 7

4. Determination of Viable Count of Bacteria 11

5. Antimicrobial Susceptibility Testing "Sensitivity" (Agar 14

Disc Diffusion Method)

6. Determination of Minimum Inhibitory Concentration 18

(MIC) (Agar Diffusion Method)

7. Determination of Minimum Inhibitory Concentration 20

"MIC" (Broth Dilution Method)

8. Determination of Minimum Bactericidal Concentration 23

"MBC"

9. Microbiological Assay of Antibiotics 25

10. Sterility test 29

11. Biological indicator for steam sterilization 30

12. Antibiotic Combinations 33

13. Detection of β- lactamase production 36

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 Pharmaceutical microbiology (practical)

Laboratory 1
SAFETY GENERAL RULES AND REGULATIONS
The following basic safety rules should be observed at all times in the laboratory:

1. Upon entering the laboratory, coats, books, and others should be placed in a specified location
and never on the bench tops.
2. At the beginning and termination of each laboratory session bench tops are to be wiped with a
disinfectant solution.
3. Smoking, eating, and drinking in the laboratory are absolutely prohibited.
4. Students should wear a lab coat while working in the laboratory.
5. Long hair should be placed inside a paper cap or tied back to minimize fire hazards or
contamination of the experiments.
6. Removal of media, equipment, and especially bacterial cultures is absolutely prohibited.
7. All cultures should be handled as being potentially pathogenic, and the following precautions
should be considered
a. Cultures must be always be carried in a test tube rack.
b. Broth cultures must never be pipetted by mouth.
c. Spilled cultures should be covered with paper towels and then saturated with
disinfectant solution. Alternatively, slant cultures must be autoclaved first then
discarded.
8. Use of glassware markers.
9. On completion of the laboratory session all cultures and materials to be discarded should be
placed in the disposal area designated by the instructor, and never discards waste materials into
the sink.
10. At the end of the laboratory session, Petri dishes, pipettes etc. should be returned to the assistant
in charge of the laboratory.
11. The microscope must be properly cleaned and the oil immersion must be cleaned with xylol
using lens paper.
12. Hands must be washed with detergent prior to leaving the laboratory.

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 Pharmaceutical microbiology (practical)

Laboratory 2

Pour Plate Technique

Objective:

To learn the pour-plate technique for using in sensitivity test and for MIC determination.

Materials:

1. Tube of nutrient agar.

2. Sterile Petri dish.
3. Bunsen burner flame.

Procedure:

The procedure will be demonstrated. Watch carefully and then do it your self, following directions
given.
1. Place a tube of sterile nutrient agar in a boiling water bath. Keep the water level at the
halfway mark.
2. When the agar is liquefied, remove the tube and allow it to cool to about 50°C.
3. Place an empty sterile petri dish before you, top side up.
4. Pick up the tube of melted, cool agar, remove its closure. Flame the mouth of the open tube.
5. With your free hand, remove the top of the petri dish and quickly pour the agar into the
dish.
6. Replace the petri dish cover. Gently rock the closed dish, or rotate it in circular fashion on
the bench top, being careful not to allow the still melted agar to wave up over the edge of
the bottom half or onto the cover.
7. Let the agar solidify without further disturbance. When it is quite firm, invert the plate.

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Pour Plate Technique

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 Pharmaceutical microbiology (practical)

Laboratory 3

Techniques for Isolation of Pure Cultures

Objective:

To perform the spread plate and/or streak plate inoculation procedure for the separation of a
mixed culture so that discrete colonies can be isolated.

Principle:

The following techniques are used for the isolation of mixed culture:
The streak plate method is a rapid qualitative isolation method. The method depends on
the spreading a loopful of culture over the surface of the agar plate. Although many types
of procedures are performed, the four-way streak is described:-
a. Place a loopful of culture on the agar surface plate in area 1. Flame and cool the
loop and streak over the surface of area 1.
b. Re-flame, cool the loop, and turn the Petri dishes 90 degrees. Then touch the loop
to a corner of the culture in area 1 and drag it across the agar in area 2.
c. Re-flame and cool the loop, and again turn the dish 90 degrees, and drag the culture
from a corner of area 3 across area 4. The loop must not touch any of the previously
streaked areas.

The spread plate technique requires that a previously diluted mixture of microorganisms
be used. During inoculation, the cells are spread over the surface of the agar with a sterile
L-shaped bent rod.
Materials:

1. Tube of nutrient agar (10 ml).

2. Sterile Petri dish.
3. Wire inoculating loop.
4. Tube containing a mixed culture of two microorganisms.
5. Permanent marker.

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 Pharmaceutical microbiology (practical)

Procedure

1. Invert the dish and by means of marker pen draw 5 groups, each with 3 parallel lines that
intersect with each other.

2. Using pour plate technique, aseptically pour the melted nutrient agar into the plate and leave
to solidify.
3. After solidification, invert the plate to avoid water condensation on the surface of agar.
4. Place a loopful of culture on the agar surface at the beginning of group 1.
5. Flame and cool the loop and drag it in one direction in each line of the group.

6. Reflame and cool the loop and turn the petri dish 90 ○. Then drag the loop in one direction

in each line of group 2 that intersect with group 1.

7. Reflame and cool the loop again and turn the petri dish 90 ○. Streak group 3 in the same

manner.
8. Repeat the same technique with group 4 and 5.

9. Incubate the plate inverted at 37 ○ C for 24h.

Note:-
• Cool the agar to 50 ○ C before pouring to avoid water condensation.
• Always drag the loop in one direction on the lines. Never return to opposite direction.
• Do not let the loop touch any of the previously streaked area.

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3 4

5 6

7 8

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Results of Experiment 2:

Observation:

Colony description Colony 1 Colony 2 Colony 3

i. Shape

ii. Color

iii. Size

iv. Edge (margin)

v. Elevation

vi. Texture

vii. Surface

viii. Optical Character

Comment:

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 Pharmaceutical microbiology (practical)

Laboratory 4

Determination of Viable Count of Bacteria

Objective:

To enumerate and count viable cells of a given culture.

Principle:

Viable count is defined as the number of living cells per 1 ml of solution. Determination
of viable cells depends on the fact that each colony represents one cell, when the cell was
allowed to grow on a solid medium. Because bacterial population is usually growing in a huge
number, a serial dilution is performed to facilitate counting of bacteria.
Viable Count (VC) = Number of Colony Forming Unit (CFU) X dilution factor

Inoculum size

Materials:

1. Bacterial suspension in a test tube.

2. Three tubes, each containing 9-ml sterile saline.
3. A sterile 1-ml pipette.
4. Sterile plates (9 plates).
5. Nutrient agar tubes.

Procedure:

1. Mark the saline tubes as 10-1, 10-2 and 10-3.

2. Divide the petri dishes into 3 groups and mark each group the same manner you follow for
the saline tubes. Thus, each group containing 3 plates will be marked as follow: 10-1, 10-2
and 10-3.
3. Shake the tube vigorously using a vortex. Then transfer 1ml aseptically from the original
suspension to the first tube.
4. Mix well by vortex and repeat the same step by transferring one ml from the first tube to
the second tube and then from the second tube to the third one.
Be careful that good mixing is very important step before transfer to the respective
dilution.

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5. After good mixing, transfer aseptically one ml from the third tube (10 -3) to each of three
plates marked as 10 -3.
-2 -1
6. Repeat the same step by transferring 1 ml from the tube marked 10 and 10 to their
corresponding plates.

7. Aseptically pour the agar into each plate and mix well. Make sure that the agar is cooled to
55 ○ C to avoid killing of microorganisms.
8. Incubate all plates at 30oC or 37oC for non-pathogenic and pathogenic organisms for an
overnight (24 h).
9. After incubation, count the colonies in plates in which the CFU ranges between
(30-300). The detected colonies are counted as colony forming unit (CFU) per ml; using
the equation:

Number of viable cells per ml = Number of colonies x dilution factor

1 (Inoculum Size in ml)

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Results of Experiment 3:

Observation:

* Discard plates out the range of 50-250 colonies (above or below).

** Number of viable cells per ml = Number of colonies x dilution factor

(Inoculum Size)

**
Mean number of viable cells per ml
=

Comment:

• In case of complete absence of bacterial growth:

• Limitations of viable count:

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Laboratory 5
Antimicrobial Susceptibility Testing "Sensitivity"
(Agar Disc Diffusion Method)

Objective:

To determine the sensitivity of a pure bacterial culture to different antibiotics.

Principle:

• This technique is also known as the Bauer Kirby Method. In this test, a number of small, sterile
filter paper discs of uniform size (6 mm) that have each being impregnated with a defined
concentration of an antimicrobial agent are placed on the surface of an agar plate, previously
inoculated with a standard amount of the organism to be tested.
• The agar medium must be appropriately enriched to support microbial growth. Using
sterile forceps, the discs are placed in even array on the plate, at equal distances from each
other. When the discs are in contact with the agar, the antimicrobial agents diffuse into the
surrounding medium and come in contact with the tested microorganism.
• After incubation, the zone of inhibition produced by the antibiotic disc is measured and the
diameter of the zone is representative for the potency of the antibiotic. The absence of a zone
of inhibition indicates that the tested organism is resistant to this antibiotic.
However, the sensitivity of the organism to the antibiotic is classified as R (resistant),
I (intermediate sensitive), and S (sensitive) according to values given by NCCLS
(National Committee for Clinical Laboratory Standard).

Materials

1. Four Wassermann tubes containing the respective antibiotic solutions.

2. Bacterial culture.
3. A nutrient agar tube.
4. Sterile filter paper discs.
5. Forceps.
6. A sterile Pasteur pipette for inoculation.
7. Beaker containing alcohol.

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Procedure:

1. Draw 4 equal sectors in the bottom of the Petri dish and mark the center of each quarter
(place of the disk).
2. Aseptically transfer 8 drops of bacterial suspension to a sterile plate.
3. Pour aseptically the melted nutrient agar and mix well, leave to solidify.
4. Dip the tips of the forceps in 70% alcohol, flame rapidly, and allow to cool.
5. Using a flamed forceps, aseptically pick up a sterile disc and immerse it (just tip) in
one of the provided antibiotic solution. Avoid overloading of the disc with antibiotic
solution by touching the disc with the inner surface of the tube to remove the excess
antibiotic solution.
6. Press the disc gently into full contact with the agar, using the tip of the forceps.
7. Dip the forceps in alcohol, flame and cool.
8. Repeat steps 5 and 6 with the other antibiotic solutions.
9. Incubate the plate without inversion at 37oC for 24 h.
10. After incubation, measure the zone of inhibition (mm) and comment on the results obtained.

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3 4

5 6

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Results of Experiment 4:

Observation:
Diameter of inhibition zone Sensitivity of the culture to the antibiotic
Antibiotic
(mm) (S ,R ,or I)

S (Sensitive) R (Resistant) I (Intermediate sensitive)

Comment:

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Laboratory 6
Determination of Minimum Inhibitory Concentration (MIC)
(Agar Diffusion Method)

Objective:

To determine the MIC of the provided antibiotic using the agar diffusion method.

Principle:

• The MIC (Minimum Inhibitory Concentration), which defined as the minimum

concentration required to kill or inhibit the growth of microorganism. MIC is used as a
valuable parameter to evaluate the antibiotic.
• The method depends on the fact that there is a direct relationship between the antibiotic
concentration and the diameter of zone of inhibition. Thus, when various dilution of antibiotic
are placed in cups in agar plate seeded with the test organism, a zone of inhibition is produced
depending on the concentration of the antibiotic.
• Plotting the diameter of zone of inhibition in mm (Y-axis) against logarithmic concentration
of the antibiotic (X-axis), a straight line will be obtained. Extrapolation of the line
to a point, where the diameter of the cup equal to the zone of inhibition, intercept with a point
equivalent to the log MIC.

Materials:

1. Four Wassermann tubes containing antibiotic concentrations.

2. Bacterial culture.
3. Nutrient agar tube.
4. A sterile cork porer.
5. An ampoule file.
6. Two sterile Pasteur pipettes.
7. Sterile Petri dishes.

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Procedure:

1. Invert the dish and by means of marker pen draw 4 equal sectors in the bottom and
mark the center (place of the cup).
2. Aseptically transfer 8 drops of bacterial suspension using a sterile Pasteur pipette to a sterile
plate.
3. Pour the nutrient agar, melted and cooled to 45 C, mix well, and then allow to solidify.
4. By the mean of a sterile cork porer, make holes in the solidified agar and remove the agar
pieces by means of ampoule file (previously immersed in 70% alcohol and
flamed).
5. Using the other sterile Pasteur pipette fill the cups with the constant equal appropriate
amount of the antibiotic solution, starting with the lowest to the highest concentration
(avoid overflow).
6. Allow diffusion at room temperature for one hour and incubate the plate (without
inversion) at 37 C for the next period.
7. Measure the zone of inhibition produced around the respective concentration.
8. Plot a graph of the zone of inhibition (mm) against the log concentration of the antibiotic

Results of Experiment 5:

Observation:

Antibiotic Diameter of inhibition zone

Log concentration
(µg/ml) (mm)

Comment:

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Laboratory 7
Determination of Minimum Inhibitory Concentration "MIC"

(Broth Dilution Method)

Objective:

To determine the MIC of the provided antibiotic using serial dilution in broth.

Principle:

• The assessment of antibiotic activity against

microorganisms, especially pathogenic-strains require determination
of the MIC.
• The broth dilution method is one of the methods used to determine the MIC. This
method is rapid, easy and economical to perform. This method depends on the serial
dilution of the antibiotic (2-fold; 100, 50, 25 etc) in a broth medium followed by inoculation
of the microorganism.
• After incubation, the observed growth is recorded, and the last dilution of antibiotic which
inhibits the microbial growth is determined MIC can be calculated from the least
concentration at which there is no visible microbial growth.

Materials:

1. Wassermann tubes.
2. Bacterial culture.
3. Solution of antibiotic.
4. A tube containing 10 ml of double strength nutrient broth.
5. Pasteur pipette.
6. Sterile 1 ml and 5 ml pipette.

Procedure:

1. Mark the Wassermann tubes from 1 to 6 and give the last test tube the symbol
(control).
2. Transfer 1 ml of the broth to each Wassermann tubes, using 5-ml pipette.
3. Transfer 1 ml of the antibiotic solution (using 1 ml pipette) to the first tube, mix well and
then transfer 1 ml to the second tube.

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4. Repeat the same procedure till the tube number 6, at which you have to discard 1 ml of the
solution; and left tube C without antibiotic.
5. Inoculate all tubes with 2 drops of the provided culture, using Pasteur pipette.
6. Mix content of the tube thoroughly and then incubate all tubes at 37 C for 18 to 24h.
7. After incubation, record the presence of growth (in the form of turbidity) and the absence
of growth (clear broth) indicated by (+) for growth and (-) for the absence of growth.
8. Read the MIC endpoint. The MIC is defined as the lowest concentration of antibiotic at
which there is no visible growth.

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Results of Experiment 7:

Observation:

Antibiotic conc. 1 2 3 4 5 6 7

Visible growth

• Record the results as follows:

(+) = growth (-) = no growth

• The initial concentration is:

• MIC = ( + )/2

MIC= µg/ml

Comment:

• In case of complete absence of growth.

• In case of the presence of growth in all tubes.

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Laboratory 8
Minimum Bactericidal concentration

Objective:

To determine the Minimum Bactericidal Concentration of antibiotic microbial strain

Definitions:

MIC: Minimum Inhibitory Concentration: This is the lowest concentration of an antimicrobial drug
that prevents visible growth of a microorganism after overnight incubation with media.

MBC: Minimum Bactericidal Concentration: This is the lowest concentration of an antibacterial

agent required to kill a particular bacterium.

Procedure:

1- Minimum Bactericidal Concentration (MBC) is measured by inoculating the broths used for
MIC determinations into drug-free medium.

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2- A pure culture of a certain microorganism is first cultivated overnight, and then the
antibacterial concentration is adjusted in a broth that supports growth

3- After serially diluting the antibacterial to be tested, an equal volume of the chosen
microorganism is used to inoculate each dilution. The test tube or microtiter plate is then
incubated for the required time and temperature.

4- The lowest concentration of bacterial growth, or MIC, was not visibly apparent, according to
observations made afterwards. Turbidity indicated the development of microorganisms in
those cases.

5- A dilution that represents the MIC and at least two additional concentrated dilutions of the
test substance are plated, and the survival CFU/ml is counted to estimate the MBC. MBC is
the lowest concentration that can inhibit the 99.9% activity of the tested bacteria

Notes:

 The MBC is the first dilution at which no growth is observed.

 Cidal drugs have MBC values that are close to the MIC value for particular organisms.

 With static agents, the MIC is much lower than the MBC.

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Laboratory 9

Microbiological Assay of Antibiotics

Objective:

To determine the relative potency of antibiotic compared to an authentic (standard) using

sensitive standard microbial strain.

Microbiological assay of antibiotics has many applications including:

1. Determination of the relative potency of the antibiotic after manufacturing.

2. During the pharmacokinetic study.
3. For monitoring and controlling antimicrobial chemotherapy.

Principle:

The method depends on the fact that when the antibiotic diffuse from the cup in the agar inoculated
with the tested organism, it produces zone of inhibition, in which the diameter of the zone is
proportional to the concentration of the antibiotic. The microorganism used in the antibiotic assay,
must be a standard culture for reproducibility of the results.

Materials:

1. Six Sterile Petri dishes.

2. Suspension of microorganism.
3. Six tubes containing 10 ml agar.
4. Three Wassermann tubes marked S1, S2 and S3 and containing the antibiotic standard in
concentration of 10, 20 and 30 units per ml respectively.
The potency of the standard is considered to be 100%.
5. Three Wassermann tubes marked T1, T2 and T3 and containing the antibiotic sample to be
assay in concentration presumed to be 5, 10, and 20 units of the antibiotic per ml
respectively.
6. A sterile cork porer.
7. Three sterile Pasteur pipettes.
8. An ampoule file.

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 Pharmaceutical microbiology (practical)

Procedure:

1. Mark the back of the plates, dividing each plate into 6 sectors and label them in the
following sequence T1, S1, T2, S2, T3, S3 (Sector S in the front of sector T).
2. Using the Pasteur pipettes put the microbial suspension into the plates.
3. Pour the melted agar (6 tubes in 6 plates), and allow to harden at room temperature.
4. Using sterile cork porer, make 6 cups in each plate, according to the pre-labeled on
the back of the plate 6X 6 = 36 cups in six plates)
5. Using a sterile ampoule file, remove the 3 plugs into the disinfectant jar.
6. Fill the cups with the test and standard solutions of antibiotic, using sterile standard
Pasteur pipette, starting with the lowest concentration.
Be careful that the cups should fill with constant equal volume of antibiotic solution, and
avoid an overflow
7. Incubate all plates without inversion at 37o C for 24 h
8. After incubation, measure the zone of inhibition in (mm) and calculate the relative potency
of the test antibiotic from the following equations.

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 Pharmaceutical microbiology (practical)

Results of Experiment 9:

Observation:

Diameter of zone of inhibition

(mm)
Plate no.

S1 S2 S3 T1 T2 T3

Mean

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Comment:

* Calculate the relative potency according to the following equations:

I (log ratio of doses) = Log 2 = 0.301

E (effect due to dose) = 1/4 [(T3-T1) + (S3- SI)]

F (effect due to test) = 1/3 [(T1+T2+T3) - (S1+S2+S3)]

B (slope) = E/I

M (potency ratio) = antilog -F/B

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Laboratory 10
Sterility Testing
Sterility testing is a GMP microbiology testing requirement used to confirm sterile products do not
contain viable microorganisms before release and patient administration. Sterility testing methods
must be as accurate as possible, due to their importance for medical devices, pharmaceutical
products, and formulations, tissue materials, and other products that claim to be sterile or free from
viable microorganisms.

Sterility testing procedures are applied to products in many industries, including food and beverage
manufacturers, but the main industries are the pharmaceutical and medical sectors where the
sterility testing of the products remains a vital and routine task for microbiologists.

In this lab we are going to demonstrate for your different sterilization equipment their principles
of sterilization.

Write your and draw your comments

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Laboratory 11
Biological indicator for steam sterilization

A-Biological indicators Introduction:

Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) is a thermophilic, aerobic
bacterium, which produces heat-resistant spores.

Spores of Geobacillus stearothermophilus are used to comply with the AAMI/American

National Standards Institute (ANSI) standard titled “Sterilization of Health Care Products-
Biological Indicators-Part 3: Biological Indicators for Moist Heat Sterilization, ST19.” Geobacillus
stearothermophilus is not harmful to humans.

G. stearothermophilus has not been observed to be pathogenic to any host. G. stearothermophilus

has a significant role in the spoilage of food, especially milk and dairy products as it can survive the
pasteurization process due to its heat-tolerance.

Geobacillus spp. have been isolated from temperate as well as hot environments including hot
springs, oilfields, deep sea sediments, sugar refineries, canned foods, dehydrated vegetables
and dairy factories.

Procedure of using BIS?

1. Place one or more Bis inside the autoclave
2. Test the most challenging area in the sterilizer as indicated in the sterilizer's instruction
manual (e. the bottom shelf near the door).
3. After completion of sterilisation process, Remove the Bl and confirm the chemical indicator
printed on the label has turned brown.
4. Always allow to cool for ten (10) minutes before crushing.
5. Activate the processed Bl within 8 hours after processing by gently crushing the inner glass
media tube using a vial crusher.
6. Incubate at 55-60°C for 24 hours checking for spore growth (visual color change from
purple to yellow) at regular intervals 3, 5 and 8 hours) or by culturing.

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Result:

No color change in the purple media indicates proper sterilization.

Any positive (growth indicated by purple to yellow color change) result, should be reported
immediately to a Supervisor and the sterilizer taken out of service until resolved. The stability of
positive growth as indicated by a yellow color change has been tested up to 48 hours.

B-Heat Resistance test

Principles
Bacteria differ in their Resistance to heat. Examples:
E. Coli can't withstand temp. above 50°C
Bacillus can't withstand temp. above 90°C
Geobacillus stearothermophilus can't withstand temp. above 100°C

Materials:
1. Petri dish
2. Nutrient agar
3. twst tubes
4. Thermometer
5. Beaker
6. Nutrient broth

Procedure:
1-Prepare three nutrient broth of [Link], Bacillus and Geobacillus stearothermophilus
2- Prepare a Nutrient agar and divide into 6 parts
3-Put all tubes into a water bath with a thermometer
4- Take a swap from each bacteria at the following temperature interval: 50°C, 90°C, 100°C after
constant exposure period and then inoculate into the nutrient agar as illustrated by your lab
instructor.
5- Incubate at 55-60°C for 24 hr.

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 Pharmaceutical microbiology (practical)

Result:
[Link] is able to grow only below 50°C
Bacillus is able to grow only below 80°C
Geobacillus stearothermophilus is able to grow only below 100°C

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Laboratory 12
Antibiotics Combination
Definitions:
Antibiotic synergism
The overall effect of antibiotic combination is greater than the sum of the individual antibiotics
(zones merge).When two bactericidal antibiotics are used in a synergism combination, One of the
two drugs must show at least 4-fold increase in antibacterial activities (or a decrease in MIC to 4).
E.g. 2+2=6
Additive Effect
The overall effect of the antibiotic combination is no greater than the sum of individual antibiotics
(own zone)
E.g. 2+2=4
Antagonism
The inhibiting or nullifying action of one antibiotic on another when combined together. Usually,
bacteriostatic antibiotics are antagonistic to bactericidal agents. (e.g., Chloramphenicol has been
shown to antagonize the bactericidal activities of penicillin). As a rule of thumb, most broad-
spectrum antibiotics are bacteriostatic, and many of the narrow spectrum antibiotics are usually
bactericidal.

Double disc synergy

Double disc synergy test was conventionally used for the detection of extended-spectrum beta-
lactamase (ESBL) production and can also be used for the detection of synergy between
antimicrobial combinations.
In this method, discs containing the antimicrobials are placed 20 mm (or sum of radii of the zone
of inhibition of each drug separately) apart over a lawn culture of the organism and incubated at
37°C. Synergy is indicated by an increase in the zone diameter of ≥2 mm compared to the single
agent or bridging of the zone of inhibition. An increase of < 2 mm in the zone of inhibition is
classified as weak synergy, and antagonism is indicated by truncation of the zone of inhibition at
the junction of the two antimicrobials. For P. aeruginosa, this method was shown to give more
synergism for a combination of antimicrobials than CB assay.
In addition, double disc synergy test was observed to show more synergy than other methods such
as agar-based and broth-based dilution method. Despite the simplicity and easy interpretation of
results, this method has not been widely used because of its qualitative nature and subjective
interpretation.
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 Pharmaceutical microbiology (practical)

Double disk synergy test. (a) Synergy (bridging of zone of inhibition); (b) synergy
(appearance of zone of inhibition in between agent A and B); (c) antagonism
(flattening of zone of inhibition); (d) indifference/additive )no effect on zone of
inhibition)

Advantages of Antibiotic Combination:

1- Extension of spectrum
2- Reduce the incidence of antibiotic resistance
3- Minimization of antibiotics resistance
4- Minimization of toxicity

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Laboratory 13
Detection of β-lactamase production
Objective

Detection of β-lactamase production by β-lactam resistant bacteria Principle:

This test is performed by iodometric overlay method (IOM). This method is based on the reaction
of penicilloic acid with starch and iodine. The acid reduces iodine and prevent the formation of
starch-iodine complex. In the absence of β- lactamases no acid is produced, and iodine combines
with the starch to produce a blue color.

Materials:
1. Starch reagent (1% of soluble starch in boiling water).
2. Penicillin G reagent (2% of penicillin G in distilled water).
3. Iodine reagent.
4. Toluene reagent.
Procedure:
1. The tested bacterial isolates are tooth picked and applied onto the surface of nutrient agar
plates.
2. After overnight incubation at 37 C, the plates are overlaid with 1% molten agarose
Containing 0.2% soluble starch, 1% penicillin G and 2% toluene.
3. Then, the plates are left for 15 min at room temperature.
4. The iodine solution is poured onto the surface of the agar plates to cover the overlay
uniformly for 10 sec.
5. The residual solution is damped.
6. The plates are left at room temperature until discoloration zones appear around the β-
lactamase producing colonies.

Results of Experiment 13:

Observation:

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 Pharmaceutical microbiology (practical)

β-lactamase producing colonies on the left of the plate have a clear zone
them with the bluish background of the medium.

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