Asignatura: Microbiología

Departamento: Química

Profesores: Pedro F. de Brito Brandão

Carlos Yesid Soto O.

Laboratorio: Bioquímica 330

Practica 1. Técnicas asépticas y métodos básicos para cultivo de microorganismos.

Objectives
Students will learn basic aseptic technique used universally in microbiology
laboratories, as well as safety precautions. Students will test different sources of
bacterial growth, from their own body or their environment, to find out where bacteria
considered “normal flora” are growing.

Basic aseptic techniques and rules in the Microbiology Laboratory

The most important goal when working with microorganisms is to prevent the
organisms from causing an infection. The word aseptic means preventing sepsis, the
presence of bacteria, their toxins and their waste products in our blood and tissues. One
of the major advances in microbiology and medicine of the 19th Century was the
development of these aseptic techniques to prevent infection in patients and health care
workers.

In this class we will only work with common bacteria that we encounter on a regular
basis with no harmful effects. However, when we grow bacteria in pure culture, they
will be present in huge numbers. Introduction of these bacteria into inappropriate
locations (under your skin by a piece of broken glassware or into your eye by careless
rubbing) could result in a nasty infection. Safety is always the number one concern in
laboratory, but that concern is magnified when working with bacteria.

Following is a short list of dos and don'ts of aseptic techniques that we will follow in
this laboratory. Many of these will seem like common sense, but you would be amazed
how often these rules get broken by careless or inattentive lab workers.

• Absolutely no food or drink in the laboratory. This includes gum.

• Never allow anything to get near your face, especially your hands. Many germs
are transmitted by the hands, especially when the eyes are rubbed.
• Use a disinfectant solution to clean your work area before and after the
laboratory exercise.
• Never wave around in the air the tools you'll be using to move bacteria. This will
cause sterile instruments to become contaminated or, if there are bacteria on the
instruments, they will be tossed about in the air.
• Know where all contaminated samples are to be discarded and discard used
materials promptly and to the correct locations. Specific instructions will be
provided by your instructor

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Preliminary preparation:
1) Wash hands and table tops before beginning any lab protocols today
2) Wash hands after swabbing anything
3) Wash hands and table tops after lab protocols are complete

(A) Loop Transfer (transferencia con el asa microbiológica)

The purpose of this exercise is to learn “aseptic technique,” a method used in every
microbiology lab setting. Aseptic technique is a method that:
(1) prevents bacterial contamination of the environment (including you);
(2) prevents environmental contamination of the bacterial culture.
It may feel tedious and awkward, especially if you are not dexterous, but you should
strive for excellence in this method above all others. Here, we will use a LOOP to
transfer media from one test tube to another test tube.

Materials:
1) Each student will receive two tubes
with Tryptic Soy Broth (TSB)

Procedures (see figure 1):

1) Label your two tubes

2) Hold loop like a pencil and flame

whole wire portion red hot. Cool 10
seconds.

3) Pick up a tube in other hand and

remove cap with pinky of hand
holding loop (DO NOT SET CAP
DOWN.)

4) Flame mouth of tube 2-3 seconds and

then place loop down tube to touch the
liquid media. Carefully withdraw loop
and re-flame mouth. Replace cap.

5) Set down first tube. Pick up second

tube. Remove cap, flame lip, and
deposit loop contents.

6) Flame loop and repeat procedure,

passing back and forth 2-3 times.

7) Place all tubes in the incubator.

Figure 1 – Loop transfer technique.

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(B) Streak Plating (Siembra por estría)
As you will discover from your ubiquity plates, there are LOTS of different kinds of
bacteria everywhere. In order to study a particular species or strain, microbiologists
need to isolate a “pure culture” of the organism of interest. This means physically
spreading a population of organisms on an agar plate in such a way that a single
organism can grow into an individual colony, significantly away from neighbours. Each
colony is a population of “clones” that represent one bacterium which has separated
from others and divided. For this exercise, we will make a streak plate from a liquid
broth culture that contains Escherichia coli.

Materials:
1) Each student will receive 1 Tryptic Soy Agar (TSA) plates
2) Liquid culture with E. coli

Streak-Plating and Pure Cultures Procedure (see Figure 2 and 3):

1) Work individually in your group.
2) Label the base of the plate with your initials along the edges
3) Flame and cool your loop before collecting a loop of E. coli from the liquid tube
labeled as such
4) Follow the diagram of figure 3e which can be drawn on the plate if desired
5) Apply bacteria to first region and flame/cool loop
6) Touch first region three times and then spread beyond; flame/cool loop
7) Touch second region three times and then spread beyond; flame/cool loop
MAKE SURE TO FILL THE WHOLE PLATE!!!!
8) Place plates in incubator

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 b)
a)

c)
d)

e)

Figura 3 - There are a number of different methods for mechanically diluting microbes
on a streak plate. The most common method is spreading microbes across a plate as
shown in the first four figures (a, b, c, d). As the concentration of microbes increases so
do the number of phases. Irrespective of the number of phases, loop is flamed between
each one. The fifth plate (e) shows an alternative method, where the streaks are not
continuous, but are a series of parallel lines.

(C) Dally Rod Plating (Siembra por extensión)

The dally rod instrument is specifically designed to spread liquid dropped onto a plate
around so that the plate is completely covered and whatever is living in the liquid can
adequately grow.

Materials:
1) Each student will receive 1 TSA plate
2) Dally rod
3) Water sample
4) Alcohol for flame sterilizing

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Procedures:
Flame-sterilization means dipping steel tools in alcohol, alighting them and burning off
flame. We will use this technique to spread bacterial culture onto the plate with a tool
called a dally rod which has been flame sterilized.

1) Work individually in your group.

2) Label the plate with your initials on the media containing side
3) Use alcohol to flame your dally rod and have it ready before hand. Set down
without it touching anything.
4) Use a pipette to place 0,1 - 0,2 ml of water on to the media side of the plate.
5) Immediately take the dally rod and swirl it around on the plate, while at the
same time moving the plate around in circles. (This must be done quickly so that water
does not settle in the middle of the plate)
6) When done and water is spread, leave plate for approximately 5 minutes with
media side down so that water will not move, and then turn over.
7) Incubate plates.

(D) Survey Emphasizing Different Kinds of Media (Gram+/Gram-)

This exercise will demonstrate that bacteria are just about everywhere on and around
you. It is important that we are very careful about our collection procedures with this
lab and use aseptic technique. In this exercise, you will use a rich solid medium called
“Triptic Soy Agar” (TSA). It is designed to grow many organisms and as such is called
“non-selective.” As we work through this class, you will be exposed to other media –
each more selective than TSA.

Materials:
1) Sterile “swabs” (autoclaved Q-tips)
You will receive per group:
2) Two TSA plates
3) Two MacConkey plates
4) Two Mannitol Salt plates

Procedures:
1) Label the plate BASE (media containing side) with your name, the date, and
what the plate contains around the edge (not in the middle)
2) Using your imagination, go out and sample something
a) ONE FROM YOUR BODY (i.e.: THROAT or BETWEEN TOES)
b) ONE CAN BE FROM ANOTHER SOURCE
3) Swab your sample on the whole surface of each different media plate.
4) Place plates media-side up in incubator. (Plates are grown upside-down to avoid
condensation “raining” down on plate surface.)

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You can use the following web sites to get more information on the methods and
procedures to be used in this class:

http://en.wikipedia.org/wiki/Sterile_technique

http://www.sumanasinc.com/webcontent/animations/content/streakplate.html

http://www2.hendrix.edu/biology/CellWeb/Techniques/Tech%20Manual%20TOC.html

http://www.microbiology.mtsinai.on.ca/manual/default.asp#general

http://www.microbes.info/index.html