Date Performed: January 14, 2014

Date Submitted: February 4, 2014

Exercise Number 10
Bacterial Motility
Herlu Carol Jean H. Lucero

ABSTRACT
Bacterial motility is achieved through the use of flagella
that are found in some bacteria. They come in a variety of forms
that is unique in every flagella-bearing species. This makes the
flagella not only as a structure for motility but also for
classification. Four representative bacteria were observed by
microscopy. Escherichia coli, Bacillus subtilis and Salmonella
typhi were found to exhibit the true motility while Staphylococcus
aureus exhibited the Brownian movement. It was also observed
that each bacterium differ in the speed of their movement. This
may be due to some intervening factors such as temperature and
pH. In relation to this, their response to some stimuli such as the
chemotactic response may also be affected.


INTRODUCTION
Motility allows a microorganism to locate life-sustaining nutrients and to migrate toward
positive stimuli such as sunlight; it also permits avoidance of harmful substances and stimuli
(Talaro and Chess, 2012). In bacteria, motility is achieved by the use of an external appendage
which is the flagellum. A flagellum is a long sheathed cylinder containing regularly spaced
hollow tubulesmicrotubulesthat extend along its entire length (Talaro and Chess, 2012).
Many bacteria show no motion and are termed non-motile. However, in an aqueous environment,
the same bacteria appear to be moving erratically. This erratic movement is due to Brownian
movement (Harley and Prescott, 1996). For the motile bacteria, their movement is called the true
motility. For this reason, it is therefore the objective of this exercise to observe true motility
among representative bacterial species and therefore differentiate it from Brownian movement.
Materials and Methods
Four representative bacterial species were to be observed for the exercise. These were 24-
hour cultures of E.coli, B. subtilis, S. aureus and S. typhi grown in agar slants. After hand
disinfection, a depression slide was handled using forceps then alcohol was poured on it before
passing it on the flame of an alcohol lamp until all of the alcohol had evaporated. The same
procedure was done on the cover slip. A glass dropper was then sterilized by gently passing it to
the flame and when already cool, sterile water was obtained from a screw cap tube. A drop was
placed at the center of the cover slip. An inoculating loop was also sterilized until red hot and
then cooled still near the flame. Using asceptic technique, a little amount of one bacterial species
was obtained from one of the four culture slants using the sterile loop and then mixed with the
water on the cover slip. The inoculating loop was again sterilized and cooled. Loopfuls of sterile
water were then placed on the four corners of the cover slip. The depression slide was inverted
over the cover slip such that the drop of water with bacteria was on the center of the concavity.
The slide was then mounted and observed under oil immersion objective. The motility of the
bacteria was noted. For the other three species of bacteria, the same steps were employed.
Results and Discussion
Bacterial flagella are fine, threadlike organelles of locomotion. They are slender (about
10 to 30 nm in diameter) and can only be seen directly using the electron microscope (Harley
and Prescott, 1996). There are four different flagella arrangements among bacteria as shown in
Figure 1.


(a) Monotichous (b) Amphitrichous


(c) Lophotrichous (d) Peritrichous
Figure 1. Flagella arrangements in bacteria.
Monotrichous bacteria possess a single polar flagellum, whereas amphitrichous have flagella at
both ends. Lophotrichous bacteria contain a tuft of flagella at one end, and peritrichous bacteria
have flagella all over their surface area (Grover-Lakomia and Fong, 1999). This possession of
flagella by certain bacteria may also be used for classification since flagellated bacteria are
grouped according to the number and arrangement of their flagella as these characteristics are
unique for each flagellated species (Grover-Lakomia and Fong, 1999).
Table 1. Motility of four representative bacteria
Organism Motility
Escherichia coli
Staphylococcus aureus
Bacillus subtilis
Salmonella typhi
true motility
Brownian movement
true motility
true motility
From Table 1, we can see that S. aureus has exhibited the Brownian movement while the
rest exhibited true motility. This is because true motility is seldom observed in cocci (Smith,
1980). The observed motility of the other bacteria also varies in the speed. S. typhi for example,
is able to progress at a rate of 200 times its own length per hour (Smith, 1980). This is due to
factors that affect the rate of speed of their motility. One study showed how temperature may
influence the chemotactic response of Vibrio anguillarum. They found out that cells of the V.
anguillarum were motile at all growth temperatures tested, and also after temperature up- and
down-shift as observed by microscopy. The swimming speed increased significantly from 25 m
s
-1
at 5C to 36 and 40 m s
-1
at 15 and 25C, respectively (Larsen et al., 2004). This motility is
relative to their chemotactic response as the authors have concluded that temperature has a more
pronounced effect of chemotaxis than on motility. Another study showed that intracellular pH
may affect the swimming speed of bacteria. The swimming speed of E. coli and Salmonella sp.
were measured in the presence and absence of a weak acid. The swimming speed of E. coli was
measured in over an external pH range from 5.0 to 7.8. The cells swam at ca. 24 m/s in pH 7
motility medium without 34 mM potassium acetate. Over the pH range of 5.0 to 7.8, the average
swimming speed varied only slightly, In contrast, in the presence of acetate, the swimming speed
did not change much over the pH range of 7.0 to 7.8 but decreased sharply between 7.0 and 5.0
(Minamino et al., 2003). The same results were obtained for Salmonella sp. In the absence of
benzoate, the average swimming speeds were ca. 13.7, 15.9, and 16.4 m/s at external pH values
of 7.0, 6.3, and 5.5, respectively. In contrast, the cell speed decreased significantly when the
external pH was decreased in the presence of benzoate, ca 12.8, 8.6, and 5.9 m/s at pH 7.0, 6.3,
and 5.5, respectively (Minamino et al., 2003).
The change in the direction of bacterial swim may be triggered by a stimuli and the
mechanism by which a change in cell behavior is brought in response to a change in the
environment is called sensory transduction (Jawetz et al., 1989). Sensory transduction is
responsible for chemotaxis that is the movement of an organism towards or away from a
chemical. Positive chemotaxis is movement towards a chemical (attractant, i.e. a substrate).
Negative chemotaxis is movement away from a chemical (repellent). Bacterial movement can be
divided into runs where the organism moves in one direction (caused by counterclockwise
flagellar rotation) and twiddles where the organism randomly tumbles (caused by clockwise
rotation of the flagella) (Nicklin et al., 1999).
Conclusion
Bacterial motility is exemplified by the possession of flagella that is found in some
bacterial species and since not all bacteria have this structure, flagella may also serve for
classification purposes as each bacterial species possess unique flagella. Four types of flagella
are observed in bacteria namely monotrichous, amphitrichous, lophotrichous and peritrichous.
They vary in the number and in the position on which they are observed in a bacterial cell.
Bacterial motility also differs in the speed in every species as they are affected by factors such as
temperature and intracellular pH. In relation to this, their response to some environmental stimuli
may also be affected such as chemotaxis which is a movement of bacteria towards or away from
a chemical.
References
Grover-Lakomia L.I. and Fong E. (1999). Microbiology for Health Careers, 6
th
Ed.
Delmar Publishers

Harley J.P. and Prescott L.M. (1996). Laboratory Exercises in Microbiology, 3
rd
Ed. Wm.
C. Brown Publishers

Jawetz E., Melnick J.L., Adelberg E.A., Brooks G.F., Butel J.S. and Ornston L.N. (1989).
Medical Microbiology, 18
th
Ed. Appleton & Lange
Larsen M.H., Blackburn N., Larsen J.L. and Olsen J.E. (2004). Influences of temperature,
salinity and starvation on the motility and chemotactic response of Vibrio
anguillarum.Society for General Microbiology

Minamino T., Imae Y., Oosawa F., Kobayashi Y. and Oosawa K. (2003). Effect of
Intracellular pH on Rotational Speed of Bacterial Flagellar Motors

Nicklin J., Graeme-Cook K., Paget T. and Killington R. (1999). Instant Notes in
Microbiology. BIOS Scientific Publishers

Smith A.L. (1980). Microbiology and Pathology, 12
th
Ed. C.V. Mosby Company

Talaro K.P. and Chess B. Foundations in Microbiology, 8
th
Ed. McGraw-Hill Companies,
Inc.









Date Performed: January 7, 2014
Date Submitted: February 4, 2014
Exercise Number 8
Bacterial Cytology
Herlu Carol Jean H. Lucero

ABSTRACT
Some physical characteristics of bacteria may be observed
with the aid of first staining them. There are two types of staining,
the simple staining and differential staining. The simple staining
may be advantageous over the differential as it is easier to perform
it when the aim is only to observe the shape, size and arrangement
of cells. But when it comes to diagnostic laboratory, the
differential becomes more valuable. One of these differential
staining methods is the Gram stain. In the exercise, gram-positive
Bacillus amyloliquifaciens and gram-negative Escherichia coli
underwent this staining to confirm it. The bacterial species are also
subjected to KOH method which is a confirmatory test of Gram
stain.

INTRODUCTION
To better observe cellular characteristics of bacteria such as the size and the shape,
microbiologists employ the use of stains. One type of staining method used is the Gram stain
named after its developer Hans Christian Gram. This is a positive staining method and classified
under differential which uses two different-colored dyes. An effective differential stain uses dyes
of contrasting color to clearly emphasize differences between two cell types or cell parts (Talaro
and Chess, 2012). The Gram stain, even today, is an important diagnostic staining technique for
bacteria. It permits ready differentiation of major categories based on the color reaction of the
cells: gram-positive, which stain purple, and gram-negative, which stain red. This difference is
staining quality is due to structural variations found in the cell walls of bacteria. The Gram stain
is the basis of several important bacteriologic topics, including bacterial taxonomy, cell wall
structure, identification, and drug therapy (Talaro and Chess, 2012).
The first step in the procedure involves staining with the basic dye crystal violet. This is
the primary stain. It is followed by treatment with an iodine solution, which functions as a
mordant; that is, it increases the interaction between the bacterial cell and the dye so that the dye
is more tightly bound or the cell is more strongly stained. The smear is then decolorized by
washing with an agent such as 95% ethanol. Finally, the smear is counterstained with a basic
dye, different in color than crystal violet. This counterstain is usually safranin (Harley and
Prescott, 1996).
Materials and Methods
A slide was first sterilized using alcohol then gently flamed until the alcohol evaporated.
An inoculating loop was also sterilized until red hot and then used to inoculate a loopful of
sterile water from a screw cap tube and then placed on the pre-sterilized slide. Another loopful of
water was obtained and then placed beside the first loopful water on the slide. Using asceptic
techniques, the culture tube of B. amyloliquifaciens was inoculated and the inoculum was mixed
with the sterile water on the slide. The loop was sterilized and when cool enough, the culture
tube of E. coli was also inoculated and the inoculum was mixed with the other sterile water on
the slide. The loop is again sterilized. Using forceps, the slide was handled and gently passed on
the flame until all of the water had evaporated. The bacterial smears were flooded with crystal
violet for a minute and then rinsed and drained. They were now treated with an iodine solution
for another minute and then rinsed and drained. They were decolorized afterwards with 95%
ethanol until no more crystal violet is washed off and then rinsed and drained. Using safranin, the
smears were counterstained for a minute and then rinsed and dried. The slide was then mounted
under LPO and HPO for observation.
After the Gram staining, a confirmatory test was also done on the bacteria that is the
KOH method. It was done by putting a loopful of 3% KOH on a slide and using a sterile loop, a
small amount of one of the bacterial species was transferred to the KOH drop. After mixing,
clotting was observed for a minute. The same was done on the other species.
Results and Discussion
Before proceeding to the staining, a bacterial smear is first prepared by fixation through
heat. The use of heat indicates a physical change as water evaporates from the smear. When only
one type of dye is used in staining together with an uncomplicated procedure, it is classified as
simple staining in contrast to differential staining. Most simple staining techniques take
advantage of the ready binding of bacterial cells to dyes like malachite green, crystal violet, basic
fuchsin, and safranin. Simple stains cause all cells in a smear to appear more or less the same
color, regardless of type, but they can still reveal bacterial characteristics such as shape, size and
arrangement (Talaro and Chess, 2012)
Although the simple staining can reveal some of the physical characteristics of bacteria,
the Gram stain still appears to be more valuable than the simple staining when it comes to
diagnostic laboratory. This staining method remains an important basis for bacterial
classification and identification. It permits differentiation of four major categories based upon
color reaction and shape: gram-positive rods, gram-positive cocci, gram-negative rods, and
gram-negative cocci. The gram stain can also be a practical aid in diagnosing infection and in
guiding drug treatment. For example, Gram staining a fresh urine or throat specimen can help
pinpoint the possible cause of infection, and in some causes, it is possible to begin drug therapy
on the basis of this stain (Talaro and Chess, 2012). The simple stain only becomes preferable to
the Gram stain when information about cell shape, size and arrangement is desired. Its chief
value lies in its simplicity and ease of use (Harley and Prescott, 1996).
The Gram stain does not always yield clear results. Species will differ from one another
in regard to the case with which the crystal violet-iodine complex is removed by ethanol. Gram-
positive cultures may often turn gram-negative if they get too old. Thus, it is always best to
Gram stain young, vigorous cultures rather than old ones (Harley and Prescott, 1996). It seems
likely that the difference between gram-positive and gram-negative bacteria is due to the
physical nature of their cell walls. If the cell wall is removed from gram-positive bacteria, thay
become gram-negative. The peptidoglycan itself is not stained; instead it seems to act as a
permeability barrier preventing loss of crystal violet ( ). Relating this to old cultures, their
cell walls are possibly already degraded making them to fail in exhibiting typical gram reactions.
The gram stain may also be valuable in establishing a pure culture of bacteria as it can reveal if
cells from one culture may differ from the vast majority of the other cells within the culture.
Conclusion
Gram staining is one staining method that is important in diagnostic laboratory. This is
one edge of this differential staining over the simple staining method. The cell wall of bacteria
are of great importance in the staining procedure as these structures are responsible to the results
of the staining. B. amyloliquifaciens was confirmed to be gram-positive bacteria while the E. coli
was gram-negative. The E. coli was also positive in the KOH method that is it exhibited
agglutination which further confirmed that it is gram-negative. B. amyloliquifaciens was negative
in the KOH method.

References
Harley J.P. and Prescott L.M. (1996). Laboratory Exercises in Microbiology, 3
rd
Ed. Wm.
C. Brown Publishers
Talaro K.P. and Chess B. Foundations in Microbiology, 8
th
Ed. McGraw-Hill Companies,
Inc.