Lab 2
|
|
|
| 1. Colony morphology | 1. Tortora, Chapter 4, pages 76-111 |
| 2. Handling cultures | 2. Pierce, pages 5-17; 20-21; 34-35; 40-41 |
| 3. Gram stain | 3. Leboffe, pages 1-4; 9; 27-28; 52-53 |
| 4. Handout |
WHODUNIT???
Don't be clueless!
Just as clinically different disease states present in sometimes distinctive fashion (a sign or symptom that literally "shouts the name" of the disease is said to be 'pathognomic' for that disease), different bacteria, although unable to shout their name, also leave tell-tale clues that distinguish the one from the other. It is your job to hone your powers of observation and to pay attention to patterns. Then, you, too, will be able to correctly identify the etiologic agent, the perpetrator, of the clinical crime.
According to Leboffe's A Photographic Atlas for the Microbiology Laboratory, "identification of a microbe begins with determining some basic characteristics of the organism:"
Gross Appearance of a Microbe's Growth and Colony Morphology
Colony Configuration and Margins
Look at your colony's configuration and its margins.
Round and smooth?
Round and concentric?
Round and wrinkled? Round and branching?
Irregular and spreading?
Branching 
Irregular 
Lobulated
Colony Elevations
Look at the elevations of the colonies.
Overall Appearance of Colony
Look at the appearance of the colonies.
And, finally, without sniffing directly from the plate, note if there is any aroma associated with the colony.
Environmental Requirements of Bacterial Colonies
According to Tortora, metabolism "is the sum of all chemical reactions within a living organism, including anabolic and catabolic reactions." For bacteria to thrive and
colonize, their environment must support their metabolic requirements.
Handling Cultures and Colony Transfer to Agar, Agar Slants, and Broth
Reference material for this portion of the lab is found on pages 4-18 in Pierce & Leboffe's Exercises for the Microbiology Laboratory.
Materials:
Inoculating loop
Glass slide
Slide holder
Wax pencil
Glass-marking pen
Test tube rack
Petri dish containing culture from last week
Rodac dish containing culture from last week
Bunsen burner
Flint lighter
Sterile petri dish, sterile slant, sterile nutrient broth
Instructions for Agar Streak Method Using Aseptic Technique for Colony Transfer:
1. Using the wax pencil, label the sterile petri dish, sterile slant, and sterile nutrient broth with your initials or some other unique identifier, today's date, and the lab section number.
2. Place the sterile slant and nutrient broth in the test tube rack. Lay the sterile petri dish on the rack.
3. Using the glass marking pen, label two glass slides with your initials and lay these on the rack.
4. Turn on the Bunsen burner.
5. Sterilize the inoculating loop by flaming it in the Bunsen burner flame. Allow it to cool.
6. Look at the petri dish containing the culture from last week. You will be holding the petri dish in your non-dominant hand. Find a single colony of bacteria that is not touching any other colony.
7. Holding the sterile inoculating loop by the black handle in your dominant hand, like a spoon, carefully pick up the selected colony. Now return the petri dish to its lid.
8. With your non-dominant hand, hold the new sterile petri dish, open. Begin to streak. You will re-sterilize the inoculating loop between each step of the streaking process.
9. To streak, do NOT follow the instructions from Pierce and Leboffe. Instead:
a. Visually (in your mind only) separate the agar into four quadrants. We will call these quadrants 'a,' 'b,' 'c,' and 'd.'
b. Beginning with quadrant 'a,' streak with your loop in a zigzag fashion, making sure to stay inside this quadrant, moving forward and never backwards and not lifting your loop up until done.
c. Lift up the loop. Turn the agar 90o. Move the loop ONCE through quadrant 'a,' dragging a line into quadrant 'b.' Zigzag forward, being careful to stay inside quadrant 'b' and never going back into quadrant 'a' and not lifting your loop up until done.
d. Lift up the loop and turn the agar another 90o. Again, move the loop ONCE through quadrant 'b,' dragging a line into quadrant 'c.' Zig zag forward, being careful to stay inside quadrant 'c' and never going back into quadrant 'b' and not lifting your loop up until done.
e. Lift up the loop, and for the last time, turn the agar another 90o. Move the loop ONCE through quadrant 'c,' dragging a line into quadrant 'd.' Zigzag forward, being careful to stay inside quadrant 'd' and not lifting your loop up until done.
10. Return the dish to its lid.
11. Sterilize your inoculating loop and rest it in your test tube rack.
See the illustration below for help with the instructions above.
What is the purpose of the agar streak method? By performing the streaking of the bacterial colony in the above manner, the mass of the bacteria on the loop is diluted out through the four quadrants. It is greatest in quadrant 'a,' and least in quadrant 'd.' When performed correctly, quadrant 'd' will yield a number of isolated colonies upon which biochemical testing for identification can be performed. In addition, antibiotic susceptibilities and serologic studies can be performed as well. This area is also useful for gram staining samples and for observing colony morphology. The above method is termed streaking for isolation.
Instructions for Slant Streaking Method Using Aseptic Technique for Colony Transfer:
1 Look at the petri dish containing the culture from last week and find another single colony of bacteria that is not touching any other colony. Replace the lid.
2. Sterilize the inoculating loop by flaming it in the Bunsen burner flame.
3. Holding the sterile inoculating loop by the black handle in your dominant hand, like a spoon, allow it to cool and then carefully pick up the selected colony.
4. With your non-dominant hand, lift up the nutrient slant.
5. While still carefully holding the inoculating loop, use your ring and pinky fingers to curl around the cap to the slant and pull it off.
6. Flame the mouth of the slant.
7. Carefully insert the inoculating loop to the butt-end of the slant. Do not pierce the slant.
8. Without piercing the agar, zigzag upwards (towards the mouth of the tube), covering the slant wedge.
9. Flame the mouth of the slant and replace the cap.
10. Return the slant to the test tube rack.
11. Sterilize your inoculating loop and rest it in your test tube rack.
What is the purpose of the slant streak method? The main purpose for streaking a colony of bacteria onto a nutrient slant is for storage of microbiologic stock. 'Stock' here means a stock of a pure culture of bacteria. Microbiologists must have a supply of certain strains of bacteria. In order to maintain a constant stock source, they must be careful to re-streak new slants before the nutrients in the present stock are consumed by the bacteria. If they fail to do that, they risk losing that particular line of bacteria.
Instructions for Broth Streaking Method Using Aseptic Technique for Colony Transfer:
1 Look at the petri dish containing the culture from last week and find another single colony of bacteria that is not touching any other colony. Replace the lid.
2. Sterilize the inoculating loop by flaming it in the Bunsen burner flame.
3. Holding the sterile inoculating loop by the black handle in your dominant hand, like a spoon, allow it to cool and then carefully pick up the selected colony.
4. With your non-dominant hand, lift up the nutrient broth.
5. While still carefully holding the inoculating loop, use your ring and pinky fingers to curl around the cap to the broth and pull it off.
6. Flame the mouth of the broth.
7. Carefully insert the inoculating loop into the broth, near the its surface.
8. Stir the loop around and rub the loop against the side of the tube in order to dislodge the colony.
9. Remove the inoculating loop, flame the mouth of the broth, and replace the cap.
10. Place the tube of broth back into the test tube rack.
11. Sterilize your inoculating loop and rest it in your test tube rack.
12. Gently agitate the tube of broth in order to suspend the bacteria (see page 7 of Pierce and Leboffe's Fundamental Skills for the Microbiology Laboratory).
What is the purpose of the broth streak method? The main purpose for streaking a colony of bacteria into a tube of nutrient broth is so that large quantities of bacteria can be grown in a very short period of time.
Staining Characteristics of Bacteria
Bacteria are microscopic and transparent. Thus, a microscope and stain are necessary in order to visualize them. There are different types of stains and different reasons for using particular stains. Probably the most important stain is the Gram stain. It is a differential stain. 'Differential' means that the stain is used to differentiate or distinguish bacteria, based on their response to the stain.
Why do some cells take up the stain (are Gram positive) and others become decolorized and counterstained (are Gram negative)?
Take Out Food for the Brain:
According to the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, nurses and doctors wash their hands only 30% of the time after seeing patients or performing patient procedures. The CDC also reports that greater than 2,400,000 patients develop nosocomial infections each year in our country. These infections are the direct cause of death for 30,000 of these patients and are indirectly linked to 70,000 other patients' deaths. Annual costs for extended care and treatment of nosocomial infections total $4.5 billion dollars.
According to Health, November/December 1996: "Medicine's Dirty Little Secret," "more Americans die from hospital infections every year than from car wrecks and homicides combined."
Is there a relationship between poor handwashing techniques and hospital-acquired infections? You tell me!
Take Home Thought
-- US CENTERS FOR DISEASE CONTROL
--