Exercise 1: The Compound Microscope (10 marks)
At the conclusion of this exercise you should be able to:
recognize the major components of a compound microscope
understand the functions of these components in distinguishing specimens at different magnifications.
Questions
Using this diagram of the microscope, label the following components:
__E_Ocular lens(es)
__F_Arm
__M_Base
_C__On/off switch
_N__Light adjustment
__J_Stage
_I__Condenser
__K_Aperture (iris diaphragm)
__D_Lamp
__A_Coarse focus knob
__G_Fine focus knob
__L_Slide adjustment knob
_B__Objective lenses
__H_Field diaphragm (field iris)
Complete the table below to describe the function of the following components.
Component Function
Ocular lenses Magnifies the image of the specimen being examined
Light adjustment Control the brightness of the light passing through the specimen.
Stage This is platform in order to light pass through from the light source to the lens.
Condenser To focus light on the specimen.
Aperture (iris diaphragm or condenser diaphragm) Hole in the stage which the light source can reach the stage.
Coarse focus knob Moves the stage up and down to focus on the specimen.
Fine focus knob This is inside the course adjustment knob and brings the specimen to a sharper focus, under lower power.
Slide adjustment Moves the slide which the specimen is on around, to look at all parts of the specimen.
10X objective Lowest objective lens, to analyze glass slide samples
100X objective Oil immersion objective lens to observe details of individual cells.
If the ocular lens has a magnification of x10, what would be the magnification of a specimen examined using the x40 objective lens?
40×10= 400x magnification power.
Exercise 2: The Gram Stain (20 marks)
At the conclusion of this exercise you should be able to:
understand the Gram Stain procedure for the identification of bacteria.
distinguish the processes of the Gram Stain in relation to bacterial structures.
Learning Activities
Read “Gram Stain” Section 6 in A photographic atlas for the microbiology laboratory.
For this activity, you will work through a virtual, interactive lab to perform a Gram Stain to identify a suspension containing a mixture of a Gram positive (S.aureus) and Gram negative (E.coli) organisms.Use the link below to access the module and follow the steps to perform your Gram Stain. Once you are done, your resulting Gram Stain will appear for your observations. This module should take no more than 15 minutes to complete.
To access the interactive exercise of performing a Gram Stain go to http://learn.chm.msu.edu/vibl/content/gramstain.html and click on the module tab.
Click “start”
Heat fix the slide: click on the Bunsen burner, pass the slide gently two or three times (1-2 seconds) through the flame. Do not overheat – this will cause distortion of the cells.
Flood the slide with crystal violet for 1 minute
Rinse with H20
Flood the slide with iodine for 1 minute
Rinse with H20
Decolorize with alcohol for 5-10 seconds
Rinse with H20
Flood the slide with safranin for 1 minute
Rinse with H20
View slide you prepared using the microscope tab
Questions
If the Gram stain was performed accordingly, describe what you would normally observe using x1000 magnification?
The x1000 magnification means that a 100x objective lens was used as the eyepiece is 10x. If this gram stain was performed accordingly, then at a high magnification, gram positive bacteria will be purple as it retains the crystal violet colour due to the thick peptidoglycan layer. Gram negative bacteria will appear pink, as it does not retain the crystal violet colour.
Did you see the same results (you may wish to attach a picture of the result to help with your discussion)? If not, what did you observe? List some procedures of the Gram stain that may have contributed to your first attempt.
The pink microorganisms were gram negative E-coli and the purple, gram-positive S. Aureus organisms. The one problem with the procedure is that the slide was heated for 3 seconds, instead of two seconds, which could have contributed to some problems in the overall results. Overall, my results looked the same as was shown.
Explain the cell wall characteristics that enable you to distinguish between E.coli and S.aureus.
Since E-coli is a gram negative, rod shaped bacteria, the cell wall contains a thin peptidoglycan layer, with an outer membrane consisting of lipopolysaccharides, periplasmic space and a peptidoglycan layer, as well as surface proteins. S. aureus is a gram-positive cell wall, it consists of a thick peptidoglycan layer.
Examine the “Examples” section of this module and describe in full the characteristics and arrangement of one Gram negative specimen and one Gram positive specimen.
Streptococcus pyogenes is a facultative, gram positive bacteria that has a cocci shape and occurs in a chain arrangement. This bacteria causes infections such as scarlet fever, tonsillitis, and rheumatic fever. This bacteria is also non-motile and does not form endospores.
Neisseria gonorrhoeae is a gram negative bacteria in a diplococci arrangement. It is non-motile and also non-spore forming. This bacteria causes pharyngitis and gonorrhea.
