BACTERIOLOGY LABORATORY ○ Suffering percutaneous

1ST SEM A.Y. 2023-2024 inoculation, that is, being

punctured by a needlestick
OUTLINE
o LABORATORY SAFTEY STANDARD PRECAUTION
o BACTERIOLOGY BASICS
MORPHOLOGY, CLASSIFICATION • In 1987, the CDC published
AND STAINING METHODS guidelines known as Universal
o SPECIMEN COLLECTION AND Precautions, to reduce the risk of
PROCESSING HBV transmission in clinical
laboratories and blood banks. In
ROUTES OF INFECTION 1996, these safety recommendations
• Mucous Membrane contact - rubbing became known as Standard
the eyes (conjunctiva) or nose with Precautions. These precautions
contaminated hands require that blood and body fluids
• Airborne - inhalation of aerosols from every patient be treated as
produced during centrifugation of potentially infectious
unstoppered tubes (M. tuberculosis, o The essentials of Standard
Brucella) Precautions and safe laboratory
• Ingestion - failure to wash hands work practices are as follows:
after work, eating, drinking and o Do not eat, drink, smoke, or apply
mouth pipetting (Salmonella and cosmetics (including lip balm).
Shigella) o Do not insert or remove contact
• Direct Inoculation - puncture by lenses.
contaminated needles and broken o Do not bite nails or chew on pens.
glass (Hepatitis virus) o Do not mouth-pipette.
• o Limit access to the laboratory to
BIOSAFETY trained personnel only.
● Individuals are exposed in o Assume all patients are infectious
various ways to laboratory acquired for HIV or other blood-borne
infections in microbiology pathogens.
laboratories. o Use appropriate barrier
● These involve the following: precautions to prevent skin and
○ Rubbing the eyes or nose with mucous membrane exposure,
contaminated hands including wearing gloves at all
○ Inhaling aerosols produced times and masks, goggles, gowns,
during centrifugation, or aprons if there is a risk of
vortexing, or spills of liquid splashes or droplet formation.
cultures o Thoroughly wash hands and
○ Accidentally ingesting other skin surfaces after gloves
microorganisms by putting are removed and immediately
pens or fingers in the mouth after any contamination.
o
 BIOLOGICAL SAFETY LEVEL spp., Rickettsiea,
Arboviruses
Biological Safety Level: Classification ● BSL-4
depend on the degree of the hazard • Organism sait to cause life
that may cause. threatening diseases
● BSL-1 • Organisms handled in
• Organism with no known research facilities
potential of infecting healthy • RITM
individual o EX. Ebola
• Could cause minimal threat to BILOGICAL SAFETY CABINETS
laboratory workers ● Biological Safety Cabinets:
o Ex. Bacillus subtilis, • Devices that enclose a work
Mycobacterium space
gordonae • Main purpose is to sterilize air
• BSL use in School with infections materials
● BSL-2 through filtration using HEPA
• Associated with laboratory filter
acquired infections • To protect lab worker
• Could cause moderate threat ● Class I
to lab workers • Air velocity 75 linear feet/min
• BSL use in Hospital • Exhaust air thru HEPA filter;
o EX. Staphylococci, • Product (culture)
Salmonella, Shigella, contaminant
Bacillus anthracis, ● Class II
Yersinia pestis • Air velocity 75 to 100
(Bioterorrism agent) linear/min- vertical laminar
airflow
● BSL-3 • No product contamination
• Associated with laboratory • Exhaust and recirculate air
acquired infections thru HEPA filter
• Organism with a potential for • IIa= exhausts air inside the
a aerosol transmission room
• The mold phase of systemic • IIb= exhausts air outside the
fungI building
o EX. Histoplasma • MUST for MICRO
capsulatum, LAB/HOSPITALS!!!
Coccidiodes immitis, ● Class III
Blastomyces • upply and exhaust air thru
dermatitidis, HEPA filter
o Paracoccidiodes • For BSL level IV (Viruses)
brazilienze
o EX. Myvcobacterium
tuberculosis, Brucella
METHODS OF MICROBIAL CONTROLS
• STERILIZATION: refers to the
destruction of ALL forms of
microbial life including their
spores
• DISINFECTION: refers to the
destruction of pathogens but
not necessarily ALL
microorganism and their
spores.
STERILIZATION
● Physical Methods of Sterilization
● Moist heat
• Autoclave= 121 degree
celsius 15 lbs psi 15 mins.
“Steam under pressure”
• Inspissation= 75-80 degree
celsius 2 hours for 3 days
“Thickening through
evaporation”
• Tyndallization= 100 degree
celsius 30 mins for 3 days
• Boiling= 100 degree celsius 30
mins
• Pasteurization = 63 degree
celsius 30 mins, 72 degree
celsius
● Dry heat
• Hot Air Oven= Drying 170-180
degree celsius (Q.C Bacillus
subtilis var niger)
• Incineration= 870-980C
heating @ high disposing of
infectious waste
• Cremation= burning material
into ashes
• Flaming= needles
• Gas- ETHYLENE OXIDE= HEAT
LABILE (Q.C Bacillus subtilis
var globijii)
● Filtration
• Seitz-filter= used to filter heat
labile fluid
• Membrane filter or cellulose
filter
• Millipore filter= .22um can
give 100%
● Ionizing Radiation= Exposure to
gamma rays, carried out for the
disposal of syringes, catheters, and
gloves
● CHEMICAL METHODS OF
STERILIZATION
o Ethylene oxide= most
commonly used chemical
sterilant, used in gaseous
form for sterilizing heat
sensitive objects
o Formaldehyde vapor and
vapor phase hydrogen
peroxide= used sterilize
HEPA filter
• Glutaraldehyde (2%) sporicidal,
kills spores in 3 -10 hours, for
medical equipment
• Paracetic acid
DISINFECTION
• Physical Methods of Disinfection
o Pasteurization- 63C for
30 mins: 72C For 15secs
o Boiling- 100C 15 to 30
mins
o UV- non-ionizing
radiation using UV light
• Chemical Method of Disinfection
o Antiseptic: chemical
germicide for use on the
skin or tissue and not to
be substituted for a
disinfectant
o Alcohol- 70 % ethyl
alcohol/isopropyl
alcohol
o Iodophors- iodine +
detergent
o Chlorhexidine
 o Hexachlorophene
o Disinfectants: for non
living things
o HALOGENS: chlorox/ Na
Hypochlorite/ household
bleach (BEST)
o Heavy metals- mercury
o Aldehydes- not use
surfaces because fumes
are irritating
o QUATS- quarternary
ammonium compounds
EX. Benzalkonium
chloride/ zephiran
o Phenolics
o Vinegar- alternative if
NaCl are not available
BACTERIOLOGY BASICS MORPHOLOGY,
CLASSIFICATION AND STAINING METHODS
● Microorganisms – several classes of
living beings
● Based on the organization of their
cellular structures, all living cells can
be divided into two groups:
eukaryotic and prokaryotic
○ Eukaryotic cell types -
Animals, plants, fungi,
protozoans, and algae
○ Prokaryotic cell types -
bacteria & blue green algae
• Antoni van Leeuwenhoek
o Leeuwenhoek is called "the
inventor of the microscope"
o Created a “simple”
microscope that could
magnify to about 275x, and
published drawings of
microorganisms in 1683
o Could reach magnifications of
over 200x with simple ground
lenses
• The Light Microscope
o many types
o bright-field microscope
o dark-field microscope
o phase-contrast microscope
o fluorescence microscopes
o compound microscopes
o image formed by action of >2
lenses
• The Compound Microscope
o The Optical System
o Objective Lens: the lens
closest to the specimen;
usually several objectives are
mounted on a revolving
nosepiece.
o Parafocal: when the
microscope is focused with
one objective in place,
another objective can be
rotated into place and the
specimen remains very nearly
in correct focus.
o Eyepiece or Ocular Lens: the
lens closest to the eye.
o Monocular: a microscope
having only one eyepiece
o Binocular: a microscope
having two eyepieces.
• Phase Contrast Microscopy
o light rays through objects of
different h ® change in
phase, not intensity
o special ring-shaped
condenser diaphragm
o special glass disc in objective
o change phase differences to
intensity differences
o can view transparent
 o objects as dark on • Protein synthesis (ribosomes, RER,
light Golgi)
o background (without • Mitochondria; chloroplasts
staining) • Lysosomes
o Right; human brain glial cells • Plasma membranes have different
• The Bright-Field Microscope modifications
o Produces a dark image • Cytoskeleton
against a brighter background
o Has several objective lenses Character Prokaryotes Eukaryotes

o par focal microscopes remain Nucleus Nuclear Absent Present

membrane
in focus when objectives are Nucleolus Absent Present
changed Chromosome One circular One or more paired
o total magnification Cell division Binary fission
and linear
Mitosis
o product of the Cytoplasmic Structure and fluid phospholipid fluid phospholipid
magnifications of the ocular membrane Composition bilayer, lacks sterols bilayer containing
sterols
lens and the objective lens Function Incapable of Capable of
endocytosis endocytosis and
• Fluorescence Microscopy (phagocytosis and exocytosis
pinocytosis) and
o Illuminate specimen with UV exocytosis

® visible fluorescence (filter Character Prokaryotes Eukaryotes

removes harmful UV) Cytoplasm Mitochondria Absent Present

o View auto-fluorescent
objects (e.g., chloroplasts) Lysosomes Absent Present

o Stain with specific fluorescent Golgi apparatus Absent Present

dyes, which absorb in region Endoplasmic Absent Present

230-350 nm & emit orange, reticulum

yellow or greenish light Vacuoles Absent Present

o Images appear coloured Ribosomes 70 S 80 S

against a dark background

Character Prokaryotes Eukaryotes

Cell Wall Present Animals & Protozoans –

Prokaryotes Absent

• Prokaryotes represent two domains,

Plants, Fungi & Algae -
Present

bacteria and archaea. Composition Peptidoglycan –

complex
Cellulose or chitin

• Archaea live in Earth’s extreme Locomotor

carbohydrate
Flagella Flagella/ Cilia
environments. organelles

• Bacteria are the most abundant and

diversified organisms on Earth.
Eukaryotes have organelles
• Much larger; more complex than
prokaryotes
• Processes compartmentalized into
organelles
• Nucleus
 ● Prokaryotic Cells OTHER SHAPES
● Much smaller (microns) and
more simple than eukaryotes
● Prokaryotes are molecules
surrounded by a membrane
and cell wall.
● They lack a true nucleus and
don’t have membrane bound
organelles like mitochondria, ARRANGEMENT OF BACTERIA: COCCI
etc.
● Large surface-to-volume ratio
: nutrients can easily and
rapidly reach any part of the
cells interior

Size of Bacteria
● Unit of measurement in bacteriology
is the micron (micrometre, µm)
● Bacteria of medical importance
● 0.2 – 1.5 µm in diameter
● 3 – 5 µm in length Reproduction
Shapes of Bacteria ● Prokaryotic cell division is binary
● Cocci – fission.
spherical/ oval ○ Single DNA molecule that first
shaped major replicates.
groups ○ Attaches each copy to a
● Bacilli – rod different part of the cell
shaped membrane.
● Vibrios – ○ Cell begins to pull apart.
comma ○ Following cytokinesis, there
shaped are then two cells of identical
● Spirilla – rigid genetic composition.
spiral forms
● Spirochetes – ARRANGEMENT OF BACTERIA: BACILLI
flexible spiral
forms
● Actinomycetes
– branching
filamentous
bacteria
● Mycoplasmas – lack cell wall
ANATOMY OF BACTERIAL CELL ○ pili
■ attachment
○ capsule
■ protection and
biofilms

Anatomy of A Bacterial Cell

● Outer layer – two components:
● Rigid cell wall
● Cytoplasmic (Cell/ Plasma)
membrane – present beneath cell
wall
Bacterial Taxonomy: Classification
● Cytoplasm – cytoplasmic inclusions,
● Orderly arrangement :
ribosomes, mesosomes and nucleus
Kingdom – Division – Class – Order –
● Additional structures – plasmid,
Family – Tribe – Genus – Species
slime layer, capsule, flagella, fimbriae
q Phylogenetic classification –
(pili), spores
represents a branching tree like
Structure of Bacteria
arrangement. One characteristic
● All cells have 3 main components:
being used for division at each
○ DNA (‘nucleoid”)
branch or level
■ genetic instructions
q Molecular or Genetic
● surrounding membrane
classification – based on the
(“cytoplasmic membrane”)
degree of genetic relatedness of
○ limits access to the cell’s
different organisms
interior
q Intraspecies classification – based
● cytoplasm, between the DNA and the
on biochemical properties
membrane
(biotypes), antigenic features
○ where all metabolic reactions
(serotypes), bacteriophage
occur
susceptibility (phage types)
○ especially protein synthesis,
which occurs on the
Bacterial Taxonomy: Nomenclature
ribosomes
● Two kinds of name are given to
● Bacteria also often have these
bacteria
features:
● Casual / common name – for
○ cell wall
local use, varies from country
■ resists osmotic
to country e.g. “typhoid
pressure
bacillus”
○ flagella
■ movement
 ● Scientific / International
Name – same all over world,
consists of two words (in
Italics)
e.g. Salmonella typhi,
Staphylococcus aureus
● Microscopy helps to Measure and
Observe the Bacteria
● Measurement
● Microorganisms are very
small
● Use metric system
● Metre (m) : standard unit
● Micrometre (µm) = 1 x10-6 m
● Nanometre (nm) = 1 x10-9 m
● Angstrom (Å) = 1 x10-10 m
Why we should be Stain Bacteria
• Bacteria have nearly the same
refractive index as water, therefore,
when they are observed under a
microscope they are opaque or
nearly invisible to the naked eye.
• Different types of staining methods
are used to make the cells and their
internal structures more visible
under the light microscope.
What is a Stain
● A stain is a substance that adheres to
a cell, giving the cell color.
● The presence of color gives the cells
significant contrast so are much
more visible.
● Different stains have different
affinities for different organisms, or
different parts of organisms
● They are used to differentiate
different types of organisms or to
view specific parts of organisms
Simple Staining
● Methylene blue, Basic fuchsin
● Provide the color contrast but
impart the same color to all the
organisms in a smear
● Loffler's ethylene blue: Sat. solution
of M. blue in alcohol - 30mlKoH,
0.01% in water -100mlDissolve the
dye in water, filter. For smear: stain
for 3’. For section: stain
● Simple Staining Easier to Perform
But has Limitations
● Simple easy to use; single staining
agent used; using basic and acid
dyes.
● Features of dyes: give coloring of
microorganisms; bind specifically to
various cell structures
Differential Stains
● Differential Stains use two or more
stains and allow the cells to be
categorized into various groups or
types.
● Both techniques allow the
observation of cell morphology, or
shape, but differential staining
usually provides more information
about the characteristics of the cell
wall (Thickness).
Gram staining
● Named after Hans Christian Gram,
differentiates between Gram-
positive purple and Gram-negative
pink stains and is used to identify
certain pathogens.
Gram staining - Principles
● Gram staining is used to determine
gram status to classify bacteria
broadly. It is based on the
composition of their cell wall. Gram
staining uses crystal violet to stain
cell walls, iodine as a mordant, and a
fuchsin or safranin counterstain to
mark all bacteria. Gram status is
important in medicine; the presence
or absence of a cell wall will change
the bacterium's susceptibility to
some antibiotics.
● Gram-positive bacteria stain dark
blue or violet. Their cell wall is
typically rich with peptidoglycan and
lacks the secondary membrane and
lipopolysaccharide layer found in
Gram-negative bacteria
● Gram-positive bacteria stain dark
blue or violet. Their cell wall is
typically rich with peptidoglycan and
lacks the secondary membrane and
lipopolysaccharide layer found in
Gram-negative bacteria
Gram Staining Steps
● Crystal violet acts as the
primary stain. Crystal violet
may also be used as a simple
stain because it dyes the cell
wall of any bacteria.
● Gram’s iodine acts as a
mordant (Helps to fix the
primary dye to the cell wall).
● Decolorizer is used next to
remove the primary stain
(crystal violet) from Gram
Negative bacteria (those with
LPS imbedded in their cell
walls). Decolorizer is
composed of an organic
solvent, such as, acetone or
ethanol or a combination of
both.)
● Finally, a counter stain
(Safranin), is applied to stain
those cells (Gram Negative)
that have lost the primary
stain as a result of
decolorization
● GRAM-POSITIVE BACTERIA
● GRAM-POSITIVE BACTERIA are
characterized by having as part of
their cell wall structure
peptidoglycan as well as
polysaccharides and/or teichoic
acids. The peptidoglycans which are
sometimes also called murein are
heteropolymers of glycan strands,
which are cross-linked through short
peptides.
What are Gram Negative Bacteria
● Gram-negative bacteria are
those bacteria that do not
retain crystal violet dye in the Gram
staining protocol. In a Gram stain
test, a counter
stain (commonly safranin) is added
after the crystal violet, coloring all
Gram-negative bacteria with a red or
pink color. The test itself is useful in
classifying two distinct types of
bacteria based on the structural
differences of their cell walls. On the
other hand, Gram-positive
bacteria will retain the crystal violet
dye when washed in a decolorizing
solution.
Gram negative bacteria
● On most Gram-stained preparations,
Gram-negative organisms will appear
red or pink because they are
counterstained. Due to presence of
higher lipid content, after alcohol-
treatment, the porosity of the cell
wall increases, hence the CVI
complex (Crystal violet -Iodine) can
pass through. Thus, the primary stain
is not retained.
Gram Negative Bacteria
● Also, in contrast to most Gram-
positive bacteria, Gram-negative
bacteria have only a few layers of
peptidoglycan and a secondary cell
membrane made primarily of
lipopolysaccharide
ACID FAST STAINING
● Acid-fast cells contain a large amount
of lipids and waxes in their cell walls
● primarily mycolic acid
 ● Acid fast bacteria are usually
members of the genus
Mycobacterium or Nocardia
● Therefore, this stain is important to
identify Mycobacterium or Nocardia
Ziehl-Neelsen stain
● Ziehl-Neelsen staining is used to stain
species of Mycobacterium
tuberculosis that do not stain with
the standard laboratory staining
procedures like Gram staining.
● The stains used are the red colored
Carbol fuchsin that stains the
bacteria and a counter stain like
Methylene blue or Malachite green.
Acid-Fast Organisms
● Primary stain binds cell wall mycolic
acids
● Intense decolorization does not
release primary stain from the cell
wall of AFB
● Color of AFB-based on primary stain
● Counterstain provides contrasting
background
AFB Staining Methods
● Zeihl Neelsen’s-hot stain
● Kinyoun’s-cold stain
● Modifications
BASIC PRINCIPLES OF SPECIMEN
COLLECTION
• Fundamentals
o Collect the specimen in the
acute phase of the infection
o Select the correct anatomic
site
o Package the specimen that
will maintain organism’s
viability
• Collection Procedures
o Blood Culture - highest
concentration occurs before
the fever spikes. Draw 2 sets
from right and left arms,
respectively >1 hr apart. >20
mlper set is collected in adults
and 5-10 ml per set in
children
• CSF & Body Fluids - collect >1mL by
needle aspiration and place in sterile,
screw-cap tube or anaerobic
transporter
• Gastrointestinal Tract
o Gastric Biopsy - rapid urease
test or culture for H. pylori
o Rectal Swab or Feces - for
isolation of E. coli & Vibrio
spp. Plated on enrichment or
selective enteric media
• Genital Tract
o Female - cervix, urethra,
vagina; Male - prostate,
urethra. For isolation of N.
gonorrhoeae
o Collected using swab
moistened with transport
medium. Plated in Thayer
Martin Medium
• Lesion/wound/abscess
o Superficial - swab along outer
edge using swab moistened
with transport medium
o Deep - aspirate with needle
and syringe and place in an
anaerobic transport system
• Lower Respiratory Tract
o Sputum - gargle with water,
cough deeply into sterile,
screw cap container (AFB,
Legionella and Nocardia)
• Upper Respiratory Tract
o Collected using swab
moistened with transport
medium
o Nasopharynx: Whooping
cough - B. pertussis
 o Pharynx (Throat): Strep Sputum Nasopharynx,
Throat - S. pyogenes; Throat
Epiglotitis - H. influenzae; 2. Anticoagulant: 0.025% Sodium
Oral gonorrhea – N. polyanethol sulfonate (SPS) and Heparin
gonorrhoeae 3. Holding or Transport Medium: Stuarts &
• Urine A ie’s (Dacron, Rayon or Calcium Alginate
o Clean Catch Mid Stream, swabs) or JEMBEC System
Catheter, Suprapubic aspirate • Specimen Receipt and Processing
o Placed directly in a sterile, • Criteria for Rejection
screw-cap container o Unmatched requisition and
o For diagnosis of lower UTI specimen label
(cystitis, urethritis) and upper o Specimen transported at
UTI (glomerulonephritis) improper temperature, fixative
• Labeling and Requisitions and media or has exceeded 2 hrs.
o Specimen Label: Patients o Specimen collected in improper
name, age and gender; areas.
identification number; o Quantity not sufficient (QNS),
patient’s room number or leaking or dried specimen.
location; requesting o Sputum with <25 WBC’s and
physician; culture site; date >10EC/lpf.
and time of collection • Macroscopy: Swab or Aspirate; Stool
o Requisition Form: Patient’s (consistency); presence of blood, clot
name, age and gender; and mucus; volume and turbidity
patient’s room number or • Specimen Preparation: Homogenization;
location; physician’s name; Concentration (centrifugation or
address; culture site; date filtration); Decontamination
and time of collection; clinical
diagnosis or patient history; MICROSCOPIC EXAMINATION OF INFECTED
name of individual MATERIALS
transcribing orders Introduction
• Confirm that the specimen is
PRESERVATION, STORAGE & TRANSPORT representative
• Specimen Storage • Identify agents using direct visual
Refrigerator Room Temp. Body detection using stains
temp. (4C) (22C) Temp. • To guide the workup of specimen for
(37C) culture.
Foreign Abscess, CSF Preparation of Samples
Devices lesion, Smear Specimen Fixation
wounds Wet Preps Fluids or a. Slide
Feces Body Fluids semisolids warmer at
Urine Genital Drop Clear, pus, 60C for >10
Samples swab rinse, mins.
 tissue b. Flooding
homogenate with 95%
Pellet Blood methanol
culture, for 1 min.
Dilute
Rolled Swab
material
(Pus)
Pull-apart Thick,
granular,
mucoid
Stains
• Simple stain: colors the forms and
shapes. E.g. Methylene Blue
• Differential stain: colors specific
components. E.g. Gram Stain, Acid Fast
Stain
• Antibody or Probe-mediated stain:
directed specifically at identification of
an organism.
• General Morphology
o Wright-Giemsa Sample with
cellular background
• Selected Morphology
o Methylene Blue: Metachromatic
granules
o Acid Fast Stains: Mycolic Acid
o Gram Stain: Cell Wall
o India Ink: Capsules
o Schaefer Fulton: Endospores
o Leifson: Flagella
• Genus (Species)-Specific Stains
o Antibody or DNA probe stains
§ Intracellular organism
§ Lacks cell wall
§ Not resolved by light
microscope

Reference: