MLS 109 – Clinical Bacteriology

PREPARATION OF
3. Let the smears dry. Do not blow on the slides, as this
will move the bacterial suspension. Do not flame the
slide, as flaming will distort the cell shapes.
BACTERIAL SMEARS AND 4. Hold the slide with forceps and heat-fix the smears
by passing the slides quickly through the blue flame
SIMPLE STAINING 2-3 times. Do not heat-fix until the smear is
completely dry.
SMEAR PREPARATION

➢ Bacterial smear preparation is a foundational

technique in microbiology, essential for the
examination and identification of bacterial species
under the microscope.
➢ This process involves spreading a thin layer of
bacterial cells onto a slide, which is then fixed and
stained to enhance visibility.
➢ Proper smear preparation is crucial for achieving
clear, distinct images of bacterial morphology and
arrangement, allowing for accurate analysis and
diagnosis.

PROCEDURE:

1. Clean your slides well with abrasive soap or cleanser,

then rinse and dry.
2. Handle clean slides by the end or edge. Use a marker
to make a dime-sized circle on each slide, on the
bottom of the slide so they will not wash off.
3. Label each slide according to the specimen used.

SPECIMEN PREPARATION: SIMPLE STAINING

A. FROM BROTH CULTURE ➢ Simple staining is a fundamental technique in
1. Shake the culture tube and with an inoculating loop, microbiology used to enhance the visibility of
aseptically. bacterial cells under a microscope.
2. Transfer 1-2 loopfuls of bacteria to the center of the ➢ By applying a single stain or dye, such as methylene
slide. blue or crystal violet, to a prepared bacterial smear,
3. Spread this out to about a ½-inch area. this method allows for the clear visualization of cell
morphology, size, and arrangement.
B. FROM SLANT OR PLATE CULTURE: ➢ Simple staining is particularly useful for quickly
assessing bacterial samples and differentiating
1. Place a loopful of distilled water in the center of the
between various shapes, such as cocci, bacilli, and
slide.
spirilla.
2. With the inoculating needle/loop, aseptically pick up
a very small amount of culture and mix into the drop
PROCEDURE
of distilled water and spread.

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 MLS 109 – Clinical Bacteriology
1. Stain one side at a time. Place it on a staining rack. A. Label the Slide: Label a clean microscopic slide
2. Flood the smear with Methylene blue or Crystal with the sample identification.
violet and leave for 30-60 seconds. B. Apply the Bacteria: Using a sterile inoculating loop,
3. Carefully wash the excess stain off with distilled place a small drop of distilled water on the slide if
water from a wash bottle. Let the water run down using a solid culture. Aseptically transfer a small
the titled slide. amount of bacterial culture to the drop of water and
4. Gently blot the smear with a paper towel or spread it to create a thin smear. If using a liquid
absorbent paper and let it dry. culture, apply a loopful directly onto the slide and
5. Examine your stained smears microscopically using spread it.
the 3 objectives. Once in the OIO, put the oil directly C. Air Dry: Allow the smear to air dry completely.
on the smear. Record your observation with labeled
drawings. 2. HEAT FIXATION
6. Blot the oil from the objective lenses with lens ➢ Pass the air-dried slide through the flame of an
paper and return your microscope to its proper alcohol lamp 2-3 times, smear side up. This process
location. Clean your slides well. kills the bacteria and fixes them to the slide.
7. Stained bacterial slides can be stored in a slide box.
Remove the oil from the slide by blotting with paper 3. STAINING
towel. Any residual oil won’t matter. A. Primary Stain: Flood the smear with crystal violet
and let it sit for 1 minute.
B. Rinse: Gently rinse the . slide with distilled water to
GRAM STAINING remove the excess stain.
C. Mordant: Cover the smear with Gram’s iodine and
➢ Differential staining uses two or more stains and let it sit for 1 minute. The iodine binds with crystal
allows for the identification of specific groups of violet to form a complex.
organisms. D. Rinse: Gently rinse the slide with distilled water.
o This technique was discovered by Hans Christian E. Decolorization: Hold the slide at an angle and apply
Gram in 1884, when he attempted to stain cells the decolorizer dropwise until the runoff is clear
and found that some lost their color when excess (about 10-20 seconds). This step differentiates
stain was washed off. Gram-positive from Gram-negative based on cell
➢ The Gram stain is a differential technique which uses wall structure.
the principle of timed, sequential applications of a F. Rinse: Immediately rinse the slide with distilled
primary stain, mordant, decolorizer, and finally, a water to stop the decolorization process.
counterstain which differentiates between Gram- G. Counterstain: Flood the smear with safranin or
positive and Gram-negative bacteria by virtue of their fuchsin and let it sit for 30 seconds. This stains the
difference in the cell wall component. Gram-negative bacteria.
o It also provides information for the necessary H. Final Rinse: Gently rinse the slide with distilled water.
workup of organisms recovered from the sample
for further identification. 4. DRYING
o Violet or purple = for positive ➢ Blot the Slide: Gently blot the slide with bibulous
o Pink or Red = for negative paper or let it air dry. Do not rub the slide as this
may remove the smear.
PROCEDURE
5. MICROSCOPY
1. PREPARATION OF THE BACTERIAL SMEAR

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 MLS 109 – Clinical Bacteriology
➢ Examine the Slide: Place the slide on the microscope o Kingella
stage and observe under oil immersion (100x ➢ ALL BACILLI ARE GRAM NEGATIVE, except:
objective). MACCBELL
➢ Interpretation: Gram-positive bacteria will appear o Mycobacterium
purple due to the retention of the crystal violet-iodine o Actinomyces
complex, while Gram-negative bacteria will appear o Clostridium
pink/red due to the counterstain. o Corynebacterium
o Bacillus
6. SAFETY PRECAUTION o Erysipelothrix rhusiopathiae
➢ Adhere to laboratory safety protocols while handling o Listeria
bacterial cultures and staining reagents, ensuring o Lactobacillus
accurate and safe execution of the procedure.
➢ Gram stain is not applied to the following
THEORIES ON GRAM STAINING organisms:
o Mycoplasma and Ureaplasma – lacks cell
A. MAGNESIUM RNA-IODINE COMPLEX THEORY wall
o Rickettsia and Chlamydia – special or
➢ Gram (+) bacteria contains Mg++RNA which
unique organisms
combines with iodine forming a compound which is
o Spirochetes – gram variable results “may
not readily soluble to alcohol
time ga show as gram (+) or gram (-) but
➢ Gram (-) bacteria do not contain Mg++RNA-Iodine
most of the time (-).”

B. BENIAN THEORY / PERMEABILITY TO IODINE -

ALCOHOL COMPLEX

➢ Gram (+) bacteria can form crystal violet–gram’s

iodine complex that is less permeable to alcohol.
➢ Gram (-) bacteria do not form crystal violet–gram’s
iodine complex, therefore, easily permeable to
alcohol and thus easily decolorized

C. STEARN & STEARN THEORY / ISOELECTRIC

POINT THEORY

➢ Gram (+) bacteria have lower isoelectric point thus

ACIDIC and combine firmly with basic dyes
➢ Gram (-) bacteria have higher isoelectric point thus
BASIC and does not readily combine with basic
dyes.

RULES IN GRAM STAINING

➢ ALL COCCI ARE GRAM POSITIVE, except: NVMAK

o Neisseria
o Veilonella
o Moraxella/Branhamella
o Acinetobacter

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 MLS 109 – Clinical Bacteriology

ACID FAST STAINING

• Prepare a smear of the sample (e.g., sputum,
tissue, or culture) on a clean microscope
slide.
• Acid-fast staining is a specialized technique used • Allow the smear to air dry completely.
to identify and differentiate bacterial species with • Heat-fix the smear by passing the slide, smear
unique cell wall characteristics, particularly those side up, through the flame of an alcohol lamp
with high lipid content such as mycobacteria. 3-4 times or by placing it on a slide warmer at
• This staining method is crucial for diagnosing 60-70°C for 2 hours.
infections caused by acid-fast bacteria, such as 2) PRIMARY STAIN → Application of Carbol Fuchsin:
Mycobacterium tuberculosis (bacilli), the causative (10 minutes)
agent of tuberculosis. • Place the slide on a staining rack.
• Flood the smear with carbol fuchsin stain.
MYCOBACTERIUM/ACID FAST BACILLI:
• Heat the slide gently by passing a flame under
➢ Cell wall: contains mycolic acid (a lipid component it until steam rises (do not boil or let it dry out).
tas daw wax ni sa) Alternatively, you can place the slide on a
o If heat is applied, mycolic acid will melt so slide warmer at 60-70°C for 5 minutes.
that in staining, the carbol fuchsin is • Cooling Step: Allow the stain to sit for 5-10
absorbed and is taken up by the cell wall = minutes, ensuring it remains moist.
that’s why red ang color. o For solidifying kay na diba na melt na
o Mycolic acid is also responsible for the acid- sa. Then, whoa ga balik ang mycolic
fastness of the mycobacteria. acid, then ma trap ang color sang
• The most common acid-fast staining methods are carbol fuchsin (red).
the Ziehl-Neelsen (Physical; hot method) and the 3) Rinsing:
Kinyoun methods (Chemical; cold method). • Rinse the slide gently with running tap water
until the water runs clear.
MATERIALS 4) Decolorization:
• Flood the slide with acid-alcohol (3%
1) Forceps
hydrochloric acid in ethanol or 1% sulfuric
2) Acid Fast Stains (C – A – M)
acid) for about 3 minutes or until the smear is
o Carbol Fuchsin
sufficiently decolorized.
o Acid Alcohol
o With the presence of acid alcohol,
o Methylene Blue
nd matunaw ang mycolic acid.
3) Glass Slides
• Rinse the slide again with running tap water to
4) Wash bottle
stop the decolorization process.
5) Alcohol lamp
5) SECONDARY STAIN → Counterstaining:
6) Staining rack
• Flood the slide with methylene blue
7) Distilled water
counterstain for 1-2 minutes.
8) Compound light microscope
• Rinse the slide gently with running tap water.
9) Immersion oil
6) Drying:
10) Sputum specimen
• Blot the slide gently with absorbent paper or
PROCEDURES: allow it to air dry.
7) Microscopic Examination:
1. Preparation of the smear from the sputum
sample:

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 MLS 109 – Clinical Bacteriology
• Examine the slide under a microscope using PROCEDURES
an oil immersion objective (1000x
magnification). A. PREPARING AGAR PLATES
• Acid-fast bacteria will appear red or pink 1. Prepare the agar medium.
against a blue or green background a) Weigh the Agar and Nutrients:
(depending on the counterstain used). • Measure out the appropriate amount of
• Non Acid-Fast bacteria/bacilli = appears agar powder and any other components
blue. (e.g., peptone, yeast extract) according to
o have no mycolic acid on their cell wall. the specific recipe. A common formulation
o Colorless after acid alcohol is 15-20 grams of agar per liter of medium.
(decolorizer). b) Dissolve the Agar:
o Methylene blue is taken up • Add the measured agar powder to the
8) Safety Precautions: Always handle specimens appropriate amount of distilled water in a
and reagents with care, using appropriate personal heat-resistant container.
protective equipment (PPE) such as gloves, lab • Stir the mixture thoroughly to distribute the
coat, and eye protection. agar powder evenly.

MEDIA PREPARATION c) Heat to Dissolve:

• Heat the mixture while stirring
continuously until the agar is completely
➢ Is the process of mixing nutrients, agents for
dissolved. This can be done using a hot
buffering and maintaining the osmotic balance, as
plate or microwave. Be cautious to avoid
well as selective inhibitors or indicators to create an
boiling over.
agar or broth that supports the growth and
2. Sterilize the Agar Medium
differentiation of microorganisms since they usually
1. Autoclave the Mixture:
depend on several factors, such as nutrients,
a. Pour the dissolved agar medium into an
oxygen, moisture, and temperature.
autoclave-safe bottle or flask, leaving
➢ Culture media preparation is one vital procedure in
some space at the top to prevent boiling
the isolation and identification of bacteria.
over.
➢ These media encourage the growth of bacteria,
b. Autoclave the medium at 121°C (250°F) for
allowing their characteristics to be noted.
15-20 minutes to sterilize it.
➢ Bacteria are grown on agar plates or tube media,
3. Pour the Agar Plates
either of which is equally important in the process.
a) Cool the Medium:
• After autoclaving, allow the agar medium
MATERIALS
to cool to approximately 45-50°C. This
temperature is cool enough to handle but
still warm enough to remain liquid.
b) Pour the Plates:
• Working near a flame or in a sterile
environment (e.g., laminar flow hood),
carefully pour the cooled agar medium
into sterile Petri dishes. Aim to cover the
bottom of the dish with about 15-20 mL
of agar.
c) Avoid Condensation:

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 MLS 109 – Clinical Bacteriology
• To minimize condensation, keep the lids • Place the filled and sealed test tubes in an
slightly ajar until the agar solidifies, then autoclave.
close the lids. Alternatively, you can pour • Sterilize at 121°C (250°F) for 15-20 minutes.
the plates in a warm room or use a drying Ensure that the caps are loose if using screw
oven set to a low temperature to reduce caps to prevent pressure build-up.
condensation.
4. Store the Agar Plates 4. Cool and Solidify (for Agar Media)
1. Label and Seal: 1. Cool the Tubes:
• Label the plates with relevant information • Allow the tubes to cool and solidify in an
(e.g., date, type of medium) and wrap them upright position if preparing solid media.
in plastic wrap or place them in a sealed • Solidifying Agar: For agar slants, tilt the
plastic bag to prevent contamination. tubes at an angle while cooling to create a
2. Refrigerate: slanted surface.
• Store the prepared agar plates upside
down (agar side up) in a refrigerator at 4°C 5. Store the Media
to prevent moisture from dripping onto the 1. Label and Store:
agar surface. • Label the tubes with relevant information
(e.g., date, type of medium).
• Store the prepared culture media in a cool,
B. PREPARING TUBE MEDIA
dry place or refrigerate them at 4°C if long-
1. Prepare the Culture Medium term storage is needed.
1. Weigh the Medium Components:
CULTURE MEDIA
• Measure the required amount of culture
medium powder according to the • Food for the organisms
manufacturer's instructions
• Nutrients
2. Dissolve the Medium:
• Agents (agar)
• Add the medium powder to the appropriate
volume of distilled water in a heat-resistant • Water
container.
• Stir the mixture thoroughly until the medium PROCEDURE:
is completely dissolved. 1) Clean
3. Heat to Dissolve (if using agar):
2) Wrap in paper
• If preparing solid media with agar, heat the 3) Autoclave
mixture while stirring continuously until the
agar is completely dissolved. COMPUTURE FOR THE AMOUNT OF CM: NUTRIENT
2. Dispense into Test Tubes
AGAR
1. Measure and Pour:
• Using a sterile pipette or measuring cylinder,
dispense the desired volume of the medium
into each test tube (typically 5-10 mL per
tube).
2. Seal the Tubes:
• Seal the test tubes with cotton plugs or
screw caps.
3. Sterilize the Media
1. Autoclave the Tubes:

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 MLS 109 – Clinical Bacteriology
INOCULATION OF PLATED • If you are using a broth culture, gently mix the broth
by swirling the tube.
• If you are using an agar slant or colony from
CULTURE MEDIA another plate, ensure you have a small, well-
isolated colony.In
(STREAK PLATE METHOD) 4. TRANSFER OF INOCULUM:
• Using the cooled, sterilized loop or needle, pick up
The manner of inoculating bacteria on agar plates follows
a small amount of the culture.
specific patterns. These patterns allow isolation of bacteria
• If you are using a sterile swab, dip it into the culture
or allowing them to grow so one would know their cultural
or moisten it with sterile saline/broth, then touch it
characteristics. Most bacteriological work requires pure
to the culture.
cultures or clones of bacteria.
5. INOCULATION OF AGAR PLATE:
• The isolation method most commonly used to get pure • Streak Plate Method
cultures is the streak plate method. o Lift the lid of the agar plate slightly to avoid
➢ A sterile inoculating loop is dipped into a mixed contamination.
culture that contains more than one type of o Starting at the edge of the plate, streak the
microbe and is streaked in a pattern over the inoculating loop back and forth across the
surface of the nutrient medium. surface of the agar in a small section of the
• As the pattern is traced, bacteria are rubbed off the loop plate.
far enough apart to grow into isolated colonies. o Flame the loop, let it cool, and rotate the plate
slightly. Streak the loop through the first
• These colonies can be picked up with an inoculating
section and into a new section.
loop and transferred to a test tube of nutrient medium to
o Repeat this process to create several streaks,
form a pure culture containing only one type of
which will help to isolate single colonies.
bacterium.
6. INCUBATION
MATERIALS • Invert the inoculated agar plates to prevent
condensation from dripping onto the agar surface.
1) Inoculating wire loop
• Place the plates in an incubator set to the
2) Alcohol lamp
appropriate temperature for the microorganism
3) Broth culture
being studied (e.g., 37°C for many human
4) Agar plates
pathogens).
PROCEDURES • Incubate for the recommended time period
(usually 24-48 hours).
1. PREPARATION
7. OBSERVATION AND ANALYSIS:
• Ensure your work area is clean and organized.
• After incubation, observe the plates for microbial
• Label the bottom of the agar plate with relevant growth.
information (e.g., date, type of medium, organism,
• Note the appearance of colonies (size, shape,
your initials).
color, etc.) and any other relevant observations.
2. STERILIZATION
8. DISPOSAL:
• Light the Bunsen burner or alcohol lamp to create
• Properly dispose of used cultures and materials
a sterile field.
following your laboratory's biosafety protocols.
• Sterilize the inoculating loop or needle by passing
it through the flame until it glows red-hot. Allow it
to cool for a few seconds.
3. INOCULUM PREPARATION:

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 MLS 109 – Clinical Bacteriology
Note!
INOCULATION OF TUBE
• Always work near the flame to maintain a sterile

•
environment.
Do not leave the inoculating loop in the flame for too
CULTURE MEDIA:
long to avoid weakening the metal. AGAR SLANT, AGAR DEEP TUBE,
• Ensure all materials are sterile before use to avoid
contamination. AGAR BUTT-SLANT, LIQUID MEDIA
• Follow your laboratory’s safety guidelines, including
wearing appropriate personal protective equipment • Inoculation of tube culture media is a common
(PPE). procedure in microbiology used for cultivating
and maintaining microorganisms.
• Tube culture media can include:
✓ Broths
✓ Agar slants
✓ Agar deeps
• Slant tubes are tubes containing a nutrient medium
plus agar.
➢ The medium has been allowed to solidify at an
angle in order to get a flat inoculating surface
(slant).
➢ A loop is used to streak the surface of the slant.
• Stab tubes (deeps) are tubes of agar medium which
are inoculated by "stabbing" the inoculum into the
agar using sterile needle.
➢ The inoculating needle is stabbed into the
medium without touching the walls of the tube
and should only penetrate mid to its depth.
• For semi-solid media, a needle is used to inoculate a
stab medium, for example mannitol motility medium.
• As for liquid media, broths and other liquid media are
inoculated using a sterile wire loop, or Pasteur pipette
depending on whether the inoculum is colonial growth
or a fluid culture or specimen.

MATERIALS:

PROCEDURES:
1. PREPARATION:
• Ensure your work area is clean and organized.

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 MLS 109 – Clinical Bacteriology
• Label the tube with relevant information (e.g., 6. INCUBATION:
date, type of medium, organism, your initials). • Place the inoculated tubes in an incubator set to
2. STERILIZATION: the appropriate temperature for the
• Light the Bunsen burner or alcohol lamp to create microorganism being studied (e.g., 37°C for many
a sterile field. human pathogens).
• Sterilize the inoculating loop or needle by passing • Incubate for the recommended time period
it through the flame until it glows red-hot. Allow it (usually 24-48 hours).
to cool for a few seconds. 7. OBSERVATION AND ANALYSIS:
3. INOCULUM PREPARATION: • After incubation, observe the tubes for microbial
• If you are using a broth culture, gently mix the broth growth.
by swirling the tube. • Note the appearance of growth (turbidity in broth,
• If you are using an agar slant or colony from colony formation on slant, or growth pattern in
another plate, ensure you have a small, well- deep) and any other relevant observations.
isolated colony. 8. DISPOSAL:
4. TRANSFER OF INOCULUM: • Properly dispose of used cultures and materials
• Using the cooled, sterilized inoculating loop or following your laboratory's biosafety protocols.
needle, pick up a small amount of the culture.
• If you are using a sterile swab, dip it into the
culture or moisten it with sterile saline/broth,
then touch it to the culture.
5. INOCULATION OF TUBE CULTURE MEDIA:
• Broth Culture:
o Remove the cap of the broth tube and flame
the neck of the tube.
o Insert the inoculating loop or needle with the
culture into the broth, gently agitate to
disperse the microorganisms, and remove.
o Flame the neck of the tube again before
capping it.
• Agar Slant:
o Remove the cap of the agar slant tube and
flame the neck of the tube.
o Streak the surface of the slant in a zigzag
pattern with the inoculating loop or needle.
o Flame the neck of the tube again before
capping it.
• Agar Deep:
o Remove the cap of the agar deep tube and
flame the neck of the tube.
o Insert the inoculating needle straight down
into the center of the agar deep and withdraw
along the same path.
o Flame the neck of the tube again before
capping it.

Francine Lourdiette Castro-Madria MT3D