PRACTICAL 2

I. EXAMINATION OF CULTURES FOR MOTILITY

Introduction: The motility of bacteria is determined by the presence of flagella. Flagella are a long
thread like structures. We can classify the organism by their motility. There are two types of
determining motility, hanging drop method and soft agar stab

Objective

– To identify and differentiate between true motility and Brownian motion

– To familiarize and perfect students aseptic techniques in handling aseptic transfers and
isolating a pure culture.

Materials

Per pair of students

4 soft agar bijous

2 cavity slides

2 wire loops

2 straight wires

Cover slips

Per bench

1 broth culture of Pseudomonas aerugenosa

1 broth culture of Staphylococcus aureus

Plasticine

Procedure

(A) Hanging Drop preparation

1. A tiny amount of plasticine was placed at each corner of the clean cover slip
2. One or two loopfuls of the broth culture was placed in the centre of the cover slip.
3. Concave area of the cavity was placed over the centre of the cover slip. The slip was
press down gently and was inverted.
4. Under the low power objective and reducing light, the edge of the drop was centred
5. The movement of cells was observed. The movement of true motility and Brownian
motion was differentiated.
(A) Soft agar ‘stab’
1. A straight wire was dip into the broth culture by using antiseptic technique.
2. One single stab was made in the centre of the soft agar down to the bottom.
3. The soft agar was not disturbed, shacked and was placed upright for incubation
 I. CULTURE TECHNIQUE

Introduction: As most microorganisms are potentially pathogenic it is essential that they be handled
by procedures which avoid contamination of the microbiologist. Furthermore, since microorganisms
are so ubiquitous it is necessary to avoid contamination of microbial culture by stray microorganisms.

Objective

– To become familiar with the basic movements of aseptic culture technique and our own
technique.

1) Inoculums transfer
(A) Technique of transferring culture from one agar plate to another by
streaking

Materials

1 wire loop

Plate culture of Escherichia coli

1 nutrient agar plate

Bunsen burner

Procedure

Remember that the agar is a fragile medium and only gentile pressure with the oop is
required.

1. The Bunsen burner was lighted and the wire loop was holed by right hand.
2. The wire loop is then sterilized by flaming it in the Bunsen burner until it is red heat. The
wire loop was allowed for 5 seconds to cool it down.
3. The lid of Petri dish containing E.coli was opened by using left hand. Then, small loop
was removed from the inoculums and the lid was closed immediately.
4. The second Petri dish was opened with left hand and the inoculum was spread at the small
area at the top edge of the agar and spread it over a small area (primary inoculums). Then
the loop was flamed.
5. After the loop was cooled make four or five single strokes starting from the primary
inoculums. The loop was flamed again and overlapping series of stroke covering anther
area was made until all the surface of the agar was covered.
6. The plate was closed and was placed inverted for incubation at 37’C for 24-48 hours.

(A) Technique of transferring culture from an agar plate to a bottle of nutrient

broth
Materials

1 wire loop

Plate culture of Escherichia coli

1 bottle of nutrient broth

Bunsen burner

Procedure

1. The wire loop was sterilized and small loop was removed from the inoculum in the plate
(as in 1) a) 3.)
2. The bottle was taking by using left hand and twist the cover off the bottle by using right hand
(while holding the wire loop) and the top of the bottle was flamed evenly near the Bunsen
burner.
3. With the right hand, the loop was dip in to the nutrient broth and was mix gently with
minimum movement.
4. The bottle was flamed as in step 3 and its cap was screwed back.
5. The loop was carefully flamed by placing in the cooler part of the Bunsen flame and
gradually drawn to the hotter region of the flame. (to reduce amount of splattering).
6. The bottle was labelled and incubated at 37’C for 24-48 hours

(A) Isolating a pure culture from a mixed culture

Materials

A plate of mixed culture of Bacillus subtilis and Staphylococcus aureus

1 wire loop

A plate of nutrient agar

1 bottle of nutrient broth

Procedure

1. The mix culture was streaked on the nutrient agar plate to obtained a pure culture of Bacillus
subtilis and Staphylococcus aureus
2. The mixed culture was transferred into the bottle of nnutrient broth
3. The bottle and plate was labelled and incubated at 37’C for 24-48 hours.

Discussion:
1. EXAMINATION OF CULTUER FOR MOTILITY
 Motile bacteria can move using flagella, bacterial gliding, twitching
motility or changes of buoyancy. In twitching motility, bacterial use
their pili as a grappling hook, repeatedly extending it, anchoring it
and then retracting it with remarkable force (>80 pN).

Bacterial species differ in the number and arrangement of flagella on their

surface, some have a single flagellum (monotrichous), a flagellum at each
end (amphitrichous), clusters of flagella at the poles of the cell
(lophotrichous), while others have flagella distributed over the entire
surface of the cell (peritrichous).

a) Brownian movement is caused by the molecules of the suspending

liquid colliding with the organism and moving it around in an
irregular, jerky pattern. This is not considered to be true motility.
While on the other hand true motility's movement in some
consistent direction or path, even though there may be many twists
and turns.

The result are:

Staphylococcus aureus - non-motile cocci

Pseudomonas aeruginosa- unipolar motility

Staphylococcus aureus
Pseudomonas aeruginosa

The hanging drop method has several advantages and

disadvantages:

Advantages:
-The hanging drop method allows the organism to live longer because it
does not dry out as quickly. -The hanging drop method also offers a
better view of true motility.
-The Vaseline-sealed depression or sometimes using plasticine,also slows
down the drying-out process, so the organisms can be observed for longer
periods.

Disadvantages:
-The hanging drop method is also far too risky to use with highly
pathogenic organisms.Human will get infection easily.

b) Motility Test medium

Semi-solid Motility Test medium may also be used to detect motility.

When a non-motile organism is stabbed into Motility Test medium, growth
occurs only along the line of inoculation. Growth along the stab line is very
sharp and defined . When motile organisms are stabbed into the soft agar,
they swim away from the stab line. Growth occurs throughout the tube
rather than being concentrated along the line of inoculation. Growth along
the stab line appears much more cloud-like as it moves away from the
stab. If a tetrazolium salt (TTC) is incorporated into the medium. Bacterial
metabolism reduces the TTC producing formazan which is red in color.
The more bacteria present at any location, the darker red the growth
appears.

Pseu. Aeruginosa empty Staph. aureus

However no color indicator used in this experiment.The advantages and

the disadvantages are:

Advantages: This technique is useful for determining the motility of

highly pathogenic bacteria. It is also easier to read than the other
techniques.
Disadvantages: This technique takes longer to provide results than the
wet mount or hanging drop.
 1. CULTURE TECHNIQUE
Cell culture is the process by which cells are grown under controlled
conditions. In practice the term "cell culture" has come to refer to the
culturing of cells derived from multicellular eukaryotes, especially animal
cells. The historical development and methods of cell culture are closely
interrelated to those of tissue culture and organ culture.

1) Inoculums transfer

a) Technique transferring culture from one agar plate to another by

streaking.
Culturing bacteria using broth culture is a good technique for
determining pure culture.

E. coli in agar medium

The advantages and disadvantages of this technique are:

Advantages:
-Growth of bacteria on solid media has advantages over use broth
cultures because it allows isolation of bacteria in pure culture. It is
because a colony well separated from others can be assumed to rise
from a single organism cluster (CFU).
Disadvantages:
-Anaerobic bacteria will not grow.
- It does not always provide the clear separation of inoculums /
bacteria desired.
- Also a chance of carry-over and contamination from one portion to
another.
- Additionally, it is often difficult to determine what portion of the
sphere has already been used.
- Essentially a storage place for your bacteria.
b) Technique transferring culture from one agar plate to a bottle of
nutrient broth.

Culturing bacteria using broth culture is a easy and good technique.

E. coli in broth medium

The advantages and disadvantages of this technique are:

Advantages:
-Allows for rapid and large volume of growth bacteria.
- It suitable for long-term storage.
Disadvantages:
-We cannot count bacterial colony directly, it must use another
method such as miles-misra method, pour plate method, most
probable number method, or other method to isolate and count
colony formed.

2) Isolating a pure culture from mixed culture

Part 1
a. Observe and sketch the distribution of growth:

Mix Culture
 b. No. It is because I do not streak the culture properly. Maybe
third or fourth streak had touch first streak. Dilution process
cannot occur. Cannot form isolated colonies. But the colonies
same with original plate.

c. Broth Culture:

Before After
Before inoculate it is clear, after inocculate it become cloudy
or turbid.It is because present microorganism in the broth
medium. Less light penetrate the broth medium because most
of light had been absorbed or reflect by the microorganism.

d. Definitely no, because we cannot determine the separated

colonies in the medium. The colonies mixed evenly in broth
medium.

Part 2

a. I am not able to separate the 2 organisms successfully. It is because

I am late to observe it. We observed after 4 days although I must
observe after 1 day. It also due to error during streaking process.
Maybe I had touched the first streak during forth streak. Diluting
process failed. Cannot form pure culture colony.

Mix culture: Shape-irregular and filamentous

Texture - smooth

Color - white colony

Elevation - raised from medium surface

E. coli : Shape-irregular

Texture - smooth

Color - white colony

 Elevation - raised from medium surface

b. When the culture is being contaminated, we can see many different

colony occur that determined by different color and shape.
c. We incubate the inoculated in inverted position because to prevent
culture from being contaminated easily.

Refferences: : -Tortora, Funke, Case’s Microbiology: An introduction.

-Levinson & Jawetz’s Medical Microbiology & Imunology.

-www.answers.com

-www.wikipedia.com

-http://in.answers.yahoo.com/question/index?
qid=20061024232001AAP0ikl