BSML 3116 BACTERIOLOGY LABORATORY

ANTIMICROBRIAL SUSCEPTIBILITY TESTING

________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

Several key steps must be completed for an Antimicrobial agents that inhibit bacterial growth, but
antimicrobial agent to successfully inhibit or kill an generally do not kill the organism, are known as
infecting microorganism bacteriostatic agents. Effectively reducing the growth
rate of an organism provides adequate protection in
 First, the agent must be in an active form. This individuals whose immune system is capable of
is ensured through the pharmacodynamic removing the agent of infection.
design of the drug, which takes into account
the route by which the patient receives the Agents that usually kill target organisms are said to be
agent (e.g., orally, intramuscularly, bactericidal. Bactericidal agents are more effective
intravenously) against organisms that are more difficult to control in
 Second, the antibiotic must be able to achieve combination with the host’s immune system.
sufficient levels or concentrations at the site of
infection so that it has a chance to exert an
antibacterial effect (i.e., it must be in anatomic
approximation with the infecting bacteria).
o The ability to achieve adequate levels
depends on the pharmacokinetic
properties of the agent, such as rate of
In patients who are allergic to penicillin
absorption, distribution, metabolism, and who have a group A -hemolytic
and excretion of the agent’s streptococcal (Streptococcus pyogenes)
infection, erythromycin (or another
metabolites. macrolide) is the drug of choice.
Occasionally group A -hemolytic
o Some agents, such as ampicillin and streptococci are resistant to this agent.
Consequently, testing of erythromycin is
ceftriaxone, achieve therapeutically warranted
effective levels in several body sites,
whereas others, such as nitrofurantoin
and norfloxacin, are limited to the
urinary tract.
o Therefore knowledge of the site of
infection can substantially affect the
selection of the antimicrobial agent for
therapeutic use.
 The remaining steps in antimicrobial action
relate to direct interactions between the
antibacterial agent and the bacterial cell.

ANTIMICROBRIAL SUSCEPTIBILITY TESTING

 BSML 3116 BACTERIOLOGY LABORATORY
ANTIMICROBRIAL SUSCEPTIBILITY TESTING
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

suspending colonies grown overnight

Antimicrobial susceptibility testing is performed on on an agar plate directly in broth or
bacteria and fungi isolated from clinical specimens to saline.
determine which antimicrobial agents might be Pure inocula are obtained
 this direct inoculum
by selecting four or five
effective in treating infections caused by these colonies of the same suspension preparation
morphology, inoculating
organisms. Only organisms that are likely to be them into a broth medium, technique is preferred for
and allowing the culture to bacteria that grow
contributing to an infection should be tested. Testing achieve active growth
organisms that are not involved in an infection is For most organisms this
unpredictably in broth (e.g.,
misleading to the physician and could lead to a more requires 3 to 5 hours of fastidious bacteria)
incubation. Alternatively, four
serious infection with development of antimicrobial to five colonies 16 to 24  because it does not rely on
hours of age may be selected growth in an inoculum broth,
resistance. One of the major purposes of the clinical from an agar plate and
suspended in broth or 0.9% the use of fresh (16- to 24-
microbiology laboratory is to identify organisms that saline solution to achieve a
turbid suspension.
hour) colonies is imperative
are the cause of infections.

 Selection of Antibiotics Two Methods for Inoculum Preparation

 Generally, laboratories choose 10-15

1. Direct Colony Suspension
antibiotics to test susceptibility for Gram-
o Colonies must not be older than 18-24
Positive organisms and another 10-15 minutes
hours
for Gram-Negative organisms.
o Suspend the colonies in saline or broth
 Each laboratory should have a battery of
2. Saline Phase Growth
antibiotics ordinarily used for testing
o Used for most organisms that grow
 Drug formulary decided by medical staff,
rapidly except Staphylococci
pharmacists and medical technologists
o Log phase growth occurs after 2-8
hours incubation
Ideal Antimicrobial Agent

 Highly toxic to the microbe McFarland Turbidity Standards

 Non-toxic to the host
 Inoculum concentration of bacteria to be
 Not interfere with the ability of the host to
tested must be standardized
fight other diseases
 False-susceptible results may occur if too few
 Not lead to the development of drug resistance
bacteria are tested, and false-resistant results
may be the outcome of testing too many
Standardization of AST bacteria
 Inoculum Preparation  The most widely used method of inoculum
o most critical steps in susceptibility standardization involves comparing the
Properly prepared inocula testing turbidity of the inoculum preparation with
are the key to any o prepared by adding cells from four to McFarland turbidity standards
antimicrobial susceptibility
testing method. five isolated colonies of similar colony  McFarland standards can be prepared by
Inconsistencies in inoculum
preparation may lead to morphology growing on a adding specific volumes of 1% sulfuric acid and
inconsistencies and noninhibitory agar medium to a broth 1.175% barium chloride to obtain a barium
inaccuracies in
susceptibility test results. medium and then allowing them to sulfate solution with a specific optical density
The two important
requirements grow to the log phase  The most commonly used is the McFarland 0.5
for correct inoculum
o four to five colonies, rather than a standard, which contains 99.5 mL of 1%
preparation are use of a
pure culture single colony, are selected to minimize sulfuric acid and 0.5 mL of 1.175% barium
and use of a
standard-sized inoculum. the possibility of testing a colony that chloride
might have been derived from a  The McFarland 0.5 standard provides turbidity
susceptible mutant comparable with that of a bacterial suspension
o can also be prepared directly by containing approximately 1.5 × 108 CFU/mL
ANTIMICROBRIAL SUSCEPTIBILITY TESTING
 BSML 3116 BACTERIOLOGY LABORATORY
ANTIMICROBRIAL SUSCEPTIBILITY TESTING
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan
The newly inoculated bacterial suspension and McFarland standard are
compared by examining turbidity against a dark background.
concentration of an antibiotic is placed on the
Inoculum Standardization agar surface
 Using 0.5 Mcfarland standard (1.5 × 108  McFarland 0.5 standardized suspension of
CFU/mL) bacteria in Mueller-Hinton broth is swabbed
 To standardize the inoculum, the inoculated over the surface of a standardized Mueller-
broth or direct suspension is vortexed Hinton agar plate, and paper disks containing
thoroughly specific concentrations of antimicrobial agent
 Then, under adequate lighting, the tube is are placed onto the inoculated surface.
positioned side by side with the McFarland 0.5  after incubation of 16 to 18 hours, the
standard against a white card containing diameters of the zones produced by
several horizontal black lines antimicrobial inhibition of bacterial growth are
 The turbidities are compared by looking at the measured, and the result is interpreted as
black lines through the suspensions nonsusceptible, susceptible, intermediate, or
 The suspension is too dense if it is more resistant to a particular drug according to
difficult to see the lines through the inoculum preset criteria depth: 3-5 mm
diameter: 150 mm
suspension than through the McFarland 0.5
standard Interpretation
 Once standardized, the inoculum suspensions  Susceptible “S” 17 mm
should be used within 15 minutes of o Interpretive category that indicated an
preparation organism is inhibited by the
 A convenient and more precise alternative to recommended dose, at the infection
visual adjustment to match the McFarland site of an antimicrobial agent
standard is the use of a nephelometric or  Intermediate “I” 14-16 mm
spectrophotometric device o Interpretive category that represents
an organism that may require a higher
Components in Standardization of AST dose of antibiotic for a longer period of
 Bacterial inoculum size time to be inhibited
 Growth medium  Resistant “R” 13 mm
o pH o Interpretive category that indicated an
o Cation concentration organism is not inhibited by the
o Blood and serum supplements recommended dose, at the infection
o Thymidine content site of an antimicrobial agent
 Incubation atmosphere
 Incubation temperature
 Incubation duration
 Antimicrobial concentrations

Disk Diffusion Method (Kirby Bauer Method)

 widely used in clinical laboratories since 1966,
when the first standardized method was
described
 more suitable for routine testing in a clinical
laboratory where a large number of isolates
are tested for susceptibility to numerous
antibiotics
 an agar plate is uniformly inoculated with the
test organism
 a paper disk impregnated with a fixed
ANTIMICROBRIAL SUSCEPTIBILITY TESTING

1-2 mL: macrodilution 0.05-1 mL: microdilution shelf life of agar: 1 week at 2-8 degrees celcius
 BSML 3116 BACTERIOLOGY LABORATORY
ANTIMICROBRIAL SUSCEPTIBILITY TESTING
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

which allows networking with other BD

microbiology instruments and a bidirectional
Automation of AST interface for connection with an LIS.
 Detect growth in microvolumes of broth with
various dilutions of antimicrobials MicroScan WalkAway
 Detection via photometric, turbidimetric or
fluorometric methods four instruments are available that  The MicroScan WalkAway plus and MicroScan
 Types: are capable of generating rapid (5
to 15 hours) or overnight (16 to 24 SI (Beckmann Coulter) consist of a large, self-
o BD Phoenix hours) susceptibility test results. contained incubator-reader unit with capacity
o Microsan Walkaway for 40 or 96 test panels, LabPro information
o TREK Sensitive management software, and printer.
o Vitek 1 and 2  The WalkAway uses microdilution panels that
are hydrated and inoculated with a hand-
BD Phoenix System operated inoculator device.
 Panels are then placed in one of the positions
 The newest automated susceptibility testing in the large incubator module.
instrument is the BD Phoenix automated  The type of test to be performed is indicated
microbiology system (BD Diagnostic Systems, on an instrument-readable bar code label
Sparks, MD). It is marketed in Europe, Japan, located on the end of each panel.
Canada, and the United States.  The instrument incubates the panels for the
 The Phoenix system consists of an upright appropriate period (depending on the type of
instrument with a built-in keyboard and bar- panel and organism), robotically positions the
coding station that can accommodate 100 test trays to add reagents if needed, and moves
panels simultaneously, along with a separate them under the central photometer station to
printer. perform the final readings of growth end
 The specially designed test cartridges contain points at the conclusion of the tests.
136 small wells to test as many as 25 different  Standard dried panels are read
antimicrobial agents, alone or in combination turbidimetrically by the Walk Away after 16 to
with wells containing biochemical substrates 18 hours of incubation with 24-hour holds for
for simultaneous identification of common specific bacteria (e.g., oxacillin for
gram-positive or gram negative bacteria, Staphylococcus and vancomycin for
including streptococci. Enterococcus; consult manufacturer’s
 The test panels are inoculated manually by a directions).
simple, gravity-fed transfer of inoculated  The instrument offers the possibility of early,
medium throughout the disposable cartridge read-when-ready interpretations of the
after pouring inoculated broth through an standard panels in 4.5 to 6.5 hours if an isolate
opening in the test device. is unequivocally susceptible or highly resistant.
 The Phoenix system uses a redox indicator  Results are available in 2 to 2.5 hours.
system (similar to resazurin) to measure  This older rapid technology, however, is being
bacterial growth in the susceptibility test wells. phased out in favor of the read-when-ready
 The indicator is added to the broth at the time approach.
of organism inoculation.  The instrument includes a rules-based expert
 This approach allows susceptibility system (LabPro AlertEX) and can be purchased
determinations after approximately 6 to 8 with a software package called LabPro, which
hours of incubation. allows networking with other MicroScan
 The Phoenix instrument includes a rules-based analyzers and workstations and a bidirectional
expert system (BDXpert) and can be purchased interface with an LIS.
with a data management personal computer
and software package called BD EpiCenter,

ANTIMICROBRIAL SUSCEPTIBILITY TESTING

 BSML 3116 BACTERIOLOGY LABORATORY
ANTIMICROBRIAL SUSCEPTIBILITY TESTING
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

hold the test cards, a robotic system to

TREK Sensititre manipulate the cards, a photometer for
 The Sensititre automated incubator reader turbidimetric measurement of growth once
(ARIS 2X, TREK Diagnostic Systems) was the per hour, and a computer module with a video
first system to be marketed in the United display terminal and printer for viewing and
States that used a fluorometric detection printing results.
system for detecting growth end points of  VITEK 1 also offers an information
common, rapidly growing bacteria using MIC management system for storing and retrieving
or breakpoint formats with a 5- or 18-hour test data for a variety of statistical reports.
incubation period.  A newer instrument is the VITEK 2 which
 Subsequent experience using rapid fluorogenic automates the initial sample processing steps
substrate detection of growth with this system to a greater degree than the VITEK 1.
and one other system demonstrated that some  It facilitates adjustment of the inoculum
resistance mechanisms cannot be accurately density, transfer of inoculum suspension to the
detected in 5–hours. cards, and sealing of the cards in a single
 For this reason, TREK has FDA clearance for instrument module.
only the 16- to 24-hour incubation version of  The VITEK 2 cards are slightly thicker than
the instrument in the United States for those of the VITEK 1 and contain 64 wells to
susceptibility testing. allow testing of more drugs in a single card.
 The Sensititre ARIS 2X is a fully automatic,  The VITEK 2 incorporates bar code labeling of
bench top incubating and reading system. It cards and storage of isolate data programmed
fits onto the AutoReader and uses an internal by the smart carrier workstation in a computer
bar code scanner to identify each plate type chip embedded in a cassette that moves the
and assign the appropriate incubation time; cards through the instrument.
when the assigned time has elapsed, the plate  The VITEK 2 cards are read turbidimetrically
is then transported to the AutoReader for every 15 minutes and analyzed according to
fluorescence measurement, with no manual several computer algorithms specific to each
intervention. drug and organism.
 The AutoReader holds as many as 64 plates.  The VITEK 2 can test S. pneumoniae in addition
to the nonfastidious, aerobic gram-positive
and gram-negative bacteria.
The VITEK  The VITEK 2 offers one of the highest levels of
automation currently available in microbiology
 This system (bioMérieux Vitek, Hazelwood, instrumentation.
MO) was originally designed for use in the U.S.
space exploration efforts of the 1970s as an The Mueller-Hinton preparation is the standard agar-base medium used for
testing most bacterial organisms, although certain supplements and substitutions
onboard test system for spacecraft exploring are required for testing fastidious organisms.
other planets for life.
 Because of its original design intention, it was If the agar is too thick, the antimicrobial agent diffuses down through the agar
as well as outward, resulting in smaller zone sizes, which may cause
highly automated and relatively compact. errors in interpretation (e.g., false-resistance); if the agar is too thin, the
Small plastic reagent cards (similar in size to a inhibition zones are larger, which may result in a false-susceptible interpretation.

credit card) contain microliter quantities of

In most instances, a maximum of 12 antibiotic disks may be applied to the
various concentrations of antimicrobial agents surface of a single 150-mm Mueller-Hinton agar plate
in 45 wells for susceptibility testing. Most commonly tested bacteria (e.g., Enterobacteriaceae, P. aeruginosa,
 The VITEK (now called the VITEK 1) can be staphylococci, and enterococci), the environmental condition consists of room air at
35°C. Fastidious bacteria, such as H. influenza and Neisseria gonorrhoeae, require
configured to accommodate 30, 60, 120, or incubation in 5% to 10% carbon dioxide (CO2)
240 cards. A dark background and reflected light are used to examine disk diffusion plat
 VITEK 1 hardware consists of a filling module For long-term storage, disks are stored at 20° C or below in a non–frost-free freezer. A working
supply of disks can be stored in a refrigerator at 2° to 8° C for at least 1 week. The container should
for inoculation of the cards, an incubator- be allowed to warm to room temperature before it is opened to prevent condensation from forming on
the disks when warm room air contacts the cold disks
reader module that incorporates a carousel to
ANTIMICROBRIAL SUSCEPTIBILITY TESTING
 BSML 3116 BACTERIOLOGY LABORATORY
STAPHYLOCOCCI, STREPTOCOCCI, ENTEROCOCCI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

General Characteristics Note:

 Gram-positive cocci (facultative)  Study Table 13-3 on Baileys

o Singly, in pairs, or in clusters (“bunches  Table 14.4 on Mahon
of grapes”)  Table 13-4 on Baileys
 Catalase positive  Table 14.6 on Mahon
 Coagulase positive (differentiates micrococci)  Table 13-5 on Baileys
 Non-motile, non-spore forming and aerobic or  Table 13-6 on Baileys
facultative anaerobic (some are obligate  Table 13-7 on Baileys
anaerobes)  Figure 14.8 on Mahon
 Colonies
o Appear cream colored, white or rarely
light gold, and “buttery looking” Additional Test
o 18-24 hours of incubation at 35 to 37  Serological test
degrees Celsius  Molecular testing
 Beta-hemolytic  Matrix assisted laser desorption ionization
 Small colony variants (SCVs) time of flight mass spectrometry (maldi-tof ms)
 Coagulase negative staphylococci (CoNS) – MRSA
o Novobiocin susceptibility  PCR/Electrospray ionization-mass
spectrometry (PCR/ESI-MS)
Specimen Collection and Processing
Antimicrobial Susceptibility testing
 No special consideration are required
 Proper procedure should be implemented  Methicillin-resistant staphylococcus aureus
(MRSA)
 Penicillin (oxacillin and Nafcillin)
Macroscopic Examination
 mecA gene – beta lactams
 Gram-positive cocci (spherical)  Cefoxitin is a better inducer of mecA mediated
 Pairs, tetrads and ultimately irregularly resistance
clusters  All susceptible to b-lactam antibiotics
 Seen along with polymorphonuclear cells in (carbapenems) except fifth generation
purulent exudates cephalosporin
 Vancomycin resistant staphylococci
Cultivation  Macrolide test
 5% sheep blood agar (primary) and chocolate
agars To differentiate staphylococci in enterococci and
o 35-37 degrees Celsius in carbon streptococci, use catalase test
dioxide (CO2) or ambient air (24 hours)
 Nutrient broth (thioglycolate, dextrose broth Susceptible: zone inhibition (positive)
and brain heart infusion)
Resistant: no zone inhibition (negative)
 Selective media
o Phenylethyl Alcohol (PEA)
o Columbia Colistin-Nalidixic Acid (CAN)
o Mannitol Salt Agar (MSA)
 48 to 72 hours
o Chromoagar

STAPHYLOCOCCI
 BSML 3116 BACTERIOLOGY LABORATORY
STAPHYLOCOCCI, STREPTOCOCCI, ENTEROCOCCI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

General Characteristics (Streptococci, Enterococci) Note:

 Gram positive cocci (in pairs or in chains)  Table 14-5 on Baileys

 Catalase negative  Table 15.2 on Mahon
 Facultative anaerobes = aerotolerant  Table 14-6 on Bailey
anaerobes  Figure 14-7 on Bailey
 Carbohydrate fermentation (lactic acid)  Figure 15.15 on Mahon
 Capnophilic  Table 15.3 on Mahon
 Colonies (small and translucent)- enhanced by
blood, serum and glucose
Antimicrobial Susceptibility testing
Specimen Collection and Processing  Penicillin (except streptococcus pneumoniae)
 No special consideration are required  Erythromycin
 Proper procedure should be implemented  Narrow spectrum cephalosporin
 Vancomycin
Microscopy
 Gram positive cocci in singly and in chains
 Round, oval-shaped or rod-like

Cultivation
 5% sheep blood agar and chocolate agars
(except abiotrophia and granulicatella)
 Selective
o Columbia agar with colistin and
nalidixic acid (CNA) and phenylethyl
alcohol agar (PEA)
 Enrichment – pyridoxal (Vitamin B6) –
abiotrophia and granulicatella
 Nutrient broths, such as thioglycollate or brain
heart infusion
 Supplementation –
trimethoprimsulfamethoxazole (SXT) – GAS
 Todd Hewitt broths (LIM) contain
antimicrobials (gentamicin, nalidixic acid, or
colistin and nalidixic acid) – GBS
 Chromoagar- GBS
enterococcosel agar is a selective differential
medium based on the esculin hydrolysis
 Preferring a CO2 enriched environment
 5-10% carbon dioxide

Classification Schemes
 Hemolytic pattern on SBA
 Physiologic characteristics
 Serologic grouping or typing of carbohydrate
 Biochemical characteristics
STAPHYLOCOCCI
 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

Corynebacterium Spp.

 Non-spore-forming, nonbranching, catalase- often must be incubated for at least 48

positive bacilli hours before growth is detected
 Gram stain: slightly curved o However, growth is enhanced if lipids
o The term diphtheroid, meaning are included in the culture medium
“diphtheria-like,” is sometimes used in  The most significant pathogen of the group, C.
reference to this Gram staining diphtheriae, has been extensively studied and
morphology is well characterized
 Gram positive: club shape or coryneform o Disease caused by C. diphtheriae is
 Culture: loeffler medium and cystine-tellurite referred to as diphtheria
blood agar (CTBA) C. diphtheriae is a highly pleomorphic (many shapes)
 Most of the species are found as normal biota gram-positive bacillus that appears in palisades (cells lie
on the skins and mucous membranes of in parallel rows) or as individual cells lying at sharp
angles to another in “V” and “L” formations.
humans and animals
 Some species are found worldwide in the Facultative anaerobe. It grows best under aerobic
conditions and has an optimal growth temperature of 37°
environment C, although multiplication occurs within the range of 15°
 Corynebacteria can be divided into to 40° C
nonlipophilic and lipophilic species
o Lipophilic corynebacteria are often
considered fastidious and grow slowly
on standard culture media; cultures

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

 L. monocytogenes can resemble Streptococcus

C. diphtheriae C. C. ulcerans when found in the coccoid form and
pseudotuberculosis Corynebacterium when the bacillus forms
+ Tinsdale + Tinsdale Halo + Tinsdale prevail
Halo Halo  Organisms are not usually seen on the CSF
V: B- + B-hemolysis V: B- smear
hemolysis hemolysis  The optimal growth temperature for L.
- - + Gelatin monocytogenes is 30° to 35° C, but growth
occurs over a wide range (0.5° to 45° C)
 Because L. monocytogenes grows at 4° C, a
S. pseudodiphtheriticum and S. ulcerans are REVERSE
technique called cold enrichment
for CAMP test  L. monocytogenes is hippurate hydrolysis
S. jeikeium and S urealyticum are POSITIVE for positive like S. agalactiae, but unlike S.
LIPOPHILIC agalactiae, it is catalase positive, bile esculin
Non lipophilic hydrolysis positive, and motile at room
Corynebacterium amycolatum
Corynebacterium striatum
temperature
 L. monocytogenes exhibits tumbling motility
Lipophilic (end-over-end motility) when viewed
Corynebacterium urealyticum
Corynebacterium jeikeium microscopically.
o In motility medium, the characteristic
“umbrella” pattern is seen when the
organism is incubated at room
temperature (22° to 25° C) but not at
35° C
 L. monocytogenes produces a positive CAMP
reaction
 L. monocytogenes produces a “block”-type
hemolysis with the CAMP test
o This type of hemolysis is in contrast to
the “arrowhead” type (reverse)
 L. monocytogenes, and other Listeria species,
are identified well by MALDI-TOF
 Listeria monocytogenes

 Gram-positive coccobacillus
 Culture: prefers a slightly increased carbon
dioxide tension – cold enrichment
 L. monocytogenes is widespread in the
environment and has been recovered from
soil; water; vegetation; and animal products,
such as raw milk, cheese, poultry, and
processed meats
 L. monocytogenes produces many products
that have been proposed as virulence factors.
o hemolysin (listeriolysin O)
o catalase
o superoxide dismutase
o phospholipase C
o surface protein (p60)

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

Listeria monocytogenes are motile “kiat” Stab motility: Umbrella

Gram stain in strep: Gram positive cocci
Gram stain in Corynebacterium and listera: Wet Preparation: Tumbling motility
Gram positive bacilli o Trichomonas vaginalis - parasite nga kiat
Catalase
o Both are positive
Esculin Hydrolysis: Erysipelothrix rhusiopathiae
o + Listeria
o – Corynebacterium  Gram positive (variable)
Motility  Catalase negative
o + Listeria  Non-spore-forming
o V: Corynebacterium  Pleomorphic rod – long filaments
B hemolysis and growth in 6.5 NaCl  Culture: nutrient both with 1% glucose and
o + Listeria incubated in 5% carbon dioxide at 35°C
o V: Corynebacterium  It is found worldwide and is a commensal or a
pathogen in a wide variety of vertebrates and
invertebrates, including domestic swine, birds,
and fishes
 E. rhusiopathiae produces three types of
disease in humans:
o Erysipeloid: which is a localized skin
disease
o Septicemia: which is often associated
with endocarditis
o Generalized: diffuse cutaneous
infection
 E. rhusiopathiae is a thin, rod-shaped, gram-
positive organism that can form long filaments
 It is arranged singly, in short chains, or in a “V”
shape

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

Gardnerella vaginalis

 Short, pleomorphic gram-positive rod or

coccobacillus
 Gram variable or gram negative – “clue cells”
 Culture: 5%-7% carbon dioxide at a
temperature of 35-37°C (24 hours) – human
blood bilayer tween (HBT) agar
 G. vaginalis is primarily known for its
association with bacterial vaginosis (BV) in
humans

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
AEROBIC GRAM-POSITIVE BACILLI
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

Bacillus

 Spore forming, nonbranching, catalase-

positive bacilli
 Aerobic or facultative anaerobic bacilli that for
endospores
 Members of the genus Bacillus are
metabolically diverse, and some species are
thermophiles that grow best at 55° C or higher
 Most species grow well on SBA and other
commonly used enriched media but typically
do not grow on Columbia colistin-naladixic acid
agar

AEROBIC GRAM-POSITIVE BACILLI

 BSML 3116 BACTERIOLOGY LABORATORY
MYCOBACTERIA
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

 General characteristics Identification

 Slender, slightly curved or straight, rod-shaped  Rate of growth

organism o slowly growing species are typically
 Although acid-fast, they are considered to be raised, with a dry, rough appearance
gram positive o Optimal growth occurs at 35° to 37° C
 Strictly aerobic but enhanced its growth with  Colony morphology
increased carbon dioxide concentration o The colonies are nonpigmented and
o The pathogenic mycobacteria grow classically described as being buff
more slowly than most other bacteria colored
pathogenic to humans  Pigmentation
o Most mycobacteria associated with  Nutritional requirement
disease require 2 to 6 weeks of  Optimal incubation temperature
incubation on complex media at  Biochemical test results
specific optimal temperatures.

Advancement
Mycobacterium spp. have been classified according to
 Broth-based culture system
phenotypic characteristics.  Polymerase chain reaction
 High performance liquid chromatography
Some species may display a branching morphology.

They are non-motile and do not form spores. Laboratory Safety Considerations

The cell wall has extremely high lipid content; thus  Personnel Safety
mycobacterial cells resist staining with commonly used o Provided with adequate safety
basic aniline dyes, such as those used in Gram stain, at equipment
o Trained in safe laboratory procedures
room temperature.
o Informed of the hazards associated
Mycobacteria take up dye with increased staining time with the procedures
or application of heat but resist decolorization with acid o Prepared for action following an
ethanol. This characteristic, referred to as acid unexpected accident
fastness—hence the term acid-fast bacilli (AFB) o Monitored regularly by medical
personnel
distinguishes mycobacteria from most other genera.
o Laboratory personnel must use
appropriate safety equipment and
The rapidly growing species generally grow on simple
follow established procedures
media in 2 to 3 days at temperatures of 20° to 40° C. o A skin test (Mantoux) with PPD should
be administered on the first day of
employment and thereafter regularly
 Classification to persons previously skin test
negative
 Mycobacterium tuberculosis complex and non- o Individuals known to be previously skin
tuberculosis mycobacteria test positive should be counseled
 Rapid growers and slow growers regularly and referred for medical
 Prominent pathogens: evaluation if their health status
o Mycobacterium tuberculosis complex changes
o Mycobacterium leprae o The minimum level of respiratory
o Mycobacterium ulcerans protection is a respirator that contains
a National Institute for Occupational

MYCOBACTERIA
 BSML 3116 BACTERIOLOGY LABORATORY
MYCOBACTERIA
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

Safety and Health–certified N series 30 minutes

filter with a 95% efficiency rating (N- o Sodium hypochlorite loses
95) effectiveness in the presence of a large
o In addition to being trained to use the amount of protein material
respirator, laboratory staff must be fit- o Phenol-soap mixtures containing
tested ortho-phenylphenol, such as Amphyl
 Ventilation (Reckitt Benckiser North America,
o The mycobacteriology laboratory Wayne, NJ), or other phenolic
should be separate from the derivatives are effective with contact
remainder of the laboratory and have periods of 10 to 30 minutes
a non-recirculating ventilation system o Solutions of 5% phenol are no longer
o The area in which specimens and recommended because of toxicity to
cultures are processed should have humans
negative air pressure in relation to
other areas; that is, the air flow should Specimen Collection
be from clean areas, such as corridors,
into less clean areas  Sterile, wide-mouthed cup with a tightly fitted
(mycobacteriology laboratory) lid
o Six to 12 room air changes per hour  Special sterile receptacles containing a 50-mL
effectively remove 99% or more of centrifuge tube for sputum collection are also
airborne particles within 30 to 45 commercially available
minutes.  Because of small sample volumes, the use of
o A much higher number of room air swabs for clinical specimens is discouraged
changes per hour can cause problems  As with all specimens for microbiological
of air turbulence within the biological examination, aseptic collection is important
safety cabinets
 Biological Safety cabinet
o To prevent the dispersal of infectious
aerosols into the laboratory area, all
potentially infectious materials should
be tightly covered when they are
outside the biological safety cabinet
o Specimens should be centrifuged in
aerosol-free safety carriers, and the
tubes should be removed from the
safety carriers only inside the
biological safety cabinet
o To avoid aerosols, an alcohol-sand
flask can be used to clean the waxy
culture material from the wire before
flaming it in a Bunsen burner
o BSC II
 Use of proper disinfectant
o Sodium hypochlorite is effective at a
concentration of 0.05% to 0.5% (e.g., a
1:50 to 1:10 dilution of most
household bleaches)
o The solution should be made fresh
daily, and contact time should be 10 to

MYCOBACTERIA
 BSML 3116 BACTERIOLOGY LABORATORY
MYCOBACTERIA
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

MYCOBACTERIA
 BSML 3116 BACTERIOLOGY LABORATORY
MYCOBACTERIA
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

 Rejection Criteria

 Insufficient volume
 Contamination with saliva
 Dried swabs
 Pooled sputum or urine
 Broken or leaking
 Delayed transportation

 Staining for Acid-Fast bacilli

 Ziehl-Neelsen and Kinyoun

 Auramine stain (Auramine-Rhodamine
Fluorochrome stain)

MYCOBACTERIA
 BSML 3116 BACTERIOLOGY LABORATORY
NEISSERIA SPP. AND MORAXELLA CATARRHALIS
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

NEISSERIA SPP. AND MORAXELLA CATARRHALIS

 BSML 3116 BACTERIOLOGY LABORATORY
NEISSERIA SPP. AND MORAXELLA CATARRHALIS
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

General Characteristics
 Obligate aerobes, non-motile and non-
hemolytic
 Fastidious, capnophilic and grow optimally in a
moist environment
 Most species are carbohydrate fermenters
 Grows best on media containing blood and
cholesterol
 All species are cytochrome oxidase and
catalase positive except for N. elongata subsp.
nitroreducens and N. bacilliformis, which are
catalase negative
 The natural habitats of Neisseria spp. are the
mucous membranes of the respiratory and
urogenital tracts
 Neisseria gonorrhoeae (often called gonococci)
and Neisseria meningitidis (meningococci) are
the primary human pathogens of the genus
 N. gonorrhoeae is not considered part of the
normal biota and is always pathogenic
 Both N. gonorrhoeae and N. meningitidis
require iron for growth
 Gram-negative diplococci that are coffee-bean
or kidney bean shaped
 Cultures: small, grayish-white, opaque, convex Moraxella Catarrhalis
and glistening
 Branhamella Catarrhalis
o The medium of choice for cultivation
 Non-motile, fastidious, b-lactamases and
of N. gonorrhoeae is CHOC agar
encapsulated with common pili
o
 Gram-negative intracellular diplococci/
 “Hockey puck” or “wagon wheel” appearance
Specimen Collection
(colonies)
o The organism grows on SBA and CHOC
 Sensitive to drying and temperature extremes
agar
 Transport media (contain with charcoal, with 6
 M. catarrhalis is an opportunistic pathogen
hours) = JEMBEC
and is recognized as a cause of upper
 Cotton swabs are not advised
respiratory tract infection in otherwise healthy
 The swab containing the specimen should be
children and in older adults
rolled in a “Z” pattern on the medium
 Blood culture is not advised due to the activity
of SPS

NEISSERIA SPP. AND MORAXELLA CATARRHALIS

 BSML 3116 BACTERIOLOGY LABORATORY
NEISSERIA SPP. AND MORAXELLA CATARRHALIS
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

NEISSERIA SPP. AND MORAXELLA CATARRHALIS

 BSML 3116 BACTERIOLOGY LABORATORY
NEISSERIA SPP. AND MORAXELLA CATARRHALIS
________________________________________________________________________
____ YEAR 1st SEM: S.Y 2023 – 2024
THIRD Bachelor of Science in Medical Laboratory Science
LECTURER: PATRICK TUDAS Gwyn Daphne Mancawan

NEISSERIA SPP. AND MORAXELLA CATARRHALIS