CLINICAL BACTERIOLOGY LABORATORY

1st Semester | A.Y. 2024-2025

Lecturer: Ma’am Ivy Iola E. Reyes
By: Aimee G. Montes

 Expect for facilities that manipulate

Lecture 1
Mycobacterium tuberculosis and viruses
THE MICROBIOLOGY LABORATORY (Biosafety Level 3), clinical microbiology
In a typical clinical microbiology laboratory, space laboratories usually operate ay Biosafety
is allocated for each of the following functions: Level 2.
 Specimen __receiving__
 Specimen _accessioning_/labelling and
ESSENTIAL INSTRUMENTS IN
processing
 Staining_ and light microscopy MICROBIOLOGY LABORATORY
 Dark room (for dark-field and fluorescence
microscopy, if applicable) 1. MICROSCOPE
 waste disposal  Microscopy is a critical technique in
 Media preparation and glassware wash microbiology labs as it provides
area essential information about the
 Isolation room (for acid-fast bacilli and morphology, structure, and behaviour
fungi processing, if applicable) - Separate of microorganisms.
rooms for specialized molecular-based tests  Light Microscopes - the most commonly
 Open benches for routine specimen workup used microscopes in microbiology labs
 Reagents/Materials storage area because they are capable of magnifying
 Space for expansion, if applicable microorganisms up to 1,000 times.
 They are primarily used for the
observation of minute particles which
The Centers for Disease Control and Prevention cannot be observed with naked eyes.
(CDC) recommends 200 sq.ft. to accommodate two  Types of Microscopy
to three technologists.  Bright field microscopy
 Electron microscopy
SAFETY CONSIDERATIONS
 Emergency showers should be centrally  Phase contrast microscopy – used
located, ideally within 10 seconds and 100 ft. for wet preparation/wet mount
of each work area. They should be supplied  Polarizing microscopy – for lipid
with cold water for the following reasons: containing substances
o Adding cold water slows down the  Dark field microscopy – used to
reaction rate of splashed chemicals. demonstrate the presence of
o It constricts the blood vessels and spirochetes of Treponema pallidum,
minimizes the circulation of an the causative agent of Syphilis
absorbed chemical.  Fluorescence microscopy – used
o It slows down the cellular metabolism
and enzyme reaction rates.
for fluorescently-stained specimens
o It helps alleviate the pain of chemical  Interference-contrast microscopy
contact. – for 3D microscopy

 The Bureau of Fire Protection (BFP) should 2. ANALYTICAL BALANCE

be consulted for guidelines in the proper  used or the measurement of mass in the
storage of chemicals. sub-milligram range
 Fire extinguishers and blankets should be  Made with a measuring pan enclosed in
readily available throughout the work area. a transparent covering that prevents
 A spill cart that contains first-aid supplies, small particles or air currents from
personal protective equipment (PPE) and kits getting collected on the pan.
to clean up acid, alkali, radioactive,  As they are highly precise and based on
corrosive and infectious materials should be advanced technology, analytical
readily accessible. balances are explicitly used in
 Automatic fire/smoke__ detection systems laboratories for the effective completion
and sprinklers should be installed in the of tasks like weighing test materials and
laboratory, and there should be two fire exits. sampling amounts, formulation, density
All electrical outlets must be grounded. determination, purity analysis, quality
 control testing, and material and
conformance testing.
5. COLONY COUNTER
3. AUTOCLAVE  Used to estimate the
 A pressurized chamber used for the density of a liquid
process of sterilization and disinfection culture by counting
by combining three factors: time, the number of CFU
pressure and steam (colony forming
 Sterilization agent: Geobacillus units) on an agar or
stearothermophilus - a biological culture plates.
indicator of autoclave that gives us info if  This instrument can
the sterilization is successful. accommodate
 Mostly used for the sterilization of different sizes of
medical or laboratory equipment with plates which are scanned on top with
the capacity of sterilizing a large number UV, white light and/or fluorescent
of materials at once. illumination.
 They are commonly used for the  One can accomplish the counting either
preparation of _______________ during manually with the touch pressure or
laboratory applications. with a digital counter.
 An autoclave works on the principle of  A colony counter is primarily used for
pressure and temperature to kill counting the number of colonies
unwanted microorganisms that can present on a culture plate to estimate
cause lab contaminations. the concentration of microorganisms in
 a type of moist heat sterilization liquid culture.

6. DEEP FREEZER
 Deep freezers are based on the principle
4. BUNSEN BURNER that under extremely low
 A standard temperatures, there is minimum
tool used in microbial growth which allows for the
laboratories, protection and preservation of
named after different substances.
Robert  Based on this principle, we can even
Bunsen. It is preserve cultures over a long period of
a gas-fuelled time without any change in the
single open concentration of the microorganisms.
flame.  They are used for maintaining
 Made with a metal tube on a flat base temperatures as low as -80 C (-112 F),
with a gas inlet at the bottom of the ensuring the longevity of
tube, which may have an adjustable microorganisms.
valve. On the sides of the tube are  Essential in the preservation of valuable
openings that can be adjusted with a and rare microorganisms, which can be
collar to control the amount of air that used for research and development of
can enter. new treatments and technologies.
 It is commonly used for processes like Without the use of deep refrigerators, the
sterilization, combustion, and viability and integrity of microorganisms
heating. can be compromised, resulting in
 In medical or microbiology laboratories, inaccurate or unreliable experimental
it is commonly used for micro-loop results.
sterilization.  Low temperature inhibit the growth of
 Coolest flame – yellow and orange bacteria
 Medium flame – blue  To preserve specimen/culture for future
 Hottest flame – roaring blue flame use
characterized by a clear blue cone in the  Refrigeration can also be used; In clinical
middle. microscopy, urine samples left in room
 The tip of the cone is the hottest part of temp for 4 hours, bacteria can multiply;
the flame. there are parameters that increases when
 used for dry heat procedure urine is left in room temp
 Additional info: urine specimen for urine
cs - mid stream clean catch
7. CENTRIFUGE cold air at the bottom. This allows
 Works on the principle of for the adequate heating of
sedimentation, where the high speed of materials inside the oven.
the rotation causes the denser particles  Static Air Hot Oven - The heat is
to move away from the center while produced by coils present at the
smaller, less dense particles are forced bottom of the oven with no fan. The
towards the center. hot air rises and doesn’t allow the
 Thus, the denser particles settle at the effective sterilization of the
bottom (precipitate) while the lighter materials.
particles are collected at the top
(supernatant). 10. INCUBATOR
 It can be used for the separation of cell  Used in laboratories for the growth and
organelles, nucleic acid, and blood mainteenance of microorganisms and
components. cultures. Incubator provides an optimal
temperature, pressure, and moisture
8. HOT PLATE among other things required for the
 A stand-alone appliance used in growth of microorganisms.
microbiology laboratories as a tabletop  The incubator is based on the principle
heating system. of maintaining a proper atmosphere
 Unlike the traditional ways of producing for the growth of microorganisms.
heat through fire, a hot plate produces  • Temperature: 35 to 37°C
heat by the flow of electricity.
 Electricity runs through the coils which 11. LAMINAR FLOW HOOD AND BIOSAFETY
have a high level of electrical resistance. CABINET
The resistance in the coils converts the  They provide a sterile and protected
electrical energy into heat energy which environment for working with
causes the coils to release heat. hazardous materials, prevent
 Laboratory device used to heat samples, contamination, and protect lab
solutions, and materials uniformly personnel. With the increasing need for
without the danger associated with the accurate and safe research practices, the
open flame at precise temperatures. The use of these instruments has become an
use of hot plate is safer as the heat comes integral part of modern microbiology
from the electricity and not from the open labs.
fire.  Laminar flow hood are used to create a
clean working environment for sensitive
9. HOT AIR OVEN work in microbiology lab experiments.
 an electrical device used for They operate by providing a constant
sterilization of medical equipment or flow of HEPA-filtered air that creates a
samples using dry heat. positive-pressure environment. This
 used to sterilize materials like glassware prevents airborne contaminants from
or metal equipment. entering the work area, making it an
 allows for destruction of microorganisms ideal environment for sensitive
as well as bacterial spores. procedures such as cell culture or tissue
 Indicator: spores of Bacillus culture.
atropheaus (formerly Bacillus  HEPA filter – high efficiency
subtilis) particulate air filter
 the commonly-used temperature and  Biosafety Cabinet – a primary
time that hot air ovens need to sterilize containment that encloses a working
materials is: area to protect workers from aerosol
 170 °C for 30 minutes a board exam exposure and or infectious disease
 160 °C for 60 minutes “must-know” agents. It is also known as a ventilated
 150 °C for 150 minutes cabinet. It is considered as the standard
 Two types based on the working device used in the academic and clinical
principle: laboratory to contain hazardous
 Forced Air Hot Oven - the heated biological agents and its products.
air inside the oven is distributed  laminar flow hood protects the
throughout the oven with a fan. sample/specimen from contamination
This prevents the rising of hot air while biosafety cabinet protects us from
towards the top while keeping the potential hazard
12. ANAEROBIC CHAMBER  The ultimate purpose of an SOP is to:
 The cultivation of anaerobic bacterial ensure operations are performed safely
species requires an anaerobic chamber. and in the correct manner.
 This special chamber is a closed  Should contain all tests done by a
environment without oxygen where the laboratory with details pertinent to the
microbiologist can work with and quality processing of all requests received.
cultivate obligate anaerobes without  should include contact details of agencies
exposing them to oxygen. or institutions that can be considered for
 Some bacteria needs special treatment as send-out requests.
it dies from prolonged exposure to air; this  should be used to communicate laboratory
chamber helps us isolate the kind of instructions to his/her personnel.
bacteria.  Details in the MOP should include the
13. CANDLE JAR ffg.:
 A large screw-capped container into o Test name (procedure title)
which the medium is placed along with a o appropriate specimen type to submit
candle. o minimum specimen requirement
 The candle is lit and the jar is sealed. o appropriate container (need for
 The candle will consume most of the anticoagulant or preservative) or
oxygen in the jar. transport/handling medium and
 principle – inaalis ang oxygen sa loob ng transport conditions (wet ice, room
jar to isolate anaerobic bacteria temperature), if applicable
o collection instructions, including
14. INOCULATING LOOP patient preparation, if applicable
o specimen storage in the laboratory
(room temperature, 4°C, -20°C, -
70°C)
o criteria for specimen rejection
(unacceptable specimens)
o principle and methodology used
(procedures written in a step-by-step
format) and interpretation of results.
o comment section indicating turn-
around times or other pertinent
information such as whether testing
is done in batches or sent to a
 Often referred to as a smear loop, reference laboratory
inoculation wand, or microstreaker, is a  A well-written MOP is validated when a
basic instrument used largely by microbiologist from another facility is able to
microbiologists to take and transfer a read and perform any procedure done by
small sample (inoculum) of a microbe the laboratory.
culture, for instance, to strip on a
culture plate. LABORATORY REQUEST FORM
 It is a tool often constructed of nichrome  An important tool for clinicians to inform
or platinum wire, with a tip with a tiny them of the array of examinations that a
loop with a diameter of around 2 mm to clinical microbiology laboratory provides.
5 mm.  It varies from one institution to the other
 blue colored inoculating loop – used for but should generally contain the following:
liquid specimens o Laboratory’s name, address, and
 red colored inoculating wand (needle-end) contact numbers
– used for sterilizing o Accreditation and licensure number
o Client/Patient’s name, address, age,
MANUAL OF OPERATING PROCEDURES (MOP) gender, birthdate, and contact
numbers (room number if an in-
 also known as STANDARD OPERATING patient)
PROCEDURES (SOP) o requesting physician
 Helps to ensure a safe work environment by o Patient’s medical history as
documenting the key risks associated with indicated by the physician
an activity and how the risks can be (antimicrobial therapy, if any,
controlled.
 immunization history, and clinical
syndrome or suspicious agent)
o Information about the biological
sample source or type and
collection date and time.
o List of laboratory examinations that
the clinical microbiology laboratory
performs.
 The personnel who set up the biological
samples in the laboratory should always
carefully check the accuracy and
consistency of the request form to verify
that the correct specimen is received
promptly and is in good condition.

LABORATORY WORKBOOK/LOGBOOK
 A legal document that can be used to
reconstruct the testing process.
 Should contain the following:
o date when the examination process
was conducted
o patient’s name
o specimen type and source
o accession number
o name of microbiologist in charge of
the processing
o all notes made by the microbiologist
during a sample workup (including
records of telephone calls, faxes, or
any mode of communication to the
patient, technician, or clinician
concerned)
o the test results
o a hard copy of the results (the
thermal paper printouts should be
photocopied because they fade away
over time)
 Lecture 2 BIOSAFETY LEVELS
BIOSAFETY BSL  no known  Bacillus subtilis
Principle: 1 potential for  M.gordonae
The fundamental objective of any biosafety program infecting health
is the containment of potentially hazardous people
biological agents and toxins. BSL  all common “SSSHY BA?”
2 agents of  Salmonella
Basic Concepts of Infection Control infectious  Shigella
Infectious agents, depending on their environmental disease  S.pneuomoniae
stability, can spread through different ways:  HIV
 not air-borne
 Y.pestis
Direct contact From contaminated food,  P. anthracis
intravenous solutions BSL  cause serious “MS FB”
Indirect Patient to patient, healthcare 3 disease  M.tuberculosis
contact professionals’ hands  air-borne  Systemic fungi
Droplet Inhalation of droplet nuclei that o Coccidiodes
contact cannot travel more than 3 ft. immitis –
Airborne Inhalation of droplets that can valley fever
contact travel large distances on air o Histoplasma
currents. capsulatum
Vector borne From animal hosts like –
contact mosquitoes, ticks and fleas. histoplasmo
sis and
Darling’s
INFECTION CONTROL COMMITTEE disease
 prevent and control communal infections  F.tularensis
 composed of Microbiologist, Infection  Brucella
control personnel, Hospital BSL  cause serious  Arbovirus
epidemiologist, Pharmacist 4 disease often  Arena virus
untreatable  Flavovirus
PREVENTING THE SPREAD OF INFECTION  air-borne  Smallpox virus
 Healthcare workers should wash their
hands in between contacts with different BIOSAFETY CABINET
patients and before and after laboratory  Encloses a workspace to protect individuals
work. from aerosol exposure to infectious disease
 Infected patients should be in private or agents.
semiprivate rooms with a cohort of patients  Should be used properly to process all
with the same diagnosis. (separated on case- specimens.
to-case basis)  Turn on UV light to disinfect interior of
 PPE should be worn when caring for cabinet when not in use.
infected patients and conducting laboratory  Also known as VENTILATED CABINET.
work.  In BSC, the air that contains the infectious
 Contaminated articles should be placed in material is sterilized, either by heat, UV light,
biohazard bags before being taken out of or by passage through HEPA filter that removes
the room for proper sterilization or disposal. particles larger than 0.3µm
 All isolation rooms should be cleaned and  8 to 10 inches opened; at armpit level
disinfected after a patient is discharged.
 Cards specifying the type of isolation and Class I  open front with negative pressure
instruction for visitors and nursing staff
should be placed on a patient’s door.  sterilize only the air to be
exhausted
PERSONAL PROTECTIVE EQUIPMENT Class II  sterilize circulating air and air to
 Gloves, laboratory coats, masks, respirators, be exhausted
face shields, safety glasses Class IIa – self-contained
A1 – 30% of air exhausted in
 Must NEVER BE WORN OUTSIDE the
room
laboratory.
A2 – 30% of air exhausted
outside the room
Class IIb – for radioisotopes and
 carcinogens  The speed or revolution per minute (rpm)
B1 – 70% of air exhausted must be checked twice a year using
outside a Tachometer.
B2 – 100% of air exhausted
outside 4. Culture Media
Class III  system is entirely closed; require  All media should be checked based on their
use of rubber gloves performance and sterility, and records
 highest level of safety should be kept for at least TWO years.
 sterilize air circulating and
5. Reagents
entering
 Reagents should be tested DAILY with both
 sterilize air to be exhausted POSTIVE and NEGATIVE controls.
 Reagents that require quality control
LABORATORY ACQUIRED INFECTION monitoring: Catalase, coagulase, gelatin,
 Mostly transmitted through needlesticks or hippurate, nitrate, oxidase, Kovac's, PYR, p-
contaminated sharps, spills and splashes, Lactamase, Voges-Proskauer, X and V strips
ingestion, and inhalation. for Haemophilus, and antibiotic discs.
 The infectious agents that pose the greatest risk
are those that are transmitted by aerosols. 6. Antimicrobial Susceptibility
 The five most frequently acquired laboratory  Susceptibility testing of control organisms is
infections are Shigellosis, Salmonellosis, usually done DAILY for 20 to 30 days.
Tuberculosis, Brucellosis, Viral Hepatitis
 Hand washing is the cornerstone for preventing 7. Personnel competence
the spread of infections and diseases including  All tests performed on patients must be
LAIs. subjected to proficiency testing TWICE a
year.
 Participation in continuing education is
INFECTION CONTROL
another form of QC.
 All laboratory-related accidents must be
reported immediately to the HEAD OF THE
LABORATORY (Pathologist or Chief Medtech
in the absence of pathologist) and SAFETY
OFFICER.
 Bench tops and work surfaces should be
decontaminated. Sodium hypochlorite – used
to disinfect with 1:10 ratio
 The BIOHAZARD SYMBOL should be visibly
seen on all equipment and instruments that
process and contain infectious and potentially
infectious samples and materials.

QUALITY CONTROL IN MICROBIOLOGY

INSTRUMENTS AND REAGENTS REQUIRING
QUALITY CONTROL MONITORING

1. Thermometer Calibration
 Thermometers should be checked
periodically against a reference thermometer
from the National Institute of Standards
and Technology.
 Thermometers should be checked daily for
the presence of gas bubbles to ensure
accuracy of reading.

2. Carbon Dioxide Incubator

 The percentage of carbon dioxide must be
checked daily.

3. Centrifuge
 Lecture 3 These samples should be rejected for anaerobic
SPECIMEN MANAGEMENT cultures,
 The validity of any test result is primarily  Gastric washings
dependent on the QUALITY of specimen  Urine other than suprapubic aspirate
received.  Stool (except for culture of C. difficile) for
epidemiologic studies or for diagnosis of
bacteria associated with food poisoning.
Specimen Collection and Handling Principles
 Oropharyngeal specimens, except deep
Time of Collection
tissues obtained during surgical procedures
 Specimens should be collected during the
 Swabs of ileostomy or colostomy sites
acute or early phases of an illness.
 Superficial Skin specimens
 If possible, specimens should be collected
before antibiotics are administered.
Note: Never assume that a patient knows how to collect
ARD – antibiotic removal device – removes antibiotic sample correctly. Always give them proper instructions.
in the sample from the patient that was given to
patient prior to sample collection. Specimen Transport

Specimen Volume  Ideally, most specimens should be

transported to the laboratory immediately
 The quantity of the collected specimen and preferably within 30 minutes of
should be sufficient for diagnostic testing. collection and not longer than two hours
o QNS – quantity not sufficient; there’s not for both aerobic and anaerobic culture.
enough specimen to perform the test; must
 For CSF and other body fluids – must be
return the specimen (repeat collection) to
the patient if the patient collected it transported within 15 minutes
 Ideally, swab specimens should come in  All specimen containers should be placed in
pairs. sealable, leak-proof plastic bags.
 Swab specimens are not acceptable for  Specimen bags should be marked with a
cultures of anaerobe, mycobacteria, and biohazard symbol.
fungi.
o anaerobe is not acceptable because if ma-
expose sa air, mamamatay leading to Sample Preservatives
false negative results
o mycobacteria is best detected if the  The sodium polyanethol sulfonate (SPS) in
specimen is sputum, tissue specimens, or 0.025% (most common) to 0.050%
aspirates concentration is the preferred anticoagulant
o fungi – difficult to grow in culture or
added into blood culture media.
difficult to identify; pwedeng ma-damage
 SPS is used as anticoagulant in blood culture
ang structure ng fungi sa culture
bottle because can inhibit phagocytosis and
 Calcium alginate, cotton, and wooden complement activation. SPS can neutralize
shaft swabs are toxic to the herpes simplex the activity of aminoglycoside antibiotics and
virus (HSC), N. gonorrhoeae and C. can neutralize the bactericidal effect of
trachomatis, respectively. plasma. SPS is not a type of antibiotic removal
device. It has anti-microbial property but it is not
Biological Samples for Rejection designed to remove antibiotics from a sample (ARD
is designed to ABSORB antiobiotics from sample).
Clinical microbiology laboratories should reject  samples should be placed in
these samples,
 Mismatched specimen with the examination  Boric acid is used to maintain colony
request form counts in urine that come from distant
 Specimen received in formalin areas.
 24-hour-old sputum collection  Stool that will not be processed within two
 Specimen in a leaking container hours should be placed in the Cary-Blair
 Specimen in a dried-out agar plate transport medium.
 Specimen contaminated with barium,
chemical dyes, or oily chemicals
 Foley catheter tips
 Duplicate specimens (except blood culture)
received in a 24-hour period
 Bloody catheter tips from patients with no
concomitant positive blood cultures
Sample Storage Conditions

Room Temperature Refrigerate

Conjunctival/Corneal Samples
o Abscess, lesions, o Catheter (IV) tips
wounds, body o CSF for viruses Conjunctiva Corneal scrapings
fluids o Outer ear samples
o Inner ear o Feces (unpreserved) o Samples should be o The clinician should
samples o Feces for C. difficile toxin obtained from both administer
o Genital samples (up to 3 days; >3 days eyes using two local anesthetic before
o Nasal, throat store at -70°C separate swabs. collecting the
o Bone, tissue o Sputum specimen.
o Urine (preserved) o Urine (unpreserved) o Using a spatula,
scrape the discharge
Note: CSF sample should always be at incubator and inoculate directly
temperature only if it is for bacterial identification. (if virus onto/into medium.
ang hinahanap -> refrigerate)

Sample Types and Details Nasopharyngeal Samples

Blood Samples  Nasopharyngeal aspirates and washings are
 Ideally, blood should be drawn during the preferred over swabs (except for C.
time of febrile (fever) episode. trachomatis)
 To collect: disinfect using 70% alcohol, then 2% iodine.  Polyester-tipped swab – used for detection
According to Clinical and Laboratory Standards Institute of C. trachomatis and C. pneumoniae and
(CLSI), their recommended skin disinfectant for BC is is placed in 2-sucrose phosphate medium
chlorhexidine gluconate for infants (2 months old) and
patient sensitive to iodine with antimicrobials or M4.
 Calcium alginate swab – used for B.
Cerebrospinal Fluid (CSF) Samples pertussis
 Collection is done by a lumbar spinal  Dacron swab – preferred in a PCR analysis.
puncture or shunt.  Polyester swab – sufficient for detecting S.
 Required volume: 10 mL aureus
 CSF is incubated for bacterial analysis and
stored in the refrigerator for a short time Sputum
for viral recovery.  Collection of sputum is done in an open area
 CSF samples are used to detect conditions with open air or inside a sputum
affecting the CNS. In bacte, it is used to detect induction chamber to avoid the spread of
meningitis, encephalitis, and neurosyphylis. infection.
Autoimmune disease can also be detected  A first-morning specimen is preferred for an
(multiple sclerosis and Guillain-Barre syndrome)
acid-fast bacilli (AFB) microscopy.
 If not processed immediately, incubate at 35°C up
to 6 hrs for bacterial analysis.  Methods of collection: Deep cough
(expectorated sputum) or aerosol-induced
Other Body Fluids (induced sputum)
 Pericardial, thoracic, and peritoneal (ascitic)  Bartlett’s sputum sample criteria
cavities and joint (synovial) fluids are Sputum Saliva
collected by aseptic aspiration using a
needle and syringe. PMN: 10-15/LPF PMN: <10/LPF
 1 to 5 mL is needed for routine
bacteriologic analyses. SEC: <10/LPF SEC: >10/LPF
 10 to 15 mL is required for the recovery of
fungi. PMN – polymorphonuclear leukocytes – WBC typically
present in sputum samples of patients with bacterial
infection in lower respiratory tract
Ear Discharge Samples SEC – squamous epithelial cell – originate from the mouth
 A sterile swab or syringe and needle are (saliva) and oropharynx
used to collect middle ear fluid. LPF – per low power field
Sputum is collected deep within the bronchi
If maraming SEC ang specimen, it is saliva
If madaming PMN ang specimen, it is sputum
UNSATIS – unsatisfactory sample, subjected to repeat
collection
 Lecture 4
Urine SLIDE PREPARATION
 First-morning urine is preferred. Wet Mount  useful for studying morphology
 Specimen must be collected in a clean-catch Preparation and motility of organisms
midstream. Spread Smear  this technique is often
Technique employed when performing
Stool
stains such as the Gram stain,
 The specimen of choice for detecting acid-fast stain, or simple stain.
gastrointestinal pathogens.
 The spread smear allows the
 Stool samples should not be retrieved from Note:
mechanical fixation; bacteria to be evenly
the toilet. para hindi ma-washout distributed and fixed to the
 Samples should be transported in a modified ang specimen during slide so that they can be
Stuart’s medium within two hours. staining
stained and observed clearly
 Cary-Blair should be used if further delay is under the microscope.
anticipated. Concentrated  This is especially important in
(Cytocentrifuged) diagnostc microbiology when
PANIC VALUES Technique detecting pathogens from fluid
(these results are life-threatening) samples like urine, sputum, or
 Positive blood cultures cerebrospinal fluid (CSF).
 Positive CSF Gram stain or culture Smear  Swab samples are often taken
 S. pyogenes in surgical wounds Preparation from body sites such as the
 Gram stain for large box-car Gram-negative from Swab throat, nose, wounds, genital
rods Samples areas, or from environmental
o these are Clostridium perfringens – surfaces.
causes gas gangrene  The smear technique allows
 Positive acid-fast stain (M.tuberculosis) microorganisms collected on
 Positive blood smear for malaria the swab to be spread on a
 Positive cryptococcal antigen test for culture slide for staining and
 S. agalactiae or herpes simplex virus (HSV) from microscopic examination.
the genital site of a pregnant patient. Touch  A method used in microbiology,
 Detection of Legionella, Brucella, vancomycin- Imprint Slide histopathology, and cytology to
resistant S. aureus and Enterococcus, Preparation transfer cells or tissue samples
and methicillin-resistant S. aureus directly onto a glass slide by
 Positive for E. coli K1 antigen – common cause of gently pressing the sample
neonatal meningitis against the slide.
 This technique is commonly
used for rapid cytological
evaluation of tissues, such as
biopsies or surgical specimens,
and for detecting infections or
neoplastic cells.
Smear  A commonly used technique in
Preparation microbiology to observe
of Plated bacterial morphology and to
Bacterial perform staining procedures
Colonies (e.g., Gram staining) for
identification purposes. This
involves transferring bacteria
from a solid culture medium
(such as an agar plate) onto a
glass slide to create a smear for
microscopic examination.
Smear  Smear preparation from broth
Preparation cultures is a common
for Broth technique in microbiology to
Cultures observe bacterial morphology
or perform staining
procedures, such as Gram
staining, from liquid media.
  Myxococcus xanthus, a soil-dwelling
Hanging Drop Method bacteria
 A technique that allows to observe
microorganisms in their natural state by 4. Swarming Motility
suspending cells in a suitable medium – water,  A rapid and coordinated movement of
saline, or broth – that temporarily maintains bacterial cells across a moist surface. It
viability and provides space and a medium for usually occurs in bacteria with peritrichous
locomotion and production. flagella.
 this technique helps to discern between false  Proteus mirabilis is well-known for its
motility or Brownian movement and true swarming motility on agar plates, forming
motility characteristic concentric rings (swarm
 Brownian movement – vibratory motion colonies)
observed in a cell suspended in a solution. This
is due to the constant interaction of the cell with 5. Spirochete Motility
the water molecules surrounding it.  Spirochetes are a group of bacteria that
exhibit a corkscrew-like motion, allowing
them to move through viscous
Types of Bacterial Motility
environments, such as mucus or connective
1. Flagellar Motility tissue.
 most common form of motility in bacteria  Spirochetes have endoflagella that run along
 uses flagella – long whip-like structures that the length of the cell, within the periplasmic
rotate to propel the bacteria through liquid space. The twisting motion of these
environments endoflagella propels the bacterium forward
 Types of Flagellar Arrangements: in a corkscrew fashion.

6. Sliding Motility
 Sliding is a passive form of motility where
bacteria spread across surfaces without the
use of appendages like flagella or pili.
 Mycobacterium smegmatis and certain
Bacillus species are known for sliding
motility

7. Brownian Motion (non-motile)

 While not a true form of motlity, non-motle
bacteria may appear to move due to
Brownian motion, which is the random
Atrichous No flagella Spirochetes, movement of particles (including bacteria) as
Lactobacillus they are bombarded by molecules in a
Monotrichous Single flagellum at V.cholerae liquid.
one pole  Bacteria without active motility, such as
Lopotrichous Tuft of flagella at Helicobacter S.aureus or Klebsiella pneumonia, exhibit
one pole pylori Brownian motion
Amphitrichous Single flagella at Campylobac
each pole -ter jejuni SUMMARY
Peritrichous Flagella distributed E.coli,
Type of Motility Structure Example
over the entire Proteus
Involved Organisms
surface mirabilis
Flagellar Flagella E. coli
Amphilophotric- Tuft of flagella on
V. cholera
hous both poles
Twitching Type IV pili Pseudomonas
aeruginosa
2. Twitching Motility
Neisseria
 Bacteria use type IV pili to move in a jerky,
gonorrhoeae
twitching manner across solid surfaces
Gliding Surface Myxococcus
 Pseudomonas aeruginosa and Neisseria
proteins, slime xanthus,
gonorrheae display twitching motility
Cytophaga
Swarming Flagella Proteus
3. Gliding Motility
mirabilis
 Some bacteria move smoothly over surfaces
Salmonella
without the use of flagella or pili
Spirochete Endoflagella Treponema
pallidum Lecture 5
Berrelia DIRECT MICROSCOPIC EXAMINATION
burgdorferi
Sliding Surface tension, Mycobacterium
GRAM STAINING (CGAS)
growth smegmatis
Bacillus species
REAGENT FUNCTION GRAM (+) GRAM (-)
Brownian none Staphylococcus
Crystal Primary Purple/Blue Purple/Blue
Motion (non- aureus
Violet stain
motile) Klebsiella
Gram’s Mordant Purple/Blue Purple/Blue
pneumoniae
Iodine
Other Types of Motility Acetone Decoloriz Purple/Blue Colorless
alcohol er
 Shooting star motility – unique motility of
V.cholerae Safranin Counter Purple/Blue Red/Pink
stain
 Tumbling motility – Listeria monocytogenes
 Darting motility – Campylobacter jejuni
Note:
 Twitching motility – Bartonella Gram (+) – has thick peptidoglycan CW – teichoic acid
 Spinning motility – Leptospira Gram (-) – has thin peptidoglycan CW –
 Gliding Motility – Mycoplasma pneumonia lipopolysaccharide
 Falling leaf-like – Giardia lamblia The decolorizer destroys lipopolysaccharide in gram
(-) bacteria that’s why it appears colorless
Simple staining – used in hema
Differential stain – Gram stain – differentiates gram
(+) and gram (-)

Falsely Gram (-) Falsely Gram (+)

 Over-decolorization  under-
 missed iodine decoloroization
 old/dying colonies  thick smear

Gram Stain Reactions

Gram Negative Cocci Gram Positive
Bacilli
All COCCI are GRAM All BACILLI are
POSITIVE, except: GRAM NEGATIVE,
(NBMV) except: (BL2AC2MEN)
 Neisseria  Bacillus
 Branhamella  Lactobacillus
 Moraxella  Listeria
 Veilonella  Actinomyces
 Clostridium
 Corynebacterium
 Mycobacterium
 Erysiphelothrix
 Nocardia
ACID-FAST STAINING
Function ZIEHL-NEELSEN KINYOUN AURAMINE-RHODAMINE
Primary Carbol fuschin Carbol fuschin Auramine-Rhodamine
Stain
Mordant Heat Phenol
Tergitol
Decolorizer 3% acid alcohol 3% acid alcohol 0.5% acid alcohol

Counterstain Methylene blue Malachite green Potassium permanganate

Results Acid-fast Non acid-fast Acid-fast Non acid-fast Acid-fast Non acid-fast
Red Blue Red Blue/Green Fluoresce Not fluoresce
Remarks Hot technique Cold technique Fluorescent technique
Most sensitive

Lecture 6
SIGNIFICANT MEDIA FOR ROUTINE BACTERIOLOGY

Types of Culture Media

Non-Selective  adequately supports growth of microorganisms TSA
Note: any bacteria can grow

Enriched  contains growth enhancers 5% sheep’s blood or

vitamins

Selective  selects for growth of a group of organisms by MAC for facultative gram (-)
adding inhibitory susbtances (dyes, alcohols, organisms
acids, microbials)
CAN for gram (+) cocci and
bacilli

Differential  support growth of a group or groups of bacteria MAC – can differentiate

and differentiate within the group between lactose fermenters
and non LF

LF – pink/red – acidic
NLF – colorless – alkaline

Commonly Used Non-selective Media

SHEEP BLOOD AGAR  supports growth of most bacteria
 exceptions: N.gonorrhoeae, H.influenza, Legionella
CHOCOLATE AGAR  Fastidious bacteria
 examples: Neisseria and Haemophilus
BUFFERED CHARCOAL YEAST  Recovery of Legionella species
EXTRACT (BCYE) AGAR  contains cysteine and iron supplementation
 activated charcoal helps bind and sequester growth inhibitors
MUELLER-HINTON AGAR  antimicrobial susceptibility testing of many common bacteria
(MHA)
THIOGLYCOLATE BROTH  cultivation of bacteria, including microaerophilic and obligate
anaerobes
 Commonly Used Selective Media
Medium Basis of Selectivity Purpose
MacConkey Agar  Bile Salts  Cultivation of hardy enteric
 Crystal Violet gram (-) rods
Eosin Methylene Blue  Aniline dyes  Cultivation of hardy enteric
gram (-) rods
Campylobacter Blood Agar  Cephalotin  Recoveery of Campylobacter
 Vancomycin species
 Trimethoprim
 Amphotericin B
 Polymyxin B
Hektoen enteric Agar  Bile salts  Enhanced recovery of
 Bromthymol blue Salmonella and Shigella
 Acid fuschin
Salmonella-Shigella Agar  Bile salts  Recovery of Salmonella and
 Sodium citrate Shigella
 Brilliant Green
Selenite broth  Sodium selenite  Recovery and enrichment of
Note: used only for Salmonella Salmonella
TCBS Agar  Bile salts inhibit Gram (+)  Recovery of Vibrio spp.
Note: used only for V.cholerae  pH inhibits most enterics
Cefsulodin-Irgasan-  Antimicrobials  Recovery of Yersinia spp.
Novobiocin Agar (CIN Agar)  Crystal violet
Anaerobic Colistin-Nalidixic  Antimicrobials  Reovery of Streptococci
Agar
Lim broth  Antimicrobials  Recovery of S.agalactiae
Regan-Lowe Medium  Antimicrobials  Recovery of Bordatella
pertussis and Bordatella
parapertussis
Thayer-Martin Agar  Antimicrobials  Recovery of Neisseria spp. from
non-sterile sites

Commonly Used Differential Media

MEDIUM BASIS OF DIFFERENTIATION LACTOSE NON-LACTOSE
FERMENTERS FERMENTERS
MacConkey Medium  Differentiation between lactose- Pink/Red colonies Colorless
fermenting and non-lactose fermenting
enterics
Eosin Methylene Blue  Differentiation between lactose- Purple-black w/ Colorless
(EMB) fermenting and non-lactose fermenting green metallic
enterics sheen
HEA  Differentiation between lactose and/or LF & SF Colorless
sucrose-fermenting and non-lactose or Yellow/Orange
sucrose nonfermenting enterics
SSA  Differentiation between lactose- Pink/Red colonies Colorless
fermenting and non-lactose fermenting
enterics
TCBS  Differentiation between sucrose- Yellow Colorless
fermenting and non-sucrose
fermenting vibrios
CIN  Differentiation between mannitol- MF NMF
fermenting and non-mannitol Bull’s eye colonies Colotrless
fermenting Yersinia (colorless w/ red
center)
Motility Test Agar  Diferentaton between motile and Motile: Non-motile:
nonmotile bacteria Growth away from Stay within the
the line of line of inoculation;
inoculation; agar is clear
clouding of agar
 Lecture 7 SPREAD PLATE METHOD
BACTERIAL TRANSFER
STREAK PLATE TECHNIQUE
 a dilution technique done by streaking a loopful
of supposedly mixed culture several times over
the surface of an agar plate

POUR PLATE METHOD

Number of Organisms can be graded:

4+ Many, heavy growth
If growth is out to the fourth quadrant
3+ Moderate growth
If growth is out of third quadrant
2+ Few or light growth
Growth in second quadrant
DILUTION
1+ Rare
If growth is the first quadrant  created by measuring a volume of a sample and
adding it to a volume of sterile water, thus
making the resulting solution less
concentrated than the original
 the repeated process for each new tube, making
a series of more and more dilute samples is
called SERIAL DILUTION

A. Dilution streak technique for isolation and

semi-quantitation of bacterial colonies.
B. Actual plates show sparse or 1+, bacterial
growth that is limited to the first quadrant.
C. Moderate or 2+, bacterial growth that
extends to the second quadrant.
D. Heavy or 3+, bacterial growth that extends
to the fourth quadrant.
 Note:
 Fermentation always start at the butt
 Fermentation is an anaerobic process; not exposed to air. In
the tube, the butt part is the area that is not exposed to air.
Lecture 8  The changes in color of agar indicate that there is
CLINICAL AND BIOCHEMICAL fermentation, thus the environment becomes acidic.
 K-alkaline
IDENTIFICATION OF PATHOGENS  A-acid
TRIPLE SUGAR IRON AGAR SLANT Tube Control tube
1
 a differential agar; a butt slant agar
Tube A/A  Yellow
 generally used for identification and 2 with  All sugar has fermented
differentiation of enteric bacteria gas  Gas production
 used to distinguish between product  Possible organisms with this reaction
ion o Klebsiella
Enterobacteriaceae and other gram-negative
o Enterobacter
intestinal bacilli Tube A/A  Black agar, yellow slant
 used to determine whether a bacteria can 3 H2S +  Hydrogen sulphide production
produce gas, can produce hydrogen sulphide  Glucose, lactose, sucrose have fermented
and/or can ferment sugar (glucose, lactose, Tube K/A  Black agar, red slant – hydrogen
sucrose) 4 H2S + sulphide production
 Note: ALL MEMBERS OF Tube A/A  Black agar, yellow slant
ENTEROBACTERIACEAE CAN FERMENT 5 H2S +  Hydrogen sulphide production
 Glucose, lactose, sucrose have fermented
GLUCOSE BUT NOT ALL CAN FERMENT
Tube K/A  Black agar, red slant – hydrogen
LACTOSE AND SUCROSE. 6 H2S + sulphide production
 used to determine which bacteria Tube K/A  Yellow butt, Red slant
ferments glucose only 7  Glucose has fermented, but not lactose
 composition of TSI: and sucrose
 Possible organisms with this reaction:
o 0.1 % glucose o Shigella
o 1% sucrose o Serratia
o 1% lactose o Providencia
 pH indicator: phenol red o Yersinia
o determines whether a bacteria TSI Reactions of Commonly Encountered Species
is sugar fermenter or non- Organism Butt Slant Gas H2 S
sugar fermenter Enterobacter A A + -
o Fermenter – acid – yellow Escherichia A A/K + -
o Non-fermenter – alkaline – red Klebsiella A A + -
Citrobacter A A/K + V
(original color of TSI, no change in Proteus A A/K + +
color) vulgaris
 indicator for H2S production: Ferrous sulphate Edwardsiella A K + V
o production of H2S – black Morganella A K + -
Serratia A A/K V -
Shigella A K - -
Salmonella A K - +
typhi

Some ENTERIC BACTERIA and their TSI Color Reaction

Organism Slant Butt H2 S Details
Shigella Food infection
dysenteriae Dysentery
Salmonella w/ gas Food poisoning
production
typhimurium
Salmonella Typhoid fever
typhi
Escherichia w/ gas Gastrointestinal
coli production flora
Citrobacter w/ gas Implicated in UTI
freundii production
Proteus w/ gas Genitourinary
vulgaris production tract infection
Motile
Klebsiella w/ gas Nosocomial
pneumonia production infection &
pneumonia
Pseudomonas Nosocomial
aeruginosa infection, wound
infection,
genitourinary
tract infection
Alcaligenes Opportunistic
fecalis bacteria

LYSINE IRON AGAR SLANT Tube K/K with H2S Salmonella

 for cultivation and differentiation of 1 (-) lysine deaminase Edwardsiella
(+) lysine decarboxylase
Enterobacteriaceae based on their
Tube K/A Enterobacter
ability to decarboxylate lysine and form
2 (-) lysine deaminase cloacae
H2S
(-) lysine decarboxylase Shigella
 has lysine, peptones, a small amount Glucose fermenter Citrobacter
of glucose, ferric ammonium citrate,
Tube K/K Klebsiella
and sodium thiosulfate
3 (-) lysine deaminase Serratia
 a differential agar slant (+) lysine decarboxylase E.coli
 to test the ability of an organism to Enterobacter
deaminate lysine or decarboxylate aerogenes
lysine
Tube R/A Proteus
 lysine decarboxylation takes place in 4 (+) lysine deaminase Providencia
the butt area; anaerobic process (-) lysine decarboxylase Morganella
 glucose fermentation takes place in the butt Glucose fermenter
 lysine deamination takes place in slant area;
aerobic process
 pH indicator: Bromcresol purple
 yellow – sugar has fermented – acid
 purple – no fermentation - alkaline

Lysine deamination
 Product: alpha keto acid
 Alpha keto acid reacts with iron salt present in
the surface of the agar. Once the reaction took
place, it will produce a red compound.
 (+) lysine deamination = red/burgundy
 (-) lysine deamination = purple
Lysine decarboxylation
 product: Cadaverine
 Cadaverine neutralizes the acid (from glucose
fermentation). Once neutralized, the resulting
product will be in alkaline state which is purple
in color.
 (+) lysine decarboxylation = purple
 (-) lysine decarboxylation = yellow
 Lecture 9  Stenotrophomonas maltophila
BIOCHEMICAL TESTS  Streptococcus pyogenes
Gram-Positive  Plesiomonas shigelloides
 Aeromonas hydrophila
CATALASE TEST  Moraxella catarrhalis
 used to differentiate (-) control Escherichia coli
Staphylococcus from
Streptococcus

Reagent 3% H2O2
(+) result Effervescence/bubble formation
(-) result No or few bubbles
(+) control S.aureus BACITRACIN (TAXO A) SUSCEPTIBILITY TEST
(-) control Streptococcus
 used to differentiate Streptococcus pyogenes
from other β -hemolytic Streptococci
COAGULASE TEST Reagent Bacitracin disk, 0.04 units (TAXO A)
 used to 5% sheep blood agar plate
differentiate (+) result Susceptible = Any zone of inhibition
Staphylococcus around the bacitracin disk
aureus from ≥10 mm
coagulase (-) (-) result Resistant = Uniform lawn of growth
staphylococcus up to the edge of the disk
(+) control Susceptible: Streptococcus pyogenes
(-) control Resistant: Streptococcus agalactiae

Slide Method Tube Method

Detects Bound/clumping Unbound/free
factor coagulase
Reagent EDTA rabbit plasma
(+) result Clumping Clot formation
(-) result No clumping No clot
(+) control S.aureus
(-) control S.epidermidis

DEOXYRIBONUCLEASE/DNAse TEST
CAMP (CHRISTIE, ATKINS, AND MUNCH-
 used to detect production of an active DNAse
PETERSEN) TEST
exoenzyme by aerobic bacterial species
Reagent  Differentiate Streptococcus agalactiae from
 DNAse agar with indicator dye
other β-hemolytic streptococci
(toluidine blue or methylene
Reagent β-Lysin–producing Staphylococcus
green)
aureus
 DNAse agar without indicator dye
Sheep blood agar plate
(+) result Methyl green = green to colorless
(+) result Arrowhead-shaped area of enhanced
Toluidine blue = blue to rose pink
hemolysis where the two
No indicator dye used = clear halo
streaks (staphylococcal and
around the colony
streptococcal) approach each other
(-) result Methyl green = green
(-) result No enhanced hemolysis
Toluidine blue = blue
(+) control Streptococcus agalactiae
No indicator dye used = no clearing
Other CAMP(+): Listeria
around colony
monocytogenes
(+) control Staphylococcus aureus
(-) control Streptococcus pyogenes
Other DNAse (+) = SSSPAM
CAMP inhibition reaction (reverse CAMP-
 Serratia spp.
positive)
 Inhibition of hemolysis by S. aureus where the Other Hippurate hydrolysis (+):
two streaks approach each other  Gardnerella vaginalis
 This reaction is characteristic of  Listeria monocytogenes
Arcanobacterium haemolyticum caused by  Campylobacter jejuni
phospholipase D. (-) control Streptococcus pyogenes
 Other reverse CAMP +: Clostridium perfringens
OPTOCHIN (TAXO P) TEST
 Susceptible and a presumptive identification of
S. pneumoniae (sensitive) from Viridans
(resistant)
Reagent 6ug or 10ug Taxo P
Sheep blood agar or chocolate agar
plate
(+) result Zone of inhibition
(-) result No zone of inhibition
(+) control Streptococcus pneumonia
(-) control Streptococcus viridans
PYRROLIDONYL-α-NAPTHYLAMIDE
HYDROLYSIS / PYR TEST
 Presumptive identification of the β-hemolytic
Streptococcus pyogenes and the nonhemolytic
group D streptococci Enterococci.
 Detects the ability of organism to produce L-
pyrrolidonyl arylamidase, also called
pyrrolidonyl aminopeptidase
Reagent PYR disk and N,N-
methylaminocinnamaldehyde NEUFELD—QUELLUNG TEST (CAPSULAR
(+) result Bright red or pink to cherry color SWELLING TEST)
(-) result No color change  Identify Streptococcus
(+) control Streptococcus pyogenes pneumoniae (possess capsule)
Other PYR (+): Enterococcus faecalis
(-) control Streptococcus agalactiae

Reagent Methylene blue solution

Polyvalent S. pneumoniae antisera
Rabbit serum
(+) result Swelling of Capsule
(-) result No swelling of Capsule
(+) control Streptococcus penumoniae
(-) control Streptococcus viridans
ENCAPSULATED BACTERIA
HIPPURATE HYDROLYSIS TEST
 Pseudomonas
 Differentiate Streptococcus aeruginosa
agalactiae from other β-hemolytic  Neisseria meningitidis
streptococci
 Klebsiella pneumoniae
 Determines the ability of organism
 Streptococcus
to produce hippuricase which
pneumoniae
splits hippuric acid into glycine
 Haemophilus influenzae
and benzoic acid
 Cryptococcus
Reagent 1% Sodium Hippurate
neoformans
Ninhydrin (detects glycine)
 Salmonella typhi
Ferric chloride (detects benzoic acid)
Sheep blood agar plate
BILE SOLUBILITY TEST
(+) result Deep purple color—indicates
Hippurate hydrolysis  Determines the ability of bacterial cells to lyse
in the presence of bile salts;
(-) result No color change or very slight purple
color  It is used to differentiate Streptococcus
(+) control Streptococcus agalactiae pneumoniae (bile-soluble) from other α-
 hemolytic streptococci NOTE: Negative result should be
(bile-insoluble) incubated for an additional
24-hour period
(+) control Group D Streptococci
(-) control Streptococcus pyogenes
Streptococcus viridans

Reagent Sodium deoxycholate

(+) result Lysis of colonies
Clearing of tube
(-) result No lysis produced
No autolysis tube remains cloudy
(+) control Streptococcus penumoniae
(-) control Streptococcus viridans

LEUCINE AMINOPEPTIDASE / LAP TEST SALT TOLERANCE

 Presumptive identification of catalase negative, 6.5% SALT TURBIDITY TEST
gram-positive cocci  Differentiate gram-positive cocci that grow in
Reagent LAP Disks (Disks impregnated with 6.5% NaCl from those that are inhibited by this
leucine-β-naphthylamide) salt concentration
(+) result Red color Reagent Nutrient broth base
(-) result No change in color 6.5% NaCl broth
(+) control Enterococcus fecalis Bromcresol purple
(-) control Leuconostoc spp. (+) result Turbidity or change in color from
Aerococcus purple to yellow
(-) result No turbidity
(+) control Enterococcus faecalis
(-) control Streptococcus pyogenes
Streptococci viridans
MODIFIED OXIDASE / MICRODASE TEST
 Rapidly differentiate staphylococci from
micrococci. Most staphylococci test negative,
whereas micrococci test positive
Reagent Microdase Disk
6% tetramethyl-p-phenylenediamine
BILE ESCULIN TEST dihydrochloride with dimethyl
 To differentiate group D streptococci and Sulfoxide
enterococci from other catalase negative, gram- Kovac’s oxidase test uses a 0.5% or
positive cocci 1% aqueous solution of tetramethyl-
 Group D streptococci and enterococci grow in ρ-phenylenediamine dihydrochloride
the presence of bile and also hydrolyze esculin (+) result Dark blue/deep purple/lavender
to esculetin and glucose. Esculetin diffuses into within 10 to 15 seconds
the agar and combines with ferric citrate in the (-) result Absence of dark blue/deep
medium to produce a black complex or brown- purple/lavender within 10 to 15
black precipitate seconds
Reagent Bile esculin agar (Esculin, 40% bile, (+) control Micrococcus
Ferric citrate) or Bile esculin slant (-) control Staphylococcus
(+) result Blackening of the agar
Brown to black precipitate
Presence of growth (can tolerate
40% bile)
NOTE: Positive result is often seen
within 4 hours. Growth alone does
not constitute a positive result.
(-) result No blackening of the agar
Absence of Brown to black precipitate
No growth
 Lecture 10 METHYL RED TEST
BIOCHEMICAL TESTS  Detects the ability of
organism to produce and
Gram-Negative
maintain stable acid end
products from glucose
OXIDASE TEST
fermentation (mixed acid
 Differentiation of production)
nonfermenters; aids
Reagent MRVP broth/peptone glucose broth
in identification of
Indicator: Methyl red
Neisseria,
Aeromonas, Vibrio, (+) result Red
and Campylobacter (-) result Yellow
spp; Detects enzyme Cytochrome-c oxidase (+) control  E.coli
Reagent 0.5-1.0% Tetramethyl-para- (-) control  Klebsiella
phenylenediamine or
paminodimethylaniline oxalate VOGES-PROSKAUER
(+) result Dark purple within 10-15 to 30 secs
(-) result No color change/absence of color  Detects the ability of organism
(+) control  Pseudomonas aeruginosa to produce neutral red end
Other oxidase +: products (acetoin or acetyl
 Moraxella methyl carbinol) from glucose
 Aeromonas fermentation.
 Neisseria
 Pasteurella Reagent  MVP Broth
 Campylobacter
 40% KOH or NaOH
 Vibrio
 Alpha-naphthol
(-) control  Enterobacteriaceae (+) result Red
NOTE:
 Do not use Nichrome loop as it contains iron (-) result Yellow
that might yield FALSE POSITIVE reaction use
platinum loop or wooden applicator stick.
(+) control  E.coli
 Excess reagent might cause weak reaction of
Oxidase positive bacteria.
(-) control  Klebsiella
 Media containing glucose may result in FALSE
POSITIVE reaction oxidase activity because of CITRATE UTILIZATION TEST
fermentation.
 Determines whether an organism can use
 Colorless colonies on selective media may result
sodium citrate as a sole carbon source
in FALSE POSITIVE reaction
Reagent  Simmons citrate agar
 MHA or nutrient agar may give inconsistent
(bromthymol blue as indicator) or
results
 Christensen citrate medium
(phenol red as indicator)
INDOLE TEST
(+) result Simmons citrate agar (green into
 Detects determine an
blue)
organism’s ability to form
Christensen citrate medium (yellow to
indole from tryptophan
pink)
with the enzyme
(-) result No growth or no change in color
tryptophanase
(+) control  E.coli
(-) control  Klebsiella

CETRIMIDE TEST
Reagent Tryptophan containing media (SIM,  Determines ability of oragnism to tolerate
tryptone/ peptone broth) cetrimide or also known as cetyl trimethyl
Kovac’s reagent/Ehlrich reagent (p- ammonium bromide or hexadecyltrimethyl
dimethylaminobenzaldehyde) ammonium bromide which is highly inhibitory
(+) result Red ring (pink to wine colored ring) to Pseudomonas aeruginosa and growth of
(-) result No red ring formation many bacteria.
(+) control  E.coli Reagent  Cetrimide agar (Pseudosel agar)
(-) control  Klebsiella medium or slant
(+) result Presence of growth (+) control  Proteus mirabilis
(-) result No growth (-) control  Acinetobacter baumannii
(+) control  Pseudomonas aeruginosa
(-) control  Escherichia coli N, N-dimethyl-a- Reacts or detect nitrite
naphthylamine (NO2) only
Sulfanilic acid =NITRATE REDUCER

(+) = Red
(-) = Colorless
Zinc powder Reacts or detects nitrate
(NO3) only
=NITRITE REDUCER

(+) = Colorless
(-) = Red

4-METHYLUMBELLIFERYL-B-D-
GLUCURONIDE/MUG TEST
 Detects ability of organism to produce enzyme
β-Glucuronidase;
 Presumptive identification of E. coli and
Streptococcus anginosus group;
enterohemorrhagic E. coli is negative
Reagent  MUG disk impregnated with 4-
methylumbelliferyl-β-glucuronide
(+) result Electric blue or bright blue
Reagent REAGENT A: Sulfanilic acid fluorescence
REAGENT B: N, N-dimethyl-α- (-) result Lack of fluorescence
naphthylamine (+) control  Escherichia coli
Zinc powder (to confirm true negative (-) control  Pseudomonas aeruginosa
result)
NOTE: Zinc powder is added to
confirm presence of nitrate or
reduced only.
(+) result Red color after addition of N, N-
dimethyl-α-naphthylamine and
Sulfanilic acid
(-) result Colorless after addition of N, N-
dimethyl-α-naphthylamine and
Sulfanilic acid
(+) control Escherichia coli
(-) control Acinetobacter baumannii

NITRITE NO2 REDUCTION TEST

 Determines whether an organism has the
ability reduce nitrite further to nitrogen gas
(N2)
Reagent REAGENT A: Sulfanilic acid
REAGENT B: N, N-dimethyl-α-
naphthylamine
Zinc (to confirm true negative result)
NOTE: Zinc powder is added to
confirm presence of nitrate or
reduced only.
(+) result Colorless
(-) result Red color (Nitrate not converted in the
first place)