Introduction

In natural environments, microorganisms usually exist as mixed

populations. However, if we are to study, characterize, and identify
microorganisms, we must have the organisms in the form of a pure
culture.
A pure culture is one in which all organisms are descendants of
the same organism. Techniques for obtaining pure cultures from a
mixed population will be described in this Lab.
In working with microorganisms we must also have a sterile
nutrient-containing-medium in which to grow the organisms.
Anything in or on which we grow a microorganism is termed a
medium.
A sterile medium is one which is free of all life forms.
It is usually sterilized by heating it to a temperature at
which all contaminating microorganisms are destroyed.

Finally, in working with microorganisms, we must have a method

of transferring growing organisms from a pure culture to a sterile
medium without introducing any unwanted outside contaminants.
This method of preventing unwanted microorganisms from gaining
access is termed aseptic technique.
For the most part, bacterial physiology only can be
studied in pure cultures.
The best way to obtain a pure culture is to start with a
single bacterial cell.

This cell then divides quickly, and may produce millions of cells
within 24 hours.
A single unwanted contaminant cell can do the same thing in an
otherwise pure culture, making the culture useless.

For this reason, and to protect

against disease, strict sterile
procedures must be used.
Aseptic Technique

 The most commonly used device for moving bacteria is the

inoculating loop.

 This is simply a piece of nichrome (an alloy

of nickel and chromium) or platinum wire
with a loop at one end and a handle at the
other.
 A similar instrument is the inoculating
needle, essentially the same as the loop,
but with just a straight wire
 Sterilize both instruments by holding the wire
portions in a flame until they glow red. The
instruments should be allowed to cool in the air
for 10-20 seconds before using them to avoid
killing the inoculum.
 In this way all contaminants on the wire are incinerated.

 Do not blow on the instruments to cool them

 Do not touch the instruments to agar to cool them
 Do not lay the loop down once it is sterilized or it may again
become contaminated.
Procedure For Aseptically Transfer

1) Flame the loop.

2) Without setting the loop down, open the first culture tube
and flame the mouth. Do not set the cap on the bench. The
cap should be held in the same hand as the loop.
3) Insert the loop into the culture medium, then withdraw it.
4) Flame the mouth of the first culture tube again, and replace
the cap.
5) Open the second culture tube and flame the mouth. Do not
set the cap on the bench. The cap should be held in the same
hand as the loop.
6. Insert the loop into the second culture tube and
spread the culture suspension (on the loop)
inoculum into/onto the second culture medium.
7. Flame the mouth of the second culture tube, then replace
the cap.
8. Flame the loop and set on the bench.
9. When in doubt about the sterility of an instrument or
container, sterilize it.
 Remember
Bacteria
 Are everywhere!
 On every surface of the body
 Including digestive tract
 Pathogenic
 Absorb nutrients and release toxins that damage cells and tissues.
 Bacterial toxins can cause disease even when bacteria are destroyed
 Bacteria are Prokaryotes.
Five Basic Techniques of Culturing

1) Inoculate
2) Incubate
3) Isolation
4) Examination
5) Identification
 Pure Culture Concept

 Attempts to identify bacteria in a clinical sample cannot be

done unless isolated colonies are used.

 To obtain well-isolated colonies, it is essential to disperse the

inoculum (sample) on the surface of an enriched agar plate so
that individual bacteria are well separated from each other.
 Contaminants: other microorganisms present in the
sample.
 Isolated colonies: a population of millions of cells
that are identical and are descendent from a single
founder cell.
 Stock Culture: a culture that already contains cells.It is used a
source of cells from which to inoculate new cultures.
 Culture Medium: rich/selective
 Growth/ inhibitors
 liquid/solid
 temperature
 source of energy
 sources of carbon, nitrogen, ...
 Aseptic technique:
 sterilization of medium and equipment
 proper handling
Necessary equipment
Procedure
1) With the loop, spread the inoculum back and forth across the
upper 1/4 of the plate, keeping the lines of inoculation very close
together (area 1 in figure below).
2) Isolated colonies are not expected in this area. Do not use strong
pressure, which will break the surface of the agar. Use the end of
the loop, not its side when streaking. Dispose of the loop in the
biohazard bucket on the bench.
3) Turn plate approximately 90oC. Streak the plate as indicated in
the figure (area 2) across about 1/4 of the plate. Dispose of the
loop.
4) Repeat step 2 one or two times more.
5) In area 3 and/or 4 single colonies should appear.
6) Label plates on the bottom and incubate inverted
at 37oC.

Note: Lids on test tubes are loose.

Always hold the glass test tube (not the lid)
when carrying them.
Forms Of Culture Media

1) Broth tube: are tubes containing a liquid medium. A typical

nutrient containing broth medium such as Trypticase Soy broth ,
nutrient broth. After incubation, growth (development of many
cells from a few cells) may be observed as one or a combination of
three forms:

a) Pellicle: A mass of organisms is floating on top of the broth.

b) Turbidity: The organisms appear as a general cloudiness
throughout the broth .
c) Sediment: A mass of organisms appears as a deposit at the
bottom of the tube.
2) Slant tubes: are tubes containing a nutrient medium plus a
solidifying agent, agar-agar. The medium has been allowed to
solidify at an angle in order to get a flat inoculating surface.
3) Stab tubes (deeps): are tubes of hardened agar medium which
are inoculated by "stabbing" the inoculum into the agar.
4) Agar plates: are sterile petri plates that are aseptically filled with
a melted sterile agar medium and allowed to solidify. Plates are
much less confining than slants and stabs and are commonly used
in the culturing, separating, and counting of microorganisms.
END OF LECTURE