MLS-Lab 411 − The immersion oil has the same refractive index as the

Clinical Bacteriology glass slide, so the oil becomes part of the optics of the
Lesson 1: Proper Use of the Brightfield Microscope in glass.
Bacteriology − Increases the RI when used properly. Limited
Lecturer: Prof. Ab Chua, RMT
disadvantages: slides prepared with immersion oil
techniques work best under higher magnification where
Learning Resources: Brightfield Microscope oils increase refraction despite short focal lens.
Immersion Oil
− When the IO is used, the light rays do not refract when
Lens Paper
as they enter the air from the slide therefore improves
Prepared Slides
the resolving power of the lens. If oil is not used in OIO
Introduction
lens, the image is fuzzy or with poor resolution.
− Bacteriology is a branch of science the deals with the
study of bacteria. Bacteriology is a branch of
microbiology in which bacteriologists study and learn
more about the characteristics, structures, genetics,
biochemistry, ecology, and mechanism through which
they cause diseases in humans and animals.
− Bacteria are minute organisms which are too small to
be seen with the naked eye.
− In order to visualize bacteria, one must use a good
compound microscope. The simplest type of compound
microscope is the Brightfield Compound Microscope
− The word COMPOUND from BCM means that a
specimen is positioned properly on the stage of a
microscope and illuminated by a light source will be
magnified by two-lens system.
− The image formed by the objective lens will be magnified
again by the ocular lens system. Therefore, the
magnification is compounded.
− The term BRIGHTFIELD refers to the fact that magnified
objects appear as dark objects against a dark
background.
− A sufficient contrast must exist between the magnified
object and the brightfield background for the objects to
be visible.

Structural Components
1. Head/Body - houses the optical parts in the upper part of
the microscope
2. Base - supports the microscope and houses the illuminator
3. Arm - connects to the base and supports the microscope
head. It is also used to carry the microscope

Optical Components
How does it work?
− The magnification is achieved when the light rays from
an illuminator or light source passes through a
condenser which has lens that directs the light rays to
the specimen.
− The light rays then pass into the objective lenses; the
lens closest to the specimen then the image of the
specimen is magnified again by the ocular lens or the
eyepiece.
Importance of using Immersion Oil
− In order to preserve the direction of lightrays at the
highest magnification, immersion oil is placed between
the glass slides and the oil immersion objective lens.
1. Eyepiece/Ocular – remagnifies the image formed by the
objective lens. Typically, standard eyepieces have a
magnifying power of 10X.
2. Eyepiece tube – holds the eyepieces in place above the
objective lens
3. Objective Lenses – primary lenses that magnifies the
microscope
a. Scanner – often marked with 4X
b. Low Power Objective (LPO) – often marked with 10X. it
is usually shorter than the other two objectives and it
usually forms the general outline or wider portion of the
object
c. High Power Objective (HPO) – often marked with 40X.
It is longer than the LPO and it forms a bigger image of
the object in focus. In most cases, it is used to enlarge
specimens that are so small under the LPO. They are
 spring loaded which means that they retract if the object
in the lens hit the slide which prevents damage to the
lens and slide.
d. Oil Immersion Objective (OIO) – often marked with
100X. It has the highest degree of magnification and
is used to examine stained smear preparations of
microorganisms using oil immersion as the medium
4. Nose Piece – a rotating turret that houses the objectives
5. Coarse Adjustment Knob – a bigger wheel used to adjust
the LPO in focusing. It is also used for initial focusing of the
specimen
6. Fine Adjustment Knob – a smaller wheel used for delicate
and final focusing of the specimen when using the HPO and
OIO. It is also used to make the specimen more vivid.
7. Stage – a square platform with an opening at the center. It
is where the slide/specimen is placed during focusing.
8. Stage Clips – paired structures found on either side of the
stage. It is used to hold the slide in place.
9. Illuminator – the light source for microscope, typically
located at the base.
10. Condenser – a lens found beneath the hole of the stage.
It is used to collect and focus the light from the
illuminator on the specimen. Useful in attaining sharp
images at 400 X magnifications and above. If the max
power of your microscope is 400X, a stage mounted 0.65
NA or greater, condenser is ideal since it gives you greater
clarity without being focused separately. If your
microscope goes to 1000X or above, focusable condenser
is 1.25 NA or greater is needed.
11. Iris Diaphragm – located above the condenser and below
the stage. It controls the amount of light reaching the
specimen. Can be adjusted to vary the intensity and size
of the cones of light that is projected. The setting depends
on the transparency and degree of contrast you want.
12. Condenser Focus Knob – moves the condenser up or
down to control the lighting focus on the specimen.
Care of the Brightfield Compound Microscope
To maximize the function and use of the BCM, proper care for
the microscope is very essential. The following are the general
guidelines in handling a BCM:
1) Get the microscope from the stockroom by taking the arm
with one hand and supporting the instrument at the base
with the other hand.
2) Place the microscope at least six inches from the edge of
the laboratory table with the microscope arm facing you.
3) Wipe all lenses with lens paper before and after use. If the
oil immersion lens is sticky, moisten a piece of lens paper
with 95% alcohol to wipe it clean.
4) Never use tissue or cloth when cleaning the objective lens
or oculars.
5) Keep the stage clean and dry.
6) Do not tilt the microscope; instead, adjust the position of
your chair so that you can comfortably use the instrument.
Tilting leads to distort wet mounts or objects covered with
oil.
Correct Manipulation of the Brightfield Compound Microscope
1) Place the specimen slide on the stage and secure it with
the stage clips. Arrange the portion of the slide to be
examined over the central opening in the stage.
2) Rotate the LPO into place under the body tube. You will
hear a “click” sound when it is correctly in place.
3) Raise the condenser as high as it goes.
4) Rotate the coarse adjustment knob clockwise to bring
down the LPO close to but not touching the slide, until the
specimen is seen through the eyepiece.
5) Regulate the intensity of light by opening or closing there
is diaphragm.
6) Sharpen the focus by turning the fine adjustment knob.
7) Turn the LPO to HPO without elevating the body tube.
Notice that it will be almost in focus because most
microscopes are parfocal. Little adjustments with fine
adjustment knob will only be needed to clearly view the
object in focus.
8) To focus on the oil immersion lens, swing the OIO halfway
towards the specimen in focus. Place a drop of immersion
oil on the specimen. Swing the OIO in place. Using the
coarse adjustment, bring the objective down until it
touches the oil. Find the object by gently turning the
coarse adjustment knob upwards. Sharpen the focus with
the fine adjustment knob.
9) Compare the relative sizes from those seen under the
LPO, HPO, and OIO.
10) Clean the stage and the lenses after each use, before
returning the microscope to the stockroom.
Calculating the Total Magnification
This is computed as follows:
TOTAL MAGNIFICATION = Magnifying power of the
eyepiece X Magnifying power of the objective used
EXAMPLE:
Magnifying power of eyepiece = 10
Magnifying power of the objective lense = 100 (OIO)
Total Magnifcation = 10 X 100 = 1000X
NOTES TO REMEMBER!!
− PARFOCAL – the objective lenses are mounted on the
microscope so that they can be interchanged without
having to appreciably vary the focus.
− RESOLVING POWER or RESOLUTION – the ability to
distinguish objects that are close together. The better the
resolving power of the microscope, the closer together
two objects can be and still be seen as separate.
− MAGNIFICATION – the process of enlarging the size of
an object, as an optical image
− TOTAL MAGNIFICATION – it is the product of the
objective and ocular lenses
MICROSCOPE TROUBLESHOOTING
Check to see if your microscope
is plugged in.
Check to see if the bulbs are
installed correctly. You may have
to install or reinstall the
microscope bulb.
If the bulbs are installed, check
My microscope will not turn on.
to make sure they are not loose,
which sometimes happens
during shipment
check the rheostat (high intensity
control) on the side of the
microscope/
Check the fuse
Make sure you have removed
the protective covering such as
I cannot see anything through my lens covers.
microscope. Ensure that the eyepieces are
installed
Check that the light is on.
 The height of the microscope MLS-LAB 411 | CLINICAL BACTERIOLOGY
condenser may be set too high LESSON 2: THE CORRECT MORPHOLOGICAL
or too low. CLASSIFICATION: TOOL FOR BACTERIA IDENTIFICATION
Make sure your microscope USING DIFFERENT STAINING METHODS
My microscope is out of focus. objective lenses are screwed into
the body of the microscope all
the way.
BACTERIAL MORPHOLOGIC TYPES / SHAPES AND
Your microscope cover slip may ARRANGEMENTS
be too thick. - Cocci
- Bacilli
PROBLEM POSSIBLE CAUSE SOLUTION - Spiral
Condenser may be too Raise the *By simply determining the bacterial shape and arrangement, or
low condenser how they look like once they are stained, it gives the doctor a
The field is dim.
Condenser iris may be Open the general idea on the diagnosis and possible intervention or
closed diaphragm treatment for that specific disease.
Dark shadows in Eyepiece may be dirty Clean the eyepiece
the field which Cocci
move when Surface of the eyepiece A new eyepiece - Once you have prepared them in a glass slide
eyepiece is may be scratched may be needed
and stained them properly, it will look like this:
moved
The smeared portion of Turn the slide over - SPHERICAL or ROUND in shape
the slide may be upside - In GREEK, it means “BERRY”
down. - Come in different arrangements:
There may be an air Move the 100X o Coccus – singly
bubble in the oil. lens from side to
Image is not
side
clear
The oil may be of poor Use only good o Diplococcus – in pairs
quality quality immersion
oil
There may be dirt in the Clean the lens o Streptococcus – chain
lens ▪ Streptococcus pneumoniae – causative
There may be oil in the Clean the lens agent of pneumonia
lens
The image
There may be dust on the Clean the leans
through LPO is
upper surface of the lens
not clear
The lens may be broken A new lens may be o Tetrad – 4, making up a square
needed
A.

o Sarcinae – cuboidal pockets of 8 or more

o Staphylococcus – irregular or grape-like

▪ Staphylococcus aureus – causative agent of
skin infections; S. pneumoniae

Bacilli
- Otherwise known as “rods”
- Under the microscope, they look like this:
- Straight, sausage, cigarette-shaped, elongated
- In LATIN, it means “LITTLE STICKS”
- Arrangements:
o Bacillus – singly
▪ Clostridium group
 • Clostridium botulinum – causative ▪ If we stain spores without using special stains,
agent of botulism or food the spores will appear as transparent spots
poisoning within the cell
▪ The ability of the spores to resist usual
o Coccobacillus – similar cocci but stains may be attributed to their chemical
chubby and elongated composition since it consists a large amount of:
▪ Lactobacillus casei strain Shirota –
bacteria found in Yakult
▪ Can present itself as elongated bacilli or
event coccobacilli
o Diplobacillus – in pairs; two bacilli
stuck on one end

o Streptobacillus – in chain
o Palisades or Chinese Letter –
palisades = fences; Chinese letter =
strokes in various strokes • Calcium
▪ Corynebacterium diphtheria – • Dipicolinic Acid
causative agent of diphtheria o A substance that is not
found in the vegetative
Spiral cell
- Curved rods, cork-screw, hellicoidal Calcium –Dipicolinic o O
- Arrangements: Acid – Peptidoglycan nly synthesized during
o Comma – curved rods or bacilli that can sometimes go spore formation
Complex
beyond its comma-like appearance and forms an S • Peptidoglycan
shape, but the curvatures do not render a complete - As a rule, only one spore is formed from a single bacterium
spiral - Spore formation among bacteria is NOT a process of
▪ Vibrio multiplication, but rather, a DEFENSE MECHANISM
• Vibrio cholera - Arrangements:
o Spirilla o Terminal spore – if the endospore is
▪ Cork-screw found at the tip of a bacterial cell
▪ Once the spirallization is complete, it is now o Subterminal spore – near the tip of a
called bacterial cell
spirilla o Central spore – if it is located at the
▪ Rigid – stiff and nonflexible middle of a bacterial cell
o Spirochete
▪ Much more flexible Capsule
▪ Wiggle when they move - Gycocalyx (sugar coat)
• Treponema pallidum o A general term used for substances
surrounding the cell
BACTERIAL STRUCTURES o Sticky, gelatinous substance that the cell
- Actual body parts of a bacteria secretes externally
- NOT ALWAYS present in all types of bacteria o Made up of either a polysaccharide, a
- The presence of these structures makes a bacterium polypeptide, or both
unique o Varies from one bacteria to another
- Significantly narrows down the list of the possible identities o Has 2 TYPES:
of the bacteria that we are examining ▪ Capsule
- Cannot be demonstrated using the routine staining • Much more organized and
methods. Hence, special stains should be used firmly attached to the cell wall
o Spore compared to the slime layer
o Capsule • Responsible for protecting
o Granule pathogenic bacteria from
o Flagellum phagocytosis
• Like spores,
Spore capsules do not stain readily
- Vaguely speaking, a structure that – we will need to stain
forms inside the bacteria the background of the
- “Endospore” – endo- means inside, cell with that capsule
hibernating part which is the resting part of the
structure that is highly resistant to conditions
that would otherwise kill the bacteria
• Ex: Streptococcus pneumoniae
o Desiccation
▪ Slime layer
o Heat
• For adhesion to surfaces
o Nutrient Depletion
o Chemicals
Granule
- Inclusion bodies
o If there is an abundance of nutrients, our
bacterial cells store some within their cytoplasm and
they use these deposits for future purposes
o A very good example: granules
o Their composition demonstrated by staining
and other chemical means contributes to our
knowledge of the physiology of the cell and their
probable role
o Many of these granules are carbohydrates such as
the case for Corynebacterium. When
they are with iodine, they appear to
brown. Hence, they are known as
IOGEN GRANULES
▪ Their presence makes it easier to
depict what bacterium is being examined.
Flagellum
- “Little Whips”
- Delicate hair-like processes that
are often longer than the
bacteria from which they arise
- Moving in a wavelike pattern using
bundles of FLAGELLIN
o Protein subunits
o Come together to make up one
whole flagella
o Contractile similar to the
contractile proteins found in the
muscle tissues
o Undergoes elastic contractions and expansions,
therefore accounting for the flagellar movements
(wavelike rhythm)
- Can be found anywhere in any part outside the bacteria
- Different types of bacterial flagellation
o Monotrichous – flagellum on one pole only
o Lophotrichous – has a tuft of flagella on one end only
o Amphitrichous – both ends have flagella
2. Place them on a drop of either NSS or deionized water that
is already found on the slide.
3. Cover it with a coverslip.
4. Examine it under the microscope. No need to dry it.
Hanging Drop Preparation
- Essentially the same as the Wet Mount Preparation
- Difference: How you are going to stick the coverslip with the
glass slide and with the type of glass slide that will be used
o GLASS SLIDES WITH RINGS
▪ Paraffin wax can be used wherein a ring is
drawn on the center of the glass slide
▪ Commercially prepared glass slide can also
be used wherein they is already a hollow on the
center
1. Put a drop of bacteria on the coverslip.
2. Allow the glass slide to stick with the coverslip
3. Turn it back up.
o The drop of bacterial suspension which is found on the
coverslip will be hanging from the coverslip and into
the hollow area on the glass slide.
Fixed-Stained Smear
- The bacteria should not be moving.
- The staining procedures are going to kill them rendering
them immobile and perfect for the morphology examination.
- Bacterial Smear
o A type of preparation that bacteriologists use to start
the determination of bacterial identity
o Has to made in order to FIX the cells to the slide first
▪ A term used in bacteriology wherein we heat
the bacteria for them to stick to the slide
▪ HEAT FIXATION – kills the bacteria, makes
them stick to the slide, and will make the
cytoplasmic proteins coagulate so you can see
them better when they are stained; it should be
done fast and enough for the cells to stick on
the glass slide and for them not to wash off
during the entire staining procedure.

o Peritrichous – flagella can be found all over the

bacteria
o Atrichous – no flagella

LABORATORY PREPARATION FOR THE

EXAMINATION OF MOTILITY AND
MORPHOLOGY OF BACTERIA
- Wet Mount FOR MOTILITY
- Hanging Drop
Preparation
- Fixed-Stained Smear → FOR MORPHOLOGY

Wet Mount
1. You take a sample of bacteria from a broth (liquid substance
that contains bacteria) OR from an agar/agar plate (solid
substance wherein we allow the bacteria to be cultivated).
 MLS 411 LAB – CLINICAL BACTERIOLOGY o Initial/Primary Stain – first to use and imparts
WEEK 3: STAINING colors
o Mordant – forms a bridge between the cell and the
All about staining initial stain
- Organic or aqueous preparations of dyes that impart ▪ Physical Mordant – heat or cold
a variety of colors to tissue or microorganisms ▪ Chemical Mordant – iodine or Fe2+
- Vast majority of the bacterial cells are nearly or o Decolorizer – removes the color
completely transparent. Thus, the visualization of ▪ Removes the initial stain for differentiation
microorganisms or bacteria in the living state is most o Counterstain/Secondary Stain
difficult.
- To provide better visualization and identification on the DIRECT STAINING PROCEDURE
microorganism or bacteria SIMPLE Staining
- Staining allows the bacteriologist to further enhance the - The stain is flooded across the smear
visibility of the microscopic bacteria o Only ONE TYPE of dye is used
- Study the structures and differentiate the bacteria into a o No special STRUCTURE of the cell is
specific group for diagnostic purposes distinguished
- Stain is not enough; and stains are not created equal - 1. Flood with stain for 1 minute
- The stains to be use will vary on: - 2. Wash it with water
o What do you want to know - 3. Allow it to dry – blot dry
o How they look like - 4. Observe in microscope
o what are their shape or arrangement o Ex.: Methylene Blue
o Identify the structure
SELECTIVE/SPECIAL Staining
NATURE OF DYES - ENDOSPORE stain – dormant and non-reproductive;
- Contains substances that is made up of ions not easily colorized and decolorized
- Dyes have salts o Coated with keratin layer
- Ions function depends on function o Dipicolinic, calcium and peptidoglycan layer
- Ions assume different roles in different dyes - CAPSULAR stain – protective outer covering of the
CHROMOPHORE Negatively bacteria that would protect it from phagocytosis
(ions that brings the COUNTERION charged @ pH 7 o Increases the pathogenicity of the bacteria
coloring material or (no color)
contains the color) o The presence of the capsule will help in
determining the proper medical intervention to
Basic combat the pathogenic feature
(+) ion (-) ion
Stain o Do not absorb the stain
- GRANULAR stain
(-) ion (+) ion
Acidic - FLAGELLAR stain
Stain
I. ENDOSPORE stain
➢ Basic stains = if the chromophore is positive
SCHAEFFER-FULTON stain
➢ Acidic stain = if the chromophore is negative
1. Prepare a bacterial smear.
- Examples of basic:
ACIDIC: 2. Flood the smear with the primary stain (Malachite
o Methylene blue
Eosin green)
o Crystal Violet
Picric acid 3. Heat thee flooded smear to gentle steaming. (5
o Malachite Green Nigrosine Minutes)
o Safranin Red India Ink 4. Pour off excess stain. Rinse thoroughly with tap
o Carbolfuchsin
water.
➢ If it will not stain the bacteria, it will stain the background
5. Flood the smear with the secondary stain (Safranin).
STAINING PROCEDURE: DIRECT AND INDIRECT
(1 minute)
DIRECT Staining Procedure
6. Rinse thoroughly with tap water. Blot dry
o It directly stains the bacteria
7. Examine under OIO
o Simple Staining – use of only one type of dye
o Selective/Special Staining – complicated;
II. CAPSULAR stain
demonstrate specific structure
DRY INDIA INK/NIGROSINE stain [indirect]
o Differential Staining – higher order; differentiate
1. Suspend the organism in a drop of 6% glucose
bacteria spp. from one to another
(further demonstrate the presence of capsule)
▪ Gram Stain
solution placed on a glass slide.
▪ Acid Fast Stain
2. Place an equal volume of INDIA INK and
NIGROSINE
3. Mix well. Perform blood smear technique.
INDIRECT Staining Procedure
4. Allow the smear to air dry. DO NOT HEAT FIX!
o Not stain the bacteria instead the bckgnd
➢ Heating cause the capsule shrink!
REAGENTS used in SELECTIVE/SPECIAL &
5. Cover the smear with SAFRANIN (10 seconds)
DIFFERENTIAL Staining
 6. Wash off the excess stain. Rinse thoroughly with tap MLS 411 - BACTERIOLOGY LABORATORY - WEEK 4
water. Air dry. GRAM STAINING: An important procedure
7. Examine under OIO. (Halo effect)
DIFFERENTIAL STAINING
III. GRANULAR stain - Also called multiple staining, because it used at least 2
ALBERT’S stain types of stain.
1. Prepare a bacterial
- Important: it can see the difference between two groups
2. Stain with LOEFFLER’S
based on their varying reactions to certain techniques.
METHYLENE BLUE (3-5
minutes) GRAM STAIN
a. Metachromatic stain INTRODUCTION:
3. Rinse thoroughly with tap water. Air dry. ● Devised by Hans Christian Gram
4. Examine under OIO. ● Allows us to differentiate between two major group of
organism based on their actions:
IV. FLAGELLAR stain ○ Gram-positive (+)
LEIFSON’S stain ○ Gram-negative (-)
1. Wash the growth from a bacterial culture with 3 mL of ● Identifying if it is gram + or -, will help us on what are the
sterile distilled water (bacterial suspension) and next steps that you have to do in the bacterial
gently agitate. Let it stand [to unravel the flagella]. (7 identification process.
minutes) ○ Example: Gram (+) cocci → catalase test →
2. Make a ring of 1-2cm in diameter on a glass slide. (3-5
coagulation test → DNA Test → until you
minutes)
identify what particular organism.
3. Allow a drop of the bacterial suspension to run across
the ringed surface of the slide. Air dry but do not heat fix. ○ It can also give the physician what possible
4. Cover the ringed area with LIEFSON’S stain (10 treatment they can give.
minutes) ■ Gram (-) are much harder to treat that
5. Wash with water and blot dry. Gram (+)
6. Examine under OIO. ● Principal stain used for microscopic examination of
Also demonstrated in Hanging Drop Technique bacteria
- Almost all of the organism or bacteria can
NEGATIVE/RELIEF Staining demonstrate in gram stain
- The background is stained instead for contrast - Except:
- DRY INDIA INK/NIGROSINE stain - intracellular organism
o Halo effect
- organism that lack cell wall
(mycoplasma and ureaplasma)
- Infinitesimal organisms that you cannot
see under a light microscope
(spirochetes)
● Must-know for all medtech students
TWO TYPES:
❖ DIRECT GRAM STAIN
➢ Directly from clinical specimen
➢ urine/sputum → Smear → gram stain
❖ GRAM STAIN FROM CULTURE (Indirect gram stain)
➢ Grow, plate, cultivate the organism → colony →
do gram stain.
PRINCIPLE

GRAM POSITIVE CELL WALL

● Very thick peptidoglycan/murein layer - difficult to
decolarizer.
 ●Absent LPS 3. Flood the slide with Gram’s iodine for 60 seconds to
●Has teichoic acid - makes gram (+) resistance to increase affinity of crystal violet. Rinse with tap water,
decolorization. shaking off all excess.
GRAM NEGATIVE CELL WALL - Form CV-Iodine complex that is larger than CV
● Very thin murein layer - decolorizer can create holes. molecule alone.
● Has LPS (lipopolysaccharides) - Lipid will get - Adhere more to bacterial cells.
dehydrated by alcohol → loses its integrity → leakable 4. Flood the slide with decolorizer (hacker’s modification)
cell wall. for ~10 seconds and rinse off immediately with tap
● Easily decolorized water. Repeat this procedure until the blue dye on longer
runs off the slide with the decolorizer. Rinse with tap
water and shake off excess.
- The smear → no color grossly.
- Most crucial: because under decolorization the
Gram (+) becomes Gram (-) or vice versa.
- Use tap water to stop the decolorizer
5. Flood the slide with counterstain and allow it to remain
on the surface without drying for 30 seconds. Rinse with
tap water and gently blot the slide dry with paper towels
or bibulous paper or air dry.
6. Examine microscopically under an oil immersion lens at
1000x for phagocytes, bacteria, and other cellular
material.
- Fixed stained: max. Magnification should be
MATERIALS: 1000x
● Primary stain: Crystal Violet VIAS = Crystal Violet → Iodine → Acetone-alcohol → Safranin
● Mordant (enhances the effect of CV): Gram's Iodine
○ CV-Iodine complex → adhere more on the cell
wall
● Decolorizer (removes the primary stain): 95% ethyl
alcohol (originally), but in the lab it uses hacker’s
modification of gram stain (acetone-alcohol
combination)
● Counterstain: Safranin
● Glass slides
● Racks
● Droppers

PROCEDURE
1. Create smear. Fix material on slide with methanol or
heat. If the slide is heat fixed, allow it to cool to the touch
before applying stain.
- Solid colony: dilute it on NSS
- Fixing: allows samples to adhere on the slide and
don't get washed away when doing washing.
- Methanol fixation: 95% methanol, used only
on direct and bloody samples. Allows to
preserve organism and human cell
- Heat Fixation: pass the smear 2-3 times
above the alcohol lamp/ bunsen burner to kill
REPORTING
the organism and allow it to adhere.
1. Gram Stain from culture
2. Flood slide with crystal violet and allow it to remain on
● Take note of:
the surface without drying for 60 seconds. Rinse the
○ Gram reaction
slide with tap water for not more than 5 seconds,
○ Shape (cocci, bacilli, or spirilla)
shaking off all excess.
○ Arrangement of bacterial chain
- Water or washing must not be forceful.
● Example: Gram positive (reaction) cocci (shape)
in clusters union (arrangement)
 ACID-FAST STAINING
- Not a routine procedure.
- Commonly for pulmonary diagnosis (TB)

INTRO AND PRINCIPLE

1. The acid-fast stain is specifically designed for a subset
of bacteria whose cell walls contain long chain fatty
(mycolic) acids.
- Because mycolic acid is much harder to stain.
2. Mycolic acids render the cells resistant to decolorization,
even with acid
alcohol
decolorizers.
3. Mycobacteria,
Nocardia,
Corynebacteria
(shortest mycolic
acid), and
Cryptosporidium

2 TYPES OF ACID-FAST STAINING

1. Ziehl-Neelsen
- Hot method
- Commonly performed
2. Kinyoun
2. Direct Smear
- Uses wetting agents
● Take note of:
- Cold method
○ Gram reaction
MATERIAL:
○ Shape (cocci, bacilli, or spirilla)
● Primary stain: Carbol Fuchsin (basic fuchsin + phenol
○ Arrangement of bacterial chain
in a form of carbol)
○ Host cell (human cell)
- Phenol component of Carbol Fuchsin allows the
- inflammatory cells
stain to penetrate the fatty waxy mycolic acid.
○ Location of organism (sometimes)
● Acid alcohol - main drawer of difference for the acid-
- some have organism are inside the cells.
fast reaction.
● Counterstain: Methylene blue (most common) /
Malachite green
● Glass slides
● Applicator stick

PROPER SPUTUM SAMPLE: (common)

TAKE NOTE!
1. Gram-positive organisms that have lost cell
wall integrity because of antibiotic
treatment, old age, or action of autolytic
enzymes may allow the crystal violet to
wash out with the decolorizing step and
may appearance of gram variable .
2. However, for identification purposes, these
organisms are considered to be truly Gram
Postive .
3. On the other hand, gram-negative bacteria
rarely, if ever, retain crystal violet.
● SPUTUM not saliva
- Mucoid thick secretion directly coming from
lungs.
● Acquire through deep expecturation.
● Sputum Characteristic: Mucoid-purulent
● Amount: 2 samples
○ 1st morning cough
○ 2nd sample 2 hours after the first sample/
anytime of the day
PROCEDURE:
1. Make a smear appropriate for acid-fast staining.
- Accurate dimension: measure 2 x 3 cm using
template.
- Use applicator stick to get the sample → spread
it on glass slide (coiling manner)
- Coiling: to liberate the trap organism.
 - Heat Fixation: pass the smear 2-3 times above
the alcohol lamp/ bunsen burner to kill the
organism and allow it to adhere.

2. Flood smears with carbolfuchsin (primary stain) and

heat to almost but not boiling by performing the
procedure on an electrically heated platform or by
passing the flame of a Bunsen burner/alcohol lamp
underneath the slides on a metal rack.
- Pag boiling → mali na siya. It should stop when
it streamed
- Heating allows to penetrate the waxy mycolic
acid
The stain on the slides should steam. Allow slides to sit
for 5 minutes after heating; do not allow them to dry out.
Wash the slides in distilled water (note: tap water may
contain acid-fast bacilli).

Drain off excess liquid. ● ACID-FAST POSITIVE WILL NOT BE DECOLORIZED!

(even using strong acids)
● ACID-FAST NEGATIVE will become colorless when
apply decolorizer → will become blue with counterstain
METH BLUE/MALACHITE GREEN

READING AND REPORTING

● Imagine 2 horizontal line (Red and Green on the photo)
→ it is where you read
3. Flood slides with 3% HCI in 95% ethanol (decolorizer)
● Each horizontal line is composed of ~150 oil immersion
for Approximately 25-30 seconds.
fields.
Check to see that no more red color runs off the surface
● Over all 300 fields
when the slide is tipped.
Wash thoroughly with water and remove the excess.
4. Flood slides with methylene blue (counterstain) and
allow them to remain on the surface of slides for 10
seconds.

Wash with distilled water and stand slides upright on

paper towels to air dry.
- Do not blot dry. The paper my pick up the
organism
5. Examine microscopically, screening at 400x
magnification and confirm all suspicious (i.e., red) ← one oil immersion field.
organisms at 1000x magnification using an oil-immersion
lens.

← you need to count acid-fast

each field.
REPORTING: (follows the WHO scale) BACTERIOLOGY LAB
IUATLD/ WHO SCALE Conventional Light CULTIVATION OF BACTERIA
Microscopy
LEARNING ACTIVITY NO. 3
0 No ARB seen in 300 oil
Skillful Demonstration of the Different Cultivation
immersion field (OIF) Techniques
Confirmation required*
+n 1-9 AFB seen in 100 OIF SPECIFIC LEARNING OBJECTIVES
1+ 10-99 AFB seen in 100 OIF At the end of this set of activities, the BMLS 3rd year student
can:
2+ 1-10 AFB/OIF in at least 50
1. Demonstrate the different streaking, isolation and cultivation
fields techniques routinely done in bacteriology;
3+ >10 AFB/OIF in at least 20 2. Identify various culture media appropriate for the type of
fields bacterial pathogens;
Example: 3. Identify important conditions and requirements of bacterial
● 9 AFB in 100 field → report as +9 (take note of the plus growth;
4. Describe cultural characteristics of bacteria;
sign)
5. Precisely prepare the routinely employed culture media in a
● Automatic 3+ if you have seen more than 10 AFB in at clinical laboratory;
least 20 fields. 6. Appreciate the significance of practicing aseptic technique in
bacterial culture.
Result form:
● Details of the Px EXPECTED ENTRY-LEVEL COMPETENCIES
● Characterics of sputum The 3rd-year BMSL student can:
a. Skillfully perform streaking, isolation, and cultivation
● Reading (if zero write negative)
techniques
b. Aseptically prepare culture media
TAKE NOTE! c. Perform aseptic transfer techniques
d. Explicitly describe cultural characteristics of bacteria
• PRESUMPTIVE DIAGNOSIS ONLY
e. Identify routinely employed culture media
– TB result should not be based alone on
the acid fast stain. LEARNING RESOURCES
– CULTURE → ideal to diagnose TB. • Reagents & solutions:
– However culture, they are slow growers ➢ Culture Media:
( at least 2 weeks ) o Blood Agar Plate (BAP) base
– “Gipa tubo nimo ang organism 2 weeks o MacConkey Agar (Mac)
o Eosin and Methylene Blue (EMB)
before nitubo ang organism, imong Px o Mueller-Hinton Agar (MHA)
kamatyonon na” (severe case) o Mannitol Salt Agar (MSA)
– Today they use PCR → for genetic o Nutrient Agar (NA)
identification of mycobacterium. o Tryptic Soy Broth Brain Heart Infusion (BHI) broth
• RESULT LARGELY DEPENDS ON THE SPUTUM o Triple Sugar Iron (TSI) agar
o Methyl Red & Vogues Proskauer Urea agar slant
SAMPLE
o Simmons citrate medium
• MOST OF THE TIME FOR MTB BUT CAN BE o Sulfide-Indole-Motility (SIM) medium
USED FOR OTHERS ORGANISM o Lysine Iron Agar (LIA)
• Not all RMT can do the ACID-FAST STAIN o DNase test agar
o Bile Esculin Agar

o Distilled water
o Alcohol (lamp)
o Lysol or Clorox (specimen treatment)
o Defibrinated type O whole blood (250 ml blood bag)
or Sheep’s blood
o Mixed population of bacteria in broth culture (screw
capped tube)

− Materials:
➢ Disposable Petri plates (sterile)
➢ Magnifying lens Marker
➢ Erlenmeyer flask (1L, 500 ml)
➢ 150x15 mm Petri plates
➢ Stirring rod
➢ Test tubes
➢ Rubber hand/hot hand
➢ Alcohol lamp or Bunsen burner
 ➢ Filter paper
➢ Inoculating loop
➢ Sterile cotton swab
➢ Masking tape
➢ Test tube rack
➢ Cotton plugs
➢ Autoclave tape
− Equipment
➢ Hot plate
➢ Incubator
➢ Refrigerator
➢ Biosafety Level 2 cabinet
➢ Autoclave
INTRODUCTION
Bacteria are microscopic organisms. However, when
mass multiplication occurs, bacteria can be seen
macroscopically, as seen in culture or in colony. In this exercise,
the students will learn the different techniques of inoculation or
simply the planting or more appropriately the seeding of a
sample suspiciously harboring bacteria. This is called cultivation.
Cultivation is the process of growing microorganisms in culture
by taking bacteria from the infection site by some means of
specimen collection and growing them in the artificial
environment of the laboratory. Nutrients are incorporated into
culture media on or in which bacteria are grown.
Various types of culture media have been developed for
use in diagnostic microbiology because different pathogenic
bacteria have different nutritional needs. For certain bacteria that
require complex and exceptional media components such as
growth factors and vitamins, these bacteria are said to be
fastidious.
Alternatively, the nutritional needs of most clinically important
bacteria are relatively basic and straightforward and are
considered nonfastidious.
Media Classifications and Functions
In diagnostic bacteriology there are four general
categories of media: enrichment (or enriched), supportive
(general), selective, and differential.
Enrichment media contain specific nutrients required
for the growth of particular bacterial pathogens that may be
present alone or with other bacterial species in a patient
specimen. This media type is used to enhance the growth of a
particular bacterial pathogen from a mixture of organisms by
using nutrient specificity. One example of such a medium is
buffered charcoal-yeast extract agar, which provides Lcysteine
and other nutrients required for the growth of Legionella
pneumophila, the causative agent of legionnaire’s disease. Two
enrichment broths generally used for stool specimens are
alkaline peptone water (APW) for alkaline-loving bacteria like the
Vibrio species and Selenite-F to enhance Salmonella and
Shigella growth.
Supportive media contain nutrients that support growth
of most nonfastidious organisms without giving any particular
organism a growth advantage, i.e. Nutrient Agar.
Selective media one or more agents that are inhibitory
to all organisms except those being sought. In other words,
these media select for the growth of certain bacteria to the
disadvantage of others, inhibitory agents used for this purpose
include dyes, bile salts, alcohols, acids and antibiotics. An
example of a selective medium is phenylethyl alcohol agar,
which inhibits the growth of aerobic and facultatively anaerobic
gram-negative rods and allows gram-positive cocci to grow.
Differential media employ some factor (or factors) that
allows colonies of one bacterial species or type to exhibit certain
metabolic or culture characteristics that can be used to
distinguish them from the other bacteria growing on the same
agar plate. One commonly used differential medium is
MacConkey agar, which differentiates between gram-negative
bacteria that can and cannot ferment the sugar lactose.
Of importance, many media used in diagnostic
bacteriology provide more than one function. For example,
MacConkey agar is both differential and selective because it will
not allow most gram-positive bacteria to grow. Another example
is sheep blood agar. This is the most commonly used supportive
medium for diagnostic bacteriology because it allows many
organisms to grow. However, in many ways this agar is also
differential because the appearance of colonies produced by
certain bacterial species is readily distinguishable.
Culture Transfer Techniques
In several microbiology experiments, you will need to
transfer microorganisms from one medium to another by
subculturing. This aseptic technique also is commonly used
when preparing and maintaining stock cultures, and when
carrying out a number of microbiological test procedures.
Microorganisms are everywhere, including in the air of
your course lab, as well as on the floors, bench tops, and
equipment. If appropriate precautions are not taken, these
microbes could end up in one of your subcultures; that is,
microbes could contaminate your materials. To prevent
contamination by unwanted microbes, the microbes you want
must be transferred using proper aseptic techniques.
Aseptic transfer techniques are not difficult to learn and
perform, although some eye-hand coordination and practice may
be needed. To simplify the process: make sure all needed
materials, cultures, and media are ready, and the wire of the
transferring device (inoculating loop or needle is straight.
Label appropriately all media into which microbes will be
transferred so you can identify your cultures
during the lab period, or if incubated, when the next lab period
meets.
Aseptic Transfer Techniques
The different steps of the aseptic transfer process are
described below and different ways are shown in
Figure 3.2 for transferring bacteria. The description and figure
show the use of the inoculating loop, which
is typically used for broth and agar/slant/plate transfers. Read
through the process completely before attempting the
procedures described below.
➢ Holding the tubes and sterilizing the inoculating loop
o If you are right-handed, hold the stock culture tube (from
which you will make the transfer) and the tube to be
inoculated in the palm of your left hand.
o To sterilize the inoculating loop, hold the handle of the
instrument in your free hand like you wound a pen or
pencil. Place the loop in the hottest portion of the Bunsen
burner flame, which is the top of the blue flame. In a few
seconds, the entire loop will turn red hot. When it does,
move the rest of the wire rapidly through the flame. Once
sterilized, the loop should not be placed on the lab bench.
Simply hold it in your hand and allow the loop or needle to
cool for 10 or 20 seconds.
Quick procedure aseptic transfer technique:
1. Sterilize inoculating loop
2. Transfer broth film or small bacterial sample on loop from
stock tube or agar plate to appropriate sterile broth tube or
streak on agar slant or plate
3. Re-sterilize loop.
LEARNING ACTIVITY NO. 3A
PREPARATION OF CULTURE MEDIA
1. Prepare the different types of culture media assigned to you
by the instructor. Get the agar container and read carefully the
directions found on the label. Follow directions. Some media do
not need autoclaving and some need the addition of blood or
urea after the sterilization process.
2. Measure agar and distilled water into clean flask or beaker.
Weigh accurately.
3. Flame sterilizes a clean glass rod to stir the medium as it
melts.
4. While wearing heat resistant hand protection, hold the flask or
beaker over the flame. Swish or stir the mixture constantly while
heating.
5. Boil the mixture for 1 minute. Remove from heat.
6. Cover with cotton plug and then with paper tied around the
neck of the flask. Do not forget to label with your class section,
group number, name of media, date prepared. Submit to the
stockroom for autoclaving.
7. After autoclaving. Place a sterile lab thermometer in the
mixture and monitor the temperature until it falls to approximately
45 - 50° C or if a lab thermometer is not available, cover and let
stand a few minutes.
8. Pour enough melted agar into each sterile plastic petri dish to
cover the bottom - about 1/8" to 1/4" deep. Replace the lid
immediately.
9. Place agar plates on a counter top to cool and set. Agar
medium will set like stiff gelatin at room temperature.
10. The agar medium is now ready for storage or use.
Storage: Stack agar plates upside down in the refrigerator. Do
Not Freeze! The purpose of placing the plates upside down is to
prevent condensation from dripping down onto the agar surface
which could then facilitate movement of organisms between
colonies.
Take note of some special procedure or condition in the
preparation of the certain culture media, i.e. Blood agar plate,
chocolate agar plate, urea agar slant.
YOUTUBE VIDEO BY TEACHER JULIE
Step 1: Disinfect the working area.
Step 2: Prepare the materials.
Step 3: Check expiry date of the culture medium in the bottle.
Step 4: Follow the directions on how to prepare the medium.
Step 5: Weigh accurately the medium powder using a digital
weighing scale
− Switch on
− Press “TARE” to start at 0.00
Step 6: Pour medium powder into the flask
Step 7: Add first, half volume of the distilled water then mix to
dissolve
− Add full volume of the distilled water
− MIX
− Drop the magnetic stirrer into the flask
Step 8: Heat to boil the medium on a hot plate and turn on
“STIR” for continuous mixing of the solution. When medium has
cleared, remove the flask from the hot plate. Use the magnetic
stick to remove the magnetic stirrer from the solution.
Step 9: Put “cotton plug” in the mouth of the flask. Cover with
foil. Place an “autoclave tape” and label the name of the
medium.
Step 10: Bring the media for sterilization.
Step 11: Check “The Autoclave’s” water level at the bottom
Step 12: Place the basket
− Cover
− Lock
− Switch on
Step 13: Wait for about 1 HOUR for complete sterilization of the
culture media
− AUTOCLAVE PRINCIPLE:
o 121C for 15 minutes at 15 psi
 o Ensure autoclave is at zero pressure before
opening
Step 14: Take out the sterilized culture media
Step 15: Dispensing media into plates
− Use 100mm X 15mm Petri plates for MAC & BAP
− Use bigger Petri plates 150mm X 15mm for MHA
Step 16: Use “hot hand” to hold flasks
− Before dispensing, heat the mouth of the flask
− Gently swirl the plate for even distribution of the agar
− Swirl the flask every now and then
− Allow to cool and solidify the agar plates
Step 17: For BAP, allow about 45C temperature before adding
5% sheep blood (12.5 ml of blood for 250ml volume used in the
video). NOTICE: hot hand is no longer used while dispensing the
BAP. Dispensing must be fast enough as pouring will be difficult
when the agar has begun to solidify.
Step 18: Label the name of the medium at the bottom of the
plate.
Step 19: Store at refrigerated temperature in an inverted
position.
Step 20: Perform Quality Control
1. Media Sterility Test
− 5%-10% of the in-house, new batch prepared
media are incubated at 37C for 18-24hrs to
ascertain that there is no growth or
contamination
− If any colonies develop after incubation, the
whole batch needs to be discarded
2. Growth Promotion Test
− Standard control strains of organisms are
inoculated into the test medium and observed
for typical reactions
− If the strain of E. coli ATCC 25922 is inoculated 3. Replace the lid. Sterilize the loop to destroy any remaining
into a sterile MacConkey agar plate, after proper bacteria. To ensure that it is cool, touch the loop to the center of
incubation, it should show lactose fermenting the plate or between the agar and the edge of the plate. Pass the
pink colonies loop one time across the previous streaks to pick up some
bacteria, and continue streaking into a second area of the plate,
as illustrated in Figure 3.4B. Replace the lid.
4. Sterilize the loop as before, then be certain that it is cool. To
pick up some bacteria, pass the loop one time through the
second area, and continue streaking into a third area (Figure
3.4C).
Replace the lid.
5. Sterilize the loop as before, and streak some bacteria from the
third area of the plate into the fourth area (Figure 3.4D). Be sure
to use up the remaining space on the plate. Replace the lid.
Sterilize the loop.
LEARNING ACTIVITY NO. 3B
CULTIVATION (STREAK PLATE) TECHNIQUE 6. To obtain practice, repeat the streak plate technique using
additional plates and different mixed populations. The instructor
1. Select a nutrient agar plate or a culture medium as directed by may recommend alternative streaking or cultivation methods.
the instructor. Label the bottom side of the plate with your name, 7. Additional practice can be obtained by attempting to isolate
the date, and the designation “streak plate.” Obtain a broth bacteria on selective media.
culture containing a mixed population of bacteria. Before 8. Invert all the plates and incubate them for 24-48 hours at the
beginning the streak plate technique, read through steps 2 appropriate temperature. The plates are inverted so that
through 4 and review Figure 3.2 to familiarize yourself with the moisture accumulates on the lid rather than on the agar surface,
technique. where it may cause colonies to run together. After incubation,
2. Aseptically obtain a loopful of the mixed population, and lightly refrigerate the plates in the inverted position until the next
streak it several times along one area (quadrant) of the plate, as laboratory session to preserve the bacterial growth and prevent
shown in Figure 3.4A. Try not to cut into the agar surface and drying of the medium.
avoid airborne contamination by lifting the lid of the Petri dish 9. Examine the plates for well-isolated and separated colonies
only enough to permit entry of the loop. and enter a representation of a good streak plate in the
Results/Lab Report section. Add appropriate labels and brief
explanations to produce a “talking picture.” Describe several
isolated colonies by numbering the colonies and noting their
size, color, and characteristics, with reference to the standard
terminology provided in Figure 3.5.
➢ Size of a colony may be determined by measuring the
colony diameter in millimeters if rulers are available.
Describe also the hemolytic pattern (alpha, beta, or
gamma) of colonies on BAP. Consistency of colonies are
classified as smooth, rough, or mucoid.
➢ On MAC or EMB, indicate further if colonies are lactose
or nonlactose fermenters. Record your observations.
10. At the direction of the instructor, use a bacteriological needle
or loop to select samples from various colonies, and inoculate
nutrient agar slants to obtain pure cultures. Stained smears may
also be made from the colonies to determine the morphological
characteristics of the organisms.

Note: Never touch the bacterial colonies on a plate with your

fingers. Each colony contains millions of live organisms.

LEARNING ACTIVITY NO. 3C

STREAKING TECHNIQUE DEMONSTRATION

Aseptic transfer and streaking techniques practical quiz.

From a stock culture, perform aseptic and pure culture

techniques onto BAP, EMB, or MAC or as directed by the
instructor.