Microscopic study of Bacteria
• Spirillum
• Microscopic morphology
- The size, shape, intracellular inclusions,
cellular appendages, and
arrangement of cells when observed
with the aid of microscopic
magnification.
• SIZE
-is measured in micrometer that is equal
to 1/25.000 of an inch or 0.001 nm
• Haemophilus -smallest pathogenic
bacillus with cell wall
• Mycoplasma -smallest pathogenic
bacteria without a cell wall
• Bacillus anthracis -largest pathogenic
bacillus
• SHAPE AND ARRANGEMENT
• COCCI - spherical in shape
• BACILLI - rod-shaped
• SPIRAL - coiling (ex. SPIROCHETES -
treponema pallidum)
• Cocobacilli - very short bacilli
• Filaments - long threads of bacilli
which have not separated into single
cells
ARANGEMENTS OF COCCI
• Coccus - single
• Diplococci - two
• Staphylococci - clusters
• Streptococci - chains
• Sarcinae/octet - cube
• Tetrad - four
ARRANGEMENTS OF BACILLUS
• Bacillus
• Diplobacillus
• Streptobacillus
• Trichome - straight
• Palisade (ex. Corynebacterium
diphtheriae
• Cocobacillus - very short
ARRANGEMENTS OF SPIRAL
• Spirochete
OTHERS
• Fusobacterium - fuseform
• Vibrio
• B dellovibrio - comma shape
• Corynebacteriaceae - club rod
(chinese letter arrangement
• Helicobacter pylori - helical
• Borrelia burgdorgeri - corkscrew
• Filamentous - very long
• Spirochete
DIRECT BACTERIAL EXAMINATION IN
LIVING STATE
• SALINE WET MOUNT -to determine
biologic activity of microorganisms,
including motility or reactions to certain
chemicals or serologic reactivity in
specific antisera. *NSS - Normal saline
solution
• HANGING DROP METHOD
-same purpose as the saline mount
-Analysis of living microorganisms
-Modified version of wet mount
method
-Fixing the microbial suspension
into a drop of liquid
*petroleum jelly - serves as a SEAL
*makes use of depression slide
• PROCEDURE
- The glass slide that you have
prepared or concavity slide
upsidedown (concavity downwards)
over the drops on the coverslip to
ensure that the vaseline is sealed the
slideslip within the concavity.
- The slide should be turned over so
that it is on the top.
- Let the organisms “settle" for a
minute. The drop is visible hanging
across the concavity.
• DIFFERENT MOTILITY
- Darting: Vibrio cholerae
- Tumbling: Listeria monocytogenes at
20 - 25 deg. C
- Stately: Clostridia
- Corkscrew: T. pallidum
- Lashing: Borrelia
- Gliding: Mycoplasma
- Swarming: Proteus mirabilis, P.
vulgaris • DARKFIELD
PREPARATION
- used to visualize certain delicate
microorganisms that are invisible by
a brightfield optics and stain only
with great difficulty.
- It is particularly useful in
demonstrating spirochetes from
suspicious syphilitic chancres for
Treponema pallidum
- *Dark-ground microscope -special
type of compound light microscope
; contains dark-field condenser (most
important part) that functions to
illuminate the object with a cone of
light
- uses reflected light
- appear brightly stained against a
dark background
USES OF THE DARK FIELD MICROSCOPE
- It is useful for the demonstration of
very thin bacteria not visible under
ordinary illumination since the
reflection of the light makes them
appear larger.
- This is a frequently used method for
rapid demonstration of Treponema
pallium in clinical specimens.
- It is also useful for the demonstration
of the motility of flagellated bacteria
and protozoa.
BACTERIAL EXAMINATION AT FIXED
STATE
- Process of artificially coloring the
microorganisms with dyes
- Make cells more visible and to
demonstrate differences between cells
- 3 steps
1. Preparation of smear
2. Fixation (heat/alcohol)
3. Application of staining solution
SIMPLE STAINING
- The coloration of bacteria by
application of a single solution of stain
to a fixed smear.
- BASIC STAINS (w/ positively
charged chromogen) stains/attract
negatively charge like nucleic acids and
other/some cell wall components
- e.g., Methylene blue, Safranin,
and Crystal violet
DIFFERENTIAL STAINING
- Elicit differences between
bacterial cells or parts of a bacterial
cell.
- It is more elaborate that the simple
staining technique in that the cells may
be exposed to more than one dye
solution or staining reagent.
- Would impart 2 or more different
colors
- Positive: Purple, Negative: Red
(Gram Staining)
- e.g., Gram Staining, Acid-fast
staining
GRAM STAINING
- introduced by Hans Gram in 1884
- most important and widely used
staining technique in microbiology
PURPOSE:
• A true STAT test in microbiology
• Judge adequacy of a specimen
• Recognition of specific
morphologies
• Indicate need for additional tests
• Expand clinical diagnostic picture
LIMITATIONS:
• Only partial bacterial
identification
• Some organisms do not stain
• no organisms seen does not rule
out infection
• Normal flora can mask pathogens
• Human error
• Organism do not stain as
expected
GRAM-POSITIVE
• Thick peptidoglycan layer
• Teichoic Acids prevents
discoloration
GRAM-NEGATIVE
• Thinner peptidoglycan layer
• Decolorizer washes out
GRAM STAINS
BACTERIA GROUP ACCDG TO
GRAM'S REACTION
- Bacteria group according to its
Gram's Reaction
- All cocci are gram positive
EXCEPT: Neisseria, Veillonella,
Branhamella
- All bacilli are gram negative
EXCEPT: Bacillus, Mycobacterium
Clostridium, Corynebacterium
- Listeria, Erysipelothrix,
Lactobacillus, Rothia (different
staining technique
- Spirals: Hard to stain (e.g.
Treponema pallidum)
- Mycoplasma/ Ureaplasma: Gram
negative, they do not have cell
wall/peptidoglycan layer
- Mycobacterium: Gram positive
(very hard/difficult to stain with
gram staining, acid-fast stain is
preferred because of its high
concentration of mycolic acid in
the cell wall, resist decolorization
- Gram-positive and Gram-negative
cells lose their cell walls and grow
as L Forms in media
COME IN AND STAIN
Crystal violet
Iodine
Acid alcohol
Safranin
ACID-FAST STAINING
- Used for Mycobacterium spp.
- The presence of higher alcohol,
glycerol, fatty acid, and especially
mycolic acid in the cell wall has
been found responsible for
keeping the acid-fast property of
bacteria.

REPORTING OF AFB
- By International Union against
Tuberculosis and Lung Disease
(IUATLD) – the most accepted,
most common reporting

No organism seen: Negative

1-9/100 OIF (oil immersion field):

Exact number

10-99/100 OIF: +

1-10/OIF: ++

10/OIF: +++

MODIFICATION OF ACID-FAST
STAINING