Clinical Bacteriology Laboratory

OLFU Laboratory Safety and Microscopy LAB 20202 nd SEM
-2021
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 1 LAB
Batch 2023 Date: February 5, 2021 (HIV), hepatitis B virus (HBV), and other blood-borne
microorganisms that can cause disease in human beings.”
❖ Understanding how microorganisms are transmitted (chain of
infection) is essential to preventing infection
Outline
At the end of the session, the student must be able to learn: Chain of Infection
I. Laboratory Safety Infectious Agents Consist of bacteria, fungi, parasites and
A. Biologic Hazards viruses
• Chain of infection Reservoir Location of potentially harmful
• Handwashing and Standard Precaution
microorganisms, the place where the
• Biologic Waste Disposal
B. Sharp Hazards infectious agent can live and possible
C. Chemical Hazards multiply
• Material Safety Data Sheets Portal of Exit Way to exit the reservoir to continue the
D. Radioactive Hazards chain of infection
E. Electrical Hazards Mode of Transmission Direct Contact, Indirect Contact,
F. Fire/ Explosive Hazards Droplets, Airborne etc.
• Types of fires and fire extinguishers
Portal of Entry Same as the portal of exit, which include
II. Microscopy
A. Microscope Definition
the mucous membranes of the nose,
B. Types of Microscope mouth and eyes, breaks in the skin, and
C. Parts and Functions of the Microscope open wounds
D. Proper use of the Microscope Susceptible Host Can be another patient during invasive
E. Magnification Formula for Microorganism Size procedures, visitors, and healthcare
personnel when exposed to infectious
specimens or needlestick injuries
I. LABORATORY SAFETY
❖ Safety
➢ To work safely in this environment, laboratory personnel must
learn what hazards exist, the basic safety precautions
associated with them, and how to apply the basic rules of
common sense required for everyday safety for patients,
co-workers, and themselves
Types of Safety Hazards

Type Source Possible Injury
Biologic Infectious agents Bacterial, fungal, viral or
parasitic infections
Sharps Needles, lancets, Cuts, punctures, or bloodborne
broken glass pathogen exposure
Chemical Preservatives Exposure to toxic,

and reagents carcinogenic or caustic agents
Radioactive Equipment and Radiation exposure
radioisotopes ❖ Proper hand hygiene, correct disposal of contaminated
Electrical Ungrounded Burns or shock materials, and wearing personal protective equipment (PPE) are
or wet of major importance in the laboratory
equipment; ❖ Handwashing
frayed cords 1. Stand in front of the sink. Do not lean on the sink with
Fire/ Explosive Open flames, Burns or dismemberment clothes
organic 2. Use paper towel to cover the water control and turn on the
chemicals water
Physical Wet floors, heavy Falls, sprains or strains 3. Wet hands thoroughly. Allow the water to flow from arms to
boxes, fingertips
patients 4. Apply soap to hands
5. Wash the palm, back and wrist of each hand using strong,
A. Biologic Hazards frictional and circular movements
6. Interlace fingers and thumbs and move hands back and
forth for ten seconds
7. Rub nails against the palm
❖ According to the CDC concept of Standard Precautions, 8. Rinse hands thoroughly
➢ “All human blood and other body fluids are treated as 9. Dry hands well
potentially infectious for human immunodeficiency virus 10. Use paper towel to turn the water off
Page 1 of 3
➢ Information contained in an MSDS includes the following:
1. Physical and chemical characteristics
2. Fire and explosion potential
3. Reactivity potential
4. Health hazards and emergency first aid procedures
5. Methods for safe handling and disposal
6. Primary routes of entry
7. Exposure limits and carcinogenic potential
D. Radioactive Hazards
➢ Radioactivity may be encountered in the clinical laboratory

when procedures using radioisotopes are performed
Personal Protective Equipment ➢ Exposure to radiation during pregnancy presents a danger
to the fetus; personnel who are pregnant or think they may
Donning Removing
be should avoid areas with this symbol
1. Gown 1. Gloves
2. Mask 2. Headcap
3. Headcap 3. Gown E. Electrical Hazards
4. Gloves 4. Mask
➢ Equipment should not be operated with wet hands
[BACT211] 1.01 Laboratory Safety and Microscopy I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Standard Precautions ➢ Designated hospital personnel monitor electrical equipment
➢ Hand hygiene includes both hand-washing and the use of closely; however, laboratory personnel should continually
alcohol-based antiseptic cleansers observe for any dangerous conditions, such as frayed cords
▪ Perform hand washing if your hands are visibly soiled and overloaded circuits, and report them to the supervisor
▪ Use alcohol if your hands are not visibly soiled ➢ Equipment that has become wet should be unplugged and
➢ Personal protective equipment allowed to dry completely before reusing. Equipment also
➢ Patient care equipment should be unplugged before cleaning
➢ Environmental control ➢ When an accident involving electrical shock occurs, the
➢ Prevent injuries when using needles, scalpels and other electrical source must be removed immediately
sharp instruments or devices ➢ This must be done without touching the person or the
➢ Respiratory hygiene/cough etiquette equipment involved to avoid transferring the current
❖ Biological Waste Disposal ➢ Turning off the circuit breaker, unplugging the equipment, or
➢ All biological waste except urine, must be placed in moving the equipment using a nonconductive glass or wood
➢ Disinfection of the sink using a 1:5 or 1:10 dilution of object are safe procedures to follow
sodium hypochlorite should be performed daily F. Fire/ Explosive Hazards
▪ Sodium hypochlorite dilutions stored in plastic
bottles are effective for 1 month if protected from light
after preparation
➢ When a fire is discovered, all employees are expected to
take the actions in the acronym RACE:
B. Sharp Hazards ▪ Rescue – Rescue anyone in immediate danger
▪ Alarm – Activate the institutional fire alarm system
➢ Includes needles, lancets and broken glassware ▪ Contain – Close all doors to potentially affected areas
➢ All sharp objects must be disposed in punctured- ▪ Extinguish/ Evacuate – Attempt to extinguish the fire,
resistant containers if possible or evacuate, closing the door
➢ The biohazard sharp containers should not be
overfilled and must always be replaced when the
safe capacity mark is reached
C. Chemical Hazards
➢ Every chemical in the workplace should be
presumed hazardous
➢ When skin contact occurs, the best first aid is to
flush the area with large amounts of water for at
least 15 minutes, then seek medical attention
➢ The National Fire Protection
Association (NFPA) has developed the standard
system for the identification of the Fire Types of Fires and Fire Extinguishers
Hazards of Materials, NFPA 704.14
➢ This symbol system is used to inform firefighters Fire Type Composition of Type of Fire Extinguishing
of the hazards they may encounter with fires in a Fire Extinguisher Material
particular area Class A Wood, paper or Class A Water
➢ The diamond-shaped, color-coded symbol clothing
contains information relating to health, Class B Flammable Class B Dry chemicals,
flammability, reactivity, and personal organic chemicals carbon dioxide,
protection/special precautions foam or halon
❖ Material Safety Data Sheets Class C Electrical Class C Sand or dry powder
Page 2 of 3
Class D Combustible None Dry chemical
metals Class ABC
❖ Brightfield Microscope
Class K Grease, oils, fats Class K Liquid designed to ➢ Used to observe morphology of microorganisms such as
prevent splashing
bacteria, protozoa, fungi, and algae in living (unstained) and
and cool the fire
nonliving (stained) state
▪ Background is bright, organism is dark
A student successfully completing basic microbiology will ❖ Darkfield Microscope
demonstrate the ability to explain and practice safe ➢ Unstained organisms are observed against a dark
background
➢ Useful for examining thin spirochetes
▪ Background is dark, organism is bright
1. Microbiological Procedures including: ❖ Phase-contrast Microscope
a. Reporting all spills and broken glassware to the ➢ Can be used to observe unstained living microorganisms
instructor and receiving instructions for cleanup ❖ Fluorescence Microscope
b. Methods for aseptic transfer ➢ Fluorescent dye attached to organism
c. Minimizing or containing the production of aerosols ➢ Primarily an immunodiagnostic technique
and describing the hazards associated with aerosols (immunofluorescence)
d. Washing hands prior to and following laboratories and ➢ Used to detect microbes in cells, tissues and clinical
at any time contamination is suspected specimens
e. Never eating or drinking in the laboratory
f. Using universal precautions
g. Disinfecting lab benches prior to and at the conclusion
of each lab session
h. Identification and proper disposal of different types of
waste
• Microorganisms: Soak in 1:10 bleach or spray with
Lysol and Autoclave.
i. Never applying cosmetics, including contact lenses, or
placing objects (fingers, pencils) in the mouth or
touching the face
j. Reading and signing a laboratory safety agreement
indicating that the student has read and understands
the safety rules of the laboratory
k. Good lab practice, including returning materials to
proper locations, proper care and handling of
equipment, and keeping the bench top clear of
extraneous materials
2. Protective procedures, including:
a. Tying long hair back, wearing personal protective
equipment (eye protection, coats, closed shoes;
glasses may be preferred to contact lenses), and
using such equipment in appropriate situations
b. Always using appropriate pipetting devices and
understanding that mouth pipetting is forbidden
3. Emergency procedures, including:
a. Locating and properly using emergency equipment
(eyewash stations, first-aid kits, fire extinguishers,
chemical safety showers, telephones, and emergency
numbers)
b. Reporting all injuries immediately to the instructors
c. Following proper steps in the event of an emergency
[BACT211] 1.01 Laboratory Safety and Microscopy I Prof. Rochelle D. Darlucio, RMT, MPH
II. MICROSCOPY
❖ Microscope
➢ An optical instrument that is used to observe tiny objects,

often objects that cannot be seen at all with the unaided
human eye
(the “naked eye”)
❖ Anton van Leeuwenhoek (1632 – 1723)
➢ The first person to see live bacteria and protozoa
➢ “father of microbiology, bacteriology, protozoology”
➢ During his lifetime, he made more than 500 single-lens
microscopes or simple microscopes
B. Types of Microscope
Page 3 of 3
(M & N) Fine and On the arm of Used to focus the objective lenses
Coarse the microscope Fine Adjustment Knob: Higher
Adjustment near the base Magnification (HPO, Oil immersion
Knobs objective)
Coarse Adjustment Knob: Scanner
and low power objective
(O) Arm Supports the binocular body and the
revolving nosepiece; held with one
hand when carrying the microscope,
with the other hand beneath the base
to support the weight of the
microscope
(P) Binocular Body Holds the ocular lenses in their
proper locations
Note:
Scanner = 4x
Low power objective = 10x
High power objective = 40x
Oil Immersion Objective = 100x
D. Proper Use of the Microscope
1. Carry microscope with two hands, supporting the base with

one hand
2. Always hold the microscope in vertical position
3. Only clean optical surfaces with good quality lens tissue
and commercial lens cleaner
4. Do not use the 10x and 40x objectives with oil
5. Clean the oil immersion after use
6. Always remove slides with the low-power objective raised
7. Store the microscope with the scanner objective in position
and the stage centered
C. Parts and Function of Microscope ❖ Oil Immersion Objective: (Cedarwood Oil)
Component Location Function E. Magnification Formula for Total Magnification

(A) Ocular lens At the top of the The ocular lens is an x10 magnifying
(also known as an microscope lens
eyepiece); a
monocular
microscope has ❖ Total Magnification = Eyepiece x Objective
one; a binocular ➢ Example: Eyepiece Magnification = 10x
microscope has Objective Magnification = high power field (40x)
two Total Magnification = (10x) (40x) = 400x total
(B) Revolving Above the stage Holds the objective lenses magnification
nosepiece
❖ Actual Size (um) = Magnification of Objective Lenses / Image
(C) Objective Held in place Used to magnify objects placed on
Size (um)
Lenses above the stage the stage
by the revolving ➢ Example:
nosepiece Oil Immersion Field of View = 0.20mm →
(D) Stage Directly beneath Flat surface on which the specimen 200um
the nosepiece is placed Image size measured = 1000um
and objective Estimate Size (um) = 200um/1000um = 0.20um
lenses
actual size
Stage adjustment Beneath the Used to move the stage and
knobs (not shown) stage microscope slide
(E) Iris Diaphragm On the Used to adjust the amount of light
Control Arm condenser passing through the condenser
(F) Condenser Beneath the Contains a lens system that focuses
stage light onto the specimen
(G) Collector Lens Beneath the Controls the amount of light entering
with Field condenser the condenser
Diaphragm
(H) Rheostat Front side of the Controls the amount of light emitted
Control Knob base from the light source
(I) Field Attached to the Used to adjust the amount of light
Diaphragm Lever field diaphragm passing through the collector lens
(J) On/ Off Switch On the side of Turns the light source on and off
the base
(K) Base Contains the light source
(L) Condenser Beneath and Used to adjust the height of the

Control Knob behind the condenser
condenser
Page 4 of 3
2020-
ASEPTIC TECHNIQUE LAB 2021 2nd
SEM
CLINICAL BACTERIOLOGY 2 BACT21
Transcriber: GAD. MPL. 1
Date: February 19, 2021 LAB
[TRANS] UNIT 2: ASEPTIC TECHNIQUE
LOOPS AND
NEEDLES
I. Introduction
II. Transfer from Broth Culture to Another
Broth
III. Transfer of Bacteria•fromAaloop
Slantor
IV. Working with Agar Platesneedle is
V. Pure Culture Techniquessterilized by
A inserting it
Streak Plate Method
i. into a Bunsen
Radiant Streak iii. Continuous Streakburner,
Italicized – additional information
incinerator
INTRODUCTI flame or
ON alcohol lamp
 Aseptic until it is red-
Technique hot.
means using • This will
practices and incinerate any
procedures to contaminating
prevent organisms
contamination that may be
. It involves present.
applying the • Allow the loop
strictest rules to cool
to minimize completely
the risk of *not cooling
infection. the loop may
kill the
WORK AREA bacteria
DISINFECTION before picking
up inoculum.
This will
ensure that
• The work
viable cells
area is first
are
treated with a
transferred.
disinfectant to
kill any • Also called
microorganis inoculating
ms that may loop/needle
be present. • Loops – circle
on the end
• Ensure
• Needle –
organism
straight on
does not
the end
contaminate
• Proper
the handler.
Handling
• Can use any
 Assu
different type
me
of disinfectant
the

loop/
Sodium
need
hypochlorite or
le is
Lysol
a
[BACT211] TRANS: ASEPTIC TECHNIQUE | Prof. Rochelle D. Darlucio, RMT, MPH
ballp inoculated by
en drawing the
 Use loop up the
domi surface of the
nant slant from the
hand bottom of the
on slant to its
holdi top.
ng • For stab
• In sterilizing cultures, a
the needle is
loop/needle, inserted into
pass through the agar
flame starting medium by
to tip to base stabbing it
or base to tip into the agar.
• Open the
tube by the
use of
dominant
hand pinky
finger, while
the non-
dominant
hand is
holding the
tube
CULTURE
TUBE FLAMING
AND
INOCULATION
• Prior to
inserting a
cooled loop or
needle into a
culture tube,
the cap is
removed and
the mouth of
the tube may
be flamed.
• If the tube is a
broth tube,
the loop is
inserted into
the tube and
twisted
several times
to ensure that
the organisms
on the loop
are delivered PETRI PLATE INOCULATIONS
to the liquid.
• If the tube is
• The plate
an agar slant,
cover is
the surface of
raised
the slant is
PAGE 2 OF 6
*slightly open • It is important
only and held not to gouge
diagonally or disturb *do
over the plate not press the
to protect the agar, mild
surface from streaking only
any the surface of
contamination the agar with
in the air. the loop.
• The loop • The cover is
containing the replaced and
inoculum is the loop is
then streaked flamed.
gently over
the surface of
the agar.
PAGE 3 OF 6
PAGE1OF6
PAGE 4 OF 6
container.
12. Incubate the cultur
48 hours.
 Disinfect the table

 Sterilize the inocula
 Hold the broth and
 Open the broth by
the tube to flame
 Get a colony – one
 Pass through flame
FINAL FLAMING OF THE LOOP OR NEEDLE

 After the inoculation is complete, the loop or needle is
flamed to destroy any organisms that remain on these
8. Grasp a tube of sterile nutrient broth with
your free hand, carefully remove the cap
with your little finger, and flame the mouth
of this tube.
9. Without flaming the loop, insert it into the
sterile broth, inoculating it. To disperse

the organisms into the medium, move the
loop back and forth in the tube.
10. Remove the loop from the tube and flame
the mouth. Replace the cap on the
11. Sterilize the loop by flaming it. Return the
loop to its
• The loop or needle is then returned to its

receptacle for storage.
• It should never be placed on the desk
surface.
PAGE 5 OF 6
FINAL DISINFECTION OF THE WORK
AREA
 When all work for the day is complete, the
work area is treated with disinfectant to
insure that any organism that might have
been deposited during any of the
procedures is killed.
TRANSFER FROM BROTH

CULTURE TO ANOTHER
BROTH
MATERIALS
• Broth culture of Escherichia coli
• Tubes of sterile nutrient broth
• Inoculating loop
• Bunsen burner or incinerator
• Disinfectant for desktop and paper
towels
• Marking pen
PROCEDURE
1. Prepare your desktop by swabbing
down its surface with a disinfectant.
Use a sponge or paper towels.
2. With a marking pen, label a tube of
sterile nutrient broth with your initials
and E. coli
3. Sterilize your inoculating loop by
flaming it until it becomes bright red.
The entire wire must be heated.
4. Using your free hand, gently shake
the tube to disperse the culture
5. Grasp the tube cap with the little
finger of your hand holding the
inoculating loop and remove it from
the tube. Flame the mouth of the
tube.
 Note: if an incinerator is used,
the tube is not flamed.
6. Insert the inoculating loop into the
culture
7. Remove the loop containing the
culture, flame the mouth of the tube
again, and recap the tube. Place the
culture tube back on the test tube
rack.
PAGE 6 OF 6
T
R
A
N
S
F
E
R
O
F
B
A
C
T
E
R
I
A
F
R
O
M
S
L
A
N
T
MATERIALS
• Agar slant culture of E. coli
 Sterilize the inoculating loop

 Open the tube, pass through flame
 Get colony by twirling the loop
 Pass through flame and close the tube
 Get your slant, open and pass through flame
 Inoculate in slant by streaking (zigzag motion) from
bottom to top. Do not put too much pressure
 Pass through flame before closing
 Sterilize the loop
PAGE 7 OF 6
• Sterile nutrient agar slant
• Inoculating loop
• Bunsen burner or incinerator 
Marking pen
PROCEDURE
1. If you have not already done so,

prepare your desktop by swabbing
down its surface with a disinfectant.
2. With a marking pen, label a tube of
nutrient agar slant with your initials
and E. coli.
3. Sterilize your inoculating loop by
holding it over the flame of a Bunsen
burner until it becomes bright red.
The entire wire must be heated.
Allow the loop to cool completely.
4. Using your free hand, pick up the
slant culture of E. coli and remove
the cap using the little finger of the
hand that is holding the loop.
5. Flame the mouth of the tube and
insert the cooled loop into the tube.
Pick up some of the culture on the
loop and remove the loop from the
tube.
6. Flame the mouth of the tube and
replace the cap, being careful not to
burn your hand. Return tube to rack.
7. Pick up a sterile nutrient agar slant
with your free hand, remove the cap
with your little finger as before, and
flame the mouth of the tube.
8. Without flaming the loop containing
the culture, insert the loop into the
tube and gently inoculate the surface
of the slant by moving the loop back
and forth over the agar surface, while
moving up the surface of the slant.
This should involve a type of
serpentine or zigzag motion.
9. Remove the loop, flame the mouth of
the tube, and recap the tube.
Replace the tube in the rack.
10. Flame the loop, heating the entire
wire to red hot, allow to cool, and
place the loop in its container.
11. Incubate the inoculated agar slant at
37C for 24-48 hours.
WORKING WITH AGAR PLATES
• Loops and Needles

 Loops are routinely used when
streaking agar plates and slants.
When used properly, a loop will not
gouge or tear the agar surface.
Needles are used in transfers
involving stab cultures.
• Plate Handling
 Media in plates must always be
protected against contamination. To
prevent exposure to air
contamination, covers should always
PAGE 8 OF 6
be left closed. When organisms are
removed from a plate culture, the
cover should be only partially
opened.
 Slightly open the cover only
 To prevent contamination, work near
the flame
• Flaming the
Procedures inoculate
 Inoculatin d
g loops or organism
needles s.
must be  The label
flamed in must be
the same not too
manner big or not
that you too small
used  Name of
when organism
working and date
with
previous
tubes.
One
difference
when
working
with
plates is
that
plates are
never
flamed!
• Plate
Labeling and
Incubation
 Petri
plates
containin
g
inoculate
d media
are
labeled
on the
bottom of
the plate.
Inoculate
d plates
are
almost
always
incubated
upside
down. PROCEDURE
This
prevents 1. If you
moisture
have not
from
done so,
condensi
swab
ng on the
your work
agar
area with
surface
disinfecta
and
nt. Allow
spreading
PAGE 9 OF 6
area to contamin
dry. ation.
2. Label a Always
sterile close the
nutrient lid once
agar slant you have
with your removed
name and organism
organism s from the
to be plate.
transferre 5. With your
d. free hand,
3. Flame an pick up
inoculatin the sterile
g loop nutrient
until it is agar slant
red-hot. tube.
Allow the Remove
loop to the cap
cool. by
4. Raise the grasping
lid of a the cap
petri plate with the
sufficientl little
y to finger of
access a the hand
colony that is
with your holding
sterile the loop.
loop. Do 6. Flame the
not gouge mouth of
the agar the tube
with your and insert
loop as the loop
you pick into the
up tube to
organism inoculate
s. Simply the
allow the surface of
loop to the slant,
gently using a
glide over serpentin
the e motion.
gelatin Avoid
like disrupting
surface of the agar
the agar. surface
Do not with the
completel loop.
y remove 7. Remove
the lid the loop
while from the
inoculatin tube and
g or flame the
removing mouth of
organism the tube.
s from the Replace
agar the cap
plate. on the
This will tube.
expose 8. Flame the
the agar loop and
surface to place it in
air and its
potential container.
PAGE 10 OF 6
9. Incubate PURE
CULTURE
the TECHNIQUES
nutrient STREAK
PLATE
agar METHOD
slant • Pure culture

 Singl
e
at
kind
of
37C orga
nism
for • Mix culture
 More
24-48 than
hours. one
kind
of
micr
oorg
anis
m
PAGE 11 OF 6
QUADRANT STREAK
 The difference of method A to B is that, B ends with a
PAGE 12 OF 6
 90̊rotation is impo
1. Streak one loop full of microorganism and apply on

quadrant 1.
2. Sterilize the loop
3. Streak again on quadrant 2 by touching quadrant 1.
5. Streak again on quadrant 3 by touching quadrant 2.
7. Streak line on quadrant 4 by touching quadrant 3.
 Do not put too much pressure

 Streak fast as possible
 Keep the plate cover half open when streaking
 90̊rotation if quadrant method
zigzag.
PAGE 13 OF 6
RADIANT STREAK
 Start streaking on the top to the middle/half of the

plate
 Rotate 180̊
 Streak again
 Reminder that there is no need to sterilize the
inoculating loop and no touching to the previous
quadrant
PAGE 14 OF 6
CONTINUOUS STREAK
PAGE 15 OF 6
SMEAR PREPARATION & LAB 2020-
2021
SIMPLE STAINING 3 2nd SEM

BACT21
CLINICAL BACTERIOLOGY 1
Transcriber: GAD. MPL.
[TRANS] UNIT 3: SMEAR PREPARATION
OUTLINE
Bacterial Smear A. From
Broth Cultures
B. From Plates and Slants
II. Simple Staining
Note:
 (3) Internal structures ( endospores )
FROM BROTH CULTURES

BACTERIAL SMEAR
• Dried preparation of bacterial cells on a glass slide.
• Different from blood smear
PROPERLY PROCESSED SMEAR
• The bacteria are evenly spread out on the slide in such a

concentration that they are adequately separated from one
another
• The bacteria are not washed off the slide during staining
• Bacterial form is not distorted ( can be identify if cocci or
baccili )
GOOD SMEARS ARE CRITICAL FOR DISCERNING

• (1) The morphology of cells ( if cocci or baccili )
• (2) The arrangement of cells ( if singly or pairs )
1. Wash a slide with soap and hot water, removing all dirt and grease. Handle the clean slide by its edges.
2. Write the initials of the organism or organisms on the left hand side of the slide with a marking pen.
3. To provide a target on which to place the organisms, make a circle on the bottom side of the slide, centrally located, with a marking
pen. Later on, when you become more skilled, you may wish to omit the use of this “target circle.”
4. Shake the culture vigorously and transfer two loopfuls of organisms to the center of the slide over the target circle. Be sure to flame
the loop after it has touched the slide.
5. Spread the organisms over the area of the target circle.
6. Allow the slide to dry by normal evaporation of the water. Don’t apply heat.
7. After the smear has become completely air dried, place the slide in a clothespin and pass the slide several times through the
Bunsen burner flame.
PAGE1OF2
[BACT211] TRANS: SMEAR PREPARATION | Prof. Rochelle D. Darlucio, RMT, MPH
CAUTION
•
Be sure to cool the loop completely before inserting it into a medium.
A loop that is too hot will spatter the medium and move bacteria
into the air.
• Avoid prolonged heating of the slide as this can result in the slide
shattering and injuring you. The underside of the slide should feel
warm to the touch
• Broth cultures can be directly placed into slides.
• Plate and Slant cultures must first placed a sterile distilled water
or sterile normal saline solution (NSS) before emulsifying.
• Heat fixing
• To adhere bacterial smear to slide
• To prevent washing off during staining
FROM PLATES AND SLANTS 1.
1.
Wash a slide with soap and hot water, removing all dirt and
grease. Handle the clean slide by its edges.
2. Write the initials of the organism or organisms on the left
hand side of the slide with a marking pen.
3. Mark a “target circle” on the bottom side of the slide with a
marking pen.
4. Flame an inoculating loop, let it cool, and transfer two
loopfuls of water to the center of the target circle.
5. Flame an inoculating needle and then let it cool. Pick up a
very small amount of the organisms, and mix it into the water on the slide. Disperse the mixture over the area of the target
circle. Be certain that the organisms have been well emulsified in the liquid. Be sure to flame the inoculating needle before
placing it in its holder.
6. Allow the slide to dry by normal evaporation of the water. Don’t apply heat.
7. After the slide has become completely dry, place it in a clothespin and pass it several times through the flame of a Bunsen
burner. Avoid prolonged heating of the slide as it can shatter from excessive exposure to heat.
SIMPLE STAINING
• The use of a single stain to color a bacterial cell

• Commonly used dyes for performing simple staining are methylene
blue, basic fuchsin, and crystal violet.
• These are referred to as basic dyes because they have color bearing
ionic groups (chromophores) that are
positively charged (cationic).
PAGE2OF2
GRAM STAINING &

I Gram Staining
OUTLINE LAB 2020-
A Gram Stain Procedure 2021
II Acid Fast Staining Kinyoun Method
Note:
ACID-FAST STAINING
4 2nd SEM
GRAM STAINING BACT21
CLINICAL BACTERIOLOGY
1
Transcriber: GAD. MPL. LAB
Date: March 4, 2021
[TRANS] UNIT 4: GRAM STAINING & ACID FAST STAINING
• Differential Stain
 Prolong decolorization, gram positive might appear as gram
negative
GRAM STAIN PROCEDURE

• Two kinds of cells, gram positive and gram negative, are
differentiated
• The Gram stain was first used in 1884 by Hans Christian Gram
• In differential staining, 2 dyes/reagent are required:
o Crystal Violet o Safranin Red
SEVERAL FACTORS CAN AFFECT THE OUTCOME OF THE PROCEDURE
1. It is important to use cultures that are 16-18 hours old

2. It is critical to prepare thin smears
3. Decolorization is the most critical step in the Gram stain procedure.
1. Cover a heat fixed smear with crystal violet and let stand for 30 seconds
2. Briefly wash off the stain, using a wash bottle of distilled water. Drain off excess water.
3. Cover the smear with Gram’s iodine solution and let it stand for 1 minute. (Your instructor may prefer only 30 seconds for this step.)
Wash off the Gram's iodine.
4. Hold the slide at a 45 degree angle and apply the decolorizer, allowing it to flow down the surface of the slide. Do this until the decolorizer is
colorless as it flows from the smear down the surface of the slide. This should take no more the 15 seconds for properly prepared smears.
*quick on rinse Note: Thick smears can take longer for decolorization. Stop decolorization by washing the slide with a gentle stream of
water.
5. Cover the smear with safranin for 1 minute.
6. Wash gently for a few seconds, blot dry with bibulous paper, *tissue and air dry.
7. Examine the slide under oil immersion.
Page 1 of 3
[BACT211] TRANS: GRAM STAINING & ACID FAST STAINING | Prof. Rochelle D. Darlucio, RMT, MPH
ACID FAST STAINING KINYOUN METHOD Mycolic Acid

• A complex lipid that is composed of fatty acids and fatty
Acid Fast Staining alcohols that have hydrocarbon chains up to 80 carbons in
• An important diagnostic tool in the identification of Mycobacterium length.
tuberculosis, the causative agent of tuberculosis, and Mycobacterium
leprae, the bacterium that causes leprosy in humans.
• Affects the staining properties of bacteria and prevents them from
being stained by many of the stains routinely used in microbiology.
Page 2 of 3
REPORTING
1. Indicate the gram stain reaction ( positive or negative)

2. Indicate the shape and morphology (cocci or bacilli)
3. Indicate the arrangement (singly, pairs, chains, pairs, or clusters
[BACT211] TRANS:
GRAM STAINING & ACID FAST STAINING | Prof. Rochelle D. Darlucio, RMT, MPH
[BACT211] TRANS: MICROBIOLOGICAL CULTURE MEDIA PREPARATION & STERILIZATION | Prof. Rochelle D. Darlucio, RMT, MPH
Mixed cultures under the microscope
PAGE 2 OF 3
Reporting:
 Gram positive cocci in clusters
Reporting:
PAGE OF 3
 If many arrangements:
 Gram positive cocci in singly, pairs, and clusters
PAGE 2 OF 3
Reporting:
 Gram positive bacilli in singly
Gram-stained Smear from specimens
Reporting:
 Gram negative cocci in pairs (diplococci)
 Gram negative bacilli in singly
Reporting:
 Gram negative bacilli in clusters
 Gram negative bacilli in singly
Reporting:
 Gram positive bacilli in clusters
PAGE OF 3
Page 3 of 3
Reporting:
 Gram positive bacilli
PAGE 2 OF 3

Dark violet - spores
PAGE OF 3
MICROBIOLOGICAL
CULTURE MEDIALAB 2020-
2021
PREPARATION
AND STERILIZATION 5 2nd SEM
CLINICAL BACTERIOLOGY
BACT21
PAGE 2 OF 3
1
Transcriber: GAD. MPL. Date: March 19, 2021 LAB
[TRANS] UNIT 5: MICROBIOLOGICAL CULTURE,

MEDIA PREPARATION & STERILIZATION
PAGE OF 3
OUTLINE CULTIVATION
I Cultivation
i. 3 Main Purposes  of Cultivating
Process Bacteria
of growing
ii. Nutritional Requirements
microorganisms in culture by
A Terms to Remember taking bacteria from the
II Culture Media infection site (in vivo) and
A Classification growing them in the culture
i. According to Consistency
media of the laboratory (in
ii. According to Chemicalvitro)
Composition
iii. According to Function
B Culture Media Preparation Steps
C
3 MAIN PURPOSES OF
Culture Media Calculations
III Sterilization CULTIVATING BACTERIA:
i. Three Basic Ways of Sterilization of Media
IV Inoculating Techniques
Note:
PAGE 2 OF 3
1. To grow 3. To obtain sufficient
and growth of clinically
isolate all relevant bacteria to allow
bacteria identification and
present in characterization
the
clinical NUTRITIONAL
specimen.
2. To determine which of REQUIREMENTS
the bacteria that grow
is most likely causing • Water
infection and which
are likely causing
• Ions
infection and which are • Nitrogen
likely contaminant. • Gases
PAGE OF 3
• Sources of carbon medium  Pure culture -
Latter requirements: contains only one specie
carbohydrate & protein  Mix culture -
contains several
species
TERMS TO REMEMBER:
 Contaminated
1. Culture Media -
culture - contains
nutrients prepared for unwanted species
bacterial growth or organisms
2. Inoculum - suspension 5. Colony - visible growth of
of microorganism microorganism on the
3. Inoculation - surface of culture media
introduction of bacteria 6. Fastidious Organism -
into culture medium nutritional needs are
4. Culture - bacteria relatively complex and
growing on culture
PAGE 2 OF 3
exceptional • Solid, liquid or
components used for semi-solid
the growth designed to
7. Non-fastidious support the
Organism - nutritional growth of
needs are relatively microorganisms
basic and CLASSIFICATION
straightforward.
I. ACCORDING TO
CULTURE MEDIA
CONSISTENCY
• Nutrient preparations that
are used for culturing 1. Solid – solidifying
microorganisms agent is added (1.5 3% of agar)
PAGE OF 3
• Agar is the most  For pure
commonly used as culture
solidifying agent isolations
• Melt: ≥95 C   Storage of
Resolidify: <50 C cultures
 To observe
• Uses:
specific
 For the biochemical
surface reactions.
growth of
microorganis Solid media can be poured
ms in order into either a test tube or petri
to observe plate (dish)
colony
appearance • In tube:
PAGE 2 OF 3
1. Agar Slant -  Agar Plate –
the medium in containing
the test tube is about 15 to 16
allowed to ml of media.
harden in a • 20 ml (standard)
slanted
position.
2. Agar deep -
the tube is
allowed to
harden in an
upright
position.
• In Plate:
PAGE OF 3
2. Liquid / Broth microor
dissolved in water ganism
• Uses: s in
 fermen
Can tation
be studies
used  For
to various
prop bioche
agat mical
e tests.
large • Turbid -
num presence of
bers microorganism
of
PAGE 2 OF 3
a. Ex. Sulfide
Indole Motility (SIM) Medium
 Uses:
 Fe
rm
en
tati
on
stu
3. die
solidifying agent is added (<1.5% s
of agar)  In
det
PAGE OF 3
e ial
r mo
m tilit
i y 
n
i
Pr
n
om
g
oti
ng
b an
a aer
c obi
t c
e gro
r
PAGE 2 OF 3
w  Appearance: spreading
t of the colony along the
h site of innoculation
 Negative (non-motile)
 Appearance: straight
line
1
 Uninocoluted II. ACCORDING TO
Appearance: clear

CHEMICAL COMPOSITION
 Manner of
innoculation: Stab
(half way) using the
incolutating needle 1. Synthetic
 Positive for motility  Chemically defined
PAGE OF 3
 Use to culture broth, Nutrient agar,
algaes or EMB
nonfastidious
heterotrophs
III. ACCORDING TO
2. Non-synthetic
(complex) FUNCTION
 Chemical
composition is not
specifically 1. Enrichment media
defined; extracts  Contains specific
of yeasts, meat or nutrients required
plants. (Bacteria for the growth of
are usually particular bacterial
grown in this pathogens
type of media)
Ex: Nutrient
PAGE 2 OF 3
 Used to enhance  Favour the growth
the growth of of a particular
particular bacterium by
pathogen from a inhibiting the growth
mixture of of undesired
organism by bacteria and
using nutrient allowing the growth
specificity of desired bacteria.
• Example: Buffered-charcoal • Example: EMB, MAC, SSA,
yeast extract agar (BCYE) HEA, MSA
that contains L-cysteine
required for the growth of 2. Enriched Medium
Legionella pneumophilia  Enriched usually by
adding blood, or
2. Selective Media eggs.
PAGE OF 3
 Contain nutrients to
support growth of
most nonfastidious
organisms without
giving any particular
organism a growth
advantage
 General culture
media
3. Differential Medium
• Example: Blood agar plate  Used to distinguish
and Chocolate agar plate them from other
2. Supportive / bacteria growing on
Nonselective Medium the same agar plate
 Contains indicator
PAGE 2 OF 3
Example: EMB, MAC, SSA,
HEA, MSA
PAGE OF 3
CULTURE MEDIA
PREPARATION STEPS
TUBE (WDDS) PLATE (WDSD)
1. Weight 1. Weigh
2. Dissolve 2. Dissolve
3. Dispense 3. Sterilize
4. Sterilize 4. Dispense
 Instructions,
procedures,
directions is in
the bottle on how
PAGE 2 OF 3
to prepare the CULTURE MEDIA
agar.
CALCULATIONS
 There is a
specific grams
per 1000 mL of • We should be reading
distilled water. “directions” listed on
 Approximateof media base container
20 carefully
mL in • For example, on the
every container of nutrient
plate. agar the following
(Standard directions are listed:
amount)  Example:
• Suspend 28 g of
powder in 1000 mL of
PAGE OF 3
D.W., then dissolve dissolve 28.0 grams
by heating until of media powder in
boiling. Autoclave 1000 mL of distilled
for 20 min, then water.
cool and pour in • The amount of media poured
Petri-dishes.
in each Petri-dish is about 20
 FORMULA FOR
mL.
DESIRED
• If we don't need 1000 mL of
VOLUME:
media; we should multiply the
• 20 ml x (desired number of dishes needed by
plates) 20, then consider it as the
 So, if we need to total volume (V) of media
prepare 1000 mL needed in the following
(1 L) of nutrient equation.
agar medium, we
should to be
PAGE 2 OF 3
𝑊. 𝑜 𝑚𝑒 𝑒𝑚𝑊𝑖 ()
()
=
𝑜 . . 𝑒𝑚𝑊𝑖 (𝑚)
. . 𝑚 (𝑚)
 Ex.
28
=
1000 𝑚
400 𝑚
PAGE OF 3
= 11.2
 All volumes should be converted to (mL), and
weights to (g) before beginning calculations.
 From (L) to (mL) multiply by 1000, while from (mL) to (L)
divided by （1000).
 From (Kg) to (g) multiply by 1000, while from (g) to (Kg)
divided by （1000).
• If the directions: suspend 28 g of powder in 1000 mL of D.W.,

then dissolve by heating with frequent agitation until boiling.
Autoclave for 20 min, then cool and pour in Petri-dishes. THREE BASIC WAYS OF STERILIZATION OF MEDIA:
• If we need (14) Petri-shish instead of entire 1000 mL, the volume • Autoclaving
of media we need to prepare calculated as (14 x 20 = 280 mL) of  Exposure to steam at 121 C and 15 lbs of pressure for
media. 15 minutes or longer, depending on the nature of the
item.
28  Microorganisms, even endospores , will not survive
= longer than about 12 to 13 minutes
1000 𝑚 280 𝑚
28 × 280 𝑚
= = 7.48
1000 𝑚
 7.48 g of powder should be dissolve in 280 mL of

D.W.
 After computation, use aluminum foil to weigh the powder.

 Weigh it using the analytical balance.
 Measure the water using graduated cylinder then place it
in Erlenmeyer flask. • Ultraviolet (UV) radiation
 REMEMBER: Transfer the first half of the water into the  Around 260 nm is quite lethal to many microorganisms
Erlenmeyer flask. but does not penetrate glass, dirt films, water, and other
substances very effectively.
 Add the powder, mix until it dissolves then pour the
remaining half of the water. • Ethylene oxide
 Sterilize.  Both microbicidal and sporicidal and kills by covalently
attaching to cell proteins.
 Dispense. (work near the flame)  ALWAYS wear a
 It is a particularly effective sterilizing agent because it
mask in preparing the agar. rapidly penetrates packing materials, even plastic wraps.
BLOOD AGAR PREPARATION INOCULATION TECHNIQUES
1. Weigh  Tube
2. Dissolve 1. LIQUID MEDIA
3. Sterilize  Plain Broth, TSB
4. Make sure the agar is cooled down. (to avoid the lysis of RBC or the  Material: Inoculating loop
creation of chocolate agar)  Manner of inoculation: Twirl / Mixing
5. Add 5% defibrinated blood (avoid bubbles) : Blood is agitated using
glass beads (marbles) o How to compute the 5%?
 5% of the total volume
 Ex. 200ml of water. 5% = 10ml
6. Dispense
• If the medium lacks agar, the powder will usually dissolve without
heating.
• If it contains agar, you must heat the medium until it starts to
simmer or boil in order to completely dissolve the agar.
2. SLANTED MEDIA
STERILIZATION  Simmon's Citrate, Urea
• The process of rendering a medium or material free of all forms of  Material: Inoculating needle / Wire loop  Manner of
life. inoculation : Streak / Line
PAGE 2 OF 3
3. SEMI SOLID BUTT MEDIA
 SIM
 Material: Inoculating needle / Wire loop
 Manner of inoculation: Stab Halfway
4. BUTT SLANTED MEDIA

 TSI, LIA
 Material: Inoculating needle / Wire loop
 Manner of inoculation: Stab & Streak
 CHROMagar
OLFU LABORATORY
• Staphylococci
round, smooth,
produce
white,
DIAGNOSIS
STAPHYLOCOCCI 2021nd
creamy colonies on SBA
after 18 to 24 hours of
incubation at 35C to 37 C
College of Medical 6 2 SEM
SPECIMEN
COLLECTION AND
• S. aureus can produce
hemolytic zones around the
HANDLING colonies and may rarely
Laboratory Science CLINICAL exhibit pigment production

Clinical materials collected
with extended incubation.
BACTERIOLOGYTranscriber: GAD. MPL. from infected sites should
BACT211be transported to the
laboratory without delay to
prevent drying, maintain the
Batch 2023 Date: March 19, 2021 proper environment, and
LAB minimize the growth of
contaminating organisms. o
[TRANS] UNIT 6: STAPHYLOCOCCI
scalded skin syndrome Turbidity of the
specimen - presence of
microorganism
OUTLINE:
I. Staphylococcus
II. Micrococcus MICROSCOPIC
III. Laboratory Diagnosis EXAMINATION
A Specimen Collection & Handling
B Microscopic Examination
C Cultivation • Gram positive cocci
i. Media of Choice • Gram stains should be
D Catalase Test performed on young
E Coagulase Test cultures
F Slide Coagulase Test
G Tube Coagulase Test
H Bacitracin Susceptibility Test
I Novobiocin Disk Test
Note:
(SSS), and toxic shock
syndrome (TSS), are also
associated with this
organism.
STAPHYLOCOCCUS
• Derived CATALASE TEST
from the • This test differentiates
Greek term catalase positive micro-
staphle, coccal and staphylococcal
meaning CULTIVATION species from catalase
“bunches of negative streptococcal
grapes species
MEDIA OF CHOICE
• Catalase • Principle: The enzyme,
producing, catalase, is capable of
Gram • Grow on 5% sheep blood converting hydrogen
positive and chocolate agars peroxide to water and
cocci that oxygen. The presence of
• They also grow well in broth
appear in singly, in pairs, MICROCOCCUS blood culture systems and enzyme in bacterial isolate
and in cluster. • Catalase producing, causes rapid elaboration of
common nutrient broths
• Non-motile , non-spore coagulase negative, gram bubbles.  Reagent :3%
forming, and aerobic or positive cocci
Selective Media: Hydrogen peroxide(H2O2)
facultatively anaerobic • They are often recovered • Results:
except for S. with staphylococci and can  Phenylethyl
Saccharolyticus, which is an alcohol (PEA)  Positive: Bubbles
be differentiated easily from
obligate anaerobe  Columbia colistin (Rapid bubbling) /
coagulase negative
nalidixic acid Effervescence
• Toxin induced diseases, staphylococci (CoNS)
(CNA) agars  Negative: No
such as food poisoning,
 Mannitol Salt Agar bubbles
PAGE 2 OF 3
PAGE 1 OF 3
Method
[BACT211] TRANS: STAPHYLOCOCCI | Prof. Rochelle D. Darlucio, RMT, MPH  Slide Test (Detection of Bound Coagulase or Clumping
Factor)
• Purpose  1. Place a drop of coagulase plasma (preferably rabbit plasma

 This test differentiate catalase-positive micrococcal and with ethylenediaminetetraacetic acid (EDTA) reagent on the
staphylococcal species from catalase-negative reaction provided by the manufacturer.
streptococcal species 2. Place a drop of distilled water of saline next to the drop of
• Principle plasma in an adjacent reaction well as a negative control.
3. Place a drop of coagulase plasma reagent in a third adjacent
 Aerobic and facultative anaerobic organisms produce two toxins
reaction well as a positive control.
during normal metabolism, hydrogen peroxide (H 2O2) and
superoxide radical (O2-). These bacteria have two enzymes that 4. With a loop, straight wire, or wooden stick, emulsify a portion of
detoxify the products of normal metabolism. One of these the isolated colony being tested in the rabbit plasma reagent.
enzymes, catalase, is capable of converting hydrogen peroxide Try to create a smooth suspension.
to water and oxygen. The presence of the enzyme in a bacterial 5. Mix well with a wooden applicator stick.
isolate is evidenced when a small inoculum introduced into 6. With a loop, straight wire, or wooden stick, emulsify a known
hydrogen peroxide (30% for the side test causes rapid Staphylococcus aureus in the positive and negative control
elaboration of oxygen bubbles. The lack of catalase is evident by walls.
a lack of or weak 7. Mix all samples well with a new wooden applicator stick for
bubble production  each sample
8. Rock the slide gently for 5 to 10 seconds. Expected Results
• Method
 Positive: Macroscopic clumping in 10 seconds or less in
1. Use a loop or sterile wooden stick to transfer a small amount of coagulated plasma drop positive control, unknown clinical
colony growth to the surface of a clean, dry glass side. isolate along with no clumping in saline or water drop
2. Place a drop of 30% hydrogen peroxide (H2O2) onto the medium.  Negative: No clumping in the unknown clinical isolate well
(3% can also be used for most organisms.)
3. Observe for the
B. TUBE COAGULASE TEST
S. aureus colonies are incubated with plasma • Confirmatory test
evolution of oxygen • Detects the presence of free
bubbles.  Reagent: Rabbit’s plasma or human plasma from EDTA *do not get from
citrate since it may cause a false positive result. coagulase  Results:
• Expected Results
 Positive: Copious
bubbles are produced as long as the positive and negative controls demonstrate
 Negative: No or few bubbles are produced appropriate reactions as described.
Note: All negative side tests must be confirmed using the tube test.
• Limitations
extracellular protein enzyme that causes the formation of a clot when
 Some organisms (enterococci) produce a peroxidase that slowly
catalyzes the breakdown of H2O2, and the test may appear A. SLIDE COAGULASE TEST
weakly positive. This reaction is not a truly positive test. Positive: Negative: MicrococcusS. aureus
 False positives may occur if the samples is contaminated with
• Screening test
blood agar.
• Quality Control
Purpose
 Positive: Staphylococcus aureus (ATCC25923)   The test is used to differentiate Staphylococcus aureus (positive)
 from coagulase-negative staphylococci
 Negative: Streptococcus pyogenes (ATCC19615) (negative). 
• Whether positive or
negative it must
COAGULASE TEST proceed to Tube
• The test is used to differentiate Staphylococcus aureus (positive) from coagulase test
coagulase negative staphylococci (negative). • For the detection of
• Principle: Bound coagulase, or “clumping factor,” is bound to the bacterial Bound
cell wall and reacts directly with fibrinogen. 
• The presence of bound coagulase correlates with free coagulase, an
Principle
 S. aureus produces two forms of coagulase bound and free. Bound
coagulase, or “clumping factor” is bound to the bacterial cell wall and
reacts directly with fibrinogen. This results in precipitation of fibrinogen
on the staphylococcal cell, causing the cells to clump when a bacterial
suspension is mixed with plasma. The presence of bound coagulase
correlates with true coagulase, an extracellular protein enzyme that
causes the formation of a clot when S. aureus colonies are incubated coagulase or Clumping factor
with plasma. The clotting mechanism involves activation of a plasma • Results:
coagulasereacting factor (CRF), which is a modified or derived thrombin  Positive: Macroscopic clumping 
molecule, to form a coagulase-CRF complex. This complex in tum reacts Negative: No clumping
with fibrinogen to produce the fibrin clot.
b
b
i
t
p
l
a
s
1. Emulsify several m
colonies of the a
unknown clinical
isolate in
0.5mL of rabbit plasma (with EDTA) to give a milky r
suspension. e
a
2. Repeat the same
g
process with a known
e
positive and negative n
control organism. t
PAGE 2 OF 3 a
n
d
[BACT211] TRANS: STAPHYLOCOCCI | Prof. Rochelle D. Darlucio, RMT, MPH w

a
t
• Limitations e
Slide Test: r

E o
q r
u
i
v s
o a
c l
a i
l n
: e
C c
l o
u n
m t
p r
i o
n l
g
d
i r
n o
p
s
b
o
t i
h n
d
i
t c
h a
e t
e
r
a
t i
h d
a e
t
c
t o
h a
e g
u
l
o
a
r
s
g
e
a
n
i t
s e
m s
t
.
a
u
t Tube Test:
o 1. Te
- s
a t
g
g
l r
u e
t s
i u
n l
a t
t s
e
s c
a
a n
n
d b
e
i
s p
o
u s
n i
s t
u i
i v
t e
a
b a
l t
e
1
f
o
t
r
o
t
4
h
e
h
o
s
u
l
rs small amount of bacitracin
an (0.04 units) is placed on an
d agar plate, allowing the
the antibiotic to diffuse into the
n medium and inhibit the growth
rev of susceptible organisms.
ert • Results:
to  Positive: >10mm = susceptible
ne
 Negative:<10mm = not susceptible
gati
ve
aft 1. U
er s
24 i
ho n
urs g
.
2. If
a
ne n
gati
ve
at i
4 n
ho o
urs c
, u
inc l
ub a
ate t
at i
roo n
m g
te
mp l
era o
tur o
e p
ove ,
rni
ght
an s
d t
che r
ck e
ag a
ain k
for
clot t
for w
ma o
tion
.
Qu o
alit r
y
Co t
ntr h
ol r
 Positive: Staphylococcus aureus (ATCC25923) e
e
BACITRACIN SUSCEPTIBILITY TEST

s
• It is used to distinguish staphylococci
u
species (resistant) from micrococci
s
(susceptible).
p
• Principle: The antibiotic bacitracin e
inhibits the synthesis of bacterial cell c
walls. A disk (TaxoA) impregnated with a
t a
t
e
c
d
o
l
o f
n o
i r
e c
s e
p
s
o
,
f
p
a
l
a
p c
u e
r
e
a
c
b
u
a
l
c
t
i
u
t
r
r
e
a
c
o i
n n
t
o
d
i
a s
k
b
l i
o n
o
d
t
h
a e
g
a
f
r
i
r
p s
l t
a
t
q
e
u
.
a
d
2. U r
s a
i n
n t
g
(
h a
e r
e c
a o
n
t
o
a
f
c
t
h
e
w
a
i
v
t
i
h
e
s
t t
h
e
g
r
o a
w g
t a
h r
)
.
s
u
G r
e f
n a
t c
l e
y .
t 3. I
a n
p c
u
t b
h a
e t
e
d
i t
s h
k e
t p
o l
a
t
e e
n
s
u f
r o
e r
a 1
d 8
e
q t
u o
a
t
2
e
4
h 5
o %
u
r
t
s
o
a
1
t
0
%
3
5
c
°
a
C
r
b
t o
o n
3 d
7 i
° o
C x
i
d
i
e
n
(
a
C
m
O
b
2
i
)
e
n
t f
o
r
a
i
r s
t
r
f
e
o
p
r
t
o
s c
t o
a c
p c
h i
y
l
d
o
i
c
f
o
f
c
e
c
r
i
e
n
a t
n i
d a
t
i i
n o
n
.
4. L
o
o
k
f
o
r
z
o NOVOBIOCIN DISK TEST
n • Used to differentiate Coagulase Negative Staphylococci
e • 5ug
 Positive: Zone of Inhibition >11mm
o  Negative: Zone of Inhibition <16mm
f
i
n
h
i
b
i
t
i
o
n
a P
r A
o G
u E
n
d
3
t
O
h F
e
3
d
i
s
k
.
• Expected Results
 Positive: Any zone of inhibition greater than 10 mm:
susceptible
 Negative: No zone of inhibition; resistant
 They are classified using the Lancefield Group Antigen
OUTLINE
I Streptococci and Enterococci
A Gram Stain Procedure
II Hemolytic Patterns A Beta
Hemolytic Groups
i. Group B Streptococci
ii. Group C Streptococci
B Alpha Hemolytic Groups
i. Streptococcus Pneumoniae
ii. Viridans Streptococci Group
iii. Group D Enterococci
iv. Group D Non-Enterococci
III Laboratory Diagnosis
A Alpha Hemolysis
B Optochin Disk (Taxo P)
C Bile Solubility Test
2020-
D
E
Bile Esculin Test Streptococci & Enterococci:
6.5% NaCl / Salt Tolerance Test
LAB 2nd SEM
F Isolation and Identification
Differentiation of GAS to GBS
7 BACT21
G Bacitracin Susceptibility Test 1
H CAMP Test I CLINICAL
Hippurate HydrolysisBACTERIOLOGY
Test LAB
Transcriber: GAD. MPL.
Note: Date: March 26, 2021
[TRANS]
Italicized – additional UNIT 7: STREPTOCOCCI
information AND ENTEROCOCCI: ISOLATION AND IDENTIFICATION
STREPTOCOCCI AND ENTEROCOCCI HEMOLYTIC PATTERNS
• Gram positive cocci
 Alpha (a)
• Facultative anaerobes and generally considered non-motile
▪ Partial lysis of RBCs around colony
• Occur singly or in pairs, however, they are best known for their
▪ Greenish discoloration of area around colony
characteristic formation of long chains
 Beta (β)
• Some species are capnophilic o Placed in a “candle jar” serves ▪ Complete lysis of RBCs around colony
as a dessicator ▪ Clear area around colony
• Pyogenic causing bacteria o Pus-producing bacteria  Nonhemolytic
▪ No lysis of RBCs around colony
 Streptococci and Enterococci differ to Staphylococci and ▪ No change in agar
Micrococci in:
 They occur in chains rather than clusters
 Lacks enzymes catalase
BETA HEMOLYTIC GROUPS
GROUP A STREPTOCOCCI
• Streptococcus pyogenes , the main representative of group A

streptococci, is by far the most serious rapidly injure cells and
tissues.
o Most common
o Only sensitive and susceptible to Bacitracin Test
• In order to differentiate S. pyogenes from other streptococci and
enterococci, isolates are tested for resistance to bacitracin.
• If a bacterial isolate is beta hemolytic and sensitive to bacitracin, it
is presumed to be S. pyogenes.
 Has a clear zone of hemolysis
PAGE 1 OF 4
[BACT211] TRANS: STREPTOCOCCI AND ENTEROCOCCI: ISOLATION & IDENTIFICATION | Prof. Rochelle D. Darlucio, RMT, MPH
 Spherical cocci (gram +) GROUP D NON-ENTEROCOCCI
GROUP B STREPTOCOCCI
• S. Bovis
o Found in human’s intestinal tract, cow and sheep
• S. agalactiae • In blood agar, the colonies of S. bovis appear large, mucoid (many
• This pathogen may be found in the pharynx, skin, and rectum; strains have a capsule), and either non-hemolytic or alpha
however, it is more likely to be found in the genital and intestinal hemolytic.
tracts of healthy adults and infants.
• In culture S. bovis grows in pairs and short chains.
• S. agalactiae colonies are large, with a • Key reactions for this group are a positive bile esculin test and
narrow zone of beta hemolysis negative salt broth test.
• Preliminary identification of this species relies heavily on a positive
CAMP reaction. LABORATORY DIAGNOSIS
 Important cause of neonatal infection 
Has a positive reaction in CAMP TEST 1. GRAM STAIN - Gram positive cocci in pairs or in chains
2. Growth on BAP & CAP
GROUP C STREPTOCOCCI  GROUP A
- Grayish white
- Transparent to translucent - Matte or glossy
• Uncommon human pathogens but may be involved in zoonosis. - Large zone of beta hemolysis
• The organism of importance in this group is S. dysgalactiae, and  GROUP B
infections from this species account for less than 1% of all - Larger than Grp A
bacteremias. - Translucent to opaque
• S. dysgalactiae produce large colonies with a large zone of beta - Flat or glossy
hemolysis on blood agar. - Narrow zone of beta hemolysis
 GROUP C
• Presumptive differentiation of S. dysgalactiae from other beta - Grayish white
hemolytic streptococci (S. pyogenes and S. agalactiae) is based - Glistening
primarily on resistance to bacitracin and a negative CAMP test. - Wide zone of hemolysis
 Can cause disease like pharyngitis and endocarditis 3. CATALASE TEST - NEGATIVE
ALPHA HEMOLYTIC GROUPS  A biochemical test
 Ifusing a 30% Hydrogen peroxide (reagent) it
STREPTOCOCCUS PNEUMONIAE is a
Superoxol Test
 Staphylococcus
• A significant human pathogen.  Appears in pin head colony 
• Colonies appear smooth, mucoid, and surrounded by a zone of Streptococcus/Enterococcus
greenish discoloration (alpha hemolysis).  Appears in pin point colony
• In culture these cells usually grow as diplococci, but they can also HEMOLYTIC PATTERNS
occur singly or in short chains.
• Presumptive identification of S. pneumoniae can be made with a
positive optochin susceptibility test.
VIRIDANS STREPTOCOCCI GROUP
• When grown on blood agar, viridans colonies appear very small,

gray to whitish gray, and opaque.
• In culture they appear rod like and grow in chains.
• The viridans group can be differentiated from the pneumococci
and enterococci by a negative result in the bile esculin ALPHA HEMOLYSIS
hydrolysis test, the salt tolerance test, and the optochin
 S. Pneumoniae and Viridans streptococci
susceptibility test.
GROUP D ENTEROCOCCI
• When grown on blood agar, enterococci form large colonies that

can appear non-hemolytic, alpha hemolytic, or rarely beta
hemolytic.
• In culture they grow as diplococci in short chains.
• Colonies of E. faecalis appear either nonhemolytic or beta
hemolytic, depending on the strain and the type of blood agar
used, while colonies of E. faecium generally appear alpha
hemolytic.
 E. Faecalis and E. faecium the natural inhabitant of GIT
PAGE 2 OF 4
STREPTOCOCCI AND ENTEROCOCCI: ISOLATION & IDENTIFICATION | Prof. Rochelle D. Darlucio, RMT, MPH
BILE ESCULIN TEST

• Used for the presumptive ID
for Enterococcus and Group D
Streptococcus
• To differentiate Enterococcus & Group D FROM Viridans Strep 
Principle:
OPTOCHIN DISK (TAXO P) - Gram (+) are inhibited by bile salt in this medium.
• Used to determine the effect of Optochin (ethyl hydrocupreine Organisms capable of growth in the presence of 4% bile
hydrochloride) in an organism and able to hydrolyze esculin to esculetin. – Esculetin
reacts with Fe3+ and forms dark brown to
• Optochin lyses Pneumococci (POSITIVE) black precipitate
 Principle: Optochin interferes with the ATPase and  Result:
production of ATP in microorganisms (+) Growth or Blackening of the agar
 Result: A zone of 14 16 mm is considered susceptible (-) No blackening of the agar.
and presumptive identification for Streptococcus
pneumoniae.
 Limitations: Equivocal: Any zone of inhibition less than
14 mm is questionable for pneumococci, the strain is
identified as a pneumococcus with confirmation by a
positive bile solubility test.
 Note: It is important to remember that pneumococci are
sometimes optochin resistant.
 S. pneumoniae (+)
6.5% NACL / SALT TOLERANCE TEST

• To determine the ability of an organism to grow in high
concentrations of salt
• Used to differentiate enterococci from non-enterococci 
Principle:
BILE SOLUBILITY TEST - Enterococci are resistant to high salt concentration -
Heart infusion broth w/ 6.5% NaCl use as test medium. -
• Confirmatory test for Streptococcus pneumoniae This broth contains a small amount of glucose &
• Used to differentiate S. pneumoniae from Viridans strep  bromcresol purple as an indicator for acid production.
Principle:  Result:
- Sodium desoxycholate (bile salt) rapidly lyses (+) turbidity
pneumococcal colonies. (-) no turbidity(Nonenterococcus)
- Lysis depends on the presence of an intracellular
autolytic enzyme
[BACT211] TRANS:
 Reagents: Plate (10% Sodium desoxycholate, Tube

(2% Sodium desoxycholate
 Result:
Tube Plate
(+) No turbidity (+) Lysed colonies
(-) Remains Turbid (-) Intact colonies
PAGE 3 OF 4
[BACT211] TRANS: STREPTOCOCCI AND ENTEROCOCCI: ISOLATION & IDENTIFICATION | Prof. Rochelle D. Darlucio, RMT, MPH
HIPPURATE HYDROLYSIS TEST

• Principle : determine the capability of organism
SCHEMATIC DIAGRAM FOR DIFFERENTIATION OF to hydrolyze hippurate
GAS FROM GBS • Hippuric acid is hydrolyzed by HIPPURICASE ENZYME that will
result to glycine and benzoic acid Glycine is deaminated to by
NINHYDRIN (oxidizing agent)
RESULT:
 (+) deep purple color (S. agalactiae)
 (-) colorless / slightly pink yellow in color (S. pyogenes)
BACITRACIN SUSCEPTIBILITY TEST (TAXO A DISK)

• Use for presumptive ID and differentiation of β haemolytic
Group A from other β haemolytic streptococci
 Principle: inhibits the synthesis of bacterial cell wall
 Result:
(+) >10mm zone of inhibition (S. pyogenes)
(-) No zone of inhibition (S. agalactiae or other group)
CAMP TEST
• The Christie, Atkins, and Munch Peterson test is used to

differentiate Group B Streptococci from other Strep spp.
 Principle: Certain organisms produce a diffusible
extracellular hemolytic protein (CAMP factor) that acts
synergistically with the beta lysin of Staphylococcus
aureus to cause enhanced lysis of RBCs.
 Result:
• (+) arrowhead shaped zone of betahemolysis at the
juncture of the two organisms (S. agalactiae)
• (-) No hemolysis - (S. pyogenes)
PAGE 4 OF 4
OUTLINE
I Family Enterobacteriaceae
II Laboratory Diagnosis
A Specimen Collection CULTURE
B Gram Stain
2020-
C Culture Biochemical Identification ofLAB 2021 2nd •
D Collonial Appearance SEM
i. MacConkey Agar Gramnegative Bacteria 8 BACT21
G
ii. Eosin Methylene Blue
iii. Hektoen Enteric CLINICAL
Agar BACTERIOLOGY 1
Transcriber: GAD. MPL. LAB
iv. Xylose-Lysine-Desoxycholate
Date: April 16, 2021
III Biochemical Tests
A
[TRANS] UNIT
Carbohydrate Utilization
8: BIOCHEMICAL IDENTIFICATION OF GRAM NEGATIVE BACTERIA
i. Oxidation Fermentation Test row at an optimal temperature of 35 C to 37 C
ii. Triple Sugar Iron • Low temperatures (1C to 5C, such
iii. Ortho-Nitrophenyl-β-Dgalactopyranoside Test as Serratia and
B Glucose Metabolism and Its Metabolic Products Yersinia)
i. Methyl Red Test
• Or tolerate high temperatures (45 C to 50 C, such as E.
ii. Voges-Proskauer Test
C Amino Acid Utilization coli)
i. Lysine Iron Agar • Visible after 18-24 hours of incubation
ii. Decarboxylase Test (Moeller’s Method) • BAP, CAP, MacConkey Agar and other selective medium
iii. Phenylalanine Deaminase Test (EMB, Hektoen,SSA)
D Miscellaneous Test
i. Sulfide Indole Motility Agar COLONIAL APPEARANCE
ii. Citrate Utilization
• All Enterobacteriaceae produce similar growth on blood
iii. Urease Test
iv. DNA Hydrolysis and chocolate agars; colonies are large, gray, and smooth.
v. Gelatin Liquefaction • Klebsiella or Enterobacter may be mucoid o due to the
vi. Malonate Utilization polysaccharide capsule
vii. Nitrate and Nitrite Reduction • P. mirabilis, P. penneri, and P.vulgaris “swarm” on blood
viii. Oxidase
and chocolate agars.
Note: o Swarming motility in BAP
Italicized – additional information • Y. pestis on 5% sheep blood agar are pinpoint at 24 hours
FAMILY ENTEROBACTERIACEAE but exhibit a rough, cauliflower appearance at 48 hours.
• Gram negative bacilli and cocobacilli, non-spore forming, • Broth cultures of Y. pestis exhibit
facultatively anaerobic a characteristic
• DO NOT produce cytochrome C oxidase “stalactite pattern”
except for • Y. enterocolitica produces bull’s-eye colonies
Plesiomonas
• ALL ferment glucose  Mucoid colony of Klebsiella or Enterobacter
• MOTILE at body temperatures except for
Klebsiella,
Shigella, and Neisseria.
• “Enterics” - found in the Gastrointestinal tract of humans
and animals
LABORATORY DIAGNOSIS
SPECIMEN COLLECTION
• To ensure isolation of both opportunistic and fastidious
pathogens, laboratories must provide appropriate transport
media, such as Cary Blair, Amies, or Stuart media.
• Fecal specimen from the patient o Acute phase of illness
o Before the administration of antibiotics
GRAM STAIN
• Direct smear examination of stool samples is not
particularly helpful in identifying enteric pathogens but may
reveal the presence of inflammatory cells.
PAGE 1 OF 7
[BACT211] TRANS: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA | Prof. Rochelle D. Darlucio, RMT, MPH
Formation of a “string”
 EOSIN METHYLENE BLUE

 Bull’s eye colonies of Y. enterocolitica
• Selective media: gram negative enteric

MACCONKEY AGAR bacilli  Differential media:
 Lactose fermenter - Dark violet
• Best used to characterize gram negative
• Hafnia, Serratia, Citrobacter
rods
• Salmonella arizonae
• Lactose fermenters can be differentiated
• Shigella sonnei
with non-lactose fermenters
• Yersinia enterocolitica
• Selective media: gram negative enteric
 Non-lactose fermenter - Colorless
bacilli  Differential media:
• All Salmonella except S. arizonae
 Lactose fermenter - Dark pink
• All Shigella except S. sonnei
• Hafnia, Serratia, Citrobacter
• All Yersinia except enterocolitica
• Salmonella arizonae
• Proteus,Providencia,Morganella,
• Shigella sonnei
Edwardsiella
• Yersinia enterocolitica
 Non-lactose fermenter - Colorless
• All Salmonella except S. arizonae
• All Shigella except S. sonnei
• All Yersinia except enterocolitica
Edwardsiella
PAGE 2 OF 7
 Methyl red and Voges-Proskauer tests to determine end
products of glucose fermentation
 Indole test to determine whether indole is formed from
tryptophan by tryptophanase
 Urease test to determine hydrolysis of urea
 Simmons' citrate to determine whether citrate can be used
as the sole carbon source
 Carbohydrate fermentation
 Lysine iron agar (LIA) to determine lysine decarboxylase
activity
HEKTOEN ENTERIC AGAR  Sulfide-indole-motility (SIM) or motility-indole-
orn1thine
• Selective media: gram negative enteric (MIO) media
bacilli  Differential media: CARBOHYDRATE UTILIZATION
 Lactose fermenter - Orange  Two enzymes are necessary for a bacterium to take up
• Hafnia, Serratia, Citrobacter lactose and to cleave it into monosaccharides :
• Salmonella arizonae  B-galactoside permease
• Shigella sonnei  B-galactosidase
• Yersinia enterocolitica  LFs- possess both β galactoside β
 Non-lactose fermenter - Blue/Green permease and galactosidase
• All Salmonella except S. arizonae  NLFs- do not possess either enzyme
• All Shigella except S. sonnei  LLFs- lack β galactoside permease but possess β
• All Yersinia except enterocolitica galactosidase
Edwardsiella OXIDATION FERMENTATION TESTS
• OF Basal Medium
 pH indicator: bromthymol blue
 Uninoculated Medium: green
 Acid Environment: yellow
 Alkaline Environment: blue
• When O/F tests are performed, two tubes of Hugh
Leifson OFBM are inoculated; one is overlaid with sterile
mineral oil to create an anaerobic environment (closed), and
the other tube is left aerobic (open), without mineral oil overlay.
 Reactions:
 Acid produced on both tubes: Oxidizer
XYLOSE-LYSINE-DESOXYCHOLATE and fermenter
 Acid only in closed tube: Fermenter and
possible obligate anaerobe
 Acid in open tube: Oxidizer
BIOCHEMICAL TEST
TRIPLE SUGAR IRON (TSI)
 Purpose: Triple sugar iron (TSI) agar is used to

determine whether agram-negative rod ferments glucose
and lactose or sucrose and forms hydrogen sulfide (H,S).
The test is used primarily to differentiate members of the
TRADITIONAL BIOCHEMICAL TESTS FOR Enterobacteriaceae family from other gram-negative rods.
IDENTIFICATION OF GRAM-NEGATIVE BACTERIA
 Expected Results
 Triple sugar iron (TSI) agar or Kligler iron agar (KIA) to  Alkaline slant/no change in the butt (K/NC):
determine glucose and lactose, or sucrose, utilization glucose,lactose,and Sucrose nonutilizer,this may
(sucrose in TSI only) and hydrogen sulfide production also be recorded as K/K (alkaline slant/alkaline butt)
PAGE 3 OF 7
 Alkaline slant/acid butt  4th tube - K/A, H2S, gas production
(K/A):glucose fermentation only.  5th tube - K/K
 Acid slant/acid butt (A/A):glucose,sucrose,and/or
lactose fermenter
Note: A black precipitate in the butt indicates production of

ferrous sulfide and H,S gas (H,S+)(Figure 12-41, B). Bubbles
or cracks in the tube indicate the production of CO,or H2.
• Kligler Iron Agar (KIA) - Used to determine

glucose and lactose or sucrose utilization and
H2S production, gas
production
 Composition: 10 parts lactose, 10 parts sucrose,
1 part glucose, peptone
 pH indicator: phenol red
ORTHO-NITROPHENYL-Β-D-GALACTOPYRANOSIDE
 H2SIndicator: Ferrous sulfate and TEST (ONPG TEST)
sodium thiosulfate
• ** Reactions should be read within an 18 to  Purpose: This test is used to determine the ability of an
24 hour incubation period; otherwise, organism to produce β-galactosidase, an enzyme that
erroneous results are possible. hydrolyzes the substrate o-nitrophenyl-β-
Dgalactopyranoside (ONPG) to form a visible (yellow)
product, ortho-nitrophenol.The test distinguishes late
lactose fermenters from non-lactose fermenters of
Enterobacteriaceae
 Positive:Yellow (presence of β-galactosidase)
 Negative:Colorless (absence of enzyme)
• β Galactosidase hydrolyzes ONPG, a colorless compound,

• Reactions on TSI agar o K- alkaline (red) o into galactose and o nitrophenol, a YELLOW compound.
A- acid (yellow) • ONPG remains colorless if the organism is an NLF.
1. K/K (Alkaline(red) slant/alkaline(red) butt) – No • The test can be performed by making a heavy suspension
fermentation (not member of enterobacteriaceae) of bacteria in sterile saline and adding commercially
2. K/A (Alkaline (red) slant/acid (yellow) butt) – prepared ONPG disks or tablets.
Glucose fermenter (possible member of • The suspension is incubated at 35 C, and positive results
enterobacteriaceae) can generally be seen within 6 hours.
3. A/A(Acid(yellow)slant/acid (yellow) butt) • Positive result: yellow
–
Glucose, lactose, sucrose fermenter
4. H2S production - (K/A, H2S) or
(A/A H2S) black precipitate
 Bacterium (acid environment) + sodium
thiosulfate -> H2S
 H2 S + ferric ions -> Ferrous
sulfide (black precipitate)
• H2S PRODUCER o Salmonella o Proteus o
Arizona o Citrobacter o Edwardsiella
5. Gas production – Bubbles, cracks, spaces, splitting of
the medium.
GLUCOSE METABOLISM AND ITS METABOLIC
PRODUCTS
METHYL RED TEST
 If glucose is metabolized by the mixed acid fermentation,

stable acid end products are produced, which results in a
low pH.
 Reagent: 5-6 drops of Methyl red
 Positive Result: Red
 Negative Result: Yellow or no color change
 1st tube - A/A, gas production
 2nd tube - A/A, H2S production
 3rd tube - K/A
PAGE 4 OF 7
VOGES PROSKAUER TEST  Negative: Purple
• Lysine Decarboxylase (Butt)
 Positive: Purple
• Detects the organism’s ability to convert the acid products
 Negative: Yellow (acidic glucose is fermented)
to acetoin and 2,3-butanediol. (Butylene glycol pathway)
• METHOD:
• After incubation, is added first as a catalyst or color
1. With a straight inoculating needle, inoculate LIA by
intensifier. Next, 40% potassium hydroxide (KOH) or
twice stabbing through the center of the medium to the
sodium hydroxide (NaOH) is added.
bottom of the tube and then streaking the slant.
 Reagent: 0.6 ml (6 drops) a-naphthol
2. Cap the tube tightly and incubate at 35 37C in ambient
 Positive Result: Red
air for 18 24 hrs.
 Negative Result: Yellow
 Alkaline slant ( purple)/alkaline butt) = K/K =
 Purpose
Lysine decarboxylation and no fermentation of
The combination test methyl red (MR) and Voges-Proskauer
glucose
(VP) differentiates members of the Enterobacteriaceae family.
 Alkaline slant (purple)/acid butt (yellow) = K/A
 Principle = Glucose fermentation
This test is used to determine the ability of an organism to  Red slant/acid butt (yellow) = R/A = Lysine
produce and maintain stable acid end products from glucose deamination and glucose fermentation
fermentation,to overcome the buffering capacity of the system,
and to determine the ability of some organism to produce DECARBOXYLASE
neutral end products (e.g.,2,3-butanediol or acetoin) from
glucose fermentation. The methyl red detects mixed acid
fermentation that lowers the pH of the broth. The MR indicator
is added after incubation. MR is red at pH 4.4 and yellow at pH
6.2.A clear red is a positive result; yellow is a negative result;
and various shades of orange are negative or inconclusive.The
VP detects the organism's ability to convert the acid products to
acetoin and 2,3-butanediol.Organisms capable of using the VP
pathway produce a smaller amount of acid during glucose
fermentaticn and therefore do not produce a color change when
tho MR indicator is added. A secondary reagent is added,
alpha-naphthol,followed by potassium hydroxide (KOH):a
positive test result is indicated by a red color complex.
AMINO ACID UTILIZATION TEST (MOELLER’S METHOD)

 Purpose: This test is used to differentiate
LYSINE IRON AGAR decarboxylaseproducing Enterobacternaceae from other
gram-negative rods.
 Principle: This test measures the enzymatic ability
• Purpose: To differentiate gram negative bacilli based on (decarboxylase) of an organism to decarboxylate (or
decarboxylation or deamination of lysine and the formation hydrolyze) an amino acid to form an
of hydrogen sulfide. amine.Decarboxylation, or hydrolysis, of the amino acid
results in an alkaline pH and a color change from orange
to purple.
 Positive: Alkaline (purple) color change compared
with the control tube
 Negative: No color change or acid (yellow) color in
test and control tube. Growth in the control tube.
PHENYLALANINE DEAMINASE (PAD) TEST
• Principle: The medium has anaerobic slant and anaerobic  Purpose: This test is used to determine the ability of an
butt. When glucose is fermented, the butt of the medium organism tooxidatively deaminate phenylalanine to
becomes acidic (yellow). If the organism produces lysine phenylpyruvic acid.The genera Morganella, Proteus,and
decarboxylase, is formed. Cadaverine neutralizes the Providencia can be differentiated from other members of
organic acids formed by glucose fermentation, and the butt the Enterobacteriaceae family.
of the medium reverts to the alkaline state (purple). If the  Expected Results
decarboxylase is not produced, the butt remains acidic  Positive: Green color develops on slant after ferric
(yellow). If oxidative deamination of lysine occurs, it forms chloride is added .
a burgundy color on the slant. If deamination does not
 Negative: Slant remains original color after the
occur, the LIA slant remains purple.
addition of ferric chloride
 pH indicator: Bromcresol purple
• Determines whether an organism possesses the enzyme
• Lysine Deaminase (Slant)
that deaminates phenylalanine to phenylpyruvic acid.
 Positive: Burgundy/ Red
PAGE 5 OF 7
• Contains 0.2% phenylalanine
• Reagent: 10% ferric chloride
• The surface of the slant is inoculated with a bacterial
colony. After incubation, addition of a 10% ferric chloride
reagent results in a green color if phenylpyruvic acid is
present
• METHOD:
1. Inoculate Simmons citrate agar lightly on the slant by
touching the tip of the needle to a colony that is 18 24
hrs old. Do not inoculate from a broth culture, because
the inoculum will be too heavy.
2. Incubate at 35-37C for up to 7 days.
3. Observe for growth and the development of blue color,
denoting alkalinization
MISCELLANEOUS TESTS • Results:
 POSITIVE: Growth on the medium, with or
SULFIDE INDOLE MOTILITY AGAR without change in the color of the indicator. (blue)
 NEGATIVE: Absence of growth (green)
• Sulfide H2S – Black precipitate

UREASE TEST
• Indole - Organisms that possess the enzyme
tryptophanase are capable of deaminating tryptophan with
the formation of the intermediate degradation products of • Determines whether a microorganism can hydrolyze urea
indole, pyruvic acid, and ammonia. • Urease hydrolyzes urea to form ammonia, water, and
 Reagent:0.5 ml of Ehrlich reagent CO2
~ PDAB – para- • Christensen's Urea Agar
dimethylaminobenzaldehyde  Positive: Change in color from Light orange (pH
 (+) = Pink to red color 6.1) to Magenta (pink) (pH 8.1)
 Motility: Cloudiness spreading from the  Negative: No change in color
inoculation line
 Inoculation: Stab-halfway (center)
DNA HYDROLYSIS (DNASE TEST AGAR)

CITRATE UTILIZATION
• Purpose: This test is used to differentiate organisms
• Purpose: It determines whether an organism can use as a based on the production of deoxyribonuclease. It is used
sodium citrate as a sole carbon source. The test is part of to distinguish Serratia sp. (positive) from Enterobacter sp.,
a series referred to as IMViC indole, methyl red, Vogues Staphylococcus aureus (positive) from other species, and
Proskauer , and citrate), which is used to differentiate Moraxella catarrhalis (positive) from Neisseria sp.
Enterobacteriaceae from other gram negative rods.
• Principle: The test is used to determine the ability of an
• Principle: Bacteria capable of growth in this medium use organism to hydrolyze DNA. The medium is pale green
the citrate and convert ammonium phosphate to ammonia because of the DNA methyl green complex. If the organism
and ammonium hydroxide, creating an alkaline pH. The pH growing on the medium hydrolyses DNA, the green color
change turns bromthymol blue indicator from green to blue. fades and the colony is surrounded by a
 Simmon's Citrate Agar contains ammonium salts
as the sole nitrogen source
 pH indicator: Bromthymol blue
 Green to blue
 Use light inoculum.
PAGE 6 OF 7
colorless zone. MALONATE
 (+) = liquefaction
• METHOD:
1. Inoculate the DNase agar with the organism to be UTILIZATION NITRATE
tested and streak for isolation.
2. Incubate aerobically at 35-37C for 13-24 hrs.
• RESULTS:
 POSITIVE: Medium will turn colorless around the
test organism.
 NEGATIVE: If no degradation of DNA occurs, the
medium remains green. 
GELATIN LIQUEFACTION
 Purpose: The production of gelatinases capable of

hydrolyzing gelatin is
used as a presumptive test for the identification of various
organisms,including Staphylococcus spp.,Enterobacteniaceae,
and some gram-positive bacilli.
 Principle: This test is used to determine the ability of an
organism to produce extracellular proteolytic enzymes
(gelatinases) that liquefy gelatin,a component of
vertebrate connective tissue.Nutrient gelatin medium
differs from traditional microbiology media in that the
AND NITRITE REDUCTION
solidifying agent (agar) is replaced with gelatin. When an  pH indicator: Bromthymol blue
organism produces gelatinase, the enzyme liquefies the • Bacteria able to use sodium malonate as a sole carbon
growth medium. source also use ammonium sulfate as a nitrogen source.
 Expected Results • A positive test results in increased alkalinity from utilization
 Positive: Partial or total liquefaction of the of the ammonium sulfate , changing the indicator from
inoculated tube (the control tube must be completely green to blue
solidified) at 4°C within 14 days
 Negative:Complete solidification of the tube at 4°C • The nitrate reduction test determines whether an organism
• Numerous bacteria produce gelatinase – proteolytic has the ability to reduce nitrate to nitrite and reduce nitrite
enzymes that break down gelatin into amino acids. further to nitrogen gas (N2)
• Gelatinase activity is detected by loss • After 24 hours of incubation, N,N-dimethyl α naphthylamine
of gelling and sulfanilic acid is added.
(liquefaction) of gelatin. • A red color indicates the presence of nitrite.
OXIDASE
 The oxidase test determines the presence of the
PAGE 7 OF 7
cytochrome oxidase system that oxidizes reduced
cytochrome with molecular oxygen.
• Kovac’s oxidase test uses a 0.5% or 1% aqueous
solution of tetramethyl ρ phenylenediamine
dihydrochloride.
 Positive: development of a lavender color within 10
to 15 seconds.
PAGE 8 OF 7
2020-
Antimicrobial Susceptibility Testing LAB 2021 2nd
SEM
CLINICAL BACTERIOLOGY 9 BACT21
Transcriber: GAD. MPL. 1
[TRANS] UNIT 9: ANTIMICROBIAL SUSCEPTIBILITY TESTING
OUTLINE
I Definition
II Antimicrobial Susceptibility Testing
A The Standardized Components of AST
B Reasons and Indications for Performing AST
Factors to Consider
III Traditional Antimicrobial Susceptibility Testing
A Disc Storage
i. Inoculum Preparation
ii. McFarland Turbidity Standards
iii. McFarland 0.5 Standard
iv. Inoculum Standardization
B Methods of AST
IV Kirby-Bauer Method
A Principle
B Step by Step Procedure
V Definition of Susceptibility Testing Interpretive Categories
Note:
DEFINITIONS
• Anti-microbial are compounds
that kill or inhibit ANTIMICROBIAL
microorganisms. SUSCEPTIBILITY
o The compounds can be
natural, synthetic or TESTING
semisynthetic. • Performed on bacteria and
fungi isolated from clinical
• Anti-biotic are antimicrobials,
specimens to determine which
usually of low molecular
antimicrobial agents might be
weight, produced by
effective in treating infections
microorganisms that inhibit or
caused by these organisms.
kill other microorganisms.
o Has different modes of • Only organism that is
actions contributing to disease should
be tested.
• The 1st line of defense
• PRIMARY GOAL: to determine
• Fatal diseases became whether the bacterial isolate is
manageable because of capable of expressing
Antibiotics resistance to the antimicrobial
agents selected for treatment
• Often performed by a disk
diffusion (usual method) or
dilution minimal inhibitory
concentration [MIC]) method.
• MIC – lowest anti-microbial that
completely inhibits visible
bacterial growth.
• Standards that describe these
methods are published and
frequently updated by the
Clinical and Laboratory
Standards Institute formerly
the National Committee for
Clinical Laboratory Standards
[NCCLS]).
[BACT211] TRANS: ANTIMICROBIAL SUSCEPTIBILITY TESTING | Prof. Rochelle D. Darlucio, RMT, MPH
THE STANDARDIZED on decreases in the
susceptibility of bacteria to
COMPONENTS OF antimicrobials
ANTIMICROBIAL
SUSCEPTIBILITY FACTORS TO CONSIDER
TESTING WHEN DETERMINING
• Bacterial inoculum size WHETHER TESTING IS
 Disk diffusion – 1.5x10
CFU/mL WARRANTED
• Body site from which the
• CFU – Colony Forming
bacterium was isolated
Units
 Broth microdilution – • Presence of other organisms
5x105 CFU/mL and quality of the specimen
from which the organism was
• Growth medium (typically a grown
Mueller Hinton base)  pH:  AST isolation of
7.2 – 7.4 organism is always on pure culture
• Agar depth: 3-5 mm (one organism)
• Cation concentration • Host’s status o
 Calcium = 25mg/L Immunocompromised -
 Magnesium = 12.5 their normal flora
mg/L can cause infection
• Blood and serum supplements TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY
 Added for fastidious TEST METHODS
organism
DISC STORAGE
• Thymidine content • Long term storage: - 20C or
• Incubation atmosphere  below in a non-frost free
Ambient air freezer
• Incubation temperature • A working supply of disks can
 35C – standard be stored in a refrigerator at 2C
temperature to 8C for at least 1 week.
• Incubation duration • Disks should always be stored

in a tightly sealed container
PAGE1OF5
 16-18 hours (Disk with desiccant.
diffusion) ; • The container should be
 16-20 hours (Broth allowed to warm to room
microdilution) temperature before it is opened
• Antimicrobial concentrations o to prevent condensation
Bacitracin - 0.04 units o
Novobiocin - % units
 Lowest concentration
 To avoid resistance
of organism
REASONS AND
INDICATIONS FOR
PERFORMING
ANTIMICROBIAL INOCULUM PREPARATION
SUSCEPTIBILITY TESTS AND USE OF MCFARLAND
STANDARDS
• Antimicrobial susceptibility
testing should be performed on INOCULUM PREPARATION
a bacterial isolate from a
clinical specimen if the isolate
is determined to be a • One of the most critical steps
probable cause of the in susceptibility testing.
patient’s infection and the (Depends on the consistency
susceptibility of the isolate to and accuracy)
particular antimicrobials • Prepared by adding cells from
cannot be reliably predicted four to five isolated colonies
based on previous of similar colony morphology
experience with the bacteria growing on a non-inhibitory
at a specific health care agar medium to a broth
facility. medium and then allowing
• Susceptibility testing of isolates them to grow to the log phase
can also provide information (preferred organism).
PAGE 2 OF 5
• Can also be prepared directly 1. Kirby Bauer Diffusion
by suspending colonies grown Method (most common)
overnight on an agar plate 2. Agar Cup Diffusion
directly in broth or saline. Method
3. Agar Cylinder Diffusion
• This direct inoculum
suspension preparation Methods
technique is preferred for 4. Epsilometer or Gradient
bacteria that grow Diffusion Method
unpredictably in broth. Because
it does not rely on growth in an DILUTION METHOD
inoculum broth, the use of fresh
(16 to 24 hour) colonies is
imperative. 1. Macrobroth Method or
Tube Dilution Method
• Use of a standard inoculum 2. Microtube Dilution Method
size is as important as culture
purity and is accomplished by
comparing the turbidity of the KIRBY BAUER METHOD
organism suspension with a • Also known as Agar
turbid standard. diffusion method or disk
diffusion method.
MCFARLAND TURBIDITY o The growth of
STANDARDS microorganism should be
homogeneous
• Used to determine the
• False susceptible results may sensitivity or resistance of a
occur if too few bacteria are bacterium to an antimicrobial.
tested.
• False resistant results may be
the outcome of testing too
many bacteria.
• McFarland standards can be
prepared by adding specific
volumes of 1% sulfuric acid
and 1.175% barium chloride.
MCFARLAND 0.5 STANDARD
 99.5 mL of 1% sulfuric acid
 0.5 mL of 1.175% barium chloride PRINCIPLE
• A standardized suspension of
organism is inoculated into
MHA (Mueller Hinton Agar)
• Paper disk impregnated with
specific antibiotics
concentration are placed into
the agar
• After 16 20 hours incubation,
the diameters of the zone of
inhibitions are measured
• Results are compared to
determine susceptibility or
INOCULUM STANDARDIZATION resistance
• The inoculated broth or direct
suspension is vortexed STEP BY STEP
(instrument that mixes the PROCEDURE
specimen) thoroughly. • Preparation of pure inoculum
• Under adequate lighting, the o USING ANY OF THE
tube is positioned side by side FOLLOWING
with the McFarland 0.5  Mueller Hinton Broth
standard against a white card  Trypticase Soy Broth
containing several horizontal  Sterile Distilled Water
black lines  Natural Saline
Solution
METHODS OF  Brain Heart Infusion
Broth
ANTIMICROBIAL
SUSCEPTIBILITY TESTING • Standardize pure inoculum o
USING 0.5 MCFARLAND
DIFFUSION METHOD STANDARD
PAGE 3 OF 5
 If standard more
turbid than inoculum
→ add colonies to
inoculum or incubate
inoculum.
 If inoculum more
turbid than standard
→ add distilled water
to inoculum
• Streaking
 Streak the pure
inoculum into the
medium (MHA)
 Use a sterile cotton
swab and streak with
no space in between.
• Incubate
 Normally 35 C for 16
20 hours.
• Measure the zone of
inhibition
 Instrument: Ruler or
microcaliper/vernie
r caliper
 Unit: mm

 Apply antibiotic discs

 Using forceps
aseptically
 Space at least 15
mm each antibiotic DEFINITIONS OF
SUSCEPTIBILITY
TESTING
INTERPRETIVE
CATEGORIES
Susceptible (S)
• Indicates that the antimicrobial
agent in question may be an
PAGE 4 OF 5
appropriate choice for treating  Use as an
the infection caused by the interpretive safety
organism. Bacterial resistance margin to prevent
is absent or at a clinically relatively small
insignificant level. changes in test
results from leading
Intermediate (I) to major swings in
• Indicates a number of interpretive category
possibilities, including: (e.g., resistant to
susceptible or vice
 The potential utility of
versa).
the antimicrobial
agent in body sites Resistant (R)
where it may be • Indicates that the antimicrobial
concentrated (e.g., agent in question may not be
the urinary tract) or if an appropriate choice for
high concentrations treatment, either because the
of the drug are used. organism is not inhibited with
 Possible serum achievable levels of the
effectiveness of the drug or because the test result
antimicrobial agent highly correlates with a
against the isolate, resistance mechanism that
but possibly less so indicates questionable
than against a successful treatment.
susceptible isolate.
PAGE 5 OF 5
PAGE 6 OF 5
PAGE 7 OF 5
Antibiotic Cells affected Cell target/specific site
Penicillin G+;G- Cell wall/B-lactamase,
peptidoglycan synthesis – amino
acid side chain
Vancomycin G+ Cell wall/peptidoglycan
synthesis
Bacitracin G+ Cell wall/Transport of
peptidoglycan monomer
Isoniazid Mycobacterium tuberculosis Cell wall/mycolic acid synthesis
in mycobacterium
Fluroquinolones G+;G- DNA/Topoisomerase unwinding
of DNA in DNA synthesis
Rifamycins G+; and some G- RNA/RNA polymerase in RNA
synthesis
Tetracyclines G+;G- Protein synthesis/30s subunit of
70s ribosomes
Streptomycin G+;G- Protein synthesis/30s subunit of
70s ribosomes
Chloramphenicol G+;G- Protein synthesis/30s subunit of
70s ribosomes
Sulfa drugs G+;G- Structural analogue of
paraamino benzoic acid (PABA)-
inhibit enzyme linking pteridine
to PABA in folic acid synthesis
Variable Standard Comments

Inoculum Use adequate McFarland
turbidity standard (0.5 for disk
diffusion) when preparing direct
suspension (without incubation),
do not use growth from plates
>1 day old
Formulation Prepare in house or purchase
from reliable source
Perform media quality control
to verify acceptability before
use for patient test
Ca, Mg content Increase concentration result in
decreased activity of
aminoglycosides against
pseudomonas aeruginosa and
decreased activity of tetrcylines
against all organism (decreased
concentration have the opposite
effect)
Thymidine content Excessive concentration can
result in false resistance to
sulfonamides and trimethroprim
pH Decreased pH can lead to

decreased activity of
aminoglycosides, erythromycin
and clindamycin and increased
activity of tetracylines
(increased pH has the opposite
effect)
Agar depth (Disk diffusion) Possibility for false susceptibility
if >3 mm or false resistance if >5
mm
Atmosphere CO2 incubation decreases pHm
which can lead to decreased
activity of aminoglycosides,
erythromycin and clindamycin
and increased activity of
tetracyclines
Temperature length 24 h for staphylococci with Some MRSA may go undetected
oxacillin and vancomycin and if >35 degrees c
for enterococci with vancomycin Some MRSA may go undetected
and gentamicin HLAR ; 48 h if <24 degrees C
enterococci with streptomycin Some vancomycin resistant
HLAR ; 24 h sometimes needed enterococci may go undetected
for fastidious bacteria if <2 h with disk diffusion Some
HLAR (gentamicin) enterococci
may go undetected if <24 h
(broth microdilution) Some
HLAR (Streptomycin)
enterococci may go undetected
if <48 h (broth microdilution)
Test Purpose
Antimicrobial concentration test (assay) Measure amount of antimicrobial agent in serum
or body fluid
Minimum bactericidal concentration test Measure of lowest concentration of antimicrobial
agent that kills a bacterial isolate
Serum bactericidal test Measure of highest dilution or titer of a patient’s
serum that is inhibitory to the patient’s own
infecting bacterium and highest dilution or titer
that is bactericidal
Synergy test Measure susceptibility of a bacterial isolate to a
combination of two or more antimicrobial agents
Time kill assay Measure of rate of killing of bacteria by an
antimicrobial agent (as determined by examining
the number of variable bacteria remaining at
various intervals after exposure to the agent)

Clinical Bacteriology Laboratory

Uploaded by

Copyright:

Available Formats

Clinical Bacteriology Laboratory

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Clinical Bacteriology Laboratory

Uploaded by

Copyright:

Available Formats

What are the different types of safety hazards discussed?

What are the different types of safety hazards discussed?

What are the steps of proper handwashing?

What are the steps of proper handwashing?

OLFU Laboratory Safety and Microscopy LAB 20202 nd SEM

College of Medical CLINICAL BACTERIOLOGY BACT211

Laboratory Science Transcriber: Riyoma Surell 1 LAB

Types of Safety Hazards

Chemical Preservatives Exposure to toxic,

➢ Radioactivity may be encountered in the clinical laboratory

➢ An optical instrument that is used to observe tiny objects,

D. Proper Use of the Microscope

1. Carry microscope with two hands, supporting the base with

C. Parts and Function of Microscope ❖ Oil Immersion Objective: (Cedarwood Oil)

Component Location Function E. Magnification Formula for Total Magnification

(L) Condenser Beneath and Used to adjust the height of the

 Disinfect the table

FINAL FLAMING OF THE LOOP OR NEEDLE

sterile broth, inoculating it. To disperse

• The loop or needle is then returned to its

TRANSFER FROM BROTH

 Sterilize the inoculating loop

1. If you have not already done so,

• Loops and Needles

slant • Pure culture

 The difference of method A to B is that, B ends with a

1. Streak one loop full of microorganism and apply on

 Do not put too much pressure

 Start streaking on the top to the middle/half of the

SIMPLE STAINING 3 2nd SEM

 (3) Internal structures ( endospores )

FROM BROTH CULTURES

PROPERLY PROCESSED SMEAR

• The bacteria are evenly spread out on the slide in such a

GOOD SMEARS ARE CRITICAL FOR DISCERNING

FROM PLATES AND SLANTS 1.

• The use of a single stain to color a bacterial cell

GRAM STAINING &

[TRANS] UNIT 4: GRAM STAINING & ACID FAST STAINING

GRAM STAIN PROCEDURE

SEVERAL FACTORS CAN AFFECT THE OUTCOME OF THE PROCEDURE

1. It is important to use cultures that are 16-18 hours old

ACID FAST STAINING KINYOUN METHOD Mycolic Acid

1. Indicate the gram stain reaction ( positive or negative)

Gram-stained Smear from specimens

 Gram negative bacilli in singly

AND STERILIZATION 5 2nd SEM

[TRANS] UNIT 5: MICROBIOLOGICAL CULTURE,

• If the directions: suspend 28 g of powder in 1000 mL of D.W.,

 7.48 g of powder should be dissolve in 280 mL of

 After computation, use aluminum foil to weigh the powder.

BLOOD AGAR PREPARATION INOCULATION TECHNIQUES

4. BUTT SLANTED MEDIA

• Purpose  1. Place a drop of coagulase plasma (preferably rabbit plasma

[BACT211] TRANS: STAPHYLOCOCCI | Prof. Rochelle D. Darlucio, RMT, MPH w

BACITRACIN SUSCEPTIBILITY TEST

BETA HEMOLYTIC GROUPS

• Streptococcus pyogenes , the main representative of group A

VIRIDANS STREPTOCOCCI GROUP

• When grown on blood agar, viridans colonies appear very small,