HES032 (Microbiology and Parasitology)

STUDENT ACTIVITY SHEET

BS NURSING / SECOND YEAR
Session # 6

LESSON TITLE: Controlling Microbial Growth in Vitro Materials:

LEARNING OUTCOMES: Book, pen, notebook, and paper

At the end of the lesson, the nursing student can:

1. Define culture, inoculum, and culture media; Reference:
2. Differentiate enriched, enrichment, selective, and differential
media and cite an example of each; Engelkirk, P., & Engelkirk, J. (2015). Burton's
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3. Enumerate the steps in preparing a culture medium in plate Microbiology for the Health and Sciences. 10
or in a tube; ed., Philadelphia: Lipincott Williams & Wilkins
4. Explain the importance of “aseptic technique” in the Tortora, G., F.B. (2016). Microbiology an
microbiology laboratory; th
Introduction. 12 ed., United States of
5. Differentiate among sterilization, disinfection, and America: Pearson Education Inc.
sanitization;
6. Discuss the physical and chemical methods of inhibiting
microbial growth in vitro.

Daily Productivity Tip:

Repetition is the key. If you repeat something several times, you are most likely to remember it just like the
terms used in this module.

LESSON PREVIEW / REVIEW (5 minutes)

Recall from the last session the phases of bacterial growth by labelling the illustration below:

STATIONARY PHASE

DEATH PHASE

LOG PHASE

LAG PHASE

Next, list down five (5) factors affecting the microbial growth:
Energy and Carbon Sources Requirement
1. ____________________
Temperature Requirement
2. ____________________
pH Requirement
3. ____________________
Oxygen Requirement
4. ____________________
Salt Requirement
5. ____________________

Awesome! Now, you can proceed with today’s session.

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MAIN LESSON (50 minutes)

Lesson 1: Encouraging the Growth of Microbes (Culture Media)

There are many reasons why the growth of microbes is encouraged in microbiology laboratories. In microbiology research
laboratories, scientists must culture microbes so that they can learn more about them, harvest antibiotics, and other
microbial products, test new antimicrobial agents, and produce vaccines.
In the laboratory, however, a pure culture of a single species of bacteria can usually be maintained if the appropriate
nutrients, growth medium, and environmental conditions are provided.

Microorganisms that are difficult to grow in the laboratory are said to be fastidious.
Other microorganisms that will not grow on artificial media include Treponema pallidum (the
bacterium that causes syphilis) and Mycobacterium leprae (the bacterium that causes leprosy)

Culture Medium – is a liquid, semi-solid or solid medium utilized to observe growth patterns of microorganism as well as
for transport and storage
Inoculum - microbes that are introduced into a culture medium to initiate growth.
Culture - the microbes that grow and multiply in or on a culture medium.

Types of Culture:
i. Pure Culture
ii. Mixed Culture
iii. Stock Culture
Classification of Culture Medium

A. According to Consistency
i. Liquid Medium (Broth)
ii. Semi-solid Medium
iii. Solid Medium
B. According to Composition
i. Synthetic or Defined Medium
ii. Non-Synthetic Medium
iii. Tissue Culture Medium
C. According to Use
i. Simple Media/General Purpose/Supportive Media
ii. Enrichment Media
iii. Enriched Media/Nonselective Media
iv. Differential Media
v. Selective Media
vi. Special Media
D. According to Dispensing or Distribution
i. Plated Media
ii. Tubed Media

The media (sing., medium) that are used in microbiology laboratories to culture bacteria are referred to as artificial media
or synthetic media, because they do not occur naturally; rather, they are prepared in the laboratory.

Chemically defined medium one in which all the ingredients are known because this is
prepared in the laboratory in a certain amount (e.g.,
carbohydrates, amino acids, salts).
Complex medium one in which all the ingredients are unknown from animal
organs (e.g.,heart, brain, liver), fish, yeasts, and plants.
Enriched medium broth or solid medium containing a rich supply of special
nutrients that promotes the growth of fastidious organisms.
Ex. Blood agar and Chocolate agar

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 Enrichment medium usually liquid that favor the growth of a particular microbe
but not others. In this sense, it is also a selective medium,
but it is designed to increase very small numbers of the
desired type of organism to detectable levels
Ex. Selenite F. for Salmonella typhi
Selective medium has added inhibitors that discourage the growth of certain
organisms without inhibiting the growth of organism being
sought.
Ex. MacConkey agar, CNA, PEA
Differential medium permits the differentiation of organisms that grow on the
medium.
Ex. MacConkey agar

Inoculation of Culture Media

Inoculation of a liquid medium involves adding a portion of the

specimen to the medium. Inoculation of a solid or plated medium REMEMBER HOW TO
involves the use of a sterile inoculating loop to apply a portion of the PREPARE YOUR
specimen to the surface of the medium; a process commonly referred CULTURE MEDIA:
to as streaking.
PAD = Plate Autoclave
Incubation
then Dispense
After media are inoculated, they must be incubated (i.e., they must be DAT = Dispense then
placed into a chamber [called an incubator] that contains the appropriate Autoclave Tube
atmosphere and moisture level and is set to maintain the appropriate
temperature). This is called incubation. To culture most human
pathogens, the incubator is set at 35°C to 37°C.

Preserving Bacterial Cultures

Two common methods of prescribing microbial cultures for long periods are deep-freezing and lyophilization.
Differentiate the two methods below:
i. Deep-freezing
ii. Lyophilization (Freeze-drying)

Think and Learn: How to culture fungi, protozoa, viruses and other obligate intracellular pathogens?

Lesson 2: Inhibiting the Growth of Microbes in Vitro

In hospitals, nursing homes, and other healthcare institutions, for example, it is necessary to inhibit the growth of
pathogens so that they will not infect patients, staff members, or visitors. Before discussing the various methods used to
destroy or inhibit the growth of microbes, let us first understand these following terms in microbiology:

Sterilization - involves the destruction or elimination of all microbes, including cells, spores, and viruses.
Disinfection - the elimination of most or all pathogens (except bacterial spores) from nonliving objects.
Pasteurization – a method of disinfecting liquids. It is used today to eliminate pathogens from milk and most
other beverages.
Disinfectant - chemicals used to disinfect inanimate objects, such as bedside equipment and operating rooms.
Antiseptics are solutions used to disinfect skin and other living tissues.
Sanitization is the reduction of microbial populations to levels considered safe by public health standards,
. such as those applied to restaurants.

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 Microbicidal agents - The suffix -cide or -cidal refers to “killing”. Bactericidal agents specifically kill bacteria,
but not necessarily endospores. Sporicidal agents are required to kill bacterial endospores. Fungicidal agents
(fungicides) kill fungi, including fungal spores. Algicidal agents (algicides) are used to kill algae in swimming
pools and hot tubs. Viricidal agents (or virucidal agents) destroy viruses. Pseudomonicidal agents kill
Pseudomona spp., and tuberculocidal agents kill M. tuberculosis.
Microbistatic agents - drug or chemical that inhibits reproduction of microorganisms, but does not necessarily
kill them. A bacteriostatic agent is one that specifically inhibits the metabolism and reproduction of bacteria. Ex.
lyophilization and rapid-freezing.
Sepsis - presence of pathogens in blood or tissues.
Asepsis - absence of pathogens.
Aseptic techniques – used to eliminate and exclude pathogens.
Antisepsis - is the prevention of infection.
Sterile technique - practiced when it is necessary to exclude all microorganisms from a particular area, so
that the area will be sterile such as the operating room in the hospital.

Two categories of aseptic technique are medical asepsis and surgical asepsis

Think and Learn: What is the difference between bacteremia and septicemia?

Medical Asepsis – the absence of pathogens in patient’s environment. Remember to observe aseptic
Surgical Aspesis – the absence of microorganisms in a surgical environment. technique and a Good
Microbiological Practice
(GMP) at all times.
Physical Methods to Inhibit the Microbial Growth in Vitro

I. Heat is the most practical, efficient, and inexpensive method of sterilization of those inanimate objects and materials
that can withstand high temperatures. Two factors that determine the effectiveness of heat for sterilization:
a. Temperature
b. Time
Thermal Death Point is the lowest temperature that will kill all the organisms in a standard pure culture within a
specified period.
Thermal Death Time is the length of time necessary to sterilize a pure culture at a specified temperature.
A. Dry Heat provides effective sterilization of metals, glassware, some powders, oils, and waxes.
Ex. Hot air oven, Incineration.
B. Moist Heat. Heat applied in the pressure of moisture, as in boiling or steaming, is faster and more effective than dry
heat.
1. Autoclaving - uses steam under pressure to completely destroy all microbial life. Commercially available
strips or solutions containing bacterial spores can be used as quality-control measures to ensure that
autoclaves are functioning properly.
2. Boiling - the vegetative forms of most pathogens are quite easily destroyed by boiling for 30 minutes. Boiling
is not always effective, because heat-resistant bacterial endospores, mycobacteria, and viruses may be
present.
II. Cold. Most microorganisms are not killed by cold temperatures and freezing, but their metabolic activities are slowed,
greatly inhibiting their growth.
NOTE: Refreezing of thawed foods is an unsafe practice, because it preserves the millions of microbes that might be
present. (C. botulinum, C. perfringens causes food poisoning)
III. Desiccation means “drying”. Dried viable pathogens may be present in dried matter, including blood, pus, fecal
material, and dust that are found on the floors, in beddings, on clothing, and in wound dressings.
IV. Radiation. Sun is not a particularly reliable disinfecting agent because it kills only those microorganisms that are
exposed to direct sunlight. Example of radiation are X-rays, and gamma and beta rays. Can cause cancer and used
in treatment of some cancer.
V. Filtration filters of various pore sizes are used to filter or separate cells, larger viruses, bacteria, and certain
microorganisms from the liquids or gases in which they are suspended.

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 NOTE: Dry gauze and paper masks prevent the outward passage of microbes from the mouth and nose, at the
same time protecting the wearer from inhaling airborne pathogens and foreign particles that could damage the
lungs.

Incineration (burning) is an effective means of destroying contaminated disposable materials.

Chemical Methods to Inhibit the Microbial Growth in Vitro

I. Use of Disinfectants
II. Use of Antiseptics Think and Learn: How do you call antimicrobial chemical agents
that can safely be applied to skin?

TAKE A QUICK BREAK. BREATHE SLOWLY. WRITE FREELY.

CHECK FOR UNDERSTANDING (30 minutes)

You will answer and rationalize this by yourself. This will be recorded as your quiz. One (1) point will be given to correct
answer and another one (1) point for the correct ratio. Superimpositions or erasures in you answer/ratio is not allowed. You
are given 30 minutes for this activity (20 points):

1. It would be necessary to use a tuberculocidal agent to kill a particular species of:

a. Clostridium.
b. Mycobacterium.
c. Staphylococcus.
d. Streptococcus.
ANSWER: A
RATIO: Tuberculocidal indicates that the disinfectant has been shown capable of killing Mycobacterium tuberculosis
var. bovis.

2. Pasteurization is an example of what kind of technique?

a. antiseptic
b. disinfection
c. sterilization
d. surgical aseptic
ANSWER: B
RATIO: Pasteurization is a method of disinfection to save milk from disease-causing pathogens and it increases milk
safety for consumers.

3. The combination of freezing and drying is known as:

a. desiccation.
b. lyophilization.
c. pasteurization.
d. tyndallization.
ANSWER: B
RATIO: Lyophilization is a water removal process typically used to preserve perishable materials, to extend shelf life or
make the material more convenient for transport. Lyophilization works by freezing the material, then reducing
the pressure and adding heat to allow the frozen water in the material to sublimate.
4. Organisms that live in and around hydrothermal vents at the bottom of the ocean are:
a. acidophilic, psychrophilic, and halophilic.

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b. halophilic, alkaliphilic, and psychrophilic.
c. halophilic, psychrophilic, and piezophilic.
d. halophilic, thermophilic, and piezophilic.
ANSWER: D
RATIO: Organisms that live in and around hydrothermal vents at the bottom of the ocean are halophilic, thermophilic,
and piezophilic.

5. When placed into a hypertonic solution, a bacterial cell will:

a. take in more water than it releases.
b. lyse.
c. shrink.
d. swell.
ANSWER: C
RATIO: When a bacterial cell is placed into a hypertonic solution which have a high concentration of solute, the _cell
will shrink because of osmosis. The high concentration of water molecule inside the cell will add to the other
hypertonic solution making the cell shrink.
6. To prevent Clostridium infections in a hospital setting, what kind of disinfectant should be used?
a. fungicidal
b. pseudomonicidal
c. sporicidal
d. tuberculocidal
ANSWER: C
RATIO: Sporicidal agent means a chemical or physical agent that destroys bacterial and fungal spores when used in
sufficient concentration for a specified contact time. It is expected to kill all vegetative microorganisms.

7. Sterilization can be accomplished by use of:

a. an autoclave.
b. antiseptics.
c. medical aseptic techniques.
d. pasteurization.
ANSWER: A
RATIO: Sterilization can be achieved by autoclaving.

8. The goal of medical asepsis is to kill , whereas the goal of surgical asepsis is to kill .
a. all microorganis, pathogens
b. bacteria, bacteria and viruses
c. nonpathogens, pathogens
d. pathogens, all microorganisms
ANSWER: Medical asepsis if for the purpose of killing pathogens, whereas the surgical asepsis is to kill all microorganism.
RATIO:

9. Which of the following types of culture media is selective and differential?

a. blood agar
b. MacConkey agar
c. phenylethyl alcohol agar
d. Thayer-Martin agar
ANSWER: B
RATIO: MacConkey Agar is a culture media that is selective and differential.

10. All the following types of culture media are enriched and selective except:
a. blood agar.
b. colistin–nalidixic acid agar.
c. phenylethyl alcohol agar.
d. Thayer-Martin agar.

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ANSWER: A
RATIO: Blood Aga is not an enriching or selective form of culture media.

RATIONALIZATION ACTIVITY (THIS WILL BE DONE DURING THE FACE TO FACE INTERACTION)
The instructor will now rationalize the answers to the students. You can now ask questions and debate among yourselves.
Write the correct answer and correct/additional ratio in the space provided.

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LESSON WRAP-UP (5 minutes)

You will now mark (encircle) the session you have finished today in the tracker below. This is simply a visual to help you
track how much work you have accomplished and how much work there is left to do.

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You are done with the session! Let’s track your progress.

Exit Ticket: One-minute paper

a. How do you feel about today’s session? __ Happy __ Satisfied __ Sad __ Confused

b. What question(s) do you have as we end this session? NONE

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Reading Assignment: For the next session, read chapter 9 of Burton’s Microbiology for the Health Siences, 10 ed.

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