Clinical Bacteriology Laboratory

○ For example, the

Lesson 1 urine sample will
Instrumentation in Bacteriology be centrifuged
before coiling or
smearing.
A Glasswares
Culture Tubes ● Cylindrical tube of clear
1. Plain Test Tubes glass, with screw cap at
2. Culture Tubes the top and rounded or
3. Durham Tube flat at the bottom.
4. Erlenmeyer Flask ● Thicker than the plain
tubes
5. Stirring Rod and Spatula
Uses:
6. Beaker
● For storing and maintaining
7. Plain Slide living adherent or
8. Hanging Drop Slide non-adherent cell cultures.
9. Coverslip or Cover Glass ● Proper use, opening and
closing the lids of culture
10. Petri Dish
tubes must be done to
B Equipment prevent contamination.
1. Autoclave
Most Common Uses of Culture
2. Hot Air Oven
Tubes
3. Incubator
4. Candle Jar Lysine Iron Triple Sugar
5. Anaerobic Jar Agar (M377) Iron Agar
(M021)
6. Water Bath
7. Alcohol Lamp
8. Caliper
9. Biosafety Cabinet
➔ Class I
➔ Class II
➔ Class III 1.Control 1.Control

C Safety Level of Biological Agents

*After *After
1. Biosafety Level 1
Incubation Incubation
2. Biosafety Level 2
3. Biosafety Level 3 2. Escherichia 2. Escherichia
4. Biosafety Level 4 coli ATCC coli ATCC
25922 25922

3. Citrobacter 3. Salmonella
Glassware freundii ATCC Typhi ATCC
Plain Test Tubes ● Cylindrical tube of clear 8090 6539
glass, usually open at one
end and rounded at the 4. Proteus 4. Proteus
other mirabilis ATCC vulgaris ARCC
Uses: 25933 13315
● Holds a small amount of
chemical/liquid 5. Salmonella 5. Citrobacter
● For transferring liquids. Typhimurium freundii
● To heat and hold reagents ATCC 14028 ATCC 8090
for observing chemical
reactions. 6. Salmonella
● Used for Centrifugation in Typhimurium
Microbiology Section ATCC 14028
Clinical Bacteriology Laboratory
storage, and other
liquid-handling processes.
7. Shigella
● In the microbiology
flexneri
laboratory or in the
ATCC 12022
bacteriology laboratory,
*This is used to differentiate various this is used more
microorganisms in the laboratory. commonly in preparing
*In every organism, there’s a agar medium for mixing
different reaction on each agar. the solutions for agar
medium.
Carbohydrate
● In microbiology, this is
fermentation
where we dissolve a
powder.
● For example, put the
Methyl red powder in the Erlenmeyer
Flask, then mix it. Biol it,
sterilize, and transfer.
Stirring Rod ● Stirring rods are clear
and Spatula straight glass that is used
for stirring liquids such as
Citric acid
reagents or chemicals
utilization
● Spatulas are mainly
porcelain or metal with
spoon-like features
Hydrogen ○ Mainly for mixing
sulfide either powder skin
production or liquid
sometimes.
● Dissolve the powder using
stirring rod.
Durham Tubes ● Smaller test tubes inserted
Beaker ● Cylindrical container with
upside down inside
a flat bottom.
another test tube
● Most also have a small
Use:
spout (or "beak") to aid
● To detect production of
pouring, as shown in the
gas by microorganisms
picture.
such as in fermentation
Use:
reaction.
● It is used for heating liquid
*** Gas Production - if there is a
and for preparing reagent
space in the upper area of the
solution.
tube.
*If red, negative in carbohydrate
fermentation. (It cannot ferment)
*If yellow, positive
*If it evaporates, the
microorganism can produce gas

Erlenmeyer ● Aka. conical flask or a

Flask titration flask.
● It features a flat bottom, a
conical body, and a
cylindrical neck.
Use:
● To contain liquids and for
mixing, heating, cooling,
incubation, filtration,
Clinical Bacteriology Laboratory
Plain Slide ● A microscope slide is a thin DIAGRAMMATIC
flat piece of glass, typically PRESENTATION HOW TO USE
75 by 26 mm (3 by 1 HANGING DROP SLIDE
inches) and about 1mm
thick, used to hold objects
for examination under a
microscope.
Uses:
● Hold objects for
examination under a
microscope.
● Used for staining bacterial
smears for observation
under a microscope.
***Frosted edge - utilized in labeling These steps are followed in order to
specimens. (Use pencil to label) observe better motility afterwards.
***If Plain Slide, use micropore to
label the slide ● When viewing any slide
***Diamond pen knife - carve out Coverslip or with a microscope, this is a
the label. Cover Glass small square thin glass.
(Usually placed over a
specimen.)
Use:
● To protect the microscope
and prevent the slide from
*Sputum specimen drying out when it is
*For sputum, use the examined.
inoculating loop to coil
Hanging Drop ● Looks like a microscope Petri Dish ● A flat, shallow transparent
Slide slide with almost the same dish made of glass or
thinnest but only with plastic with a suitable lid.
concavity on the center. ● Generally, a plastic petri
Use: dish is disposable.
● For hanging drop Use:
preparation to observe ● For culturing cells by
motility of bacteria. providing storage space
and preventing them from
Why did hanging drop slides used getting contaminated.
to observe motility? ● Since the dish is also
● The concavity presents a transparent, it is also easy
space for microscopic to observe the growth
cultures or bacterial stages of microorganisms.
cultures to be placed ● For growing bacteria
inside the concavity.
● Through this, there is a 2 TYPES of Petri Dish
space or area for the 1. Disposable - 1 time use
bacteria to freely move so 2. Glass
that we can properly
observe their motility. Note: Petri Dish is transparent - for
us to easily take note the colony (if
nag-grow na). To avoid
contamination.

For example, 24-hour in the

incubator, check if there is a
colony
Clinical Bacteriology Laboratory
How do we store culture media
when inside a petri dish? Right side
up or upside down?
- Upside down. To prevent
contamination and
condensation.

Inoculation ● Inoculation loop is a simple

Loop or tool used to take and
Needle transfer a small sample of
a microorganism culture Hot Air Oven ● For sterilization of
from a culture of glassware, such as test
microorganisms by tubes, pipettes and petri
smearing. dishes.
● Inoculation needles are *Autoclave and Hot Air Oven are
used to transfer inoculum both for sterilization
by stabbing Incubator ● An insulated and enclosed
Use: device that provides an
● For transferring inoculum optimal condition of
by stabbing using the temperature, humidity,
pointed end of the needle. and other environmental
● Get a sample from serum conditions required for the
or colonies growth of organisms.
● Colonies - if the petri dish is ● Where microorganisms
incubated, then may grow
nag-grow na bacteria
● Needle - for stabbing Temperature: 37°C or Body
(culture tubes) Temperature
Equipment ● The incubator mimics the
normal body temperature
Autoclave ● A pressurized chamber
wherein any of these
used for the process of
microorganisms can grow
sterilization and disinfection
or thrive.
by combining three
● Some microorganisms
factors:
would need a different
1. time
temperature but this is the
2. pressure
common temperature
3. steam
used.
(TPS)
Use:
Candle Jar ● A container into which a lit
● To sterilize liquid substances
candle is introduced
such as prepared media
before sealing the
and saline (diluents)
container's airtight lid.
solutions, and also to
● The candle's flame burns
sterilize glassware.
until extinguished by
oxygen deprivation, which
creates a carbon
dioxide-rich, oxygen-poor
atmosphere in the jar.
Clinical Bacteriology Laboratory
● (Used for ANAEROBIC ● The residual oxygen left
CULTURES.) - requires the behind is converted to
absence of Oxygen water using Spongy
palladium or platinum
Not all microorganisms can multiply catalyst.
into a certain environment. ● The catalyst acts as a
catalyzing agent causing
Process - put the culture tubes and a slow combination of
petri dish in the jar, then after that, hydrogen and oxygen to
put the candle with flame. Then, form water.
close tightly. ● Reduced methylene blue
is generally used an
***Low oxygen, increase CO2 indicator (mixture of
High oxygen, decrease CO2 NaOH, methylene blue,
and glucose).
Capnophiles organism - requires ● It becomes colorless
CO2-rich environment anaerobically but regains
● Principle of evacuation blue color on exposure to
Anaerobic Jar and replacement, where oxygen.
the air inside the chamber
is evacuated and Notes:
replaced with a mixture of ● If it becomes colorless, the
gasses consisting of environment is anaerobic
OXYGEN & HYDROGEN to ● If it remains blue, it is
form water. aerobic.
Water Bath ● Used for chemical
INDICATORS: (CONTROL) reactions that require a
- Methylene blue. controlled environment at
a constant temperature.
INTERPRETATION: ● Temperature: 35°C - 37°C
● Colorless - The area inside
is anaerobic; no oxygen
● Blue - There is a presence Alcohol Lamp ● Used for heating
of oxygen. inoculation loops and used
while transferring culture
Detailed Description of Anaerobic samples.
Jar ● Sterilization of inoculating
loop
Caliper ● To measure the zone of
inhibition
● Ruler in microbiology
● Unit of measurement:
millimeters (mm)
● For culture and sensitivity
testing.

● It is practically impossible
to evacuate all the air so
some amount of oxygen
will still be left behind. CULTURE AND SENSITIVITY TESTING
Clinical Bacteriology Laboratory
● When you want to
(Open system) (Open System) Cabinet
measure the inhibition
(Closed
zone size, you should
System)
measure the zone
diameter to the edge of
the growth
● An antibiotic disk is placed
in the Mueller-Hinton agar
and incubates the
Open fronted Also known as Provides the
organism they want to see.
type with the laminar highest level of
● If a zone of inhibition is
negative flow cabinet. safety to the
observed, it means or it
pressure. Allows worker.
indicates that the
room air to Most
antibiotic is effective
enter the commonly A closed
against that certain
cabinet,
bacteria. used BSC in the system. The air
circulate and
clinical coming in and
filtered out with
Notes: microbiology out of the
a High
Susceptible - Antibiotic is puwede laboratory. cabinet passes
Efficiency
pa through a HEPA
Particulate air
Resistant - Antibiotic is hindi na filter as well
Absorbing filter Utilized when
puwede.
handling and rubber
microorganisms gloves are
We need to determine bacteria for
under biosafety attached and
us to determine antibiotics we
should administer to our patient. level 2. sealed to the
cabinet.
If mag-da-drop ng antibiotics, Type A: 30% of
dapat hindi dikit-dikit. May space the air is Microorganisms
dapat beh! - Zone of inhibition exhausted and we handle here
70% is are biosafety
level 4
recirculated
Biosafety ● The primary purpose of microorganisms
Cabinet biosafety cabinets is to .
Type B: 70% is
protect the laboratory
exhausted and *Attach by
personnel and the
30% would be rubber gloves
environment from the
recirculated *Expensive shit
pathogenic microorganism
inside the area.
as aerosols might be
formed during the
processing of such High-Efficiency Particulate Air Filter
microorganisms. ● Functions to remove organisms larger than 0.3
● (For example, during micra
centrifugation, there is an ● Generally used in Class II and Class III
aerosol formation. So it is
best to use a biosafety
cabinet to protect SAFETY LEVEL OF BIOLOGICAL AGENTS
ourselves.)
BIOSAFETY LEVEL 1
● No known potential for infecting health people
● Non-pathogenic; hindi ka mamamatay
● Non-pathogenic microorganisms
● Ex. Bacillus subtilis, Naegleria gruberi

BIOSAFETY LEVEL 2
Class I Cabinet Class II Cabinet Class III
Clinical Bacteriology Laboratory
● Common agents of infectious diseases
acquired by ingestion, percutaneous, exposure
of mucous membranes
● Ex. Salmonella, Shigella, HIV (causative agent
of AIDS), Bacillus anthracis, Yersinia pestis
(causative agent of Bubonic plague)
● COVID-19 Throat Swab or Nasopharyngeal
Swab

BIOSAFETY LEVEL 3
● Potential agents for aerosol transmission
● Agents can be acquired via inhalation
● Ex. Mycobacterium tuberculosis, Francisella
tularensis, Brucella spp.
● COVID-19 falls under here when it is viral. Lesson 2
Control of Bacterial Growth
BIOSAFETY LEVEL 4
● Agents responsible for life-threatening STERILIZATION
infections ● Removal of all forms of life including bacterial
○ Generally, if microorganisms handled spores
in the laboratory are exposed outside,
there will be a worldwide pandemic or DISINFECTION
an epidemic if controlled. ● Removal, inhibition, or killing of microorganisms
● Maximum containment and decontamination including potential pathogens by using
of all personnel chemical agents usually on inanimate objects;
● Aerosol Transmission is possible does not remove spores
● Ex. Arbovirus, Arenavirus, Filovirus, and smallpox ● Except bacterial spores
virus ● Alcohol and safeguard (99.9%)

Bacterial Spores - the protective barrier of the bacterial

cell. Protects the bacteria. Thick wall.

Sterilization Vs Disinfection
STERILIZATION ● Total destruction of all
microorganisms (whether or
not pathogenic) and their
spores, usually through the
use of drastic methods
● Uses more robust methods
such as high heat or
chemicals
● Uses chemicals, heat, high
pressure, filtration, and
irradiation
● A method that gives
extreme cleanliness
● Destroys both living
organisms and their resistant
structures
● Used in the
decontamination of food,
medicine and surgical
instruments

● Removal of all forms of life

including bacterial spores
Clinical Bacteriology Laboratory
● Downside of sterilization is ● Are for an inanimate object
you need to use equipment (non-living things)
because it is not as handy
or as easy as using chemical Antiseptic agents
agents. However, it ● Are for living tissues (hands,
guarantees 100% site for venipuncture, etc.)
elimination of any form of ● Also a 75% isopropyl alcohol
life.
DISINFECTION ● Cleaning of something using WHAT ARE SPORES?
chemicals that kill bacteria Spores
and other very small living ● In microbiology, these are
things that cause disease the dormant form of the
● Uses moderately effective bacteria because not all
methods bacteria are capable
● Uses detergents, hydrogen of forming spores, only a
peroxide, alcohols, bleach, few bacteria are able to
halogens like chlorine, form spores.
phenolic disinfectants,
heavy metals, heating and Why do bacteria create
pasteurization spores?
● A method that gives an ● Spores are highly resistant
adequate cleanliness substances against
● Destroys only the living destruction. They are built
organisms very specifically in order to
● Mostly used to withstand a lot of
decontaminate surfaces environmental factors.
and air.
● Removal, inhibition, or killing IF YOU ARE WONDERING
of microorganisms including WHY MOST
potential pathogens by ANTIBACTERIAL SOAPS,
using chemical agents ALCOHOL, OR
usually on inanimate DISINFECTANTS ARE
objects; does not remove CAPABLE OF
spores KILLING 99.99% OF BACTERIA
● Mostly use chemical agents ONLY
that would allow killing NOT 100%
whatever microorganism ● Because disinfectants such
that is usually on an as bleach (Lysol, isopropyl
inanimate object (working alcohol, etc.) are unable to
bench, chairs, non-living kill off or remove spores,
things) they don’t have the
● Main thing about mechanism in order to
DISINFECTION is that it does remove or kill off spores,
not remove spores. which is why they kill 99.99%
of germs killing action only.
70% or 75% isopropyl alcohol
● The most common
disinfectant
● Example of disinfectant but DRY HEAT MOIST HEAT
could also be an antiseptic STERILIZATION STERILIZATION
DEFINITION Dry heat sterilization Moist heat
DIFFERENCE BETWEEN
is a method of sterilization is a
DISINFECTANT
sterilization that uses method of
AND ANTISEPTIC:
high temperature sterilization that
Disinfectant
under dry uses high
conditions temperature
Clinical Bacteriology Laboratory
and pressure needs to reach a ● The process is
generated by higher faster than dry
water temperature that heat
TYPE OF HEAT DRY HEAT MOIST HEAT requires also a sterilization and
higher amount of you would
time when it need a shorter
DRY DRY CONDITIONS WET
comes to the period of time
CONDITIONS/ CONDITIONS
operation of a because moist
WET
very specific heat or steam
CONDITIONS
sterilizer. is able to
MAIN METHODS FLAMING, Steam under
spread more
INCINERATION, HOT pressure. stream
Type of heat: evenly.
AIR OVEN, ETC. at atmospheric
● Dry heat
pressure
Type of heat:
(non-pressurized
Dry conditions or Wet ● Moist heat
). boiling.
conditions? Dry conditions or Wet
pasteurization,
● Dry conditions conditions?
etc.
● Wet conditions
KILLING EFFECT Protein Mainly by
Main methods:
denaturation, denaturation
● Flamming, Main methods:
oxidative damage and
incineration, hot ● Steam under
and toxic effect of coagulation of
air oven, etc. pressure,
elevated level of proteins.
stream at
electrolytes.
Killing effect: atmospheric
MOST EFFECTIVE HOT AIR OVEN AUTOCLAVE
● Protein pressure
METHOD ● Mas ● Mas
denaturation, (non-pressurize
mababa mataas
oxidative d), boiling,
ang ang
damage, and pasteurization,
temperatur temper
toxic effect of etc.
e (160 - 180 ature
elevated level of
degree (121
electrolytes Killing effect:
Celsius degree
● Mainly by
● More time s
Most effective method: denaturation
(2 hours) Celsius)
● Hot air oven and
● Lesser
coagulation of
time (15
Time taken for sterilization: proteins
minutes
● More time
)
Most effective method:
TIME TAKEN FOR MORE TIME LESS TIME ● Autoclave
STERILIZATION
Time taken for
sterilization:
DIFFERENT METHODS FOR STERILIZATION ● Less time
Dry Heat Sterilization Moist Heat Sterilization
● Dry heat ● Moist heat
sterilization is a sterilization is a
method of method of MOIST HEAT PROCEDURES
sterilization that sterilization that AUTOCLAVE ● Based on moist heat
uses high uses high sterilization or steam
temperature temperature under pressure.
under dry and pressure ● Most effective method
conditions generated by for sterilization
● Use the usual heat water steam. ● Biological Indicator:
● The process is ● Use of water in Bacillus
longer than moist order to create stearothermophilus
heat since it moisture.
Clinical Bacteriology Laboratory
● for media, liquids, ● It is used to test the
instrument: performance of the
● for infectious medical equipment or the
waste: particular procedure or
method of sterilization
● A very important piece that you are trying to
of equipment in perform
bacteriology because it ● With the use of biological
is considered the most indicator, it will tell us if
effective method of the autoclave is working
sterilization because of properly or if not then
how it operates. there may be a
calibrations/fixing/troubl
3 factors are utilized in the use of eshooting that needs to
autoclave: be done for the
1. Time autoclave to work
2. Pressure properly.
3. Steam
Performed at:
Explanation: ● Temperature of 121
● Those 3 factors work degrees Celsius for 15
together in order to kill minutes at 15 psi (pounds
off the organism per square inch)
because with the ● Mas mataas ‘yung
pressure exerted and temperature
with the steam created ● psi - pounds per square
by water or from the inch
water, and then the
pressure exerted that all Infectious Medical Waste
of those things ● 132 degrees Celsius
combined in order to ● 30-60 minutes
give the best result when ● 15 psi
it comes to the
penetration of the heat Principle of autoclave:
within the bacteria. ● Steam under pressure.

Biological indicator: ***Don’t forget to refill the water

● Spores of Bacillus in an AUTOCLAVE***
stearothermophilus
● Proper writing - Genus VERTICAL AUTOCLAVE
(capital letter); Specie
Type (small letter)

What is a biological indicator?

● Assess the performance
of autoclave (if kaya ● Common autoclave that
niyang pumatay ng is present in the
highly resistant laboratory also the kind
microorganism) of autoclave that they
● It indicates life have in normal medical
● The purpose of having a centers
biological indicator is it ● It can hold more
should be a living materials and also
organism, especially deeper and the basket
when the case is about or the chamber can
bacteria contain more equipment
Clinical Bacteriology Laboratory
sterilizing very specific
AUTOCLAVE TAPE type of substances or
materials

Used for
● High protein media (ex.
Lowenstein Jensen)

*therefore, we have culture

● It looks like normal tape media that are specified with the
but has a yellow to white higher amount of a protein
diagonal lines at the example is:
beginning
Lowenstein Jensen- is a culture
Purpose of autoclave tape: media that is specifically fortified
● It looks like normal tape in high protein content for the
but has a yellow to white cultivation of mycobacterium
diagonal lines at the tuberculosis (a big bacteria that
beginning. needs a higher amount of
● It is used to stick the protein) If you expose them in a
materials that you would temperature higher than 90, they
load into the autoclave can start to denature or can
and autoclave it the undergo the process of
usual way and after if denaturation Denaturation
you need to observe for
a transition in color. Performed at:
● 70-80 degrees celsius for
What are we expecting to 2 hours for 3 consecutive
observe? days
● The diagonal lines would turn
black (which indicates that the PASTEURIZATION ● Used to destroy possible
temperature of 121 degrees pathogens in milk and
Celsius has been reached) beverages
● Used to lower bacterial
BOILING ● Destroys vegetative count by 95-99%
bacteria but not all
spores or viruses ● 2 types:
● Performed at: 100C for a. FLASH – 74 degrees Celsius for
10 to 15 mins 3-5 seconds (MABILIS)
TYNDALLIZATION (Fractional Sterilization) b. BATCH – 62 degrees Celsius
● Used to sterilize media for 30 minutes
containing milk or serum
● Used to kill heat-resistant at 118 Degrees Celsius
endospores ● Proteins begin to
● Performed at: flowing denature
steam for 30 mins using
temperature of 100C for Low-Temp Pasteurization
3 days ● More enzymes and
● Needs to perform proteins remain intact
multiple times on when milk is heated
different days below these
INSPISSATION ● Involves thickening temperatures
through evaporation
● Useful when you are High-Temp Pasteurization
using a very specific or if (Danger)
you are going to be ● 161 degrees Celsius
Clinical Bacteriology Laboratory
● Kills enzymes, much of and infected laboratory
the healthy animal.
microorganisms, and ● Recommended for
denatures the proteins. eliminating prions
This milk is difficult to ● Hazardous materials are
digest burned to ashes at 870 to
980C.
Ultra Pasteurization (Danger) ● If you want to maintain the
● 280 degrees Celsius, infection as much as
usually for 2 to 3 seconds possible without any risk of
time contamination, you need
● Kills potentially harmful to perform incineration
bacteria in the milk, but
also damages all of the Prions
vitamins, minerals, and ● Not necessarily infectious
other nutrients originally but it does infect a specific
contained in the milk host, it is actually an
abnormal protein that if it
DRY HEAT PROCEDURES infects or it is able to enter
OVEN ● Used to sterilize glassware, a host or a specific living
instrument which are not organism, it can affect the
permeable to water structure of the proteins of
● Kills microbial life by that living organism as well
oxidation
Example: mad cow disease
Biological Indicator:
● Bacillus subtilis variant Mad cow disease
niger ● Affects animals,
particularly cow and is
Performed at: called the
● 160-180C for 1-2 hours Creutzfeldt-Jakob disease
FLAMING ● Used for aseptic technique for a human
to prevent contamination ● Affects brain the most
of bacteria.
● Alcohol lamp can be used
in heating the inoculating PRECAUTIONS IN USING DRY HEAT OVEN
loop or needle Following precautions should be observed when using
● Used for inoculating to hot air oven:
prevent contamination of 1. The oven must not be overloaded space must
bacteria be left for circulation of air through the articles.
● Using a bunsen burner or 2. It must first be loaded and then heated up to
an alcohol lamp. sterilization temperature in the courses for 1 to 2
Specifically used for hours.
inoculation or inoculating 3. Holding period of one hour at 160 degrees
needles or inoculating Celsius is required for sterilization. It means one
loops (to sterilize them) hour after attaining degrees Celsius.
● Technique is you need to
hold the inoculating loop OTHER METHOD OF STERILIZATION
under or within the flame if FILTRATION
exposing it to the flame ● A process that separates solid particles from a
make sure that it turns red liquid or gas by passing the mixture through a
hot (‘wag ipadaan daan filter
lang) to make sure that it ● Method of choice for sterilization of antibiotic
has been properly sterilized solutions, toxic chemicals, vaccines and
INCINERATION ● Most common method of carbohydrates.
treating infectious waste ● Air Filtration
Clinical Bacteriology Laboratory
How to prepare 1 is to 10 concentration?
Examples: ● Combination of 1 part bleach and 9 parts of
● HEPA filter (High Efficiency Particulate Air water
● Filter)
○ Designed to remove organisms larger
than 0.3 um Lesson 3
○ Most bacterias are around 0.3 um SPECIMEN COLLECTION,
TRANSPORT, and PROCESSING
MOST COMMON DISINFECTANTS THAT WE HAVE IN THE
LABORATORY MAIN GOAL - AVOID FALSE POSITVE DUE TO
CONTAMINATION

1. 70-75% isopropyl alcohol RESPIRATORY TRACT

Throat swab ● Swab in the posterior
Why is it only 70-75% and not 100%? pharynx, tonsils and
● Because it is specified or it is inflamed areas.
concentrated at a specific ● Kunin mo ang sample
concentration of 75% because we sa inflamed area.
actually need the addition of the ● After swabing, put it
diluent (sterile water/distilled water) on the NSS (Normal
because it helps the isopropyl alcohol Saline Solution)
do its job in killing the organisms.
Used for:
Characteristic/s of 100% alcohol: ● Streptococcus
● They can easily evaporate pyogenes
● They are volatile ● Candida albicans
● Haemophilus
TAKE NOTE!!!! influenzae
● more water in the isopropyl alcohol Nasal Swab ● Moistened swab in
increases the contact time, therefore the nose.
nagagawa ng mas maayos ni alcohol ● Sa ilong lang
yong job to kill microorganisms
● Aside from that, water can actually Used for:
insert hydrophilic or hydrolytic bonds ● Streptococcus aureus
that couldMfurther the killing of the Nasopharyngeal ● Flexible swab inserted
organisms swab through the nose and
● Water also acts as a catalyst to into posterior
jump-start the reaction they did in nasopharynx and
order for the alcohol to do its job in then rotated for 5
killing off the organism secs.
● If you are going to disinfect something, ● Pinapasok sa loob ng
make sure that the alcohol has ilong
cleaning contact within the surface
● Don’t expect for the organism to die Used for:
right after you drop/spread the ● Neisseria meningitidis
alcohol/disinfectant. You have to give and Bordetella
it time to do its job. pertusis

2. Sodium hypochlorite (bleach)

● Used for a lot of time when you are disinfecting
certain materials/instances including the
bacteriology section

Concentration
● One is to ten concentration
Clinical Bacteriology Laboratory
Sputum ● Most common URINE ● Used to diagnose
specimen for upper or lower UTI
respiratory tract ● Preferred type of
● Collected in the urine specimen:
morning (More Midstream Clean
Concentrated) Catch
● 3 Specimens needed ● First morning urine
● 2 Specimens ● Catheterized urine
needed** based on may be collected
SOP by inserting a
● Rinse or gargle w/ catheter into the
water then instruct bladder, then allow
patient to deep passage of first
cough and 15ml, before
expectorate into collecting
sterile container remainder
● Bakit water? If the ● Upper UTI - above
patient is gargled the bladder
with a mouthwash, (kidney;
the bacteria will be pyelonephritis)
eliminated. Our goal ● Lower UTI - below
is to grow a bacteria. the bladder
Bronchial ● Organisms are seen (bladder)
washings and on a gram stain, but ● Patient in ICU - use
bronchoalveolar no growth occurs catheterized urine.
lavage (BAL) aerobically and Pagka-insert ng
anaerobically catheter, discard
● May be caused by the 1st 15 mL. Then
inhibition by collect the sample.
antimicrobial therapy
Example:
OTHER SPECIMENS ● There’s a urinalysis
GASTROINTESTINAL ● Stool, gastric and Culture and
TRACT secretions and Sensitivity Testing.
rectal swab The patient
● Used to diagnose submitted 1 urine
the cause of sample. Perform
gastroenteritis first the C&S, to
● 3 specimens, one avoid
per day for 3 days contamination.
should be ALWAYS CHECK IF
collected to rule THERE IS A C&S.
out infection. Then, ibalik sa CM
● When collecting to urinalysis.
stool, it should be
directly
● Rectal Swab - insert
it into the rectal as
deep as you can.
Dapat ‘yung
cotton may tae.
Clinical Bacteriology Laboratory
BLOOD ● Used to diagnose ● Aerobic then
septicemia and anaerobic. (the
bacteremia needle has a
● Collection of Blood vacuum, thus,
CUlture and there’s a presence
Routine of oxygen. Since
Venipuncture is mauuna ‘yumg
different. aerobic (okay lang
● Blood Culture - na malagyan siya
Alcohol - iodine ng oxygen galing
(eliminate normal sa vacuum), then
floras) followed by
● Routine anaerobic.
Venipuncture - ● Adult: 5-10 ml/
alcohol bottle

4S Anticoagulants PROCEDURE:
1. SPS A. Collect directly into the
2. Sodium citrate bottles using a butterfly
3. Sucrose needle.
4. Sodium I. Fill the aerobic first.
amylosulfate II. Avoid backflow by
keeping the culture
bottle lower than
the collection site.
III. Fill aerobic and
anaerobic bottles
with 5-10 ml of
blood.
Aerobic - green cap IV. Fill pediatric bottles
Pediatric - yellow cap with 1-3 ml of
Anaerobic - red cap blood.

PROCEDURE:
1. Clean the site.
a. Clean with antiseptic-
70% isopropyl alc. For a
minimum of 30 seconds.
b. Use a tincture of iodine
for 30 sec or a
povidone-iodine swabstick
for 60 seconds to cleanse
the site.*
c. Allow the site to air-dry.

2. Remove the
protective flip-flop
covering the
rubber septum and
clean the tops of
the bottle with 70%
alcohol or iodine.
● Use buttlerfly
needle
Clinical Bacteriology Laboratory
CEREBROSPINAL ● Collected through WOUND AND Exogenous wound
FLUID lumbar puncture or ABSCESSES infection:
spinal tap ● Animal/human
● Don’t Discard!!! bites, burns,
● Colorless traumatic wounds
● Used to diagnose (gunshot, stabbing)
meningitis (can be
bacterial, fungal, or Endogenous wound
viral) infection:
● CSF tubes (3 tubes ● Dental infection,
are collected) septic arthritis
● 1st tube: ● Needle and syringe
Chemistry/ aspirate
Serology
● 2nd tube: GENERAL GUIDELINES FOR SPECIMEN COLLECTION
Microbiolo ● Should be collected at the correct
gy for anatomic site
culture ● Should be collected in the acute phase of
● 3rd tube: illness (w/in 2-3days for viral infection)
Hematolog ● Should be collected before the initiation of
y for cell antibiotic therapy (perform before the
count antibitic therapy)
● Timing of collection should be considered
Bakit second tube ay ● Should be collected under sterile and
microbiology? aseptic conditions
● Thru lumbar ● The quantity of material must be adequate
puncture or spinal (the more, the better)
tap, para tayong ● The specimen must be taken to the
magsasalok. May laboratory and examined promptly (2 hours)
skin na (habang tumatagal ‘yung specimen, the
madadaanan microogranism will multiply and can lead to
muna ‘yung falsely increase result)
needle. ● Must be labeled accurately with patient
information, date, and the specific
What if isa lang ‘yung tube? anatomic site (If sa right arm kinuhanan,
● Microbiology first. indicate on the culture tube, RIGHT ARM)
● Delayed ● Should have a direct microscopic exam
processing: (Gram staining and Acid-Fast Staining)
○ Room ● Always talk to the requesting physician
Temp before discarding unacceptable specimens
(<30mins) ● If cannot be processed ASAP, they must be
○ 37C stored
(>30mins) ○ Refrigerator temp (4C)
GENITAL TRACT ● Used to determine ○ Ambient rom temp (22C)
the cause of ○ Body temp (37C)
vaginitis, urethritis ○ Freezer temp ( -20 degrees Celsius
and cervicitis, as and -70 degrees Celsius)
well as childbirth ● Place specimen in containers designed to
infections maintain viability of organism and avoid
● Used to diagnose hazard that result from leakage (DO NOT PUT
sexually-transmitted OR RECEIVE IF THE SPECIMEN IN THE SALAD
disease CAP)

SPECIMEN TRANSPORT AND PROCESSING

Clinical Bacteriology Laboratory
● The specimen transport time exceeds 2
hours post collection and unpreserved
specimen
● The specimen was received in a fixative
(formalin) which can kill any
microorganisms present (In Bacteriology
we don’t use fixative; formalin)
● The specimen has been received for
anaerobic culture from a site known to
have anaerobes as part of the normal
flora (vagina and mouth)
1. STUART’S SYSTEM/MEDIUM ● The specimen is dried up
○ Respiratory Tract (Nose, ● Processing the specimens that would
Nasopharengeal, etc.) produce information of questionable
COMPONENTS OF STUART’S MEDIUM medical value
Methylene Blue Oxidation – reduction
indicator Medium appears
Lesson 4
colorless when reduced and
Examination of Living Organism
blue when oxidized
Thioglycollate Is added to create a ● An important aspect of bacteriology because
reduced environment, we are examining living organisms under here
improving the recovery of depends on what state they are in.
anaerobic organism ● For example, in the activity, we examined living
Sodium To maintain the pH of the organisms, particularly in a liquid sample.
glycerophosphate medium

2. AMIES Wet Mount Hanging Drop Method

○ Modified Stuart’s medium
○ Contains charcoal to prolong the ● The simplest way ● Similar to the
viability of pathogenic organism or method of wet mount we
3. CARY-BLAIR MEDIUM observing living are using liquid
○ Stool specimens (enteric pathogens) microorganisms samples here
4. CALCIUM ALGINATE/DACRON SWABS under a but we are using
○ Preferred over cotton tipped swabs microscope. a very specific
○ Microbiology - Dacron Swab ● Utilized in other slide.
○ Virus - Cotton Swab sections as well. ● Depression slide
● Most common or the hanging
method utilized drop slide is
in the laboratory utilized.
to observe living ● There is a
organisms or concavity on
observe the middle of
CRITERIA FOR SPECIMEN REJECTION elements within the slide. It is
a specific where the
● The information on the label does not
sample. sample will
much the information on the request
● It is preferred hang.
form (If not the same, reject.)(You can
when it comes ● Instead of
call the nurse if in-patient; call the
to the other putting the
attention, if out-patient)
method sample directly
● The specimen has been transported at
because of its on the slide, we
the improper temperature and not in
simplicity. It is place it on a
proper medium
easier to cover slip.
● The quantity of the specimen is
perform and ● We use
insufficient for testing (considered QNS)
very direct. petroleum jelly
● The specimen is leaking
● You simply or vaseline as a
prepare your seal or a sealant.
Clinical Bacteriology Laboratory
sample then ● It is called a
place a drop of hanging drop
whatever because the
suspension that samples are
will be on a slide allowed to
and put in a hang. Basically
coverslip. Then the samples are
you can view allowed to hang
the slide under a in this setup.
microscope. ● You have a drop
of liquid hanging For solid/viscous process
For example, upside down
examination of urine or from the under
WET MOUNT VS HANGING DROP
performance of surface of the
urinalysis. Basically, you coverslip.
will prepare a wet mount WET MOUNT TECHNIQUE HANGING DROP
for that similarly with TECHNIQUE
fecalysis (stool).
1. Centrifuge the The rate of movement The rate of movement
Urine slows down because is constant because
2. Decant the wet mount slide the petroleum jelly
3. Use the dries due to the heat keeps the hanging
sediment from the light of the drop mount hydrated
4. Put it in the slide microscope
5. Put a cover slip
6. Observe it under The microorganisms’ The microorganisms
the microscope movement is limited can move freely
because they are because of the
pressed in between the curvature on the slide
flat surface of the slide which provides them
and the cover slip space to move

WET MOUNT ADVANTAGES AND DISADVANTAGES

ADVANTAGES
● Simplicity - no need to use petroleum jelly. No
need to use a depression slide. You will just
1. Place a Drop of directly transfer the sample and observe it
sterile water on under the microscope.
slide
2. Place the DISADVANTAGES
specimen on ● It dries out too quickly. With the microscope we
water Drop by are using, it uses light. Therefore, it will generate
using a loop heat. The heat that comes from the
3. Put the coverslip microscope that actually enhances the drying
over the glass of the slide.
slide including ● NOTE: If you cannot see a proper image under
the mount the microscope, one reason may be because
4. Observe the the sample might have already dried down.
Slide under the Therefore, the movement and observation of
Microscope the specific microorganism slows down.
For Liquid process ● One of the reasons why we examine
something, particularly a living organism is to
detect movement because we are examining
a living organism.
● When it comes to movement it is very limited.
Clinical Bacteriology Laboratory
We won’t be able to observe the movement of ● The ability to move or the motility of the
a specific organism properly because the microorganism is one clue.
coverslip is pressed in between the flat surface ● Not all bacteria can move. Therefore, if we are
of the slide. able to observe that they are moving then
● Upon the preparation, our sample is very that’s another clue to write down for us to
compressed in between the slide and the identify what would be the final identification
cover slip. of the organism.

What makes an organism capable of moving?

HANGING DROP MOUNT ADVANTAGES AND ● Through the use of a flagellum or flagella.
DISADVANTAGES ● We generally refer to them as locomotory
organ.
ADVANTAGES ● The locomotory organ of a microorganism
● It provides high quality observation allows them to move.
● The observance of movement would be better ● If a microorganism can move, then it means
because it is more hydrated. that the bacteria possesses a special type of
● Hydration - all because of the presence of the flagella.
sealant as well as the shape of the actual slide. ● Examples of locomotory organs:
● Through the concavity, it will not easily ○ Cilia - hairlike structure
evaporate or dry up the content of the slide. ○ Pseudopods - fake feet
● Therefore, the time to observe the sample is ● Cilia and Pseudopods are mostly found in
much longer under the microscope. parasites.
● There is an ample amount of space for the ● For bacteria, only flagella and cilia can be
sample because of the concavity as well as found in them.
the coverslip.
● If there are any motiles under the sample, we 1. BROWNIAN MOVEMENT
will be able to observe them because of the
✓ Brownian motion is random,
presence of additional space in between
uncontrolled movement of
samples.
particles in a fluid as they
constantly collide with other
DISADVANTAGES
molecules.
● It takes more time. You need additional
✓ Movement of microscopic
equipment and it involves the use of other
particles suspended in liquids or
steps.
gases resulting from the impact of
molecules of the surrounding
IMAGES UNDER THE MICROSCOPE
medium.
WET MOUNT VS HANGING DROP

NOTE: Brownian movement is NOT true motility.

Hindi lahat ng bacteria ay motile. Most bacteria are
non-motile.

Paano tayo nakakakita ng bacteria na gumagalaw?

● Brownian Movement
○ Liquid or gas molecules parang
nababangga niya ‘yung mga
bacteria.
○ For example, the dagat with lots of
Why is it important to observe the motility of the kalat, gumagalaw raw ‘yung kalats
microorganism? whenever there’s a wave.
● The ability of the organism to move is already
the clue of the identity of the microorganism. ● It is just the random and uncontrolled
One of our main goals in the bacteriology movement of particles.
section is for us to identify the organism and ● It looks like it is moving, but in reality, it is the
bacteria that may be present in the sample. particles that are moving.
● So we need as many clues as possible that can ● Specifically, the water or liquid particles that
give us an idea on the identity of the sample. are diluting or suspending the organism.
Clinical Bacteriology Laboratory
● The presence of water molecules gives the 2. Rods - bacilli or bacillus
impression that the organism is moving. 3. Spiral - spirochetes
● Basically, it is just the background or water ● Only a little number of bacteria manifests this
suspending the sample that is moving. shape
3 Shapes
In this case, how can you make sure that the organism ● Cocci, Bacilli, and Spiral
is really moving?
● Through the presence of the locomotory organ ARRANGEMENT OF COCCI
of the organism.

2. TRUE MOTION
● Independent movement brought about by
different mechanism of locomotion (e.g.
flagella, cilia)
● Bacterias that have a motility
● The movement is NOT random and
uncontrolled.
● Basically there is an actual direction of
movement brought bY the presence of a
locomotory organ.
● It may move up and down, left or right, or
circular.
● Ex. Flagella, Cilia, and Pseudopod

Notes:
Diplococci - 2/combined
Staphylococci - grape-like clusters

ARRANGEMENT OF BACILLI

Listeria monocytoges - common bacteria

- For tumbling motility

Lesson 5
Bacterial Cytology and Morphology

Bacteria
● Very diverse in their characteristics, the
diseases that they cause, and their actual
structure.

3 Basic Shapes of Bacteria

1. Spherical - cocci or coccus
Clinical Bacteriology Laboratory
Notes:
Initial Identification:
Palisades - Chinese Letter arrangement. Not arranged
● Streptococcus
like bacilli
pneumoniae

ARRANGEMENT OF SPIROCHETES
Chain - streptococci
except for Streptococcus
pneumoniae

Streptococcus
pneumoniae is a
streptococci bacteria

Biochemical Test - used

to identify what type of
bacteria it is.

If may makikita na
streptococci na bacteria
(chain) - Streptococcus
spp.

Rod shaped -
Notes: ● bacilli
Spirochetes - don’t have external flagella; corkscrew
na mas mahaba Arrangement:
Spirilla - with flagella; corkscrew na short ● Pallisading
Vibrio - comma-shape bacilli

Identification:
DESCRIPTION OF THE MORPHOLOGY OF THE ● Corynebacteriu
MICROORGANISM m diptheriae

Morphology
Rod-shaped ● Palisades or
● bacilli Chinese letter
arrangement
Has terminal spores
Shape
Identification: ● Spiral -
● Clostridium Comma-shaped
tetani
Arranngement
Disease (Causative ● Vibrio
Agent) ● Has a presence
● Tenanus of flagella

Looks like a lollipop Identification:

● Vibrio cholerae
Shape:
● Spherical -
cocci

Arrangement:
● In pairs -
diplococci
Clinical Bacteriology Laboratory
Spherical: biochemical test siya -
● cocci use Blood Agar

Arrangement:
● in cluster - ● Spiral
staphylococci
Arrangement
● Spirochetes
Hindi mo siya
ma-iidentyfy the 3 Genus classifies as
stapylococcus areus spirochetes:
siya agad, may ibang 1. Treponema
staphylococcus such as 2. Borrelia
epidermitis and 3. Leptospira
saprophyticus
Axial filament -
If may mukhang ● responsible for
staphylococcus the shape
(clustains/chain)-
Staphylococcus spp. Initial Identification:
● Treponema
Spherical: pallidum
● cocci
Arrangement:
● in pairs -
diplococci

Lesson 6
Big circles -
Smear Preparation and Staining
● WBCs or
neutrophils
STEPS OF READING STAINS OR SMEARS
1. Identify the shape
Intracellular
2. Identify the arrangements
● important to add

All of these will give us very important information for us

● Intracellular
to determine the presumptive or initial identification of
diplococci
the bacteria.

Initial Identification:
SMEAR PREPARATION
● Genus -
Neisseria
Wet mount - just check up on the presence of a
gonorrhea
specific constituent or an element
● We do not use this usually in bacteriology
Ininfect niya ang cervix
section
ng babae kaya nasa
● Most common in clinical microscopy (fecalysis
loob ng ephithelial cells.
and urinalysis)

Smear - Looking for specific type of morphology and

Spherical:
structure in the case bacteria
● cocci
● What we prepare is the smear
● This is the very vital procedure
Arrangement:
● In bacteriology, we need to create a smear to
● in chains -
have an initial identification or initial clue for
streptococci
what is the identity of the microorganism.
● A smear is basically a sample fixed on the slide
Hindi agad ma-iidentify
with a stain or dye,
kung anong
Clinical Bacteriology Laboratory
ASEPTIC TECHNIQUE How do we essentially create a smear?
● It would depend on what sample we are going
to use if it is liquid or solid

● Big and crucial part in smear preparation most

especially in clinical bacteriology
● The inoculating loop should be red hot under
the flame before moving to another portion
● Proper flaming technique is a must Smear from Liquid Medium

Why do we flame the innoculating loop? 2 Types of Liquid Sample

● To prevent contamination 1. Viscous - sputum, endotracheal aspirate,
● ALWAYS FLAME BEFORE USING AGAIN peritoneal fluid, pleural fluid, pericardial fluid
○ It will lead to false positive ● In this case you will directly get a
● Falme all the like-wired sample

Aseptic Technique NOTE: Coiling should be done. You have to spread it in

● To minimize any possibility or risk of a circular motion so that it will coil in the slide.
contamination. It is important to perform the
aseptic technique properly. 2. Thinner - liquid consistency
● How? By using a flame an alcohol lamp or ● Centrifuge it first to have a sediment as a
Bunsen burner. Just pass the inoculating loop to sample
the flame until it is red hot. ● Next is to decant it
● It depends on the strength of the flame for the ● Ex: urine, cerebrospinal fluid
time it takes for it to be sterilized.
● When using any sterile container that has a lid, NOTE: The most essential procedure when
NEVER LEAVE THEM OR PUT THEM ON TOP OF THE creating a thin sample in a slide is to spread out
TABLE OR WORKING BENCH. thinly.
○ HUWAG ILALAPAG ‘YUNG CAP EXCEPET ● If you are going to be doing something which
IF NASA BIOSAFETY CABINET involves the microscope, the sample should be
○ IT WILL BE CONTAMINATED IF ILALAGAY as thin and as evenly as possible
SA KUNG SAAN-SAAN
● Why? Because that can contaminate the lid. If Why do we have to do this in this manner?
the lid is contaminated, put it in the container. ● Because we are going to observe it under the
Then that may cause another form of microscope.
contamination. ● And the bacteria are very small organisms so a
● OPEN THE PETRI DISH BY NOT LAPAG SA TABLE, little bit of sample can go a long way.
NOT UNLESS NASA BIOSAFETY CABINET KA. ● Otherwise, if your sample is very thick, they
● HERE’S THE CORRECT WAY TO OPEN THE PETRI tend to be crowded or overlapped to one
DISH another under the microscope.
○ If this happens, you cannot identify
their shape properly.

● Needs centrifugation to yield a more

concentrated product. Otherwise, if you do not
centrifuge it, the pathogen or microorganism
that you are isolating will be lesser since the
sample is not concentrated.
● For the sample, you need to obtain an aliquot
and transfer it to a test tube then centrifuge it,
Clinical Bacteriology Laboratory
decant, and use the sediment (the one that ● We need to create a liquid suspension.
settles at the bottom of the tube) as a main ● Why? Because when you spread it in the slide,
sample for the preparation of the smear. it comes out uneven.
● Aspirate a small portion of the sediment onto ● Ex: Colony
the center of the slide or just directly drop it in
the test tube. Different Types of Liquid Suspension
● Concentrate the sample at the center of the 1. Distilled or Sterile Water
slide then evenly spread it out thinly. 2. Normal Saline Solution (NSS)
● It is important to control the amount of
samples you are putting in there. Sometimes, Steps
they tend to put a lot of samples resulting in a 1. Add a drop of a suspension agent or diluent
thicker slide. 2. Get a sample and emulsify it up until the solid
● To spread it evenly, you have to spread it out. sample is evenly distributed within the
● Use the inoculating loop to spread it thinly after suspending agent or the liquid portion
sterilizing it through flaming. 3. Spread it out gently and thinly

Viscous
● Centrifugation is NOT needed because it is
already thick.
● You can directly use it as a sample. Using the
inoculating loop right after aseptic technique.
Allow it to cool down for a few seconds before
using it to obtain a sample.
● Do not dip the inoculating loop directly
because what happens is that you get a very Allow the smear to air dry
big portion of the sample. ● Wait for it to be dried up
● You can just do it little by little, small amounts ● If your sample is thinner, it is easier for it to dry
per batch depending on the sample you need. up. Otherwise, it would take several minutes to
● Concentrate the sample at the center of the dry if you create a thicker sample
slide.
Lastly, we should fix the sample after it is already dry
Method to create a very good sample of acid fast ● Make sure the sample have stuck on the glass
staining using a sputum sample: slide properly
● Recommended shape: oblong with 2 by 3 ● If you will not fix the sample, it might be washed
measurement out when we stain it
● Method: Coiling ● The water might wash it out
○ Creating a very defined coil within
the spaces using the sample. Just drag How to fix a sample?
the sample and coil it. ● Through the use of heat.
○ Do not be too gentle. Resulting in a ● Heat fixation - just let the slide pass several
non-defined coil. Put a pressure in the times through the flame until you observe the
inoculating loop so that it will be well sample from opaque to more of a color (white)
defined. or structure.

● The quality of the smearing preparations that Usually refer to the colonies
we will create depends on how you will make ● When you isolate the organism, the next step is
your smear. to perform staining techniques
● If you make your smear too thick, too thin, or ● Since you are dealing with a solid sample, you
too little sample, that will have direct have use a liquid diluent for you to emulsify the
consequences on what will be your final output sample properly
at the end of the staining. ● Diluents: Sterile water, distilled water, normal
saline solution (NSS)
Smear from Solid Medium ● It is possible to actually stain a colony. Just
drop the emulsifying agents that we will use
Solid Samples then transfer a small amount of culture and mix
● We need to dissolve it first in a liquid suspension. it evenly within the liquid agent that we used.
Clinical Bacteriology Laboratory
● Similarly, just spread it out thinly. ● Sa wet mount and hanging drop technique,
● We need to wait for the smear to air-dry. hindi niya ma-identify kung anong bacteria
● Make sure no traces of liquid are left to the siya, sa staining, malalaman if bacteria talaga
smear. siya. There are special principles na didikit
‘yung stains sa bacteria.

HEAT FIXATION Goals in Bacteriology:

✓Heat fixing kills the bacteria in the smear 1. Culturing of bacteria: Isolate the organism that
✓Firmly adheres the Smear to the slide may have caused the disease.
✓Allows the sample to more readily take up stains 2. Identify the bacteria: to give relevant
● PARA MAMATAY ‘YUNG BACTERIA information to the physician as to what would
● HEAT FIX AFTER SMEARING be the possible causative agent experienced
● 3-5x ang recommended ang heat fixation by the patient.
● For example, above is the specimen and 3. To be able to give further information on the
below is the slide, heat fix it 3-5x collection of antimicrobial agents that would
● What happens if over heat fix? best treat the patient.
○ Mag-dedenature ‘yung morphology
ng bacteria. Means, wala ka ng Through staining, we can identify the bacteria, which
bacteria na makikita because gives us information about the bacteria.
nasunog na silang lahat, beh. ● It is important to have staining techniques in
○ Denature - tatanggalin lahat ng order to visualize the morphology, the internal
contents or structures kung anong structures of the cells properly, which give us
meron sa bacteria information or clues as to what will be the
possible identity of the microorganism.
NOTE: It is okay that the bacteria is dead because we
are only concerned in the observation of morphology In LPO, the light should be weaker.
which are the shape and arrangement as well as the
other characteristics.
STAIN
Fixation ● Is a dye that is used to impart color in a living or
● After air drying the sample. non living organism.
● General term to allow the smear to adhere ○ The presence of color gives a
better on the glass slide. significant contrast so they are much
● In the bacteriology section, we use the heat more visible.
fixation method. ○ Through this, we can contrast them
○ Just pass the slide through the flame several versus the actual background.
times. ● Most common microscope: Bright field
○ With this, no need to pass it too long. ○ Why a bright field? Because the
○ Left, right, up, and down is enough. background is white, organism is
● How do we know it is heat fixed properly? opaque or transparent (depends)
○ When the smear turns more opaque. ○ Dark field - the white one is the
organism while the black is the
Lesson 7 background.
Staining Techniques: Gram Stain and Acid-Fast Stain ● Control the light that passes or enters through
the microscope.
Why should we stain bacteria? ○ LPO: Use less light
● Bacteria have nearly the same refractive index ○ Why? Because the element is already
as water, therefore, when they are observed opaque, the light is very bright, so you
under a microscope they are opaque or nearly cannot see it anymore.
invisible to the naked eye ● Staining imparts an artificial coloration to the
● Different types of staining methods are used to smear materials that allows them to be seen
make the cells and their internal structures using the magnification provided by a
more visible under the light microscope microscope.
● To provide contrast and observe the bacteria ● The presence of color gives the cells significant
properly. contrast so are much more visible
Clinical Bacteriology Laboratory
TYPES OF STAIN pathogens, and it gives important
clues related to the quality of a
ACIDIC BASIC NEUTRAL
specimen.
Negatively Positively Includes both ○ It gives valuable information
charged acid charged basic acidic and towards the identity of the
radicals that radicals that basic stains microorganism.
combines with combines with therefore ○ CNS usually takes a longer
positively negatively imparting color time. 3-5 days is the
charged charged to both positive turnaround time.
cellular particles in the and negative ● Gram staining will only take 5 minutes.
components cytoplasm cellular ● It is important to properly prepare a gram stain
components specimen.
● In bacteriology, if not CNS, Gram stain is usually
<7 pH >7 pH = 7 pH requested.

Examples: Eosin, Examples: Examples: ● Provides a mechanism for the rapid

Malachite Hematoxylin, Romanowsky presumptive identification of pathogens, and it
green, Nigrosin, Crystal Violet stain (Giemsa gives important clues related to the quality of a
India Ink stain, Wright specimen.
stain)
How is knowing the gram stain of the patient leads to
the rapid presumptive identification?
Why do we have a basic and acidic stain? ● Because most organisms can be classified as
● With a pH being lower, they are negatively either gram positive or gram negative
charged. depending on the reaction at the end or how
you view them under the microscope.
Acidic stains ● In bacteria, it is classified as gram positive or
● Acidic stains cause lower pH, assuming a gram negative.
negative charge. ● Ex. You can already eliminate gram negative
● SIGNIFICANCE: An acidic stain is negatively bacteria if you already know that the bacteria
charged, they will only combine with positively is already gram positive.
charged cellular components. ● Therefore, it provides us with a rapid
● Example: Nucleus presumptive identification.
● Most often, acidic stains stain the nucleus of ● We eliminate in order for us to determine the
the cells. bacteria causing the infection.

Basic stains ● Most of the time, gram stain ang rinerequest ng

● Because of its basic pH, it assumes a positive mga physician.
charge. ● Gram-positive and gram-negative
● It combines with negatively charged particles. ● Gram staining is not used for identification of
● Example: Cytoplasm of the cell bacteria, this is used for diagnosis only
● Diagnosis - clue kung anong meron sa sakit ng
Neutral stains patient na iyon.
● Able to stain all the components of the cell
● Utilized when staining peripheral blood smear
● You can see platelets, WBCs, RBCs, etc. HANS CHRISTIAN GRAM
● The Gram stain was devised by
the Danish physician, Hans
GRAM STAINING Christian Joachim Gram, while
● Gram stain is the principal stain used for working in Berlin in 1883. He later
microscopic examination of bacteria and is published this procedure in 1884.
one of the most important bacteriologic At the time, Dr. Gram was
techniques within the microbiology laboratory. studying lung tissue sections
○ Why is it considered an important from patients who had died of pneumonia.
bacteriologic technique? It provides a
mechanism for the “rapid”
presumptive identification of
Clinical Bacteriology Laboratory
● Pinapakapit ni mordant yung stain na
GRAM STAIN COMPONENTS ginagamit mo onto whatever material that is
stained.
● Specific structure stained: Cell wall of bacteria’
STEP REAGENTS Time
● In gram staining, the structure that we are
PRIMARY STAIN Crystal Violet 1 min primarily concerned with is the cell wall.
Adhere to the ● The mordant’s job is to adhere or fix the crystal
cell membrane violet towards the cell wall (peptidoglycan
of the bacteria layer) of the organism.
(stain)
3. Decolorizer
MORDANT GRAM’S IODINE: 1 min ● It decolorizes the color or the stain that was
FIXES CRYSTAL previously applied to the primary stain.
VIOLET TO CELL ● The differentiation between a gram + and
WALL gram - happens in the decolorization step.
To support sa
pagdikit ng 4. Counter Stain or Secondary stain
Crystal Violet ● Responsible for forming the final color of the
organism depending on the organisms’
To stick the characteristics.
stain. For
betterment of NOTE: Apply each component and at every after
the attachment application, rinse it with running water.
of the stain
RINSING
DECOLORIZER Acetone 30 secs ● Do not put the smear directly under the running
Remover of the water.
color ● It is better to let your hand run through it and
from your hand, the water falls through it.
COUNTER STAIN Safranin 1 min ● Why do we rinse? To remove the excess stain
The color pink that did not stick to the cell wall of the
To determine organism to end up with a cleaner output.
the bacteria if it
is gram-positive After applying all the gram stain components, blot or
or wipe the sides and back of the slides to remove excess
gram-negative water and allow it to dry completely.
● By that time you've already performed gram
staining on your sample.

GRAM STAINING PRINCIPLE

● The difference in composition between
gram-positive cell walls, which contain thick
peptidoglycan with numerous teichoic acid
cross-linkages, and gram-negative cell walls,
which consist of a thinner layer of
peptidoglycan, accounts for the Gram staining
differences between these two major groups of
Gram-positive - violet bacteria.
Gram-negative - pink ● Meanwhile Gram-negative bacteria are
1. Primary Stain decolorized and take up the counter stain
● Forms the initial color of the organism therefore staining red.
● Gram-Positive - thick peptidoglycan with
2. Mordant numerous teichoic acid cross-linkages
● A substance used in staining that allows the ● Gram-Negative - thinner layer of
stain to stain to stick to the material better. peptidoglycan
Clinical Bacteriology Laboratory
● Walang teichoic acid ang gram-negative,
okay? GRAM STAINING PROCEDURE

The difference when it comes to the gram-positive and

to the gram-negative bacteria lies within their
difference in structure within their cell wall. Most
especially, the peptidoglycan layer.
● Gram-positive: Thick peptidoglycan layer
● Gram-negative: Thin peptidoglycan layer

Which one of them will be decolorized easily?

● Gram-negative because it has a thinner
peptidoglycan layer.
● The crystal violet stain will be completely rinsed
off for the gram-negative.

STRUCTURE OF GRAM-POSITIVE AND GRAM-NEGATIVE

EXPLANATION:
● The cell wall which is the peptido glycan, ‘yung
stain na crytal violet, mag-sstay siyang violet sa
gram-negative. If linagyan na ng decolorizer,
hindi na matatangal ‘yung stain sa
gram-positive. As for the gram-negative,
matatanggal na siya using the decolorizer.
● Safranin, magiging violet pa rin ‘yung
gram-positive since roon na nag-stay ‘yung
violet dye. For the gram-negative, since
na-decolorize na ‘yung crystal violet, magiging
pink na siya.
● Gram-negative are all bacteria.
● Most gram-negative are bacilli
● Most gram-positive are cocci

Explanation sa procedure:
1. The first stain na gagamitin is crystal violet. Put it
on all parts of the slide except on the frosted
area because we will put the label there. After
applying, 1 min ang waiting time, then, wash
off.
2. Next is Gram’s iodine, apply it, and the waiting
time is 1 min, then, wash off.
3. Put the decolorizer, wait for 5-10 seconds, then,
wash off.
4. Safranin, for 45 seconds, then wash off.
Clinical Bacteriology Laboratory
● Is it positive (PURPLE) or negative (PINK)
● 1 and 2 slides will be rejected because the
smear is too thick. 2. Morphology of the bacteria
● 3rd slide can be accepted because may mga ● We do not report in pairs and singular
part na puwedeng basahin.
EVALUATION OF GRAM STAIN
● When we evaluate the morphology of an
STANDARD OPERATING PROCEDURE (SOP) organism we use an oil immersion objective
● Depends on what institution or what brand they
will assign on how the procedure should be EXPECTED RESULTS
performed.
● The instructions on the manufacturer’s of
staining kits should be followed as SOP.
● Different labs have different protocols so
consult with the laboratory admin first.

Send out - no equipment or needs secondary

equipment
1. Fix material on slide with methanol or heat. If the slide
is head fixed, allow it to cool to the touch before
applying stain.

2. Flood slide with crystal violet (purple) and allow it to

remain on the surface without drying for 10 to 30
seconds. Rinse the slide with tap water, shaking off all
excess.

3. Flood the slide with iodine to increase affinity of

crystal violet and allow it to remain on the surface
without drying for twice as long as the crystal violet was
NOTE: It is important to report the arrangement in cocci
in contact with the slide surface (20 seconds of iodine
because we can classify them in their arrangement.
for 10 seconds of crystal violet, for example). Rinse with
tap water, shaking off all excess.
In bacilli, we do not put importance in their
arrangement UNLESS it is pallisading or in chains
4. Flood the slide with a decolorizer for 10 seconds or
because there is a specific group of bacteria or rods
less ( optimal decolorization depends on chemical
that assume in those arrangements.
used) and rinse off immediately with tap water. Repeat
this procedure until the blue dye no longer runs off the
Oil immersion objective
slide with the decolorizer. Thicker smears require more
● Always wipe of the lens after use to avoid
prolonged decolorizing. Rinse with tap water and
clogging that would result in damage to the
shake off excess.
lens of the objectives.

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. For delicate
smears, such as certain body fluids, air drying is the best
method.

6. Examine microscopically under an oil immersion lens

at 1000x for phagocytes, bacteria, and another cellular
material.

REPORTING THE GRAM STAIN RESULTS

1. Gram staining reaction
Clinical Bacteriology Laboratory
reactions difficult. Gram-negative
GOOD TO KNOW organisms may not decolorize properly.
● On smears that have been correctly prepared ○ Masyadong think sila, hindi tama
and stained host cells, such as red and white ‘yung pag-sho-show ng kulay ng
blood cells (phagocytes), allow the crystal gram stain
violet stain to wash out with decolorization and ● Cultures older than 16 to 18 hours will
should appear pink. contain living and dead cells. Cells that
● Fungal cells and elements generally stain are dead will be deteriorating and will
gram-positive. not retain the stain properly.
● Gram stains from patients on antibiotics
● RBC and WBC should appear pink. Because or antimicrobial therapy may have
na-wawash out ang crystal violet. They don’t altered Gram stain reactivity due to the
have the same structure for the gram-positive. successful treatment.
○ If with antibiotic - useless
CASE ANALYSIS because we can’t see bacteria
● In the case of sputum, sometimes the infection
is not caused by a bacteria. It may be caused Limitations
by a fungus because it also stains purple which 1. Overdecolorization may result in the
indicates gram-positive. identification of false gram-negative results,
● Fungal cells can either be branching or whereas underdecolorization may result in the
spherical. identification of false gram-positive results.
● If circular in form, it may be mistakenly ● Decolorization step is very important
identified as bacteria. towards the actual reaction at the end
● The difference is that fungal cells are larger because it totally depends on that
than the bacteria. step if that is gram positive or negative.
● Therefore, it is important to perform this
step properly. Do not exceed in 30
LIMITATIONS seconds because it causes
● Overdecolorization may result in the overdiscolorization.
identification of false gram-negative ○ It will appear false
results, whereas underdecolorization gram-negative because the
may result in the identification of false crystal violet might be washed
gram-positive results. out because of the longer
○ If nasobrahan sa decolorization, contact time.
mas lalong tinatanggal ‘yung
stain. Then, after applying the 2. Smears that are too thick or viscous may retain
safranin, nagging pink na siya. too much primary stain, making identification
○ For example, gram-positive of proper Gram stain reactions difficult.
‘yung bacteria, overdecolorized Gram-negative organisms may not decolorize
siya, then, natanggal ‘yung mga properly.
crystal violet. After applying ● Thicker = more stain
Safranin, naging pink na siya, ○ Appears false gram positive
leading to false gram-negative
results. 3. Cultures older than 16 to 18 hours will contain
living and dead cells. Cells that are dead will
○ Underdecolorization - There’s a be deteriorating and will not retain the stain
stain as crystal violet/ properly.
○ For example, gram-negative ● Use younger colonies. Maximum is 30
‘yung specimen niya, since hours.
underdelorized siya, hindi ● If more than 30 hours, this is not a very
natanggal lahat ng mga crystal good sample to use because by this
violet stains on the time this will now contain both living
gram-negative na specimen. and dead cells.
● Smears that are too thick or viscous may ● It might contain more dead cells
retain too much primary stain, making because of the lack of nutrients.
identification of proper Gram stain
Clinical Bacteriology Laboratory
● It will deteriorate the cells and or infect cells of the host in order for
deteriorated cells will not retain the them to live, grow, and multiply.
stains properly because of the ● Outside of the cell, they cannot live.
damaged cell wall. ● Therefore, if they are inside the cell you
● It will make the interpretation of the won’t be able to properly stain them.
stain difficult.
3. Bacteria that have unusual or different shape
4. Gram stains from patients on antibiotics or that makes them hard to stain with most
antimicrobial therapy may have altered Gram staining technique
stain reactivity due to the successful treatment. ● Ex. Treponema pallidum (causative
● What if your patient already took agent of syphilis)
antibiotics before collecting a sample? ○ An example of a spirochete
○ It is relevant and important ● We use a different technique to adequately
information to take note observe them properly under the microscope
because some antibiotics work which is through the process of dark field
by destroying or damaging the microscopy.
cell wall of the bacteria. ○ Bacteria - lighter,
○ Specific antimicrobial agents Background - black
specifically target the cell wall ● If there is contrast in color, it is more easily
of the microorganism. observed.
○ If the cell wall of the bacteria ● Technically, Treponema pallidum is a gram
has already been damaged, positive bacteria.
the stain will not be effective.
○ You can no longer accurately 4. Acid fast organism
identify if the microorganism is ● Clinically significant microorganism, so
gram positive or gram microbiologists developed a specific staining
negative. method to stain this bacteria

L-forms
● These are bacteria who naturally have a cell ACID FAST STAIN
wall but lose it due to: ✓ The acid-fast stain is specifically designed for a
1. Antibiotic therapy subset of bacteria whose cell walls contain long- chain
2. Exposure to the outside environment mycolic acid.
✓ Mycolic acids render the cells resistant to
NOTE: We need to define this properly because there decolorization, even with acid alcohol decolorizers.
are some bacteria that naturally do not have a Thus, these bacteria are referred to as being acid-fast.
cell wall. ✓ Mycobacteria are the most commonly encountered
acid-fast bacteria
Most bacteria can be classified as gram positive
or gram negative.
● There are some bacteria cannot be sufficiently
be identified as gram + or gram -

4 GROUPS OF ORGANISMS THAT DOES NOT USUALLY

IDENTIFIED AS GRAM + OR GRAM -
1. Bacteria that naturally do not have cell walls.
● Ex. Mycoplasma, Ureaplasma
● Mycolic Acid is resistant to any chemical
2. Obligate intracellular bacteria components (e.g., WBC - hindi tumatabla)
● Obligate - strictly they are intracellular ● For example, Acid Alcohol used for Acid-Fast
● Ex. Chlamydia, Rickettsia Staining, hindi tumatabla for acid-fast bacteria
● These are very special types of or bacillus
bacteria that need to infect a cell at ● Acid-Fast also known as Acid-Resistant
all times in order for them to live. ● For example, Mycobacterium tuberculosis -
● They refer to these as parasitic Acid Fast na nag-red
bacteria because they need to invade
Clinical Bacteriology Laboratory
ACID FAST STAIN COMPONENTS

STEP REAGENTS

PRIMARY STAIN Carbolfuchsin

MORDANT Heat (Zeihl-Neelsen)

DECOLORIZER Acid Alcohol

EXPLANATION:
COUNTER STAIN Methylene blue, ● For example, the acid fast, the Carbolfuchsin is
Malachite green red, then hindi gumana ;yung decolorizer, at
then end red pa rin siya, means acid-fast. After
applying of methylene blue, gumana siya, turns
blue, then it is non-acid fast.

ACID FAST STAIN PROCEDURE

METHODS OF ACID-FAST STAIN

STEP ZEIHL-NEELSEN KINYOUN

(HOT METHOD) (COLD
METHOD)

PRIMARY STAIN Carbolfuchsin Carbolfuchsin

(higher
concentration EXPLANATION:
of Phenol ● Carbolfuchsin red, waiting time for 10 mins,
then, wash off
MORDANT Heat Detergent ● For the decolorizer, 3 mins, then, wash off
(Zeihl-Neelsen) ● For counterstain, 1 mins, then, wash off
● NOTE: if usok na, stop na (Zeihl-Neelsen)
DECOLORIZER Acid Alcohol Acid Alcohol
ACID-FAST STAIN SMEAR
COUNTER STAIN Methylene Methylene
blue, Malachite blue, Malachite
● Non-acid fast because it is blue
green green

● Carbolfuchsin - penetrate the cell wall of the

bacteria
● Mordant - used to support and aid; using heat
to destroy the cell wall of the bacteria
(hanggang sa may makitang usok)
● Detergent - It helps the Carbolfuchsin die na
kumabit sa cell wall ng acid-fast bacteria
● Decolorizer - determine wether acid-fast ba
siya or nag-acid-fast bacilli. Bakit? Resistant to
acid; magiging red na siya.
● Resistant to acid = red
● Color of non-acid fast fasy bacillus/bacteria =
methylene blue or malachite green
Clinical Bacteriology Laboratory
EXPECTED RESULTS
DECOLORIZER 0.5% Acid Alcohol (5
mins)

COUNTER STAIN 0.5% Potassium

Permanganate (1 min)

EXPECTED RESULTS

● The second picture is a Mycobacterium

Tuberculosis because mukha siya bacilli

ACID-FAST ORGANISMS
● Mycobacterium (bacteria)
● Isospora (parasite)
● Legionella micadei
● Yellow - positive
● Cryptosporidium (parasite)
● Green - negative
● Nocardia (bacteria)
● Anong bacteria ang merong Mycolic Acid?
○ Acid-Fast Bacilli

NATIONAL STANDARD REPORTING SCALE BARLETTE’S CRITERIA

3+ More than 10 AFB/OIF in

at least 20 fields

2+ 1-10 AFB/OIF in at least

50 fields

1+ 10-99 AFB/100 OIF

+n 1-9 AFB/100 OIF ● We can identify here if sputum ba ‘yung

specimen or saliva.
0 No AFB seen in 300 OIF ● For the SPUTUM - >25 PMN/LPF and <10
squamous epithelial cells.
● OIF - Oil Immersion Field ● For SALIVA - <10 PMN/LPF and >25 squamous
● For example, pagtingin sa microscope, walang epithelial cells
nakikita, then pagka-move ng slide (second
field) - Polymhorphonuclear - a type of WBC na they have
● Field - per move of slide three nucleus. From the term poly, meaning many.
Morpho, morphology. Nuclear, nucleus.

AURAMINE-RHODAMINE STAIN Sa saliva, walang masyadong WBC sa mouth, more

● Auramine-rhodamine fluorochrome staining squamous epithelial cells.
also known as “Truant method of staining”,
bind to the mycolic acid present in them and Neutrophil and Macropages ay nakatambay sa lungs
impart bright yellow or orange fluorescence to protect
against a greenish background when viewed
using a fluorescent microscope ADDITIONAL NOTES

AURAMINE-RHODAMINE STAIN COMPONENTS EXAMINATION MORPHOLOGY SMEAR

PREP STAINING TECHNIQUES PART TWO
STEP REAGENTS
ACID-FAST STAIN
PRIMARY STAIN Auramine Rhodamine
solution (20 mins) ● The acid-fast stain is specifically designed for
Clinical Bacteriology Laboratory
a subset of bacteria whose cell walls contain Sputum Smear Microscopy)
long-chain mycolic acid. ● The most efficient, most common, and the
cheapest way to diagnose tuberculosis is via
DSSM
● The technique to read this under the
microscope is to stain it using the acid-fast
staining

ACID-FAST STAINING PROCEDURE

4 Components

Mycolic acid
● An example of lipid
● In some other references, they refer
acid-stain fast organism as bacteria that
contains high lipid content within the cell
wall.
● With the presence of mycolic acid, they are
not easily washed out or decolorized.
● Mycolic acids render the cells resistant to
decolorization, even with acid alcohol
decolorizers. Thus, these bacteria are PRIMARY STAIN
referred to as being acid-fast. ● Carbolfuchsin
○ (application time is 10-20 minutes)
○ Magenta in color
Mycobacteria ○ No need to rinse right away
● Are the most commonly encountered
acid-fast bacteria MORDANT
● A group of bacteria that has mycolic acid ● Heat (for Ziehl-Neelsen method)
within their cell wall ○ With the use of flame source or
● They are the most clinically significant alcohol lamp
acid-fast bacteria, therefore, when a doctor ○ Put the alcohol lamp under the slide
requests for acid-fast staining they usually ○ Apply heat until steam forms
want to diagnose or determine if the ○ Countdown starts at 10-20 minutes
organism has mycobacteria within their right after the application of
respiratory system. Therefore the most mordant
common sample is sputum ○ After is you have to rinse it with
running water before putting
Request for acid fast staining decolorizer
● To see if there is a presence of ● Cold (for Kinyoun method)
mycobacteria, or specifically ● Depending on what technique because
mycobacterium tuberculosis there are two types of method

Mycobacterium tuberculosis DECOLORIZER

● Causative agent of tuberculosis ● Acid Alcohol
● What you need to achieve is to have the
DSSM (Direct Sputum Smear Microscopy) primary stain remove for it to be sufficient
● Within the Philippine setting or majority of before applying methylene blue
hospitals that do acid-fast staining is for the
diagnosis of tuberculosis or DSSM (Direct COUNTER STAIN
Clinical Bacteriology Laboratory
● Methylene Blue (most common), Malachite ● Application of
green Carbolfuchsin
● Application is 5 to 10 minutes (primary stain)

*Acid-fast staining will take you a lot of time because

of the application of specific stains that needs to last
longer to account for the presence of the mycolic
acid because it renders.

METHODS OF ACID-FAST STAIN

● Application of
STEP ZEIHL-NEELSEN KINYOUN heat (mordant)
(HOT METHOD) (COLD
METHOD)

PRIMARY STAIN Carbolfuchsin Carbolfuchsin

(higher
concentration
of Phenol

MORDANT Heat Detergent

(Zeihl-Neelsen)
● Application of
DECOLORIZER Acid Alcohol Acid Alcohol Acid Alcohol
(decolorizer)
COUNTER Methylene Methylene
STAIN blue, blue,
Acid Alcohol
Malachite Malachite
● Combination of
green green
acid and
alcohol
Kinyoun method’s mordant (detergent) particularly in
● Tergitol the form of
hydrochloric
Ziehl-Neelsen (Hot method) acid
● ●Most common and usually used in the ● The
laboratory concentration of
acid is lesser
Kinyoun (Cold method) than the
● Not usually used but is advantageous if concentration of
dealing with samples that can not be alcohol.
applied heat with (ex. Tissue sample)
● Application of
*If you are going to apply heat or Ziehl-Neelsen on a Methylene Blue
tissue, it will affect the morphology or the structure of (counter stain)
the sample.

ACID-FAST STAINING PROCEDURE

ACID-FAST STAINING PROCEDURE

Clinical Bacteriology Laboratory
● Because of the presence of the mycolic
acid because it renders the organism hard
to be decolorized

NON-ACID FAST
● Color blue
1. Fix smears on the heated surface (60°C for at least
● If there are no bacterias seen therefore the
10 minutes).
result is negative

2. Flood smears with carbol-fuchsin (primary stain)

Why is it blue in color?
and heat to almost boiling by performing the
● Because they are easily decolorized by the
procedure on an electrically heated platform or by
decolorizer (acid alcohol)
passing the flame of a Bunsen burner underneath the
slides on a metal rack. 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.

3. Flood slides with 3% HCI in 95% ethanol

(decolorizer) for approximately 1 minute. Check to
see that no more red color runs off the surface when
the slide is tipped. Add a bit more decolorizer for very
thick slides or those that continue to "bleed" red dye.
Wash thoroughly with water and remove the excess. EXAMPLE OF ACID-FAST BACILLI

4. Flood slides with methylene blue (counterstain)

and allow them to remain on the surface of the slides
for 1 minute. Wash with distilled water and stand
slides upright on paper towels to air dry. Do not blot
dry.

5. Examine microscopically, screening at 400x

magnification and confirm all suspicious (i.e., red)
organisms at 1000x magnification using an
oil-immersion lens.

EXAMPLE OF NON-ACID FAST BACILLI

EXPECTED RESULTS

ACID FAST
● Color red or pink
● If there is a red rod shaped bacilli therefore it
is positive

Why is acid-fast red in color?

Clinical Bacteriology Laboratory
*Sputum Sample (1 minute)

NATIONAL STANDARD REPORTING SCALE

EXPECTED RESULTS OF APPLICATION OF FLUORESCENT
3+ More than 10 AFB/OIF in at least 20 STAIN
fields

2+ 1-10 AFB/OIF in at least 50 fields

1+ 10-99 AFB/100 OIF

+n 1-9 AFB/100 OIF

0 No AFB seen in 300 OIF

● Glow in the dark because of the application of the

ABBREVIATION/S: fluorescent stain
● They use a black background to make the sample
OIF- Oil Immersion Field stand out
PMN- Polymorphonuclear
LPF- Low Power Field

BARTLETT’S CRITERIA
AURAMINE-RHODAMINE STAIN

● Auramine-rhodamine fluorochrome staining

also known as “Truant method of staining”
● Directly bind to the mycolic acid present in
them and impart bright yellow or orange
fluorescence against a greenish background
when viewed using a fluorescent
microscope.
● An example of fluorochrome stain
● Uses a fluorescent microscope and a ● In the hospital, in order for you to accept the
specific fluorescent stain which is in the form sample it should be gram stained and the reading
of auramine-rhodamine stain should adhere towards the bartlett’s criteria. It should
contain more than 25 PMN/LPF and Less than 10
squamous epithelial cells so that the sample won’t
be more on saliva
EXPECTED RESULTS OF APPLICATION OF FLUORESCENT
STAIN

Primary Auramine Rhodamine

Stain solution
TAKE NOTE!!!
(20 minutes)
● Oil Immersion Field is for gram staining or red
● Low Power Field is for evaluation of the quality of
Decolorizer 0.5% Acid Alcohol
the sputum
(5 minutes)

Counter 0.5% Potassium

Stain Permanganate
Clinical Bacteriology Laboratory
PARTIALLY ACID FAST ORGANISM

Parasites: Partially acid fast

● Nocardia
● Actinomycetes
● Intestinal Coccidians

Bacteria: Not usually significant but are

partially acid fast
● Rhodococcus
● Tsukamurella