Objective: To estimate the microbial count of air of different quality and source; i.e.

- Bathroom air and

laboratory air, two types of water; tap and drain water and soil sample.

Principle: The atmosphere contains all the major groups of microbes ranging from the algae to the viruses
and it is never pure. Outdoor air, especially with the changing season of the year contains a variety of pollen
grains, spores of fungi, algae, ferns, mosses, bacteria and viruses. Air is not a medium in which microorganism
can grow but is a carrier of particulate matter, dust and droplets, which may be laden with microbes. The
numbers and kinds of microorganisms present in the air are determined by the sources of contamination in the
environment, e.g. microorganisms are sprayed by coughing and sneezing from the human respiratory tract,
circulation of dust particles from the surface of earth by air curents. The bacterial count of air depends on
" Increased density of human and animal population
" Nature of soil
" Amount of vegetation.
" Atmospheric conditions such as humidity. temperature, wind, rainfall and sunlight.
The level of bacterial contamination is expressed as the number of bacteria carrying particles per meter cube
or per feet cube. Since an adult male inhales about 1Sm' and a baby about I m' of air in a day, the bacterial
content ofair is important particularly when the air contents have pathogens. Possibility of any person acquiring
infection will be more ifh is exposed to high concentration of air borne pathogens. Air contamination standards,
should not exceed the upper limit of:
" 10/ f in operation theaters
" 1/A in operation theaters for neurosurgery and burns
" 50/t in factories, offices and homes.
A bacteriological examination of air is required in surgical operation theaters, hospital wards, store houses or
premises of food particles or pharmaceutical preparations. As air cannot be diluted, so conventional plating
techniques cannot be applied here. Instead we use settle plate or sedimentation plate technique for enumeration
of bacterial count of air or estimation of air quality to verify with standard.
Water examination is needed since it is associated with all sorts of food processing and cleaning operations.
Water is one of the main components which is taken by every organisms. So, if water is polluted or mixed with
microorganisms, then it may cause severe water borne diseases. That is why all international and National
organizations and governments bave regulations for delivering safe water.
Soil being the natural layer on the carth cnust gets mixed with raw food and every other material. It can get
transported via air, water, humans, animals, or various other medium and deposits on the surface. Microbe
infested soil can produce different infections and also affect water surrounding or nearby.
Estimation of microbial quality of these three elements of nature belps us to understand how microorganism
are distributed in nature, their natural characteristics and habitat.

Materials required:
" Petri plates.
" Nutrient agar media.
"Test tubes

Instruments required:
" Incubator.
" Colony counter
 Instruments required:
" Incubator.
" Colony counter

Procedure:
For Air:

" Note the time at which experiment is starting.

" Take two sterile Petri plates with solidified agar media.
" Keep one Petri plate open in the lab air and other plate in bathroom air for 15-20 minutes for allowing
the particles to settle down in the solid agar media.
"Roaming in the assigned place is recommended for better estimation and study.
" After that, keep the petri plates inside the incubator for growth of microorganism for 24 hr at 37°C in
inverted position.
" Observe the plates after 24 h with the aid of colony counter.
For Water:
Collect one test tube containing tap water and another test tube containing drain water.
" For tap water take up to l0>dilution and drain water up to 104 dilution. So take three test-tubes for tap
water and four test tubes for drain water and label the test tubes10-, 102,10,10+ etc. by a marker pen.
" Take 9nml of distilled water in to a test-tube and add Iml of tap water sample in to that test tube by
pipetting to prepare 10! dilution.
" After that take 9ml of distilled water in to another test tube and add Iml of 10-! dilution sample by pipetting
in to that test tube to prepare 10 dilution.
" Similar to 10-'and10- dilution prepare up to 10- dilution for tap water and10+dilution for drain water.
Use 10- dilution and 10+ dilution to culture bacteria from tap water and drain water respectively for pour
plate technique and spread plate technique.
"For performing spread plate technique, take solidified agar media in a Petri plate and take 0.1 ml each of
10'and 10+ dilution of the two water sample.
" Add it into two Petri plates and spread it on the media by means of an L-shaped spreader.
"After incubation observe for colonies after 24 h
For Soil:
"Take 1 gm soil sample and serially dilute it in 0.1% sterile peptone water.
" Plate the aliquots (0.1 ml) Nutrient Agar medium.
"The culture must be spread properly with the heip of a spreader in the media surface.
" Incubate the plates at 37°C for 24 h.
" After incubation observe for colonies after 24 h.

Observation:

Result and Discussions:

Name of the Experiment: Preparation and inoculation of synthetic medium for yeast and mould and their
identification.

Objective: To prepare Potato dextrose agar and to inoculate it with standard strains of yeast and mold and
identify colony characteristics.

Principle: Yeast is a single cell organism, which is usually a thread and filamentous in shape, appear white
or colorless and divide asexually. On the other hand, molds are multi-cellular, appearing round or oval shape
and appear in various color; they divide sexually or asexually. There are about 1500 types of yeast and 400,000
types of molds present. Yeasts are used in industry for baking purposes, as additive, in alcohol production or
making food beverages. It is very common and can be found in fruits, vegetables, on the skin of mamnmals and
among other places. Molds are used in industry especially for cheese making and also in some other processes.
Though mnolds can be sometimes hazardous and causes respiratory infections and allergies. They are usually
found in damp. dark or steam-filled areas. Both Yeast and Molds are eukaryotic microorganisms, and belongs
to the kingdom fungus. In spite of being in the same kingdom, both differ in function, appearance, colour, and
mode of reproduction.

Potato Dextrose Agar (PDA) is used for the cultivation of fungi. It is a general pupose medium for ycasts and
molds that can be supplemented with acid or antibiotics to inhibit bacterial growth. It is recommended for plate
count methods for foods, dairy products and testing cosmetics. PDA can be used for growing clinically
significant yeast and molds. The nutritionally rich base (potato infusion) encourages mold sporulation and
pigment production. Composition of PDA can be tabulated as follows:

Potato infusion 200 gm

Dextrose 20 gm

Agar 20 gm
Distilled water | liter

Fiual pH 5.6 + 0.2.

It is to be noted that, 200 gm of potato infusion is equivalent to 4.0 gm of potato extract.

Materials required:
" Sterile petri plates
" Potatoces
" Dextrose
" Agar
" Distilled water
" 0.1 (N) Hydrochloric acid
" 0.1 (N) sodium bydroxide
" Peptone
" Test tubes
" Micropipette
"Spreader
" Sachharomces cerevisiae
" Aspergillus oryzae
Instruments required:
" Weighing balance
" Heater
" Autoclave

" Colony Counter

" Laminar air flow chamber
" Incubator

Procedure:
" Prepare potato infusion by boiling 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min.
" Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated
form).
" Mix with Dextrose, Agar and Water and boil to dissolve.
" Adjust the pH using acid or base.
" Autoclave for 15 min at 121°C.

" Dispense 20-25 ml portions into sterile petri plates and allow for solidification.
" Prepare serial dilution of the pure cultures of Sachharomyces cerevisiae and Aspergillus oryzae by
taking 0.1 % sterile peptone water as the diluents.
" Dispense 0.1 ml of both the cultures in to respective petri plates and spread them thoroughly using a
spreader.
"Keep the plates inside the incubator at 25-30°C for 48 to 72 hrs.
" After the incubation period, observe the colony characteristics.

Observation:

Result and Discussions:

Name of the Experiment: Study of autoclave, Preparation and sterilization of nutrient
broth and agar.

Objective: To prepare both solid and liquid nutrient media and sterilize by operating
Autoclave and then comparing it with a non sterile media.

Principle: The autoclave is an apparatus in which saturated steam under pressure affects
sterilization. Sterilization can thus be achieved at temperatures considerably above the boiling
point of water; laboratory autoclaves are commonly operated at steam pressure of 1Slb/in sq,
which is above atmospheric pressure and corresponds to a temperature of 121°C.
The autoclave is a double walled, cylindrical metal vessel made of stainless steel, one end of
which is open to receive the materials to be sterilized. The base of the inner cylinder is
concave, which holds water that can be heated by an in built immersion heater. The lid is
provided with pressure gauze, a safety valve, a steam- outlet and metal handles with screws.
Inside the vessel cavity, there is a wire basket with triple stand where the materials are
packed using autoclave bags before the start of sterilization. All articles that have to be
sterilized, needs to be cotton plugged well, covered with newspapers and tied properly.
In preparing a culture media for any microbial growth our goal is to provide a balanced
mixture of the required nutrients, at concentrations that will permit good growth. No
ingredient should be given in excess because many nutrients may become growth inhibitory
or toxic as their concentration is raised. A medium composed entirely of chemically defined
nutrients is termed as synthetic medium. One that contains ingredients of unknown chemical
composition is termed a complex medium. For different purposes in laboratory either solid or
liquid mnedia is used.

Composition of liquid or broth media:

Nutrient broth: Nutrient broth is one of the basic media used for the study of different types
of microbes. It is one of the most important liquid media used for bacteriological growth
purposes. It usually contains
Beef extract - 3 gm. Distilled water - 1000ml.
Peptone -5 gm. pH -6.8 -7.0
Sodium chloride 5gm.

Composition of solid or agar medium:

Nutrient agar: Nutrient agar is an important medium for bacteriological growth purposes. It is
simply nutrient broth solidified by the addition of agar. Due to its solid consistency, this
medium serves as a good media for the culture of bacteria on a solid surface. It usually
contains
Beef extract - 3gm. Peptone -5gm.
 Sodium chloride -5gm Distilled water -1000ml.
Agar - 20 gm. pH-6.8 to 7.0

Materials required:
Conical Flask Distilled water
Cotton
Measuring cylinder.
Glass beaker pH paper
Beef extract Paper
Rubber band/ thread
Peptone
Sodium chloride Autoclave bag
Agar -agar
Instruments required:
Digital weighing balance. Autoclave

Procedure:
To make 1000ml nutrient broth, weigh 3g of beef extract, 5 g of Sodium chloride and
5gms of peptone and transfer to a conical flask.
Add 1000ml of distilled water mix the ingredients thoroughly.
Adjust the pH by adding a little acid or alkali (if required).
Plug the flask with cotton wool and paper.
To make 1000ml nutrient Agar, all the steps are same as nutrient broth along with 20
g of agar-agar which must be added separately after heating and mixing in a separate
beaker.
Keep the cotton plugged conical flasks inside an autoclave bag for sterilization in
autoclave.
Put the autoclave bag inside after checking the water level inside the autoclave.
Close the lid by tightening the screws and steam valve is kept open at first.
Allow all air inside to be replaced by steam which can be indicated by release of
steam from the steam valve.
Close the valve and allow the pressure inside to rise which is indicated at the pressure
gauge reading.
Keep the autoclave for 15 minutes time once the pressure reaches 15 psi inside having
corresponding temperature at 121° C.
Switch off the autoclave after this and wait for it to cool down.
Open the steam valve to release the steam inside.
Take out all the sterilized articles after the autoclaving operation is over.

Observation:
Result and Discussions:
.End
Name of the Experiment: Dilution and Plating by spread plate, streak plate and pour plate techniques.

Objective: To dilute sample by serial dilution method and plate microorganisms by spread plate and pour
plate techniques using different water samples.

Principle: There is enormous Dumber of microorganisms present in water which is impossible to calculate
in their natural state. So to calculate them, different plating techniques like pour plate technique and spread
plate techniques have been suggested. These techniques usually give an estimation of the total number of
microorganisms present in a very minute volume of the sample and are not exact. The plate count technique is
based on the principal that when sample containing bacteria is cultured, every bacterium develops in to a visible
colony on a nutricnt agar media.
Serial dilution is the process of stepwise dilution of the sample. For a logarithmic ten-fold serial dilution on a
Iml scale, test tubes are filled with 900 microlitres of diluents and 100 microlitres of sanple. It is then
repeatedly serially transferred in the same fashion where each time the original sample gets diluted ten times.
The use of this technique is to reduce the concentration of microorganisms in a sample so that they can be
casily counted on a cultured petri plate.

Sugenon

Figure I: Serial dilution technique Figure 2: Spread plate and pour plate techniques

Spread plate technique is the method of addition of sample to growth media where a very small volume of
sample (0. Il to I ml) is injected on to the surface of previously solidified agar plates. The sample is then spread
over using an L-shaped rod called spreader uniformly on the agar surface. Mainly aerobic or surface growth
of colonies is seen in this type of plating technique.
Streak plate techniq1ue is the method of addition of sample in solidified agar media by using a loopful of culture
with a nichrome loop. The nichrome loop can be moved in a zigzag or parallel fashion to get streaked culture
onto the surface of media. Mainly surface organisms can grow by this technique, bhowever colony counting
cannot be done by this method.
Pour Plate technique is the method of addition of sample into molten agar media kept at 45°C to allow thorough
distribution of inoculums. The inoculated media is the dispersed into the petri plates and allowed to solidify
resulting in surface or subsurface growth of colonies. Almost all types of organisms can grow by this technique.
All these techniques are employed for the bacterial cell enumeration, separation of a dilute, mixed population
of microorganisms: so that individual colonies can be isolated. The colonies can be counted and estimated
based on the formula of colony forming unit which is given as
Colony Forning Unit, CFU = (no. of colony/amount plated)*dilutlon factor
Materials
required:
. Water sample (tap ldrain watcr)
"Diuent ( water or 0.1% peptone water)
Nutrient agar media
" Measuring cylinder
Petri plates
" Pipette
Micro pipettes
Spirit lamp
 Spreader
Test tubes

Instruments required:
"Autoclave
Laminar air flow chamber
BOD Incubator
Colony counter

Procedure:
" All the techniques are done inside the laminar air flow chamber with a spirit lamp and the blower on.
" Sterilize the chamber previously by switching on the ultraviolet light for 10-15 minutes and then wiping
it with ethyl alcohol.
"Collect one test tube containing tap water and another test tube containing drain water.
"For tap water take up to 10- dilution and drain water up to l04 dilution. So take three test-tubes for tap
water and four test tubes for drain water and label the test tubes10-',10-2,10-3,104 etc. by a marker pen.
" Take 9ml of distilled water in to a test-tube and add Iml of tap water sample in to that test tube by
pipetting to prepare 10-! dilution.
" After that take 9ml of distilledwater in to another test tube and add Iml of 10! dilution sample by pipetting
in to that test tube to prepare 10: dilution.
" Similar to 10'and10² dilution prepare up to 10 dilution for tap water and10dilution for drain water.
"Use 10 dilution and 10+ dilution to culture bacteria from tap water and drain water respectively for pour
plate technique and spread plate technique.
"For performing spread plate technique, take solidified agar media in a Petri plate and take 0.1 ml each of
10'and 10+ dilution of the two water sample.
" Add it into two Petri plates and spread it on the media by means of an L-shaped spreader.
" Keep the petri plates inside the incubator for incubation or growth of microorganism for 24 h at 37°C.
"For performing streak plate technique, take a loopful of culture into the nichrome loop and streak onto the
surface of agar in a zigzag motion.
" Keep the petri plates inside the incubator for incubation or growth of microorganism for 24 h at 37°C in
the line of streaking.
" For performing pour plate technique, take molten agar media in a Petri plate and talke l ml cach of 103
and 104 dilution of the two water sample.
" Rotate the plate in clock wise and anti-clock wise direction to spread the sample all over the Petri plate in
uniform manner.
" Then allow some time for solidification.
" After solidification keep it inside an incubator for growth of microorganism for 24 h at 370C.
" Count and observe all plates after 24 hrs.

Observation:

Result and Discussions:

..End
Name of the Experiment: Study of a compound microscope.
Objective: To study the functions of different parts of a microscope and observe certain
morphological features of micrOorganisms.

Principle: A microscope is an instrument which gives a magnified image of the object and
is used to examine minute objects that are not visible through naked eyes using the principle
of reflection of light. Effective magnification and resolving power of any microscope will
depend the ability to distinguish between two adjacent objects as separate and distinct
images, rather than as a single blurred image. The total magnification of the microscope is
calculated by multiplying the magnification of the objective lens by the magnification of the
Ocular lens.

Introduction to different parts: Any simple microscope consists of a single lens

system while compound microscope consists of two or more lens system. Depending upon
the source of illumination the microscope can be classified as:
Light microscope.
Electron microscope.
In light microscope, the specimen is illuminated by visible light is under the course of our
study. They include bright field, dark field, phase contrast and fluorescent micrOscopes.
A typical bright -field microscope consists of the following basic parts:
A. Mechanical Parts
The mechanical parts of a compound microscope includes
) Stand- It is the main supporting part of the instrument and consists of Base, Pillar
and Arm.
The base is the V or U shaped, solid, metallic part with which the
instrument is placed on the table.
The Pillar is the short vertical continuation of base on which the remaining
part rests.
The Arm is a curved part, the lower end of which is attached to the upper
end of the pillar with the help of an inclination screw.
ii) Body tube and Draw ube- The Body tube is a wide cylindrical part attached
vertically to the upper end of the arm that holds the nose piece. Another narrow
tube, called Draw tube holds the eye piece.
Nose Piece- It is the revolving disc like part attached to the lower end of the Body
tube; the objective lenses remain mounted on it.
iv) Stage- It is the rectangular platform attached to the lower end of the arm and
positioned above the pillar. The slide that is to be examined is placed on it. The

stage has a central hole for passage of light and a pair of simple side clips or
mechanical stage clips for holding the slide. With the help of the clips, the slide
can be moved smoothly by using stage knobs.
V) Adjustment Screws- Two paired adjustment screws are present on the upper part
of the arm, one of which is larger called Coarse Adjustment Screw and the other is
smaller called the Fine adjustment Screw. These can move either the Body tube of
the stage to adjust the distance between the object and the objective lens.
B. Optical Parts
The Optical part consists of
i) Mirror- A compound microscope contains a plano-concave mirror attached to the
pillar. The mirror can be rotated in any direction to reflect light to the condenser.
The Plano- concave mirror is used when the object is examined under low power
objective and the concave mirror is used for examining the object in high power
objective.
ii) Sub-stage Condenser- It is the lens system provided with a diaphragm which is
placed either within or beneath the stage. The Condenser lens helps to converge a
cone of light on the object present on the slide. The diaphragm, which is either
movable or fixed, is used to control the amount of light incident on the condenser
lens.
ii) Objective- it contains two or three objective lenses of different magnifying power,
mounted on nose piece. These are Low power lens (10X), High power lens (40X)
and Oil immersion lens (100X).
iv) Eye Piece or Ocular lens- It is the short, hollow cylinder having two lenses
mounted on its either end. In an advanced type of compound microscope called
Binocular microscope, there are two Eye Pieces.

Ocular lenses

revolving nosepiece carrying hande

objectve lens

slide hokder

stage
coarse adustment
condenser aperture lever
condenser

oht -fino adjustment

ight intensity control

Differentparts of a compound Microscope

5
Name of the Experiment: Gram Staining and Study of morphology of bacterial cells.

Objective: To study the morphological structure of some microorganisms in a given

sample and learn to differentiate Gram Positive and Gram negative Bacteria under
microscopic observation.

Principle: Microorganisms are semitransparent and difficult to see in the unstained state.
Stains are used - (i) to render microscopic and semitransparent objects visible (i) to reveal
their shape and size (iii) to show the presence of various internal and external structures and
(iv) to produce specific physical & chemical reactions.
Staining is a procedure increasing visibility and to reveal additional information for
identifying microbes to differentiate them into specific groups or genera or species. The terms
stains and dyes are generally used interchangeably by biologists; but they are not the same. A
coloring agent for general purpose use is called a dye whereas the coloring agent used for
biological operations is called a stain. One of the most important and widely used differential
staining techniques in microbiology is Gram staining. This technique was introduced by
Christian Gram in 1884. This staining focuses on differentiating two different groups of
Bacteria- Gram positive and Gram negative.
In Gram staining process, two different stains- crystal violet and safranin are used. Gram
positive bacteria can retain the crystal violet stain and hence appear deep violet
whereas
Gram negative bacteria loses the crystal violet stain and instead take the counter stain
safranin which makes them red to purple in appearance. The differences in staining responses
are relatedto the chemical and physical differences in their cell wall. The Gram Negative
Bacteria have higher lipid content and lower protein content than Gram Positive
Bacteria
which do not allow them to retain the primary crystal violet stain and thus they take
the
counter stain Safranin and hence its color. On the other hand, the Gram Positive
Bacteria can
retain the crystal violet stain as CVIcomplex within their cell wall and thus they take the
violet color of the Crystal violet stain.

Materials required:
24 hr culture.
Staining tray.
Gram staining reagents. Droppers.
1. Crystal violet ( primary stain)
2. Gram's lodine solution (mordant)
Inoculating needle or loop.
Glass slides.
3. 95%Alcohol (decolorizing agent)
4. Safranin (counter stain)
Blotting paper.
Bunsen burner or spirit lamp.

6
Instruments required:
Compound Microscope.

Procedure:
glass slides and make them grease free using cotton dipped in alcohol.
Clean
using inoculation loop.
Make thin smears of microorganism on glass slide
Let the smear dry in air.
spirit lamp.
Heat-fix the smear by moving them over the flame of
and keep it for 30-60 seconds.
Cover each smear drop wise with crystal violet
excess stain.
Wash each slide with water for a few seconds to remove
solution and keep it for 30-60
Cover each smear drop wise with Gram's lodine
seconds.
no more
Wash off the lodine solution with 95% Ethyl alcohol drop by drop until
color flows from the smear.
Wash the slide with water.
Apply safranin drop wise to smear and keep it for 30-60 seconds (counter staining).
Wash with water and blot dry with absorbent paper.
Observe the slide in oil under oil immersion lens of a compound microscope.

Observation:

Result and Discussions:

.End