Name: JUSTINE MARIE G.

SISON Score:
Year & Section: BSBIO - 4C Date: ________

Activity # 5:
ASEPTIC TECHNIQUE AND CULURE MEDIA PREPARATION

I. Introduction

When working with microorganisms it is desirable to work with a

pure culture. A pure culture is composed of only one kind of
microorganism. Occasionally a mixed culture is used. In mixed culture,
there are two or more organisms that have distinct characteristics and
can be separated easily. In either situation, the organisms can be
identified. When unwanted organisms are introduced into the culture they
are known as contaminants. The aseptic technique is a method that
prevents the introduction of unwanted organisms into an environment.
When changing wound dressings aseptic technique is used to prevent
possible infection. When working with microbial cultures aseptic
technique is used to prevent the introduction of additional organisms into
the culture. Microorganisms are everywhere in the environment. When
dealing with microbial cultures it is necessary to handle them in such a
way that environmental organisms do not get introduced into the culture.
Microorganisms may be found on surfaces and floating in air currents.
They may fall from objects suspended over a culture or swim in fluids.
The aseptic technique prevents environmental organisms from entering a
culture. This technique is achieved by growing the microorganisms in
artificial chemical preparations referred to as culture media. They can be
prepared in both liquid forms called broth and in a solid form called agar.
Culture media need to be sterilized by autoclaving, heating, or by
membrane-filtration when it is prepared.

II. Objectives:

At the end of the activity, the students will be able to:

1. demonstrate aseptic technique.
2. acquire and develop skills in the preparation of different types of culture
media for the cultivation of microorganisms.
III. Materials:
1. Gauze or bandage
2. Cotton
3. Masking tape
4. Nutrient Agar (450 mL)
5. Nutrient Broth (50 mL)
6. 0.9% Saline Solution (220 mL)
7. Eosin Methylene Blue Agar (250 mL)
8. Potato Dextrose Agar (250 mL)
9. Mueller Hinton Agar (450 mL)
10. Erlenmeyer flasks (500 mL, 3 pcs)
11. Beaker (150 mL, 2 pcs)
12. Glass petri dish (40 pcs)
13. Test tubes and test tube rack (26 PCS)
14. Aluminum foil
15. Weighing scale
16. Distilled water
17. Graduated cylinder (500 mL)
18. Stirring rod
19. Glass pipette and aspirator
20. Alcohol lamp

IV. Procedure:
1. Make cotton plugs for Erlenmeyer flasks and test tubes.
2. Preparation and sterilization of culture media and glass wares:
a. Nutrient Agar (NA). Weigh fourteen (14) grams of NA,
place in an Erlenmeyer flask, and dissolve in one liter of
distilled water with heating until the solution became clear
yellow. Cover the flask with a cotton plug and aluminum foil.
Sterilize the medium in an autoclave at 121 oC for 15
minutes or a pressure cooker at 15 pounds per square
inches (psi) for 30 minutes. The sterile NA is allowed to cool
and then pour on Petri dishes for colony characterization of
the isolates. Petri dishes must be covered immediately after
pouring to avoid contamination. Wait for the agar to solidify.
When preparing NA for slants, transfer a desired
volume of NA into the test tubes and cover them with cotton
plugs and aluminum foil before autoclaving.
 After sterilization, place the test tubes in a slanted
position (avoid touching the cotton) and wait for the agar to
solidify.
b. Nutrient Broth (NB). Weigh thirteen (13) grams of NB,
place in an Erlenmeyer flask, and dissolve in one liter of
distilled water, stir until the solution became clear yellow.
Cover the flask with a cotton plug and aluminum foil.
When preparing NB in test tubes, transfer a desired
volume of NB into the test tubes and cover them with cotton
plugs and aluminum foil before autoclaving. Sterilize the
medium in an autoclave at 121oC for 15 minutes or a
pressure cooker at 15 psi for 30 minutes. The sterilized NB
in test tubes is used for serial dilution and inoculation of
samples.
c. Eosin Methylene Blue Agar (EMBA). Weigh thirty-six (36)
grams of EMBA, place in an Erlenmeyer flask, and dissolve
in one liter of distilled
water with heating until the granules are dissolved. Cover
the flask with a cotton plug and aluminum foil. Sterilize the
medium in an autoclave at 121oC for 15 minutes or a
pressure cooker at 15 pounds per square inches (psi) for 30
minutes. The sterile EMBA is allowed to cool and then pour
on Petri dishes for the detection of coliform and E. coli. Petri
dishes must be covered immediately after pouring to avoid
contamination. Wait for the agar to solidify.
d. Potato Dextrose Agar (PDA). Weigh thirty-nine (39) grams
of PDA, place in an Erlenmeyer flask, and dissolve in one
liter of distilled water with heating until the solution becomes
clear creamy white color. Cover the flask with a cotton plug
and aluminum foil. Sterilize the medium in an autoclave at
121oC for 15 minutes or a pressure cooker at 15 pounds per
square inches (psi) for 30 minutes. The sterile PDA is
allowed to cool and then pour on Petri dishes for the
isolation of fungi. Petri dishes must be covered immediately
after pouring to avoid contamination. Wait for the agar to
solidify.
e. 0.9% Normal Saline Solution (NSS). Weigh nine-tenths
(0.9) grams of NaCl, place in a beaker, and dissolve in one
hundred milliliters of distilled water. Transfer 9 mL into test
tubes and cover with cotton plug. Place all test tubes in one
beaker and cover it with aluminum foil.
 Sterilize in an autoclave at 121 oC for 15 minutes or a
pressure cooker at 15 pounds per square inches (psi) for 30
minutes. The sterile NSS is allowed to cool.
f. Materials and glasswares such as Petri dishes, Erlenmeyer
flasks, pipettes, stirring rods, and beakers, can be sterilized
in two ways:
i. Using a laboratory oven: Materials and glasswares
are sterilized without any cover at 100oC for one
hour.
ii. Using an autoclave or a pressure cooker:
Materials and glasswares are wrapped with
newspaper or a bond paper and are sterilized in an
autoclave at 121oC for 15 minutes or a pressure
cooker at 15 psi for 30 minutes.

Sterilization of glass wares and culture media was very

important to kill unwanted objects, microorganisms, and
contaminants that could possibly give an effect on the result of
the experiment or the study.

V. Guide Questions:

1. Why is it important to correctly fit the cotton plug?

- to preserve specimen integrity.

2. What is the purpose of using agar plate, agar slant and broth in
culture for microorganisms? Explain.
- Agar can sustain a variety of chemical conditions, making it
suitable for different types of microorganisms, including bacteria,
fungi, and algae.

3. Why should prepared media be stored in the lower

compartments of the refrigerator and not in the freezer?
- Culture media should not be stored at or below 0 °C as freezing
could damage the media performance. Culture media are
hygroscopic in nature where they tend to absorb moisture from the
surrounding. Sealed plastic containers are unaffected by normal
laboratory humidity.
Name: JUSTINE MARIE G. SISON Score: _______
Year & Section: BSBIO – 4C Date: ________

Activity # 6:
ISOLATION OF MICROORGANISMS

I. Introduction

Microbial life forms are widely distributed in the biosphere. They

are ubiquitous and can be found widespread in the ecosphere – the
atmosphere (gas), the hydrosphere (water), and the lithosphere (rocks
and soil). They are present in ocean depths of over a kilometer, far below
the limits of light penetration; far up in the atmosphere in the form of
spores, cysts, either in inactive or dormant cells; down deep in the soil
strata beyond the root zones of plants and where oxygen supply is
absent. As long as inorganic or organic material is available for energy
metabolism, hardly is there a nook or corner in the biosphere where
microorganisms are not to be found. The only exception is where there is
total sterility, as in sterilized media in test tubes and flasks, a condition
which, nevertheless, is ended sooner or later by microbial
recontamination and recolonization.

II. Objectives:
At the end of the activity, the students will be able to: 1. isolate

microorganisms from known clinical and environmental

samples.

2. enumerate and practice the standard methods for isolating and

purifying microorganisms.

III. Materials:
1. Samples (urine, soil, and tap water)
2. Prepared Media
3. Inoculating loop
 4. Sterile cotton swab
5. Glass Petri dish (2 pairs)

IV. Procedure:
1. Prepare and borrow the needed materials.
2. Collect samples before the experiment and label them:

A. Urine sample – collect 50 mL of urine in a specimen cup.

B. Soil sample – collect approximately 10 g of soil in a specimen
cup.
C. Tap water sample – collect 50 mL of tap water in a specimen
cup.
3. Methods of isolation and purification:

Serial Dilution – a systematic reduction of a bacterial

concentration through successive re-suspension of the
sample (sample0) into fixed volumes of a liquid diluent
(broth/solution). Transfer 1mL of the sample into the first test
tube (10-1) containing 9mL of NB or 0.9% NSS and mix with
vortex. Further dilution is done through 9 mL of NB or 0.9%
NSS in a second tube (10-2), third tube (10-3), and so on until
the tenth tube (10-10), each

with a corresponding pipette for use to transfer 1 mL aliquot

from one tube to another tube.
A. Streak plate - designed to isolate pure cultures of bacteria, or
colonies, from mixed populations by simple mechanical
separation. It is the most commonly used isolation technique. A
sterile inoculating loop is used to spread an inoculum across the
surface of nutrient agar, care being taken not to cut the agar
surface in the streaking process. The loop is also used to lightly
streak a set of patterns that gradually dilutes the sample to a point
during the appropriate period of time called incubation, colonies
develop from each isolate. The loop is sterilized between streaks.
The various types of organisms present are distinguished from
one another by differences in colonial characteristics.

B. Pour plate - The sample volume to be plated should be between

0.1 and 1.0 mL from a dilution series using a micropipette. Agar
pours are then added to each plate. Next, cover the plate and mix
the sample with the agar by gently swirling the plate. Allow the
agar to thoroughly solidify before inverting the plate for
incubation.

C. Spread plate – The original sample is diluted several times to

decrease or dilute the population sufficiently. A 0.1 mL of each
 dilution is then dispensed using a micropipette into the surface of
an agar-containing Petri plate. Spread the suspension using a
sterile glass spreader. Sterilization of glass spreader is done by
dipping it into a beaker of 70% ehanol or denatured alcohol. Drain
and ignite excess alcohol by passing it through the flame.
Remember to cool it down before using it. Isolated cells grow into
colonies and can be used to establish pure culture.

4. Perform isolation techniques for the following samples/source:

Note: Label all the plates and indicate the medium used and the sample
streaked or swabbed.
A. Urine: Apply serial dilution and pour plate method. Dispense 1
mL from 10-9 dilutions in the center of a blank sterile plate using a
micropipette. Pour about 20 mL of Nutrient Agar (NA) and mix
the sample with the media by gently swirling the plate. The plate
 is generally swirled in an “8” shape and then clockwise and
counterclockwise three times. Prepare another NA plate for 10-
10
dilution.
B. Soil: Apply serial dilution and spread plate method. Dispense 0.1
mL from 109 dilution onto the surface of a Potato Dextrose Agar
(PDA) and another NA plate for 10 -10 dilution. Soak the glass
spreader in a beaker with denatured alcohol. Let it cool for 15
seconds. Spread the suspension using a sterile glass spreader
onto the surface of the agar to ensure distribution.

C. Air: Select a place in (laboratory room, CR, corridor) or out

(garden, park) of the building and expose the Nutrient Agar (NA)
plate for 1 hr (lids off).
D. Surface: Moisten the sterile cotton swab with NB and swab to a
10 cm squared area of any surface (Cellphone, tabletop,
doorknob, bowl, sink) and then streak onto the Nutrient Agar (NA)
plate in a single stroke.
E. Deep well/tap water: Streak a loopful of the water sample onto
the Eosin Methylene Blue (EMB) Agar plate.

Incubation of plates: Seal all plates with masking tape, stack the plates on
top of one another, place upside down (Inverted position) and incubate at
37oC for 24 hrs.

V. Guide Questions:

1. How should agar plates be incubated? Why?

- Seal all plates with masking tape, stack the plates on top of one
another, place upside down (Inverted position) and incubate at 37oC
for 24 hrs. To reduce the risk of growing pathogens

2. What advantage(s) does the streak-plate method have over the

pour-plate method?
- The streak-plate method is preferred over the pour-plate method
primarily because it provides isolated colonies, is faster, requires
less equipment, and offers greater precision. These advantages
make it a more efficient technique for isolating and identifying
microbial species in microbiology

3. Why is the loop flamed before it is placed in a culture tube? Why

is it flamed after completing the inoculation?
 - To sterilize the loop before and after use . Flaming the
loop before and after use in a culture tube is essential for
sterilization and to prevent microbial contamination, ensuring
aseptic conditions in microbiology labs.

Name: JUSTINE MARIE G. SISON Score: _______

Year & Section: BSBIO – 4C Date: ________

Activity # 6:
ISOLATION OF MICROORGANISMS

ANTIMICROBIAL ASSAY

I. Introduction

Antimicrobial is a general term for compounds that kill or inhibit

microorganisms. Antibiotics, disinfectants, and antiseptics are examples
of antimicrobials.

Microorganisms produce antibiotics that target specific cellular

processes to inhibit or kill other microorganisms. The mode of action of
antibiotics is specific to certain kinds of cells. Disinfectants and
antiseptics, on the other hand, do not possess that specificity and
destroy structures in a broad range of cells, including those in the body.
Due to this characteristic, disinfectants are used to inhibit or destroy
microbial cells on inanimate objects. A subgroup of disinfectants, known
as antiseptics, prevent the growth of microbes on human tissue or kill
them.

It is important to test the effectiveness of antimicrobial agents

against specific organisms to identify their range of activity or its effect.
The Kirby-Bauer disk diffusion test is a widely used method to
determine the susceptibility of a microorganism to various antimicrobial
agents and drugs. This test has been in use for a long time and serves
as a starting point for determining the effectiveness of different drugs
against a given microorganism. The disk diffusion method of Kirby and
 Bauer has been standardized and is a viable alternative to broth dilution
methods for laboratories without the resources to utilize the newer
automated methods for broth microdilution testing.

A Kirby-Bauer test involves the placement of a small filter disk

containing a specific concentration of antibiotic and disinfectant on a
plate. The antimicrobial agents on the disk diffuse into the agar during
incubation, resulting in the formation of a concentration gradient.

The sensitivity level to the particular agent can be determined by

measuring the Inhibition Zone (IZ), which is the clearing zone around
the disk where the bacteria did not grow.

The Mueller Hinton Agar is often used as a medium for

susceptibility testing of non-fastidious bacteria due to the following
reasons: its susceptibility testing has acceptable batch-to-batch
reproducibility, it contains low amounts of sulfonamide, trimethoprim,
and tetracycline inhibitors, it supports the satisfactory growth of most
non-fastidious pathogens and a wealth of data and experience has been
collected regarding susceptibility tests performed using this medium.

II. Objectives:
At the end of the activity, the students will be able to:
1. describe and differentiate how the Kirby-Bauer disk diffusion test
determines the susceptibility of Staphylococcus aureus to different
antimicrobial agents
2. learn how to set up the Kirby-Bauer disc diffusion test
3. determine the sensitivity and resistance of Staphylococcus aureus
to certain antibiotics
4. measure the zone of inhibition/sizes and interpret the data
gathered based on the effectiveness of the antimicrobial agents

III. Materials:
1. 24-hr old bacterial culture
2. Prepared sterile Mueller Hinton Agar plates
3. Antimicrobial agents (70% isopropyl alcohol, antibacterial liquid
hand soap, iodine)
4. Disc
5. Forceps
 IV. Procedure
Kirby-Bauer disk diffusion susceptibility test protocol (Adapted from the
American Society of Microbiology).

I. Inoculation
a. Standardize the bacterial suspension to 0.5 McFarland.
b. Inoculate the plate with the test organism by streaking
the swab in a back-and-forth motion very close together
as you move across and down the plate. Rotate the
plate 60° and repeat this action. Rotate the plate once
more and repeat the streaking action.
c. This ensures an even distribution of inoculum that will
result in a confluent lawn of growth.
The diagram illustrates the pattern the swab should
follow as it is drawn across the plate.
d. Rim the plate with the swab by running the swab around
the edge of the entire plate to pick up any excessive
inoculum that may have been splashed near the edge.
The arrow indicates the path of the swab.
II. Placement of the Discs
a. Place the appropriate disks soaked in the different
antimicrobial agents such as your disinfectants and
antibiotics on the surface of the agar, using sterile
forceps.
b. Gently press the disk with the forceps to ensure
complete contact with the agar surface.
c. Seal the plates with masking tape, stack the plates
on top of one another, place upside down (Inverted
position) and incubate at 37oC for 24 hrs.

III. Measuring zone sizes

a. Following incubation, measure the zone sizes to
the nearest millimeter using a ruler or caliper;
include the diameter of the disk in the
measurement.
b. When measuring zone diameters, always round up
to the next millimeter.
c. All measurements are made with the unaided eye
while viewing the back of the petri dish. Hold the
plate a few inches above a black, nonreflecting
surface illuminated with reflected light
V. Guide Questions:

1. How do you classify the bacterial response to each antibiotic

(e.g., resistant, intermediate, or susceptible? Describe each.
-
Susceptible: A bacterial strain is said to be susceptible to a
given antibiotic when it is inhibited in vitro by a concentration
of this drug that is associated with a high likelihood of
therapeutic success.

Intermediate: The sensitivity of a bacterial strain to a given

antibiotic is said to be intermediate when it is inhibited in
vitro by a concentration of this drug that is associated with
an uncertain therapeutic effect.

Resistant : A bacterial strain is said to be resistant to a given

antibiotic when it is inhibited in vitro by a concentration of
this drug that is associated with a high likelihood of
therapeutic failure.

2. Are the results compared against standard reference charts for

each antibiotic? What are the Zone diameter standards for
Staphylococcus species?

Staphylococcus aureus, using MIC breakpoints of < or = 0.5

mg/L for susceptible and > or = 2 mg/L for resistant tentative
interpretive zone diameters of > or = 20 mm and > or = 21
mm for susceptible and < or = 17 mm and < or = 18 mm for
resistant are suggested for the 5 microg and the 10 microg
disk, respectively.
Name: JUSTINE MARIE G. SISON Score: _______
Year & Section: BSBIO – 4C Date: ________

Activity # 7:
GRAM STAINING

I. Introduction

Bacteria have almost the same refractive index as water. This

means when you try to view them using a microscope they appear as
faint, gray shapes and are difficult to visualize. Staining is one
method for making microbial cells easier to visualize. Simple stains
use only one dye that stains the cell wall of bacteria much like dying
eggs at Easter. Differential stains use two or more stains and
categorize cells into groups. Both staining techniques allow the
detection of cell morphology or shape, but the differential stain
provides additional information concerning the cell. The most
common differential stain used in microbiology is the Gram stain.

The Gram stain uses four different reagents and the results
are based on differences in the cell wall of bacteria. Some bacteria
have relatively thick cell walls composed primarily of a carbohydrate
known as peptidoglycan. Other bacterial cells have thinner cell walls
composed of peptidoglycan and lipopolysaccharides. Peptidoglycan
is not soluble in organic solvents such as alcohol or acetone, but
lipopolysaccharides are nonpolar and will dissolve in nonpolar
organic solvents.

Crystal violet acts as the primary stain. This stain can also be
used as a simple stain because it colors the cell wall of any bacteria.
Gram’s iodine acts as a mordant. This reagent reacts with crystal
violet to make a large crystal that is not easily washed out of the cell.
At this point, all cells will have the same color. The difference in the
cell walls is displayed by the use of the decolorizer, a solution of
acetone, and alcohol is used on the cells. The decolorizer does not
affect those cell walls composed primarily of peptidoglycan but those
with the lipid component will have large holes develop in the cell wall
where the lipid is dissolved away by the acetone and alcohol.
 These large holes will allow the crystal violet-iodine complex
to be washed out of the cell leaving the cell colorless. A
counterstain, safranin, is applied to the cells which will dye the
colorless cells.

The cells that retain the primary stain will appear blue or purple and
are known as Gram-positive. Cells that stain with the counterstain will
appear pink or red and are known as Gram-negative. The
lipopolysaccharide of the Gram-negative cell not only accounts for
the staining reaction of the cell but also acts as an endotoxin. This
endotoxin is released when the cell dies and is responsible for the
fever and general feeling of malaise that accompanies a Gram-
negative infection. When reporting a Gram stain, you must indicate
the stain used, the reaction, and the morphology of the cell. Round,
purple (blue) cells would be reported as Gram-positive cocci; and
rod-shaped, purple (blue) cells would be reported as Gram-positive
bacilli.

In order to survive some bacteria, produce endospores that

are highly resistant to harsh environmental conditions. The
malachite green staining procedure is a differential staining that is
used to distinguish between vegetative cells and endospores.

Bacteria that retain the blue stain are known as gram-positive;

those that do not are known as gram-negative. Organisms that
sometimes retain the blue color and sometimes do not are known as
gram-variable. Typical gram-positive bacteria are those staphylococci
that produce boils; typical gram-negative bacteria are the bacilli that
cause whooping cough; typical gram-variable bacteria are the bacilli
that cause tuberculosis.

II. Objectives:

At the end of the activity, the students will be able to:

1. Differentiate gram-positive and gram-negative

2. Identify examples or representatives of the bacteria
III. Materials:
1. Bacterial culture
2. Microscope
3. Glass slides & cover slips 4. Wash bottle with distilled water
5. Reagents: a. Crystal violet
b. Iodine
c. 95% alcohol or acetone
d. Safranin

IV. Procedure:

1. Smear preparation of the bacterial culture. (Properly labeled)

i. Put a drop of water on a glass slide. ii. Using a sterile
needle or an inoculating loop, slightly touch the colony and
mix it into the drop of water. iii. Spread the bacterial
suspension thin on the slide with a cover slip and air dry.
2. Fixation: pass the slide upright three times over the flame of
an alcohol lamp 3. Gram Staining:
i. Flood with Gram's Crystal Violet for 1 min. Wash
with distilled water and air dry.
ii. Flood the smear with Gram’s Iodine for 1 min.
Rinse with distilled water and air dry.
iii. Tilt the smear and add drop-by-drop of the decolorizer
(95% ethanol) for 5-10 sec only or until no more blue
dye runs off. Rinse with distilled water.
iv. Counterstain with Safranin. Allow it to remain for 45-60
sec. Rinse with distilled water.
v. Allow the slide to air dry or blot dry between sheets of
clean paper. vi. Examine the finished smear under
HPO.
 VI. Worksheet
1. Picture the bacteria you observed. Paste in the circle provided.
3 points
2. Below the circle, provide the following (10 points):
a. Gram stain reaction
b. Color
c. Shape
d. Arrangement

Bacteria A Bacteria B

3. What is the mordant used in the gram staining procedure and

what is the function?

Gram’s iodine.

4. Why is counterstain necessary when using a differential

staining technique such as gram stain?

Safranin
5. What step in the Gram stain procedure is most critical?

Smearing