Experiment No.

Study of Laboratory Instruments

Aim: To study Laboratory instruments:

 Laminar air flow

 Fermentor/Bioreactor
 CO2 incubator
 Inverted Microscope

Laminar Air Flow

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed
bench designed to prevent contamination of biological samples, or any particle sensitive
device. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow
towards the user. The cabinet is usually made of stainless steel with no gaps or joints
where spores might collect.

Such hoods exist in both horizontal and vertical configurations, and there are many
different types of cabinets with a variety of airflow patterns and acceptable uses. NSF49 is
the commonly accepted regulatory standard for these cabinets.

Laminar flow cabinets may have a UV-C germicidal lamp to sterilize the shell and contents
when not in use.

A high efficiency particulate Air, or HEPA filter is a type of high-efficiency air filter. HEPA
filters are composed of a mat of randomly arranged fibres. The fibres are typically
composed of fiberglass and possess diameters between 0.5 and 2.0 micrometer. HEPA
filters remove at least 99.97% of airborne particles 0.3 micrometers (µm) in diameter. The
filters maximum resistance to airflow, or pressure drop, is usually specified around 300 Pa
at its nominal flow rate.
 Fermentor/Bioreactor

A bioreactor may refer to any device or system that supports a biologically active
environment. In one case, a bioreactor is a vessel in which a chemical
process(fermentation) is carried out which involves organisms or biochemically active
substances derived from such organisms. This process can either be aerobic or anaerobic.
These bioreactors are commonly cylindrical, ranging in size from liters to cubic meters,
and are often made of stainless steel.

A bioreactor may also refer to a device or system meant to grow cells or tissues in the
context of cell culture. These devices are being developed for use in tissue engineering.

On the basis of mode of operation, a bioreactor may be classified as batch, fed batch or
continuous (e.g. a continuous stirred-tank reactor model).

Bioreactor design is a relatively complex engineering task, which is studied in the

discipline of biochemical engineering. Under optimum conditions, the microorganisms or
cells are able to perform their desired function with 100 percent rate of success. The
bioreactor's environmental conditions like gas (i.e., air, oxygen, nitrogen, carbon dioxide)
flow rates, temperature, pH and dissolved oxygen levels, and agitation speed/circulation
rate need to be closely monitored and controlled.

Most industrial bioreactor manufacturers use vessels, sensors and a control system
networked together.
 CO2 Incubator

An incubator is a device used to grow and maintain microbiological cultures or cell

cultures. The incubator maintains optimal temperature, humidity and other conditions
such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside. Incubators
are essential for a lot of experimental work in cell biology, microbiology and molecular
biology and are used to culture both bacterial as well as eukaryotic cells.

The simplest incubators are insulated boxes with an adjustable heater, typically going up
to 60 to 65 °C (140 to 150 °F), though some can go slightly higher (generally to no more
than 100 °C). More elaborate incubators can also include the ability to lower the
temperature (via refrigeration), or the ability to control humidity or CO 2 levels. This is
important in the cultivation of mammalian cells, where the relative humidity is typically
>95% and a slightly acidic pH is achieved by maintaining a CO2 level of 5%.

Most incubators include a timer; some can also be programmed to cycle through different
temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units the
size of small rooms.

Inverted Microscope

An inverted microscope is a microscope with its light source and condenser on the top,
above the stage pointing down, while the objectives and turret are below the stage
pointing up. It was invented in 1850 by J. Lawrence Smith, a faculty member of Tulane
University.

Inverted microscopes are useful for observing living cells or organisms at the bottom of a
large container (e.g. a tissue culture flask) under more natural conditions than on a glass
slide, as is the case with a conventional microscope.

The stage on an inverted microscope is usually fixed, and focus is adjusted by moving the
objective lens along a vertical axis to bring it closer to or further from the specimen. The
focus mechanism typically has a dual concentric knob for coarse and fine adjustment.

Depending on the size of the microscope, four to six objective lenses of different
magnifications may be fitted to a rotating turret known as a nosepiece. These microscopes
may also be fitted with accessories for fitting still and video cameras, fluorescence
illumination, confocal scanning and many other applications.

Experiment No. 2

Establishment of Primary cell culture

Introduction

The growth of explants taken directly form the living organism (e.g. biopsy material) is
also known as primary cell culture. The culture consists of mixed population of cell types.
Frequently the some of the cells may survive without proliferating and will therefore be
lost in the increasing population of those which are able to multiply in the conditions
supplied in vitro. Cells from explants may sometimes be converted to cell lines by passage.
These may continue to proliferate for a number of cell generations. In some instances the
primary cells are fused with so-called immortal (cancer) cells to produce a hybridoma line.
Many of the explanted cells will only survive for one or a few passages before dying.

Apart from study of the primary cells per se there are a number of other uses for these
cells including the use of primary fibroblasts as feeder layers for the growth of some
embryonic stem cell types.

Requirement :

 Chick embryo (approximately 8 days old)

 70% (v/v) ethanol for swabbing
 Sterile scissors, forceps and probes
 Sterile petri plates
 Phosphate buffered saline (PBS)
 Trypsin, cold sterilized in a 125 ml sterile erlenmeyer containing a magnetic stirring
bar
 Minimum Essential Medium
 Fetal Calf Serum
 Clinical centrifuge with sterile capped centrifuge tubes
 Culture flasks
 Inverted phase contrast microscope (Optional)

Procedure *
1. Candle an 8 day old egg to ensure that it is alive. This is easily accomplished by
holding the egg in front of a bright light source; the embryo can be seen as a
shadow. Circle the embryo with a pencil.
2. Place the egg in a beaker with the blunt end up, and wash the top with a mild
detergent, followed by swabbing with ethanol.
3. Carefully puncture the top of the egg with the point of a pair of sterile scissors and
cut away a circle of shell, thus exposing the underlying membrane (the
chorioallantois).
4. With a second pair of sterile scissors, carefully cut away and remove the
chorioallantoic membrane, exposing the embryo.
5. Identify and carefully remove the embryo by the neck, using a sterile metal hook or
a bent glass rod, and place the embryo in a 100mm petri dish containing phosphate
buffered saline (PBS). Wash several times with PBS by transferring the embryo to
fresh petri plates. After removal of all yolk and/or blood, move the embryo to a
clean dish with PBS.
6. Using two sterile forceps, remove the head, limbs, and viscera. Be sure to remove
the entire limb by pulling at the proximal end. Move the remaining tissues of the
embryo to yet another dish and wash with PBS.
7. Mince the embryo finely with scissors and transfer the minced tissue to a flask
containing PBS. Allow the tissue pieces to settle.
8. Remove the PBS with a sterile pipette and add 25 ml of trypsin, a proteolytic
enzyme. Stir the solution gently at 37° C for 15-20 minutes.
9. Allow the larger, undigested tissue pieces to settle and decant the supernatant into
an equal volume of Minimal Essential Medium (MEM) + 10% Fetal Calf Serum
(FCS). FCS contains protease inhibitors which will inactivate the trypsin.
10. Centrifuge the cells in MEM at 1000 rpm for 10 minutes in a standard clinical
centrifuge. Remove the supernatant and resuspend the pellet in 25 ml of fresh MEM
+ 10% FCS.
11. Remove 0.1 ml of the culture and determine cell concentration and viability as
directed in “cell viability count practical”.
12. Seed two 25 cm plastic culture flasks containing 25 ml of MEM + 10% FCS to a
final concentration of 10 cells/ml.
13. Label and place your cultures in the tissue culture incubator at 37° C and examine
daily for cell density and morphology.
14. Note any changes in the color of the media. Tissue Culture media has a pH
indicator (Phenol Red) added in order to check on the growth of cells. The media
initially is a cherry red (with slight blue haze) and turns orange and then yellow as
the cells grow, thereby reducing the media. Should this color change occur within
24 hours, the culture is most likely contaminated and should be disposed of.
15. Examine the cultures using an inverted phase contrast microscope. This will allow
observation of the cells without opening or disturbing the growth.
16. Make cell density determinations at 10 X magnification using a square ocular grid,
as explained in Chapter One for the determination of area.
17. Plot the cell density on a log scale vs. time of culture.
18. Diagram the shape of the cells at each phase.
Notes

The cultures will develop differently than the suspension cultures. The viable cells will
grow out of the trypsinized pieces of tissue and will remain in contact with the bottom of
the culture flask. They will continue to divide and migrate until the entire bottom of the
flask is covered with a single layer of cells (contact inhibition and the formation of a
monolayer).

*Modified from Freshney, R. Ian. Culture of Animal Cells: A Manual of Basic Technique. Alan R. Liss,
Inc. New York, 1983.

Experiment No. 3

Bacteria as bio-pesticides-Bacillus thuringiensis

Aim: To evaluate the biopesticide/biocontrol potential of Bacillus thuringiensis on

lepidopterian insects

Introduction

Insects suffer from diseases caused by pathogens (bacteria, viruses, fungi, nematodes and
protozoa). Sometimes natural outbreaks of diseases occur that control the pest population.
Microbial control aims to use pathogens as tools to suppress pest. This involves finding of
pathogens specific to pest: its easy production; storage of pathogens in ineffective state;
application of pathogens in effective manner. This also involves knowledge of the biology
of pathogen to use it as effective control tool.
Many of the bacterial that infect insects are lethal only in stressed insects. Because the
bacteria, lacking effective means of escaping from the hosts gut after ingestion, are unable
to enter its body cavity. Out of four Bacillus thuringiensis, B. sphaericus, B. popililae and
Serratia entomophila, only Bacillus thuringiensis has been used widely as pesticide. This
produces toxic crystalline proteins inside its spores. These crystals bind to the gut
membrane and degrade it, allowing bacteria to penetrate the body cavity and kill the host.
New strains of the said bacteria can be used against several insect species. These pathogens
are commercially produced in fermentation media without any living hosts rendering it
cheaper. Application of Bacillus thuringiensis is advantageous in forests, where residues of
insecticides are objectionable as it is harmful to wild life.

This is also suitable in IPM, wherein natural enemies are required to stay in the field.
Genes from Bacillus thuringiensis that code for toxic proteins, have been isolated and
inserted into plants, where they are expressed and produce insecticidal proteins in plant
tissues and pollen. Transgenic varieties of major crops like corn, Soya bean and Cotton (Bt)
now exist. Other species of bacteria (B. sphaericus) are used against some species of
mosquito larvae. B. popillae has been used for Japanese beetle. This bacterium requires a
living host for its production. S. entomophila can be produced in fermentation media apart
from a living host.
Requirement:

 Commercial preparation of Bacillus thuringiensis named as DELFIN ®WG

 Susceptible larvae of Plutella sp., Heliothis sp., Spodoptera sp., etc

Protocol

 Prepare various dilutions of the powdered Bacillus thuringiensis in distilled water

 Spray it on the feeding larvae or feed of the larvae by grouping in appropriate

number with control group exposed to distilled water spray.

 Periodically observe the effect of spray on the larval population and report it in the
form LD50 .
 Experiment No.4

Title: Cell viability assay by trypan blue exclusion method

Aim: To determine cell viability using trypan blue exclusion assay.

Introduction:

Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo
dye. Live cells or tissues with intact cell membranes are not coloured. Since cells are very
selective in the compounds that pass through the membrane, in a viable cell trypan blue is
not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are
shown as a distinctive blue colour under a microscope. Since live cells are excluded from
staining, this staining method is also described as a dye exclusion method. The reactivity
of trypan blue is based on the fact that the chromopore is negatively charged and does
not interact with the cell unless the membrane is damaged. Therefore, all the cells which
exclude the dye are viable. Trypan blue is so-called because it can kill trypanosomes, the
parasites that cause sleeping sickness.

Fig: Structure of Trypan blue

Principle:

Requirement:

 0.4% trypan blue solution

  Culture cell suspension/Chick embryo

 Hemocytometer

Pocedure: Trypan Blue Staining of Cells

1. Centrifuge an aliquot of cell suspension being tested for viability 5 min at 100 ×g and
discard supernatant. the size of the aliquot depends on the approximate number of cells
present. the aliquot should contain a convenient number of cells to count in a
hemacytometer when suspended in 1 ml pbs and then diluted again by mixing with
0.4% trypan blue (e.g., 5 × 105 cells/ml).
2. Resuspend the cell pellet in 1 ml pbs or serum-free complete medium. Serum proteins
stain with trypan blue and can produce misleading results. Determinations must be
made in serum-free solution.
3. Mix 1 part of 0.4% trypan blue and 1 part cell suspension (dilution of cells). allow
mixture to incubate 3 min at room temperature. cells should be counted within 3 to 5
min of mixing with trypan blue, as longer incubation periods will lead to cell death and
reduced viability counts. mixing can be performed in a well of a microtiter plate or a
small plastic tube using 10 to 20 µl each of cell suspension and trypan blue.
4. Apply a drop of the trypan blue/cell mixture to a hemacytometer. Place the
hemacytometer on the stage of a binocular microscope and focus on the cells.

5. Count the unstained (viable) and stained (nonviable) cells separately in the
hemacytometer. To obtain the total number of viable cells per ml of aliquot, multiply the
total number of viable cells by 2 (the dilution factor for trypan blue). To obtain the total
number of cells per ml of aliquot, add up the total number of viable and non-viable cell.
Calculate the percentage of viable cells as follows:

viable cells(%)=
total number of viable cells per ml of aliquot x 100
total number of cells per ml of aliquot

Reference:

1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p.

117, Alan R. Liss, Inc., New York.

Comment :

Dye exclusion is a simple and rapid technique measuring cell viability but it is subject to
the problem that viability is being determined indirectly from cell membrane integrity.
thus, it is possible that a cell's viability may have been compromised (as measured by
capacity to grow or function) even though its membrane integrity is (at least transiently)
maintained. conversely, cell membrane integrity may be abnormal yet the cell may be able
to repair itself and become fully viable. another potential problem is that because dye
uptake is assessed subjectively, small amounts of dye uptake indicative of cell injury may
go unnoticed. in this regard, dye exclusion performed with a fluorescent dye using a
fluorescence microscope routinely results in the scoring of more nonviable cells with dye
uptake than tests performed with trypan blue using a transmission microscope. a more
sophisticated method of measuring cell viability is to determine the cell's light scatter
characteristics or propidium uptake (unit 5.4). however, this technique is far more time
consuming and is necessary only when precise measurements on the number. of dead cells
in a cell mixture must be obtained trypan blue exclusion, as described in the above
protocol, can be performed in 5 to 10 min

Experiment No.5

Positive and Negative selection

Aim : To select desired clone among countless bacteria, transformed & untransformed
using Blue white screening.

Requirements :

Genei Transformation Teaching Kit ( 107385 or 106220) provided by Ms. Bangalore

Genei.

Principle:

Screening of transformants:

Selection of cells containing foreign DNA is done basedon the selection marker carried by
this DNA. Example, pUC plasmid has ampicillin resistance factor that enables only
transformed cells to grow on LB-Amp plates. Non-transformants, which are ampicillin
sensitive, do not produce colonies on the selective medium. Transformants and non-
transformants are therefore easily distinguished.

Screening of recombinants:

Identification of recombinants among the transformed cells is generally done by

insertional-inactivation. With most cloning vectors, insertion of DNA fragment into the
plasmid destroys the integrity of one of the genes present on the molecule. As a result, the
characteristic coded by the inactivated gene is no longer displayed by host cells and this is
called insertional-inactivation. For example, pUC18 is a high copy number plasmid of size
2686 bp, with Col E1 origin of replication. It carries a 54 bp polycloning region and
ampicillin resistance marker, along with coding information for the first 146 amino acids
(amino terminal of β-galactosidase (Lac Z) gene. Some strains of E. coli bear a deletion at
the amino terminal end of Lac Z gene and thus synthesize an inactive C-terminal fragment
On transforming such competent bacterial strains with pUC18 the host and plasmid
encoded fragments associate to form an enzymatically active protein. This type of
complementation is known as α-complementation. Lac+ bacteria that result from α-
complementation can be recognized as they form blue colour colonies in presence of X-gal
(chromogenic substrate for β-galactosidase) and IPTG (inducer for the expression of the
enzyme).

However, insertion of a fragment of foreign DNA into the polycloning site of plasmid
results in production of an amino terminal fragment that is not capable of α
complementation. Hence, cells carrying recombinant plasmid will form white colonies.
This is also referred to as Blue-White Screening.

Procedure:

As per manufacturers instruction.

Observation & Result :

As given in the manual

 (Use only for teachers reference and should not be included in Journal)

Experiment No.5

Positive and Negative selection

A central problem of cloning is the identification of a desired clone among countless

bacteria, transformed & untransformed.

In the simple cloning system we have used so far, we used antibiotic resistance to select
the desired clones. This was possible because our desired clones all carried antibiotic
resistance genes that untransformed bacteria did not.

But what if you need to select for MORE than just antibiotic resistance? In Labs 18-20,
we are cloning a PCR-amplified gene into a plasmid vector (pBLU). After ligation and
transformation, we are faced with

two variables:

• Tranformed vs untransformed bacteria: Bacteria transformed by pBLU are selected

from untransformed cells by resistance to ampicillin (pBLU carries the ampR gene).

• Bacteria carrying empty pBLU vs bacteria carrying pBLU+PCRproduct (the desired

clones): All of these cells will be able to grow on ampicillin. To identify desired clones,
use Blue/White screening.

Blue/White screening: How does it work?

pBLU, and other specially designed cloning vectors, make use of the lac operon. pBLU
carries the gene for β-galactosidase (also known as lacZ). This enzyme catalyzes the
breakdown of lactose as a food source. It can also degrade an artificial substrate called x-
gal, which turns BLUE when it is broken down by β-galactosidase.

• Colonies that produce β-galactosidase and are fed x-gal will turn BLUE.
 • Colonies that do NOT produce β-galactosidase remain white in color, even in the
presence of x-gal.

Plasmid vectors designed for blue/white screening have a multicloning site carefully
placed early within the coding region of lacZ. Successful ligation of a foreign DNA into
this multicloning site interrupts lacZ and abolishes production of functional β-
galactosidase.

Therefore:

Untransformed bacteria: No growth on amp

Bacteria transformed with

original pBLU: Blue colonies on amp + x-gal

Bacteria transformed with

pBLU + insert DNA: White colonies on amp + x-gal

Note that blue/white screening is not selection (it does not kill the unwanted bacteria); it is
screening.

In some blue/white screening systems, an additional reagent must be used: IPTG

(isopropylthiogalactoside). IPTG is an inducer that de-represses lacZ expression (it turns
the gene on). In some cases, without IPTG, not enough β-galactosidase is produced to turn
the colony blue even if the lacZ gene is intact.
 Experiment No.6

Culture of bacteria in Liquid medium and on agar plate

Aim- To culture bacteria in Liquid medium and on agar plate

Introduction:

Microorganisms must have a constant nutrient supply if they are to survive. Free-living
organisms acquire nutrients from the environment and parasitic organisms acquire nutrients
from their host. When trying to grow microbes in the lab adequate nutrition must be
provided using artificial media. Media may be liquid (broth) or solid (agar). Any desired
nutrients may be incorporated into the broth or agar to grow bacteria. Agar is the
solidifying material used in solid media. It is an extract of seaweed that melts at 100 oC and
solidifies at about 42oC. Most pathogenic bacteria prefer to grow at 37oC so agar allows for
a solid medium at incubator temperatures. Since agar remains solid until reaching 100 oC,
thermophiles (heat -lovers) that prefer temperatures above 50 oC for growth can still be
grown on solid media. Organisms grown in broth cultures cause turbidity, or cloudiness, in
the broth. On agar, masses of cells, known as colonies, appear after a period of incubation.
Certain techniques will allow bacterial cells to be widely separated on agar so that as the
cell divides and produces a visible mass (colony), the colony will be isolated from other
colonies. Since the colony came from a single bacterial cell, all cells in the colony should
be the same species. Isolated colonies are assumed to be pure cultures.

Requirements:

 Cultures: Isolated microorganisms or any natural source of microorganism

 Biochemicals : Peptone, beef/yeast/meat extract, NaCl, agar agar

 Apparatus : Petriplate, Conical flask, beaker, test tube(rimless), nichrome wire

loop(Himedia), Permanent marker, Swab, etc

 Equipments: Autoclave, hot air oven, digital balance, pH meter,

 Nutrient Agar Composition (100ml)

Peptone------------------------ 1g
NaCl--------------------------- 0.5g
Yeast Extract----------------- 0.3 g
Distilled Water-------------- 100 ml
pH----------------------------- 7.2
Agar -------------------------- 2.5 g
Nutrient broth lack agar from above composition.

Principle:
Most microbiological laboratory procedures require the use of living organisms, hence
microorganism can be cultured on artificial media by supplying nutritional need through
variety of media components in liquid and on solid medium. Culturing techniques provide a
means for maintaining adequate nutrition for the organisms so they can continue to survive.
As organisms grow in a culture they consume the available nutrients and periodically need
to be transferred to fresh media to continue to grow. Certain culturing techniques not only
provide the organisms with a fresh supply of nutrients but also allow for the separation of
bacterial cells to obtain isolated colonies. These culturing procedures are known as
isolation techniques.
Streak plates allow for the growth of isolated colonies on the surface of the agar. An
isolated colony is a colony that is not touching any other colonies and is assumed to be a
pure culture. These colonies are easily accessible for performing staining and identification
procedures. They also show colonial morphology that may be useful in identifying the
organism. Part of the identification of any organism includes a description of colonial
morphology. Since organisms may grow differently on different media, the type of media
used must be included as a part of any colonial morphology. The cultivation of
microorganism often involves preparation of culture media, inoculation of medium,
incubation and growth observation & interpretation using parallel control experiments.
Procedure:
This is divided in to 4 phases
A) Preparation and Sterilization of Microbial media.
1. Appropriately weighted of the media components is added in distilled water one
after another except agar agar powder.
2. The pH(7.2) of the medium is adjusted using dilute HCl or NaOH.
3. Agar agar is added and digested by direct heating until agar powder dissolves.
4. The flask is plugged with cotton and wrapped well using paper and autoclaved at
15lbs pressure for 15 minutes.
5. For preparation of Nutrient broth agar addition step is not followed. The flask is
directly autoclaved.
6. The agar plates were prepared under aseptic condition (between flames of burner or
in Laminar Air Flow) by pouring 15 to 20 ml approximate quantity of the medium
in sterile empty agar plates. The agar solidifies below 400C hence pouring should be
well above this temperature. Very hot medium pouring will lead to condensation of
moisture on the lid of the plate.
7. The Nutrient agar plates are allowed to solidify by keeping at R.T. for 30 minutes.
B) Inoculation & Incubation.
1. The Nutrient agar plates are spot inoculated/streaked/spread using nichrome wire
loop by following appropriate aseptic technique.

2. The Nutrient broth medium is inoculated using nichrome loop or measured aliquots
is added using sterile pipettes.

3. The plated were kept inverted to avoid dripping of moisture on the surface of agar.

4. The inoculated media is incubated in incubator or shaking incubator at temperature

depending on temperature optima of bacteria being studied.

C) Observation and interpretation of growth

1. The well isolated bacterial colonies on agar surface are observed. Their cultural
characteristic are noted(Fig 1.1).

2. In broth medium turbidity pattern (Fig 1.1) is observed compare to un-inoculated

medium. The turbidity may be measured at 600nm using colorimeter.
Observation table
Culture Growth Isolation

Broth Turbidity(yes/no) No

Slant Colonial growth(yes/no) No

Streak plate Colonial growth(yes/no) Yes/No

Spread plate Colonial growth(yes/no) Yes/No

Fig. Streak pattern on agar plate

 Growth in broth media

Figure 1.1 Cultural characteristics of bacteria

 Experiment No.7

Antibiotic Sensitivity/Resistance Test

Aim: To evaluate antibiotic sensitivity of microorganisms by multidisc technique

Introduction:

When a filter paper disc impregnated with a chemical is placed on agar the chemical will
diffuse from the disc into the agar. This diffusion will place the chemical in the agar only
around the disc. The solubility of the chemical and its molecular size will determine the
size of the area of chemical infiltration around the disc. If an organism is placed on the
agar it will not grow in the area around the disc if it is susceptible to the chemical. This
area of no growth around the disc is known as a “zone of inhibition”.

Requirements:

Cultures : Any Gram positive or Gram negative bacteria.

Media : Muller Hinton Agar No.2 or Nutrient Agar plates
Nutrient Agar Composition(100ml)
Peptone------------------------ 1g
NaCl--------------------------- 0.5g
Yeast Extract----------------- 0.3 g
Distilled Water-------------- 100 ml
pH----------------------------- 7.2
Agar -------------------------- 2.5 g
Nutrient broth lack agar from above composition.

Antimicrobial Sensitivity disc: Multidisc of antibiotic from any manufacturer(DynaMiro)

Principle:

Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics.

Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic
will be most successful in treating a bacterial infection in vivo. Testing for antibiotic
sensitivity is often done by the Kirby-Bauer method which is filter paper disc-agar
diffusion method. This method allows for the rapid determination of the efficacy of a drug.
The susceptibility of microorganism to a drug is determined by the size of inhibition zone
(no growth around disc), which is dependent on the ability of the ability and the rate of
diffusion of antibiotic into the medium and its interaction with the test microorganisms,
number, growth rate and microbial sensitivity to the antibiotic.

Procedure:
A) Preparation of inoculum:

1. A bacterial colony growth from agar plate or slant is inoculated in Nutrient Broth
and grown overnight i.e. 16-18hrs.

2. The optical density of the culture is adjusted to 1.0 at 600nm using sterile Nutrient
broth medium.

B) Seeding Nutrient Agar Plates

2. The sterile Nutrient agar plate is labeled with the name of the test organism to be
inoculated

3. Using sterile technique agar plates with their respective test organisms is inoculated
as follow:

a) Dip sterile cotton swab into a well mixed inoculom and remove excess inoculums
by pressing the saturated swab against inner wall of the tube.

b) Using swab streak the entire agar surface horizontally, vertically and around the
outer edge of the plate to ensure heavy growth over the entire surface.

4. Allow culture plates to dry about 5 minutes.

C) Multi disc transfer

1 The forceps dipped in alcohol and flamed is used to transfer multi-disc on the
surface of agar under aseptic condition.

2 Gently each disc is pressed on the agar surface to ensure its adherence to agar
surface using forceps.

3 Incubate inverted culture plates in incubator for 24 to 48hrs at 37 0C.

4 Measure the zone diameter using scale or special antibiotic zone scale (HiMedia
Cat. no. PW096).

5 Interpret the result as sensitive or resistant according to the multi disk manufacturer
instructions provided.

Observation Table :

Antibiotic Disk Content Zone diameter Result

(Microgram) (mm) (Sensitive/Resistant/Intermediate)
Miscellaneous