EXPT 1 PREPARATION OF STERILE CULTURE MEDIUM

Roll no; NAME

Date / /2023 Time 2.00 to 5.00pm

Aim To prepare a sterile simple medium for growth of microorganisms.

Theory Microorganisms consume substance, called nutrients, from the environment for the
synthesis of their cellular components and generation of metabolic energy. Culture
medium is an artificial media can be used in the form of liquid or solid. solid culture
medium is prepared by the addition of solidifying agent agar to the liquid up to 90͘͘ c Agar
medium is useful for isolation of pure cultures. Different types of culture media have been
developed and are classified into synthetic, complex, selective, differential etc. Based on
their properties.

To culture a pure culture the growth environment must be free of other microbes and
protected from external contamination Sterilization is a treatment that frees the treated
object of all living organisms. Dry heat, autoclave, membrane filtration, ethylene oxide
and radiation are some of the sterilization methods.

Protocol Simple Medium

 Dissolve the component of nutrient medium in 100 ml distilled water in a 250 ml conical
flask.
 Calculate the agar into the flask and melt it by micro oven or water bath.
 Cool to 50* c and transfer 5 ml aliquots to 2 test tubes (use glass pipette).
 Plug all the flasks and test tubes with cotton wool and sterilize at 15 Ibo for 20 min using an
autoclave.
 Allow the test tubes and flasks containing agar medium to cool down to 45-50*c
 Pour 20-25 ml of agar medium from the conical flask into each of the petri plates inside the
sterile chamber and leave them to solidify.
 Leave all the sterilized media overnight incubate at the 37*c.
 Next day observe the plate sterility. Further store the plate 4*c for exp#2.
 Expt 2 ISOLATION MICROORGANISMS FROM SOIL

Roll no Name
Date / / 2023 Time 2.00 to 5.30pm

Aim To isolated microorganisms from soil.

Theory Microorganisms are present everywhere in including soil, water, air, etc. The
microorganisms in these sources can be culture by exposure of sterile growth
medium to sample from any of these sources the function of the microorganisms in
nature are diverse including food digestion. Disease causatives and antibiotic
producers using solid medium like agar petri plate pure microorganisms can be
isolated from sources like soil. Still there are more types of microorganisms in the
environment that cannot be easily cultured under the laboratory conditions. Study
of the entire community of microbes reveals the magnitude of diversity of the
microbial world and their function in the ecosystem. .The microbial population can
be identified and categorized based on the differences in appearance, morphology
and metabolism.

Protocol SOIL
 Add 200 mg of soil sample to 1 ml of sterile saline water, Mix
 Roughly for 5 min.Allow to solids to settle.
 Pipette out 100µlof appropriately diluted liquid onto corresponding agar plates in sterile hood.
 Spread the plates using sterile L-rod.

WATER

 Collect 1 ml of tap water in a sterile test tube.

 Add 100 µl of tap water to the Nutrient agar plates in sterile hood.

AIR

 Open the sterile Nutrient agar plates and expose to atmosphere for 10 min and close. Keep
one plate without inoculation using for control.
 Leave all the plates at 37*c for 24 hrs. Observe and note morphology colonies Wrap the plates
with parafilm and store at 4*c for next experiment.
 EXPT 3 MICROSCOPY AND GRAM STANING
Roll no Name
Date; / / 2023 Time; 2pm to 5.30 pm

Aim Staining and visualization of bacterial cell through microscope.

Theory Microorganisms are unicellular and too small to be seen by the naked human eye.
Microbial cells can be visualized through microscope by optical magnification of the
cell size. A microscope must possess a resolving power sufficient to permit the
perception as separate objects of adjacent points is an image. The Magnification
denial of a microscope depends on the resolving limit which calculated from the
equation = (0͘͘.5ʎ) (N sin ά).Where ʎ is the refractive index of the medium between
the specimen and front of the objective lens. And N is the refractive index of the
medium between the specimen and front of the objective lens. The denominator (N
sin ά) is commonly called numerical aperture (NA).The valve of N can be increased
by the intervening space with oil. Which has higher refractive index than air.

Protocol Smear Preparation

Place a loop of sterile water on a glass slide.

 Suspend cells from plate culture in water on slide and spread.
 Air dry the cell spread.
 Heat-fix the cells by passing the rear side of the slide through flame.
Gram Staining
 Flood the smear with 3-4 drops of the primary dye (Crystal violet) and
Leave it for 30 sec.
 Wash gently with running tap water.
 Add few drops of iodine solution and leave it for 1 min.
 Wash the excess stain with drop wise addition of alcohol for 20-30 sec.
 Flood the slide with counter stain Safranin and leave it for 1 min.
 Wash the slide gently with the running tap water and air dry.
 Examine the stained smear under the microscope.

Observation of stained culture slides

Place the pre stained slide on the stage.
With 5 x objective of high magnification (10x to 100x) and bring the desired region of the cells in to focus.
Place a drop of oil on the slide and exam in the cells under microscope (100x)
 EXPT 4 ISOLATION OF PURE CULTURE
Roll no Name
Date / /2023 Time; 2 to 5.30pm

AIM To isolated pure culture from the mixed culture.

Theory Pure culture is a microbial population with a single species. All environments
contain many kinds of microorganism called mixed cultures when sample of
mixed culture suspension is spread on solid growth medium. Each colony consists
of same type of microbial cells. Individual colonies are picked and propagated
separately and each culture is called pure culture representing one type of
microorganism.
Streak plates is a simple and rapid method .When the loop is streaked
across the agar surface microbial population is diluted and separated as
individual organisms on the agar surface that on growth discrete colonies
are formed.

Pour plate, where the mixed culture is mixed thoroughly with melted agar
and then poured into an empty plate and allowed to solidify. On incubation
discrete colonies are formed on the agar and in the agar.

Spread plate, where the culture sample is diluted in sterile water and
aliquots of diluted samples are spread on the surface of agar plates Dilution
of the culture sample decreases the microbial population which enables
formation of more single colonies.

Protocol Streak plate

 Pick single colonies with a sterile wire loop and streak on fresh agar plates as in Fig.
 Sterilize the loop by heating to red-hot in flame and cool to room temperature.
 From the first streak do second streak as shown in fig.
 Repeat the previous two steps to do 3rd and 4th streaks as in fig.
Patch plate
Pick individual colonies from the petri plates using sterile tooth picks and patch on
fresh sterile agar plates. Keep all plates in incubator.
 EXPT 5 COTTON BLUE STAINING
Roll no Name;
Date; / /2022 Time; 2 to 6 pm

Aim To identify fungi with cotton blue staining procedure.

Principle The lacto phenol blue (LCB) preparation is widely used to stain yeast and
fungi. Cotton blue is an acid dye that stains chitin, a nitrogenous substance
present in the cell walls of most fungi. The high concentration of the phenol
kills any organisms and deactivates lytic cellular enzyme this keeping the cells
intact. Lactic acid preserves fungal structures.

Materials required LCB Stain composition of Stain; Methyl blue/ cotton blue/aniline blue 0.075g
phenol 20 g acetic acid 10 ml, Glycerol 40 ml, make up the final volume to 100
ml with water). Glass slides, dropper, inoculation loop, cover slip.

Procedure

* Place a drop of LCB stain on a clean and dry glass slide.

* Transfer a fragment of the culture on to the slide. With an inoculation loop or
tooth pick spread culture carefully into a thin smear.
* Place a cover slip edge on the drop and slowly lower it. Avoid trapping air
bubbles under the cover slip.
* Wait for 5 mints and observe under a low power microscope.

Observation

* Observe the fungal elements (hyphae, fruiting body) stained blue.

 EXPT 6 DETERMINATION OF CELL NUMBER

Roll no Name;

Date; / / 2023 Time; 2 to 5.30 pm

Aim To determine the number of viable cells in the culture sample.

Theory A microbial population may contain both living and dead cells. The number of
viable cells can be determined by spread plate culture method. When” the culture
sample is spread on agar plate only the viable cells grow as colonies and dead
cells do not grow. Thus the number of colonies obtained on agar plate gives the
number of viable cells in a bacterial population. The sample is diluted before
plating to obtain well separated and countable colonies on plates. Based on the
concentration of the culture sample the magnitude of dilution can be varied.
Generally the culture sample is diluted serially to various folds and spread on
agar plates. After incubation of the plates for 24hrs. The number of colonies
formed on the plates was counted which gives the number of viable cells in the
sample.

Protocol
• Take 5 sterile test tubes and label from number 1 to 4.Transfer 4.5 ml of
sterile saline solution or 450 µl sterile saline solution into each test tube.
• Add 0.5 ml or 50µl of the culture to test tube #1 and mix well.
• Transfer 0.5 ml culture from tube#1 into test tube #2 and mix well.
» Similarly follow culture transfer until the test tube #5 is reached.
• Spread 0.1 ml of culture from each tube on nutrient agar plates. ,
• Incubate the plates for 24 hrs.
* Select the plates containing 30-300colonies and count them.
 EXPT 7 ANTIBIOTIC SENSITIVITY
Roll no Name;
Date; / / 2022 Time; 2pm to 5.30 pm

AIM To determine antibiotic sensitivity of bacterial strains and minimal

Inhibitory concentration.

Principle Antibiotics are commonly used for the treatment of infectious diseases.
Antibiotics inhibit the growth of microorganisms by inhibiting synthesis
of cell wall and protein. Sensitivity of microorganism to various antibiotics
can be determined by disk- diffusion method. When the test strain is spread on agar
plate with antibiotic impregnated paper disk on the surface, the growth is inhibited
resulting in circular zone of clearance. The zone of inhibition is dependent on
factors like diffusion of antibiotic and susceptibility of the strain. Discs with
different concentrations of antibiotic are used to determine the minimal inhibitory
concentration (MIC} which is the lowest concentration of the antibiotic required to
inhibit the growth of.

Protocol
 Spread the test culture on nutrient agar plates to form lawn growth.
 Leave the plates for 5 minutes to air dry.
 Using sterile forceps place the discs impregnated with antibiotic on the surface of pre-
inoculated agar plates.
 Gently press the discs to make good contact with agar.
 Incubate the plates for 18 hrs. At 37‘C.

MIC;
 Measure the zone of clearance around the discs. Minimal inhibitory concentration
 Follow the above steps using different concentrations of antibiotic
 Find out the MIC of the antibiotic
 EXPT 8 CHARACTERIZATION OF BACTERIA
Roll no Name;

Date; / / 2023 Time; 2 pm to 5.30 pm

To determine lactose fermentation and extracellular enzyme synthesis

Theory. MacConkey agar; It is a selective and differential media. This medium

inhibits the growth of Gram positive bacteria. This test also determines the
fermentation of lactose.

Starch hydrolysis: Starch is a polysaccharide that can be hydrolyzed into smaller

oligosaccharides and glucose by enzyme amylase. Iodine solution complexes with
soluble starch to form blue color but not with oligosaccharides. Hydrolysis of
starch will be observed as a clear zone surrounding the colony after addition of
iodine solution to the agar plate

Gelatin hydrolysis: Gelatin is a protein produced by hydrolysis of collagen.

Proteolysis cleavage by gelatinize enzyme liquefies gelatin. The medium
becomes liquid if gelatinize is produced by the culture.

Tributyrin agar; Triglycerides serve as bacterial carbon end energy source.

Some bacteria produce enzyme called lipases that hydrolyse triglycerides. The
synthesis of lipases is identified as clear zone around the colony.

Protocol
• Streak the organism on Starch agar plate.
• Inoculate the gelatine deep tube by means of stab inoculation.
• Incubate at 37oC for 24 h.
• Flood starch agar plates with iodine solution. Zone of clearance around the culture.
Indicates amylase production.
• Observe the liquefaction of gelatine tubes and clear zone on tributyin agar.
 EXPT 9 CHARACTERIZATION OF BACTERIA

ROLL NO ; Name;
Date / / 2023 Time; 2pm to 5.30 pm

Aim: Determination of motility and biochemical properties

Theory Triple Sugar iron agar; TSI agar contains lactose, sucrose and glucose and phenol red is
incorporated to detect carbohydrate fermentation. Slant-red (alkaline) and butt-yellow (acidic):
Glucose fermentation. Slant-yellow and butt-yellow: lactose and sucrose fermentation. Slant-
red and butt-red: No carbohydrate fermentation, catabolism of peptone forms ammonia which
forms red colour.
Indole formation: Tryptophan is an essential amino acid that can be metabolized through the
formation of iodole and this enzymatic reaction is present only in selective organisms. The
presence of iodole is detected by the addition of Kovac’s reagent which forms cherry red color.

MRVP test- The Voges-Proskauer test determines the capability of some organisms to produce
acetylmethylcarbinol from the organic acids that results from glucose fermentation. This compound
formation is detected by of addition Barrett’s reagent which forms rose color medium.

Motility test; this test is to determine the actual motility of the organism. When the
growth of culture spreads and not confined to lone of inoculation it indicates the motility
of culture.
Citrate; Utilization of bitrate as carbon source is determined. The citrate medium is
supplemented with bromothymol blue indicated.Which changes from green to Prussian
blue if nitrate is metabolized.

Protocol
• Inoculate a tubes of tryptophan broth with one loop of culture.
• Incubate for 24 to 48 hrs.
• Indole: Add 0.5ml of Kovac's reagent p-dimethyl aminobenzaldehyde in
acidic butanol) to approximately 5 ml of the broth. Let it stand for 1 to 5
min. Appearance of dark red surface layer indicates iodole synthesis
• MRVP: Divide the culture into two parts.
Methyl red: To one part of culture add five drops of methyl red indicator and
observe the colour of the medium. Red colour indicates high concentration
of acidic products formed from fermentation.-
VP test: To another part of culture add ten drops of Barritt’S reagent and
shake the culture. Rose colour formation indicates positive.
• Observe motility and colour change for citrate utilization in the respective media.
 EXPT 1 0 CHARACTERIZATION OF BACTERIA

Roll no Name

Date ; / / 2023 Time 2 to 5.30 pm

To test biochemical characteristics.

Nitrate reduction: To determine the ability of some organisms to reduce nitrate. This can
be determined by cultivating organisms in a nitrate booth. Following the incubation of
Theory culture addition of sulfanilic acid and alphanaphthalamine form a cherry red color indicating
the nitrate reduction.
Urease: To determine the ability of microorganism to degrade urea. Urease hydrolyses
urea and forms ammonia. Synthesis of urea is detected by using pH indicator phenol red in
the medium which turns to deep pink.

Catalase: To determine the ability of some organisms to degrade hydrogen peroxide by

producing catalase. Aerobic respiration of microorganisms produce hydrogen peroxide which
are detrimental to the organisms. Organisms capable of producing catalase rapidly degrade
hydrogen peroxide to water and oxygen .catalyse production can be determined from the
formation of bubbles of free oxygen gas by addition of hydrogen peroxide to cells.

Oxidase: To test the culture for by molecular oxygen. The oxidase activity can be
determined using discs containing chromogenic substrate Change in disc color to purple in
20-30 sec on addition of culture to the discs indicates oxidase activity.
Protocol
*Inoculate the bacterial culture into the respective media
*Incubate at 3 C for 24 h. examine the culture
*Add reagents to appropriate culture and observe the results