Aim of the experiment: To study the different parts of a microscope.
Theory: microscopes are employed as a basic tool to observe microorganisms and to study
the structural details of the cells. Nowadays, highly sophisticated microscopes such as phase
contrast, dark field and electron microscopes are available but simple and compound
microscopes remain as a basic tool in routine microbiology research work. A simple
microscope is just a hand lens and magnifying glass whose magnification power is relatively
lower. In order to achieve higher magnification power, combination of lenses is used as
objective lens and ocular lens in components are grouped into two categories:
I.
Structural components
II.
Optical components
I.
II.
Structural components: three structural components of microscope are head, arm and
base. Head contains the optical parts in the upper part of the microscope. Base
supports it and contains eliminator (condenser). The arm is fitted with the coarse and
fine adjustment knobs and help in easy handling of microscope.
Optical components: there are 2 types of optical structures:
a. Eye piece/ocular: it is a cylinder containing 2 or more lenses. Its function is to
bring the image into focus for the eye. Typical magnification values for the
ocular lens is 2x, 10x and 50x.
b. Objective lens: objective lens is usually a cylinder containing lens attached to
the circular disk with the movable head of the microscope. Magnification of the
objective lens ranges from 10x, 40x to 100x. Three to four objective lenses are
attached to the piece.
c. Stage: stage is the flat platform used to place the slide. An eliminator is
provided under the stage to provide the sufficient light which passes through the
holes on the stage for focussing the contents of the slide.
d. Condenser: it is used to collect the light from the light source.
Working:
The objective lens is placed close to the object to be viewed and ocular lens is placed
closed to the eyes. The primary enlargement of the object is produced by objective
lens. The image produced thus is transmitted to the ocular where the final enlargement
occurs. Therefore, the magnifying capacities of the compound microscope are the
product of magnification of the objective and the ocular lens. For e.g.; using an
objective lens of 40x and ocular lens of 10x will produce a magnification of 400x. The
primary image produced by the objective lens is real and inverted image.
Aim of the experiment: To study various tools and instruments required in the
microbiology lab.
Theory:
1. Autoclave: it is an apparatus in which saturated steam under pressure affects the
sterilization. The increased pressure increases the boiling point of water and produces
steam due to which microbial cells are destroyed. The temperature, time and pressure
conditions are 121°C at 15 psi for 15 minutes. It is the most efficient and the most
commonly used instrument for the sterilization of solid state and liquid media,
glassware and rubber products. It is double-valued cylindrical vessel made up of steel
and the top is opened to receive the materials to be sterilised. Autoclave is provided
with pressure gauge for recording pressure and steam cork rises for release of steam.
The articles to be sterilized are kept in the basket provided with holes all round for
free circulation of steam.
2. Laminar flow: it is used for reducing danger of contamination while working with the
infectious microbes. It works on the principle of application of fibrous filters usually
HEP filter i.e. high efficiency particulate air filters. Room air is filtered before
entering the working chambers and it moves in a single direction. UV light is also
provided to reduce the risk of spores if present.
3. Incubator: it consists of an insulated, fitted with the heating element at the bottom.
The temp of the incubator can be maintained at a desired level by an aromatic device
called thermostat which cuts the connection when the temp falls below that point.
They are provided with double doors, the inner one is made up of glass so that the
contents maybe viewed from outside. It helps in culturing of the microbes at a constant
temp and helps in growth and development of various organisms. it uses dry heat of
maintaining temperature.
4. Water bath: a water bath is an insulating metal box filled with an electric heating
mechanism and thermostat which maintains the temperature at a desired level. The
bath is filled with water until it reaches almost to the top of the brim, as it contains
racks for holding test tubes and flasks. Water bath is mainly used for melting up of
various microbes in Wassermann’s test.
5. Magnetic hot plate stirrer: it is an extremely useful in making media as well as to
stir to make a suspension. It is fitted with stirrer and heat. A control stirring is done by
creation of magnet kept in the beaker to spin resulting in stirring of the medium.
6. Spectrophotometer: it is used for counting population of bacteria based upon the
principle of turbidity or optical density. Turbidity is the cloudiness of the suspension.
The more turbid suspension is, less light will be absorbed and is proportional to the
mass of cells in the light part. Bacteria may grow in broth, clean broth may become
turbid. Since, the turbidity increases, the indicator is used for determination of
bacterial density in the broth. Turbidity is also used for standardising of bbacteria
structures of chemical significance.
7. pH meter: it is used to determine pH that maybe acidic or alkaline of the solution of
unknown pH as well as setting of pH. Various media used for cultivation and testing
of biochemical activities of microbes. The measurement of ph and ph meter is done
electrometrically and depends upon development of membrane potential by a glass
electrode. A pH meter consists of another electrode known as reference electrode. This
consists of metallic internal element typically of mercury i.e. HgCl2 or AgCl2
immersed in an electrolyte. Usually, a saturated solution of KCl is used. The function
of electrolyte is to form a conductive salt bridge between the metallic element and the
sample solution in which electrodes are placed to keep stable electrically
communication between the internal metallic element and sample solution. A liquid is
present in the tip of the outer body of reference electrode.
8. Oven: an oven is based on the principle of sterilization which is accomplished by dry
heat or hot air. Hot air oven are mostly used for sterilisation of glassware like
petridishes, pipette and metallic instruments. An oven consists of an insulated cabinet
which is held at a constant temperature by means of thermostat. It is fitted with fan to
keep hot air circulation. The shelves are perforated for normal sterilisation, the oven
will be operated at 160°C will require the period of two hours for total sterilisation.
Aim- To prepare culture media.
Requirements- Nutrient broth, Nutrient Agar, Distilled Water, Autoclave, flask etc.
TheoryMost common media used for culturing the micro-organism is nutrient broth and nutrient
agar.
Nutrient broth consists of:
Composition
g/l
Peptones
5
NaCl
5
Yeast Extract
2
Beef Extract
1
13g of nutrient broth for 10 ml of medua is added.It is dissolved in distilled water to prepare
1000ml of media onlyh if it is available otherwise nutrient broth can be supplemented with
2% of agar agar to prepare nutrient agar.
Procedure:
1- Weighed point 0.6g of nutrient broth and mixed with 50ml of distilled water.
2- Cotton plug the flask
3- 4.2g of nutrient agar was weighed and it was added to 150ml of distilled water. Again
cotton plug the flask.
4- Nutrient broth and agar was autoclaved at 1210C for 15minutes at 15 psi pressure.
5- After autoclaving, the media were cooled to 450C.
Aim- To prepare pour plate, spread plate & streak plate method for
isolation and enumeration of micro-organism.
Theoryi.
ii.
iii.
STREAK PLATE TECHNIQUE: By means of a transfer loop, a portion of the
mixed culture is placed on the surface of an agar medium and streaked across the
surface. The manipulation thins out the bacteria are separated from each other.
When streaking is done properly the colonies grow sufficiently far apart showing
no merge of colonies. The assumption is made that colony is derived from a single
cell &therefore the colony is a clone.
POURPLATE TECHNIQUE: In this mixed culture is diluted directly in tubes of
liquid agar medium. The medium is made liquid state at a temperature of 45 0C to
allow thorough distribution of the inoculum. The inoculated medium is dispensed
into petri-dishes, allowed to solidify and then incubated.
SPREAD-PLATE METHOD: The mixed culture is not diluted in the culture
medium instead it is diluted in a series of tubes containing a sterile liquid. A
sample is removed from each tube, placed onto the surface of an agar plate by
means of bent glass rod. One plate of the series bacteria will be in numbers
sufficiently low as to allow the development of well separated colonies.
AIM: Isolation and enumeration of micro-organisms from soil using serial dilution
method.
REQUIREMENTS : Soil sample, nutrient agar, saline solution , test tubes , autoclave , laminar air flow ,
incubator , weighing balance , micropipette , micro tips.
PRINCIPLE : various methods are available to isolate and enumerate micro-organisms from soil sample ,
food stuffs , milk and water. The serial dilution method is one of the commonly used procedures for
isolation and enumeration. This method is based on the principle that when material containing microorganisms is cultured, each viable micro-organism will develop into colony. Hence , the number of colonies
appearing on plates presents the number of living organisms present in the sample.
In this method a known amount of sample is dissolved in a sterile water to make Microbial suspension.
Serial dilutions from 10-1 to onwards are made by pipetting the measured volume (1ml) into previously
sterile saline solution. Finally, 1ml aliquot of various dilutions are added to sterile petriplates to which 15-20
ml of cooled molten media (nutrient agar) is added. Upon solidification the plates are incubated at 25 .C for
3-7 days. The number of colonies appearing on the plates are counted and number of cell per ml of sample is
calculated by using formula :
Cfu/ml or Number of cells/ml/g =
Colonies
dilution factor X aliquot taken
PROCEDURE: 1. Collect the soil sample at random for microbial analysis.
2. Make the dilutions upto 10-5 with the help of sterile saline solution (0.85%) and add the sample in it.
3. Pipette 1ml of the sample in the petriplate and add nutreient agar in it.
4. Upon solidification , incubate the plates at 25.C for 3-7 days.
5. Results were recorded in terms of cfu/ml.
AIM : To perform gram staining of given sample.
MATERIAL REQUIRED : Glass slides, bunsan burner , cotton , sample , microscope.
REAGENTS REQUIRED: Crystal violet dye, iodine, alcohol (95% ethyl alcohol), saffranin dye
PRINCIPLE: Gram staining is most widely staining technique used in m/o examination. It was discovered
by Danish scientist and physician Hans Christain Joachin Gram in 1884. This technique differentiates
bacteria in 2 groups i.e. Gram positive and Gram negative bacteria. The procedure is based on the ability of
m/o to retain colour of the stain during Gram reaction. Gram negative bacteria are decolourised by alcohol
losing the colour of primary stain , purple. Gram positive bacteria are not decolourised by alcohol and will
remain as purple. After decolourisation stop , a counter stain is used to impart pink colour to the gram
negative m/o.
Gram positive bacteria have a thick mesh like cell wall which is made up of peptidoglycan (50-90%) of cell
wall, which stain purple. Gram negative bacteria have a thinner layer of peptidoglycan (10% of cell wall)
and lose the crystal violet iodine complex during decolourisation with alcohol rinse but retain the counter
stain safarin thus appearing reddish or purple.
STAIN REACTION :
1. Application of crystal violet to heat fixed smear :
CV dissociates in aqueous solution into CV+ and Cl- ions. These two penetrate the cell wall and cell
membrane of both gram positive and gram negative . CV+ interact with negative component of
bacterial cell and stain it purple.
2. Addition of gram iodine :
Iodine acts as a mordant and a trapping agent. A mordant is a substance that increase the affinity of
cell wall for a stain by binding to primary stain , thus forming a insoluble complex that get trapped in
cell. During the reaction CV-I compex is formed and all the cells turn purple.
3. Decolourization with ethyl alcohol :
Alcohol dissolve the lipid outer membrane of gram negative bacteria, thus leaving the petidoglycan
layer exposed and increase the porosity of cell wall. The CV-I complex is then washed away from
the peptidoglycan layer leaving gram negative bacteria colourless.In gram positive bacteria , alcohol
has dehydrating effect on cell wall causing cell wall to shrink , then CV-I complex get tightly bound
into multi layered leaving the cell with purple colour.
4. Counter stain with safranin dye :
The decolourised gram negative cell can be visible with a suitable counter stain which is usually
positively charged safarnin, which stained it pink.
PROCEDURE :1. Prepared very thin smear of sample on glass slide and heat fixed it.
2. Flooded the smeared slide with crystal violet dye. Avoid over flooding and kept it for 1 minute.
3. Washed the slide under running tap water.
4. Applied iodine solution gently all over the slide and kept for 1 minute.
5. Washed it under tap water.
6. Applied 95% ethyl alcohol all over the slide drop wise and kept for 10 second.
7. Immediately rinsed with water.
8. Finally, flooded the sample with saffranin dye to counter stain and kept for 45 seconds.
9. Washed the slide with running water.
10. Observed it under microscope.
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