MODULE 6: DETERMINATION OF MICROBIAL MOTILITY Hanging Drop Technique for Bacterial Specimen

To prepare the specimen for observation, remove a loopful

Hanging Drop Technique
1. Place four small drops of petroleum jelly on the edge corner of
the coverslip.
2. Place two loopfuls of pondwater at its center.
3. Rapidly invert the coverslip and place it on the depression slide.
The drop must hang into the depression but should not touch the
bottom of the depression slide.
4. Position the hanging drop on the stage so that the specimen is
over the light hole.
5. Adjust the condenser so that a minimum amount of light hits the
object. Observe under the HPO.
6. Note the direction of the movement of the microorganisms.
7. Observe again the motility of microorganisms in wet mount
preparations.
from a culture tube using a wire loop following the aseptic technique
outlined below:
1. Place the culture tube to rest on the palm of the left hand just
between the index and the middle fingers and steady it with the
thumb.
2. Hold the loop with the right hand as you would hold a pencil.
3. Heat it to redness over the flame of an alcohol lamp or Bunsen
burner and then flame the lower part of the handle.
4. Allow the loop to cool for about three seconds. The loop should
not come in contact with anything.
5. While holding the sterilized cooled loop, remove the cotton plug
from the mouth of the tube by grasping the plug with the little finger
of the right hand.
6. Flame the mouth of the culture tube, insert the loop into the broth
culture and draw out a loopful.
7. Flame the mouth of the tube again and carefully return the plug.

8. Put the loop of culture on the cover slip.

Demonstration of Brownian Movement
The oscillation or quivering motion exhibited by all very
minute particles suspended in water or other fluids is known as
Brownian movement. The movement of the particles is due to the
bombardment by the molecules of the dispersion medium. In
contrast, motility of microorganisms is an independent, true
movement of translation brought about by organs of locomotion or
gliding.
9. Flame the loop and handle before laying it on the table.
10. Invert the depression slide and center its well over the drop of
dye.
11. Carefully lower the slide onto the cover slip so that when contact
is made, the moisture outside the depression will form a film and
hold the cover slip in place.

1. Clean a depression slide and a cover slip with detergent.
2. With a wire loop, place a loopful of water at each corner of the
cover slip, then place a loopful of carmine dye at its center. (The
wire loop is a tool consisting of a piece of 24-gauge wire either
platinum-iridium or nichrome, 5 cm long, inserted into a light but rigid
wire holder. The platinum-iridium or nichrome wire is chosen
because it heats easily, cools fast and is inert.)
3. Invert the depression slide and center its well over the drop of
dye.
4. Carefully lower the slide onto the cover slip so that when contact
is made, the moisture outside the depression will form a film and
hold the cover slip in place.
12. Turn the slide right side up quickly so that the specimen will not
spread over the cover slip. There should be a water seal outside the
depression, and the hanging drop should not touch this seal nor the
bottom of the depression.
13. Examine the hanging drop with the LPO. Reduce the amount of
light with the iris diaphragm in order that the drop may be seen
distinctly. Arrange the slide so that the edge of the drop is within the
field of vision. The edge serves as a convenient reference for the
observer to move the specimen into proper view.
14. Swing the HPO objective into place. Use the fine focus knob for
sharp focusing.

5. Turn the slide right side up quickly so that the specimen will not
spread over the cover slip. There should be a water seal outside the
depression, and the hanging drop should not touch this seal nor the
bottom of the depression.

6. Examine the hanging drop with the LPO. Reduce the amount of
light with the iris diaphragm in order that the drop may be seen
distinctly. Arrange the slide so that the edge of the drop is within the
field of vision. The edge serves as a convenient reference for the
observer to move the specimen into proper view.

7. Swing the HPO objective into place. Use the fine focus knob for
sharp focusing. Indicate the movement exhibited by the carmine dye
particles.
Hanging drop of Protozoan culture from Hay Infusion
1. Place four small drops of petroleum jelly on the edge corner of
the coverslip.
2. Place two loopfuls of hay infusion at its center.
3. Rapidly invert the coverslip and place it on the depression slide.
The drop must hang into the depression but should not touch the
bottom of the depression slide.
4. Position the hanging drop on the stage so that the specimen is
over the light hole.
5. Adjust the condenser so that a minimum amount of light hits the
object. Observe under the HPO.
6. Note the direction of the movement of the microorganisms.
Note: If the organisms are too motile for close observation,
they may be immobilized to a great extent by adding a drop of 2 %
methyl cellulose solution to the preparation.
Wet Mount Preparations
A. For broth culture, place a drop of the culture on a plain glass slide
and carefully set the cover slip on top of the drop.
B. For surface growth, get a small amount of culture and place it on
the slide containing a drop of water. Mix specimen and liquid. Place
the coverslip.
C. Place a cover slip over the mount, lowering one edge before the
other so that air bubbles can escape.
Observation of Motility Band (Culture Method)
A. Preparation of the Bacterial Suspension
1. Transfer bacterial culture to NA slant. Incubate at
temperature 5 ͦ C below optimum for 24-48 h.
2. Add 2-3 mL sterile distilled water to slant and incubate for
10 minutes.
B. Inoculation of the Motility Medium using Drop method
1. With a pipette, get one drop of bacterial suspension
prepared in part IIIA and carefully add to MM.
2. Incubate as above.
3. After two days of incubation, observe motility band and
measure distance traveled from point of inoculation.
C. Inoculation of the Motility Medium using Stab method
1. Stab the medium to within 1 cm of the bottom of the test
tube.
2. See to it that the needle is aligned as it is inserted and
removed from the medium.
3. Incubate as above.
4. After two days of incubation, observe motility band or the
dispersion of the bacteria from the line of streak outward
and measure distance traveled from point of inoculation.
Positive: Diffuse, hazy growths that spread throughout the medium
rendering it slightly opaque.
Negative: Growth that is confined to the stab-line, with sharply
defined margins and leaving the surrounding medium clearly
transparent.
MODULE 7: FACTORS AFFECTING MICROBIAL GROWTH
Microorganisms have adapted to a variety of extremely
variable environmental conditions. The study of microorganisms
from natural environments requires a variety of physical and
chemical growth conditions for isolation, identification and analysis.
Physical factors include temperature and osmotic
pressure, whereas chemical factors include pH and oxygen
requirement, among others. Temperature is one of the most
important factors influencing the activity of enzymes. There are
groups of microorganisms capable of growing in different
temperature ranges. Psychrophile can grow between 0 – 20˚C,
mesophiles can grow in optimum environmental conditions, and
thermophiles can thrive in between 50 to 90˚C. pH is a measure of
hydrogen ion concentration in an organism’s environment. It limits
the activity of enzymes.
Microorganisms can also optimize their growth at varying
pH concentrations. Acidophiles can grow below pH 6.5, alkalophiles
at pH above 7.5, and neutrophiles can thrive between pH 6.5-7.5.
Increasing solute concentration can affect the amount of
water availability and further affects the survival of microorganisms.
However, there are microorganisms capable of growing in the
presence of high solute concentration. Osmophiles require high
solute concentration whereas osmotolerant can grow in higher
solute concentration.
Microorganisms are divided into five categories depending
on their need to molecular oxygen. Obligate aerobes use oxygen as
the final electron acceptor while obligate anaerobes generate their
energy through fermentative processes or anaerobic respiration and
thus do not require oxygen as their terminal electron and hydrogen
ion acceptor. Both facultative and aerotolerant anaerobes may or
may not require oxygen but the former prefer the presence of it.
However, there are microorganisms that require low concentration
of oxygen (2-10%) and these are microaerophile.
Growth Requirements
1. Physical requirements
- Temperature
- pH
- Osmotic Pressure
2. Chemical requirements
- CHONPS
- Micronutrients
- Growth factors
 Temperature o Micrococcus luteus (ML) into 3 NB tubes with diff.
pH
Minimum growth temperature • Label with the specimen name:
- the lowest temperature at which a species will grow. o Incubate the tubes at 37 ⁰C, 24-48 hrs.
• Result interpretation:
Optimum growth temperature o Turbid - with growth (+, ++, +++)
o Clear - No growth (-)
- the temperature at which it grows best.

Maximum growth temperature

- the highest temperature at which growth is possible.

Procedures

1. Microbial Response on Temperature

2. Microbial Response on pH

3. Microbial Response on Osmotic Pressure

4. Effect of Oxygen

Microbial Response to Temperature

• Inoculate 1 loopful of: Microbial Response on Osmotic Pressure

o Bacillus subtilis (BS) into 3 nutrient broth tubes
o Micrococcus luteus (ML) into 3 nutrient broth 1. NaCl Concentration
tubes
o Staphylococcus aureus (SA) into 3 nutrient broth • Inoculate (by single streak):
tubes o B. stearothermophilus (BS)
• Label with the specimen name and incubation o E. coli (EC)
temperature: o M. luteus (ML)
o Place the tubes in the respective temperature o S. aureus (SA)
storage: at 4⁰C (Ref.), 37 ⁰C and 55 ⁰C • Each into a Nutrient agar plate with salt concentration.
(incubators) • Label with the specimen name:
o Incubate: 24-48 hrs. • Incubate: 37 ⁰C, 24-48 hrs.
• Result interpretation: • Result interpretation:
o Turbid - with growth (+, ++, +++) o Growth in the streak line (+, ++, +++)
o Clear - No growth (-) o Clear (-)

Microbial Response to pH

• Inoculate 1 loopful of:

o Escherichia coli (EC) into 3 NB tubes with diff. pH
o Staphylococcus aureus (SA) into 3 NB tubes with
diff. pH
2. Sucrose Concentration Microbial Response on Oxygen

• Inoculate (by single streak): 1. Melt 5 Thioglycollate butt (TA).

o Serratia mascescens (SM) 2. Inoculate the following organisms: M. luteus (ML), E. coli
o E. coli (EC) (EC), S. aureus (SA), P. aeruginosa (PA) and B.
o M. luteus (ML) stearothermophilus (BS) into TA tubes.
o S. aureus (SA) 3. Incubate at 37˚C for 48 hrs.
• Each into a Nutrient agar plate with sucrose concentration: 4. After 48 hrs, look for the evidence of growth on each tube
5%, 10%, 25% and answer items 9-10 in the worksheet.
• Label with the specimen name:
• Incubate: 37 ⁰C, 24-48 hrs.
• Result interpretation: Results and Discussion
o Growth in the streak line (+, ++, +++)
o Clear (-) 1. Temperature
• a very common and effective way of controlling
microorganisms
• Temperatures below the minimum usually have a
Effect of Oxygen
static action on microorganisms. They inhibit
- Thioglycollate agar (TA) tubes microbial growth by slowing down metabolism but
- Melted and cooled to ~45 ⁰C (liquid) do not necessarily kill the organism.
• Inoculate (1 loopful): • Temperatures above the maximum usually have
o M. luteus (ML) a cidal action, since they denature microbial
o E. coli (ML) enzymes and other proteins.
o S. aureus (SA)
o P. aeruginosa (PA)
o B. stearothermophilus (BS)
• Each into TA tubes.
• Label with the specimen name:
• Incubate: 37 ⁰C, 24-48 hrs.

Thioglycollate agar

- Has sodium thioglycollate in order to trap the oxygen:

creates oxygen tension in the medium.

Microbial Responses to Osmotic Pressure (NaCl)

A. Effect of Sodium Chloride

1. Divide the nutrient agar plate without salt (0% NaCl) into Psychrophiles
four parts.
2. Inoculate each part with the following bacteria: M. luteus • Minumum temp – below 0°C
(ML), E. coli (EC), B. subtilis (BS) and S. aureus (SA). • Optimum temp – 10-15 °C or lower
3. Inoculate the remaining plates having 2.5%, 5.0, and 7.5% • Maximum temp – around 20 °C
NaCl respectively. • Ex. Flavobacterium, Achromobacter, Alcaligenes,
4. Incubate the plates at 37˚C for 48 hrs. Chlamydomonas sp. (algae)
5. After 48 hrs, observe for evidence of growth and answer • Readily isolated from Arctic and Antarctic habitats
item 7 in the worksheet. • Their enzymes, transport systems and protein synthetic
B. Effect of Sucrose mechanisms function well at low temp.
• Cell membranes have high levels of unsaturated fatty
1. Divide the nutrient agar plate without sugar (0% sugar) into acids and remain semifluid when cold.
four parts. • At temp. higher than 20 °C, psychrophiles begin to leak
2. Inoculate each part with the following bacteria: M. luteus cellular constituents because of cell membrane disruption.
(ML), S. marcescens (SM), E.coli (EC) , and S. aureus
(SA).
3. Inoculate the remaining plates having 5%, 10% and 25 %
Psychrotrophs
sugar in the same way as in salt.
4. Incubate the plates at 37˚C for 48 hrs. • or facultative psychrophiles
5. After 48 hrs, observe for evidence of growth and answer • Minimum temp. - 0°C
item 8 in the worksheet. • Optimum temp – between 20 – 30 °C
• Maximum temp – about 35 °C
• Major factors in the spoilage of refrigerated food.
 o Pseudomonas fluorescens pH
o Bacillus psychrophilus
- Measure of hydrogen ion concentration
- Limits the activity of the enzyme

Mesophiles

• Minimum temp. – 10-15 °C Response of Different Bacteria on Varying pH

• Optimum temp. – 30-40 °C
• Maximum temp. 45 °C or lower
• Almost all human pathogens - 37°C
• Staphylococcus aureus, Escherichia coli, Enterococcus
faecalis, Neisseria gonorrhea

Thermophiles

• Minimum temp. - 45°C

• Optimum temp. - 55°C or higher
• Maximum temp. - 75°C
o Microorganisms that flourish in composts, self-
heating hay stocks, hot water lines, hot springs
• Have more heat stable enzymes and protein synthesis Optimum pH preferences
systems.
• Have membrane lipids that are more saturated and have • Acidophiles – between pH 0 amd 5.5
higher melting points – remain intact at higher temps. • Neutrophiles – between pH 5.5 and 8
• Examples: • Alkalophiles – between pH 8.5 to 11.5
o Bacillus flavothermus • Extreme alkalophiles – pH 10 or higher
o Bacillus stearothermophilus • Drastic variations in pH can harm by:
o disrupting the plasma membrane
o inhibiting the activity of enzymes and membrane
Hyperthermophiles transport systems.

• Minimum temp. - 55°C

• Optimum temp. – between 80°C and about 113°C. Osmolarity
• Marine hyperthermophiles found in hot areas of the - determined by solute concentration in the environment.
seafloor. - inversely related to water activity (Aw), which is more like
• Pyrococcus abyssi a measure of the concentration of water (H2O) in a
• Pyrodyctium occultum. solution.
- Increased solute concentration means increased
osmolarity and decreased Aw.

Osmotic Pressure

- affects the amount of water availability and further affects

the survival of the microorganism
- Water availability can be reduced by interaction with solute
molecules.

Response of Different Bacteria on Varying sugar concentration

Response of Different Bacteria on Varying Salt concentration • Anaerobic
o Inorganic substances as the final electron
acceptor
o Cannot tolerate gaseous O2. Its presence is
lethal for them.
• Facultative Anaerobe
o Prefer presence of oxygen, but can grow without
it.
• Aerotolerant Anaerobe
o Ignore O2 and grow equally well with or without
oxygen
o Can tolerate O2 pressures less than atmospheric
pressures.
• Microaerophilic
o Requires 2 to 10% oxygen – very low conc. of O2
o Both absence and higher conc. are lethal for
them.

Halophiles vs. Halotolerant

Available Oxygen
• Halophiles are microorganisms that require some NaCl
for growth. • Aerobes, facultative anaerobes and aerotolerant
o Mild halophiles require 1-6% salt, anaerobes must have the enzymes that afford protection
o Moderate halophiles require 6-15% salt; from toxic O2 products:
o Extreme halophiles require 15-30% NaCl for • Superoxide dismutase
growth. • Catalase
• Halotolerant bacteria are able to grow at moderate salt • Peroxidase
concentrations, even though they grow best in the absence
of NaCl.
Thioglycollate Agar tube:

Halophilic microorganisms • There is a concentration gradient of dissolved oxygen .

• Organisms will grow only on that part of the tube where the
• Require NaCl for growth oxygen concentration meets their needs.
• Vibrio cholerae - require as little as 5 to 15 mM NaCl for
optimal growth
• Halotolerant microorganisms
Facultative anaerobes
• Grow in the presence of NaCl
• Staphylococcus aureus - can grow in aerobic or anaerobic conditions although tend
to grow better in aerobic conditions.
- Ex. E. coli and some yeasts.
Oxygen

• Main hydrogen acceptor in cell respiration Aerotolerant anaerobes

o It plays an important role in oxidation-reduction - don’t use O2 but tolerate it – have SOD or some similar
reactions of the cell. enzyme.
• The presence or absence of oxygen alters the oxidation- - Many ferment carbohydrate to lactate, which inhibits
reduction potential of the cell. growth of aerobes.

Oxygen Requirement Microaerophilic aerobes

• Oxygen accepts electrons and is readily reduced because
its 2 outer orbitals are unpaired.
o Formation of reduction products
▪ Superoxide radicals
▪ Hydrogen peroxide
▪ Hydroxyl radicals
• Aerobic
o Oxygen as the final electron acceptor.
o presence of O2 is essential for continued growth
and existence.
- require low O2 concentrations; they are sensitive to
oxygen-derived free radicals.
Obligate anaerobe
- cannot withstand O2.
- They do not have the enzymes that will facilitate their
utilization of oxygen.
- They will die in the presence of O2.
 Oxygen Requirement MODULE 9: CONTROL OF MICROBIAL CONTROL

Micrococcus luteus

- aerobic Understanding how microorganisms can be destroyed is

one of the most important concerns in microbiology. Because of
Escherichia coli their pathogenic effects to humans and other living forms, a number
of procedures introducing chemical compounds as antimicrobial
- Facultative anaerobic
agents have been developed. In the study of microbial control, two
Streptococcus lactis methods are often considered. These are the physical and chemical
methods.
- Anaerobic
Scrubbing and handwashing with soap are efficient ways
Pseudomonas aeruginosa of removing microorganisms from the body through physical
methods. In hospitals, the use of surgical gloves and gowns helps
- Aerobic
prevent infection of patients as well as the hospital personnel.
Bacillus stearothermophilus
Another method in controlling the growth of
- Aerobic microorganisms is the use of chemicals or antimicrobial agents.
These are substances that inhibit the growth of unwanted forms.
The effectiveness of an antimicrobial agent is frequently compared
to that of a phenol. A phenol coefficient (PC) is then measured. If
Culture Media Used the PC of the chemical used is greater than 1, it would indicate that
the chemical being tested is more effective.
Nutrient Broth
This activity emphasizes the control of microbial growth
- With different pH values
using handwashing. It also determines the effectivity of chemicals
Nutrient Agar through measurement of phenol coefficient using Staphylococcus
aureus as the test organism.
- With different concentration of salt and sugar

Thioglycollate Medium
Effect of Handwashing
- For Oxygen requirement
- Has Sodium Thioglycollate in order to trap the oxygen 1. Allow the students to shake the hands of at least 5 students in the
class.

2. Then, soak a cotton pledget into sterile distilled water and swab
Conclusion the palm. Spread it over the entire NA plate.
Microorganisms have different responses on 3. Ask the student to wash his hands with soap and water for 1
environmental conditions and should always be optimized. Their minute.
growth depends on the physical conditions and chemical
components of the culture medium. 4. Repeat step 2.

5. Incubate the plates at 37˚C for 48 hrs.

6. Check for the presence of growth in the two plates. Refer to the
colony characteristics in Figure 6.1 and answer items 1-3 in the
worksheet.

Effect of UV Light (radiation) on Microbes

1. Divide three NA plate into two quadrants each. Label one plate
with “UV for 30s”; another with “UV for 1 min”; and last with “UV for
2 mins”.

2. Inoculate each plate with coli and create a bacterial lawn.

3. Prepare the UV lamp. Expose the first plate for 30s, while
covering one half with a clean board to serve as the control
(unexposed).

4. Expose the second plate and third plate accordingly as with the
first plate.

5. Incubate the plates at 37˚C for 24 hrs.

6. Check for presence or surviving colonies.

Effect of antimicrobial agent

1. Prepare Phenol and Test chemical solutions using the following

dilutions (1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130) so that the
final volume in each tube is 50 ml. (or any range necessary to
determine the data requirement of the formula)

2. Inoculate each of these dilutions with 0.5 ml of a 24-hr broth

culture of Staphylococcus aureus.

3. At intervals of 5 and 10 mins, obtain 0.1 mL of the broth culture

and inoculate into sterile NB labelled accordingly.

4. Incubate the labelled broth tubes at 37˚C for 48 hrs. Observe for
the presence of growth i.e. turbidity.

Formula: PC = A / B

A = highest dilution (lowest concentration) of disinfectant

that kills the microorganism after 10 min but not 5 min.

B = highest dilution (lowest concentration) of Phenol that

kills the microorganism after 10 min but not 5 min.

Interpretation:

PC > 1 = indicates that the disinfectant is more effective than

Phenol.

PC = 1 indicates equal effectiveness of the two chemicals.

PC < 1 indicates less effectiveness of the test chemical.

Explanation:

Given are broth tubes inoculated with 0.1 ml of mixture of

test bacterium and the dilution of either phenol or test chemical. The
shade indicates turbidity, hence, growth of microorganisms. To find
“A”, examine the tubes under the “test” chemical and look for the
highest dilution with growth in the “5” minutes and not in “10”
minutes. For instance, 1/160 and 1/320 fit this criterion. However,
1/320 is the higher dilution, thus, A = 320. Note that the reciprocal
of the dilution is used.

For Phenol, 1/80 and 1/160 have these characteristics.

However, 1/160 is the higher dilution, thus, B= 160. Applying the
formula, Phenol Coefficient of the test chemical is 2, which says that
disinfectant is twice as more effective than Phenol.