The document describes several microscopy experiments involving microscopy, staining techniques, and aseptic techniques. The microscopy experiment involves observing samples under different magnifications of a compound microscope and identifying structures. Staining techniques are used to make microorganisms visible, including simple staining using methylene blue dye and Gram staining to differentiate bacteria. Aseptic techniques aim to minimize contamination and prepare pure cultures, including cleaning work areas, sterilizing equipment in flame, and isolation techniques like streak plating or pour plating that allow single colonies to grow separated on nutrient agar.

1. EXPERIMENT #1 MICROSCOPY
INTRODUCTION
Binocular compound microscope
Microscope is an instrument that is used to magnify any material or organism that cannot be
seen by our naked eye.
A compound microscope is a type of microscope which is very delicate and composed of many
parts that are accurately filled together. Such as
 Eyepiece lens
 Body tube
 Objective lenses
 Stage
 Condenser lens
 Iris diaphragm
 Light source
 Base
 Body arm
 Nose piece
 Coarse adjustment
 Fine adjustment
Eyepiece lens-is a lens through which the experimenter makes his/her observation. If the
microscope has only one lens we call it monocular microscope and if it has two lenses we call it
binocular microscope.
Body tube - is the optical housing for the objective lenses.
Objective lenses- a set of lenses mounted on a rotating turret at the bottom of the body tube
from 4X up to 100X magnification power.
Stage – a horizontal surface on which the slide is placed.

2. Condenser lens- a systemof lens that focuses light on your specimen.
Iris diaphragm-is used to regulate the amount of light passing to the stage since the amount of
light is very important for a higher magnification.
Light source
Base-is also called supporting stand
Body arm- is used for carrying the instrument
Nose piece-is the mounting for the objective lenses which rotates to remove the desired
objectives in to position.
Coarse adjustment-is used for rapid focusing of the specimen until the specimen is roughly in
focus.
Fine adjustment-is located at either side of the microscope which is used for the control of the
object for precise focusing.
PURPOSE (OBJECTIVE)
 The main objective of the experiment was to give us an awareness about how to use a
microscope in the laboratory with the required safety cautions and correct procedure of
observation.
MATERIALS AND METHODS
The materials used were
 compound microscope
 clean microscope slide
 cover slip
 lens paper
 pen

3. METHOD
During our experiment we have observed the magnification power of the microscope first by
using a letter written on a paper. The letter that was written on the paper was latter “e” and we
have observed this written letter under the microscope and different magnification power. We
have also observed agar plate with yeast colonies.
RESULT
As we observed using the compound microscope first we have only seen a rough unidentified
sketch or portion of the written letter with the magnification of 40X then after adjusting our
lens to a magnification power of 100X we were able to observe an inverted letter “e”. And
when we have observed the agar plate with yeast colonies we have seen the yeasts with a
round shape clustered together.
DISSCUSSION
From the overall experiment we have observed the function and method of usage of
microscope and the caution that are needed to make while using it.during the observation we
have managed to know that by adjusting our lens we can be able to get the correct image of
the sample on the specimen.

4. EXPERIMENT#2 STAINING TECHNIQUES
INTRODUCTION
Most living microorganisms are generally colorless and almost invisible because of their lack of
contrast with the water in which they may reside, staining is necessary in order to make them
readily visible for observation of intracellular structures as well as overall morphology.
Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of
the microscopic image. Stains and dyes are widely used in the scientific field to highlight the
structure of the biological specimens, cells, tissues etc.
The use of a single stain to color a bacterial organism is referred to as simple staining. Some of
the most commonly used dyes for simple staining are ethylene blue, basic fuchsine, and crystal
violet.
All of these work well on bacteria because they have color bearing ions (chromophores) that
are positively charged. Since bacteria are negatively charged there is an attraction between
them and the chromophores. Positively charged dyes are classified as basic dyes. In this
experiment a basic dye, methylene blue is used.
The staining times for most simple stains are relatively short, usually from 30sec to 2minutes,
depending on the affinity of the dye. The stained sample is examined under oil immersion. This
is useful in determining basic morphology and the presence or absence of certain kinds of
granules. One of steps in identifying this pathogen is to do a simple stain of it to demonstrate
the following characteristics: pleomorphism, metachromatic granules and palisade
arrangement of cells.
Pleomorphism: pertains to irregular of form i.e. demonstrating several different shapes.
Metachromatic granules: having the capacity to stain different elements of a cell or tissue in
different colors or shades.
Palisade arrangement: the collective shape of microorganisms

5. A cover slip is not used in a staining process. Instead a heat fixation is used. Heat fixation means
to fix the sample on the slide by passing the slide over a heat source now and again for a very
short time. Which may itself consist of several steps–aims to preserve the shape of the cells or
tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and alter
the specimen so it accepts stains.
IMPORTANCE OF A GRAM STAIN
The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria based on
staining characteristics, enabling the bacteria to be examined using a light microscope. The
bacteria present in an unstained smear are invisible when viewed using a light microscope.
Once stained, the morphology and arrangement of the bacteria may be observed as well.
Furthermore, it is also an important step in the screening of infectious agents in clinical
specimens such as direct smears from a patient.
The Gram stain procedure enables bacteria to retain color of the stains, based on the
differences in the chemical and physical properties of the cell wall.
OBJECTIVES
 To differentiate between the two major categories of bacteria: Gram positive and
Gram negative.
 To understand how the Gram stain reaction affects Gram positive and Gram
negative bacteria based on the biochemical and structural differences of their cell
walls.
 To identifying the arrangement of yeast cells using ethylene blue.

6. MATERIALS
 Petri dish containing yeast colonies
 burner
 microscopic slide
 inoculating loop
 microscope
CHEMICALS
 methylene blue
 distilled water
 disinfectant(alcohol)
PROCEDURES
1. Clean the working area by disinfectant.
2. A drop of Distilled water was placed on the slide.
3. A yeast culture sample was smeared with a spreader.
4. Then the slide was passed over a Bunsen burner for fixation of the sample.
5. A drop of Methylene blue dye was dropped on the sample. Then it was spread with a
spreader.
6. The stain was fixed by passing over the Bunsen burner.
7. The sample was mounted on the microscope under the oil immersion objective.

7. RESULT AND CONCLUSION
The experiment is kept as sterile as possible by conducting the experiment within 10 cm of the
flame from a Bunsen burner. This is to avoid contamination of the sample by microbes in the
air. The loops used to smear the microbes on to the slide are sterilized in a flame. Meanwhile,
the slid with sample on was to fix the bacteria onto the slid. Now about the stains methylene
blue dye is used to make the cells more visible.
The bacterial cells usually stain uniformly and the color of the cell depends on the type of dye
used. If methylene blue is used, some granules in the interior of the cells of some bacteria may
appear more deeply stained than the rest of the cell, which is due to presence of different
chemical substances.
Methylene blue is stains the dead cells and thus differentiate it from the living cells because the
dead cells will take up the stain easily than the live cells.

8. EXPERIMENT #3 ASEPTIC TECHNIQUES
OBJECTIVE
 Preparation of pure culture
INTRODUCTION
There are various techniques that are used to minimize the introduction of microorganisms into
media especially during transfer processes, such as, pouring of media into Petri dishes,
inoculation of culture. These techniques include:
 cleaning the bench top work areas with disinfectant solution
 washing hands before starting work
 wiping inoculating loops with cotton and alcohol and flaming it until it becomes red hot
 Other specific techniques that will be demonstrated in the lab.
I. Pure culture preparation and cell growth
Pure culture is a culture which contains one and only one species of microorganisms, and the
reason why we need pure culture is to study the morphological, physiological, nutritional,
genetic characteristic and environmental requirements etc. of a given species.
In pure culture extraction there are two important procedures, i.e. isolation and culturing of
cell.
a)Isolation
There are a number of procedures available for the isolation of pure culture from mixed cell
population. A pure culture may be isolated by use of special media with specific chemicals and
physical agents that allow the selection of one organism over another. The simpler methods
are; spread plating over solid agar media with glass spreader, streak plating with a loop. And
the purpose of both isolation method is to isolate individual cell (colony-forming units) on a
nutrient medium.

9. Both procedures (spread plating and streak plating) require understanding of the aseptic
technique. Asepsis can be defined as the absence of infectious microorganisms. However, the
term is usually applied to any technique designed to keep unwanted microorganisms from
contaminating sterile materials
II. Pour Plate Technique
In this technique, the number of bacteria per unit volume of sample is reduced by serial
dilution before the sample is spread on the surface of an agar plate. The steps are:
Step1: Prepare serial dilutions of the broth culture as shown below. Be sure to mix the
nutrient broth tubes before each serial transfer. Transfer 0.1 ml of the final three dilutions
(10-5, 10-6, and 10-7) to each of three nutrient agar plates, and label the plates.
Step2: Position the beaker of alcohol containing the glass spreader away from the flame.
Remove the spreader and very carefully pass it over the flame just once. This will ignite the
excess alcohol on the spreader and effectively sterilize it.
Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient agar plates
until the medium no longer appears moist. Return the spreader to the alcohol.
Repeat the flaming and spreading for each of the remaining plates.
Invert the three plates and incubate at room temperature until the required temperature.
III. Streak Plate Technique
The streak plating technique isolates individual microorganism cells (colony-forming units)
on the surface of an agar plate using a wire loop. The streaking patterns shown in the figure
below result in continuous dilution of the inoculum to give well separated surface colonies.
Once again, the idea is to obtain isolated colonies after incubation of the plate. The steps
are:
Step1: Prepare agar media

10. Step2: Prepare streak plates by following two of the 3 streaking patterns shown in the
figure below. Use the 10-1 dilution as inoculum.
Step3: Invert the plates and incubate at room temperature until it reach the required
temperature.
b)Culturing
After the pure culture prepared the next step is the cell growth, and it is the growing of the
isolated microbe on a specific media into large number. In the cell growth there are four
phases, i.e. lag phase, log or exponential phase, stationary phase and death phase. The
phase is represented on the next graph:
In the lag phase:
• No increase in cell number
• Induction of enzyme to utilized substrate
• Industrially very important to decrease lag period to increase productivity
In the log phase:
• Nutrient and substrate concentrations are large
• The cell growth rate is independent of nutrient and substrate concentration.
In stationary phase:
• No net growth of cell numbers
• Cell growth rate = Cell death rate
• Secondary metabolites (products) produced
• Endogenous metabolism of energy stores can result in maintaining cell viability
• Removal of inhibitors will result in further growth, if additional substrate is provided.
In death phase:
• Cell lysis may occur
• Growth can be re-established by transferring fresh media

11. IV. Cell count or cell number determination
Determination of cell numbers can be accomplished by a number of direct or indirect methods.
The methods include standard plate counts, Turbido-metric measurements, and visual
comparison of turbidity with a known standard, direct microscopic counts, cell mass
determination, and measurement of cellular activity.
1) Direct method
Direct cell count
Direct counting methods are used to determine the number of microorganisms without the
need for advanced equipment. It includes microscopic counts using a haemocytometer or a
counting chamber. It is easy to perform and doesn’t require highly specialized equipment, but
are slower than other methods.
Viable cell count
Viable cell count allows one to identify the number of actively growing dividing cells in a
sample. A viable cell is defined as a cell which is able to divide and form a population (or
colony). A viable cell count is usually done by diluting the original sample, plating aliquots of the
dilutions onto an appropriate culture medium, and then incubating the plates under proper
conditions so that colonies are formed. After incubation, the colonies are counted and, from a
knowledge of the dilution used, the original number of viable cells can be calculated. For
accurate determination of the total number of viable cells, it is critical that each colony comes
from only one cell, so chains and clumps of cells must be broken apart. However, since one is
never sure that all such groups have been broken apart, the total number of viable cells is
usually reported as colony-forming units (CFUs) rather than cell numbers. This method of
enumeration is relatively easy to perform and is much more sensitive than turbidometric
measurement. A major disadvantage, however, is the time necessary for dilutions, plating and
incubations, as well as the time needed for media preparation.

12. 2) Indirect methods
Spectrophotometry
Spectrophotometer cell suspensions are turbid. Cells absorb and scatter the light. The
higher the cell concentration, the higher the turbidity. Spectrophotometers can
measure intensity of light very accurately. The cell culture is placed in a transparent
cuvette and the absorption is measured relative to medium alone. Optical density is
directly proportional to the biomass in the cell suspension in a given range that is
specific to the cell type. Spectrophotometer’s drawback is its inability to provide an
absolute count or distinguish between living and dead cells. Before measurements can
be made, the transmittance of the test tube containing only the culture media is
measured and calibrated.
Measuring the metabolic activity of the population
It is another way of estimating the numbers of microbes. The metabolic activity can be
measured using the acid production, or oxygen consumption. For filamentous organisms
as fungi, measuring dry weight is a convenient method of growth measurement.
Direct Microscopic Count
Petroff-Hausser counting chambers can be used as a direct method to determine the number of
bacterial cells in a culture or liquid medium. In this procedure, the number of cells in a given
volume of culture liquid is counted directly in 10-20 microscope fields. The average number of
cells per field is calculated and the number of bacterial cells ml-1 of original sample can then be
computed. A major advantage of direct counts is the speed at which results are obtained.
However, since it is often not possible to distinguish living from dead cells, the direct
microscopic count method is not very useful for determining the number of viable cells in a
culture.

13. PROCEDURE:
Step1: First, 100ml distilled water was measured.
Step2: Six test tubes were filled with 9ml distilled water each.
Step3: The test tubes were covered with an aluminum foil to protect them from damage.
Step4: The petri dishes were covered with plastics.
Step5: The media was dissolved in a flask.
Step6: The dissolved media was boiled.
Step7: Then it was covered with an aluminum foil.
Step8: Then they were placed in the autoclave.
Step9: The agar media was sterilized.
Step10: Six plates were labelled from 10-1 to 10-6.
Step11: Then the media was poured on the plates.
Step12: 1 ml drop was transferred by using the pipette to the test tubes.
Step13: The test tubes were shake for 30 seconds on a vortex.
Step14: 0.1 µl of the sample was transferred from each test tube to its respective plate.
Step15: The drops were spread on the plates.
Step16: The spreader was sterilized.
Step17: The serial dilution plates were put in the incubator inverted upside down to prevent
moisture drop on the media.
Step18: Then the media was placed in the incubator for 24 hours.

14. OBSERVATIONAND CONCLUSION
After 24hours, using a direct count method five colonies were found on each Petri dish. And we
have also observed that the petri dishes containing the more diluted samples had less colonies
than the contained the less diluted ones.
Therefore, we conclude that:
1. The microbial growth is highly sensitive process and it must be conducted within a
highly sterile environment.
2. Samples that have undergone more stages of serial dilution have less microbial
population.
ANSWERS TO THE EXERCISE:
A. Results
1. No
2. –
3. Because, all the plates, except the 10-1 plate, didn’t develop any colony.
B. Questions:
1. Any microorganisms that may be present on the working area.
2. No. only the vegetative cells are destroyed.
3. Until it turns red.
4. To prevent contamination from transient and air borne microbes.