STUDY ON THE EFFECT OF

CIPROFLOXACIN AND SODIUM

CHLORIDE AND THEIR
COMBINATION ON BIOFILM
BASED ON MINIMUM INHIBITORY CONCERTATION AND ZONE OF INHIBITION

UNDER GUIDANCE OF PRESENTED BY

Dr. ABHIK SI ANSHUK BISWAS
PROFESSOR, MICROBIOLOGY 7th Semester, Final Year
Roll no. 17401912005
BIOFILM

A biofilm is an assemblage of microbial cells that is irreversibly associated (not

removed by gentle rinsing) with a surface and enclosed in a matrix of primarily
polysaccharide material.
Develops on- Non-cellular materials such as mineral crystals, corrosion particles,
clay or silt particles, or blood components, depending on the environment and also
in biofilm matrix.
The water system biofilm is highly complex, containing corrosion products, clay
material, fresh water diatoms, and filamentous bacteria.
The biofilm on the medical device, on the other hand, appears to be composed of a
single, coccoid organism and the associated extracellular polymeric substance (EPS)
matrix.
PLAQUE AS A BIOFILM:
Dental plaque is a biofilm or mass of bacteria that grows on surfaces within the mouth.
It appears as a white or pale yellow "slime layer", that is commonly found between the
teeth and along the cervical margins. Dental plaque is also known as microbial plaque,
oral biofilm, dental biofilm, dental plaque biofilm or bacterial plaque biofilm.
The microorganisms that form the biofilm are mainly Streptococcus mutants and
anaerobes, with the composition varying by location in the mouth. Examples of such
anaerobes include fusobacterium and actinobacteria. These microorganisms present in
dental plaque are all naturally present in the oral cavity, and are normally harmless.
However, failure to remove plaque by regular tooth brushing means that they are
allowed to build up in a thick layer and cause dental disease. Those microorganisms
nearest the tooth surface ferment dietary sucrose; it is in this state that they start to
produce acids.
ACTIVE SALT (SODIUM CHLORIDE)

• Sodium chloride is an ionic compound with

the chemical formula NaCl, representing a
1:1 ratio of sodium and chloride ions.
• Sodium chloride is the salt most
responsible for the salinity of seawater and
of the extracellular fluid of many
multicellular organisms.
• Commonly used as food preservative.
MINIMUM INHIBITORY CONCETRATION (MIC)
• Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an
antimicrobial that will inhibit the visible growth of a microorganism after overnight
incubation.
• MICs are used by diagnostic laboratories mainly to confirm resistance, but most often as
a research tool to determine the in vitro activity of new antimicrobials, and data from
such studies have been used to determine MIC breakpoints.
PRACTICAL WORK
• Firstly nutrient broth media is prepared.
• Transfer of microorganism from teeth and from stock culture.
• Estimation of minimum inhibitory concentration (mic) using antibiotic, active
salt and antibiotic with active salt combination.
• Antibiotics used: CIPROFLOXAIN
• Active salt used: Sodium Chloride (NaCl)
Estimation of Minimum Inhibitory Concentration (MIC) using antibiotic, active salt and antibiotic with active salt combination.

Test tube Nutrient broth Culture of micro- Antibiotic: ciprofloxacin. (In Active salt (final concentration Antibiotic: ciprofloxacin (Final concentration in Turbidity observed
no. media (ml) organism from teeth microgram / ml, Final in % w/v) microgram / ml) with final concentration of 0.05%
sample (in micro liter) concentration) w/v Active salt

1 2 100 60 Nil Nil --

2 2 100 40 Nil Nil --
3 2 100 20 Nil Nil +
4 2 100 5 Nil Nil ++
5 2 100 2.5 Nil Nil ++
6 2 100 nil Nil Nil ++
7 2 100 Nil 0.5% w/v Nil ++
8 2 100 Nil 0.25% w/v Nil ++
9 2 100 Nil 1% w/v Nil ++
10 2 100 Nil 0.05% w/v Nil ++
11 2 100 Nil Nil Nil ++
12 2 100 Nil Nil 60 + A.S --
13 2 100 Nil Nil 40+ A.S --
14 2 100 Nil Nil 20+ A.S --
15 2 100 Nil Nil 5+ A.S --
16 2 100 Nil Nil 2.5+ A.S +
17 2 100 Nil Nil Nil ++
GRAPH OF MINIMUM INNHIBITORY CONCENTRATION
2
Medium: Nutrient broth

Ciprofloxacin Cipro+NaCl
TURBIDITY

MIC[Cipro+NaCl]

MIC[Cipro]

0
0 2.5 5 20 40 60
CIPROFLOXACIN CONC. ( G/ML)
DISCUSSION AND CONCLSION OF MIC
Since it was investigated and reported that any kind of biofilm made by the
microorganisms exhibit a marked decrease in susceptibility to antimicrobial agents and
host defence systems compared to their planktonic counterparts, our main investigation
was to study on measure of degree of decreased susceptibility to certain antibiotics by
the biofilm producing microorganisms. The minimum inhibitory concentration and the
zone of inhibition study were performed using different parameter to establish that.
the MIC for only Ciprofloxacin is 40 micrograms/ml and for the combination with Sodium
Chloride, which acts as an active salt, is 5 micrograms/ml. i.e., susceptibility to combined
treatment is 8 times more potent than that of only Ciprofloxacin for the biofilm making
microorganisms.
only salt is not sufficient to reduce the growth of the microorganism. Which clearly
indicates that when bactericides are combined to a salt solution, they are capable of
killing microorganisms which are retrieved as planktonic organisms and no longer
organized as a biofilm due to the salt effect.
ZONE OF INHIBITION
 If an antibiotic stops the bacteria from growing or kills the
bacteria, there will be an area around the drug or antbiotic
where the bacteria have not grown enough to be visible.
This is called a zone of inhibition.
 The size of this zone depends on how effective the antibiotic is at
stopping the growth of the bacterium.
PRACTICAL WORK
Materials required:
1. Culture of microorganism from teeth sample.
2. Culture of Pseudomonas aeruginosa.
3. Nutrient broth.
4. Antibiotic: ciprofloxacin.
Preparation of nutrient agar media
Preparing agar plates
 MEASURMENT OF MICROBIAL ZONE OF INHIBITION
Petridish No. Hole No. Microorganism Antibiotic (ciprofloxacin) concentration* (in microgram / Average Zone of Inhibition in
ml) mm.

1 A from teeth sample 200 26.4

B 100 22.1
C 50 19.9
D 25 Nil
2 A from teeth sample 12.5 Nil
B 6.2 Nil
C Sterile distilled water Nil
D Sterile distilled water Nil
3 A from teeth sample Nil Nil
B Nil Nil
C Nil Nil
D Nil Nil
4 A Pseudomonas aeruginosa 200 42.2
B 100 35.0
C 50 30.2
D 25 24.6
5 A Pseudomonas aeruginosa 12.5 19.8
B 6.2 Nil
C Sterile distilled water Nil
D Sterile distilled water Nil
6 A Pseudomonas aeruginosa Nil Nil
B Nil Nil
C Nil Nil
GRAPH FOR ZONE OF INHIBITION
Antibiotic, C Log [C] Culture Ps.
45 Culture from Ps. Aeruginosa
(mg/mL) from aeruginosa Teeth sample
40
Teeth Nutrient agar medium
35
sample
30

ZOI (MM)
200 2.301 26.4 42.2 25

20
100 2.000 22.1 35 15

10
50 1.699 19.9 30.2
5
25 1.398 24.6 0
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
12.5 1.097 19.8 LOG[CONC( G/ML)]
DISCUSSION AND CONCLUSION FOR ZOI
 The measurement of microbial zone of inhibition experiment was carried out using
teeth sample and Pseudomonas aeruginosa sp.
 From the both result and graph it was concluded that micro orgasms from teeth
sample are much more resistant to Ciprofloxacin than Pseudomonas aeruginosa sp.
 This clearly indicates that bacterial cell undergo a number of physiological and
phenotypic changes following attachment to a solid surface. These lead to higher cell
resistance to antimicrobial compounds.
 These biofilms can induce diseases such as caries, gingivitis and periodontitis which
are among the most common infections of man.
 The antibiotic susceptibility is increased many folds when combined with active salt,
this may give a new era to treat different types of teeth disease caused by biofilm
making microorganisms.