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Exercise 2

The document describes procedures for preparing microscope slides of microorganisms in both living and dead states. It includes techniques for hanging drop mounts and slide mounts of various microbes. The goal is for students to learn to differentiate true motility from Brownian movement and examine morphological characteristics of microorganisms under the microscope.

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Andreah Baylon
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20 views

Exercise 2

The document describes procedures for preparing microscope slides of microorganisms in both living and dead states. It includes techniques for hanging drop mounts and slide mounts of various microbes. The goal is for students to learn to differentiate true motility from Brownian movement and examine morphological characteristics of microorganisms under the microscope.

Uploaded by

Andreah Baylon
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Exercise No.

2:
Microscopic Examination
of Microorganisms
Introduction

Microorganisms may be observed under the microscope either in their living


or dead state. Whenever studies on cell motility, arrangement and
morphology are conducted, the examination of microorganisms in their living
state is resorted to. For a more definite view of the appearance, shape and
arrangement of microorganisms, however, their observation in the dead state
is more useful

The plain slide and cover glass serve to protect the specimen and render it
easily visible by enclosing the specimen between plain parallel surfaces so
that it is substantially in one plane and is completely surrounded by the
mounting medium The depression slide and cover glass provide a condition
wherein the microorganisms are suspended in a clear fluid of water, saline
solution or broth. This enables the microorganisms to be examined in the
living state.

Learning Outcomes
At the end of the exercise, the students should be able to:

1. prepare a hanging drop and slide mount of various microorganisms in


living state:
2. differentiate true motility from Brownian movement through
microscopic observation.
3. examine the different morphological characteristics of
microorganisms in their unstained/living state and stained/dead
conditions.
Materials

• Alcohol lamp, wire loop, flat end needle, compound microscope, glass
slides, depression slides, cover slips, lactophenol, culture of bacteria,
molds and yeast, hay infusion, prepared slides of algae, bacteria,
molds, protozoa and yeast.

Procedure

I. Microscopic Observation in the Living State


A. Hanging Drop Technique
1) For protozoa in hay infusion

a. Clean a depression slide and cover slip.


b. With a wire loop, place two loopfuls of water around the edge of
the cavity opposite each other
c. Place a loopful of the specimen to be examined at the center of
the cover slip.
d. Invert the depression slide and carefully lower it onto the cover slip
seeing to it that the loopful of specimen is at the center of the
depression. When contact is made, the moisture around the edge
will form a film and hold the cover slip.
e. Turn the slide right side up quickly so that the drop of the
specimen will hang suspended from the cover slip.
f. Examine the hanging drop with the low power objective
g. Reduce the amount of the light with the iris diaphragm in order
that the drop may be seen distinctly.
h. Adjust the slide so that the edge of the drop runs through the
center of the field
2) Hanging drop technique of carmine dye particles in solution for
demonstration of Brownian movement.

A hanging drop slide containing carmine dye particles will be mounted on the
microscope by the instructor for the whole class.

Take a look into the eyepiece and keep in mind the movement exhibited by
the non-living carmine dye particles and compare this with true motility
exhibited by protozoans in hay infusion.
B. Slide Mount Technique - for bacteria, yeasts and molds

Use the cover slip and glass slide in examining bacteria, yeast and mold

1. Bacterial Mount
a. Place the culture tube on the palm of the left hand just between
the index and middle finger and steady it with the thumb
b. Hold the wire loop with the right hand as you would hold a pencil.
Heat to redness the tip of the loop in the flame of alcohol lamp
and flame the handle.
c. Allow the loop to cool for about 10 seconds. While cooling the
loop, it should not come in contact with anything.
d. While holding the sterilized cooled loop remove the cotton plug
from the mouth of the tube by grasping the plug with the little
finger of the right hand.
e. Flame the mouth of the tube
f. Insert loop into the mouth of tube and draw out a loopful of
bacteria.
g. Put the loopful of culture in a drop of stele water on a clean glass
slide and mix.
h. Flame again the mouth of tube and carefully return the plug.
i. Put cover slip.
j. Flame the wire loop again before laying it down on the table
k. Focus mounted specimen and examine finally at the oil immersion
objective. Draw observed bacterial cells,
2. Yeast Slide Mount

Follow steps a-k above this time using yeast culture

3. Mold Slide Mount

Follow steps a-e above except that you are to use a flat end needle instead of
wire loop

a. Insert the flat-end needle into the mouth of the tube and using the
curved end of the needle, cut through the agar and the mycelial mat of
the mold.
b. Place the agar fragment with the mold onto a clean glass with a drop
of plain lactophenol and add cover slip. Heat lightly by passing the
slide right side up 3-4x over the flame.
c. Tap lightly the cover slip.
d. Focus and examine finally at HPO Draw spores, mycelia and
sporangium of Rhizopus.

II. Microscopic Observation of Prepared Slides


A. Examination of Bacteria - bacíllus, coccus
B. Examination of Yeasts - Saccharomyces cerevisiae
C. Examination of Protozoans - Paramecium, Euglena, Amoeba
D. Examination of Molds – Penicillium, Aspergillus, Rhizopus sporangia,
Rhizopus zygospore
E. Examination of Green Algae -Volvox, Spirogyra, Nostoc
F. Examination of Golden Brown Algae- diatoms

Focus slides under the microscope and draw the different kinds of
microorganisms observed.

Study Guide Questions:


1. How can you distinguish true motility from Brownian movement?
2. How do protozoans move?
3. What is the role of vegetative hyphae?
4. Why are fungi cosmopolitan in nature?

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