Laboratory manual in microbiology

with standard procedures, techniques,

safety measures and technical
information as a reference framework
to enable efficient student practical
laboratory work

Laboratory
Manual in
Microbiology
F.Y.B.Sc. CBCS Semester I
2022-2023

St. Xavier’s College, Mapusa- Goa

 Compiled and Edited by
Ms. Ruella D’souza

Contributors:
Ms. Linette de Souza
Ms . Katelyn Gonsalves
Ms. Arina Frank
Mr. Siddhesh Menon
Dr. Marielou Ferrao
Dr. Sheryanne Velho-Pereira
Dr. Valerie Gonsalves
Dr. Trelita de Sousa
Ms. Nadine de Souza
 INDEX
Expt. Title of the experiment
No.
1 Microbiology Good Laboratory
Practices and Biosafety
2 Principle and applications of lab
instruments
3 Preparation of Culture Media
4 Monochrome staining
5 Negative Staining
6 Gram Staining
7 Isolation of Pure culture by Streak
Plate method
8 Estimation of Viable Count by spread
plate method
9 Motility – Hanging Drop method
10 Qualitative tests for carbohydrates
11 Qualitative tests for proteins
12 Qualitative tests for lipids
Experiment No. 1

MICROBIOLOGY GOOD LABORATORY PRACTICES (GLP) AND BIOSAFETY

A rewarding laboratory experience demands strict adherence to prescribed rules for personal and
environmental safety. Avoiding laboratory accidents, maintaining scrupulously clean laboratory
setting, and mastering aseptic techniques form an integral part of most microbiological
procedures. Although the virulence of microorganisms used in the academic laboratory
environment has been greatly diminished because of their long-term maintenance on artificial
media, all microorganisms should be treated as potential pathogens.

The following basic steps should be observed at all times in the laboratory:
1. Upon entering the laboratory, place coats, books in specified locations, never on bench tops.
2. Keep doors and windows closed during the laboratory session to prevent contamination from
air currents.
3. At the beginning and termination of each laboratory session, wipe bench tops with a
disinfectant solution.
4. Do not place contaminated instruments, such as inoculating loops, needles, and pipettes, on
bench tops. Loops and needles should be sterilized and pipettes should be disposed of in
designated receptacles.
5. On completion of the laboratory session, place all cultures and materials in the disposal area.
6. Wash your hands with liquid detergent and disinfectant upon entering and prior to leaving the
laboratory.
7. A laboratory coat is necessary while working in the laboratory, to protect clothing from
contamination or accidental discoloration by staining solutions.
8. Wear disposable gloves during the manipulation of test materials such as blood, serum, and
other body fluids.
9. Tie back long hair to minimize its exposure to open flames, wear closed shoes at all times in
the laboratory setting, never apply cosmetics or insert contact lenses in the laboratory.
10. Do not eat, or drink in the laboratory.
11. Carry cultures in a test-tube rack when moving around the laboratory and keep cultures in a
test-tube rack on the bench tops when not in use.
12. Report accidental cuts or burns to the instructor immediately.
13. Speak quietly and avoid unnecessary movement around the laboratory to prevent distractions
that may cause accidents.
Experiment No. 2

PRINCIPLE AND APPLICATIONS OF IMPORTANT LABORATORY INSTRUMENTS

Aim: To study the principle and applications of important instruments used in the Microbiology
Laboratory

1. BIOLOGICAL SAFETY CABINET / LAMINAR AIR FLOW (LAF)

Biological safety cabinets work by the use of in-flow laminar air drawn through one or more
High-Efficiency Particulate Air (HEPA) filters, which remove 99.97% of 0.3µ particles and are
one of the most important air filtration systems. Laminar flow workstations force air through
HEPA filters and then project a laminar curtain of sterile air across the cabinet. 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. Laminar flow cabinets generally
have a UV-C germicidal lamp to sterilize the interior and contents before usage to prevent
contamination of the experiment. Germicidal lamps are usually kept on for 15 minutes to
sterilize the interior before the cabinet is used. The light must be switched off when the cabinet is
being used, to limit exposure to skin and eyes as stray ultraviolet light emissions can cause
cancer and cataracts.

Laminar airflow biological safety cabinets are designed to create a particle-free working
environment, prevent contamination of biological samples, and provide protection to any
particle-sensitive materials. Biological safety cabinets render three levels of protection: 1)
Personal Protection from harmful agents within the cabinet; 2) Product Protection to avoid
contamination of the samples; and 3) Environmental Protection from contaminants contained
within the cabinet.
BIOLOGICAL SAFETY CABINET / LAMINAR AIR FLOW (LAF)
2. AUTOCLAVE
An autoclave is a self-sealing vessel used to perform sterilization in high-pressure and high-
temperature environments. It works on the principle of moist heat sterilization where steam
under pressure is used to sterilize the material present inside the chamber. Boiling point of water
is directly proportional to the pressure when volume is constant. When pressure is increased in a
closed vessel, the temperature increases proportionally e.g. at 15 lbs/sq.in., the temperature kept
constant at 121.6 ˚C, for 20 minutes, is sufficient to kill all the vegetative forms and spores of the
organisms.

It is a cylindrical vessel made of gun metal or copper. The cylindrical shape withstands high
pressure more easily than a box or a cube. It has a lid, which can be fastened to the body by
means of screw-clamps. The lid is provided with the steam valve, a thermometer, a safety valve
and a pressure gauge. On the rim of the cylinder is a rubber gasket to ensure an air tight closure.
The cylinder contains water up to a certain level which is heated by means of an electrical or gas
device. Above this level, there is a perforated diaphragm which is used for keeping the material
to be sterilized. Materials to be sterilized are placed on the perforated diaphragm. Test tubes and
flasks are plugged with non-absorbent cotton and covered with paper to avoid drenching of the
plugs during the release of steam, when condensation occurs.

After adjusting the water level, the autoclave is switched on. The lid is kept in position and is
closed tightly by means of the screw clamps. The steam valve is kept open until all the air is
expelled and only steam comes out. The steam valve is then closed, so that pressure begins to
rise. As the pressure rises temperature also rises accordingly. When the pressure is 15 lbs/sq.in
(temperature 121.6 ˚C) the time is noted, one of the electric coils is switched off (when two coils
are used) or the steam valve is opened partially so as to maintain the pressure constant at15 psi.
After 20 minutes the autoclave is switched off and is allowed to cool down after all the steam has
escaped from the open steam valve. Apparatus is then opened and all sterilized materials are
removed.

Autoclaves are widely used for sterilization in microbiology, medicine, veterinary medicine,
mycology, funerary practice, dentistry, and prosthetics fabrication. They vary in size and
function depending on the media to be sterilized and are sometimes called retort in the chemical
and food industries. Autoclaves are used in medical applications to perform sterilization and in
the chemical industry to cure coatings and vulcanize rubber and for hydrothermal synthesis.
Industrial autoclaves are used in industrial applications, especially in the manufacturing of
composites. In the laboratory it is used for the sterilization of laboratory culture media, aprons,
rubber tubing, etc.

AUTOCLAVE
3. INCUBATOR
An incubator is a device used to cultivate and maintain microbiological cultures or cell cultures
at constant temperature and humidity. All incubators are based on the concept that when
organisms are provided with the optimal condition of temperature, humidity, oxygen, and carbon
dioxide levels, they grow and divide to form more organisms. The incubator maintains optimal
temperature, humidity and other conditions such as the CO2 and oxygen content of the
atmosphere inside. It consists of a box with a double wall, insulated with asbestos. It is heated by
an electric current and is maintained at constant temperature by means of a thermostat. The
temperature is noted by means of a thermometer. The incubator is provided with a double door,
the inner one is glass and outer one is of the same material as the rest of the chamber. It is
generally maintained at 37 °C which is the optimum temperature of most human pathogens.

INCUBATOR
4. BOD (BIOLOGICAL OXYGEN DEMAND) INCUBATOR
The BOD incubator is the most versatile and reliable low temperature incubator which is
designed to maintain at 20°C, necessary for the determination of Biological Oxygen Demand
(BOD). BOD incubators provide accurate, uniform and controlled temperature conditions for
accelerated tests and exposures. The BOD incubator is widely used in microbiology laboratories
for the applications that include cell culture and fungal growth, BOD test, fermentation, crop and
physiology and various pharmaceutical tests etc. It is used to maintain temperature for test tissue
culture growth, storage of bacterial cultures and incubation where high degree of constant
temperature accuracy is required.
5. HOT AIR OVEN
Hot air ovens are electrical devices which use dry heat to sterilize. They were originally
developed by Pasteur. Generally, they use a thermostat to control the temperature. Their double
walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor
and outer layer being metallic. Hot air ovens use extremely high temperatures over several hours
to destroy microorganisms and bacterial spores. The ovens use conduction to sterilize items by
heating the outside surfaces of the item, which then absorbs the heat and moves it towards the
centre of the item. It consists of a chamber with double walls insulated with asbestos or glass
wool to prevent radiation of heat. The chambers are heated electrically and controlled
thermostatically. The temperature is noted on the thermometer. The temperatures are generally
used 180 °C for 30 min or 160 °C for 1 hour or148° C for 1-2 hours. This temperature is
sufficient for destruction of vegetative cells and spores. Hot air ovens are generally used for dry
sterilization of glassware e.g. test tubes, pipettes and petri plates. Culture media cannot be
sterilized by this method.

Precautions:
• Glassware should be dry before sending for sterilization
• To prevent contamination after sterilization, the test tubes, flasks and pipettes are plugged
and wrapped in paper; petri plates are also wrapped in paper
• The temperatures should not be allowed to increase above 180° C, otherwise cotton plugs
and papers may get charred
• All sterilized articles must be cooled to room temperature before removal
HOT AIR OVEN
6. LIGHT MICROSCOPE
The microscope is an instrument which is absolutely essential for microbiologists. It is a
combination of lenses so adjusted that minute objects invisible to the naked eye are magnified
and made visible. The term microscope is compounded from two Greek words micro-small and
scope –to view.

It is a complicated instrument with the different parts fitted in each other very accurately. The
eyepiece fits into a graduated draw tube which in turn slides in the body tube. A revolving nose-
piece is attached to the lower end of the body tube and to it, various objectives are fitted. The
body tube and the objectives move up and down by means of a rack and pinion arrangement
known as the course adjustment. The tube and its attachments are connected to the foot of the
microscope by means of hinged joint. Below the objective, in the centre of the microscope, is a
platform called the stage on which microscopic preparations are mounted. A mechanical stage
may be fitted for the steady and controlled movements of the slide. Below the stage is a sub-
stage, which is fitted with an Abbe’s condenser, in order to increase the intensity of illumination
at high magnification. The condenser has an iris diaphragm, which controls the intensity of the
beam of the light, entering the condenser. The condenser can be raised or lowered. Below the
condenser there is a horseshoe shaped base over which a plano-concave mirror is mounted. The
plane side of the mirror is generally used with the condenser while the concave side is used in its
absence.

The objective is of 3 types:-

Lower power lens Focal length 16mm or 2/3’’
High power lens Focal length 4 mm or 1/6’’
Oil immersion lens Focal length 2 mm or 1/12’’

The oil immersion lens must be used with a drop of cedar wood oil (preferable) for microscopic
examination of cells up to 0.22/μm. A micrometer (or micron) is the unit of linear measurement
on the microscopic scale symbolized by um and is equal to 0.001 mm. The total magnification of
the objective magnification x eye-piece magnification. The total magnification of objective can
be calculated as the ratio of mechanical tube length of the objective.
 Objective Magnification of the Total magnification Diameter of the
lens with 10 x ocular field seen
Lower power lens 160/16=10 10 x 10=100 1.55mm
High power lens 160/4=40 40 x 10=400 0.31mm
Oil immersion lens 160/2=80 40 x 10=400 0.31mm

Nowadays oil immersion lenses with 100 x magnification are used. When oil immersion lens is
used, a drop of cedar wood oil is placed on the object and the lens is immersed in it. This is
because when an oblique ray emerges out from a denser to rarer medium it is refracted away
from the normal and hence the light rays are scattered. If the space between the object and
objective is filled with immersion oil which has a refractive index similar to that of glass, the
rays do not undergo refraction and pass into the oil immersion lens giving clear, bright magnified
image of the object.

Parts of the Microscope

7. pH METER
pH is defined as the concentration of hydrogen ions in the solution. When the solution contains
more H+ ions, it is said to be acidic; while a solution that contains more OH- ions is deemed
alkaline. The pH value ranges from 1 (highly acidic) to 14 (highly basic), pH 7 being neutral.

A pH meter is used to determine the acidity or alkalinity of a solution. It contains a pH probe,

which when dipped into a solution, passes the electrical signals to the pH meter which displays
the pH value of the solution. The glass pH probe contains two electrodes: a sensor and a
reference electrode. These electrodes are in the form of glass tubes, one contains buffer 7 and the
other contains saturated potassium chloride solution. The sensor electrode bulb is made up of
porous glass or permeable glass membrane coated with silica and metal salts. A silver wire
coated with silver chloride is immersed in pH 7 buffer in the bulb. Another silver wire coated
with silver chloride is immersed in the saturated potassium chloride solution in the reference
electrode. When the probe is placed in a solution to measure the pH, hydrogen ions accumulate
around the bulb and replace the metal ions from the bulb. This exchange of ions generates some
electric flow that is captured by the silver wire. The voltage of this electric flow is measured by
the pH meter by converting it into pH value by comparing the generated voltage with the
reference electrode. Increase in acidity of the solution has a greater concentration of hydrogen
ions that increases the voltage. This increased voltage decreases the pH reading in pH meter. In
the same manner, an increase in alkalinity decreases the hydrogen ions and increases the pH
value in the pH meter.
Experiment No. 03
PREPARATION OF CULTURE MEDIA

Aim: To prepare routine laboratory media

Principle: A growth or culture medium is a solid, liquid or semi-solid designed to support the
growth of microorganisms in the laboratory. A suitable medium provides essential nutrients, an
energy source and appropriate growth conditions required by the microorganism to survive and
proliferate. Laboratory media may be simple, complex, differential, defined or selective for
specific microorganisms.

Requirements: Peptone, Meat extract, Agar-agar, glucose, sodium taurocholate, lactose, sodium
chloride, 1% neutral red indicator, 0.1 N HCl, 0.1 N NaOH, Distilled water, pH paper

Procedure:
A) Peptone Water
Composition:
Peptone -1g
NaCl - 0.5 g
Distilled water - 100 mL
pH - 7.4
Weighed amounts of peptone and NaCl are dissolved in 100 mL of distilled water. The pH is
adjusted to 7.4 by adding 0.1 N NaOH dropwise if required. The final pH of the medium is
checked using pH paper. 5 mL of the prepared medium is then distributed into each test tube.
The test tubes are plugged with cotton wool, wrapped in paper and sterilized in an autoclave at
15 psi for 20 minutes.

B) Nutrient Broth
Composition:
Peptone -1g
NaCl - 0.5 g
 Meat Extract - 0.3 g
Distilled water - 100 mL
pH - 7.4
Weighed amounts of the ingredients are dissolved in 100 mL of distilled water. The pH is
adjusted to 7.4 by adding 0.1 N NaOH dropwise if required. The final pH of the medium is
checked using pH paper. 5 mL of the prepared medium is then distributed into each test tube.
The test tubes are plugged with cotton wool, wrapped in paper and sterilized in an autoclave at
15 psi for 20 minutes.

C) Nutrient agar
Composition:
Peptone -1g
NaCl - 0.5 g
Meat Extract - 0.3 g
Agar -2g
Distilled water - 100 mL
pH - 7.4
To prepare nutrient agar, add 2 g of agar powder to 100 mL of nutrient broth and sterilize the
flask in an autoclave at 15 psi for 20 minutes.

D) MacConkey’s broth and agar

Composition:
Peptone - 1.7 g
Proteose peptone - 0.3 g
Lactose -1g
Bile salts - 0.15 g
Sodium chloride - 0.5 g
Neutral red - 0.003 g
Crystal violet - 0.0001 g
Agar - 1.5 g
 Distilled water - 100 mL
pH - 7.4
Weigh the ingredients as given in the composition except sugar, agar and indicator and dissolve
them in 90 mL of distilled water. Dissolve 1 g of lactose in 10 mL of distilled water.
Adjust the pH of the medium to 7.4. Now add the required quantity of neutral red indicator. If
MacConkey’s agar is being prepared, add 1.5 g agar powder to the medium before autoclaving.
Sterilize the medium and sugar separately by autoclaving at 15 psi for 20 minutes. After
autoclaving add the lactose solution to the molten medium.

E) Sabouraud’s broth
Composition:
Glucose -4g
Peptone -1g
Distilled water - 100 mL
pH - 5.4
Weigh the ingredients as given in the composition and dissolve them in 100 mL of distilled
water. The pH is adjusted to 5.4 by adding 0.1 N HCl dropwise if required. The final pH of the
medium is checked using narrow range pH paper. The medium is sterilized by placing in a
boiling water bath for 10-15 minutes and cooled.

F) Sabouraud’s Agar
Composition:
Glucose -4g
Peptone -1g
Agar - 1.5 g
Distilled water - 100 mL
pH - 5.4
Weigh the ingredients as given in the composition (except agar) and dissolve them in 100 mL of
distilled water. Add the agar powder. The pH is adjusted to 5.4 by adding 0.1 N HCl dropwise if
required. The final pH of the medium is checked using narrow range pH paper. The medium is
sterilized for three consecutive days by autoclaving at 10 psi for 10 minutes.
To check the sterility of the media, observe after 24 hours and up to 4 days to check for any
growth or contamination.

Results: No growth was observed in the sterilized media as preparation was done aseptically.
Experiment No. 04
MONOCHROME STAINING

Aim: To study the morphology of bacteria using simple stains.

Principle: Any basic dye such as methylene blue, safranin, or crystal violet can be used to color
the bacterial cells. These stains readily give up a hydroxide ion or accept a hydrogen ion, thus
making the stain positively charged. Since the surface of most bacterial cells and cytoplasm is
negatively charged, these positively charged stains adhere to the cell surface. The bacteria will
show up as colored spots against a white background.

Requirements:
Culture: Bacterial suspension
Labware: Glass slides
Nichrome loop
Glass marking pencil / marker
Immersion Oil
Equipment: Microscopes
Stains: Crystal violet, Basic Fuchsin, Loeffler’s Methylene blue, Safranin.
Protocol:
 Take a clean grease - free slide

Place a loopful of the suspension on the glass slide inside the marked area and
spread into a thin, even film.

Air dry the film

Heat fix

Keep the slide on a support and flood the smear with any one of the following stains and
allow to stand for the following time intervals.
Crystal violet-30 seconds Loeffler’s methylene
Basic fuchsin-2-3 minutes Safranin - 1 minute.
to 1 minute blue-2-3 minutes

Gently wash the slide using an indirect stream of tap water.

Using a filter paper, blot dry (without rubbing) the smear.

Examine the smear under oil immersion lens.

Results: Cocci, Bacilli and yeast cells were observed

Suspension Morphology Arrangement

Experiment No. 05

NEGATIVE STAINING

Aim: To demonstrate the morphology of bacteria by negative staining technique.

Principle: India ink or nigrosin is an acidic stain. The stain readily gives up a hydrogen ion
(proton) and the chromophore of the dye becomes negatively charged. Since the surface of most
bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will
stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark
background.

Requirements:
Culture: Bacterial suspension
Labware: Glass slides
Nichrome loop
Glass marking pencil / marker
Immersion Oil
Equipment: Microscopes
Stains: Nigrosine stain
Protocol:

Take a clean grease - free slide

Place a drop of Nigrosin stain toward one end of the slide

Place a loopful of inoculum into the drop of stain, mix

Prepare a thin film of stain on slide by using the edge of another slide

Air dry the film

Focus a thin area under oil immersion and observe the unstained cells
surrounded by the grey/black stain
Observation And Expected Results:

Colorless cocci, bacilli and yeast cells against a dark background

Experiment No. 06
GRAM STAINING

Aim: To determine the Gram character of the given microorganism.

Principle: The structure of the organism’s cell wall determines whether the organism is Gram
positive or negative. Cell are stained with a primary stain Crystal violet (CV) which dissociates
into CV+ and Cl– ions in aqueous solutions. These ions penetrate through the cell wall and cell
membrane of both Gram-positive and Gram-negative cells and the CV+ ion interacts with
negatively charged components of bacterial cells and stains the cells purple. Iodine (I), used as a
mordant, interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I)
within the inner and outer layers of the cell. When a decolorizer such as alcohol or acetone is
added, it interacts with the lipids of the cell membrane. Gram negative organism have
additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so Gram
negative organisms fail to retain the complex and get decolorized as the complex is washed
away. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This
closes the pores in the cell wall and thus traps the CV–I complex and prevents it from exiting the
cell. After decolorization, the Gram-positive cell remains purple and the Gram-negative cell
loses its purple color. Counterstain, which is usually positively-charged safranin or basic fuchsin,
is applied last to give decolorized Gram-negative bacteria a pink or red color.

Requirements:
Culture: Bacterial suspension
Labware: Glass slides
Nichrome loop
Glass marking pencil / marker
Immersion Oil
Equipment: Microscopes
Stains: Crystal violet (Primary stain), Gram’s iodine (Mordant), 95% alcohol (Decolourizer),
Safranine or Basic fuchsin (Counter stain)
Procedure:

Take a clear grease-free slide

Using a sterile nichrome loop make an even smear of the culture suspension

Allow the smear to air dry and heat fix (pass through a flame 2-3 times)

Flood the smear with crystal violet and keep for 1 minute

Discard the stain and gently wash with tap water

Flood the smear with Gram’s Iodine and keep for 2 minutes and then discard the excess

Treat with alcohol for 3 minutes till decolorized

Wash the slide with water

Stain the smear with safranin and keep for 1 minute

Discard the stain and gently wash with tap water

Air dry and observe under oil immersion lens

Observation And Expected Results:
Experiment No. 07

ISOLATION OF PURE CULTURE BY STREAK PLATE METHOD

Aim: To obtain isolated colonies of bacteria/ fungi on an agar plate from a consortium of cells

Principle: The application of microbial cultures to the surface of the agar and spreading them by
a loop is called streaking and the plates so prepared are called streak plates. During inoculation,
the closely packed cells at the start of the streak form colonies that run together but as streaking
continues, fewer and fewer cells remain in the droplet being carried on the loop. As this falls off
and grows on the surface, well separated colonies develop. A good plate results from progressive
movement of the loop, repeated several times.

Requirements:
Culture: Bacterial suspension
Labware: Nichrome loop
Glass marking pencil / marker
Media: Sterile nutrient Agar plates
Protocol:

Divide the agar plate into three distinct zones: a small inoculation
zone, a sterile zone and a larger isolation zone

Flame the loop

Inoculate a loopful of culture in the inoculation zone.

Flame the loop and cool it

Touch the sterile loop onto the inoculation zone to collect a small
inoculum and streak the culture in the isolation zone back as shown in
the diagrams.

Flame the loop, between streaks, and cool it

Incubate the plate upside down and observe for the appearance of
isolated colonies
Observation And Expected Results:

Isolated Colonies along the line of streaking

Experiment No. 08
VIABLE COUNT METHOD

Aim: To enumerate the bacteria in the given suspension using 1. Pour plate method and 2.
Surface spreading method

Requirements:
Culture: Bacterial suspension18-24 hour old culture of E. coli
Labware: Glass marking pencil / marker
Sterile tubes
Sterile pipettes (1ml and 10 ml)
Media: Sterile nutrient agar plates.
Sterile nutrient agar butts (10 ml)
Chemicals: sterile saline

Procedure:

SPTREAD PLATE METHOD

a) Prepare the dilutions of the given culture as mentioned.
b) Transfer 0.1 ml of each of 10-5, 10-4 and 10-3 dilutions to the surface of a sterile agar plate with
a sterile pipette.
c) Using an alcohol sterilized glass spreader spread the dilutions evenly on the agar surface.
d) Incubate the plates inverted at 37⁰C for 24-48 hours.

Result: The viable count of the given sample (E. coli) by spread plate method was found to be
________________ cfu/ml.
 Viable Count

Calculations:

Viable count= Average number of Colonies x Dilution factor

Volume of Inoculum

Units- cfu/ml
Experiment No. 09

HANGING DROP (MOTILITY TEST)

Aim: To demonstrate the motility of the given organism by means of the hanging drop
technique.

Requirements:
Culture: 24 hour old culture of organisms
Glassware: Cavity slide, cover slips, nichrome loop, vaseline.

Principle: A very small drop of bacterial suspension is hung from the centre of a cover slip into
the cavity of a cavity slide. The hanging drop is observed under a microscope using oil-
immersion objective. If the bacteria are motile, its cells can be seen to have erratic movements in
the surrounding medium. In contrast, if it is non-motile, its cells remain static in the medium
without any movement or may show Brownian movement (resulting from the bombardment of
cells with other cells or with surrounding water molecules in the medium).

Procedure:
Using a match stick, apply vaseline on the edges of a
coverslip.

Under sterile conditions, place a loopful of culture in the

centre of the coverslip.

Invert a slide on the coverslip so that the drop is in the

centre of the cavity.

Press the coverslip down gently to make an airtight seal.

Turn the slide so that the drop is now hanging downward

from the coverslip into the cavity.

Look for the edge of the drop under the low power
objective

Observe motility under high power objective with the

condenser lowered to increase contrast.
Result:
Experiment No. 10

QUALITATIVE TESTS FOR CARBOHYDRATES

1. MOLISCH TEST -

Aim: To detect the presence of carbohydrates in the sample.

Requirements:
Chemicals: conc, H2SO4, α-naphthol, distilled water.
Glassware: test tubes
Sample: Test sample

Principle: Conc. H2SO4 hydrolysis the glycosidic bonds to give monosaccharides, which are
then hydrolysed to furfural and its derivatives. These products combine with sulphonated α-
naphthol to give a purple coloured complex. This reaction is seen in general with all carobydrates
which can give furfural with conc. H2SO4.

Procedure:
a) Add 2 drops of α-naphthol solution (Molisch’s reagent) to 2ml of test solution.
b) Carefully pour about 1ml of conc. H2SO4 along the sides of the tube so as to form 2 layers.
c) Carefully observe any colour change at junction of two layers.
d) Use D/W for the control.
e) Positive test is indicated by the formation of a violet ring at the junction of two layers.

Result: Violet ring is observed at the junction of 2 layers. Therefore carbohydrates are present.
MOLISCH TEST (BLANK PAGE)
2. BENEDICT’S TEST

Aim: To detect the presence of carbohydrates (Reducing sugars).

Requirements:
Chemicals: Benedict’s solution, distilled water
Glassware: test tubes
Sample: Test sample (Glucose)

Principle: Benedict’s solution contains cupric ions (Cu+2) in a stable complex form. If the
carbohydrates have a reducing group, the cupric ion is reduced to cuprous ions (Cu+) or even
copper and this show up a rusty brown color or red precipitate.

Procedure:
a) Add 5 drops of test solution to 2 ml of Benedict’s solution and place in a boiling water bath
for 5 mins.
b) Use D/W for the control tube.
c) Positive test is indicated by the formation of a brick red color precipitate.

Result: Brick red / orange color precipitate is observed upon boiling indicating the presence of
reducing sugars.
BENEDICT’S TEST
3. IODINE TEST

Aim: To detect the presence of Starch.

Requirements:
Chemicals: Iodine solution, dil. HCl, distilled water.
Glassware: test tubes
Sample: Test sample (starch)

Principle: Iodine forms colored complexes with polysaccharides. Starch gives a blue color with
iodine. Glycogen forms wine red color upon reacting with Iodine.

Procedure:
1. Add 2 drops of Iodine solution to 1 ml of test solution and compare the color with the control
comprising of Distilled water.
2. Positive test is indicated by the formation of a bluish black color.

Result: Bluish black color is obtained on addition of Iodine indicating the presence of Starch.
IODINE TEST
Experiment No. 11
QUALITATIVE TESTS FOR PROTEINS

1. BIURET TEST -

Aim: To detect the presence of proteins in the sample by detecting the presence of peptide bonds

Requirements:
Chemicals: CuSO4, NaOH, distilled water.
Glassware: test tubes
Sample: Test sample

Principle: Proteins are made up of many amino acids linked to each other by peptide bonds.
Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give a violet
colored complex. In this reaction, the Cu+2 ions form a colored coordinate complex with the
nitrogen atoms of the peptide bond.

Procedure:
a) Add 2 drops of CuSO4 solution to 2ml of NAOH and 1 ml test solution.
b) Mix thoroughly and observe the color produced.
c) Use D/W for the control.
d) Positive test is indicated by the formation of violet color in the solution.

Result: Violet color is observed. Therefore proteins are present

BIURET TEST
2. NINHYDRIN TEST

Aim: To detect the presence of amino acids

Requirements:
Chemicals: Ninhydrin reagent, distilled water
Glassware: test tubes
Sample: Test sample

Principle: Ninhydrin is a powerful oxidizing agent. It reacts with the α-amino group of free
amino acids between pH 4 to 8 to give a purple colored complex. The amino group undergoes
oxidative deamination to liberate ammonia, CO2 and the reduced form of ninhydrin called
hydrindantin. The formed products further react with ninhydrin to produce a purple colored
complex. Amino acids like proline give a yellow colored reaction.

Procedure:
a) Take 1 ml of test solution in a test tube.
b) Add 5 drops of Ninhydrin solution and carefully boil for 2 minutes.
b) Use D/W for the control tube.
c) Positive test is indicated by the formation of a violet colored solution.

Result: Violet color is observed upon boiling indicating the presence of amino acids.
NINHYDRIN TEST
Experiment No. 12
QUALITATIVE TESTS FOR LIPIDS

1. SOLUBILITY TEST

Aim: To determine the solubility of lipids in different solvents.

Principle:
Lipids are a heterogeneous class of naturally occurring organic substances grouped together on
the basis of a physical property namely solubility and not the functional group. They are widely
distributed in both animal and plant systems and perform a wide variety of functions. These
include energy storage, structural components (e.g. cell membranes), vitamins, metabolism
regulators (e.g. steroid hormones), and emulsifying agents.

Lipids are all insoluble in polar solvents like water, but highly soluble in non-polar or weakly
polar organic solvents, including ether, chloroform, benzene, and acetone. Upon addition of
water, lipids form a milky white emulsion of fine droplets.

Lipids includes not only fats and oils, which are esters of the trihydroxy alcohol glycerol and
fatty acids, but also compounds derived from phosphoric acid, carbohydrates, amino alcohols
and steroids

Requirements:
Chemicals: alcohol, ether, distilled water
Glassware: test tubes
Sample: Test sample or oil

Procedure:
1. Place 1 ml of the test solution in 3 clean test tubes containing 2 ml of the solvents namely
water, alcohol, and ether.
2. Shake the tubes thoroughly, then leave the solution for about one minute
Result: Lipids are insoluble in water, forms an emulsion with alcohol and is soluble in ether
2. SAPONIFICATION AND SOAP FORMATION

Aim: To detect the presence of fatty acids.

Principle: Triglycerides (vegetable oils and animal fats) are greasy materials that are saponified.
These triglycerides, are mixtures derived from diverse fatty acids. Triglycerides can be converted
to soap by treating it with a strong base (e.g. lye), which cleaves the ester bond; releasing fatty
acid salts (soaps) and glycerol.

Requirements:
Chemicals: NaOH solution in alcohol (20% NaOH) Glassware: test tubes
Sample: Test sample or oil
Equipment: Boiling water bath

Procedure:
1. Place 2 ml of test solution in a large test tube
2. Add 4 ml of alcoholic sodium hydroxide.
3. Boil the solution for 3 minutes.
4. After this period, make sure the process of saponification has occurred by taking a drop of
the solution and mix it with water, (if the oil separates it indicates the non-completion of the
saponification process).
5. In this case, continue to boil until all the alcohol evaporates.
6. Take the remaining solid material (soap) and add about 30 ml of water.
7. Shake the solution after it cools and note the formation of thick foam.

Result: Precipitation of insoluble salts (soap) was observed and therefore fatty acids are present.
References:

1. Cappuccino, J.G. and Sherman, N. (2002) Microbiology: A Laboratory Manual. 6th

Edition, Pearson Education Inc.
2. Negative Staining- Principle, Reagents, Procedure and Result (microbiologyinfo.com)