Isolation of Microorganisms from an Environment of Choice

Objective
In order to demonstrate the ubiquity and diversity of microbes in the environment,
samples from immediate areas of the environment and/or from your body will be
obtained and cultured.

Introduction
Microorganisms are found throughout the environment: in the air and water; on
the surface of any object such as clothes, walls, furniture; in soil and dust; and on and in
our own bodies (skin and mucous membranes). Microbes are even found in extreme,
seemingly inhospitable environments: living and thriving microbes are common in hot
springs with temperatures in excess of 95°C, in Antarctic seawater below 0°C, and in
acid mine waters whose pH is below 4.
Ordinarily these ubiquitous microorganisms are of no threat to healthy humans,
and quite a few are actually beneficial. Normal flora on and in our body act as an initial
defense against invading pathogens. Normal flora compete with foreign microorganisms
for nutrients and space, thereby preventing them from flourishing. In the absence of the
normal flora (e.g., due to prolonged antibiotic treatment), opportunistic microorganisms
may become established, increasing the risk of developing disease. In the environment,
many organisms in soil play important geochemical roles in processing vital elements
such as phosphorus and nitrogen, making them available to other living things.
Nonetheless, there are organisms in our environment that are harmful: soil also harbors
the spores of the tetanus and botulism bacteria.

Materials
4 sterile culture swabs
Sterile water in a test tube
2 TSA plates (trypticase soy agar - general purpose nutrient medium that promotes
the growth of bacteria)
2 SDA plates [Sabouraud’s dextrose agar – a medium that promotes the growth of
fungi (yeast and molds) but which is inhibitory for many bacteria]
2 tubes of nutrient broth
A few sterile tongue depressors for throat swabs

BISC 300L, Fall 2002

Laboratory Exercise
Isolation of Microorganisms from the Environment
adapted from Martin, Fall 1999 Page 1 of 6
Possible samples:
Human body Environment
Throat Bench top (before and after disinfection)
Skin Sink basin
Scalp Water tap
Teeth or gums Floor
Ear Coin
Under the nails Doorknob
Between toes Windowsill
Armpit Air in the lab (exposed plate)

Procedure

1. Determine 2 sampling sources, one from your body and one from the
surrounding environment.

2. With a marker, label the bottom of the plates and the side of the tubes with the
date, the name of the sample to be inoculated (e.g. bench top, skin), and your
initials.

3. Open the “stick” end of the sterile culture swabs to avoid contamination of the
swab. Swab samples and inoculate each type of media (plates and broth) with
your samples. Use a fresh swab for each plate and broth.

a. For plates, gently, so as not to mess up the agar surface, rub the swab over
the entire surface of the Petri dish without going back over areas you have
already swabbed.
b. For broth, dip the swab into the media and shake it slightly (or twirl it between
your fingers) to disperse the sample into the media

- If you take a swab from a dry surface (e.g. skin, bench top) remove the sterile
cotton swab from the package and immerse it in the sterile water. Wring out
the excess water on the inside of the test tube and rub the swab over about 4-
6 cm2 of your test area.

- For a source such as the throat that is moist to begin with, it is not necessary
to pre-moisten the swab. Be especially careful with samples from human
sources. Discard the used swab in the Biohazard waste.
- For throat swabs, have your partner open his/her mouth wide. Gently,
but firmly press down on the tongue with a sterile tongue depressor
and gently inset the dry swab past the soft palate, past the uvula (the
little flap of flesh that hangs down at the back of your throat) to the
tonsil area. Touch the swab to a few spots at that area, withdraw the
swab without touching the tongue, and swab the plate

BISC 300L, Fall 2002

Laboratory Exercise
Isolation of Microorganisms from the Environment
adapted from Martin, Fall 1999 Page 2 of 6
 - For sampling the air, remove the lids from the plates and the caps from
the broth and allow them to sit uncovered for 45 min. Then replace the
lids/caps.

4. Incubate all plates inverted:

- SDA plates at 30°C
- TSA plates at 37°C

5. Incubate broth cultures at 37°C.

Analysis –Macroscopic Observation of Microorganisms (Laboratory Exercise 1)

1. Examine your plates (and the plates of your classmates).

2. Count, if possible, or estimate the total number of organisms per plate.

3. Determine the number of different organisms based on differences in colony

types (see below).

4. Describe morphology: color, size, shape, texture, margin or edge, elevation.

BISC 300L, Fall 2002

Laboratory Exercise
Isolation of Microorganisms from the Environment
adapted from Martin, Fall 1999 Page 3 of 6
Laboratory Report

1st environment sampled _________________________________________________

Number of organisms ______________ Number of different organisms _____________

Colony Description
Colony Type I Colony Type II Colony Type III

Color

Size

Shape

Texture
Margin or
Edge

Elevation

Nutrient Broth Growth Description

Colony Type I Colony Type II Colony Type III

Surface Growth

Sediment

Overall

BISC 300L, Fall 2002

Laboratory Exercise
Isolation of Microorganisms from the Environment
Page 4 of 6
2nd environment sampled _________________________________________________

Number of organisms ____________ Number of different organisms _____________

Colony Description
Colony Type I Colony Type II Colony Type III

Color

Size

Shape

Texture
Margin or
Edge

Elevation

Nutrient Broth Growth Description

Colony Type I Colony Type II Colony Type III

Surface Growth

Sediment

Overall

BISC 300L, Fall 2002

Laboratory Exercise
Isolation of Microorganisms from the Environment
Page 5 of 6
Questions

1. Which environment seems to harbor the greatest variety of microbes?

2. As the semester progresses, you will on many occasions be working with pure
cultures of various types of microorganisms. What measures can you take to
prevent microbes from the environment from contaminating your culture?

3. As mentioned in the introduction, normal flora microorganisms are those

commonly found in or on a particular area of the body. Based on your
observations, can you come to any conclusions about the normal flora of the
skin? throat? scalp? Do you know what organisms constitute the normal flora of
human blood? peritoneal fluid? lower digestive tract?

BISC 300L, Fall 2002

Laboratory Exercise
Isolation of Microorganisms from the Environment
Page 6 of 6