CLINICAL BACTERIOLOGY | LAB

2ND SEMESTER | A.Y 2022-2023 | CLIBAC

LECTURER: MS.JUNALEN AND MS.JANINE

AFB STAINING

SPUTUM EXAMINATION AFB STAINS

SPUTUM CONTAINERS  Ziehl-Neelsen Method

-hot method  convention method
 Disposable -w/steaming or heating  brightfield microscope
 Clean and sterile  C.A.M reagents
MAIN METHODS  Kinyoun Method
 Unbreakable -cold method
 Unleadable -w/out steaming/heating
 Wide-mounted w/a screw cap or tightly fiiting cap -detergent
 Auramine-Rhodamine Method
LABELING OF SPUTUM CONTAINER
-fluorescent microscope
 -bacili appear yellow/orange
 The label should be on the body, not on the  Cooper’s Modification
lid of the cup and w/ complete label. (kung  Gabbett Modification
sala ang ging himo sang patient/s, IBALIK  Muller-Chermock Carbolfuchsin Tergitol Cold stain
SAILA!)  Pappenheims
 Baumgarten’s Method
COLLECTION TECHNIQUE  Fluorochrome Stain
 Prior collection NOTE: The heat/steam drives the stain into the cells.
 During collection Once the organisms have taken up the carbolfuchsin,
they are not easily decolorized by acid alcohol, and
SPUTUM SMEAR EXAMINATION RESULTS
hence are termed ACID-FAST.
 Doubtful
GOOD SMEAR=3cmx2cm
 Negative
 Positive PROPER SCANNING=Horizontal scanning and Vertical
scanning
THE SPUTUM MUST BE COLLECTED IN THE 1 EARLY ST

MORNING AND IT MUST BE COLLECTED W/IN 2 HOURS

AFTER WAKE UP TIME.

SPUTUM SPECIMEN STORAGE

 Examine as soon as possible

 Keep in the refrigerator if delay
 keep in cool, dark place, away from the sunlight
and protected from insects or rodents
 Never leave in room temp
 The storage period of sputum can be considered
about same as for milk BACTERIA WHICH AFB (+)

IMPORTANT POINTS TO REMEMBER DURING SPUTUM  Mycobacteria

COLLECTION  Nocardia

 Instruct the patient properly ACID FAST CELL WALL

 Sputum collection should be done in an open air
 Spot sputum collection  more than 60% of the cell well is LIPID
 3 sputum specimens should be sent immediately  major lipid component is MYCOLIC ACID(strong
 Avoid exposure to sunlight hydrophobic molecule that forms a lipid shell
around the organisms and affect its permeability)
IDEAL SPUTUM SPECIMEN FOR DIAGNOSIS
(MACROSCOPIC) PRINCIPLE OF AFB

 Yellowish  acid fastness is due to the high lipid content

 Muco-purulent (mycolic acid) in the cell wall of bacteria which
 Cheesy material resist decolorization with acid alcohol

(MICROSCOPIC) INTERPRETATION OF RESULTS (AFB)

 greater than 25WBC’s(LPO) or 5WBC’s(OIO)  Positive= at least one sputum smear is + for AFB
 presence of alveolar macrophages or dust cells  Negative=both sputum smears are – for AFB

(SPUTUM-deep lung tissue contains many dust cells and RESISTANCE

desquamated ciliated cells which is good specimen
 When protected from sunlight, they remain:
from direct smear examination
-in putrefying for weeks
-in dried sputum for 6 to 8 months
 Droplets of dried sputum in the air remains
infectious for 8 to 10 days.
 Organisms from culture are killed within 2
hours when exposed to sunlight; but in
sputum, they require 20 to 30 hours exposure
before they are killed.
 They are generally more resistant to chemical
disinfection than other vegetative organisms
(requires 24 hours of exposure in 5% phenol).
 Easily killed by moist heat (boiling for
10mins), pasteurization or steam under
pressure (autoclave).

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CLINICAL BACTERIOLOGY | LAB
2ND SEMESTER | A.Y 2022-2023 | CLIBAC
LECTURER: MS.JUNALEN AND MS.JANINE

 known as Tubercle bacilli

Mycobacterium  Microincinerator or Bunsen burner
 Mycolic acid-layer on its cell wall
tuberculosis  Inoculating loop
 Aerobic, rod shaped, thin and slightly curve  staining tray
(0.2-0.6um in diameter. 1-4um length)  Glass slide
 Slow grower in Lowenstein Jensen medium (5-  Lens paper
10% carbon dioxide)  microscope.
 ph 6.5-6.8
 no capsules, spores, flagella PROCEDURE OF N.S
 describe by Robert Koch in 1882
 Place a small drop of nigrosin close to one end of a
 causative agent of tuberculosis(oldest
clean slide.
documented communicable disease
 Place a slide against the drop of suspended
 Non-motile
organisms at a 45° angle and allow the drop to
 Colonies are nonpigmented and classically
spread along the edge of the applied slide.
described as being buff-colored
 Using aseptic technique, place a loopful of
 It transmits through INHALATION
inoculum from the bacterial
DIAGNOSIS culture in the drop of nigrosin and mix.
 Push the slide away from the drop of suspended
 Direct sputum smear microscopy organisms to form a thin
 Culture (most accurate test)  smear. Air-dry.
 Tuberculin skin test  Note: Do not heat fix the slide.
 DOTS(Directly Observed Treatment Short  Examine the slides under oil immersion.
Course)-internationally recommended
strategy for TB control by the WHO and STAINS OF N.S
IUATLD and recognized as highly efficient
 India Ink
and cost-effective strategy
 Nigrosin
 Eosin

NEGATIVE STAINING

 study of morphological shape, size, and

arrangement of the bacteria cells that is
difficult to stain
 it does not stain the BACTERIAL
CELLS(due to repulsion between the
negative charges of the stains and the
negatively charged bacterial wall, the dye
stains the background) directly, instead it
strains the background and strains the
actual glass slide and it also uses a
negatively charged dye

ADVANTAGES OF N.S

 use of only 1 stain and absence of heat fixation of

the sample.
 NS employs the use of an acidic stain and due to
repulsion between the (-) charges of the stain and
bacterial surface and the dye will not penetrate
the cell
 useful for determining cell size and arrangement.
 also, can be used to stain cells that are too
delicate to be heat fixed.

PRINCIPE OF N.S

 requires an acidic dye such as INDIA INK OR

NIGROSIN (means that the stain readily gives up a
hydrogen ion (proton) and the chromophore of the
dye becomes negatively charged)
 the surface of most bacterial cells is negatively
charged, the cell surface repels the stain. The
glass of the slide will stain, but the bacterial cells
will not. The bacteria will show up as clear spots
against a dark background.

REQUIREMENT OF N.S

 Nigrosin

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CLINICAL BACTERIOLOGY | LAB
2ND SEMESTER | A.Y 2022-2023 | CLIBAC
LECTURER: MS.JUNALEN AND MS.JANINE

CONTROL OF MICROORGANISM

 Sterilization-destruction of all forms of life.

including bacterial spores
-Physical Method
-Chemical Method
 Disinfection-process that eliminates a defined
scope of microorganisms including some
spores
-Physical M
-Chemical M (Disinfectants)(Antiseptic)
-Disinfectant-chemical agent applied on
inanimate objects or surfaces
-Antispetic-chemical agent applied to
skin/tissue(animate) for the purpose of
reducing the number of bacteria

FACTORS THAT INFLUENCE THE DEGREE OF KILLING

A. Types of Organisms
-Vary deeply in their ability to withstand
treatment
-Depends on their physical and chemical
components and how they protect themselves

DIFFERENT TYPES OF MICROBES

-Prions (most resistant)

 naked pieces of protein similar to virus but

without nucleic acid
 transmitted to humans via contaminated
medical

products, therapeutic devices, body fluids ,

and food products

-Bacterial spores

-Mycobacteria

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CLINICAL BACTERIOLOGY | LAB
2ND SEMESTER | A.Y 2022-2023 | CLIBAC
LECTURER: MS.JUNALEN AND MS.JANINE

-Non-lipid viruses

-Fungi

-Bacteria

-Lipid viruses

B. Number of Organisms
-Microbial load
 aka “bioburden”
 total number of microorganisms

Note: In general, higher numbers of organisms require

longer exposure time to the killing agents

C. Concentration of disinfecting agent

-amount of disinfectant needed to destroy PHYSICAL METHODS
microbes varies with different agents
-Proper concentrations ensure: HEAT FILTRATION RADIATION
1.the inactivation of target organisms Moist heat Air-HEPA filter Ionizing
2.promote safe and cost-effective practices Dry heat Liquid-thin Non-ionizing
D. Presence of organic material membrane filters
-Organic materials, such as blood, mucus, and Pasteurization
pus, affects killing activity by: Boiling
 inactivating the disinfecting agent HEAT
 preventing full contact between
object and agent -Moist heat
E. Nature of surface to be disinfected
 heat under steam pressure
-made up of biomaterials that exclude the use
 Agent used in autoclaves
of certain disinfection or sterilization methods
 Steam under 15 psi (or 1 ATM pressure)
because of possible damage to the instruments
 Temperature and time required: 121 °C for 15
F. Contact time
mins
-Too little contact time does not allow the
 Application: method of choice for heat stable
agent work properly
objects
-Example: Betadine requires 1-2 minutes
contact time -DRY HEAT
G. Temperature
-Disinfectants are generally used at room  Requires longer exposure times and increased
temperature (20-22°C) temperature than for moist heat
-↑ activity = ↑ temperature  Temperature and time required: above 160°C
-↓ activity = ↓ temperature for 3 hours
 Applications: commonly used to sterilize glass
H. pH wares and heat-stable substances (such as
-It is critical to make sure: oils)
 at what pH the agent is active
 and the pH of the material to be
exposed to the agent
I. Biofilms
-Considered as a community of bacteria or -PASTEURIZATION
other microorganisms
-To disinfect materials that may have a biofilm  Method that achieves disinfection but not
present: sterilization
 Concentration of the disinfectant 
 Contact time  Temperature and time required:
J. Compatibility of disinfectants –Batch : 63 °C for 30 mins
-common mistake is to believe that the use of
two or more disinfectants are better than one –Flash : 72 °C for 15 secs
•But one may inhibit/inactivate the action of
another or work against each other  Application: used mostly in the food
•Example: bleach and quaternary ammonium industry - eliminates food-borne
pathogens and organisms responsible for
METHODS OF STERILIZATION food spoilage

-Physical Methods -Boiling

-Chemical Methods  Method that achieves disinfection but not

sterilization
 Temperature and time required: 100 °C
for 10 to 15 minutes

FILTRATION

RADIATION

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CLINICAL BACTERIOLOGY | LAB
2ND SEMESTER | A.Y 2022-2023 | CLIBAC
LECTURER: MS.JUNALEN AND MS.JANINE
CHEMICAL METHODS
 Just as physical methods are used mainly to
achieve sterilization, chemical agents are used as
disinfectants
 Some chemical agents may be used to sterilize
devices that comes in contact with patients – these
are known as chemosterilizers
Actions of C.M
-Reaction of components of the cytoplasmic membrane
results in cell death
-Denaturation of cellular proteins (CHON) – disrupts
the metabolism of the cells
-Reacts to thiol (-sh) group of enzymes – disrupts the
amino acid
-Damage of RNA and DNA - inhibits replication
ANTISEPTIC
 Germ Theory of Disease-one of the most
important contributions by microbiologists to
the general welfare of the worldwide
population
 medical community gradually grew aware of
the problem of nosocomial infections and the
need to practice asepsis to prevent the
contamination of wounds, dressings, and
surgical instruments.
 germ theory of disease also contributed to the
development of antimicrobial
chemotherapeutics.
Pioneers for
 Ignatz Semmelweis(1816-1865)- ‘Father of
Handwashing” “Father of Hospital epidemiology”
-he demonstrated that routine handwashing can
prevent the spread of disease
 Joseph Lister (1827-1912)- “Father of antiseptic
surgery”
-introduced handwashing and the use of phenols
for surgical wound disinfection
GOALS OF HYGIENIC HANDWASHING
 To eliminate transient flora (Contracted from the
environment or from other people)
 To protect the skin with its resident flora
Routine handwashing in health care settings is performed
in the following situations:
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 Removing physical dirt
 Before and after routine patient contact
 After contact with infected patients or their
surroundings
 In high risk units such as ICU, and burn units
 Upon entering isolation units and leaving source
isolation units
 Before antiseptic procedures
Routine Handwashing Technique:
1.Treat the hands with antiseptic products.
2.Wash the lower third of the forearm.
3.Rinse with tap water.
4.Dry w/ disposable or sterile towel.
MEDIA PREP
PRINCIPLES OF BACTERIAL CULTURE
 To grow and isolate all bacteria in a
 To determine which of the bacteria
that grow are most likely causing
infection and which are likely
contaminants or colonizers.
 To obtain sufficient growth of
clinically relevant bacteria to allow
identification and characterization
CULTIVATION-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 CULTURE MEDIA
CULTURE MEDIA-Nutrient preparations that are used for
culturing microorganisms
-Serve as artificial environment providing the same
elements found in bacteria’s natural habitat
CLASSIFICATIONS OF CULTURE MEDIA
1.According to physical state
2.According to functional type
3.According to the method of dispensing
According to physical state
- Classification of bacterial culture media on the
basis of consistency
-has a 3 physical state (ara sa table)
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CLINICAL BACTERIOLOGY | LAB
2ND SEMESTER | A.Y 2022-2023 | CLIBAC
LECTURER: MS.JUNALEN AND MS.JANINE

no agar

contains
solidifying
agent (agar)

A.LIQUID MEDIA/BROTH C.SOLID MEDIA

Applications:  Contains 5 to 10% agar

 initially used to propagate large numbers of Applications

microbes
 Surface growth to observe colony
 for fermentation studies
appearance
 biochemical studies
 Pure culture isolation
Disadvantage:  Storage of culture
 Biochemical reactions
 not recommended for motility study
Examples
Examples
-NA -BAP -MacConkey -EMB -CHOC -LIA -TSI
-TSB
ACCORDING TO FUNCTIONAL TYPE
-BHI
 Classification of bacterial culture media on their
-Thioglycolate ability to support bacterial growth

-Urea broth Four (4) Functional Types:

Positive Growth 1.Nonselective media- Support the growth of most non-

fastidious microbes
-Turbidity – cloudiness of the medium
Examples: -Nutrient agar -Nutrient broth

2.Selective media- Support the growth of one type or

group of microbes but not another

- may contain inhibitory substances such as antimicrobials,

dyes, or alcohol

Examples:

 Mac-Conkey agar is selective for enteric gram (-)

bacilli and inhibits the growth of most gram (+)
bacteria
 Lowenstein Jensen medium – for isolation of M.
tuberculosis
B. SEMI-SOLID
MEDIA 3.Differential media- Allow grouping of microbes based on
different characteristics demonstrated on the medium
 Contains 0.5 to 1.5% agar
 Contains less concentration of “agar” to allow Examples: -Blood agar -MacConkey
for the free spread of microorganisms
Media can be differential and selective

 MacConkey agar inhibits gram-positive organisms

Applications and differentiates gram-negative bacilli on the
basis of lactose fermentation
 Fermentation studies
 Sheep blood agar is nonselective but
 Bacterial motility
differentiates organisms on the basis of
 Promotes anaerobic growth
hemolysis
Positive Growth
4.Enriched media-Contain growth enhancers that
 Motile organisms extend from the are added to nonselective agar to allow fastidious
stab line and produce turbidity or organisms to flourish.
cloudiness throughout the medium
Example: -Chocolate agar is an enriched medium
Example
ACCORDING TO THE METHODS OF DISPENSING
 SIM (Sulfide Indole Motility
 While in the liquefied state, solid media can be
poured into either a test tube or petri plates
 Agar melts 100 °C and solidifies at 45 °C

A. Agar butt (deep)

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CLINICAL BACTERIOLOGY | LAB
2ND SEMESTER | A.Y 2022-2023 | CLIBAC
LECTURER: MS.JUNALEN AND MS.JANINE

-hardened in an upright position STORAGEOFMEDIA

B. Agar slant 1. refrigerator to avoid dehydration

-hardened in a slanted position 2.agar plates are air tight

C. Agar butt-slant 3.tube media with fitted capped is store at room temp

-hardened in a semi-upright/slanted position 4. agar/plate should be warmed before use

D. Agar plate or plated medium HOWTOPREPARECULTUREMEDIUM

-poured into a petri dish 1. wear your mask,labgown and gloves

CULTURE MEDIA PREPARATION 2.disinfect the laboratory table top

 During preparation of culture media, it is 3.prepare the plates,flaskand tubes (sterile)

important to take note if the medium being
prepared is a tube or plated medium. 4. prepare the media powder, distilled water etc.
 In preparing tube medium, it is necessary to
5. follow aseptic technique from preparation to dispensing.
dispense first the medium before sterilizing while
for the preparation of plated medium, it is 6.autoclave the media and store properly
important to sterilize first before dispensing in
Petri dish

Important points to remember (tube media)

 Before autoclaving, cover each tube with

cotton
 Place tubes in large beaker and cover with
foil.

STERILIZATION AND DISINFECTION METHOD

 Moist heat (Autoclave)

Advantages:

-almost all media and anything that withstand 121

°C can be sterile in this way
-rapid and dependable

Disadvantages:

-tends to etch glass wares and leaves it damp

 Dry heat

Advantage:

-commonly used to sterilize glasswares

Disadvantages:

-not as effective as moist heat

-may cause charring of cotton and papers

 Bacteriologic filter

Advantage:

-some media cannot withstand heating, can be

sterilized by passing through a filter

Disadvantage:

-there will be a high chance that the filtrate will

not be sterile

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