Expt.

Date Title of the experiment Page Teacher’s

no.
No. initials
1 Microbiology Good Laboratory Practices and Biosafety

2 Using permanent slides, study ofmorphological

characteristics of
a. protozoans
b. fungi
c. algae
3 Monochrome staining
4 Negative staining
5 Gram’s staining
6 Lactophenol-cotton blue staining
7 Staining of intracellular structure: endospore

8 Staining of intracellular structure: metachromatic

granules
9 Preparation of culture media for bacterial
cultivation; synthetic media,complex media,
Nutrient agar, MacConkey agar
10 Isolation of pure cultures of bacteria by streaking
method
11 Determination of viable count by spread plate method

12 Determination of viable count by pour plate method

13 Sterilization using physical methods: dry heat (hot air

oven), moist heat (autoclaving)
14 Testing the efficacy of sterilization
using chemical methods: Determination of phenol
coefficient
15 Study of the structure of cell organelles
through electron micrographs
16 Preservation of cultures by periodic
transfer and overlaying with mineral oil
Experiment No. 1 Date:

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 Date:
STUDY OF MORPHOLOGICAL CHARACTERISTICS OF PROTOZOANS, FUNGI,
AND ALGAE USING PERMANENT SLIDES

Aim: To study the morphology characteristics of different protozoans, fungi and algae.

Requirements: Permanent slides of various protozoan, fungi and algae.

Introduction:
A. PROTOZOA
Protozoa are eukaryotic, unicellular organisms which are larger than bacteria and hence their
internal structures can be easily observed under high power lens. They require moisture for
survival as they are prone to desiccation. They inhabit marine and freshwater environments and
are also found in soil and decaying organic matter. Protozoa derive their nutrition either by
photosynthesis, phagocytosis or by saprozoic mode wherein soluble nutrients are taken into the
cell by endocytsis, diffusion or active transport.

The protozoan cell consists of cytoplasm differentiated into gel like ectoplasm and fluid
endoplasm. Other structures include one or more nuclei, ribosomes, Golgi apparatus,mitochondria,
kinetosomes, vacuoles (contractile, secretory and food). Many protists undergo encystation during
their life cycle by forming resistant cysts. Upon return of favourable conditions, the cells undergo
excystation.

Reproduction in protozoa is mostly asexual by binary fission. Reproduction by budding and

fragmentation is also seen.

Amoeba: Amoeba has a single nucleus and free form of cytoplasm. They project their cytoplasm
into temporary protuberances called pseudopodia. They ingest food by phagocytosis and
reproduce mainly by binary fission.

Paramoecium: Paramoecium has a characteristic slipper shape. It possesses hair-like cilia which
aid in locomotion and feeding. It has two nuclei: a large macronucleus and a smaller micronucleus.
Paramoecium reproduces asexually by transverse binary fission and sexually by conjugation.

Euglena: Euglena is an elongated single celled organism covered by a protein pellicle. The
flagellum helps in locomotion. It has chloroplast and a red eyespot for photosynthesis.
Reproduction is by binary fission.
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STUDY OF PROTOZOA

AMOEBA PARAMOECIUM

EUGLENA
B. FUNGI
Structure of fungi: The main body of most fungi is made up of fine, branching, usually colourless
threads called hyphae. Each fungus will have vast numbers of these hyphae, all intertwining to
make up a tangled web called the mycelium.

Mucor
These are saprophytic fungi and grow on dead organic material. The colony of Mucor shows rapid
growth and the colour of the colony is usually white to grey and turns to brown when the culture
becomes old. Hyphae of Mucor is filamentous, aseptate or coenocytic. Sporangiophore are aerial
hyphae which originates vertically out from the prostrate hyphae. Sporangiophore swells up to
form a dome-like structure called “Columella” which can vary in both shape and size. Sporangium
is the round and thick outer covering which carries numerous spores inside it;it can be globose
to spherical. Spores are the reproductive structures forms within the sporangium which are simple,
flattened and variable in shape and size. The reproduction takes place by means of asexual and
sexual methods. It is called black mould because it producesblack spores.

Aspergillus
It is saprophytic fungus, which grows on decaying vegetables , butter, ghee, bread, rice, jams and
jellies. Aspergillus also refers to black or green mould because the majority of them form black
and green spores that grow on the substratum gives a mouldy appearance. The mycelium made
up of loosely internetwork mass of branched, septate hyphae which are divided into segments.
Each segment contains one or more nuclei. Most of the Aspergillus species reproduce asexually
by the means of conidiospores and few grow sexually by the means of ascospores. Asexual
reproduction takes place by formation of conidia or conidiospores. The long and erect
conidiophores arises from thick walled foot cells. It forms a terminal swollen vesicle on which
bottle shaped structures called sterigmata are formed all over the surfaces. Conidia are produced
in acropetal succession from the sterigmata. The conidia are circular, multinucleate and have a
rough and thick wall. Sexual reproduction takes place by formation of antheridia and ascogonium.
They are produced on the same somatic hyphae. Both are multinucleate elongated structures which
coil around each other

Penicillium
Commonly known as green or blue mould and grows as a saprophyte on decaying fruits and
vegetables. The mycelium is profusely branched with septate hyphae, composed of thin-walled
cells containing one to many nuclei. Each septum has a central pore, through which cytoplasmic
continuity is maintained. Reproduction takes place both asexually and sexually. Asexual
reproduction takes place by vegetative method and sporulation. Vegetative method takes place
by fragmentation; each fragment then grows individually like the mother mycelium. Sporulation
takes place by formation of non-motile asexual spores called the conidia produced exogenously
at the long erect, special, septate hyphae called conidiophores. The conidiophore develops as an
erect branch from any cell of the vegetative mycelium. The conidiophore may be unbranched or
becomes variously branched. The branch of the conidiophore further becomes branched to form
metulae. A number of flask-shaped phialid or sterigmata develops at the tip of each metulae.
Conidia are borne in long unbranched chains from the tip of the phialid or sterigmata. Sexual
reproduction is by antheridia and ascogonia.
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STUDY OF FUNGI
C. ALGAE

Nostoc
Nostoc; genus of blue green algae with cells arranged in bead-like chains that are grouped together
in a gelatinous mass. They range from microscopic to walnut sized masses. Nostoc maybe found
in soil and floating in quiet water. Reproduction is by fragmentation. A special thick- walled cell
(akinete) has the ability to withstand desiccation for long periods of time. After 70 years of dry
storage, the akinete of one species germinates into a filament when moistened. Like most blue
green algae , nostoc contains 2 pigments, blue phycocyanin and red phycoerythrin, as well as
chlorophyll, and has the ability to fix nitrogen in specialized cells called heterocysts. They lack
flagella. They use gas vesicles to move in the water. Many filamentous species show gliding
movements. Some filaments shift their position laterally in water.

Vegetative structures may be unicellular or form colonies of different shapes or form filaments.
The filament is composed of trichomes. Trichome is a chain of cells. Each filament is surrounded
by mucilaginous sheath. A trichome with its enclosing sheath is called filament which may be
branched or unbranched. The cell is surrounded by a cell wall which is composed of cellulose
and pectic substances. Mucilage from sheath around the cell sheath increases the water holding
capacity of cells. The colour of the sheath protects the cell from strong light. The protoplast is
divided into 2 parts; centroplasm (central colorless region) and chromoplasm (outer blue green
pigmented region).

Oscillatoria
Oscillatoria, genus of blue-green algae, common in freshwater environments including hotsprings.
This unbranched, filamentous algae, occurring singly or in tangled mats, derives itsname from
its slow, rhythmic oscillating motion, which is thought to result from a secretion of mucilage that
pushes the filament away from the direction of excretion. Reproduction is by fragmentation in
which dead concave cells (separation disks) separate sections of the filament (hormogonia) when
present, the mucilage sheath is very thin.

Vegetative structure
Its body is composed of a single row of cells. These cells form trichomes. Its trichomes are
unbranched filaments. They are covered by very thin mucilaginous sheath. All cells of a trichome
are similar in shape except apical cells. The apical cells are convex at the tip. All other cells are
broader and cylindrical. In some species, the apical cells may end in subacute point. In some cases
it may have cap or calyptras at the tip. Some species have narrow trichome. They have cylindrical
cells with their length equal or greater than the breadth.

Cell structure
Each cell has an outer cell wall. This wall consists of 3 layers. The inner layer is a thin cellular
layer, the middle is a pectic layer and outer is a mucilage layer. Protoplasm is composed of 2 parts.
The peripheral part is called chromoplasm. It contains pigments. Hence it is colored. The central
part of the protoplasm is colorless. It contains nucleus like material called central body orchromatin
granules.
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STUDY OF ALGAE
Experiment No. 03 Date:

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.

Procedure:
1. Clean the slides with soap powder, wash with tap water and dry.
2. With marking pencil, mark an area on the reverse of the slide.
3. Sterilize the nichrome loop by passing it through a flame. Allow it to cool.
4. Place a loopful of the suspension on the glass slide inside the marked area and spread into a
thin, even film.
5. Allow it to dry in air.
6. When the smear is dry, heat fix it by passing the slide quickly through a flame 2-3 times, and
then cool the slide.
7. 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.
a. Crystal violet-30 seconds to 1 minute
b. Basic fuchsin-2-3 minutes
c. Loeffler’s methylene blue-2-3 minutes
d. Safranin - 1 minute.
8. Gently wash the slide using an indirect stream of tap water.
9. Using a filter paper, blot dry (without rubbing) the smear.
10. Examine the smear under oil immersion lens.

Results:
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Staining with Crystal Violet Staining with Safranin
Experiment No. 04 Date:

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

Procedure:
1. Mix a drop of Nigrosine stain on a slide with a loopful of the bacterial suspension.
2. Using another slide, make a thin film.
3. Allow to air dry and observe under oil immersion lens.

Results:
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Dark background
Experiment No. 05 Date:

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 washedaway. 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:
1. Using a nichrome loop make an even smear of the culture suspension on a clear grease-free
slide
2. Allow the smear to dry and heat fix it (pass through a flame 2-3 times)
3. Flood the smear with crystal violet and keep for 1 minute
4. Discard the stain and gently wash with tap water
5. Flood the smear with Gram’s Iodine and keep for 2 minutes and then discard the excess
6. Treat with alcohol for 3 minutes till decolorized
7. Wash the slide with water
8. Stain the smear with safranin and keep for 1 minute
9. Discard the stain and gently wash with tap water
10. Air dry and observe under oil immersion lens

Results:
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Experiment No. 06 Date:

LACTOPHENOL COTTON BLUE STAINING

Aim: To determine the morphology of the given fungi by lactophenol cotton blue staining.

Principle: Microscopic morphology of the filamentous fungus is critical in the identification of

molds since biochemical and molecular methods are not readily available for the identification of
molds. Lactophenol cotton blue is a stain that is used to examine fungal elements following either
a tape preparation or a scraping. This stain contains phenol, which will kill the organisms, lactic
acid which preserves fungal structures, and cotton blue which stains the chitin found in
the fungal cell walls.

Requirements:
Culture: Fungal cultures on plates
Labware: Glass slides
Forceps
Glass marking pencil / marker
Equipment: Microscopes
Stains: Lactophenol cotton blue stain

Procedure:
1. Place a drop of lactophenol cotton blue onto a clean slide.
2. Pick a part of fungal colony from an agar medium and place in the drop lactophenol blue dye
3. Tease the mycelia with a sterile needle
4. Place a coverslip of the fungal preparation
5. Dab the excess stain
5. Observe under a light microscope at 45x magnification

Results:
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Aspergillus Penicillium Mucor

Experiment No. 07 Date:

STAINING OF INTRACELLULAR STRUCTURE: ENDOSPORE

Aim: To stain bacterial spores by Schaeffer and Fulton’s method.

Principle: In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the
spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity
for cellular material, so vegetative cells may be decolorized with water. Safranin is then applied to
counterstain any cells which have been decolorized. At the end of the staining process,
vegetative cells will be pink, and endospores will be dark green.

Requirements:
Culture: Bacterial suspension
Labware: Glass slides, Nichrome loop, Glass marking pencil / marker, Immersion Oil
Equipment: Microscopes
Stains: Malachite green, Safranine.

Procedure:
1. Using a nichrome loop make an even smear of the culture suspension on a clear grease-
free slide
2. Allow the smear to dry and heat fix it (pass through a flame 2-3 times)
3. Flood the smear with malachite green stain for 10-15 minutes.
4. Heat the slide from below until steam arises.
5. Discard the stain and wash the slide using an indirect stream of tap water.
6. Counterstain the smear using safranine for 1-2 mins.
7. Gently wash the slide using an indirect stream of tap water.
8. Using a filter paper, blot dry (without rubbing) the smear.
9. Examine the smear under oil immersion lens.
Results:
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Experiment No. 08 Date:

STAINING OF INTRACELLULAR STRUCTURE: METACHROMATIC GRANULES

Aim: To stain metachromatic granules by Alberts staining.

Principle: Alberts staining technique aims at detecting the presence of metachromatic granulated
bodies of Corynebacterium diphtheriae. Metachromatic granules which are intracellular inclusion
bodies found in the cytoplasmic membrane of some bacterial cells for storage of complexed
inorganic phosphate and enzymes. The granules are called so because of the property of
metachromasia wherein the granules appear in a color different from that used for staining; the
granules are also called volutin bodies. The bacterium produces the granules in abundance when
grown in nutrient rich media such as Loefflers serum slope. The granules stain reddish-brown
against the light green counter-stained cytoplasm.

Requirements:
Culture: Bacterial suspension
Labware: Glass slides, Nichrome loop, Glass marking pencil / marker, Immersion Oil
Equipment: Microscopes
Stains: Alberts Staining Solution I (Toluidine blue -0.15 g, Malachite green -0.2g, Glacial acetic
acid -1ml, 95% ethyl alcohol-2ml, Distilled water-100ml), Alberts Staining Solution II (Iodine -
2g, KI-3g, Distilled water-300ml)

Procedure:
1. Using a nichrome loop make an even smear of the culture suspension on a clear grease-
free slide
2. Allow the smear to dry and heat fix it (pass through a flame 2-3 times)
3. Flood the smear with Staining solution I and keep for 5-7 minutes.
4. Discard the excess stain but do not wash it
5. Flood the smear with Staining Solution II and keep for 2-3 mins
 6. Gently wash the slide using an indirect stream of tap water.
7. Using a filter paper, blot dry (without rubbing) the smear.
8. Examine the smear under oil immersion lens.

Results:
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Experiment No. 09 Date:

PREPARATION OF CULTURE MEDIA FOR BACTERIAL CULTIVATION

Aim: To prepare routine laboratory media for bacterial cultivation: synthetic media, complex
media, Nutrient agar, MacConkey agar

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. Synthetic Medium: M9 Medium

Part A Part B Part C Part D

Anhydrous Na2HPO4 0.6 g Glucose 2g MgSO 4 0.1203 g CaCl2 0.0111 g
Anhydrous KH2PO4 0.3g D/W 10ml D/W 10ml D/W 10ml
NaCl 0.05g
NH4Cl 0.1g 2ml 1 ml 1 ml
Agar 1.5g
D/W 100ml
Weigh the ingredients as given in the part A (except agar) and dissolve them in 100 mL of distilled
water. Adjust the pH to and then add the agar powder. To this add 2ml of Part B, 1ml each of
Part C and D. Plug the flask and sterilize the flask in an autoclave at 15 psi for 20 minutes.
B. Complex Medium: Sabouraud’s Agar
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. Adjust the pH to 5.5 and then add the agar powder. The medium is sterilized for
three consecutive days by autoclaving at 10 psi for 10 minutes.

C. Nutrient Agar
Peptone -1g
NaCl - 0.5 g
Meat Extract - 0.3 g
Distilled water - 100 mL
pH - 7.4
Agar -2g
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 / HCl dropwise if required. The final pH of the medium is checked
using pH paper / probe. To this, add 2 g of agar powder. Plug the flask and sterilize the flask in an
autoclave at 15 psi for 20 minutes.

D. MacConkey’s agar
Composition:
Part A Part B
Peptone - 1.7 g Lactose -1g
Proteose peptone - 0.3 g Distilled water - 10 mL
Bile salts - 0.15 g
Sodium chloride - 0.5 g
Distilled water - 90 mL
 pH - 7.4
Agar- 1.5 g

Part A: Dissolve ingredients in 90ml, distilled water, adjust the pH of the medium to 7.4,
and Part B, as above.
Neutral red - 0.003 g
Crystal violet - 0.0001 g
Agar - 1.5 g Distilled water - 100 mL

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.. Now add
the required quantity of neutral red indicator. If MacConkey’s agar is being prepared, add
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.

Results: No growth was observed in the sterilized media as preparation was done aseptically.
Experiment No. 10 Date:

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

Procedure:
1. Divide the agar plate into three distinct zones: a small inoculation zone, a sterile zone and a
larger isolation zone
2. Flame the loop
3. Inoculate a loopful of culture in the inoculation zone.
4. Flame the loop and cool it
5. 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.
6. Flame the loop, between streaks, and cool it
7. Incubate the plate upside down for the appearance of isolated colonies

Results:
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Experiment No. 11 Date:
DETERMINATION OF VIABLE COUNT METHOD BY SPREAD PLATE METHOD

Aim: To enumerate the bacteria in the given suspension using 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)
Glass spreader
Media: Sterile nutrient agar plates.
Chemicals: sterile saline

Procedure:
1. Weigh out 1 g of the sample (food, soil etc.) into a large test tube and add 10 ml of sterile
saline to it.
2. Shake the tube thoroughly; allow it to stand until the heavier particles settle down.
3. Transfer 1ml of supernatant to the second tube containing 9 ml of sterile saline (10-2 dilution).
Mix the contents well and similarly prepare further dilutions up to 10-4. In case of bacterial
culture take 1 ml of it and mix with 9 ml of sterile saline aseptically (10-1 dilution) and proceed
to prepare further dilutions.
4. Transfer 0.1 ml of each of 10-2, 10-3 and 10-4 dilutions to the surface of a sterile agar plate with
a sterile pipette
5. Use as sterile spreader to spread the suspension on the medium.
6. Invert and incubate the plates for 24-48 hours at room temperature.
7. Count the colonies

Result: The viable count of the given sample (E. coli) by spread plate method was found to be
cfu/ml.
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Experiment No. 12 Date:
DETERMINATION OF VIABLE COUNT METHOD BY POUR PLATE METHOD

Aim: To enumerate the bacteria in the given suspension using pour plating 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 butts (10 ml)
Chemicals: sterile saline

Procedure:
1. Prepare the dilutions of the given culture / sample
2. Transfer 1 ml of 10-6 dilution to sterile molten agar butt, cooled to 45⁰ C.
3. Mix thoroughly by rotation
4. Pour the inoculated molten agar in a petridish.
5. Use the pipette to transfer similar amounts from 10-5 and 10-4 dilution to separate sterile
molten agar butts and pour in sterile petridish
6. Allow the plates to set and incubate for 24-48 hours at room temperature.

Result: The viable count of the given sample (E. coli) by pour plate method was found to be
cfu/ml.
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Calculations
Experiment No. 13 Date:
STERILIZATION USING PHYSICAL METHODS: DRY HEAT; MOIST HEAT

Aim: To carry out sterilization using dry heat (Hot air oven) and moist heat (autoclave)
Introduction: Sterilization is the removal of all forms of microorganisms from the surface of an object.
It includes both spore and vegetative forms. Sterilization is carried out by various physical and chemical
methods such that it eliminates around 106 log colony-forming units.

Sterilization is done to avoid the growth of microorganisms which may grow on the surface of an object
if left without killing the germs. It is, however, different from disinfection or sanitization where only
reduction of the microorganisms takes place, rather than total elimination. After sterilization, an object
becomes sterile or aseptic.

Sterilization is achieved by different physical and chemical methods. Sterilization is classified into 2
types – physical sterilization and chemical sterilization

Physical sterilization includes Heat sterilization. It is the most effective method of sterilization, where
the elimination of microbes is achieved by the destruction of cell constituents and enzymes. It is done
by two methods:
a) Moist Heat Sterilization: It is one of the best methods of sterilization. Moist heat sterilization is
done with the help of an autoclave. An autoclave works on the principle of producing steam
under pressure and is also known as steam sterilization. The water is boiled in an autoclave at
121°-134℃ at a pressure of 20 psi. This leads to coagulation of proteins in the microorganism,
and they are effectively killed.

b) Dry Heat Sterilization: This method is used on objects that are sensitive to moisture. Moisture-
free heat or dry heat is applied on the surface or objects such that there is denaturation and lysis
of proteins which leads to oxidative damage, and ultimately the microbial cell dies out or may
even burn. Some methods of dry heat sterilization include incinerators, hot air ovens and flaming
techniques.
Experiment No. 14 Date:
TESTING THE EFFICACY OF STERILIZATION USING CHEMICAL METHODS:
DETERMINATION OF PHENOL COEFFICIENT

Aim: To determine the efficacy of sterilization by determining the phenol coefficient.

Principle: The effectiveness of a disinfectant or antiseptic can be determined by comparing its

effectiveness with that of phenol. This is done using either Staphylococcus aureus (Gram-positive)
or Salmonella typhi (Gram-negative). The test organism is exposed to dilutions of phenol and the test
disinfectant, for specified time. The phenol coefficient is then calculated. A phenol coefficient of 1.0
means that the chemical agent has about the same level of effectiveness as phenol.
A chemical agent with a phenol coefficient of less than 1.0 is less effective than phenol.
A chemical agent with a phenol coefficient greater than 1.0 is more effective than phenol.
Although the phenol coefficient was once a useful measure of effectiveness, it is no longer commonly
used because the conditions and organisms used were arbitrarily chosen.

Requirements:
Culture: Bacterial suspension 18-24-hour old culture of S. aureus
Glassware: Sterile tubes, Sterile pipettes
Chemicals: Phenol dilutions - 1:80 1:90 1:100
Lysol dilutions-1: 400 1: 450 1: 500
Media: Nutrient broth tubes

Procedure:
1. Label 18 Nutrient broth tubes with the name and dilution of the disinfectant, and time interval of
subculturing (E.g. phenol 1:80, 5 minutes)
2. Quickly add 100 μL of S. aureus culture into each of test tube of disinfectant at various
concentrations and mix well.
3. After the desired time of incubation, of 5, 10, and 15 minutes, remove 100 μL of sample from each
of the tubes and transfer them into the (appropriately labelled) respective sterile tube of Nutrient
broth.
4. Incubate the tubes for 24-48 hours 37 ℃.
5. The phenol coefficient is determined by dividing the highest dilution of chemical being tested that
destroyed the microorganisms in 10 minutes but not in 5 minutes by the highest dilution of phenol
being tested that destroyed the microorganisms in 10 minutes but not in 5 minutes
Result: The phenol coefficient of the tested disinfectant was found to be ________________ against S.
aureus culture.
Blank page work:

Chemical agent and Dilution Growth of culture after exposure (mins)

5 10 15
Phenol 1:80
1:90
1:100
Dettol 1:400
1:450
1:500
Key
+ Growth
- No growth
Calculations
Phenol Coefficient= Highest dilution of dettol that destroyed S. aureus in 10 minutes but not in 5mins
Highest dilution of phenol that destroyed S. aureus in 10 minutes but not in 5mins

=
Experiment No. 15 Date:
STUDY OF THE STRUCTURE OF CELL ORGANELLES THROUGH ELECTRON
MICROGRAPHS

Aim: To study the different organelles of a Eukaryotic cell

Introduction:
Eukaryotic cells are enclosed by a plasma membrane and contain a membrane bound nucleus and
organelles.

Centrioles - Centrioles are self-replicating organelles made up of nine bundles of microtubules and are
found only in animal cells. They appear to help in organizing cell division, but are not essential to the
process.

Cilia and Flagella - For single-celled eukaryotes, cilia and flagella are essential for the locomotion of
individual organisms. In multicellular organisms, cilia function to move fluid or materials past an
immobile cell as well as moving a cell or group of cells.

Endoplasmic Reticulum - The endoplasmic reticulum is a network of sacs that manufacture, process, and
transport chemical compounds for use inside and outside the cell. It is connected to the double-layered
nuclear envelope, providing a pipeline between the nucleus and the cytoplasm.

Endosomes - Endosomes are membrane-bound vesicles, formed via a complex family of processes
collectively known as endocytosis, and found in the cytoplasm of almost every animal cell. The basic
mechanism of endocytosis involves the invagination (folding inward) of a cell's plasma membrane to
surround macromolecules or other matter diffusing through the extracellular fluid.

Golgi Apparatus - The Golgi apparatus is the distribution and shipping department for the cell's chemical
products. It modifies proteins and fats built in the endoplasmic reticulum and prepares them for export
to the outside of the cell.

Intermediate Filaments - Intermediate filaments are a very broad class of fibrous proteins that play an
important role as both structural and functional elements of the cytoskeleton. Ranging in size from 8 to
12 nanometers, intermediate filaments function as tension-bearing elements to help maintain cell shape
and rigidity.

Lysosomes - The main function of these microbodies is digestion. Lysosomes break down cellular waste
products and debris from outside the cell into simple compounds, which are transferred to the cytoplasm
as new cell-building materials.

Microfilaments - Microfilaments are solid rods made of globular proteins called actin. These filaments
are primarily structural in function and are an important component of the cytoskeleton.

Microtubules - These straight, hollow cylinders are found throughout the cytoplasm of all eukaryotic
cells (prokaryotes don't have them) and carry out a variety of functions, ranging from transport to
structural support.

Mitochondria - Mitochondria are oblong shaped organelles that are found in the cytoplasm of every
eukaryotic cell. In the animal cell, they are the main power generators, converting oxygen and nutrients
into energy.

Nucleus - The nucleus is a highly specialized organelle that serves as the information processing and
administrative center of the cell. This organelle has two major functions: it stores the cell's hereditary
material, or DNA, and it coordinates the cell's activities, which include growth, intermediary
metabolism, protein synthesis, and reproduction (cell division).

Peroxisomes - Microbodies are a diverse group of organelles that are found in the cytoplasm, roughly
spherical and bound by a single membrane. There are several types of microbodies but peroxisomes are
the most common.

Plasma Membrane - All living cells have a plasma membrane that encloses their contents. In
prokaryotes, the membrane is the inner layer of protection surrounded by a rigid cell wall. Eukaryotic
animal cells have only the membrane to contain and protect their contents. These membranes also
regulate the passage of molecules in and out of the cells.

Ribosomes - All living cells contain ribosomes, tiny organelles composed of approximately 60 percent
RNA and 40 percent protein. In eukaryotes, ribosomes are made of four strands of RNA. In prokaryotes,
they consist of three strands of RNA.
STUDY OF STRUCTURE OF CELL ORGANELLES THROUGH ELECTRON
MICROGRAPHS

Electron micrograph of Mitochondria Electron micrograph of Endoplasmic Reticulum & nucleus

Electron micrograph of Golgi apparatus

Experiment No. 16 Date:
PRESERVATION OF CULTURES BY PERIODIC TRANSFER AND OVERLAYING WITH
MINERAL OIL

Aim: To preserve cultures by periodic transfer and overlaying with mineral oil.

Principle: Periodic tranfer to fresh media refers to sub-culturing the culture at regular intervals on slants
and/or stabs. Here, transfer is made to fresh medium at definite intervals before organism killed by own
waste products. The viability of the culture varies with the organism. Culture growth can be covered
with a layer of mineral /paraffin oil 1cm above tip and stored at 0 – 5 ℃. This prevents dehydration of
the medium and ensures oxygen limitation thus reducing metabolism. Viability of such cultures are
increased to 15 – 20yrs. From these tubes, growth can be picked and further sub-cultured for use.

Requirements:
Culture: Bacterial suspension18-24 hour old culture
Media: Sterile nutrient agar slants
Chemicals: sterile mineral oil

Procedure:
1. Using a sterile nichrome loop, remove a loopful of culture from a plate / tube.
2. Streak this inoculum on a sterile slant.
3. Incubate the tubes for 24 h.
4. After incubation, under sterile conditions pour mineral oil in the tubes
5. Mineral oil should be atleast 1 cm above the tip.
6. Stored the tubes at 0 – 5 ℃, till further use.

Result:.