Lab Notes for Exam 1 Section

Ex. 2-1: diversity and ubiquity of microoganisms

Purpose: Microorganisms are found every where in the environment around us. To demonstrate this and to get a taste of the different types of organisms in our environment, we can culture these microorganisms by collecting them on a swab and transferring them to an agar plate.

Media & Materials: 1 Tryptic Soy Agar (TSA) plate, 1 sterile swab and 1 tube of sterile saline per person

Procedure:

1.  We will use a simplified version of the lab manual protocol. The lab manual describes several methods for sample collection. Confer with your lab partners at your table so that each person uses a different method. Your table should have a total of 4 plates (5 if you have an extra person at your table).

2.  Label the plate with your name, today's date, and the source of inoculum to be collected (for example, desktop, doorknob, faucet, hair, skin, etc.). You may also use any public body surface except for your mouth (this means any areas of skin that you might normally expose in polite company). Transfer any microbes you may have picked up to the agar plate by gently swabbing the surface of the plate with the swab. The instructor will demonstrate the method for spreading the cells on the plate.

3.  The plates will be incubated at 37ºC until next lab period.

4.  Observe your plates during our next lab meeting. On page 487, make note of the various colors, textures, and shapes that are produced when microscopic organisms are allowed to reproduce in large numbers. The individual areas of growth you see are colonies and consist of millions of identical cells that all arose from a single parent cell.

5.  Be sure to dispose of your cultures in the Biohazard waste containers when your observations are completed.

EX 2-15: EFFECTIVENESS OF HAND SCRUBBING

Purpose: This experiment will allow you to observe the effectiveness of soap in normal hand washing. Each student will test his/her hands before and after hand washing. One student in your group will use soap when washing his/her hands, and the other student will use plain water.

Media: 2 TSA plates per pair of students

Procedure- Follow the directions on page 61 of the lab manual:

  1. Each student will use 1 plate. Divide the plate into 4 quadrants using a lab marker. Label the top two quadrants of the plate “Before” and the bottom two “After”.
  2. One lab partner will soap, the other will use plain water. Label the left and right side of the plates with your initials. Be sure that you do not touch anything besides a clean paper towel after you wash your hands and before you inoculate your plate. We will not be using sterile cotton.
  3. The plates will be incubated at 37ºC until next lab period.

Ex. 1-3: Aseptic Transfer and inoculation Techniques

Since this is the first time you will be working with bacterial cultures, the procedure for handling and transferring microorganisms will be described in detail. Aseptic technique, the procedure used to prevent contamination, is carried out so that you, your neighbors, and your belongings are not contaminated by the microbial culture and so that microbes from the environment do not contaminate your bacterial culture.

Purpose: In this experiment you will be transferring living cells grown on two different types of media: broth and slant. You will use these cells to inoculate a fresh slant and a fresh broth culture.

Cultures: a broth culture of Serratia marcescens and an agar slant culture of Serratia marcescens

Media: 1 tryptic soy agar (TSA) slant and 1 tryptic soy broth (TSB) per student

Procedure:

  1. Before beginning, label all tubes of sterile media with the name of the organism, the date, and your names or initials. ALWAYS label before inoculating.
  2. Follow the protocol described in the manual. We will be using only one organism today, Serratia marcescens. This organism produces a red pigment when incubated under the appropriate conditions. This red or pink color will help you to determine if your aseptic transfer has been successful when you look for growth next lab period. You should not see any pink or red color in your freshly inoculated tubes today.
  3. Use the broth culture of Serratia marcescens to inoculate the slant. Use the slant culture to inoculate the broth.
  4. Your instructor may demonstrate some techniques that are slightly different from the ones described in the lab manual. These variations are acceptable as long as they are safe and produce the desired results. If you feel confused by any differences, ask for clarification.
  5. After you replace the screw top on a culture tube, back off the top about 1/2 turn before placing the tube in the incubator in order to allow air to circulate. Do not incubate cultures with the top screwed down tightly. Those bugs need to breath, just like you!
  6. Incubate all cultures at 37ºC until next lab period.
  7. During the next lab meeting, make your observations on the data sheet located near the back of your lab manual. Dispose of your cultures in the appropriate Biohazard container as directed by your instructor.

EX. 1-4: STREAK PLATE ISOLATION OF PURE CULTURE (PART 1)

Purpose: A pure culture is one that contains a single type of organism. You must first isolate single colonies in order to cultivate a pure culture. Single colonies contain identical cells that have arisen from a single cell and are genetic clones of each other. Single colonies can only be obtained by spreading single cells far apart from each other on the surface of an agar plate, then allowing them to grow. There are several ways to separate single cells, but we will only be using the streak plate method using an inoculating loop.

Cultures: a mixed broth culture containing both Serratia marcescens and Staphylococcus aureus and a mixed broth culture containing both Escherichia coli and Micrococcus luteus.

Media: 2 TSA plates per pair of students

Procedure: The Streak Plate Method

  1. Each student will inoculate a mixed broth culture onto a separate agar plates according to the streak plate procedure described by the lab manual and demonstrated by the instructor. Work with your lab partner so that each of you streaks a different mixed culture.
  2. When you make the streaks across the plate, do them gently so that you do not dig into the agar with the loop. This will cause growth to occur in streaks, rather than in single colonies.
  3. Incubate your plates upside down (lid on the bottom, agar on top) in a wire basket at 37ºC for until next lab period. Baskets can be shared between lab partners. These plates are considered mixed cultures because they will contain two different types of colonies.
  4. Next lab meeting: make your observations on the data sheet pages. Use blank paper if you need additional space. Include a drawing and use the information in Ex. 2-2 to write a description of a colony from each different type of organism. Observe the plates for visible differences in colony morphology (size, color, shape, elevation, margin, texture, optical characteristics). Did you get nice isolated single colonies? They should be spaced far enough apart so that you can pick up an without touching more than one colony using a loop. Serratia marcescens colonies should be pink or reddish. Staphylococcus aureus form smaller, white colonies. Escherichia coli produces beige colonies 2 to 3 mm in diameter, while Micrococcus luteus will produce smaller yellow colonies.

EX. 1-4 (and EX. 2-2): STREAK PLATE ISOLATION OF PURE CULTURE (Part 2)

Purpose: Isolate a pure culture from mixed culture plates by restreaking a fresh streak plate from a single isolated colony

Organisms: Last period’s mixed culture plates with isolated colonies from Ex. 1-4.

Media: 4 TSA plates per pair of students

Procedure:

  1. Label fresh plates with the names of the four organisms found on your mixed culture plates, your initials, and the date. Look at the two streak plates that you and your lab partner prepared from mixed cultures the last lab period. Choose single colonies that are well separated from the others so that they are easy to pick up.
  2. From a mixed culture plate, aseptically touch the edge of the sterile loop to the colony. It is not necessary to scoop up the entire colony. You should not transfer a visible quantity of inoculum to the fresh plate. Using the streak plate dilution method that you used last week, streak a fresh, labeled plate with the appropriate colony. Remember to flame the loop in between streaking each quadrant of the plate. If the colonies are very small and close together, you can use a needle to pick up cells. Note: Make sure that you pick up your inoculum from only one colony.
  3. Use the same method to streak plates of all four organisms.
  4. Place the plates inverted in a basket. Incubate the four plates at 37ºC until next lab period.
  5. Next lab period: You will use these plates as part of Ex. 2-2, Colony Morphology. Record your observations on your data sheet. Use additional paper for drawings or other information. Each plate should have only one type of colony.

Ex. 2-3: Growth patterns on slants

Purpose: Observe possible differences in morphology between different bacterial species grown on slant media

Organisms:

• Bacillus subtilis and Mycobacterium smegmatis

• Last period’s mixed culture plates with isolated colonies from Ex. 1-4

Media: 6 TSA slants

Procedure:

1. Label each slant tube with the date, organism name, and your initials.

2. Using the aseptic technique for inoculating slants that we learned last week, inoculate each tube.

For Serratia marcescens, Micrococcus luteus, Escherichia coli, Staphylococcus aureus, carefully pick up cells of a single colony from your mixed plates. Normally, pure cultures would be used for transferring organisms to slants or broths, but we are using the mixed plates due to time considerations. If you do not have sufficient single colonies of each organism, there will be some pure culture plates available as well.

3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. For your uninoculated control, use a fresh TSA slant, but return it to the cart when finished.

Ex. 2-4: Growth patterns in broth

Purpose: Observe possible differences in morphology between different bacterial species grown in broth media

Organisms:

• Bacillus subtilis and Mycobacterium smegmatis

• Last period’s mixed culture plates with isolated colonies from Ex. 1-4

Media: 6 TSB broths

Procedure:

1. Label each broth tube with the date, organism name, and your initials.

2. Inoculate these tubes with the same cultures used in Ex. 2-3. Again, be especially careful when transferring cells from your mixed culture plates.

3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. For your uninoculated control, use a fresh TSB broth tube, but return it to the cart when finished.

Ex. 2-2: colony morphology

Purpose: Observe possible difference in colony morphology between different bacterial species grown on solid culture media.

Organisms: broth cultures of Bacillus subtilis and Mycobacterium smegmatis

Media: Two TSA plates per pair

Procedure:

1.  With a sterile loop, inoculate Bacillus subtilis and Mycobacterium smegmatis onto separate TSA plates using the streak plate method. Note: Be careful to avoid cross-contamination between plates. Be especially conscientious in flaming your loop after transferring any Bacillus species, which produces spores. Make sure that the loops are flamed long enough to glow orange. Take the time to flame the loops and go through the procedures slowly and carefully. Report any spills to the instructor so that proper cleanup can occur.

2.  You have already streaked plates with the other organisms from Ex. 1-4. Incubate all the plates 37ºC until next lab period. The Mycobacterium and Micrococcus cultures may require additional incubation time. Therefore, if the growth is scant and the colonies are very small, incubate these cultures for 1 to 2 more periods.

3.  Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. There may be demonstration plates of other organisms to view as well. Be sure to include observations on these cultures in your results.

WHEN YOU ARE FINISHED MAKING ALL YOUR OBSERVATIONS, CULTURES SHOULD BE DISPOSED OF IN YOUR RED AUTOCLAVE BAGS AND THOSE BAGS PLACED IN THE LARGE GRAY BIOHAZARD WASTE CAN.

Ex. 2-7: Fluid Thioglycollate: Atmospheric Oxygen Requirements

Purpose: Observe the growth patterns of different organisms according to their oxygen requirements

Organisms: Broth cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Neisseria sicca, and Clostridium butyricum

Media: Four tubes of thioglycollate broths and one tube of supplemented thioglycollate per pair

Procedure:

1. Thioglycollate is a reducing agent that removes oxygen from the broth. Label the tube of SUPPLEMENTED thioglycollate for the organism Clostridium butyricum (don't forget your initials and the date). Label the other tubes for each of the remaining organisms.