OLFU Laboratory Safety and Microscopy LAB

2020-2021
2nd SEM
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 1 LAB

Batch 2023 Date: February 5, 2021

Mode of Transmission Direct Contact, Indirect Contact,

Outline Droplets, Airborne etc.
At the end of the session, the student must be able to learn: Portal of Entry Same as the portal of exit, which include
I. Laboratory Safety the mucous membranes of the nose,
A. Biologic Hazards mouth and eyes, breaks in the skin, and
• Chain of infection open wounds
• Handwashing and Standard Precaution
• Biologic Waste Disposal
Susceptible Host Can be another patient during invasive
B. Sharp Hazards procedures, visitors, and healthcare
C. Chemical Hazards personnel when exposed to infectious
• Material Safety Data Sheets specimens or needlestick injuries
D. Radioactive Hazards
E. Electrical Hazards
F. Fire/ Explosive Hazards
• Types of fires and fire extinguishers
II. Microscopy
A. Microscope Definition
B. Types of Microscope
C. Parts and Functions of the Microscope
D. Proper use of the Microscope
E. Magnification Formula for Microorganism Size

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 ❖ Proper hand hygiene, correct disposal of contaminated materials, and
Biologic Infectious agents Bacterial, fungal, viral or wearing personal protective equipment (PPE) are of major importance
parasitic infections in the laboratory
Sharps Needles, Cuts, punctures, or blood- ❖ Handwashing
lancets, broken borne pathogen exposure 1. Stand in front of the sink. Do not lean on the sink with clothes
glass 2. Use paper towel to cover the water control and turn on the water
Chemical Preservatives Exposure to toxic, 3. Wet hands thoroughly. Allow the water to flow from arms to
and reagents carcinogenic or caustic agents fingertips
Radioactive Equipment and Radiation exposure 4. Apply soap to hands
radioisotopes 5. Wash the palm, back and wrist of each hand using strong,
Electrical Ungrounded or Burns or shock frictional and circular movements
wet equipment; 6. Interlace fingers and thumbs and move hands back and forth for
frayed cords ten seconds
Fire/ Explosive Open flames, Burns or dismemberment 7. Rub nails against the palm
organic 8. Rinse hands thoroughly
chemicals 9. Dry hands well
Physical Wet floors, Falls, sprains or strains 10. Use paper towel to turn the water off
heavy boxes,
patients

A. Biologic Hazards

❖ According to the CDC concept of Standard Precautions,

➢ “All human blood and other body fluids are treated as potentially
infectious for human immunodeficiency virus (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

Chain of Infection
Infectious Agents Consist of bacteria, fungi, parasites and
viruses
Reservoir Location of potentially harmful Personal Protective Equipment
microorganisms, the place where the Donning Removing
infectious agent can live and possible 1. Gown 1. Gloves
multiply 2. Mask 2. Headcap
Portal of Exit Way to exit the reservoir to continue the 3. Headcap 3. Gown
chain of infection 4. Gloves 4. Mask

Page 1 of 3
[BACT211] 1.01 Laboratory Safety and Microscopy I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Standard Precautions F. Fire/ Explosive Hazards
➢ Hand hygiene includes both hand-washing and the use of
alcohol-based antiseptic cleansers ➢ When a fire is discovered, all employees are expected to take the
▪ Perform hand washing if your hands are visibly soiled actions in the acronym RACE:
▪ Use alcohol if your hands are not visibly soiled ▪ Rescue – Rescue anyone in immediate danger
➢ Personal protective equipment ▪ Alarm – Activate the institutional fire alarm system
➢ Patient care equipment ▪ Contain – Close all doors to potentially affected areas
➢ Environmental control ▪ Extinguish/ Evacuate – Attempt to extinguish the fire, if
➢ Prevent injuries when using needles, scalpels and other sharp possible or evacuate, closing the door
instruments or devices
➢ Respiratory hygiene/cough etiquette
❖ Biological Waste Disposal
➢ All biological waste except urine, must be placed in
➢ Disinfection of the sink using a 1:5 or 1:10 dilution of sodium
hypochlorite should be performed daily
▪ Sodium hypochlorite dilutions stored in plastic bottles are
effective for 1 month if protected from light after preparation

B. Sharp Hazards
➢ Includes needles, lancets and broken glassware
➢ All sharp objects must be disposed in punctured-resistant
containers Types of Fires and Fire Extinguishers
➢ The biohazard sharp containers should not be overfilled and Fire Type Composition of Type of Fire Extinguishing
must always be replaced when the safe capacity mark is reached Fire Extinguisher Material
Class A Wood, paper or Class A Water
C. Chemical Hazards clothing
➢ Every chemical in the workplace Class B Flammable Class B Dry chemicals,
should be presumed hazardous organic carbon dioxide,
➢ When skin contact occurs, the best chemicals foam or halon
first aid is to flush the area with large Class C Electrical Class C Sand or dry powder
amounts of water for at least 15
Class D Combustible None Dry chemical
minutes, then seek medical attention
metals Class ABC
➢ The National Fire Protection
Class K Grease, oils, fats Class K Liquid designed to
Association (NFPA) has developed
prevent splashing
the standard system for the
and cool the fire
identification of the Fire Hazards of
Materials, NFPA 704.14
A student successfully completing basic microbiology will
➢ This symbol system is used to inform
demonstrate the ability to explain and practice safe
firefighters of the hazards they may
encounter with fires in a particular area
➢ The diamond-shaped, color-coded symbol contains information 1. Microbiological Procedures including:
relating to health, flammability, reactivity, and personal a. Reporting all spills and broken glassware to the instructor
protection/special precautions and receiving instructions for cleanup
❖ Material Safety Data Sheets b. Methods for aseptic transfer
➢ Information contained in an MSDS includes the following: c. Minimizing or containing the production of aerosols and
1. Physical and chemical characteristics describing the hazards associated with aerosols
2. Fire and explosion potential d. Washing hands prior to and following laboratories and at
3. Reactivity potential any time contamination is suspected
4. Health hazards and emergency first aid procedures e. Never eating or drinking in the laboratory
5. Methods for safe handling and disposal f. Using universal precautions
6. Primary routes of entry g. Disinfecting lab benches prior to and at the conclusion of
7. Exposure limits and carcinogenic potential each lab session
h. Identification and proper disposal of different types of waste
D. Radioactive Hazards • Microorganisms: Soak in 1:10 bleach or spray with
Lysol and Autoclave.
➢ Radioactivity may be encountered in the clinical laboratory when i. Never applying cosmetics, including contact lenses, or
procedures using radioisotopes are performed placing objects (fingers, pencils) in the mouth or touching
➢ Exposure to radiation during pregnancy presents a danger to the the face
fetus; personnel who are pregnant or think they may be should j. Reading and signing a laboratory safety agreement
avoid areas with this symbol indicating that the student has read and understands the
safety rules of the laboratory
E. Electrical Hazards k. Good lab practice, including returning materials to proper
locations, proper care and handling of equipment, and
➢ Equipment should not be operated with wet hands
keeping the bench top clear of extraneous materials
➢ Designated hospital personnel monitor electrical equipment
2. Protective procedures, including:
closely; however, laboratory personnel should continually
a. Tying long hair back, wearing personal protective
observe for any dangerous conditions, such as frayed cords and
equipment (eye protection, coats, closed shoes; glasses
overloaded circuits, and report them to the supervisor
may be preferred to contact lenses), and using such
➢ Equipment that has become wet should be unplugged and
equipment in appropriate situations
allowed to dry completely before reusing. Equipment also should
b. Always using appropriate pipetting devices and
be unplugged before cleaning
understanding that mouth pipetting is forbidden
➢ When an accident involving electrical shock occurs, the electrical
3. Emergency procedures, including:
source must be removed immediately
a. Locating and properly using emergency equipment (eye-
➢ This must be done without touching the person or the equipment
wash stations, first-aid kits, fire extinguishers, chemical
involved to avoid transferring the current
safety showers, telephones, and emergency numbers)
➢ Turning off the circuit breaker, unplugging the equipment, or
b. Reporting all injuries immediately to the instructors
moving the equipment using a nonconductive glass or wood
c. Following proper steps in the event of an emergency
object are safe procedures to follow
Page 2 of 3
[BACT211] 1.01 Laboratory Safety and Microscopy I Prof. Rochelle D. Darlucio, RMT, MPH
II. MICROSCOPY (B) Revolving Above the stage Holds the objective lenses
nosepiece
(C) Objective Held in place Used to magnify objects placed on
❖ Microscope Lenses above the stage the stage
➢ An optical instrument that is used to observe tiny objects, often by the revolving
objects that cannot be seen at all with the unaided human eye nosepiece
(the “naked eye”) (D) Stage Directly beneath Flat surface on which the specimen is
❖ Anton van Leeuwenhoek (1632 – 1723) the nosepiece placed
and objective
➢ The first person to see live bacteria and protozoa
lenses
➢ “father of microbiology, bacteriology, protozoology” Stage adjustment Beneath the Used to move the stage and
➢ During his lifetime, he made more than 500 single-lens knobs (not shown) stage microscope slide
microscopes or simple microscopes (E) Iris Diaphragm On the Used to adjust the amount of light
Control Arm condenser passing through the condenser
B. Types of Microscope (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
❖ Brightfield Microscope
with Field condenser the condenser
➢ Used to observe morphology of microorganisms such as Diaphragm
bacteria, protozoa, fungi, and algae in living (unstained) and (H) Rheostat Front side of the Controls the amount of light emitted
nonliving (stained) state Control Knob base from the light source
▪ Background is bright, organism is dark (I) Field Attached to the Used to adjust the amount of light
❖ Darkfield Microscope Diaphragm Lever field diaphragm passing through the collector lens
➢ Unstained organisms are observed against a dark background (J) On/ Off Switch On the side of Turns the light source on and off
➢ Useful for examining thin spirochetes the base
▪ Background is dark, organism is bright (K) Base Contains the light source
❖ Phase-contrast Microscope (L) Condenser Beneath and Used to adjust the height of the
Control Knob behind the condenser
➢ Can be used to observe unstained living microorganisms condenser
❖ Fluorescence Microscope (M & N) Fine and On the arm of Used to focus the objective lenses
➢ Fluorescent dye attached to organism Coarse Adjustment the microscope Fine Adjustment Knob: Higher
➢ Primarily an immunodiagnostic technique (immunofluorescence) Knobs near the base Magnification (HPO, Oil immersion
➢ Used to detect microbes in cells, tissues and clinical specimens 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

❖ Oil Immersion Objective: (Cedarwood Oil)

E. Magnification Formula for Total Magnification

❖ Total Magnification = Eyepiece x Objective

➢ Example: Eyepiece Magnification = 10x
Objective Magnification = high power field (40x)
Total Magnification = (10x) (40x) = 400x total
C. Parts and Function of Microscope magnification
❖ Actual Size (um) = Magnification of Objective Lenses / Image Size
(um)
Component Location Function
(A) Ocular lens At the top of the The ocular lens is an x10 magnifying
➢ Example:
(also known as an microscope lens Oil Immersion Field of View = 0.20mm → 200um
eyepiece); a Image size measured = 1000um
monocular Estimate Size (um) = 200um/1000um = 0.20um
microscope has actual size
one; a binocular
microscope has
two

Page 3 of 3
ASEPTIC TECHNIQUE LAB 2020-
2021

CLINICAL BACTERIOLOGY 2 2nd SEM

BACT21
Transcriber: GAD. MPL. 1
Date: February 19, 2021 LAB
[TRANS] UNIT 2: ASEPTIC TECHNIQUE

CULTURE TUBE FLAMING AND INOCULATION

OUTLINE:
I. Introduction
II. Transfer from Broth Culture to Another Broth  Prior to inserting a cooled loop or needle into a culture
III. Transfer of Bacteria from a Slant tube, the cap is removed and the mouth of the tube may
IV. Working with Agar Plates be flamed.
V. Pure Culture Techniques  If the tube is a broth tube, the loop is inserted into the tube
A Streak Plate Method and twisted several times to ensure that the organisms on
i. Quadrant Streak
the loop are delivered to the liquid.
ii. Radiant Streak
iii. Continuous Streak  If the tube is an agar slant, the surface of the slant is
Note: inoculated by drawing the loop up the surface of the slant
Italicized – additional information from the bottom of the slant to its top.
 For stab cultures, a needle is inserted into the agar
INTRODUCTION medium by stabbing it into the agar.
 Aseptic Technique means using practices and  Open the tube by the use of dominant hand pinky finger,
procedures to prevent contamination. It involves applying while the non-dominant hand is holding the tube
the strictest rules to minimize the risk of infection.

WORK AREA DISINFECTION

 The work area is first treated with a disinfectant to kill any

microorganisms that may be present.
 Ensure organism does not contaminate the handler.
 Can use any different type of disinfectant
 Ex. Sodium hypochlorite or Lysol

LOOPS AND NEEDLES

 A loop or needle is sterilized by inserting it into a Bunsen

burner, incinerator flame or alcohol lamp until it is red-hot.
 This will incinerate any contaminating organisms that may
be present.
 Allow the loop to cool completely *not cooling the loop
may kill the bacteria before picking up inoculum. This will
ensure that viable cells are transferred.
 Also called inoculating loop/needle
 Loops – circle on the end
 Needle – straight on the end
 Proper Handling
 Assume the loop/needle is a ballpen
 Use dominant hand on holding
 In sterilizing the loop/needle, pass through flame starting
to tip to base or base to tip

PETRI PLATE INOCULATIONS

 The plate cover is raised *slightly open only and held

diagonally over the plate to protect the surface from any
contamination in the air.
 The loop containing the inoculum is then streaked gently
over the surface of the agar.
 It is important not to gouge or disturb *do not press the
agar, mild streaking only the surface of the agar with the
loop.
 The cover is replaced and the loop is flamed.

PAGE 1 OF 6
[BACT211] TRANS: ASEPTIC TECHNIQUE | Prof. Rochelle D. Darlucio, RMT, MPH

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
container.
12. Incubate the culture you just inoculated at 37C for 24-
48 hours.

 Disinfect the table

 Sterilize the inoculating tube and let it cool down
 Hold the broth and gently shake
 Open the broth by the pinky finger and pass through
the tube to flame
 Get a colony – one loop full
 Pass through flame before closing the tube

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
implements.
 The loop or needle is then returned to its receptacle for
storage.
 It should never be placed on the desk surface.

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 2 OF 6
[BACT211] TRANS: ASEPTIC TECHNIQUE | Prof. Rochelle D. Darlucio, RMT, MPH

TRANSFER OF BACTERIA FROM A SLANT

MATERIALS
 Agar slant culture of E. coli
 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.

 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
WORKING WITH AGAR PLATES
 Pass through flame before closing
 Sterilize the loop
 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 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

PAGE 3 OF 6
[BACT211] TRANS: ASEPTIC TECHNIQUE | Prof. Rochelle D. Darlucio, RMT, MPH

5. With your free hand, pick up the sterile nutrient agar

slant tube. Remove the cap by grasping the cap with
 Flaming Procedures the little finger of the hand that is holding the loop.
 Inoculating loops or needles must be flamed in the 6. Flame the mouth of the tube and insert the loop into
same manner that you used when working with the tube to inoculate the surface of the slant, using a
previous tubes. One difference when working with serpentine motion. Avoid disrupting the agar surface
plates is that plates are never flamed! with the loop.
 Plate Labeling and Incubation 7. Remove the loop from the tube and flame the mouth
 Petri plates containing inoculated media are labeled of the tube. Replace the cap on the tube.
on the bottom of the plate. Inoculated plates are 8. Flame the loop and place it in its container.
almost always incubated upside down. This prevents 9. Incubate the nutrient agar slant at 37C for 24-48
moisture from condensing on the agar surface and hours.
spreading the inoculated organisms.
 The label must be not too big or not too small
 Name of organism and date

PURE CULTURE TECHNIQUES

STREAK PLATE METHOD

 Pure culture
 Single kind of organism
 Mix culture
 More than one kind of microorganism

PROCEDURE

1. If you have not done so, swab your work area with
disinfectant. Allow area to dry.
2. Label a sterile nutrient agar slant with your name and
organism to be transferred.
3. Flame an inoculating loop until it is red-hot. Allow the
loop to cool.
4. Raise the lid of a petri plate sufficiently to access a
colony with your sterile loop. Do not gouge the agar
with your loop as you pick up organisms. Simply allow
the loop to gently glide over the gelatin like surface of
the agar. Do not completely remove the lid while
inoculating or removing organisms from the agar plate.
This will expose the agar surface to air and potential
contamination. Always close the lid once you have
removed organisms from the plate.

PAGE 4 OF 6
[BACT211] TRANS: ASEPTIC TECHNIQUE | Prof. Rochelle D. Darlucio, RMT, MPH

QUADRANT STREAK

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

zigzag.
 90̊ rotation is important for easy streaking.

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.
4. Sterilize the loop
5. Streak again on quadrant 3 by touching quadrant 2.
6. Sterilize the loop
7. Streak line on quadrant 4 by touching quadrant 3.
8. Sterilize the loop

 Do not put too much pressure

 Streak fast as possible
 Keep the plate cover half open when streaking
 90̊ rotation if quadrant method

PAGE 5 OF 6
[BACT211] TRANS: ASEPTIC TECHNIQUE | Prof. Rochelle D. Darlucio, RMT, MPH

RADIANT STREAK

CONTINUOUS 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 6 OF 6
SMEAR PREPARATION & LAB 2020-
2021

SIMPLE STAINING 3 2nd SEM

BACT21
CLINICAL BACTERIOLOGY 1
Transcriber: GAD. MPL.
Date: February 19, 2021 LAB
[TRANS] UNIT 3: SMEAR PREPARATION

OUTLINE
I. Bacterial Smear
A. From Broth Cultures
B. From Plates and Slants
II. Simple Staining

Note:
Italicized – additional information

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 )
 (3) Internal structures ( endospores )

FROM BROTH CULTURES

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.

PAGE 1 OF 2
[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. 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).

PAGE 2 OF 2
GRAM STAINING & LAB 2020-
2021

ACID-FAST STAINING 4 2nd SEM

BACT21
CLINICAL BACTERIOLOGY 1
Transcriber: GAD. MPL. LAB
Date: March 4, 2021
[TRANS] UNIT 4: GRAM STAINING & ACID FAST STAINING

OUTLINE
I Gram Staining
A Gram Stain Procedure
II Acid Fast Staining Kinyoun Method

Note:
Italicized – additional information

GRAM STAINING

 Differential Stain
 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
 Prolong decolorization, gram positive might appear as gram
negative

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.

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

Acid Fast Staining

 An important diagnostic tool in the identification of Mycobacterium
tuberculosis, the causative agent of tuberculosis, and
Mycobacterium leprae, the bacterium that causes leprosy in
humans.
Mycolic Acid
 A complex lipid that is composed of fatty acids and fatty alcohols
that have hydrocarbon chains up to 80 carbons in length.
 Affects the staining properties of bacteria and prevents them from
being stained by many of the stains routinely used in microbiology.

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

Page 2 of 3
[BACT211] TRANS: GRAM STAINING & ACID FAST STAINING | Prof. Rochelle D. Darlucio, RMT, MPH

Mixed cultures under the microscope

Reporting:
 Gram positive cocci in clusters

Reporting:
 Gram positive bacilli
 Dark violet - spores

Reporting:
 If many arrangements:
 Gram positive cocci in singly, pairs, and clusters

Reporting:
 Gram negative bacilli in singly

Reporting:
 Gram negative bacilli in clusters
 Gram negative bacilli in singly

Reporting:
 Gram positive bacilli in clusters
 Gram positive bacilli in singly

Gram-stained Smear from specimens

Reporting:
 Gram negative cocci in pairs (diplococci)

Page 3 of 3
MICROBIOLOGICAL CULTURE MEDIA LAB 2020-
2021
PREPARATION AND STERILIZATION
CLINICAL BACTERIOLOGY 5 2nd SEM
BACT21
Transcriber: GAD. MPL. 1
Date: March 19, 2021 LAB
[TRANS] UNIT 5: MICROBIOLOGICAL CULTURE, MEDIA PREPARATION & STERILIZATION

OUTLINE
I Cultivation
i. 3 Main Purposes of Cultivating Bacteria
ii. Nutritional Requirements
A Terms to Remember
II Culture Media
A Classification CLASSIFICATION
i. According to Consistency
ii. According to Chemical Composition
iii. According to Function I. ACCORDING TO CONSISTENCY
B Culture Media Preparation Steps 1. Solid – solidifying agent is added (1.5 3% of agar)
C Culture Media Calculations  Agar is the most commonly used as solidifying agent
III Sterilization
 Melt: ≥95 C
i. Three Basic Ways of Sterilization of Media
IV Inoculating Techniques  Resolidify: <50 C
 Uses:
Note:  For the surface growth of microorganisms in order to
– additional information observe colony appearance
 For pure culture isolations
 Storage of cultures
CULTIVATION  To observe specific biochemical reactions.

 Process of growing microorganisms in culture by taking bacteria Solid media can be poured into either a test tube or petri plate (dish)
from the infection site (in vivo) and growing them in the culture  In tube:
media of the laboratory (in vitro) 1. Agar Slant - the medium in the test tube is allowed to
harden in a slanted position.
3 MAIN PURPOSES OF CULTIVATING BACTERIA: 2. Agar deep - the tube is allowed to harden in an upright
position.
1. To grow and isolate all bacteria present in the clinical  In Plate:
specimen.  Agar Plate – containing about 15 to 16 ml of media.
2. To determine which of the bacteria that grow is most likely  20 ml (standard)
causing infection and which are likely causing infection and
which are likely contaminant.
3. To obtain sufficient growth of clinically relevant bacteria to
allow identification and characterization

NUTRITIONAL REQUIREMENTS
 Water
 Ions
 Nitrogen
 Gases
 Sources of carbon
2. Liquid / Broth – dissolved in water
Latter requirements: carbohydrate & protein
 Uses:
 Can be used to propagate large numbers of
TERMS TO REMEMBER: microorganisms in fermentation studies
1. Culture Media - nutrients prepared for bacterial growth
 For various biochemical tests.
2. Inoculum - suspension of microorganism
 Turbid - presence of microorganism
3. Inoculation - introduction of bacteria into culture medium
4. Culture - bacteria growing on culture medium
 Pure culture - contains only one specie
 Mix culture - contains several species
 Contaminated culture - contains unwanted species or
organisms
5. Colony - visible growth of microorganism on the surface of
culture media
6. Fastidious Organism - nutritional needs are relatively
complex and exceptional components used for the growth
7. Non-fastidious Organism - nutritional needs are relatively
basic and straightforward.
3. Semi solid - solidifying agent is added (<1.5% of agar)
CULTURE MEDIA a. Ex. Sulfide Indole Motility (SIM) Medium
 Uses:
 Nutrient preparations that are used for culturing microorganisms
 Fermentation studies
 Solid, liquid or semi-solid designed to support the growth of  In determining bacterial motility
microorganisms
 Promoting anaerobic growth

PAGE 1 OF 3
[BACT211] TRANS: MICROBIOLOGICAL CULTURE MEDIA PREPARATION & STERILIZATION | Prof. Rochelle D. Darlucio, RMT, MPH

 Uninocoluted
 Appearance: clear
 Manner of innoculation: Stab (half way) using the
incolutating needle
 Positive for motility
 Appearance: spreading of the colony along the site of
innoculation
 Negative (non-motile)
 Appearance: straight line

II. ACCORDING TO CHEMICAL COMPOSITION

1. Synthetic
 Chemically defined
 Use to culture algaes or nonfastidious heterotrophs
2. Non-synthetic (complex)
 Chemical composition is not specifically defined;
extracts of yeasts, meat or plants. (Bacteria are
usually grown in this type of media)
Ex: Nutrient broth, Nutrient agar, EMB CULTURE MEDIA PREPARATION STEPS
III. ACCORDING TO FUNCTION TUBE (WDDS) PLATE (WDSD)
1. Weight 1. Weigh
1. Enrichment media 2. Dissolve 2. Dissolve
 Contains specific nutrients required for the growth of 3. Dispense 3. Sterilize
particular bacterial pathogens 4. Sterilize 4. Dispense
 Used to enhance the growth of particular pathogen
from a mixture of organism by using nutrient  Instructions, procedures, directions is in the bottle on
specificity how to prepare the agar.
 Example: Buffered-charcoal yeast extract agar (BCYE) that  There is a specific grams per 1000 mL of distilled
contains L-cysteine required for the growth of Legionella water.
pneumophilia  Approximate of 20 mL in every plate. (Standard
amount)
2. Selective Media
 Favour the growth of a particular bacterium by inhibiting CULTURE MEDIA CALCULATIONS
the growth of undesired bacteria and allowing the
growth of desired bacteria.  We should be reading “directions” listed on media base
 Example: EMB, MAC, SSA, HEA, MSA container carefully
 For example, on the container of nutrient agar the following
3. Enriched Medium directions are listed:
 Enriched usually by adding blood, or eggs.  Example:
 Example: Blood agar plate and Chocolate agar plate  Suspend 28 g of powder in 1000 mL of D.W.,
then dissolve by heating until boiling. Autoclave
4. Supportive / Nonselective Medium for 20 min, then cool and pour in Petri-dishes.
 Contain nutrients to support growth of most non-  FORMULA FOR DESIRED VOLUME:
fastidious organisms without giving any particular  20 ml x (desired plates)
organism a growth advantage  So, if we need to prepare 1000 mL (1 L) of nutrient agar
 General culture media medium, we should to be dissolve 28.0 grams of media
powder in 1000 mL of distilled water.
5. Differential Medium  The amount of media poured in each Petri-dish is about 20 mL.
 Used to distinguish them from other bacteria growing
on the same agar plate
 If we don't need 1000 mL of media; we should multiply the
number of dishes needed by 20, then consider it as the total
 Contains indicator
volume (V) of media needed in the following equation.
Example: EMB, MAC, SSA, HEA, MSA
��. �� ����� �� ���������� (�) �� �� ������ �ℎ���� �� ����ℎ� (�)
=
� �� �. �. �� ���������� (��) � �� ����� �. �. �� ���� (��)

 Ex.
28 � �
=
1000 �� 400 ��

PAGE 2 OF 3
[BACT211] TRANS: MICROBIOLOGICAL CULTURE MEDIA PREPARATION & STERILIZATION | Prof. Rochelle D. Darlucio, RMT, MPH

= 11.2 � THREE BASIC WAYS OF STERILIZATION OF MEDIA:

 All volumes should be converted to (mL), and
 Autoclaving
weights to (g) before beginning calculations.  Exposure to steam at 121 C and 15 lbs of pressure for
 From (L) to (mL) multiply by 1000, while from (mL) to (L) 15 minutes or longer, depending on the nature of the
divided by （1000). item.
 From (Kg) to (g) multiply by 1000, while from (g) to (Kg)  Microorganisms, even endospores , will not survive
divided by （1000). longer than about 12 to 13 minutes

 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.
 If we need (14) Petri-shish instead of entire 1000 mL, the volume
of media we need to prepare calculated as (14 x 20 = 280 mL) of
media.

28 � �
=
1000 �� 280 ��
28 � × 280 ��
�= = 7.48 �
1000 ��  Ultraviolet (UV) radiation
 Around 260 nm is quite lethal to many microorganisms
 7.48 g of powder should be dissolve in 280 mL of but does not penetrate glass, dirt films, water, and other
D.W. substances very effectively.
 Ethylene oxide
 After computation, use aluminum foil to weigh the powder.  Both microbicidal and sporicidal and kills by covalently
 Weigh it using the analytical balance. attaching to cell proteins.
 Measure the water using graduated cylinder then place it  It is a particularly effective sterilizing agent because it
in Erlenmeyer flask. rapidly penetrates packing materials, even plastic
wraps.
 REMEMBER: Transfer the first half of the water into
the Erlenmeyer flask.
 Add the powder, mix until it dissolves then pour the
INOCULATION TECHNIQUES
remaining half of the water.
 Tube
 Sterilize. 1. LIQUID MEDIA
 Dispense. (work near the flame)  Plain Broth, TSB
 ALWAYS wear a mask in preparing the agar.  Material: Inoculating loop
 Manner of inoculation: Twirl / Mixing
BLOOD AGAR PREPARATION
1. Weigh
2. Dissolve
3. Sterilize
4. Make sure the agar is cooled down. (to avoid the lysis of RBC or
the creation of chocolate agar)
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 2. SLANTED MEDIA
heating.  Simmon's Citrate, Urea
 Material: Inoculating needle / Wire loop
 If it contains agar, you must heat the medium until it starts to
 Manner of inoculation : Streak / Line
simmer or boil in order to completely dissolve the agar.

STERILIZATION
 The process of rendering a medium or material free of all forms of
life.

3. SEMI SOLID BUTT MEDIA

 SIM
 Material: Inoculating needle / Wire loop
 Manner of inoculation: Stab Halfway

PAGE 2 OF 3
[BACT211] TRANS: MICROBIOLOGICAL CULTURE MEDIA PREPARATION & STERILIZATION | Prof. Rochelle D. Darlucio, RMT, MPH

4. BUTT SLANTED MEDIA

 TSI, LIA
 Material: Inoculating needle / Wire loop
 Manner of inoculation: Stab & Streak

PAGE 2 OF 3
OLFU STAPHYLOCOCCI LAB 2020-
2021
College of Medical 2nd SEM
Laboratory Science
CLINICAL BACTERIOLOGY 6 BACT21
Transcriber: GAD. MPL. 1
Batch 2023 Date: March 19, 2021 LAB
[TRANS] UNIT 6: STAPHYLOCOCCI

CULTIVATION
OUTLINE:
I. Staphylococcus
II. Micrococcus
MEDIA OF CHOICE
III. Laboratory Diagnosis  Grow on 5% sheep blood and chocolate agars
A Specimen Collection & Handling  They also grow well in broth blood culture systems and common
B Microscopic Examination nutrient broths
C Cultivation
i. Media of Choice Selective Media:
D Catalase Test  Phenylethyl alcohol (PEA)
E Coagulase Test  Columbia colistin nalidixic acid (CNA) agars
F Slide Coagulase Test  Mannitol Salt Agar
G Tube Coagulase Test  CHROMagar
H Bacitracin Susceptibility Test
I Novobiocin Disk Test  Staphylococci produce round, smooth, white, creamy colonies on
SBA after 18 to 24 hours of incubation at 35C to 37 C
Note:
Italicized – additional information
 S. aureus can produce hemolytic zones around the colonies and may
rarely exhibit pigment production with extended incubation.

STAPHYLOCOCCUS
 Derived from the Greek term staphle, meaning “bunches of grapes
 Catalase producing, Gram positive cocci that appear in singly, in
pairs, and in cluster.
 Non-motile , non-spore forming, and aerobic or facultatively anaerobic
except for S. Saccharolyticus, which is an obligate anaerobe
 Toxin induced diseases, such as food poisoning, scalded skin
syndrome (SSS), and toxic shock syndrome (TSS), are also
associated with this organism.
MICROCOCCUS
 Catalase producing, coagulase negative, gram positive cocci
 They are often recovered with staphylococci and can be differentiated
easily from coagulase negative staphylococci (CoNS)

LABORATORY DIAGNOSIS

SPECIMEN COLLECTION AND HANDLING

 Clinical materials collected from infected sites should be transported CATALASE TEST
to the laboratory without delay to prevent drying, maintain the proper  This test differentiates catalase positive micro-coccal and
environment, and minimize the growth of contaminating organisms. staphylococcal species from catalase negative streptococcal species
o Turbidity of the specimen - presence of microorganism  Principle: The enzyme, catalase, is capable of converting hydrogen
peroxide to water and oxygen. The presence of enzyme in bacterial
MICROSCOPIC EXAMINATION isolate causes rapid elaboration of bubbles.
 Reagent :3% Hydrogen peroxide(H2O2)
 Gram positive cocci  Results:
 Gram stains should be performed on young cultures  Positive: Bubbles (Rapid bubbling) / Effervescence
 Negative: No bubbles

PAGE 1 OF 3
[BACT211] TRANS: STAPHYLOCOCCI | Prof. Rochelle D. Darlucio, RMT, MPH

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

1. Emulsify several colonies of the unknown clinical isolate in

0.5mL of rabbit plasma (with EDTA) to give a milky suspension.
2. Repeat the same process with a known positive and negative
control organism.
3. Incubate tube at 35'C to 37'C in ambient air for 1 to 4 hours.
4. Check for clot formation.
 Purpose
 The test is used to differentiate Staphylococcus aureus  Expected Results
(positive) from coagulase-negative staphylococci  Positive: Clot of any size.
(negative).  Negative: No clot

PAGE 2 OF 3
[BACT211] TRANS: STAPHYLOCOCCI | Prof. Rochelle D. Darlucio, RMT, MPH

 Limitations
Slide Test:
 Equivocal: Clumping in both the rabbit plasma reagent and
water or saline control drops indicate that the organism
auto-agglutinates and is unsuitable for the slide coagulase
test.
Tube Test:
1. Test results can be positive at 1 to 4 hours and then revert to
negative after 24 hours.
2. If negative at 4 hours, incubate at room temperature overnight
and check again for clot formation.
Quality Control
 Positive: Staphylococcus aureus (ATCC25923)

BACITRACIN SUSCEPTIBILITY TEST

 It is used to distinguish staphylococci species (resistant) from
micrococci (susceptible).
 Principle: The antibiotic bacitracin inhibits the synthesis of bacterial
cell walls. A disk (TaxoA) impregnated with a small amount of
bacitracin (0.04 units) is placed on an agar plate, allowing the
antibiotic to diffuse into the medium and inhibit the growth of
susceptible organisms.
 Results:
 Positive: >10mm = susceptible
 Negative:<10mm = not susceptible

1. Using an inoculating loop, streak two or three suspect colonies

of a pure culture onto a blood agar plate.
2. Using heated forceps, place a bacitracin disk in the first quadrant
(area of heaviest growth). Gently tap the disk to ensure adequate
contact with the agar surface.
3. Incubate the plate for 18 to 24 hours at 35°C to 37°C in ambient
air for staphylococci and in 5% to 10% carbon dioxide (CO2) for
streptococci differentiation.
4. Look for a zone of inhibition around the disk.

 Expected Results
 Positive: Any zone of inhibition greater than 10 mm:
susceptible
 Negative: No zone of inhibition; resistant

NOVOBIOCIN DISK TEST

 Used to differentiate Coagulase Negative Staphylococci
 5ug
 Positive: Zone of Inhibition >11mm
 Negative: Zone of Inhibition <16mm

PAGE 3 OF 3
Streptococci & Enterococci: LAB 2020-
2021
Isolation and Identification 7 2nd SEM
BACT21
CLINICAL BACTERIOLOGY 1
Transcriber: GAD. MPL. LAB
Date: March 26, 2021
[TRANS] UNIT 7: STREPTOCOCCI AND ENTEROCOCCI: ISOLATION AND IDENTIFICATION

HEMOLYTIC PATTERNS
OUTLINE
I Streptococci and Enterococci  Alpha (a)
A Gram Stain Procedure ▪ Partial lysis of RBCs around colony
II Hemolytic Patterns ▪ Greenish discoloration of area around colony
A Beta Hemolytic Groups  Beta (β)
i. Group B Streptococci ▪ Complete lysis of RBCs around colony
ii. Group C Streptococci ▪ Clear area around colony
B Alpha Hemolytic Groups  Nonhemolytic
i. Streptococcus Pneumoniae ▪ No lysis of RBCs around colony
ii. Viridans Streptococci Group ▪ No change in agar
iii. Group D Enterococci
iv. Group D Non-Enterococci
III Laboratory Diagnosis
A Alpha Hemolysis
B Optochin Disk (Taxo P)
C Bile Solubility Test
D Bile Esculin Test
E 6.5% NaCl / Salt Tolerance Test
F Differentiation of GAS to GBS
G Bacitracin Susceptibility Test
H CAMP Test
I Hippurate Hydrolysis Test
BETA HEMOLYTIC GROUPS
Note:
Italicized – additional information
GROUP A STREPTOCOCCI
STREPTOCOCCI AND ENTEROCOCCI  Streptococcus pyogenes , the main representative of group A
 Gram positive cocci streptococci, is by far the most serious rapidly injure cells and
tissues.
 Facultative anaerobes and generally considered non-motile
o Most common
 Occur singly or in pairs, however, they are best known for their o Only sensitive and susceptible to Bacitracin Test
characteristic formation of long chains
 In order to differentiate S. pyogenes from other streptococci and
 Some species are capnophilic enterococci, isolates are tested for resistance to bacitracin.
o Placed in a “candle jar” serves as a dessicator
 If a bacterial isolate is beta hemolytic and sensitive to bacitracin, it
 Pyogenic causing bacteria is presumed to be S. pyogenes.
o Pus-producing bacteria  Has a clear zone of hemolysis
 Spherical cocci (gram +)
 Streptococci and Enterococci differ to Staphylococci and
Micrococci in:
 They occur in chains rather than clusters GROUP B STREPTOCOCCI
 Lacks enzymes catalase  S. agalactiae
 This pathogen may be found in the pharynx, skin, and rectum;
however, it is more likely to be found in the genital and intestinal
tracts of healthy adults and infants.
 S. agalactiae colonies are large, with a narrow zone of beta
hemolysis
 Preliminary identification of this species relies heavily on a
positive CAMP reaction.
 Important cause of neonatal infection
 Has a positive reaction in CAMP TEST

 They are classified using the Lancefield Group Antigen GROUP C STREPTOCOCCI
 Uncommon human pathogens but may be involved in zoonosis.
 The organism of importance in this group is S. dysgalactiae, and
infections from this species account for less than 1% of all
bacteremias.
 S. dysgalactiae produce large colonies with a large zone of beta
hemolysis on blood agar.
 Presumptive differentiation of S. dysgalactiae from other beta
hemolytic streptococci (S. pyogenes and S. agalactiae) is based
primarily on resistance to bacitracin and a negative CAMP test.
 Can cause disease like pharyngitis and endocarditis

PAGE 1 OF 4
[BACT211] TRANS: STREPTOCOCCI AND ENTEROCOCCI: ISOLATION & IDENTIFICATION | Prof. Rochelle D. Darlucio, RMT, MPH

ALPHA HEMOLYTIC GROUPS HEMOLYTIC PATTERNS

STREPTOCOCCUS PNEUMONIAE
 A significant human pathogen.
 Colonies appear smooth, mucoid, and surrounded by a zone of
greenish discoloration (alpha hemolysis).
 In culture these cells usually grow as diplococci, but they can also
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. ALPHA HEMOLYSIS
 In culture they appear rod like and grow in chains.  S. Pneumoniae and Viridans streptococci
 The viridans group can be differentiated from the pneumococci
and enterococci by a negative result in the bile esculin
hydrolysis test, the salt tolerance test, and the optochin
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

GROUP D NON-ENTEROCOCCI
 S. Bovis
o Found in human’s intestinal tract, cow and sheep
 In blood agar, the colonies of S. bovis appear large, mucoid
(many strains have a capsule), and either non-hemolytic or alpha OPTOCHIN DISK (TAXO P)
hemolytic.  Used to determine the effect of Optochin (ethyl hydrocupreine
 In culture S. bovis grows in pairs and short chains. hydrochloride) in an organism
 Key reactions for this group are a positive bile esculin test and  Optochin lyses Pneumococci (POSITIVE)
negative salt broth test.  Principle: Optochin interferes with the ATPase and
production of ATP in microorganisms
 Result: A zone of 14 16 mm is considered susceptible
LABORATORY DIAGNOSIS and presumptive identification for Streptococcus
pneumoniae.
1. GRAM STAIN - Gram positive cocci in pairs or in chains  Limitations: Equivocal: Any zone of inhibition less than
2. Growth on BAP & CAP 14 mm is questionable for pneumococci, the strain is
 GROUP A identified as a pneumococcus with confirmation by a
- Grayish white positive bile solubility test.
- Transparent to translucent  Note: It is important to remember that pneumococci are
- Matte or glossy sometimes optochin resistant.
- Large zone of beta hemolysis  S. pneumoniae (+)
 GROUP B
- Larger than Grp A
- Translucent to opaque
- Flat or glossy
- Narrow zone of beta hemolysis
 GROUP C
- Grayish white
- Glistening
- Wide zone of hemolysis
3. CATALASE TEST - NEGATIVE
 A biochemical test
 If using a 30% Hydrogen peroxide (reagent) it is a
Superoxol Test
 Staphylococcus BILE SOLUBILITY TEST
 Appears in pin head colony  Confirmatory test for Streptococcus pneumoniae
 Streptococcus/Enterococcus  Used to differentiate S. pneumoniae from Viridans strep
 Appears in pin point colony  Principle:
- Sodium desoxycholate (bile salt) rapidly lyses
pneumococcal colonies.
- Lysis depends on the presence of an intracellular
autolytic enzyme

PAGE 2 OF 4
[BACT211] TRANS: STREPTOCOCCI AND ENTEROCOCCI: ISOLATION & IDENTIFICATION | Prof. Rochelle D. Darlucio, RMT, MPH

 Reagents: Plate (10% Sodium desoxycholate, Tube

(2% Sodium desoxycholate
 Result:

Tube Plate
(+) No turbidity (+) Lysed colonies
(-) Remains Turbid (-) Intact colonies

SCHEMATIC DIAGRAM FOR DIFFERENTIATION OF

GAS FROM GBS

BILE ESCULIN TEST

 Used for the presumptive ID for Enterococcus and Group D
Streptococcus
 To differentiate Enterococcus & Group D FROM Viridans Strep
 Principle:
- Gram (+) are inhibited by bile salt in this medium. -
Organisms capable of growth in the presence of 4%
bile and able to hydrolyze esculin to esculetin. –
Esculetin reacts with Fe3+ and forms dark brown to
black precipitate
 Result:
(+) Growth or Blackening of the agar
(-) No blackening of the agar.

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)

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:
- Enterococci are resistant to high salt concentration
- Heart infusion broth w/ 6.5% NaCl use as test medium.
- This broth contains a small amount of glucose &
bromcresol purple as an indicator for acid production.
 Result:
(+) turbidity
(-) no turbidity (Non enterococcus)
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 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 to hydrolyze
hippurate
 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)

PAGE 4 OF 4
Biochemical Identification of Gram- LAB 2020-
2021
negative Bacteria 8 2nd SEM
BACT21
CLINICAL BACTERIOLOGY 1
Transcriber: GAD. MPL. LAB
Date: April 16, 2021
[TRANS] UNIT 8: BIOCHEMICAL IDENTIFICATION OF GRAM NEGATIVE BACTERIA

OUTLINE
I Family Enterobacteriaceae
II Laboratory Diagnosis
A Specimen Collection
B Gram Stain
C Culture
D Collonial Appearance
i. MacConkey Agar
ii. Eosin Methylene Blue
iii. Hektoen Enteric Agar
iv. Xylose-Lysine-Desoxycholate
III Biochemical Tests
A Carbohydrate Utilization CULTURE
i. Oxidation Fermentation Test  Grow at an optimal temperature of 35 C to 37 C
ii. Triple Sugar Iron  Low temperatures (1C to 5C, such as Serratia and
iii. Ortho-Nitrophenyl-β-Dgalactopyranoside Yersinia)
Test  Or tolerate high temperatures (45 C to 50 C, such as E.
B Glucose Metabolism and Its Metabolic Products
i. Methyl Red Test
coli)
ii. Voges-Proskauer Test  Visible after 18-24 hours of incubation
C Amino Acid Utilization  BAP, CAP, MacConkey Agar and other selective medium
i. Lysine Iron Agar (EMB, Hektoen,SSA)
ii. Decarboxylase Test (Moeller’s Method)
iii. Phenylalanine Deaminase Test COLONIAL APPEARANCE
D Miscellaneous Test
i. Sulfide Indole Motility Agar  All Enterobacteriaceae produce similar growth on blood
ii. Citrate Utilization and chocolate agars; colonies are large, gray, and smooth.
iii. Urease Test  Klebsiella or Enterobacter may be mucoid
iv. DNA Hydrolysis o due to the polysaccharide capsule
v. Gelatin Liquefaction  P. mirabilis, P. penneri, and [Link] “swarm” on blood
vi. Malonate Utilization and chocolate agars.
vii. Nitrate and Nitrite Reduction
o Swarming motility in BAP
viii. Oxidase
 Y. pestis on 5% sheep blood agar are pinpoint at 24 hours
Note: but exhibit a rough, cauliflower appearance at 48 hours.
Italicized – additional information  Broth cultures of Y. pestis exhibit a characteristic
“stalactite pattern”
FAMILY ENTEROBACTERIACEAE  Y. enterocolitica produces bull’s-eye colonies
 Gram negative bacilli and cocobacilli, non-spore forming,
facultatively anaerobic  Mucoid colony of Klebsiella or Enterobacter
 DO NOT produce cytochrome C oxidase except for
Plesiomonas
 ALL ferment glucose
 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  Formation of a “string”
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

 Bull’s eye colonies of Y. enterocolitica

MACCONKEY AGAR
 Best used to characterize gram negative rods
 Lactose fermenters can be differentiated with non-lactose
fermenters
 Selective media: gram negative enteric bacilli
 Differential media:
 Lactose fermenter - Dark pink
 Hafnia, Serratia, Citrobacter
 Salmonella arizonae
 Shigella sonnei EOSIN METHYLENE BLUE
 Yersinia enterocolitica  Selective media: gram negative enteric bacilli
 Non-lactose fermenter - Colorless  Differential media:
 All Salmonella except S. arizonae  Lactose fermenter - Dark violet
 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
 Proteus,Providencia,Morganella,
Edwardsiella

PAGE 2 OF 7
[BACT211] TRANS: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA | Prof. Rochelle D. Darlucio, RMT, MPH

HEKTOEN ENTERIC AGAR  Sulfide-indole-motility (SIM) or motility-indole-orn1thine

 Selective media: gram negative enteric bacilli (MIO) media
 Differential media:
 Lactose fermenter - Orange CARBOHYDRATE UTILIZATION
 Hafnia, Serratia, Citrobacter  Two enzymes are necessary for a bacterium to take up
 Salmonella arizonae lactose and to cleave it into monosaccharides :
 B-galactoside permease
 Shigella sonnei
 B-galactosidase
 Yersinia enterocolitica
 LFs- possess both β galactoside permease and β
 Non-lactose fermenter - Blue/Green
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
 Proteus,Providencia,Morganella,
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 and
XYLOSE-LYSINE-DESOXYCHOLATE 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
Enterobacteriaceae family from other gram-negative rods.
TRADITIONAL BIOCHEMICAL TESTS FOR
IDENTIFICATION OF GRAM-NEGATIVE BACTERIA  Expected Results
 Alkaline slant/no change in the butt (K/NC):
 Triple sugar iron (TSI) agar or Kligler iron agar (KIA) to glucose,lactose,and Sucrose nonutilizer,this may
determine glucose and lactose, or sucrose, utilization also be recorded as K/K (alkaline slant/alkaline butt)
(sucrose in TSI only) and hydrogen sulfide production  Alkaline slant/acid butt (K/A):glucose fermentation
 Methyl red and Voges-Proskauer tests to determine end only.
products of glucose fermentation  Acid slant/acid butt (A/A):glucose,sucrose,and/or
 Indole test to determine whether indole is formed from lactose fermenter
tryptophan by tryptophanase
 Urease test to determine hydrolysis of urea Note: A black precipitate in the butt indicates production of
 Simmons' citrate to determine whether citrate can be ferrous sulfide and H,S gas (H,S+)(Figure 12-41, B). Bubbles
used as the sole carbon source or cracks in the tube indicate the production of CO,or H2.
 Carbohydrate fermentation
 Lysine iron agar (LIA) to determine lysine decarboxylase  Kligler Iron Agar (KIA) - Used to determine glucose and
activity lactose or sucrose utilization and H2S production, gas
production

PAGE 3 OF 7
[BACT211] TRANS: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA | Prof. Rochelle D. Darlucio, RMT, MPH

 Composition: 10 parts lactose, 10 parts sucrose,

1 part glucose, peptone
 pH indicator: phenol red
 H2S Indicator: Ferrous sulfate and sodium
thiosulfate
 ** Reactions should be read within an 18 to 24 hour
incubation period; otherwise, erroneous results are
possible.

ORTHO-NITROPHENYL-Β-D-GALACTOPYRANOSIDE
TEST (ONPG TEST)
 Purpose: This test is used to determine the ability of an
organism to produce β-galactosidase, an enzyme that
hydrolyzes the substrate o-nitrophenyl-β-D-
 Reactions on TSI agar galactopyranoside (ONPG) to form a visible (yellow)
o K- alkaline (red) product, [Link] test distinguishes late
o A- acid (yellow) lactose fermenters from non-lactose fermenters of
1. K/K (Alkaline(red) slant/alkaline(red) butt) – No Enterobacteriaceae
fermentation (not member of enterobacteriaceae)
2. K/A (Alkaline (red) slant/acid (yellow) butt) –  Expected Results
Glucose fermenter (possible member of  Positive:Yellow (presence of β-galactosidase)
enterobacteriaceae)  Negative:Colorless (absence of enzyme)
3. A/A(Acid(yellow) slant/acid (yellow) butt) –
Glucose, lactose, sucrose fermenter  β Galactosidase hydrolyzes ONPG, a colorless compound,
4. H2S production - (K/A, H2S) or (A/A H2S) black into galactose and o nitrophenol, a YELLOW compound.
precipitate  ONPG remains colorless if the organism is an NLF.
 Bacterium (acid environment) + sodium  The test can be performed by making a heavy suspension
thiosulfate -> H2S of bacteria in sterile saline and adding commercially
 H2S + ferric ions -> Ferrous sulfide (black prepared ONPG disks or tablets.
precipitate)
 The suspension is incubated at 35 C, and positive results
 H2S PRODUCER can generally be seen within 6 hours.
o Salmonella
 Positive result: yellow
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

 1 tube - A/A, gas production
st  If glucose is metabolized by the mixed acid fermentation,
 2nd tube - A/A, H2S production stable acid end products are produced, which results in a
 3rd tube - K/A low pH.
 4th tube - K/A, H2S, gas production  Reagent: 5-6 drops of Methyl red
 5th tube - K/K  Positive Result: Red
 Negative Result: Yellow or no color change

VOGES PROSKAUER TEST

 Detects the organism’s ability to convert the acid products
to acetoin and 2,3-butanediol. (Butylene glycol pathway)
 After incubation, is added first as a catalyst or color
intensifier. Next, 40% potassium hydroxide (KOH) or
sodium hydroxide (NaOH) is added.

PAGE 4 OF 7
[BACT211] TRANS: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA | Prof. Rochelle D. Darlucio, RMT, MPH

 Reagent: 0.6 ml (6 drops) a-naphthol  Alkaline slant ( purple)/alkaline butt) = K/K =

 Positive Result: Red Lysine decarboxylation and no fermentation of
 Negative Result: Yellow glucose
 Purpose  Alkaline slant (purple)/acid butt (yellow) = K/A
The combination test methyl red (MR) and Voges-Proskauer = Glucose fermentation
(VP) differentiates members of the Enterobacteriaceae family.  Red slant/acid butt (yellow) = R/A = Lysine
deamination and glucose fermentation
 Principle
This test is used to determine the ability of an organism to
produce and maintain stable acid end products from glucose
fermentation,to overcome the buffering capacity of the system,
and to determine the ability of some organism to produce
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 [Link]
VP detects the organism's ability to convert the acid products
to acetoin and 2,[Link] 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, DECARBOXYLASE TEST (MOELLER’S METHOD)
alpha-naphthol,followed by potassium hydroxide (KOH):a  Purpose: This test is used to differentiate decarboxylase-
positive test result is indicated by a red color complex. producing Enterobacternaceae from other gram-negative
rods.
 Principle: This test measures the enzymatic ability
AMINO ACID UTILIZATION (decarboxylase) of an organism to decarboxylate (or
hydrolyze) an amino acid to form an
LYSINE IRON AGAR [Link], or hydrolysis, of the amino acid
results in an alkaline pH and a color change from orange
 Purpose: To differentiate gram negative bacilli based on
to purple.
decarboxylation or deamination of lysine and the formation
 Expected Results
of hydrogen sulfide.
 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

 Purpose: This test is used to determine the ability of an
organism tooxidatively deaminate phenylalanine to
phenylpyruvic [Link] genera Morganella, Proteus,and
Providencia can be differentiated from other members of
the Enterobacteriaceae family.
 Principle: The medium has anaerobic slant and anaerobic  Expected Results
butt. When glucose is fermented, the butt of the medium  Positive: Green color develops on slant after ferric
becomes acidic (yellow). If the organism produces lysine chloride is added .
decarboxylase, is formed. Cadaverine neutralizes the  Negative: Slant remains original color after the
organic acids formed by glucose fermentation, and the addition of ferric chloride
butt of the medium reverts to the alkaline state (purple). If  Determines whether an organism possesses the enzyme
the decarboxylase is not produced, the butt remains acidic that deaminates phenylalanine to phenylpyruvic acid.
(yellow). If oxidative deamination of lysine occurs, it forms  Contains 0.2% phenylalanine
a burgundy color on the slant. If deamination does not
 Reagent: 10% ferric chloride
occur, the LIA slant remains purple.
 The surface of the slant is inoculated with a bacterial
 pH indicator: Bromcresol purple
colony. After incubation, addition of a 10% ferric chloride
 Lysine Deaminase (Slant)
reagent results in a green color if phenylpyruvic acid is
 Positive: Burgundy/ Red
present
 Negative: Purple
 Lysine Decarboxylase (Butt)
 Positive: Purple
 Negative: Yellow (acidic glucose is fermented)
 METHOD:
1. With a straight inoculating needle, inoculate LIA by
twice stabbing through the center of the medium to
the bottom of the tube and then streaking the slant.
2. Cap the tube tightly and incubate at 35 37C in
ambient air for 18 24 hrs.

PAGE 5 OF 7
[BACT211] TRANS: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA | Prof. Rochelle D. Darlucio, RMT, MPH

 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
 Results:
 POSITIVE: Growth on the medium, with or
without change in the color of the indicator. (blue)
 NEGATIVE: Absence of growth (green)

MISCELLANEOUS TESTS UREASE TEST

 Determines whether a microorganism can hydrolyze urea
SULFIDE INDOLE MOTILITY AGAR  Urease hydrolyzes urea to form ammonia, water, and
 Sulfide H2S – Black precipitate CO2
 Indole - Organisms that possess the enzyme  Christensen's Urea Agar
tryptophanase are capable of deaminating tryptophan with  Positive: Change in color from Light orange (pH
the formation of the intermediate degradation products of 6.1) to Magenta (pink) (pH 8.1)
indole, pyruvic acid, and ammonia.  Negative: No change in color
 Reagent:0.5 ml of Ehrlich reagent ~ PDAB –
para-dimethylaminobenzaldehyde
 (+) = Pink to red color
 Motility: Cloudiness spreading from the
inoculation line
 Inoculation: Stab-halfway (center)

DNA HYDROLYSIS (DNASE TEST AGAR)

 Purpose: This test is used to differentiate organisms
based on the production of deoxyribonuclease. It is used
to distinguish Serratia sp. (positive) from Enterobacter sp.,
Staphylococcus aureus (positive) from other species, and
CITRATE UTILIZATION
Moraxella catarrhalis (positive) from Neisseria sp.
 Purpose: It determines whether an organism can use as a  Principle: The test is used to determine the ability of an
sodium citrate as a sole carbon source. The test is part organism to hydrolyze DNA. The medium is pale green
of a series referred to as IMViC indole, methyl red, Vogues because of the DNA methyl green complex. If the
Proskauer , and citrate), which is used to differentiate organism growing on the medium hydrolyses DNA, the
Enterobacteriaceae from other gram negative rods. green color fades and the colony is surrounded by a
 Principle: Bacteria capable of growth in this medium use colorless zone.
the citrate and convert ammonium phosphate to ammonia
and ammonium hydroxide, creating an alkaline pH. The
pH change turns bromthymol blue indicator from green to
blue.
 Simmon's Citrate Agar contains ammonium
salts as the sole nitrogen source
 pH indicator: Bromthymol blue
 Green to blue
 Use light inoculum.

 METHOD:
1. Inoculate the DNase agar with the organism to be
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.


PAGE 6 OF 7
[BACT211] TRANS: BIOCHEMICAL IDENTIFICATION OF GRAM-NEGATIVE BACTERIA | Prof. Rochelle D. Darlucio, RMT, MPH

GELATIN LIQUEFACTION  A red color indicates the presence of nitrite.

 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 [Link] gelatin medium
differs from traditional microbiology media in that the
solidifying agent (agar) is replaced with gelatin. When an
organism produces gelatinase, the enzyme liquefies the
growth medium.
 Expected Results
 Positive: Partial or total liquefaction of the
inoculated tube (the control tube must be completely
OXIDASE
solidified) at 4°C within 14 days
 Negative:Complete solidification of the tube at 4°C  The oxidase test determines the presence of the
 Numerous bacteria produce gelatinase – proteolytic cytochrome oxidase system that oxidizes reduced
enzymes that break down gelatin into amino acids. cytochrome with molecular oxygen.
 Gelatinase activity is detected by loss of gelling  Kovac’s oxidase test uses a 0.5% or 1% aqueous
(liquefaction) of gelatin. solution of tetramethyl ρ phenylenediamine
 (+) = liquefaction dihydrochloride.
 Positive: development of a lavender color within
10 to 15 seconds.

MALONATE UTILIZATION
 pH indicator: Bromthymol blue
 Bacteria able to use sodium malonate as a sole carbon
source also use ammonium sulfate as a nitrogen source.
 A positive test results in increased alkalinity from utilization
of the ammonium sulfate , changing the indicator from
green to blue

NITRATE AND NITRITE REDUCTION

 The nitrate reduction test determines whether an organism
has the ability to reduce nitrate to nitrite and reduce nitrite
further to nitrogen gas (N2)
 After 24 hours of incubation, N,N-dimethyl α
naphthylamine and sulfanilic acid is added.

PAGE 7 OF 7
Antimicrobial Susceptibility Testing LAB 2020-
2021
CLINICAL BACTERIOLOGY 9 2nd SEM
BACT21
Transcriber: GAD. MPL. 1
Date: February 19, 2021 LAB
[TRANS] UNIT 9: ANTIMICROBIAL SUSCEPTIBILITY TESTING
 Standards that describe these methods are published and
frequently updated by the Clinical and Laboratory Standards
OUTLINE Institute formerly the National Committee for Clinical Laboratory
I Definition Standards [NCCLS]).
II Antimicrobial Susceptibility Testing
A The Standardized Components of AST THE STANDARDIZED COMPONENTS OF
B Reasons and Indications for Performing AST
C Factors to Consider ANTIMICROBIAL SUSCEPTIBILITY TESTING
III Traditional Antimicrobial Susceptibility Testing  Bacterial inoculum size
A Disc Storage  Disk diffusion – 1.5x108 CFU/mL
i. Inoculum Preparation  CFU – Colony Forming Units
ii. McFarland Turbidity Standards  Broth microdilution – 5x105 CFU/mL
iii. McFarland 0.5 Standard  Growth medium (typically a Mueller Hinton base)
iv. Inoculum Standardization  pH: 7.2 – 7.4
B Methods of AST  Agar depth: 3-5 mm
IV Kirby-Bauer Method  Cation concentration
A Principle  Calcium = 25mg/L
B Step by Step Procedure  Magnesium = 12.5 mg/L
V Definition of Susceptibility Testing Interpretive Categories  Blood and serum supplements
 Added for fastidious organism
Note:
Italicized – additional information  Thymidine content
 Incubation atmosphere
 Ambient air
DEFINITIONS  Incubation temperature
 Anti-microbial are compounds that kill or inhibit microorganisms.  35C – standard temperature
o The compounds can be natural, synthetic or semi-  Incubation duration
synthetic.  16-18 hours (Disk diffusion) ;
 Anti-biotic are antimicrobials, usually of low molecular weight,  16-20 hours (Broth microdilution)
produced by microorganisms that inhibit or kill other  Antimicrobial concentrations
microorganisms. o Bacitracin - 0.04 units
o Has different modes of actions o Novobiocin - % units
 The 1st line of defense  Lowest concentration
 Fatal diseases became manageable because of Antibiotics  To avoid resistance of organism

REASONS AND INDICATIONS FOR PERFORMING

ANTIMICROBIAL SUSCEPTIBILITY TESTS

 Antimicrobial susceptibility testing should be performed on a

bacterial isolate from a clinical specimen if the isolate is
determined to be a probable cause of the patient’s infection
and the susceptibility of the isolate to particular
antimicrobials cannot be reliably predicted based on
previous experience with the bacteria at a specific health
care facility.
 Susceptibility testing of isolates can also provide information on
decreases in the susceptibility of bacteria to antimicrobials

FACTORS TO CONSIDER WHEN DETERMINING

WHETHER TESTING IS WARRANTED
 Body site from which the bacterium was isolated
 Presence of other organisms and quality of the specimen from
which the organism was grown
 AST isolation of organism is always on pure culture
(one organism)
ANTIMICROBIAL SUSCEPTIBILITY TESTING  Host’s status
o Immunocompromised - their normal flora can cause
 Performed on bacteria and fungi isolated from clinical specimens
infection
to determine which antimicrobial agents might be effective in
treating infections caused by these organisms.
 Only organism that is contributing to disease should be tested. TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY
 PRIMARY GOAL: to determine whether the bacterial isolate is TEST METHODS
capable of expressing resistance to the antimicrobial agents DISC STORAGE
selected for treatment  Long term storage: - 20C or below in a non-frost free freezer
 Often performed by a disk diffusion (usual method) or dilution  A working supply of disks can be stored in a refrigerator at 2C to
minimal inhibitory concentration [MIC]) method. 8C for at least 1 week.
 MIC – lowest anti-microbial that completely inhibits visible  Disks should always be stored in a tightly sealed container with
bacterial growth. desiccant.

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[BACT211] TRANS: ANTIMICROBIAL SUSCEPTIBILITY TESTING | Prof. Rochelle D. Darlucio, RMT, MPH

 The container should be allowed to warm to room temperature 4. Epsilometer or Gradient Diffusion Method
before it is opened to prevent condensation
DILUTION METHOD
1. Macrobroth Method or Tube Dilution Method
2. Microtube Dilution Method

KIRBY BAUER METHOD

 Also known as Agar diffusion method or disk diffusion
method.
o The growth of microorganism should be homogeneous
 Used to determine the sensitivity or resistance of a bacterium to
an antimicrobial.
INOCULUM PREPARATION AND USE OF
MCFARLAND STANDARDS

INOCULUM PREPARATION
 One of the most critical steps in susceptibility testing. (Depends
on the consistency and accuracy)
 Prepared by adding cells from four to five isolated colonies of
similar colony morphology growing on a non-inhibitory agar
medium to a broth medium and then allowing them to grow to the
log phase (preferred organism).
 Can also be prepared directly by suspending colonies grown
overnight on an agar plate directly in broth or saline. PRINCIPLE
 This direct inoculum suspension preparation technique is  A standardized suspension of organism is inoculated into MHA
preferred for bacteria that grow unpredictably in broth. Because it (Mueller Hinton Agar)
does not rely on growth in an inoculum broth, the use of fresh (16  Paper disk impregnated with specific antibiotics concentration are
to 24 hour) colonies is imperative. placed into the agar
 Use of a standard inoculum size is as important as culture purity  After 16 20 hours incubation, the diameters of the zone of
and is accomplished by comparing the turbidity of the organism inhibitions are measured
suspension with a turbid standard.  Results are compared to determine susceptibility or resistance

MCFARLAND TURBIDITY STANDARDS STEP BY STEP PROCEDURE

 False susceptible results may occur if too few bacteria are tested.  Preparation of pure inoculum
 False resistant results may be the outcome of testing too many o USING ANY OF THE FOLLOWING
bacteria.  Mueller Hinton Broth
 Trypticase Soy Broth
 McFarland standards can be prepared by adding specific volumes
 Sterile Distilled Water
of 1% sulfuric acid and 1.175% barium chloride.
 Natural Saline Solution
 Brain Heart Infusion Broth
MCFARLAND 0.5 STANDARD  Standardize pure inoculum
 99.5 mL of 1% sulfuric acid o USING 0.5 MCFARLAND STANDARD
 0.5 mL of 1.175% barium chloride  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.

INOCULUM STANDARDIZATION
 The inoculated broth or direct suspension is vortexed (instrument
that mixes the specimen) thoroughly.
 Under adequate lighting, the tube is positioned side by side with
the McFarland 0.5 standard against a white card containing
several horizontal black lines

METHODS OF ANTIMICROBIAL SUSCEPTIBILITY

TESTING
DIFFUSION METHOD
1. Kirby Bauer Diffusion Method (most common)
2. Agar Cup Diffusion Method
3. Agar Cylinder Diffusion Methods

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[BACT211] TRANS: ANTIMICROBIAL SUSCEPTIBILITY TESTING | Prof. Rochelle D. Darlucio, RMT, MPH

DEFINITIONS OF SUSCEPTIBILITY TESTING

 Apply antibiotic discs INTERPRETIVE CATEGORIES
 Using forceps aseptically
 Space at least 15 mm each antibiotic Susceptible (S)
 Indicates that the antimicrobial agent in question may be an
appropriate choice for treating the infection caused by the
organism. Bacterial resistance is absent or at a clinically
insignificant level.

Intermediate (I)
 Indicates a number of possibilities, including:
 The potential utility of the antimicrobial agent in body
sites where it may be concentrated (e.g., the urinary
tract) or if high concentrations of the drug are used.
 Possible effectiveness of the antimicrobial agent
against the isolate, but possibly less so than against a
susceptible isolate.
 Use as an interpretive safety margin to prevent
relatively small changes in test results from leading to
major swings in interpretive category (e.g., resistant to
susceptible or vice versa).
Resistant (R)
 Indicates that the antimicrobial agent in question may not be an
appropriate choice for treatment, either because the organism is
not inhibited with serum achievable levels of the drug or because
the test result highly correlates with a resistance mechanism that
 Incubate indicates questionable successful treatment.
 Normally 35 C for 16 20 hours.
 Measure the zone of inhibition
 Instrument: Ruler or microcaliper/vernier caliper
 Unit: mm


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PAGE 5 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 para-
amino 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 Some MRSA may go undetected
vancomycin and gentamicin if <24 degrees C
HLAR ; 48 h enterococci with Some vancomycin resistant
streptomycin HLAR ; 24 h enterococci may go undetected
sometimes needed for if <2 h with disk diffusion
fastidious bacteria 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)