Name:
Partner's name:
Section:
Introductory Microbiology Prelab Assignment
Widespread Distribution of Bacteria
1. (1pt.) A turbid culture in liquid media signifies what?
2. (1pt.) List the two purposes for preparing a smear?
3. (1pt.) List and describe the three basic shapes of bacteria?
4. (1pt) Define simple staining.
5. (1pt) Why are most stains positively charged?
NOTE: BRING YOUR MICROSCOPY LAB WITH YOU TO THIS LAB EXERCISE. YOU WILL NEED THE
INSTRUCTIONS FOR USING THE SCOPE.
1
2
Lab Exercise
Widespread Distribution of Microorganisms
09/11/14
All cellular life, large and small, can be classified into three Domains. Microorganisms (microbes)
include a wide variety of organisms too small to be seen with the naked eye. The Domain Bacteria and
the Domain Archaea consist entirely of microscopic prokaryotes. Although prokaryotes have DNA, no
true membrane-bound nucleus is present. The third Domain, Eukarya, includes several groups containing
microorganisms). All Eukarya have a true nucleus.
The smallest microorganisms, the viruses, are neither prokaryotic nor eukaryotic. They are noncellular
microorganisms that must invade a cellular organism to replicate.
Classification of Microorganisms
Domain Bacteria: Several thousand species of Bacteria are known. Bacteria are classified in a number of
phyla, but can be characterized in the following major groups:
1. Gram negative (may include the cyanobacteria)
2. Gram positive
3. Mycoplasmas (do not have cell walls)
4. Cyanobacteria (although often referred to as blue-green algae, they are bacteria!)
Domain Archaea: Archaea have characteristics distinct from the Bacteria. Several groups are as follows:
1.
2.
3.
4.
Extreme Thermophiles (extreme heat-loving)
Thermoacidophiles
Extreme Halophiles (salt-loving)
Methane-producing
Domain Eukarya:
Kingdom Protista
There are two Subkingdoms in the Kingdom Protista: the Algae and the Protozoa. Microscopic algae, or
phytoplankton, are primitive plant-like organisms. Protozoa, on the other hand, have many characteristics of
animals. Protozoa are also microscopic (and often pathogenic) and are included in the zooplankton.
Kingdom Fungi
Fungi consist of macroscopic and microscopic forms. The microscopic fungi consist of the yeasts and the molds.
Some fungal species consist of a mold AND a yeast form depending on the environment in which the fungus is
found.
Viruses:
Viruses are the smallest microorganisms and cannot be seen with an ordinary light microscope. The International
Committee on Taxonomy of Viruses developed a classification system for viruses. Although hundreds of viruses
are as yet unassigned, over 3,600 viral species have an approved classification.
Exercise Objective: Demonstrate the widespread distribution of microorganisms. Bacteria are the most commonly
isolated microbes during this procedure.
3
The majority of the laboratory exercises performed in this course utilize cultures of bacteria. These
cultures are usually pure (only one species of bacteria is present). They must remain pure for the successful
execution of the experiment. If one is not constantly aware that bacteria (and other microbes) are present
everywhere, the possibility of contamination (unwanted introduction of microbes) is very high. In order to prevent
contamination, a collection of techniques is used. These skills are referred to as aseptic technique. Your success
in this course will be determined, to a great extent, by your mastery of aseptic technique. Aseptic technique also is
essential to the safety of the students in this lab and those students who will use the laboratory after you.
A secondary objective of the exercise is to stain (apply dyes to) bacteria for viewing under the
microscope. Since bacteria are basically transparent, applying stains is crucial in order to observe many of the
important morphological characteristics of the cell. For example, bacterial classifications include cell shape.
Bacteria have three primary shapes: bacillus (rod-shaped), coccus (spherical), and curved (comma-shaped or
spiral).
Procedure
Always label plates and tubes with all group members' initials, the lab section, type of media, and other pertinent
information required by the lab.
Day 1
Materials: per group of 2 students
1 plate of trypticase soy agar (TSA)
1 tube of trypticase soy broth (TSB)
2 sterile cotton swabs
distilled water in small beakers
1. Label the BOTTOM of your plate with your assignment, your and your partner's initials, and the lab section.
Moisten the swab with distilled water. Wipe the swab over a part of your body or some object. Gently smear the
surface of the agar with the swab.
Invert the plate, and incubate for 48 hours at 37C. Plates are always incubated in an inverted position to
prevent condensation from dripping on the colonies.
Exposure method:
2. Label the tube with your and your partner's initials and the section. Dip the swab into the distilled water.
Repeat the above procedure, but return the swab to the tube of broth. Replace the cap on the tube. You
may have to break off part of the swab's stick.
Incubate for 48 hours at 37C.
Exposure method:
Students might use the same or different exposure methods, according to the directions from the instructor. Be
sure to indicate your exposure method in the lab report.
4
Day 2
Materials: per individual student
1 microscope slide
1 inoculating loop
microscope
immersion oil
lens paper
bunsen burner and striker
DEMO: sterile tube of broth
coverslip
clothespin
KimWipes
safety glasses, apron, gloves
distilled water in small wash bottles
distilled water in small beakers
stain: methylene blue
1. After 48 hours of incubation, observe the plates and tubes. Colonies should be seen on the plates, and the tubes
should appear turbid (cloudy-indicates growth).
Compare the tube with an uncontaminated (sterile) tube.
Observe the appearance of the colonies on the plate and their number.
Number and appearance of colonies:
2. Smear preparation:
Many types of staining procedures require a smear preparation in order to kill the microbes and fix them on the
slide. At all times, aseptic technique must be observed! There are two types of smear preparations: one from
liquid media and one from solid media. ONLY PERFORM ONE OF THE PROCEDURES; either from liquid OR
from solid media. However, be prepared to perform both techniques during future labs.
Smear preparation procedure from liquid media:
1. *Flame the loop the length of the wire just until it glows red hot. Cool ~15 seconds.
*DO NOT SET THE LOOP DOWN OR IT WILL HAVE TO BE FLAMED AGAIN!
2. Gently shake the tube to disperse the organisms.
3. Remove the cap from the tube.
4. *DO NOT SET THE CAP DOWN! Immediately *flame the neck of the tube.
5. Remove one loopful of the media, and place it in the center of the slide.
6. Disperse the liquid (with the loop) in a small, dime-sized circle in the center of the slide.
7. *Flame the neck of the tube again, and replace the cap of the tube.
8. *Flame the loop.
9. Allow the smear to dry COMPLETELY at room temperature.
10. Attach one end of the slide to the clothespin, and pass the slide through the flame several times to heat-kill
and fix the organisms to the slide.
11. You may now stain the specimen.
*aseptic technique
Smear preparation procedure from solid media:
1. *Flame the loop the length of the wire only until it glows red hot. Cool ~15 seconds. *DO NOT SET THE
LOOP DOWN OR IT WILL HAVE TO BE FLAMED AGAIN!
2. Place one loopful of distilled water in the center of the slide. *Flame and cool the loop again.
3. Remove the lid of the petri plate. *DO NOT SET THE LID DOWN!
5
4. Gently touch the center of one of the bacterial colonies with the loop. ONLY A VERY SMALL AMOUNT
OF BACTERIA IS NEEDED. Replace the lid on the petri plate.
5. Disperse the bacteria on the loop in the water so that you have a dime-sized specimen in the center of the
slide. *Flame the loop again.
6. Allow the smear to dry COMPLETELY at room temperature.
7. Attach one end of the slide to the clothespin, and pass the slide through the flame several times to heat-kill
and fix the organisms to the slide.
8. You may now stain the specimen.
3. Simple staining with methylene blue
When only one stain is used, the procedure is called simple staining. Most stains are cationic (positively
charged). This allows the stain to adhere to the cells because the cells are slightly negatively charged (anionic).
Procedure:
1. Cover the smear with methylene blue for one minute over the sink or appropriate container.
2. Wash the stain briefly (2 seconds) with the distilled water in the wash bottle. Do not squirt the water directly
on the specimen! Let the water flow over the smear.
3. Blot the stain carefully with KimWipes. Be gentle. Dry the bottom of the slide.
4. Put a coverslip over the specimen on the slide.
5. View the specimen under the microscope. Follow the procedure in the Microscopy lab.
In future labs we will be making more stains which require a smear. Please bring these lab instructions with
you to each.
*aseptic technique
5
6
Name:
Partner's name:
Section:
Solid Media Exposure Method:
Liquid Media Exposure Method:
Introductory Microbiology Lab Report
Widespread Distribution of Bacteria
Complete the checklist on the following page when finished viewing. Return it to the instructor.
1. (1pt.) Draw the cells in the circle.
2. (1pt.) What color are the cells?
3. (1pt.) What shape(s) are the cells?
4. (1pt.) List two aseptic techniques used in this lab.
5. (1pt) As part of the procedure, you were not to set the lid of the petri plate or the tube cap down on the lab
bench. Why is this?
7
Student Microscope Checklist
When finished using the microscope, complete the following.
Check off each step when completed.
Name:
Date:
Lab Section:
Microscope Number: HCC
Bring the stage to its lowest level.
Click the 4X objective into place.
Remove the slide and dispose of appropriately. Wipe prepared slides until ALL oil/dirt is
removed (top and bottom).
Clean objective lenses with swabs, and, if using oil, liquid lens cleaner.
Clean eyepieces with dry swabs.
Clean condenser lens with dry swabs.
If necessary, clean the stage with a damp Kimwipe.
Turn the light switch OFF. Do not move the microscope for a few minutes
before putting it away.
Turn the light switch OFF. Do not move the microscope for a few minutes
before putting it away.
Rotate the head so that the eyepieces are facing forward (away from the arm).
Replace the dust cover.
Return the microscope to the appropriate location in the cabinet.
Check to be sure all waste is removed from the sink and floor.
8