ORO, CHARLOU J. “Module 3 post task / review question submission” SIR.

PAHAYO
DENT 3F MICROBIOLOGY LAB.

Module 3 Lesson 1: Estimation of Bacterial Growth

POST TASKS
1. Why is serial dilution usually performed with a power of 10 dilution
ANSWER:
- The first step in making a serial dilution is to take a known volume (usually 1ml)
of stock and place it into a known volume of distilled water (usually 9ml). This
produces 10ml of the dilute solution. This dilute solution has 1ml of extract /10ml,
producing a 10-fold dilution. (i.e. the amount of stock in each ml of the diluted
solution is 0.1ml.)

2.What are possible sources of error in performing serial dilutions?

ANSWER:
-Serial dilution processes face two major challenges. The first is error propagation
across columns or rows. With each sequential serial dilution step, transfer
inaccuracies lead to less accurate and less precise dispensing. The result is that
the highest dilutions will have the most inaccurate results. To compensate for this
error possibility, longer mixing times are required, which then increases the time
required to perform the serial dilution. These challenges greatly limit the
throughput capacity of an automated serial dilution system.

3.An area of application for serial dilution is in Pharmacology when Minimum

Inhibitory Concentration (MIC) of a drug needs to be determined. Read about
this and give your insights on how is it similar or different from the serial
dilution in Microbiology?
ANSWER:
-In microbiology, serial dilutions (log dilutions) are used to decrease a bacterial
concentration to a required concentration for a specific test method, or to a
concentration which is easier to count when plated to an agar plate.
- In pharmaceutical laboratories, serial dilution is performed to receive the
necessary concentration of chemicals and compounds as this method is more
effective than individual dilutions.
-Serial dilutions are used to accurately create extremely diluted solutions, as well
as
solutions for experiments that require a concentration curve with an exponential
or
logarithmic scale. Serial dilutions are widely used in experimental sciences,
including biochemistry, pharmacology, microbiology, and physics.

4.Sample problem:
Assuming that serial dilution was carried out in a laboratory experiment in 6
tubes with 9 ml of saline in each tube and 0.1ml of the stock solution was used
for initial dilution, pour plate method of inoculation was performed after the
dilution process using 1ml of the culture from the samples from the test tubes.  
It was found out that on the 6th petridish after incubation, there was a total of
16 individual colonies counted.  Compute the total number of microorganisms
present in tube one, assuming that there was no human error in the transferring
process.
ANSWERS

Equation:
DF=Volume of stock/Final Volume
DF=0.1mL/(0.1ml+0.9mL)
DF=0.1mL/9.1mL
DF=0.01 or 1x10^-2

Formula:
Dilution Factor (DF)= Volume of Stock/Final Volume
Final Dilution= (Previous Dilution x Dilution of Next Tube)
Colony/Plaque Forming Units=(# of colonies / Amt. plated (mL) x Dilution =
CFU/mL)
Given:
Saline in each tube: 9mL
Amt. plated (mL): 1mL
Stock Dilution= 0.1 mL
Total Colonies Counted= 16

Viable Count Assay:

Plates with 300+ colonies/plaques: Too numerous to count (TNTC)
Plates with less than 30 colonies/plaques: Too few to count (TFTC)

1st 2nd 3rd 4th 5th 6th

Sample sample sample sample sample sample
Dilution 10 ^ -2 10 ^ -2 10 ^ -2 10 ^ -2 10 ^ -2 10 ^ -2
Final 10 ^ -2 10^ -4 10^-6 10^ -8 10^ -10 10^ -12
Dilution

TFTC (Too few to count) because the counted colony (16) is less than 30
Colony/Plaque Forming Units= # of Colonies ÷ Amt. plated (mL) × Dilution =
CFU/mL
=16/1mL x 10 ^12= 1.6 x 10^13 CFU/mL
=1.6 x 10^13 CFU/mL
https://www.training.nih.gov/assets/Lab_Math_II_Transcript_-_508.pdf
https://www.bio.umass.edu/micro/immunology/elisa/serial.htm
http://biology.kenyon.edu/courses/biol09/tetrahymena/serialdilution2.htm/
ORO, CHARLOU J. “Module 3 post task / review question submission” SIR. PAHAYO
DENT 3F MICROBIOLOGY LAB.

LESSON 2
1. What are the stages of the bacterial growth curve? Discuss Briefly.
● Lag Phase: This initial phase is characterized by cellular activity but not
growth. The cells increase in size, but no cell division occurs in the phase.
● Exponential (Log) Phase: After the lag phase, bacterial cells enter the
exponential or log phase. This is the time when the cells are dividing by
binary fission and doubling in numbers after each generation time.
● Stationary Phase: Bacterial cell growth reaches a plateau, or stationary
phase, where the number of dividing cells equal the number of dying cells.
●Death Phase: As nutrients become less available and waste products
increase, the number of dying cells continues to rise. In the death phase, the
number of living cells decreases exponentially and population growth
experiences a sharp decline.

2. What are the factors influencing the decline of a bacterial culture?

- If a bacterial culture is left in the same media for too long, the cells use up the
available nutrients, excrete toxic metabolites, and eventually the entire
population will die. Thus bacterial cultures must be periodically transferred,
or subcultured, to new media to keep the bacterial population growing.
-At death phase (decline phase), bacteria die. This could be caused by lack of
nutrients, environmental temperature above or below the tolerance band for
the species, or other injurious conditions.

3. Would all microorganisms exhibit the same bacterial growth curve? Why?
- No. Since bacteria require certain conditions for growth, and these conditions
are not the same for all bacteria. Factors such as oxygen, pH, temperature,
and light influence microbial growth. Additional factors include osmotic
pressure, atmospheric pressure, and moisture availability. A bacterial
population's generation time, or time it takes for a population to double,
varies between species and depends on how well growth requirements are
met.

4. Why is there an existence of a stationary phase? Is it really because the

microorganisms “stop” growing or dividing?
- It’s because at some point the bacterial population runs out of an essential
nutrient/chemical or its growth is inhibited by its own waste products or
lack of physical space, causing the cells to enter into the stationary phase.

5. What are the other methods of determining cell population?

6. Differentiate the following: Optimum growth temperature, minimum
growth temperature & maximum growth temperature:
- Minimum temperature: It is the lowest temperature which will support the
growth of the microorganism. Below the minimum temperature the
microorganisms survive for a long time and such low temperatures may be
used for their storage.
- Optimal temperature: It is the temperature, or more usually the range of
temperatures, which supports the fastest growth rate.
-Maximum temperature: It is the highest temperature which will support
the growth of the microorganism. Above the maximum temperature the
microorganism is usually killed and such high temperatures may be used in
sterilization.

7. Name the different types of microorganisms based on their oxygen

requirement:
-Obligate aerobes: Bacteria that can not survive without oxygen. These
microbes are dependent upon oxygen, as they convert oxygen to energy
during cellular respiration.
-Obligate anaerobes: Bacteria that can survive without oxygen. Their
metabolic processes for energy production are halted in the presence of
oxygen.
- Facultative anaerobes: Can grow with or without oxygen. In the absence of
oxygen, they utilize either fermentation or anaerobic respiration for energy
production.
-Aerotolerant anaerobes: Utilize anaerobic respiration but are not harmed
in the presence of oxygen.

8. Different psychrophiles, mesophiles & thermophiles:

- Bacteria that grow best in cooler environments are called psychrophiles.
These microbes prefer temperatures ranging between 4°C and 25°C (39°F
and 77°F). Extreme psychrophiles thrive in temperatures below 0°C/32°F
and can be found in places such as arctic lakes and deep ocean waters.
- Bacteria that thrive in moderate temperatures (20-45°C/68-113°F) are
called mesophiles. These include bacteria that are part of the human
microbiome which experience optimum growth at or near body temperature
(37°C/98.6°F).
-Thermophiles grow best in hot temperatures (50-80°C/122-176°F) and can
be found in hot springs and geothermal soils. Bacteria that favor extremely
hot temperatures (80°C-110°C/122-230°F) are called hyperthermophiles.

9. Aside from the factors discussed in the laboratory exercise, what other
factors do you think influence bacterial growth?
Aside from temperature, oxygen, pH, and moisture, the other factors
that influence bacterial growth are:
*Osmotic pressure
- Microbes obtain almost all their nutrients in solution from
surrounding water.
- Hence factors such as osmotic pressure and salt concentration
of the solution affect the growth of bacteria.
- Bacteria by virtue of mechanical strength of their cell wall are
able to withstand a wide range of external osmotic variations.
- Organisms requiring high osmotic pressures are called
osmophilic bacteria.
*Light
- Phototrophs (bacteria deriving energy from sunlight)
- Chemotrophs (bacteria deriving energy from chemical
sources).
* Carbon dioxide
- Carbon is the structural backbone of the organic compounds
that make up a living cell. Based on their source of carbon
bacteria can be classified as autotrophs or heterotrophs.
- Autotrophs: require only carbon dioxide as a carbon source. An
autotroph can synthesize organic molecules from inorganic
nutrients.
- Heterotrophs: require organic forms of carbon. A Heterotroph
cannot synthesize organic molecules from inorganic nutrients.

10. Is there an actual theoretical value for optimum growth temperature? Why
or why not? Justify your answer:

-Yes, an actual theoretical value for optimum growth temperature exists because
it has been researched by experts that they expect from that value that it
assumes perfect or near-perfect conditions. For example, it is proven that a
“Mesophile” is a microorganism that grows best in moderate temperature,
neither too hot nor too cold, commonly ranges between 20 and 45 °C (68 and
113 °F). Some bacteria grow best in cooler environments called “Psychrophiles”
that grow best in hot temperatures (50-80°C/122-176°F) and can be an estimation
of temperatures between 4°C and 25°C (39°F and 77°F). Some grnd in hot springs
and geothermal soils called “Thermophiles”. Others, prefer to be in extremely hot
temperatures (80°C-110°C/122-230°F) are called “hyperthermophiles”. So yes, All
bacteria have their own optimum environmental surroundings and temperatures
in which they thrive the most.

SOURCES:

 https://www.thoughtco.com/bacterial-growth-curve-phases-4172692#:~:text=The%20bacterial
%20growth%20curve%20represents,metabolically%20active%20but%20not%20dividing.
 https://bio.libretexts.org/Bookshelves/Microbiology/Book
%3A_Microbiology_(Bruslind)/09%3A_Microbial_Growth#Growth_Curve
 https://slideplayer.com/slide/7470122/
 https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/bacteriological-culture-
methods/#:~:text=Growing%20Bacteria%20in%20Culture&text=If%20a%20bacterial%20culture%20is,the
%20entire%20population%20will%20die.
 https://microbenotes.com/bacterial-growth-and-factors-affecting-growth-of-bacteria/
 https://bio.libretexts.org/Bookshelves/Microbiology/Book
%3A_Microbiology_(Kaiser)/Unit_7%3A_Microbial_Genetics_and_Microbial_Metabolism/17%3A_Bac
terial_Growth_and_Energy_Production/17.2%3A_Factors_that_Influence_Bacterial_Growth
ORO, CHARLOU J. “Module 3 post task / review question submission” SIR. PAHAYO
DENT 3F MICROBIOLOGY LAB.

LESSON 3:
1. Give the biochemical principle, positive results and at least 1 organism
identified by the following tests:

BIOCHEMICAL POSITIVE ORGANISM

PRINCIPLE RESULT

A. Catalase Test The enzyme Bubbles are a Used to differentiate

catalase mediates positive result for staphylococci (catalase
the breakdown of the presence of positive) from
hydrogen peroxide catalase. streptococci (catalase
into oxygen and negative).
water. The
presence of the
enzyme in a
bacterial isolate is
evident when a
small inoculum is
introduced into
hydrogen
peroxide.

B. Coagulase Coagulase is an Macroscopic Staphylococcus

test enzyme-like clumping in 10 aureus and other
protein and causes seconds or less in animal host bacteria
plasma to clot by coagulated plasma like S.
converting drop and no pseudintermedius, S.
fibrinogen to clumping in saline intermedius, S.
fibrin. or water drop. schleiferi, S. delphini,
S. hyicus, S. lutrae,
and S. hyicus.

C. Oxidase Test Cytochrome Development of a Pseudomonas,

C containing deep purple- Neisseria, Alcaligens,
organisms blue/blue colour Aeromonas,
produce an indicates oxidase Campylobacter,
intracellular production within Vibrio, Brucella,
oxidase enzyme. 5-10 seconds. Pasteurella, Moraxella,
This oxidase Helicobacter pylori,
enzyme catalyzes Legionella
the oxidation of pneumophila, etc.
cytochrome c.
Organisms which
 contain
cytochrome c as
part of their
respiratory chain
are oxidase-
positive and turn
the reagent
blue/purple.

D. Urease Urea is the Development of Proteus spp,

product of an intense Cryptococcus spp,
decarboxylation of magenta to bright Corynebacterium spp,
amino acids. pink color in 15 Helicobacter pylori,
Hydrolysis of urea min to 24 h. Yersinia spp, Brucella
produces spp, etc.
ammonia and CO2.
The formation of
ammonia
alkalinizes the
medium, and the
pH shift is
detected by the
color change of
phenol red from
light orange at pH
6.8 to magenta
(pink) at pH 8.1.
Rapid urease-
positive organisms
turn the entire
medium pink
within 24 hours.

E. Hemolysis Certain organisms Enhanced Streptococcus

(including group B hemolysis is agalactiae
streptococci) indicated by an (ATCC13813)—enhan
produce a arrowhead shaped ced arrowhead.
diffusible zone of beta
extracellular hemolysis at the
hemolytic protein juncture of the
(CAMP factor) that two organisms.
acts synergistically
with the beta-lysin
of Staphylococcus
aureus to cause
enhanced lysis of
 red blood cells.
The group B
streptococci are
streaked
perpendicular to a
streak of S. aureus
on sheep blood
agar.

F. Hydrogen This test Positive result is The test aids in

Sulfide determines indicated by the the identification and
whether the presence of black differentiation of
microbe reduces precipitate formed members of
sulfur-containing in the medium. Enterobacteriaceae
compounds to (enterics) from other
sulfides during the Gram- bacilli. It is
process of especially helpful in
metabolism. If identifying
sulfide is Salmonella,
produced, it Francisella, and
combines with Proteus species.
iron compounds to
produce FeS, a
black precipitate.

2. What is an API system?

-The analytical profile index or API is a classification of bacteria based on
experiments, allowing fast identification. This system is developed for quick
identification of clinically relevant bacteria. Because of this, only known bacteria
can be identified.
3. Answer the ff:
Bacteria Indole test Methyl Red Voges- Citrate Test
Test Proskauer
Test
E.coli Positive Positive Negative Negative
A.aerogenes Negative Negative Positive Positive
S.dysenteriae Variable Positive Negative Negative
K.pneumonia Negative Negative Positive Positive
V.cholera Positive Negative Variable Positive