Notes for Ex. 2-1, 1-3, 1-4
Week 1
Tuesday
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 (NOTE: TSA will be used instead of the nutrient agar described in the lab manual, pg.
58).
Procedure:
1. We will use a simplified version of the lab manual protocol. 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.
2. The plates will be incubated at 37ºC for 48 hours.
3. THURSDAY: Observe your plates during our next lab meeting. 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.
4. Be sure to dispose of your cultures in the Biohazard waste containers when your
observations are completed.
Notes for Week 1
page 2
Thursday
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.
EX. 1-3: ASEPTIC TRANSFER AND INOCULATION TECHNIQUES
Purpose: In this experiment you will 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
•
An agar slant culture of Serratia marcescens
Media: 1 tryptic soy agar (TSA) slant and 1 tryptic soy broth (TSB) per student (NOTE: TSA
will be used instead of nutrient agar and nutrient broth, pg. 37)
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) 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 on Thursday. You should not see any pink or red color in your freshly inoculated
tubes today.
Common aseptic (sterile) transfer technique is described in Exercise 1-3 of the Lab
Manual (pages 18 – 24). There are some significant differences in the way I prefer for
you to handle the cultures. Please pay attention to the demonstration for the preferred
methods.
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, loosen 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 37°C for 48 hours. This week we will move your cultures to a
refrigerator for you to preserve them until next Tuesday.
Notes for Week 1
page 3
EX. 1-4: STREAK PLATE ISOLATION OF PURE CULTURE
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
•
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 37°
C. Baskets can be shared between table partners. These plates will be incubated for 48
hours, then moved to the 4º refrigerator until Tuesday of next week, when you will use
them as the stock cultures for Ex. 2-2, 2-3, and 2-4. These plates are considered mixed
cultures because they will contain two different types of colonies.
4) TUESDAY OF WEEK 2: Observe the plates for visible differences in colony
morphology (size, color, shape, elevation, margin, texture, optical characteristics – see
pg. 37). Did you get nice round 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.
5) Follow the instructions found in Lab Notes for Week 2 to complete this exercise.